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In 1798, intrigued by the fact that milk- old bacterial culture. ...... (a) Viruses: Trans- worm, being attacked by macr...

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KUBY

Immunology

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Icons Used in This Book Antigenic peptide

T cell receptor

Antibody

CD3

Immature thymocyte

TH cell

CD8

TC cell

Plasma cell

B cell

CD4

Class I MHC

Cytokine

Class II MHC

Cytokine receptor

Cytotoxic T cell

Bone marrow stromal cell

Neutrophil

Basophil

Eosinophil

Dendritic cell

Monocyte

Macrophage

Class I MHC

Erythrocyte

Antigen-presenting cell

CD4 Altered self cell

B cell

Platelets

Mast cell

Class II MHC

CD8 TC cell

Natural killer cell

TH cell

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KUBY

Immunology Judith A. Owen Haverford College

Jenni Punt Haverford College

Sharon A. Stranford Mount Holyoke College with contributions by

Patricia P. Jones

Seventh Edition

Stanford University

W. H. Freeman and Company • New York

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Publisher: Susan Winslow Senior Acquisitions Editor: Lauren Schultz Associate Director of Marketing: Debbie Clare Marketing Assistant: Lindsay Neff Developmental Editor: Erica Champion Developmental Editor: Irene Pech Developmental Coordinator: Sara Ruth Blake Associate Media Editor: Allison Michael

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Library of Congress Control Number: 2012950797 North American Edition Cover image: ©2009 Pflicke and Sixt. Originally published in The Journal of Experimental Medicine. 206:2925-2935. doi:10.1084/jem.20091739. Image provided by Holger Pflicke and Michael Sixt. International Edition Cover design: Dirk Kaufman Cover image: Nastco/iStockphoto.com

Supplements Editor: Yassamine Ebadat Senior Project Manager at Aptara: Sherrill Redd Photo Editor: Christine Buese Photo Researcher: Elyse Reider Art Director: Diana Blume Text Designer: Marsha Cohen Illustrations: Imagineering Illustration Coordinator: Janice Donnola

North American Edition ISBN-13: 978-14292-1919-8 ISBN-10: 1-4292-1919-X International Edition ISBN-13: 978-14641-3784-6 ISBN-10: 1-4641-3784-6 © 1992, 1994, 1997, 2000, 2003, 2007, 2013 by W. H. Freeman and Company All rights reserved

Production Coordinator: Lawrence Guerra Composition: Aptara®, Inc. Printing and Binding: RR Donnelley

Printed in the United States of America First printing North American Edition W. H. Freeman and Company 41 Madison Avenue New York, NY 10010 www.whfreeman.com International Edition Macmillan Higher Education Houndmills, Basingstoke RG21 6XS, England www.macmillanhighered.com/international

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To all our students, fellows, and colleagues who have made our careers in immunology a source of joy and excitement, and to our families who made these careers possible. We hope that future generations of immunology students will find this subject as fascinating and rewarding as we have.

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About the Authors All four authors are active scholars and teachers who have been/are recipients of research grants from the NIH and the NSF. We have all served in various capacities as grant proposal reviewers for NSF, NIH, HHMI, and other funding bodies as well as evaluating manuscripts submitted for publication in immunological journals. In addition, we are all active members of the American Association of Immunologists and have served our national organization in a variety of ways.

Judy Owen holds B.A. and M.A. (Hons) degrees from Cambridge University. She pursued her Ph.D. at the University of Pennsylvania with the late Dr. Norman Klinman and her postdoctoral fellowship with Dr. Peter Doherty in viral immunology. She was appointed to the faculty of Haverford College, one of the first undergraduate colleges to offer a course in immunology, in 1981. She teaches numerous laboratory and lecture courses in biochemistry and immunology and has received several teaching and mentorship awards. She is a participant in the First Year Writing Program and has been involved in curriculum development across the College.

Jenni Punt received her A.B. from Bryn Mawr College (magna cum laude) majoring in Biology at Haverford College, She received her VMD (summa cum laude) and Ph.D. in immunology from the University of Pennsylvania and was a Damon Runyon-Walter Winchell Physician-Scientist fellow with Dr. Alfred Singer at the National Institutes of Health. She was appointed to the faculty of Haverford College in 1996 where she teaches cell biology and immunology and performs research in T cell development and hematopoiesis. She has received several teaching awards and has contributed to the development of college-wide curricular initiatives. Together, Jenni Punt and Judy Owen developed and ran the first AAI Introductory Immunology course, which is now offered on an annual basis.

Sharon Stranford obtained her B.A. with Honors in Biology from Arcadia University and her Ph.D. in Microbiology and Immunology from Hahnemann (now Drexel) University, where she studied autoimmunity with funding from the Multiple Sclerosis Foundation. She pursued postdoctoral studies in transplantation immunology at Oxford University in England, followed by a fellowship at the University of California, San Francisco, working on HIV/AIDS with Dr. Jay Levy. From 1999 to 2001, Sharon was a Visiting Assistant Professor of Biology at Amherst College, and in 2001 joined the faculty of Mount Holyoke College as a Clare Boothe Luce Assistant Professor. She teaches courses in introductory biology, cell biology, immunology, and infectious disease, as well as a new interdisciplinary course called Controversies in Public Health.

Pat Jones graduated from Oberlin College in Ohio with Highest Honors in Biology and obtained her Ph.D. in Biology with Distinction from the Johns Hopkins University. She was a postdoctoral fellow of the Arthritis Foundation for two years in the Department of Biochemistry and Biophysics at the University of California, San Francisco, Medical School, followed by two years as an NSF postdoctoral fellow in the Departments of Genetics and Medicine/ Immunology at Stanford University School of Medicine. In 1978 she was appointed Assistant Professor of Biology at Stanford and is now a full professor. Pat has received several undergraduate teaching awards, was the founding Director of the Ph.D. Program in Immunology, and in July, 2011, she assumed the position of Director of Stanford Immunology, a position that coordinates activities in immunology across the university.

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Contents

SUMMARY

23

REFERENCES

23

1

USEFUL WEB SITES

23

2

STUDY QUESTIONS

24

Chapter 1

Overview of the Immune System A Historical Perspective of Immunology Early vaccination studies led the way to immunology

2

Vaccination is an ongoing, worldwide enterprise

3

Chapter 2

Immunology is about more than just vaccines and infectious disease

4

Immunity involves both humoral and cellular components

6

How are foreign substances recognized by the immune system?

Cells, Organs, and Microenvironments of the Immune System

9

Important Concepts for Understanding the Mammalian Immune Response Pathogens come in many forms and must first breach natural barriers The immune response quickly becomes tailored to suit the assault Pathogen recognition molecules can be encoded in the germline or randomly generated Tolerance ensures that the immune system avoids destroying the host The immune response is composed of two interconnected arms: innate immunity and adaptive immunity Adaptive immune responses typically generate memory

The Good, Bad, and Ugly of the Immune System

11 12 12

Cells of the Immune System

27 27

Hematopoietic stem cells have the ability to differentiate into many types of blood cells

28

Hematopoeisis is the process by which hematopoietic stem cells develop into mature blood cells

32

Cells of the myeloid lineage are the first responders to infection

32

Cells of the lymphoid lineage regulate the adaptive immune response

37

14 15

16

Primary Lymphoid Organs— Where Immune Cells Develop

41

The bone marrow provides niches for hematopoietic stem cells to self-renew and differentiate into myeloid cells and B lymphocytes

41

The thymus is a primary lymphoid organ where T cells mature

41

17

19

Secondary Lymphoid Organs— Where the Immune Response Is Initiated

48

Secondary lymphoid organs are distributed throughout the body and share some anatomical features

48

22

Lymphoid organs are connected to each other and to infected tissue by two different circulatory systems: blood and lymphatics

48

22

The lymph node is a highly specialized secondary lymphoid organ

50

Inappropriate or dysfunctional immune responses can result in a range of disorders

19

The immune response renders tissue transplantation challenging Cancer presents a unique challenge to the immune response

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Contents The spleen organizes the immune response against blood-borne pathogens

53

Signal-induced PIP2 breakdown by PLC causes an increase in cytoplasmic calcium ion concentration

75

MALT organizes the response to antigen that enters mucosal tissues

53

Ubiquitination may inhibit or enhance signal transduction

76

The skin is an innate immune barrier and also includes lymphoid tissue

56

Tertiary lymphoid tissues also organize and maintain an immune response

57

SUMMARY

60

REFERENCES

Frequently Encountered Signaling Pathways

77

The PLC pathway induces calcium release and PKC activation

77

60

The Ras/Map kinase cascade activates transcription through AP-1

78

USEFUL WEB SITES

61

PKC activates the NF-κB transcription factor

79

STUDY QUESTIONS

61

Receptor-Ligand Interactions Receptor-ligand binding occurs via multiple noncovalent bonds

80

Antibodies share a common structure of two light chains and two heavy chains

81

There are two major classes of antibody light chains

85

There are five major classes of antibody heavy chains

85

66

Antibodies and antibody fragments can serve as antigens

86

66

Each of the domains of the antibody heavy and light chains mediate specific functions

88

X-ray crystallography has been used to define the structural basis of antigen-antibody binding

90

65

How do we quantitate the strength of receptorligand interactions?

66

Interactions between receptors and ligands can be multivalent

67

Receptor and ligand expression can vary during the course of an immune response Local concentrations of cytokines and other ligands may be extremely high

Common Strategies Used in Many Signaling Pathways Ligand binding can induce conformational changes in, and/or clustering of, the receptor

80

Antibodies are made up of multiple immunoglobulin domains

Chapter 3

Receptors and Signaling: B and T-Cell Receptors

The Structure of Antibodies

Signal Transduction in B Cells

91

68

Antigen binding results in docking of adapter molecules and enzymes into the BCR-Igα/Igβ membrane complex

91

68

B cells use many of the downstream signaling pathways described above

92

69

B cells also receive signals through co-receptors

94

T-Cell Receptors and Signaling 71

95

The T-cell receptor is a heterodimer with variable and constant regions

95

Some receptors require receptor-associated molecules to signal cell activation

71

The T-cell signal transduction complex includes CD3

98

Ligand-induced receptor clustering can alter receptor location

71

The T cell co-receptors CD4 and CD8 also bind the MHC

99

Tyrosine phosphorylation is an early step in many signaling pathways

73

Lck is the first tyrosine kinase activated in T cell signaling

100

Adapter proteins gather members of signaling pathways

74

T cells use downstream signaling strategies similar to those of B cells

100

Phosphorylation on serine and threonine residues is also a common step in signaling pathways

SUMMARY

101

74

REFERENCES

102

USEFUL WEB SITES

102

STUDY QUESTIONS

103

Phosphorylation of membrane phospholipids recruits PH domain-containing proteins to the cell membrane

75

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Contents Cytokine storms may have caused many deaths in the 1918 Spanish influenza

Chapter 4

Receptors and Signaling: Cytokines and Chemokines General Properties of Cytokines and Chemokines

Cytokine-Based Therapies

105 106

REFERENCES

138

USEFUL WEB SITES

139

STUDY QUESTIONS

140

Cytokines have numerous biological functions

107

Chapter 5

Cytokines can elicit and support the activation of specific T-cell subpopulations

107

Innate Immunity

Cell activation may alter the expression of receptors and adhesion molecules

109

Signaling through multiple receptors can fine tune a cellular response

Six Families of Cytokines and Associated Receptor Molecules Cytokines of the IL-1 family promote proinflammatory signals

110

113

Hematopoietin (Class I) family cytokines share three-dimensional structural motifs, but induce a diversity of functions in target cells

116

The Interferon (Class II) cytokine family was the first to be discovered

119

Members of the TNF cytokine family can signal development, activation, or death

123

The IL-17 family is a recently discovered, proinflammatory cytokine cluster

127

Chemokines direct the migration of leukocytes through the body

Cytokine Antagonists

Anatomical Barriers to Infection

141 143

Epithelial barriers prevent pathogen entry into the body’s interior

143

Antimicrobial proteins and peptides kill would-be invaders

145

Phagocytosis 111

137 138

107

110

137

SUMMARY

Cytokines mediate the activation, proliferation, and differentiation of target cells

Cytokines are concentrated between secreting and target cells

ix

147

Microbes are recognized by receptors on phagocytic cells

147

Phagocytosed microbes are killed by multiple mechanisms

151

Phagocytosis contributes to cell turnover and the clearance of dead cells

152

Induced Cellular Innate Responses

152

Cellular pattern recognition receptors activate responses to microbes and cell damage

153

Toll-like receptors recognize many types of pathogen molecules

153

C-type lectin receptors bind carbohydrates on the surfaces of extracellular pathogens

158

Retinoic acid-inducible gene-I-like receptors bind viral RNA in the cytosol of infected cells

160

129

133

The IL-1 receptor antagonist blocks the IL-1 cytokine receptor

133

Nod-like receptors are activated by a variety of PAMPs, DAMPs, and other harmful substances

160

Cytokine antagonists can be derived from cleavage of the cytokine receptor

134

Expression of innate immunity proteins is induced by PRR signaling

160

Some viruses have developed strategies to exploit cytokine activity

134

Cytokine-Related Diseases

Inflammatory Responses 134

166

Inflammation results from innate responses triggered by infection, tissue damage, or harmful substances

167

Proteins of the acute phase response contribute to innate immunity and inflammation

168

Septic shock is relatively common and potentially lethal

135

Bacterial toxic shock is caused by superantigen induction of T-cell cytokine secretion

135

Natural Killer Cells

168

Cytokine activity is implicated in lymphoid and myeloid cancers

137

Regulation and Evasion of Innate and Inflammatory Responses

169

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Contents Innate and inflammatory responses can be harmful

169

Innate and inflammatory responses are regulated both positively and negatively

172

Complement activity is passively regulated by protein stability and cell surface composition

210

Pathogens have evolved mechanisms to evade innate and inflammatory responses

173

The C1 inhibitor, C1INH, promotes dissociation of C1 components

211

Decay Accelerating Factors promote decay of C3 convertases

211

Interactions Between the Innate and Adaptive Immune Systems

173

The Regulation of Complement Activity

210

The innate immune system activates and regulates adaptive immune responses

Factor I degrades C3b and C4b

212

174

Protectin inhibits the MAC attack

213

Adjuvants activate innate immune responses to increase the effectiveness of immunizations

175

Carboxypeptidases can inactivate the anaphylatoxins, C3a and C5a

213

Some pathogen clearance mechanisms are common to both innate and adaptive immune responses

176

Ubiquity of Innate Immunity

176

Complement Deficiencies

213

Microbial Complement Evasion Strategies

214

Plants rely on innate immune responses to combat infections

177

Some pathogens interfere with the first step of immunoglobulin-mediated complement activation

215

Invertebrate and vertebrate innate immune responses show both similarities and differences

177

Microbial proteins bind and inactivate complement proteins

215

SUMMARY

180

Microbial proteases destroy complement proteins

215

REFERENCES

181

USEFUL WEB SITES

182

Some microbes mimic or bind complement regulatory proteins

215

STUDY QUESTIONS

182

Chapter 6

The Complement System

187

The Evolutionary Origins of the Complement System

215

SUMMARY

219

REFERENCES

220

USEFUL WEB SITES

220

STUDY QUESTIONS

221

The Major Pathways of Complement Activation 189 The classical pathway is initiated by antibody binding

190

The lectin pathway is initiated when soluble proteins recognize microbial antigens

195

The alternative pathway is initiated in three distinct ways

196

The three complement pathways converge at the formation of the C5 convertase

The Organization and Expression of Lymphocyte Receptor Genes 225

200

The Puzzle of Immunoglobulin Gene Structure

C5 initiates the generation of the MAC

200

The Diverse Functions of Complement

201

Complement receptors connect complementtagged pathogens to effector cells

201

Complement enhances host defense against infection

204

Complement mediates the interface between innate and adaptive immunities

207

Complement aids in the contraction phase of the immune response

207

Complement mediates CNS synapse elimination

210

Chapter 7

226

Investigators proposed two early theoretical models of antibody genetics

226

Breakthrough experiments revealed that multiple gene segments encode the light chain

227

Multigene Organization of Ig Genes

231

Kappa light-chain genes include V, J, and C segments

231

Lambda light-chain genes pair each J segment with a particular C segment

231

Heavy-chain gene organization includes VH, D, JJ, and CH segments

232

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Contents

The Mechanism of V(D)J Recombination Recombination is directed by signal sequences Gene segments are joined by the RAG1/2 recombinase combination V(D)J recombination results in a functional Ig variable region gene V(D)J recombination can occur between segments transcribed in either the same or opposite directions Five mechanisms generate antibody diversity in naïve B cells

B-Cell Receptor Expression

232 233 234 235

239 239

242

Allelic exclusion ensures that each B cell synthesizes only one heavy chain and one light chain

242

Receptor editing of potentially autoreactive receptors occurs in light chains

243

Ig gene transcription is tightly regulated

244

Mature B cells express both IgM and IgD antibodies by a process that involves mRNA splicing

246

T-Cell Receptor Genes and Expression

247

Understanding the protein structure of the TCR was critical to the process of discovering the genes

247

The β-chain gene was discovered simultaneously in two different laboratories

249

A search for the α-chain gene led to the γ-chain gene instead

250

TCR genes undergo a process of rearrangement very similar to that of Ig genes

251

TCR expression is controlled by allelic exclusion

253

TCR gene expression is tightly regulated

253

SUMMARY

255

REFERENCES

256

USEFUL WEB SITES

257

STUDY QUESTIONS

258

Class II molecules have two non-identical glycoprotein chains

262

Class I and II molecules exhibit polymorphism in the region that binds to peptides

263

General Organization and Inheritance of the MHC

The Major Histocompatibility Complex and Antigen Presentation The Structure and Function of MHC Molecules Class I molecules have a glycoprotein heavy chain and a small protein light chain

261 262 262

267

The MHC locus encodes three major classes of molecules

268

The exon/intron arrangement of class I and II genes reflects their domain structure

270

Allelic forms of MHC genes are inherited in linked groups called haplotypes

270

MHC molecules are codominantly expressed

271

Class I and class II molecules exhibit diversity at both the individual and species levels

273

MHC polymorphism has functional relevance

276

The Role of the MHC and Expression Patterns

277

MHC molecules present both intracellular and extracellular antigens

278

MHC class I expression is found throughout the body

278

Expression of MHC class II molecules is primarily restricted to antigen-presenting cells

279

MHC expression can change with changing conditions

279

T cells are restricted to recognizing peptides presented in the context of self-MHC alleles

281

Evidence suggests different antigen processing and presentation pathways

284

The Endogenous Pathway of Antigen Processing and Presentation

285

Peptides are generated by protease complexes called proteasomes

285

Peptides are transported from the cytosol to the RER

285

Chaperones aid peptide assembly with MHC class I molecules

286

The Exogenous Pathway of Antigen Processing and Presentation

Chapter 8

xi

288

Peptides are generated from internalized molecules in endocytic vesicles

288

The invariant chain guides transport of class II MHC molecules to endocytic vesicles

289

Peptides assemble with class II MHC molecules by displacing CLIP

289

Cross-Presentation of Exogenous Antigens

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Contents Dendritic cells appear to be the primary crosspresenting cell type

292

Mechanisms and Functions of Cross-Presentation

292

Presentation of Nonpeptide Antigens

293

SUMMARY

295

REFERENCES

295

USEFUL WEB SITES

296

STUDY QUESTIONS

296

Chapter 9

T-Cell Development Early Thymocyte Development

299

Apoptosis allows cells to die without triggering an inflammatory response

318

Different stimuli initiate apoptosis, but all activate caspases

318

Apoptosis of peripheral T cells is mediated by the extrinsic (Fas) pathway

320

TCR-mediated negative selection in the thymus induces the intrinsic (mitochondria-mediated) apoptotic pathway

321

Bcl-2 family members can inhibit or induce apoptosis

321

SUMMARY

324

REFERENCES

325

USEFUL WEB SITES

326

STUDY QUESTIONS

327

301

Thymocytes progress through four double-negative stages

301

Chapter 10

Thymocytes can express either TCRαβ or TCRγδ receptors

302

B-Cell Development

DN thymocytes undergo β-selection, which results in proliferation and differentiation

303

Positive and Negative Selection

The Site of Hematopoiesis 304

Thymocytes “learn” MHC restriction in the thymus

305

T cells undergo positive and negative selection

305

Positive selection ensures MHC restriction

307

Negative selection (central tolerance) ensures self-tolerance

310

The selection paradox: Why don’t we delete all cells we positively select?

312

An alternative model can explain the thymic selection paradox

313

Do positive and negative selection occur at the same stage of development, or in sequence?

314

Lineage Commitment Several models have been proposed to explain lineage commitment Double-positive thymocytes may commit to other types of lymphocytes

Exit from the Thymus and Final Maturation

329 330

The site of B-cell generation changes during gestation

330

Hematopoiesis in the fetal liver differs from that in the adult bone marrow

332

B-Cell Development in the Bone Marrow The stages of hematopoiesis are defined by cellsurface markers, transcription-factor expression, and immunoglobulin gene rearrangements

332

334

The earliest steps in lymphocyte differentiation culminate in the generation of a common lymphoid progenitor 337 The later steps of B-cell development result in commitment to the B-cell phenotype

339

Immature B cells in the bone marrow are exquisitely sensitive to tolerance induction

344

Many, but not all, self-reactive B cells are deleted within the bone marrow

345

314

B cells exported from the bone marrow are still functionally immature

345

316

Mature, primary B-2 B cells migrate to the lymphoid follicles

349

314

316

Other Mechanisms That Maintain Self-Tolerance 316

The Development of B-1 and Marginal-Zone B Cells

351

TREG cells negatively regulate immune responses

317

B-1 B cells are derived from a separate developmental lineage

351

Peripheral mechanisms of tolerance also protect against autoreactive thymocytes

318

Marginal-zone cells share phenotypic and functional characteristics with B-1 B cells and arise at the T2 stage

352

Apoptosis

318

Comparison of B- and T-Cell Development

352

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Contents SUMMARY

354

REFERENCES

355

USEFUL WEB SITES

355

STUDY QUESTIONS

356

Chapter 12

B-Cell Activation, Differentiation, and Memory Generation 385 T-Dependent B-Cell Responses

Chapter 11

T-Cell Activation, Differentiation, and Memory 357 T-Cell Activation and the Two-Signal Hypothesis

358

Costimulatory signals are required for optimal T-cell activation and proliferation

359

Clonal anergy results if a costimulatory signal is absent

363

Cytokines provide Signal 3

364

Antigen-presenting cells have characteristic costimulatory properties

365

Superantigens are a special class of T-cell activators

366

T-Cell Differentiation

368

Helper T cells can be divided into distinct subsets

370

The differentiation of T helper cell subsets is regulated by polarizing cytokines

371

Effector T helper cell subsets are distinguished by three properties

372

Helper T cells may not be irrevocably committed to a lineage

378

Helper T-cell subsets play critical roles in immune health and disease

378

T-Cell Memory

379

xiii

388

T-dependent antigens require T-cell help to generate an antibody response

388

Antigen recognition by mature B cells provides a survival signal

389

B cells encounter antigen in the lymph nodes and spleen

390

B-cell recognition of cell-bound antigen results in membrane spreading

391

What causes the clustering of the B-cell receptors upon antigen binding?

392

Antigen receptor clustering induces internalization and antigen presentation by the B cell

393

Activated B cells migrate to find antigen-specific T cells

393

Activated B cells move either into the extrafollicular space or into the follicles to form germinal centers

395

Plasma cells form within the primary focus

395

Other activated B cells move into the follicles and initiate a germinal center response

396

Somatic hypermutation and affinity selection occur within the germinal center

398

Class switch recombination occurs within the germinal center after antigen contact

401

Most newly generated B cells are lost at the end of the primary immune response

403

Naïve, effector, and memory T cells display broad differences in surface protein expression

379

Some germinal center cells complete their maturation as plasma cells

403

TCM and TEM are distinguished by their locale and commitment to effector function

380

B-cell memory provides a rapid and strong response to secondary infection

404

How and when do memory cells arise?

380

What signals induce memory cell commitment?

381

Do memory cells reflect the heterogeneity of effector cells generated during a primary response?

381

Are there differences between CD4+ and CD8+ memory T cells?

381

How are memory cells maintained over many years?

381

SUMMARY

381

REFERENCES

382

USEFUL WEB SITES

383

STUDY QUESTIONS

383

T-Independent B-Cell Responses

406

T-independent antigens stimulate antibody production without the need for T-cell help

406

Two novel subclasses of B cells mediate the response to T-independent antigens

407

Negative Regulation of B Cells

411

Negative signaling through CD22 shuts down unnecessary BCR signaling

411

Negative signaling through the FcγRIIb receptor inhibits B-cell activation

411

B-10 B cells act as negative regulators by secreting IL-10

411

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Contents SUMMARY

412

REFERENCES

413

USEFUL WEB SITES

414

STUDY QUESTIONS

414

Naïve lymphocytes sample stromal cells in the lymph nodes

461

Naïve lymphocytes browse for antigen along reticular networks in the lymph node

461

Immune Cell Behavior during the Innate Immune Response

464

Chapter 13

Antigen-presenting cells travel to lymph nodes and present processed antigen to T cells

465

Effector Responses: Cell-and Antibody-Mediated Immunity

Unprocessed antigen also gains access to lymphnode B cells

465

Antibody-Mediated Effector Functions Antibodies mediate the clearance and destruction of pathogen in a variety of ways Antibody isotypes mediate different effector functions Fc receptors mediate many effector functions of antibodies

Cell-Mediated Effector Responses

415 416

Immune Cell Behavior during the Adaptive Immune Response

467

+

416 419 423

427

Naïve CD4 T cells arrest their movements after engaging antigens

468

+

B cells seek help from CD4 T cells at the border between the follicle and paracortex of the Lymph Node

468

Dynamic imaging approaches have been used to address a controversy about B-cell behavior in germinal centers

470

+

Cytotoxic T lymphocytes recognize and kill infected or tumor cells via T-cell receptor activation

428

CD8 T cells are activated in the lymph node via a multicellular interaction

471

Natural killer cells recognize and kill infected cells and tumor cells by their absence of MHC class I

435

Activated lymphocytes exit the lymph node and recirculate

472

A summary of our current understanding

472

The immune response contracts within 10 to 14 days

474

NKT cells bridge the innate and adaptive immune systems

441

Experimental Assessment of Cell-Mediated Cytotoxicity

444

Co-culturing T cells with foreign cells stimulates the mixed-lymphocyte reaction

444

Chemokine receptors and integrins regulate homing of effector lymphocytes to peripheral tissues

474

CTL activity can be demonstrated by cell-mediated lympholysis

445

Effector lymphocytes respond to antigen in multiple tissues

475

The graft-versus-host reaction is an in vivo indication of cell-mediated cytotoxicity

SUMMARY

480

446

REFERENCES

481

SUMMARY

446

USEFUL WEB SITES

482

REFERENCES

447

STUDY QUESTIONS

482

USEFUL WEB SITES

448

STUDY QUESTIONS

448

Immune Cell Behavior in Peripheral Tissues

474

Chapter 15 Chapter 14

The Immune Response in Space and Time Immune Cell Behavior before Antigen Is Introduced Naïve lymphocytes circulate between secondary and tertiary lymphoid tissues

451 455 455

Allergy, Hypersensitivities, and Chronic Inflammation

485

Allergy: A Type I Hypersensitivity Reaction

486

IgE antibodies are responsible for type I hypersensitivity

487

Many allergens can elicit a type I response

487

IgE antibodies act by cross-linking Fcε receptors on the surfaces of innate immune cells

487

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Contents IgE receptor signaling is tightly regulated

491

Chapter 16

Innate immune cells produce molecules responsible for type I hypersensitivity symptoms

491

Type I hypersensitivities are characterized by both early and late responses

494

Tolerance, Autoimmunity, and Transplantation

There are several categories of type I hypersensitivity reactions

494

There is a genetic basis for type I hypersensitivity

497

Diagnostic tests and treatments are available for type I hypersensitivity reactions The hygiene hypothesis has been advanced to explain increases in allergy incidence

Antibody-Mediated (Type II) Hypersensitivity Reactions Transfusion reactions are an example of type II hypersensitivity

498 501

501

Hemolytic disease of the newborn is caused by type II reactions

503

Hemolytic anemia can be drug induced

504

Immune Complex-Mediated (Type III) Hypersensitivity Immune complexes can damage various tissues Immune complex-mediated hypersensitivity can resolve spontaneously Autoantigens can be involved in immune complexmediated reactions

505 505 506 506

Delayed-Type (Type IV) Hypersensitivity (DTH)

506

The initiation of a type IV DTH response involves sensitization by antigen

507

The effector phase of a classical DTH response is induced by second exposure to a sensitizing antigen

507

The DTH reaction can be detected by a skin test

508

Contact dermatitis is a type IV hypersensitivity response

508

509

Infections can cause chronic inflammation

518

Antigen sequestration is one means to protect self antigens from attack

519

Central tolerance limits development of autoreactive T cells and B cells

520

Peripheral tolerance regulates autoreactive cells in the circulation

520

525

Some autoimmune diseases target specific organs

526

Some autoimmune diseases are systemic

529

Both intrinsic and extrinsic factors can favor susceptibility to autoimmune disease

531

Several possible mechanisms have been proposed for the induction of autoimmunity

533

Autoimmune diseases can be treated by general or pathway-specific immunosuppression

534

505

Arthus reactions are localized type III hypersensitivity reactions

Chronic Inflammation

517

Establishment and Maintenance of Tolerance

Autoimmunity 501

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509

Transplantation Immunology

536

Graft rejection occurs based on immunologic principles

536

Graft rejection follows a predictable clinical course

541

Immunosuppressive therapy can be either general or target-specific

543

Immune tolerance to allografts is favored in certain instances

545

Some organs are more amenable to clinical transplantation than others

546

SUMMARY

549

REFERENCES

550

USEFUL WEB SITES

551

STUDY QUESTIONS

551

Chapter 17

There are noninfectious causes of chronic inflammation 510

Infectious Diseases and Vaccines 553

Obesity is associated with chronic inflammation

510

Chronic inflammation can cause systemic disease

510

The Importance of Barriers to Infection and the Innate Response

554

SUMMARY

513

Viral Infections

555

REFERENCES

515

Many viruses are neutralized by antibodies

556

USEFUL WEB SITES

515

STUDY QUESTIONS

516

Cell-mediated immunity is important for viral control and clearance

556

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Contents Viruses employ several different strategies to evade host defense mechanisms

556

B-cell immunodeficiencies exhibit depressed production of one or more antibody isotypes

601

Influenza has been responsible for some of the worst pandemics in history

557

Disruptions to innate components may also impact adaptive responses

601

Complement deficiencies are relatively common

603

560

Immunodeficiency that disrupts immune regulation can manifest as autoimmunity

603

Bacteria can evade host defense mechanisms at several different stages

563

Immunodeficiency disorders are treated by replacement therapy

604

Tuberculosis is primarily controlled by CD4+ T cells

564

Animal models of immunodeficiency have been used to study basic immune function

604

Diphtheria can be controlled by immunization with inactivated toxoid

565

Bacterial Infections Immune responses to extracellular and intracellular bacteria can differ

Parasitic Infections Protozoan parasites account for huge worldwide disease burdens A variety of diseases are caused by parasitic worms (helminths)

Fungal Infections

560

565 565 567

569

Innate immunity controls most fungal infections

569

Immunity against fungal pathogens can be acquired

571

Emerging and Re-emerging Infectious Diseases 571 Some noteworthy new infectious diseases have appeared recently

572

Diseases may re-emerge for various reasons

573

Vaccines Protective immunity can be achieved by active or passive immunization

574 574

Secondary Immunodeficiencies

606

HIV/AIDS has claimed millions of lives worldwide

607

The retrovirus HIV-1 is the causative agent of AIDS

608

HIV-1 is spread by intimate contact with infected body fluids

610

In vitro studies have revealed the structure and life cycle of HIV-1

612

Infection with HIV-1 leads to gradual impairment of immune function

615

Active research investigates the mechanism of progression to AIDS

616

Therapeutic agents inhibit retrovirus replication

619

A vaccine may be the only way to stop the HIV/AIDS epidemic

621

SUMMARY

623

REFERENCES

623

USEFUL WEB SITES

624

STUDY QUESTIONS

624

There are several vaccine strategies, each with unique advantages and challenges

578

Conjugate or multivalent vaccines can improve immunogenicity and outcome

583

Chapter 19

Adjuvants are included to enhance the immune response to a vaccine

585

Cancer and the Immune System

627

SUMMARY

586

Terminology and Common Types of Cancer

627

REFERENCES

587

Malignant Transformation of Cells

628

USEFUL WEB SITES

588

DNA alterations can induce malignant transformation

629

STUDY QUESTIONS

588

The discovery of oncogenes paved the way for our understanding of cancer induction

629

Genes associated with cancer control cell proliferation and survival

630

Malignant transformation involves multiple steps

633

Chapter 18

Immunodeficiency Disorders Primary Immunodeficiencies Combined immunodeficiencies disrupt adaptive immunity

593 593 597

Tumor Antigens

634

Tumor-specific antigens are unique to tumor cells

636

Tumor-associated antigens are normal cellular proteins with unique expression patterns

636

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Contents

The Immune Response to Cancer Immunoediting both protects against and promotes tumor growth

638 639

Key immunologic pathways mediating tumor eradication have been identified

639

Some inflammatory responses can promote cancer

642

Some tumor cells evade immune recognition and activation

643

Cancer Immunotherapy

644

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Hemagglutination inhibition reactions are used to detect the presence of viruses and of antiviral antibodies 658 Bacterial agglutination can be used to detect antibodies to bacteria

659

Antibody Assays Based on Antigen Binding to Solid-Phase Supports

659

Radioimmunoassays are used to measure the concentrations of biologically relevant proteins and hormones in bodily fluids

659

Monoclonal antibodies can be targeted to tumor cells

644

ELISA assays use antibodies or antigens covalently bound to enzymes

660

Cytokines can be used to augment the immune response to tumors

646

The design of an ELISA assay must consider various methodological options

662

Tumor-specific T cells can be expanded and reintroduced into patients

647

ELISPOT assays measure molecules secreted by individual cells

663

New therapeutic vaccines may enhance the anti-tumor immune response 647

Western blotting can identify a specific protein in a complex protein mixture

664

Methods to Determine the Affinity of AntigenAntibody Interactions

664

Manipulation of costimulatory signals can improve cancer immunity

647

Combination cancer therapies are yielding surprising results

648

SUMMARY

649

REFERENCES

650

USEFUL WEB SITES

650

STUDY QUESTIONS

651

Chapter 20

Experimental Systems and Methods

653

Antibody Generation

654

Polyclonal antibodies are secreted by multiple clones of antigen-specific B cells A monoclonal antibody is the product of a single stimulated B cell Monoclonal antibodies can be modified for use in the laboratory or the clinic

Immunoprecipitation- Based Techniques

654 654 655

656

Immunoprecipitation can be performed in solution

656

Immunoprecipitation of soluble antigens can be performed in gel matrices

656

Immunoprecipitation allows characterization of cell-bound molecules

657

Agglutination Reactions

658

Hemagglutination reactions can be used to detect antigen conjugated to the surface of red blood cells

any 658

Equilibrium dialysis can be used to measure antibody affinity for antigen

665

Surface plasmon resonance is commonly used for measurements of antibody affinity

667

Microscopic Visualization of Cells and Subcellular Structures

668

Immunocytochemistry and immunohistochemistry use enzyme-conjugated antibodies to create images of fixed tissues

668

Immunoelectron microscopy uses gold beads to visualize antibody-bound antigens

669

Immunofluorescence-Based Imaging Techniques

669

Fluorescence can be used to visualize cells and molecules

669

Immunofl uorescence microscopy uses antibodies conjugated with fluorescent dyes

669

Confocal fluorescence microscopy provides threedimensional images of extraordinary clarity

670

Multiphoton fluorescence microscopy is a variation of confocal microscopy

670

Intravital imaging allows observation of immune responses in vivo

671

Flow Cytometry

672

Magnetic Activated Cell Sorting

677

Cell Cycle Analysis

678

Tritiated (3H) thymidine uptake was one of the first methods used to assess cell division

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Contents

Colorimetric assays for cell division are rapid and eliminate the use of radioactive isotopes

678

Bromodeoxyuridine-based assays for cell division use antibodies to detect newly synthesized DNA

678

Propidium iodide enables analysis of the cell cycle status of cell populations Carboxyfluorescein succinimidyl ester can be used to follow cell division

Assays of Cell Death

678 679

679

The 51Cr release assay was the first assay used to measure cell death

679

Fluorescently labeled annexin V measures phosphatidyl serine in the outer lipid envelope of apoptotic cells

680

Transgenic animals carry genes that have been artificially introduced

684

Knock-in and knockout technologies replace an endogenous with a nonfunctional or engineered gene copy

685

The cre/lox system enables inducible gene deletion in selected tissues

687

SUMMARY

689

REFERENCES

690

USEFUL WEB SITES

690

STUDY QUESTIONS

691

Appendix I

The TUNEL assay measures apoptotically generated DNA fragmentation

680

Caspase assays measure the activity of enzymes involved in apoptosis

681

Biochemical Approaches Used to Elucidate Signal Transduction Pathways

681

Biochemical inhibitors are often used to identify intermediates in signaling pathways

681

Many methods are used to identify proteins that interact with molecules of interest

682

CD Antigens

A-1

Appendix II

Cytokines

B-1

Appendix III Whole Animal Experimental Systems

682

Animal research is subject to federal guidelines that protect nonhuman research subjects

682

Inbred strains can reduce experimental variation

683

Congenic resistant strains are used to study the effects of particular gene loci on immune responses

684

Adoptive transfer experiments allow in vivo examination of isolated cell populations

684

Chemokines and Chemokine Receptors Glossary Answers to Study Questions Index

C-1 G-1 AN-1 I-1

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Feature Boxes in Kuby 7e Clinical Focus

Classic Experiment

Box 1.1

Box 2.1 Box 2.3 Box 3.1 Box 3.3 Box 6.1 Box 7.1

Box 1.2 Box 1.3 Box 2.2 Box 3.2 Box 4.2 Box 4.4 Box 5.2

Box 6.2 Box 7.3 Box 8.2 Box 8.4 Box 9.2 Box 9.3 Box 10.1 Box 11.2 Box 11.4 Box 13.1 Box 15.2 Box 15.3 Box 16.1 Box 16.2

Box 16.4 Box 17.1 Box 18.1 Box 19.1

Vaccine Controversy: What’s Truth and What’s Myth? p. 5 Passive Antibodies and the Iditarod p. 8 The Hygiene Hypothesis p. 20 Stem Cells—Clinical Uses and Potential p. 42 Defects in the B-Cell Signaling Protein Btk Lead to X-Linked Agammaglobulinemia p. 93 Therapy with Interferons p. 120 Cytokines and Obesity p. 136 Genetic Defects in Components of Innate and Inflammatory Responses Associated with Disease p. 170 The Complement System as a Therapeutic Target p. 208 Some Immunodeficiencies Result from Impaired Receptor Gene Recombination p. 255 MHC Alleles and Susceptibility to Certain Diseases p. 277 Deficiencies in TAP Can Lead to Bare Lymphocyte Syndrome p. 287 How Do T Cells That Cause Type 1 Diabetes Escape Negative Selection? p. 311 Failure of Apoptosis Causes Defective Lymphocyte Homeostasis p. 322 B-Cell Development in the Aging Individual p. 333 Costimulatory Blockade p. 364 What a Disease Reveals about the Physiological Role of TH17 Cells p. 376 Monoclonal Antibodies in the Treatment of Cancer p. 420 The Genetics of Asthma and Allergy p. 498 Type 2 Diabetes, Obesity, and Inflammation p. 511 It Takes Guts to Be Tolerant p. 523 Why Are Women More Susceptible Than Men to Autoimmunity? Gender Differences in Autoimmune Disease p. 528 Is There a Clinical Future for Xenotransplantation? p. 548 The 1918 Pandemic Influenza Virus: Should It Publish or Perish? p. 557 Prevention of Infant HIV Infection by AntiRetroviral Treatment p. 610 A Vaccine to Prevent Cervical Cancer, and More p. 637

Box 8.1

Box 9.1

Box 10.3 Box 11.1 Box 12.1

Box 13.2

Box 15.1 Box 16.3

Advances Box 4.1 Box 4.3

Box 5.1 Box 6.3

Box 10.2 Box 11.3 Box 12.2 Box 14.1 Box 14.2 Box 17.2

Evolution Box 2.4 Box 5.3 Box 7.2

Box 8.3

Variations on Anatomical Themes p. 57 Plant Innate Immune Responses p. 178 Evolution of Recombined Lymphocyte Receptors p. 240 The Sweet Smell of Diversity p. 275

Isolating Hematopoietic Stem Cells p. 29 The Discovery of a Thymus—and Two p. 46 The Elucidation of Antibody Structure p. 82 The Discovery of the T-Cell Receptor p. 96 The Discovery of Properdin p. 198 Hozumi and Tonegawa’s Experiment: DNA Recombination Occurs in immunoglobulin Genes in Somatic Cells p. 227 Demonstration of the Self-MHC Restriction of CD8⫹ T Cells p. 282 Insights about Thymic Selection from the First TCR Transgenic Mouse Have Stood the Test of Time p. 308 The Stages of B-Cell Development: Characterization of the Hardy Fractions p. 342 Discovery of the First Costimulatory Receptor: CD28 p. 362 Experimental Proof That Somatic Hypermutation and Antigen- Induced Selection Occurred Within the Germinal Centers p. 399 Rethinking Immunological Memory: NK Cells Join Lymphocytes as Memory-Capable Cells p. 442 The Discovery and Identification of IgE as the Carrier of Allergic Hypersensitivity p. 488 Early Life Exposure to Antigens Favors Tolerance Induction p. 546

Box 20.1

Methods Used to Map the Secretome p. 111 How Does Chemokine Binding to a Cell-Surface Receptor Result in Cellular Movement Along the Chemokine Gradient? p. 130 Inflammasomes p. 162 Staphylococcus aureus Employs Diverse Methods to Evade Destruction by the Complement System p. 216 The Role of miRNAs in the Control of B-Cell Development p. 336 How Many TCR Complexes Must Be Engaged to Trigger T-Cell Activation? p. 368 New Ideas on B-Cell Help: Not All Cells That Help B Cells Make Antibodies Are T Cells p. 408 Dynamic Imaging Techniques p. 452 Molecular Regulation of Cell Migration Between and Within Tissues p. 456 A Prime and Pull Vaccine Strategy for Preventing Sexually Transmitted Diseases p. 586 Flow Cytometry Under the Hood p. 674

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Preface

Like all of the previous authors of this book, we are dedicated to the concept that immunology is best taught and learned in an experimentally-based manner, and we have retained that emphasis with this edition. It is our goal that students should complete an immunology course not only with a firm grasp of content, but also with a clear sense of how key discoveries were made, what interesting questions remain, and how they might best be answered. We believe that this approach ensures that students both master fundamental immunological concepts and internalize a vision of immunology as an active and ongoing process. Guided by this vision, the new edition has been extensively updated to reflect the recent advances in all aspects of our discipline.

New Authorship As a brand-new team of authors, we bring experience in both research and undergraduate teaching to the development of this new edition, which continues to reflect a dedication to pedagogical excellence originally modeled by Janis Kuby. We remain deeply respectful of Kuby’s unique contribution to the teaching of immunology and hope and trust that this new manifestation of her creation will simply add to her considerable legacy.

2a

1

Lymph node

P

3

P

P

B

B T T

5a

T

4

B

5b Memory

2b

N

B

T

OVERVIEW FIGURE 1-9 Collaboration between innate and adaptive immunity in resolving an infection.

A new capstone chapter (Chapter 14) integrates the events of an immune response into a complete story, with particular reference to the advanced imaging techniques that have become available since the writing of the previous edition. In this way, the molecular and cellular details presented in Chapters 2-13 are portrayed in context, a moving landscape of immune response events in time and space (Figure 14-5).

Understanding Immunology As a Whole We recognize that the immune system is an integrated network of cells, molecules, and organs, and that each component relies on the rest to function properly. This presents a pedagogical challenge because to understand the whole, we must attain working knowledge of many related pieces of information, and these do not always build upon each other in simple linear fashion. In acknowledgment of this challenge, this edition presents the “big picture” twice; first as an introductory overview to immunity, then, thirteen chapters later, as an integration of the details students have learned in the intervening text. Specifically, Chapter 1 has been revised to make it more approachable for students who are new to immunology. The chapter provides a short historical background to the field and an introduction to some of the key players and their roles in the immune response, keeping an eye on fundamental concepts (Overview Figure 1-9). A new section directly addresses some of the biggest conceptual hurdles, but leaves the cellular and molecular details for later chapters.

FIGURE 14-5 A T cell (blue) on a fibroblastic reticular network (red and green) in the lymph node.

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Preface

Focus on the Fundamentals The order of chapters in the seventh edition has been revised to better reflect the sequence of events that occurs naturally during an immune response in vivo. This offers instructors the opportunity to lead their students through the steps of an immune response in a logical sequence, once they have learned the essential features of the tissues, cells, molecular structures, ligand-receptor binding interactions, and signaling pathways necessary for the functioning of the immune system. The placement of innate immunity at the forefront of the immune response enables it to take its rightful place as the first, and often the only, aspect of immunity that an organism needs to counter an immune insult. Similarly, the chapter on complement is located within the sequence in a place that highlights its function as a bridge between innate and adaptive immune processes. However, we recognize that a course in immunology is approached differently by each instructor. Therefore, as much as possible, we have designed each of the chapters so that it can stand alone and be offered in an alternative order.

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signaling, as well as to specific molecules and pathways involved in signaling through antigen receptors. Chapter 4 includes a more thorough introduction to the roles of cytokines and chemokines in the immune response. • An expanded and updated treatment of innate immunity (Chapter 5), which now includes comprehensive coverage of the many physical, chemical, and cellular defenses that constitute the innate immune system, as well as the ways in which it activates and regulates adaptive immunity. • Substantial rewriting of chapters concerned with complement (Chapter 6) and antigen receptor gene rearrangement (Chapter 7). These chapters have been extensively revised for clarity in both text and figures. The description of the complement system has been updated to include the involvement of complement proteins in both innate and adaptive aspects of immunity. • A restructured presentation of the MHC, with the addition of new information relevant to cross-presentation pathways (Chapter 8) (Figure 8-22b).

(b) DC cross-presentation and activation of CTL

Challenging All Levels

Cross-presenting dendritic cell

While this book is written as a text for students new to immunology, it is also our intent to challenge students to reach deeply into the field and to appreciate the connections with other aspects of biology. Instead of reducing difficult topics to vague and simplistic forms, we instead present them with the level of detail and clarity necessary to allow the beginning student to find and understand information they may need in the future. This offers the upper level student a foundation from which they can progress to the investigation of advances and controversies within the current immunological literature. Supplementary focus boxes have been used to add nuance or detail to discussions of particular experiments or ideas without detracting from the flow of information. These boxes, which address experimental approaches, evolutionary connections, clinical aspects, or advanced material, also allow instructors to tailor their use appropriately for individual courses. They provide excellent launching points for more intensive in class discussions relevant to the material. Some of the most visible changes and improvements include: • A rewritten chapter on the cells and organs of the immune system (Chapter 2) that includes up to date images reflecting our new understanding of the microenvironments where the host immune system develops and responds. • The consolidation of signaling pathways into two chapters: Chapter 3 includes a basic introduction to ligand:receptor interactions and principles of receptor

Exogenous antigen TLR Crossover pathway Class I MHC CD8 CD3

CD80/CD86 CD28

IL-2

Naïve TC cell

FIGURE 8-22b Exogenous antigen activation of naïve Tc cells requires DC licensing and cross-presentation

• The dedication of specialized chapters concerned with T cell development and T cell activation (Chapters 9 and Chapter 11, respectively). Chapter 11 now includes current descriptions of the multiple helper T cell subsets that regulate the adaptive immune response. • Substantially rewritten chapters on B cell development and B cell activation (Chapters 10 and 12, respectively) that address the physiological locations as well as the nature of the interacting cells implicated in these processes. • An updated discussion of the role of effector cells and molecules in clearing infection (Chapter 13), including a more thorough treatment of NK and NKT cells.

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Preface

• A new chapter that describes advances in understanding and visualizing the dynamic behavior and activities of immune cells in secondary and tertiary tissue (Chapter 14). • Substantial revision and updating of the clinical chapters (Chapters 15-19) including the addition of several new clinically relevant focus boxes. • Revised and updated versions of the final methods chapter (Chapter 20), and the appendices of CD antigens, chemokines, and cytokines and their receptors. Throughout the book, we attempt to provide a “big picture” context for necessary details in a way that facilitates greater student understanding.

Recent Advances and Other Additions Immunology is a rapidly growing field, with new discoveries, advances in techniques, and previously unappreciated connections coming to light every day. The 7th edition has been thoroughly updated throughout, and now integrates the following new material and concepts: • New immune cell types and subtypes, as well as the phenotypic plasticity that is possible between certain subtypes of immune cells. • A greater appreciation for the wide range of mechanisms responsible for innate immunity and the nature and roles of innate responses in sensing danger, inducing inflammation, and shaping the adaptive response (Figure 5-18).

2 T cell

Inhibitory cytokines

T cell

TGF␤ 1

Cytokine deprivation

IL–2R

FoxP3

3

TCR APC

Inhibiting antigen presenting cells

MHC

4

Cytotoxicity

T cell

T cell

FIGURE 9-10 How regulatory T cells inactivate traditional T cells. • The roles of the microbiome and commensal organisms in the development and function of immunity, as well as the connections between these and many chronic diseases. • A new appreciation for the micro environmental substructures that guide immune cell interactions with antigen and with one another (Figure 14-11a). Antigen delivery to T cells Subcapsular sinus (SCS)

Lymph node

DC presenting antigen

Afferent lymphatic

Antigen

B cell follicle

Bacteria Naïve TLR4 or TLR5

Dectin-1

T H1

IFN-γ

IL-12 TLR3, 7, 9

Fungi IL-6 IL-23 Virus

Naïve

TH17

IL-17

Tricellular complex (CD8+ T cell, CD4+ T cell, and DC)

FRC network

T cell zone (paracortex)

FIGURE 14-11a How antigen travels into a lymph node. IL-10

TLR2/1

Naïve

Helminth

TH2

IL-4 IL-5 IL-13

TLR2/6

Fungi IL-10 RA TGF-β

CD28

CD80/86

TCR

MHC II with peptide

Naïve

Treg

IL-10 TGF-β

FIGURE 5-18 Differential signaling through dendritic cell PRRs influences helper T cell functions.

• Regulation of immunity, including new regulatory cell types, immunosuppressive chemical messengers and the roles these play, for example, in tolerance and in the nature of responses to different types of antigens (Figure 9-10).

• Many technical advances, especially in the areas of imaging and sequencing, which have collectively enhanced our understanding of immune function and cellular interactions, allowing us to view the immune response in its natural anatomical context, and in real time (see Figure 14-5).

Connections to the Bench, the Clinic, and Beyond We have made a concerted effort in the 7th edition to integrate experimental and clinical aspects of immunology into the text. In Chapter 2, illustrations of immune cells and tissues are shown alongside histological sections or, where possible, electron

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Preface micrographs, so students can see what they actually look like. Throughout the text, experimental data are used to demonstrate the bases for our knowledge (Figure 3-4b), and the clinical chapters at the end of the book (Chapters 15 through 19) describe new advances, new challenges, and newly appreciated connections between the immune system and disease.

FIGURE 3-4b Targeted delivery of cytokines (pink).

Featured Boxes Associated with each chapter are additional boxed materials that provide specialized information on historically-important studies (Classic Experiments) that changed the way immunologists viewed the field, noteworthy new breakthroughs (Advances) that have occurred since the last edition, the clinical relevance of particular topics (Clinical Focus) and the evolution of aspects of immune functioning (Evolution). Examples of such boxes are “The Prime and Pull Vaccine strategy,” “Genetic defects in components of innate and inflammatory responses associated with disease,” “The role of miRNAs in the control of B cell development” and an updated “Stem cells: Clinical uses and potential.” We have involved our own undergraduate students in the creation of some of these boxes, which we believe have greatly benefitted from their perspective on how to present interesting material effectively to their fellow students.

Critical Thinking and Data Analysis Integration of experimental evidence throughout the book keeps students focused on the how and why. Detailed and clear descriptions of the current state of the field provide students with the knowledge, skills, and vocabulary to read critically in the primary literature. Updated and revised study questions at the end of the chapter range from simple recall of information to analyzing original data or proposing hypotheses to explain remaining questions in the field. Classic Experiment boxes throughout the text help students to appreciate the seminal experiments in immunology and how they were conducted, providing a bridge to the primary research articles and emphasizing data analysis at every step.

Media and Supplements NEW! ImmunoPortal (courses.bfwpub.com/immunology7e) This comprehensive and robust online teaching and learning tool combines a wealth of media resources, vigorous

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assessment, and helpful course management features into one convenient, fully customizable space.

ImmunoPortal Features: NEW! Kuby Immunology Seventh Edition e-Book—also available as a standalone resource (ebooks.bfwpub.com/ immunology7e) This online version of the textbook combines the contents of the printed book, electronic study tools, and a full complement of student media, including animations and videos. Students can personalize their e-Book with highlighting, bookmarking, and note-taking features. Instructors can customize the e-Book to focus on specific sections, and add their own notes and files to share with their class.

NEW! LearningCurve—A Formative Quizzing Engine With powerful adaptive quizzing, a game-like format, and the promise of significantly better grades, LearningCurve gives instructors a quickly implemented, highly effective new way to get students more deeply involved in the classroom. Developed by experienced teachers and experts in educational technology, LearningCurve offers a series of brief, engaging activities specific to your course. These activities put the concept of “testing to learn” into action with adaptive quizzing that treats each student as an individual with specific needs: • Students work through LearningCurve activities one question at a time. • With each question, students get immediate feedback. Responses to incorrect answers include links to book sections and other resources to help students focus on what they need to learn. • As they proceed toward completion of the activity, the level of questioning adapts to the level of performance. The questions become easier, harder, or the same depending on how the student is doing. • And with a more confident understanding of assigned material, students will be more actively engaged during classtime.

Resources The Resources center provides quick access to all instructor and student resources for Kuby Immunology.

For Instructors— All instructor media are available in the ImmunoPortal and on the Instructor Resource DVD. NEW! test bank—over 500 dynamic questions in PDF and editable Word formats include multiple-choice and shortanswer problems, rated by level of difficulty and Bloom’s Taxonomy level.

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Acknowledgements

Fully optimized JPEG files of every figure, photo, and table in the text, featuring enhanced color, higher resolution, and enlarged fonts. Images are also offered in PowerPoint® format for each chapter. Animations of complex text concepts and figures help students better understand key immunological processes. Videos specially chosen by the authors to complement and supplement text concepts.

• Flashcards test student mastery of vocabulary and allow students to tag the terms they’ve already learned. • Immunology on the Web weblinks introduce students to a world of online immunology resources and references.

For Students—

In this convenient space, ImmunoPortal provides instructors with the ability to assign any resource, as well as e-Book readings, discussion board posts, and their own materials. A gradebook tracks all student scores and can be easily exported to Excel or a campus Course Management System.

All of these resources are also available in the ImmunoPortal. • Student versions of the Animations and Videos, to help students understand key mechanisms and techniques at their own pace.

Assignments

Acknowledgements We owe special thanks to individuals who offered insightful ideas, who provided detailed reviews that led to major improvements, and who provided the support that made writing this text possible. These notable contributors include Dr. Stephen Emerson, Dr. David Allman, Dr. Susan Saidman, Dr. Nan Wang, Nicole Cunningham, and the many undergraduates who provided invaluable students’ perspectives on our chapters. We hope that the final product reflects the high quality of the input from these experts and colleagues and from all those listed below who provided critical analysis and guidance. We are also grateful to the previous authors of Kuby’s Immunology, whose valiant efforts we now appreciate even more deeply. Their commitment to clarity, to providing the most current material in a fast moving discipline, and to maintaining the experimental focus of the discussions set the standard that is the basis for the best of this text. We also acknowledge that this book represents the work not only of its authors and editors, but also of all those whose experiments and writing provided us with ideas, inspiration and information. We thank you and stress that all errors and inconsistencies of interpretation are ours alone. We thank the following reviewers for their comments and suggestions about the manuscript during preparation of this seventh edition. Their expertise and insights have contributed greatly to the book. Lawrence R. Aaronson, Utica College Jeffrey K. Actor, University of Texas Medical School at Houston Richard Adler, University of Michigan-Dearborn Emily Agard, York University, North York

Karthik Aghoram, Meredith College Rita Wearren Alisauskas, Rutgers University John Allsteadt, Virginia Intermont College Gaylene Altman, University of Washington Angelika Antoni, Kutztown University Jorge N. Artaza, Charles R. Drew University of Medicine and Science Patricia S. Astry, SUNY Fredonia Roberta Attanasio, Georgia State University Elizabeth Auger, Saint Joseph’s College of Maine Avery August, Penn State University Rajeev Aurora, Saint Louis University Hospital Christine A. Bacon, Bay Path College Jason C. Baker, Missouri Western State College Kenneth Balazovich, University of Michigan-Dearborn Jennifer L. Bankers-Fulbright, Augsburg College Amorette Barber, Longwood University Brianne Barker, Hamilton College Scott R. Barnum, University of Alabama at Birmingham Laura Baugh, University of Dallas Marlee B. Marsh, Columbia College Rachel Venn Beecham, Mississippi Valley State University Fabian Benencia, Ohio University Main Campus Charlie Garnett Benson, Georgia State University Daniel Bergey, Black Hills State University Carolyn A. Bergman, Georgian Court College Elke Bergmann-Leitner, WRAIR/Uniformed Services University of Health Services

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Acknowledgements Brian P. Bergstrom, Muskingum College Susan Bjerke, Washburn University of Topeka Earl F. Bloch, Howard University Elliott J. Blumenthal, Indiana University–Purdue University Fort Wayne Kathleen Bode, Flint Hills Technical College Dennis Bogyo, Valdosta State University Mark Bolyard, Union University Lisa Borghesi, University of Pittsburgh Phyllis C. Braun, Fairfield University Jay H. Bream, Johns Hopkins University School of Medicine Heather A. Bruns, Ball State University Walter J. Bruyninckx, Hanover College Eric L. Buckles, Dillard University Sandra H. Burnett, Brigham Young University Peter Burrows, University of Alabama at Birmingham Ralph Butkowski, Augsburg College Jean A. Cardinale, Alfred University Edward A. Chaperon, Creighton University Stephen K. Chapes, Kansas State University Christopher Chase, South Dakota State University Thomas Chiles, Boston College Harold Chittum, Pikeville College Peter A. Chung, Pittsburg State University Felicia L. Cianciarulo, Carlow University Bret A. Clark, Newberry College Patricia A. Compagnone-Post, Albertus Magnus College Yasemin Kaya Congleton, Bluegrass Community and Technical College Vincent A. Connors, University of South CarolinaSpartanburg Conway-Klaassen, University of Minnesota Lisa Cuchara, Quinnipiac University Tanya R. Da Sylva, York University, North York Kelley L. Davis, Nova Southeastern University Jeffrey Dawson, Duke University Joseph DeMasi, Massachusetts College of Pharmacy & Allied Health Stephanie E. Dew, Centre College Joyce E. S. Doan, Bethel University Diane Dorsett, Georgia Gwinnett College James R. Drake, Albany Medical College Erastus C. Dudley, Huntingdon College Jeannine M. Durdik, University of Arkansas Fayetteville Karen M. Duus, Albany Medical College Christina K. Eddy, North Greenville University Anthony Ejiofor, Tennessee State University Jennifer Ellington, Belmont Abbey College Samantha L. Elliott, Saint Mary’s College of Maryland Lehman L. Ellis, Our Lady of Holy Cross College Sherine F. Elsawa, Northern Illinois University

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Uthayashanker Ezekiel, Saint Louis University Medical Center Diana L. Fagan, Youngstown State University Rebecca V. Ferrell, Metropolitan State College of Denver Ken Field, Bucknell University Krista Fischer-Stenger, University of Richmond Howard B. Fleit, SUNY at Stony Brook Sherry D. Fleming, Kansas State University Marie-dominique Franco, Regis University Joel Gaikwad, Oral Roberts University D. L. Gibson, University of British Columbia-Okanagan Laura Glasscock, Winthrop University David Glick, Kings College Elizabeth Godrick, Boston University Karen Golemboski, Bellarmine University Sandra O. Gollnick, SUNY Buffalo James F. Graves, University of Detroit-Mercy Demetrius Peter Gravis, Beloit College Anjali D. Gray, Lourdes University Valery Z. Grdzelishvili, University of North Carolina-Charlotte Carla Guthridge, Cameron University David J. Hall, Lawrence University Sandra K. Halonen, Montana State University Michael C. Hanna, Texas A & M-Commerce Kristian M. Hargadon, Hampden-Sydney College JL Henriksen, Bellevue University Michelle L. Herdman, University of Charleston Jennifer L. Hess, Aquinas College Edward M. Hoffmann, University of Florida Kristin Hogquist, University of Minnesota Jane E. Huffman, East Stroudsburg University of Pennsylvania Lisa A. Humphries, University of California, Los Angeles Judith Humphries, Lawrence University Mo Hunsen, Kenyon College Vijaya Iragavarapu-Charyulu, Florida Atlantic University Vida R. Irani, Indiana University of Pennsylvania Christopher D. Jarvis, Hampshire College Eleanor Jator, Austin Peay State University Stephen R. Jennings, Drexel University College of Medicine Robert Jonas, Texas Lutheran University Vandana Kalia, Penn State University- Main Campus Azad K. Kaushik, University of Guelph George Keller, Samford University Kevin S. Kinney, De Pauw University Edward C. Kisailus, Canisius College David J. Kittlesen, University of Virginia Dennis J. Kitz, Southern Illinois University-Edwardsville Janet Kluftinger, University of British Columbia -Okanagan Rolf König, University of Texas Medical Branch at Galveston Kristine Krafts, University of Minnesota-Duluth Ruhul Kuddus, Utah Valley University Narendra Kumar, Texas A&M Health Science Center

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Acknowledgements

N. M. Kumbaraci, Stevens Institute of Technology Jesse J. Kwiek, The Ohio State University Main Camp John M. Lammert, Gustavus Aldolphus College Courtney Lappas, Lebanon Valley College Christopher S. Lassiter, Roanoke College Jennifer Kraft Leavey, Georgia Institute of Technology Melanie J. Lee-Brown, Guilford College Vicky M. Lentz, SUNY College at Oneonta Joseph Lin, Sonoma State University Joshua Loomis, Nova Southeastern University Jennifer Louten, Southern Polytechnic State University Jon H. Lowrance, Lipscomb University Milson J. Luce, West Virginia University Institute of Technology Phillip J. Lucido, Northwest Missouri State University M.E. MacKay, Thompson Rivers University Andrew P. Makrigiannis, University of Ottawa Greg Maniero, Stonehill College David Markwardt, Ohio Wesleyan University John Martinko, Southern Illinois University Andrea M. Mastro, Penn State University-Main Campus Ann H. McDonald, Concordia University Lisa N. McKernan, Chestnut Hill College Catherine S. McVay, Auburn University Daniel Meer, Cardinal Stritch University JoAnn Meerschaert, Saint Cloud State University Brian J. Merkel, University Wisconsin-Green Bay Jiri Mestecky, University of Alabama at Birmingham Dennis W. Metzger, Albany Medical College Jennifer A. Metzler, Ball State University John A. Meyers, Boston University Medical School Yuko J. Miyamoto, Elon College Jody M. Modarelli, Hiram College Devonna Sue Morra, Saint Francis University Rita B. Moyes, Texas A&M Annette Muckerheide, College of Mount Saint Joseph Sue Mungre, Northeastern Illinois University Kari L. Murad, College of Saint Rose Karen Grandel Nakaoka, Weber State University Rajkumar Nathaniel, Nicholls State University David Nemazee, University of California, San Diego Hamida Rahim Nusrat, San Francisco State University Tracy O’Connor, Mount Royal College Marcos Oliveira, University of the Incarnate Word Donald Ourth, University of Memphis Deborah Palliser, Albert Einstein College of Medicine Shawn Phippen, Valdosta State University Melinda J. Pomeroy-Black, La Grange College Edith Porter, California State University, Los Angeles Michael F. Princiotta, SUNY Upstate Medical University Gerry A Prody, Western Washington University Robyn A. Puffenbarger, Bridgewater College

Aimee Pugh-Bernard, University of Colorado at Denver Pattle Pun, Wheaton College Sheila Reilly, Belmont Abbey College Karen A. Reiner, Andrews University Margaret Reinhart, University of the Sciences in Philadelphia Stephanie Richards, Bates College Sarah M. Richart, Azusa Pacific University James E. Riggs, Rider University Vanessa Rivera-Amill, Ponce School of Medicine Katherine Robertson, Westminster College James L. Rooney, Lincoln University Robin S. Salter, Oberlin College Sophia Sarafova, Davidson College Surojit Sarkar, Penn State University-Main Campus Perry M. Scanlan, Austin Peay State University Ralph Seelke, University Wisconsin-Superior Diane L. Sewell, University Wisconsin-La Crosse Anding Shen, Calvin College Penny Shockett, Southeastern Louisiana University Michael Sikes, North Carolina State University Maryanne C. Simurda, Washington and Lee University Paul K. Small, Eureka College Jonathan Snow, Williams College Ralph A. Sorensen, Gettysburg College Andrew W. Stadnyk, Dalhousie University Faculty of Medicine Douglas A. Steeber, University Wisconsin-Milwaukee Viktor Steimle, University of Sherbrooke, Sherbrooke Douglas J. Stemke, University of Indianapolis Carolyn R. Stenbak, Seattle University Jennifer Ripley Stueckle, West Virginia University Kathleen Sullivan, Louisiana Technical College Alexandria Susmit Suvas, Oakland University Gabor Szalai, University of South Carolina Seetha M Tamma, Long Island University-C.W. Post Matthew J. Temple, Nazareth College Kent R. Thomas, Wichita State University Diane G. Tice, SUNY Morrisville Sara Sybesma Tolsma, Northwestern College Clara Tóth, Saint Thomas Aquinas College Bebhinn Treanor, University of Toronto Scarborough Allen W. Tsang, Bowman Gray Medical School Amar S. Tung, Lincoln University Lloyd Turtinen, University Wisconsin-Eau Claire Timothy M VanWagoner, Oklahoma Christian/ University of Oklahoma HSC Evros Vassiliou, Kean University Vishwanath Venketaraman, Western University of Health Sciences Kathleen Verville, Washington College Katherine A. Wall, University of Toledo Helen Walter, Mills College

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Acknowledgements Christopher Ward, University of Alberta Benjamin S. Weeks, Adelphi University Ben B. Whitlock, University of Saint Francis Robert Winn, Northern Michigan University Candace R. Winstead, California Polytechnic State UniversitySan Luis Obispo Dorothy M. Wrigley, Minnesota State University Jodi L. Yorty, Elizabethtown College Sheryl Zajdowicz, Metropolitan State University of Denver Mary Katherine Zanin, The Citadel The Military College of South Carolina Gary Zieve, SUNY at Stony Brook Michael I. Zimmer, Purdue Calumet Gilbert L. Zink, University of the Sciences in Philadelphia Patty Zwollo, College of William & Mary

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Finally, we thank our experienced and talented colleagues at W. H. Freeman and Company. Particular thanks to the production team members Philip McCaffrey, Sherrill Redd, Heath Lynn Silberfeld, Diana Blume, Lawrence Guerra, Janice Donnola, Christine Buese, and Elyse Reider. Thanks are also due to the editorial team of Lauren Schultz, Susan Winslow, Allison Michael, Yassamine Ebadat, and Irene Pech. However, a very special thanks go to our developmental editor, Erica Champion, and our developmental coordinator, Sara Ruth Blake. Erica has guided us from the beginning with a probing vision, endless patience, and keen eye for narrative and clarity. Sara kept us organized and true to deadlines with heroic resolve. The involvement of these two extraordinarily talented team members has made this edition, and its ambitious aspirations, possible.

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1

Overview of the Immune System

T

he immune system evolved to protect multicellular organisms from pathogens. Highly adaptable, it defends the body against invaders as diverse as the tiny (~30 nm), intracellular virus that causes polio and as large as the giant parasitic kidney worm Dioctophyme renale, which can grow to over 100 cm in length and 10 mm in width. This diversity of potential pathogens requires a range of recognition and destruction mechanisms to match the multitude of invaders. To accomplish this feat, vertebrates have evolved a complicated and dynamic network of cells, molecules, and pathways. Although elements of these networks can be found throughout the plant and animal kingdoms, the focus of this book will be on the highly evolved mammalian immune system. The fully functional immune system involves so many organs, molecules, cells, and pathways in such an interconnected and sometimes circular process that it is often difficult to know where to start! Recent advances in cell imaging, genetics, bioinformatics, as well as cell and molecular biology, have helped us to understand many of the individual players in great molecular detail. However, a focus on the details (and there are many) can make taking a step back to see the bigger picture challenging, and it is often the bigger picture that motivates us to study immunology. Indeed, the field of immunology can be credited with the vaccine that eradicated smallpox, the ability to transplant organs between humans, and the drugs used today to treat asthma. Our goal in this chapter is therefore to present the background and concepts in immunology that will help bridge the gap between the cellular and molecular detail presented in subsequent chapters and the complete picture of an immune response. A clear understanding of each of the many players involved will help one appreciate the intricate coordination of an immune system that makes all of this possible. The study of immunology has produced amazing and fascinating stories (some of which you will see in this book), where host and microbe engage in battles waged over both minutes and millennia. But the immune system is also much more than an isolated component of the body, merely responsible for search-and-destroy missions. In fact, it interleaves with many of the other body systems,

A phagocytic cell (macrophage, green) engulfing the bacteria that cause tuberculosis (orange). Max Planck Institute for Infection Biology/Dr. Volker Brinkmann



A Historical Perspective of Immunology



Important Concepts for Understanding the Mammalian Immune Response



The Good, Bad, and Ugly of the Immune System

including the endocrine, nervous, and metabolic systems, with more connections undoubtedly to be discovered in time. Finally, it has become increasingly clear that elements of immunity play key roles in regulating homeostasis in the body for a healthy balance. Information gleaned from the study of the immune system, as well as its connections with other systems, will likely have resounding repercussions across many basic science and biomedical fields, not to mention in the future of clinical medicine. This chapter begins with a historical perspective, charting the beginnings of the study of immunology, largely driven by the human desire to survive major outbreaks of infectious disease. This is followed by presentation of a few key concepts that are important hallmarks of the mammalian immune response, many of which may not have been encountered elsewhere in basic biology. This is not meant as a comprehensive overview of the mammalian immune system but rather as a means for jumping the large conceptual hurdles frequently encountered as one begins to describe the complexity and interconnected nature of the immune response. We hope this will whet the appetite and prepare the reader for a more thorough discussion of the specific components of immunity presented in the 1

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PA R T I

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Introduction

following chapters. We conclude with a few challenging clinical situations, such as instances in which the immune system fails to act or becomes the aggressor, turning its awesome powers against the host. More in-depth coverage of these and other medical aspects of immunology can be found in the final chapters of this book.

A Historical Perspective of Immunology The discipline of immunology grew out of the observation that individuals who had recovered from certain infectious diseases were thereafter protected from the disease. The Latin term immunis, meaning “exempt,” is the source of the English word immunity, a state of protection from infectious disease. Perhaps the earliest written reference to the phenomenon of immunity can be traced back to Thucydides, the great historian of the Peloponnesian War. In describing a plague in Athens, he wrote in 430 bc that only those who had recovered from the plague could nurse the sick because they would not contract the disease a second time. Although early societies recognized the phenomenon of immunity, almost 2000 years passed before the concept was successfully converted into medically effective practice.

Early Vaccination Studies Led the Way to Immunology The first recorded attempts to deliberately induce immunity were performed by the Chinese and Turks in the fifteenth century. They were attempting to prevent smallpox, a disease that is fatal in about 30% of cases and that leaves survivors disfigured for life (Figure 1-1). Reports suggest that the dried crusts derived from smallpox pustules were either inhaled or inserted into small cuts in the skin (a technique called variolation) in order to prevent this dreaded disease. In 1718, Lady Mary Wortley Montagu, the wife of the British ambassador in Constantinople, observed the positive effects of variolation on the native Turkish population and had the technique performed on her own children. The English physician Edward Jenner later made a giant advance in the deliberate development of immunity, again targeting smallpox. In 1798, intrigued by the fact that milkmaids who had contracted the mild disease cowpox were subsequently immune to the much more severe smallpox, Jenner reasoned that introducing fluid from a cowpox pustule into people (i.e., inoculating them) might protect them from smallpox. To test this idea, he inoculated an eight-year-old boy with fluid from a cowpox pustule and later intentionally infected the child with smallpox. As predicted, the child did not develop smallpox. Although this represented a major breakthrough, as one might imagine, these sorts of human studies could not be conducted under current standards of medical ethics. Jenner’s technique of inoculating with cowpox to protect against smallpox spread quickly through Europe. However, it

FIGURE 1-1 African child with rash typical of smallpox on face, chest, and arms. Smallpox, caused by the virus Variola major, has a 30% mortality rate. Survivors are often left with disfiguring scars. [Centers for Disease Control.]

was nearly a hundred years before this technique was applied to other diseases. As so often happens in science, serendipity combined with astute observation led to the next major advance in immunology: the induction of immunity to cholera. Louis Pasteur had succeeded in growing the bacterium that causes fowl cholera in culture, and confirmed this by injecting it into chickens that then developed fatal cholera. After returning from a summer vacation, he and colleagues resumed their experiments, injecting some chickens with an old bacterial culture. The chickens became ill, but to Pasteur’s surprise, they recovered. Interested, Pasteur then grew a fresh culture of the bacterium with the intention of injecting this lethal brew into some fresh, unexposed chickens. But as the story is told, his supply of fresh chickens was limited, and therefore he used a mixture of previously injected chickens and unexposed birds. Unexpectedly, only the fresh chickens died, while the chickens previously exposed to the older bacterial culture were completely protected from the disease. Pasteur hypothesized and later showed that aging had weakened the virulence of the pathogen and that such a weakened or attenuated strain could be administered to provide immunity against the disease. He called this attenuated strain a

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Overview of the Immune System vaccine (from the Latin vacca, meaning “cow”), in honor of Jenner’s work with cowpox inoculation. Pasteur extended these findings to other diseases, demonstrating that it was possible to attenuate a pathogen and administer the attenuated strain as a vaccine. In a now classic experiment performed in the small village of Pouilly-le-Fort in 1881, Pasteur first vaccinated one group of sheep with anthrax bacteria (Bacillus anthracis) that were attenuated by heat treatment. He then challenged the vaccinated sheep, along with some unvaccinated sheep, with a virulent culture of the anthrax bacillus. All the vaccinated sheep lived and all the unvaccinated animals died. These experiments marked the beginnings of the discipline of immunology. In 1885, Pasteur administered his first vaccine to a human, a young boy who had been bitten repeatedly by a rabid dog (Figure 1-2). The boy, Joseph Meister, was inoculated with a series of attenuated rabies virus preparations. The rabies vaccine is one of very few that can be successful when administered shortly after exposure, as long as the virus has not yet reached the central nervous system and begun to induce neurologic symptoms. Joseph lived, and later became a caretaker at the Pasteur Institute, which was opened in 1887 to treat the many rabies victims that began to flood in when word of Pasteur’s success spread; it remains to this day an institute dedicated to the prevention and treatment of infectious disease.

FIGURE 1-2 Wood engraving of Louis Pasteur watching Joseph Meister receive the rabies vaccine. [Source: From Harper’s Weekly 29:836; courtesy of the National Library of Medicine.]

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CHAPTER 1

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Vaccination Is an Ongoing, Worldwide Enterprise The emergence of the study of immunology and the discovery of vaccines are tightly linked. The development of effective vaccines for some pathogens is still a major challenge, discussed in greater detail in Chapter 17. However, despite many biological and social hurdles, vaccination has yielded some of the most profound success stories in terms of improving mortality rates worldwide, especially in very young children. In 1977, the last known case of naturally acquired smallpox was seen in Somalia. This dreaded disease was eradicated by universal application of a vaccine similar to that used by Jenner in the 1790s. One consequence of eradication is that universal vaccination becomes unnecessary. This is a tremendous benefit, as most vaccines carry at least a slight risk to persons vaccinated. And yet in many cases every individual does not need to be immune in order to protect most of the population. As a critical mass of people acquire protective immunity, either through vaccination or infection, they can serve as a buffer for the rest. This principle, called herd immunity, works by decreasing the number of individuals who can harbor and spread an infectious agent, significantly decreasing the chances that susceptible individuals will become infected. This presents an important altruistic consideration: although many of us could survive infectious diseases for which we receive a vaccine (such as the flu), this is not true for everyone. Some individuals cannot receive the vaccine (e.g., the very young or immune compromised), and vaccination is never 100% effective. In other words, the susceptible, nonimmune individuals among us can benefit from the pervasive immunity of their neighbors. However, there is a darker side to eradication and the end of universal vaccination. Over time, the number of people with no immunity to the disease will begin to rise, ending herd immunity. Vaccination for smallpox largely ended by the early to mid-1970s, leaving well over half of the current world population susceptible to the disease. This means that smallpox, or a weaponized version, is now considered a potential bioterrorism threat. In response, new and safer vaccines against smallpox are still being developed today, most of which go toward vaccinating U.S. military personnel thought to be at greatest risk of possible exposure. In the United States and other industrialized nations, vaccines have eliminated a host of childhood diseases that were the cause of death for many young children just 50 years ago. Measles, mumps, chickenpox, whooping cough (pertussis), tetanus, diphtheria, and polio, once thought of as an inevitable part of childhood are now extremely rare or nonexistent in the United States because of current vaccination practices (Table 1-1). One can hardly estimate the savings to society resulting from the prevention of these diseases. Aside from suffering and mortality, the cost to treat these illnesses and their aftereffects or sequelae (such as paralysis, deafness, blindness, and mental retardation) is immense and dwarfs the costs of immunization. In fact, recent estimates suggest

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Cases of selected infectious disease in the United States before and after the introduction of effective vaccines

Disease Smallpox

ANNUAL CASES/YR

CASES IN 2010

Prevaccine

Postvaccine

48,164

Reduction (%)

0

100 100

Diphtheria

175,885

0

Rubeola (measles)

503,282

26

99.99

Mumps

152,209

2,612

98.28

Pertussis (“whooping cough”)

147,271

27,550

81.29

Paralytic polio

16,316

0

Rubella (German measles)

47,745

5

Tetanus (“lockjaw”) Invasive Haemophilus influenzae

1,314 (deaths) 20,000

26 (cases) 3,151

100 99.99 98.02 84.25

SOURCE: Adapted from W. A. Orenstein et al., 2005. Health Affairs 24:599 and CDC statistics of Notifiable Diseases.

that significant economic and human life benefits could be realized by simply scaling up the use of a few childhood vaccines in the poorest nations, which currently bear the brunt of the impact of these childhood infectious diseases. For example, it is estimated that childhood pneumonia alone, caused primarily by vaccine-preventable Streptococcus pneumoniae (aka, pneumococcus) and Haemophilus influenzae type b (aka, Hib), will account for 2.7 million childhood deaths in developing nations over the next decade if vaccine strategies in these regions remain unchanged. Despite the many successes of vaccine programs, such as the eradication of smallpox, many vaccine challenges still remain. Perhaps the greatest current challenge is the design of effective vaccines for major killers such as malaria and AIDS. Using our increased understanding of the immune system, plus the tools of molecular and cellular biology, genomics, and proteomics, scientists will be better positioned to make progress toward preventing these and other emerging infectious diseases. A further issue is the fact that millions of children in developing countries die from diseases that are fully preventable by available, safe vaccines. High manufacturing costs, instability of the products, and cumbersome delivery problems keep these vaccines from reaching those who might benefit the most. This problem could be alleviated in many cases by development of future-generation vaccines that are inexpensive, heat stable, and administered without a needle. Finally, misinformation and myth surrounding vaccine efficacy and side effects continues to hamper many potentially life-saving vaccination programs (see Clinical Focus Box on p. 5).

Immunology Is About More Than Just Vaccines and Infectious Disease For some diseases, immunization programs may be the best or even the only effective defense. At the top of this list are infec-

tious diseases that can cause serious illness or even death in unvaccinated individuals, especially those transmitted by microbes that also spread rapidly between hosts. However, vaccination is not the only way to prevent or treat infectious disease. First and foremost is preventing infection, where access to clean water, good hygiene practices, and nutrient-rich diets can all inhibit transmission of infectious agents. Second, some infectious diseases are self-limiting, easily treatable, and nonlethal for most individuals, making them unlikely targets for costly vaccination programs. These include the common cold, caused by the Rhinovirus, and cold sores that result from Herpes Simplex Virus infection. Finally, some infectious agents are just not amenable to vaccination. This could be due to a range of factors, such as the number of different molecular variants of the organism, the complexity of the regimen required to generate protective immunity, or an inability to establish the needed immunologic memory responses (more on this later). One major breakthrough in the treatment of infectious disease came when the first antibiotics were introduced in the 1920s. Currently there are more than a hundred different antibiotics on the market, although most fall into just six or seven categories based on their mode of action. Antibiotics are chemical agents designed to destroy certain types of bacteria. They are ineffective against other types of infectious agents, as well as some bacterial species. One particularly worrying trend is the steady rise in antibiotic resistance among strains traditionally amenable to these drugs, making the design of next-generation antibiotics and new classes of drugs increasingly important. Although antiviral drugs are also available, most are not effective against many of the most common viruses, including influenza. This makes preventive vaccination the only real recourse against many debilitating infectious agents, even those that rarely cause mortality in healthy adults. For instance, because of the high mutation rate of the influenza virus, each year a new flu vaccine must

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BOX 1-1

CLINICAL FOCUS

Vaccine Controversy: What’s Truth and What’s Myth? Despite the record of worldwide success of vaccines in improving public health, some opponents claim that vaccines do more harm than good, pressing for elimination or curtailment of childhood vaccination programs. There is no dispute that vaccines represent unique safety issues, since they are administered to people who are healthy. Furthermore, there is general agreement that vaccines must be rigorously tested and regulated, and that the public must have access to clear and complete information about them. Although the claims of vaccine critics must be evaluated, many can be answered by careful and objective examination of records. A recent example is the claim that vaccines given to infants and very young children may contribute to the rising incidence of autism. This began with the suggestion that thimerosal, a mercury-based additive used to inhibit bacterial growth in some vaccine preparations since the 1930s, was causing autism in children. In 1999 the U.S. Centers for Disease Control and Prevention (CDC) and the American Association of Pediatricians (AAP) released a joint recommendation that vaccine manufacturers begin to gradually phase out thimerosal use in vaccines. This recommendation was based on the increase in the number of vaccines given to infants and was aimed at keeping children at or below Environmental Protection Agency (EPA)–recommended maximums in mercury exposure. However, with the release of this recommendation, parent-led public advocacy groups began a media-fueled campaign to build a case demonstrating

what they believed was a link between vaccines and an epidemic of autism. These AAP recommendations and public fears led to a dramatic decline in the latter half of 1999 in U.S. newborns vaccinated for hepatitis B. To date, no credible study has shown a scientific link between thimerosal and autism. In fact, cases of autism in children have continued to rise since thimerosal was removed from all childhood vaccines in 2001. Despite evidence to the contrary, some still believe this claim. A 1998 study appearing in The Lancet, a reputable British medical journal, further fueled these parent advocacy groups and anti-vaccine organizations. The article, published by Andrew Wakefield, claimed the measles-mumps-rubella (MMR) vaccine caused pervasive developmental disorders in children, including autism spectrum disorder. More than a decade of subsequent research has been unable to substantiate these claims, and 10 of the original 12 authors on the paper later withdrew their support for the conclusions of the study. In 2010, The Lancet retracted the original article when it was shown that the data in the study had been falsified to reach desired conclusions. Nonetheless, in the years between the original publication of the Lancet article and its retraction, this case is credited with decreasing rates of MMR vaccination from a high of 92% to a low of almost 60% in certain areas of the United Kingdom. The resulting expansion in the population of susceptible individuals led to endemic rates of measles and mumps infection, especially in several areas of Europe, and is credited with thou-

be prepared based on a prediction of the prominent genotypes likely to be encountered in the next season. Some years this vaccine is more effective than others. If and when a more lethal and unexpected pandemic strain arises, there will be a race between its spread and the manufacture and administration of a new vaccine. With the current ease of worldwide travel, present-day emergence of a pandemic strain of influenza could dwarf the devastation wrought by the 1918 flu pandemic, which left up to 50 million dead.

sands of extended hospitalizations and several deaths in infected children. Why has there been such a strong urge to cling to the belief that childhood vaccines are linked with developmental disorders in children despite much scientific evidence to the contrary? One possibility lies in the timing of the two events. Based on current AAP recommendations, in the United States most children receive 14 different vaccines and a total of up to 26 shots by the age of 2. In 1983, children received less than half this number of vaccinations. Couple this with the onset of the first signs of autism and other developmental disorders in children, which can appear quite suddenly and peak around 2 years of age. This sharp rise in the number of vaccinations young children receive today and coincidence in timing of initial autism symptoms is credited with sparking these fears about childhood vaccines. Add to this the increasing drop in basic scientific literacy by the general public and the overabundance of ways to gather such information (accurate or not). As concerned parents search for answers, one can begin to see how even scientifically unsupported links could begin to take hold as families grapple with how to make intelligent public health risk assessments. The notion that vaccines cause autism was rejected long ago by most scientists. Despite this, more work clearly needs to be done to bridge the gap between public perception and scientific understanding. Gross, L. 2009. A broken trust: Lessons from the vaccine–autism wars. PLoS Biology 7:e1000114. Larson, H.J., et al. 2011. Addressing the vaccine confidence gap. Lancet 378:526.

However, the eradication of infectious disease is not the only worthy goal of immunology research. As we will see later, exposure to infectious agents is part of our evolutionary history, and wiping out all of these creatures could potentially cause more harm than good, both for the host and the environment. Thanks to many technical advances allowing scientific discoveries to move efficiently from the bench to the bedside, clinicians can now manipulate the immune response in ways never before possible. For example, treatments to boost, inhibit,

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or redirect the specific efforts of immune cells are being applied to treat autoimmune disease, cancer, and allergy, as well as other chronic disorders. These efforts are already extending and saving lives. Likewise, a clearer understanding of immunity has highlighted the interconnected nature of body systems, providing unique insights into areas such as cell biology, human genetics, and metabolism. While a cure for AIDS and a vaccine to prevent HIV infection are still the primary targets for many scientists who study this disease, a great deal of basic science knowledge has been gleaned from the study of just this one virus and its interaction with the human immune system.

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Immunity Involves Both Humoral and Cellular Components Pasteur showed that vaccination worked, but he did not understand how. Some scientists believed that immune protection in vaccinated individuals was mediated by cells, while others postulated that a soluble agent delivered protection. The experimental work of Emil von Behring and Shibasaburo Kitasato in 1890 gave the first insights into the mechanism of immunity, earning von Behring the Nobel Prize in Physiology or Medicine in 1901 (Table 1-2). Von Behring and

Nobel Prizes for immunologic research

Year

Recipient

Country

Research

1901

Emil von Behring

Germany

Serum antitoxins

1905

Robert Koch

Germany

Cellular immunity to tuberculosis

1908

Elie Metchnikoff Paul Ehrlich

Russia Germany

Role of phagocytosis (Metchnikoff ) and antitoxins (Ehrlich) in immunity

1913

Charles Richet

France

Anaphylaxis

1919

Jules Bordet

Belgium

Complement-mediated bacteriolysis

1930

Karl Landsteiner

United States

Discovery of human blood groups

1951

Max Theiler

South Africa

Development of yellow fever vaccine

1957

Daniel Bovet

Switzerland

Antihistamines

1960

F. Macfarlane Burnet Peter Medawar

Australia Great Britain

Discovery of acquired immunological tolerance

1972

Rodney R. Porter Gerald M. Edelman

Great Britain United States

Chemical structure of antibodies

1977

Rosalyn R. Yalow

United States

Development of radioimmunoassay

1980

George Snell Jean Dausset Baruj Benacerraf

United States France United States

Major histocompatibility complex

1984

Niels K. Jerne Cesar Milstein Georges E. Köhler

Denmark Great Britain Germany

Immune regulatory theories (Jerne) and technological advances in the development of monoclonal antibodies (Milstein and Köhler)

1987

Susumu Tonegawa

Japan

Gene rearrangement in antibody production

1991

E. Donnall Thomas Joseph Murray

United States United States

Transplantation immunology

1996

Peter C. Doherty Rolf M. Zinkernagel

Australia Switzerland

Role of major histocompatibility complex in antigen recognition by T cells

2002

Sydney Brenner H. Robert Horvitz J. E. Sulston

South Africa United States Great Britain

Genetic regulation of organ development and cell death (apoptosis)

2008

Harald zur Hausen Françoise Barré-Sinoussi Luc Montagnier

Germany France France

Role of HPV in causing cervical cancer (Hausen) and the discovery of HIV (Barré-Sinoussi and Montagnier)

2011

Jules Hoffman Bruce Beutler Ralph Steinman

France United States United States

Discovery of activating principles of innate immunity (Hoffman and Beutler) and role of dendritic cells in adaptive immunity (Steinman)

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FIGURE 1-3 Drawing by Elie Metchnikoff of phagocytic cells surrounding a foreign particle (left) and modern image of a phagocyte engulfing the bacteria that cause tuberculosis (right). Metchnikoff first described and named the process of phagocytosis, or ingestion of foreign matter by white blood cells. Today, phagocytic cells can be imaged in great detail using advanced microscopy techniques. [Drawing reproduced by permission of The British Library:7616.h.19, Lectures on the Comparative Pathology of Inflammation delivered at the Pasteur Institute in 1891, translated by F. A. Starling and E. H. Starling, with plates by II’ya II’ich Mechnikov, 1893, p. 64, fig. 32. Photo courtesy Dr. Volker Brinkmann/Visuals Unlimited, Inc.]

Kitasato demonstrated that serum—the liquid, noncellular component recovered from coagulated blood—from animals previously immunized with diphtheria could transfer the immune state to unimmunized animals. In 1883, even before the discovery that a serum component could transfer immunity, Elie Metchnikoff, another Nobel Prize winner, demonstrated that cells also contribute to the immune state of an animal. He observed that certain white blood cells, which he termed phagocytes, ingested (phagocytosed) microorganisms and other foreign material (Figure 1-3, left). Noting that these phagocytic cells were more active in animals that had been immunized, Metchnikoff hypothesized that cells, rather than serum components, were the major effectors of immunity. The active phagocytic cells identified by Metchnikoff were likely blood monocytes and neutrophils (see Chapter 2), which can now be imaged using very sophisticated microscopic techniques (Figure 1-3, right). Humoral Immunity The debate over cells versus soluble mediators of immunity raged for decades. In search of the protective agent of immunity, various researchers in the early 1900s helped characterize the active immune component in blood serum. This soluble component could neutralize or precipitate toxins and could agglutinate (clump) bacteria. In each case, the component was named for the activity it exhibited: antitoxin, precipitin, and agglutinin, respectively. Initially, different serum components were thought to be responsible for each activity, but during the 1930s, mainly through the efforts of Elvin Kabat, a fraction of

serum first called gamma globulin (now immunoglobulin) was shown to be responsible for all these activities. The soluble active molecules in the immunoglobulin fraction of serum are now commonly referred to as antibodies. Because these antibodies were contained in body fluids (known at that time as the body humors), the immunologic events they participated in was called humoral immunity. The observation of von Behring and Kitasato was quickly applied to clinical practice. Antiserum, the antibodycontaining serum fraction from a pathogen-exposed individual, derived in this case from horses, was given to patients suffering from diphtheria and tetanus. A dramatic vignette of this application is described in the Clinical Focus box on page 8. Today there are still therapies that rely on transfer of immunoglobulins to protect susceptible individuals. For example, emergency use of immune serum, containing antibodies against snake or scorpion venoms, is a common practice for treating bite victims. This form of immune protection that is transferred between individuals is called passive immunity because the individual receiving it did not make his or her own immune response against the pathogen. Newborn infants benefit from passive immunity by the presence of maternal antibodies in their circulation. Passive immunity may also be used as a preventive (prophylaxis) to boost the immune potential of those with compromised immunity or who anticipate future exposure to a particular microbe. While passive immunity can supply a quick solution, it is short-lived and limited, as the cells that produce these antibodies are not being transferred. On the other hand, administration

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CLINICAL FOCUS

Passive Antibodies and the Iditarod In 1890, immunologists Emil Behring and Shibasaburo Kitasato, working together in Berlin, reported an extraordinary experiment. After immunizing rabbits with tetanus and then collecting blood serum from these animals, they injected a small amount of immune serum (cell-free fluid) into the abdominal cavity of six mice. Twenty-four hours later, they infected the treated mice and untreated controls with live, virulent tetanus bacteria. All of the control mice died within 48 hours of infection, whereas the treated mice not only survived but showed no effects of infection. This landmark experiment demonstrated two important points. One, it showed that substances that could protect an animal against pathogens appeared in serum following immunization. Two, this work demonstrated that immunity could be passively acquired, or transferred from one animal to another by taking serum from an immune animal and injecting it into a nonimmune one. These and subsequent experiments did not go unnoticed. Both men eventually received titles (Behring became von Behring and Kitasato became Baron Kitasato). A few years later, in 1901, von Behring was

awarded the first Nobel Prize in Physiology or Medicine (see Table 1-2). These early observations, and others, paved the way for the introduction of passive immunization into clinical practice. During the 1930s and 1940s, passive immunotherapy, the endowment of resistance to pathogens by transfer of antibodies from an immunized donor to an unimmunized recipient, was used to prevent or modify the course of measles and hepatitis A. Subsequently, clinical experience and advances in the technology of immunoglobulin preparation have made this approach a standard medical practice. Passive immunization based on the transfer of antibodies is widely used in the treatment of immunodeficiency and some autoimmune diseases. It is also used to protect individuals against anticipated exposure to infectious and toxic agents against which they have no immunity. Finally, passive immunization can be lifesaving during episodes of certain types of acute infection, such as following exposure to rabies virus. Immunoglobulin for passive immunization is prepared from the pooled

of a vaccine or natural infection is said to engender active immunity in the host: the production of one’s own immunity. The induction of active immunity can supply the individual with a renewable, long-lived protection from the specific infectious organism. As we discuss further below, this long-lived protection comes from memory cells, which provide protection for years or even decades after the initial exposure. Cell-Mediated Immunity A controversy developed between those who held to the concept of humoral immunity and those who agreed with Metchnikoff ’s concept of immunity imparted by specific cells, or cell-mediated immunity. The relative contributions of the two were widely debated at the time. It is now obvious that both are correct—the full immune response requires both cellular and humoral (soluble) components. Early studies of immune cells were hindered by the lack of genetically defined animal models and modern tissue culture tech-

plasma of thousands of donors. In effect, recipients of these antibody preparations are receiving a sample of the antibodies produced by many people to a broad diversity of pathogens—a gram of intravenous immune globulin (IVIG) contains about 1018 molecules of antibody and recognize more than 107 different antigens. A product derived from the blood of such a large number of donors carries a risk of harboring pathogenic agents, particularly viruses. This risk is minimized by modern-day production techniques. The manufacture of IVIG involves treatment with solvents, such as ethanol, and the use of detergents that are highly effective in inactivating viruses such as HIV and hepatitis. In addition to treatment against infectious disease, or acute situations, IVIG is also used today for treating some chronic diseases, including several forms of immune deficiency. In all cases, the transfer of passive immunity supplies only temporary protection. One of the most famous instances of passive antibody therapy occurred in 1925, when an outbreak of diphtheria

niques, whereas early studies with serum took advantage of the ready availability of blood and established biochemical techniques to purify proteins. Information about cellular immunity therefore lagged behind a characterization of humoral immunity. In a key experiment in the 1940s, Merrill Chase, working at The Rockefeller Institute, succeeded in conferring immunity against tuberculosis by transferring white blood cells between guinea pigs. Until that point, attempts to develop an effective vaccine or antibody therapy against tuberculosis had met with failure. Thus, Chase’s demonstration helped to rekindle interest in cellular immunity. With the emergence of improved cell culture and transfer techniques in the 1950s, the lymphocyte was identified as the cell type responsible for both cellular and humoral immunity. Soon thereafter, experiments with chickens pioneered by Bruce Glick at Mississippi State University indicated the existence of two types of lymphocytes: T lymphocytes (T cells), derived from

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BOX 1-2

White Mountain Golovin Koyuk Galena Nulato Nome Ruby Safety Elim Kaktag Cripple Shaktoolik

Nenana

Unalakleet Nikolai Ophir Takotna Rohn McGrath Skwentna Rainy Pass Yentna Finger Lake Willow Anchorage Campell Airstrip

FIGURE 1 (left) Leonhard Seppala, the Norwegian who led a team of sled dogs in the 1925 diphtheria antibody run from Nenana to Nome, Alaska. (right) Map of the current route of the Iditarod Race, which commemorates this historic delivery of lifesaving antibody. [Source: Underwood & Underwood/Corbis.]

was diagnosed in what was then the remote outpost of Nome, Alaska. Lifesaving diphtheria-specific antibodies were available in Anchorage, but no roads were open and the weather was too dangerous for flight. History tells us that 20 mushers set up a dogsled relay to cover the almost 700 miles between Nenana, the end of the railroad run, and remote

Nome. In this relay, two Norwegians and their dogs covered particularly critical territory and withstood blizzard conditions: Leonhard Seppala (Figure 1, left), who covered the most treacherous territory, and Gunnar Kaasen, who drove the final two legs in whiteout conditions, behind his lead dog Balto. Kaasen and Balto arrived in time to save many of the chil-

the thymus, and B lymphocytes (B cells), derived from the bursa of Fabricius in birds (an outgrowth of the cloaca). In a convenient twist of nomenclature that makes B and T cell origins easier to remember, the mammalian equivalent of the bursa of Fabricius is bone marrow, the home of developing B cells in mammals. We now know that cellular immunity is imparted by T cells and that the antibodies produced by B cells confer humoral immunity. The real controversy about the roles of humoral versus cellular immunity was resolved when the two systems were shown to be intertwined and it became clear that both are necessary for a complete immune response against most pathogens.

How Are Foreign Substances Recognized by the Immune System? One of the great enigmas confronting early immunologists was what determines the specificity of the immune response

dren in the town. To commemorate this heroic event, later that same year a statue of Balto was placed in Central Park, New York City, where it still stands today. This journey is memorialized every year in the running of the Iditarod sled dog race. A map showing the current route of this more than 1000-mile trek is shown in Figure 1, right.

for a particular foreign material, or antigen, the general term for any substance that elicits a specific response by B or T lymphocytes. Around 1900, Jules Bordet at the Pasteur Institute expanded the concept of immunity beyond infectious diseases, demonstrating that nonpathogenic substances, such as red blood cells from other species, could also serve as antigens. Serum from an animal that had been inoculated with noninfectious but otherwise foreign (nonself) material would nevertheless react with the injected material in a specific manner. The work of Karl Landsteiner and those who followed him showed that injecting an animal with almost any nonself organic chemical could induce production of antibodies that would bind specifically to the chemical. These studies demonstrated that antibodies have a capacity for an almost unlimited range of reactivity, including responses to compounds that had only recently been synthesized in the laboratory and are otherwise not found in nature! In addition, it was

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shown that molecules differing in the smallest detail, such as a single amino acid, could be distinguished by their reactivity with different antibodies. Two major theories were proposed to account for this specificity: the selective theory and the instructional theory. The earliest conception of the selective theory dates to Paul Ehrlich in 1900. In an attempt to explain the origin of serum antibody, Ehrlich proposed that cells in the blood expressed a variety of receptors, which he called side-chain receptors, that could bind to infectious agents and inactivate them. Borrowing a concept used by Emil Fischer in 1894 to explain the interaction between an enzyme and its substrate, Ehrlich proposed that binding of the receptor to an infectious agent was like the fit between a lock and key. Ehrlich suggested that interaction between an infectious agent and a cell-bound receptor would induce the cell to produce and release more receptors with the same specificity (Figure 1-4). In Ehrlich’s mind, the cells were pluripotent, expressing a number of

FIGURE 1-4 Representation of Paul Ehrlich’s side chain theory to explain antibody formation. In Ehrlich’s initial theory, the cell is pluripotent in that it expresses a number of different receptors or side chains, all with different specificities. If an antigen encounters this cell and has a good fit with one of its side chains, synthesis of that receptor is triggered and the receptor will be released. [From Ehrlich’s Croonian lecture of 1900 to the Royal Society.]

different receptors, each of which could be individually “selected.” According to Ehrlich’s theory, the specificity of the receptor was determined in the host before its exposure to the foreign antigen, and therefore the antigen selected the appropriate receptor. Ultimately, most aspects of Ehrlich’s theory would be proven correct, with the following minor refinement: instead of one cell making many receptors, each cell makes many copies of just one membrane-bound receptor (one specificity). An army of cells, each with a different antigen specificity, is therefore required. The selected B cell can be triggered to proliferate and to secrete many copies of these receptors in soluble form (now called antibodies) once it has been selected by antigen binding. In the 1930s and 1940s, the selective theory was challenged by various instructional theories. These theories held that antigen played a central role in determining the specificity of the antibody molecule. According to the instructional theorists, a particular antigen would serve as a template around which antibody would fold—sort of like an impression mold. The antibody molecule would thereby assume a configuration complementary to that of the antigen template. This concept was first postulated by Friedrich Breinl and Felix Haurowitz in about 1930 and redefined in the 1940s in terms of protein folding by Linus Pauling. In the 1950s, selective theories resurfaced as a result of new experimental data. Through the insights of F. Macfarlane Burnet, Niels Jerne, and David Talmadge, this model was refined into a hypothesis that came to be known as the clonal selection theory. This hypothesis has been further refined and is now accepted as an underlying paradigm of modern immunology. According to this theory, an individual B or T lymphocyte expresses many copies of a membrane receptor that is specific for a single, distinct antigen. This unique receptor specificity is determined in the lymphocyte before it is exposed to the antigen. Binding of antigen to its specific receptor activates the cell, causing it to proliferate into a clone of daughter cells that have the same receptor specificity as the parent cell. The instructional theories were formally disproved in the 1960s, by which time information was emerging about the structure of protein, RNA, and DNA that would offer new insights into the vexing problem of how an individual could make antibodies against almost anything, sight unseen. Overview Figure 1-5 presents a very basic scheme of clonal selection in the humoral (B cell) and cellular (T cell) branches of immunity. We now know that B cells produce antibodies, a soluble version of their receptor protein, which bind to foreign proteins, flagging them for destruction. T cells, which come in several different forms, also use their surfacebound T-cell receptors to sense antigen. These cells can perform a range of different functions once selected by antigen encounter, including the secretion of soluble compounds to aid other white blood cells (such as B lymphocytes) and the destruction of infected host cells.

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1-5

OVERVIEW FIGURE

An Outline for the Humoral and Cell-Mediated (Cellular) Branches of the Immune System Foreign proteins or infectious agents

Vertebrate body Humoral response (B lymphocytes)

Cell-mediated response (T lymphocytes) T-cell receptor

B cell B T-cell receptor +

T

B-cell receptor

T

+ Antigen

+ Antigen

Antigen T Antigen-selected antibodysecreting B cell

Antigenselected T cells

T Killing of infected cells

B

Cytokine secretion

Antibody

Antigen elimination

The humoral response involves interaction of B cells with foreign proteins, called antigens, and their differentiation into antibodysecreting cells. The secreted antibody binds to foreign proteins or infectious agents, helping to clear them from the body. The cell-

Important Concepts for Understanding the Mammalian Immune Response Today, more than ever, we are beginning to understand on a molecular and cellular level how a vaccine or infection leads to the development of immunity. As highlighted by

mediated response involves various subpopulations of T lymphocytes, which can perform many functions, including the secretion of soluble messengers that help direct other cells of the immune system and direct killing of infected cells.

the historical studies described above, this involves a complex system of cells and soluble compounds that have evolved to protect us against an enormous range of invaders of all shapes, sizes, and chemical structures. In this section, we cover the range of organisms that challenge the immune system and several of the important new concepts that are unique hallmarks of how the immune system carries out this task.

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Pathogens Come in Many Forms and Must First Breach Natural Barriers Organisms causing disease are termed pathogens, and the process by which they induce illness in the host is called pathogenesis. The human pathogens can be grouped into four major categories based on shared characteristics: viruses, fungi, parasites, and bacteria (Table 1-3). Some example organisms from each category can be found in Figure 1-6. As we will see in the next section, some of the shared characteristics that are common to groups of pathogens, but not to the host, can be exploited by the immune system for recognition and destruction. The microenvironment in which the immune response begins to emerge can also influence the outcome; the same pathogen may be treated differently depending on the context in which it is encountered. Some areas of the body, such as the central nervous system, are virtually “off limits” for the immune system because the immune response could do more damage than the pathogen. In other cases, the environment may come with inherent directional cues for immune cells. For instance, some foreign compounds that enter via the digestive tract, including the commensal microbes that help us digest food, are tolerated by the immune system. However, when these same foreigners enter the bloodstream they are typically treated much more aggressively. Each encounter with pathogen thus engages a distinct set of strategies that depends on the nature of the invader and on the microenvironment in which engagement occurs. It is worth noting that immune pathways do not become engaged until foreign organisms first breach the physical barriers of the body. Obvious barriers include the skin and

TABLE 1-3

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the mucous membranes. The acidity of the stomach contents, of the vagina, and of perspiration poses a further barrier to many organisms, which are unable to grow in low pH conditions. The importance of these barriers becomes obvious when they are surmounted. Animal bites can communicate rabies or tetanus, whereas insect puncture wounds can transmit the causative agents of such diseases as malaria (mosquitoes), plague (fleas), and Lyme disease (ticks). A dramatic example is seen in burn victims, who lose the protective skin at the burn site and must be treated aggressively with drugs to prevent the rampant bacterial and fungal infections that often follow.

The Immune Response Quickly Becomes Tailored to Suit the Assault With the above in mind, an effective defense relies heavily on the nature of the invading pathogen offense. The cells and molecules that become activated in a given immune response depend on the chemical structures present on the pathogen, whether it resides inside or outside of host cells, and the location of the response. This means that different chemical structures and microenvironmental cues need to be detected and appropriately evaluated, initiating the most effective response strategy. The process of pathogen recognition involves an interaction between the foreign organism and a recognition molecule (or molecules) expressed by host cells. Although these recognition molecules are frequently membranebound receptors, soluble receptors or secreted recognition molecules can also be engaged. Ligands for these recognition molecules can include whole pathogens, antigenic fragments of pathogens, or products secreted by these foreign organisms. The outcome of this ligand binding is an intracellular

Major categories of human pathogens

Major groups of human pathogens

Specific examples

Disease

Viruses

Poliovirus Variola Virus Human Immunodeficiency Virus Rubeola Virus

Poliomyelitis (Polio) Smallpox AIDS Measles

Fungi

Candida albicans Tinea corporis Cryptococcus neoformans

Candidiasis (Thrush) Ringworm Cryptococcal meningitis

Parasites

Plasmodium species Leishmania major Entamoeba histolytica

Malaria Leishmaniasis Amoebic colitis

Bacteria

Mycobacterium tuberculosis Bordetella pertussis Vibrio cholerae Borrelia burgdorferi

Tuberculosis Whooping cough (pertussis) Cholera Lyme disease

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(a) Virus: Rotavirus

(b) Fungus: Candida albicans

(c) Parasite: Filaria

(d) Bacterium: Mycobacterium tuberculosis

FIGURE 1-6 Pathogens representing the major categories

normal bacterial flora. (c) Parasites: The larval form of filaria, a parasitic worm, being attacked by macrophages. Approximately 120 million persons worldwide have some form of filariasis. (d) Bacteria: Mycobacterium tuberculosis, the bacterium that causes tuberculosis, being ingested by a human macrophage. [(a) Dr. Gary Gaugler/Getty Images;

of microorganisms causing human disease. (a) Viruses: Transmission electron micrograph of rotavirus, a major cause of infant diarrhea. Rotavirus accounts for approximately 1 million infant deaths per year in developing countries and hospitalization of about 50,000 infants per year in the United States. (b) Fungi: Candida albicans, a yeast inhabiting human mouth, throat, intestines, and genitourinary tract; C. albicans commonly causes an oral rash (thrush) or vaginitis in immunosuppressed individuals or in those taking antibiotics that kill

or extracellular cascade of events that ultimately leads to the labeling and destruction of the pathogen—simply referred to as the immune response. The entirety of this response is actually engagement of a complex system of cells that can recognize and kill or engulf a pathogen (cellular immunity), as well as a myriad of soluble proteins that help to orchestrate labeling and destruction of foreign invaders (humoral immunity). The nature of the immune response will vary depending on the number and type of recognition molecules engaged. For instance, all viruses are tiny, obligate, intracellular patho-

(b) SPL/Photo Researchers; (c) Oliver Meckes/Nicole Ottawa/Eye of Science/ Photo Researchers; (d) Max Planck Institute for Infection Biology/Dr. Volker Brinkmann.]

gens that spend the majority of their life cycle residing inside host cells. An effective defense strategy must therefore involve identification of infected host cells along with recognition of the surface of the pathogen. This means that some immune cells must be capable of detecting changes that occur in a host cell after it becomes infected. This is achieved by a range of cytotoxic cells but especially cytotoxic T lymphocytes (aka CTLs, or Tc cells), a part of the cellular arm of immunity. In this case, recognition molecules positioned inside cells are key to the initial response. These intracellular receptors bind to viral proteins present in the cytosol and

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initiate an early warning system, alerting the cell to the presence of an invader. Sacrifice of virally infected cells often becomes the only way to truly eradicate this type of pathogen. In general, this sacrifice is for the good of the whole organism, although in some instances it can cause disruptions to normal function. For example, the Human Immunodeficiency Virus (HIV) infects a type of T cell called a T helper cell (TH cell). These cells are called helpers because they guide the behavior of other immune cells, including B cells, and are therefore pivotal for selecting the pathway taken by the immune response. Once too many of these cells are destroyed or otherwise rendered nonfunctional, many of the directional cues needed for a healthy immune response are missing and fighting all types of infections becomes problematic. As we discuss later in this chapter, the resulting immunodeficiency allows opportunistic infections to take hold and potentially kill the patient. Similar but distinct immune mechanisms are deployed to mediate the discovery of extracellular pathogens, such as fungi, most bacteria, and some parasites. These rely primarily on cell surface or soluble recognition molecules that probe the extracellular spaces of the body. In this case, B cells and the antibodies they produce as a part of humoral immunity play major roles. For instance, antibodies can squeeze into spaces in the body where B cells themselves may not be able to reach, helping to identify pathogens hiding in these out-of-reach places. Large parasites present yet another problem; they are too big for phagocytic cells to envelop. In this case, cells that can deposit toxic substances or that can secrete products that induce expulsion (e.g., sneezing, coughing, vomiting) become a better strategy. As we study the complexities of the mammalian immune response, it is worth remembering that a single solution does not exist for all pathogens. At the same time, these various immune pathways carry out their jobs with considerable overlap in structure and in function.

Pathogen Recognition Molecules Can Be Encoded in the Germline or Randomly Generated As one might imagine, most pathogens express at least a few chemical structures that are not typically found in mammals. Pathogen-associated molecular patterns (or PAMPs) are common foreign structures that characterize whole groups of pathogens. It is these unique antigenic structures that the immune system frequently recognizes first. Animals, both invertebrates and vertebrates, have evolved to express several types of cell surface and soluble proteins that quickly recognize many of these PAMPs; a form of pathogen profiling. For example, encapsulated bacteria possess a polysaccharide coat with a unique chemical structure that is not found on other bacterial or human cells. White blood cells naturally express a variety of receptors, collectively referred to as pattern recognition receptors (PRRs), that specifically recognize these sugar residues, as well as other common foreign struc-

tures. When PRRs detect these chemical structures, a cascade of events labels the target pathogen for destruction. PPRs are proteins encoded in the genomic DNA and are always expressed by many different immune cells. These conserved, germline-encoded recognition molecules are thus a first line of defense for the quick detection of many of the typical chemical identifiers carried by the most common invaders. A significant and powerful corollary to this is that it allows early categorizing or profiling of the sort of pathogen of concern. This is key to the subsequent immune response routes that will be followed, and therefore the fine tailoring of the immune response as it develops. For example, viruses frequently expose unique chemical structures only during their replication inside host cells. Many of these can be detected via intracellular receptors that bind exposed chemical moieties while still inside the host cell. This can trigger an immediate antiviral response in the infected cell that blocks further virus replication. At the same time, this initiates the secretion of chemical warning signals sent to nearby cells to help them guard against infection (a neighborhood watch system!). This early categorizing happens via a subtle tracking system that allows the immune response to make note of which recognition molecules were involved in the initial detection event and relay that information to subsequent responding immune cells, allowing the follow-up response to begin to focus attention on the likely type of assault underway. Host-pathogen interactions are an ongoing arms race; pathogens evolve to express unique structures that avoid host detection, and the host germline-encoded recognition system co-evolves to match these new challenges. However, because pathogens generally have much shorter life cycles than their vertebrate hosts, and some utilize error-prone DNA polymerases to replicate their genomes, pathogens can evolve rapidly to evade host encoded recognition systems. If this were our only defense, the host immune response would quickly become obsolete thanks to these real-time pathogen avoidance strategies. How can the immune system prepare for this? How can our DNA encode a recognition system for things that change in random ways over time? Better yet, how do we build a system to recognize new chemical structures that may arise in the future? Thankfully, the vertebrate immune system has evolved a clever, albeit resource intensive, response to this dilemma: to favor randomness in the design of some recognition molecules. This strategy, called generation of diversity, is employed only by developing B and T lymphocytes. The result is a group of B and T cells where each expresses many copies of one unique recognition molecule, resulting in a population with the theoretical potential to respond to any antigen that may come along (Figure 1-7). This feat is accomplished by rearranging and editing the genomic DNA that encodes the antigen receptors expressed by each B or T lymphocyte. Not unlike the error-prone DNA replication method employed by pathogens, this system allows chance to play a role in generating a menu of responding recognition molecules. As one might imagine, however, this cutting and splicing of chromosomes is not without risk. Many B and T cells do not

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Deletion

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Clonal selection and expansion

2

1

2 Antigen 2 2

2

2

Stem cell

3

2

3 2

4

Primary lymphoid organs

FIGURE 1-7 Generation of diversity and clonal selection in T and B lymphocytes. Maturation in T and B cells, which occurs in primary lymphoid organs (bone marrow for B cells and thymus for T cells) in the absence of antigen, produces cells with a committed antigenic specificity, each of which expresses many copies of surface receptor that binds to one particular antigen. Different clones of B cells (1, 2, 3, and 4) are illustrated in this figure. Cells that do not die or become deleted during this maturation and weeding-out process move into the circulation of the body and are available to interact with antigen. There, clonal selection occurs when one of these cells

survive this DNA surgery or the quality control processes that follow, all of which take place in primary lymphoid organs: the thymus for T cells and bone marrow for B cells. Surviving cells move into the circulation of the body, where they are available if their specific, or cognate, antigen is encountered. When antigens bind to the surface receptors on these cells, they trigger clonal selection (see Figure 1-7). The ensuing proliferation of the selected clone of cells creates an army of cells, all with the same receptor and responsible for binding more of the same antigen, with the ultimate goal of destroying the pathogen in question. In B lymphocytes, these recognition molecules are B-cell receptors when they are surface structures and antibodies in their secreted form. In T lymphocytes, where no soluble form exists, they are T-cell receptors. In 1976 Susumu Tonegawa, then at The Basel Institute for Immunology in Switzerland, discovered the molecular mechanism behind the DNA recombination events that generate B-cell receptors and antibodies (Chapter 7 covers this in detail). This

Circulation through the body

encounters its cognate or specific antigen. Clonal proliferation of an antigen-activated cell (number 2 or pink in this example) leads to many cells that can engage with and destroy the antigen, plus memory cells that can be called upon during a subsequent exposure. The B cells secrete antibody, a soluble form of the receptor, reactive with the activating antigen. Similar processes take place in the T-lymphocyte population, resulting in clones of memory T cells and effector T cells; the latter include activated TH cells, which secrete cytokines that aid in the further development of adaptive immunity, and cytotoxic T lymphocytes (CTLs), which can kill infected host cells.

was a true turning point in immunologic understanding; for this discovery he received widespread recognition, including the 1987 Nobel Prize in Physiology or Medicine (see Table 1-2).

Tolerance Ensures That the Immune System Avoids Destroying the Host One consequence of generating random recognition receptors is that some could recognize and target the host. In order for this strategy to work effectively, the immune system must somehow avoid accidentally recognizing and destroying host tissues. This principle, which relies on self/ nonself discrimination, is called tolerance, another hallmark of the immune response. The work credited with its illumination also resulted in a Nobel Prize in Physiology or Medicine, awarded to F. Macfarlane Burnet and Peter Medawar in 1960. Burnet was the first to propose that exposure to nonself antigens during certain stages of life could result in an

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Introduction

immune system that ignored these antigens later. Medawar later proved the validity of this theory by exposing mouse embryos to foreign antigens and showing that these mice developed the ability to tolerate these antigens later in life. To establish tolerance, the antigen receptors present on developing B and T cells must first pass a test of nonresponsiveness against host structures. This process, which begins shortly after these randomly generated receptors are produced, is achieved by the destruction or inhibition of any cells that have inadvertently generated receptors with the ability to harm the host. Successful maintenance of tolerance ensures that the host always knows the difference between self and nonself (usually referred to as foreign). One recent re-envisioning of how tolerance is operationally maintained is called the danger hypothesis. This hypothesis suggests that the immune system constantly evaluates each new encounter more for its potential to be dangerous to the host than for whether it is self or not. For instance, cell death can have many causes, including natural homeostatic processes, mechanical damage, or infection. The former is a normal part of the everyday biological events in the body, and only requires a cleanup response to remove debris. The latter two, however, come with warning signs that include the release of intracellular contents, expression of cellular stress proteins, or pathogen-specific products. These pathogen or cell-associated stress compounds, sometimes referred to as danger signals, can engage specific host recognition molecules (e.g., PRRs) that deliver a signal to immune cells to get involved during these unnatural causes of cellular death. One unintended consequence of robust self-tolerance is that the immune system frequently ignores cancerous cells that arise in the body, as long as these cells continue to express self structures that the immune system has been trained to ignore. Dysfunctional tolerance is at the root of most autoimmune diseases, discussed further at the end of this chapter and in greater detail in Chapter 16. As one might imagine, failures in the establishment or maintenance of tolerance can have devastating clinical outcomes.

The Immune Response Is Composed of Two Interconnected Arms: Innate Immunity and Adaptive Immunity Although reference is made to “the immune system,” it is important to appreciate that there are really two interconnected systems of immunity: innate and adaptive. These two systems collaborate to protect the body against foreign invaders. Innate immunity includes built-in molecular and cellular mechanisms that are encoded in the germline and are evolutionarily more primitive, aimed at preventing infection or quickly eliminating common invaders (Chapter 5). This includes physical and chemical barriers to infection, as well as the DNA-encoded receptors recognizing common chemical structures of many pathogens (see PRRs, above). In this case, rapid recognition and phagocytosis or destruction of the pathogen is the outcome. Innate immunity also includes a series of preexisting serum proteins, collectively

referred to as complement, that bind common pathogenassociated structures and initiate a cascade of labeling and destruction events (Chapter 6). This highly effective first line of defense prevents most pathogens from taking hold, or eliminates infectious agents within hours of encounter. The recognition elements of the innate immune system are fast, some occurring within seconds of a barrier breach, but they are not very specific and are therefore unable to distinguish between small differences in foreign antigens. A second form of immunity, known as adaptive immunity, is much more attuned to subtle molecular differences. This part of the system, which relies on B and T lymphocytes, takes longer to come on board but is much more antigen specific. Typically, there is an adaptive immune response against a pathogen within 5 or 6 days after the barrier breach and initial exposure, followed by a gradual resolution of the infection. Adaptive immunity is slower partly because fewer cells possess the perfect receptor for the job: the antigenspecific, randomly generated receptors found on B and T cells. It is also slower because parts of the adaptive response rely on prior encounter and “categorizing” of antigens undertaken by innate processes. After antigen encounter, T and B lymphocytes undergo selection and proliferation, described earlier in the clonal selection theory of antigen specificity. Although slow to act, once these B and T cells have been selected and have honed their attack strategy, they become formidable opponents that can typically resolve the infection. The adaptive arm of the immune response evolves in real time in response to infection and adapts (thus the name) to better recognize, eliminate, and remember the invading pathogen. Adaptive responses involve a complex and interconnected system of cells and chemical signals that come together to finish the job initiated during the innate immune response. The goal of all vaccines against infectious disease is to elicit the development of specific and long-lived adaptive responses, so that the vaccinated individual will be protected in the future when the real pathogen comes along. This arm of immunity is orchestrated mainly via B and T lymphocytes following engagement of their randomly generated antigen recognition receptors. How these receptors are generated is a fascinating story, covered in detail in Chapter 7 of this book. An explanation of how these cells develop to maturity (Chapters 9 and 10) and then work in the body to protect us from infection (Chapters 11-14) or sometimes fail us (Chapters 15-19) takes up the vast majority of this book. The number of pages dedicated to discussing adaptive responses should not give the impression that this arm of the immune response is more important, or can work independently from, innate immunity. In fact, the full development of the adaptive response is dependent upon earlier innate pathways. The intricacies of their interconnections remain an area of intense study. The 2011 Nobel Prize in Physiology or Medicine was awarded to three scientists who helped clarify these two arms of the response: Bruce Beutler and Jules Hoffmann for discoveries related to the activation events important for innate immunity, and Ralph Steinman for his discovery of the role of dendritic cells in activating adaptive immune

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responses (see Table 1-2). Because innate pathways make first contact with pathogens, the cells and molecules involved in this arm of the response use information gathered from their early encounter with pathogen to help direct the process of adaptive immune development. Adaptive immunity thus provides a second and more comprehensive line of defense, informed by the struggles undertaken by the innate system. It is worth noting that some infections are, in fact, eliminated by innate immune mechanisms alone, especially those that remain localized and involve very low numbers of fairly benign foreign invaders. (Think of all those insect bites or splinters in life that introduce bacteria under the skin!) Table 1-4 compares the major characteristics that distinguish innate and adaptive immunity. Although for ease of discussion the immune system is typically divided into these two arms of the response, there is considerable overlap of the cells and mechanisms involved in each of these arms of immunity. For innate and adaptive immunity to work together, these two systems must be able to communicate with one another. This communication is achieved by both cell-cell contact and by soluble messengers. Most of these soluble proteins are growth factor–like molecules known by the general name cytokines. Cytokines and cell surface ligands can bind with receptors found on responding cells and signal these cells to perform new functions, such as synthesis of other soluble factors or differentiation to a new cell type. A subset of these soluble signals are called chemokines because they have chemotactic activity, meaning they can recruit specific cells to the site. In this way, cytokines, chemokines, and other soluble factors produced by immune cells recruit or instruct cells and soluble proteins important for eradication of the pathogen from within the infection site. We’ve probably all felt this convergence in the form of swelling, heat, and tenderness at the site of exposure. These events are a part of a larger process collectively referred to as an inflammatory response, which is covered in detail in Chapter 15.

Adaptive Immune Responses Typically Generate Memory One particularly significant and unique attribute of the adaptive arm of the immune response is immunologic

TABLE 1-4

Magnitude of immune response

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Repeat Antigen A Antigen A

Adaptive Innate 0

14

28 0

14

28

Time, days Primary response

Secondary response

FIGURE 1-8 Differences in the primary and secondary response to injected antigen reflect the phenomenon of immunologic memory. When an animal is injected with an antigen, it produces a primary antibody response (dark blue) of low magnitude and short duration, peaking at about 10 to 20 days. At some later point, a second exposure to the same antigen results in a secondary response that is greater in magnitude, peaks in less time (1–4 days), and is more antigen specific than the primary response. Innate responses, which have no memory element and occur each time an antigen is encountered, are unchanged regardless of how frequently this antigen has been encountered in the past (light blue). memory. This is the ability of the immune system to respond much more swiftly and with greater efficiency during a second exposure to the same pathogen. Unlike almost any other biological system, the vertebrate immune response has evolved not only the ability to learn from (adapt to) its encounters with foreign antigen in real time but also the ability to store this information for future use. During a first encounter with foreign antigen, adaptive immunity undergoes what is termed a primary response, during which the key lymphocytes that will be used to eradicate the pathogen are clonally selected, honed, and enlisted to resolve the infection. As mentioned above, these cells incorporate messages received from the innate players into their tailored response to the specific pathogen. All subsequent encounters with the same antigen or pathogen are referred to as the secondary response (Figure 1-8).

Comparison of innate and adaptive immunity Innate

Adaptive

Response time

Minutes to hours

Days

Specificity

Limited and fixed

Highly diverse; adapts to improve during the course of immune response

Response to repeat infection

Same each time

More rapid and effective with each subsequent exposure

Major components

Barriers (e.g., skin); phagocytes; pattern recognition molecules

T and B lymphocytes; antigen-specific receptors; antibodies

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OVERVIEW FIGURE

Collaboration Between Innate and Adaptive Immunity in Resolving an Infection 2a

1

Lymph node

P

3

P

P

B

B T T

5a

T

4

B

5b Memory

2b

This very basic scheme shows the sequence of events that occurs during an immune response, highlighting interactions between innate and adaptive immunity. 1. Pathogens are introduced at a mucosal surface or breach in skin (bacteria entering the throat, in this case), where they are picked up by phagocytic cells (yellow). 2a. In this innate stage of the response, the phagocytic cell undergoes changes and carries pieces of bacteria to a local lymph node to help activate adaptive immunity. 2b. Meanwhile, at the site of infection resident phagocytes encountering antigen release chemokines and cytokines (black dots) that cause fluid influx and help recruit other immune cells to the site (inflammation). 3. In the lymph node, T (blue) and B (green) cells with appropriate receptor specificity are clonally selected when

During a secondary response, memory cells, kin of the final and most efficient B and T lymphocytes trained during the primary response, are re-enlisted to fight again. These cells begin almost immediately and pick up right where they left off, continuing to learn and improve their eradication strategy during each subsequent encounter with the same antigen. Depending on the antigen in question, memory cells can remain for decades after the conclusion of the primary response. Memory lymphocytes provide the means for subsequent responses that are so rapid, antigen-specific, and effective that when the same pathogen infects the body a second time, dispatch of the offending organism often occurs without symptoms. It is the remarkable property of memory that prevents us from catching many diseases a second time. Immunologic memory harbored by residual B and T lymphocytes is the foundation for vaccination, which uses crippled or killed pathogens as a safe way to “educate” the immune system to prepare it for later attacks by life-threatening pathogens. Overview Figure 1-9 highlights the ways in which the innate and adaptive immune responses work together to

N

B

T

their surface receptors bind antigen that has entered the system, kicking off adaptive immunity. 4. Collaboration between T and B cells and continued antigen encounter occurs in the lymph node, driving lymphocyte proliferation and differentiation, generating cells that can very specifically identify and eradicate the pathogen. For example: 5a. B cells secrete antibodies specific for the antigen, which travels to the site of infection to help label and eradicate the pathogen. 5b. In addition to the cells that will destroy the pathogen here, memory T and B cells are generated in this primary response and will be available at the initiation of a secondary response, which will be much more rapid and antigen specific. (Abbreviations: T ⫽ T cell, B ⫽ B cell, P = phagocyte; N = neutrophil, a type of immune cell.)

resolve an infection. In this example, bacteria breach the mucosal lining of the throat, a skin or mucous barrier, where it is recognized and engulfed by a local phagocytic cell (step 1). As part of the innate immune system, the phagocytic cell releases cytokines and chemokines that attract other white blood cells to the site of infection, initiating inflammation (step 2b). That phagocytic cell may then travel to a local lymph node, the tissue where antigen and lymphocytes meet, carrying bacterial antigens to B and T lymphocytes (step 2a). Those lymphocytes with receptors that are specific for the antigen are selected, activated, and begin the adaptive immune response by proliferating (step 3). Activated TH cells help to activate B cells, and clonal expansion of both types of lymphocyte occurs in the lymph node (step 4). This results in many T and B cells specific for the antigen, with the latter releasing antibodies that can attach to the intruder and direct its destruction (step 5a). The adaptive response leaves behind memory T and B cells available for a future, secondary encounter with this antigen (step 5b). It is worth noting that memory is a unique

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Overview of the Immune System capacity that arises from adaptive responses; there is no memory component of innate immunity. Sometimes, as is the case for some vaccines, one round of antigen encounter and adaptation is not enough to impart protective immunity from the pathogen in question. In many of these cases, immunity can develop after a second or even a third round of exposure to an antigen. It is these sorts of pathogens that necessitate the use of vaccine booster shots. Booster shots are nothing more than a second or third episode of exposure to the antigen, each driving a new round of adaptive events (secondary response) and refinements in the responding lymphocyte population. The aim is to hone these responses to a sufficient level to afford protection against the real pathogen at some future date.

The Good, Bad, and Ugly of the Immune System The picture we’ve presented so far depicts the immune response as a multicomponent interactive system that always protects the host from invasion by all sorts of pathogens. However, failures of this system do occur. They can be dramatic and often garner a great deal of attention, despite the fact that they are generally rare. Certain clinical situations also pose unique challenges to the immune system, including tissue transplants between individuals (probably not part of any evolutionary plan!) and the development of cancer. In this section we briefly describe some examples of common failures and challenges to the development of healthy immune responses. Each of these clinical manifestations is covered in much greater detail in the concluding chapters of this book (Chapters 15–19).

Inappropriate or Dysfunctional Immune Responses Can Result in a Range of Disorders Most instances of immune dysfunction or failure fall into one of the following three broad categories:



Hypersensitivity (including allergy): overly zealous attacks on common benign but foreign antigens



Autoimmune Disease: erroneous targeting of selfproteins or tissues by immune cells



Immune Deficiency: insufficiency of the immune response to protect against infectious agents

A brief overview of these situations and some examples of each are presented below. At its most basic level, immune dysfunction occurs as a result of improper regulation that allows the immune system to either attack something it shouldn’t or fail to attack something it should. Hypersensitivities, including allergy, and autoimmune disease are cases of the former, where the immune system attacks an improper

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target. As a result, the symptoms can manifest as pathological inflammation—an influx of immune cells and molecules that results in detrimental symptoms, including chronic inflammation and rampant tissue destruction. In contrast, immune deficiencies, caused by a failure to properly deploy the immune response, usually result in weakened or dysregulated immune responses that can allow pathogens to get the upper hand. This is a good time to mention that the healthy immune response involves a balancing act between immune aggression and immune suppression pathways. While we rarely fail to consider erroneous attacks (autoimmunity) or failures to engage (immune deficiency) as dysfunctional, we sometimes forget to consider the significance of the suppressive side of the immune response. Imperfections in the inhibitory arm of the immune response, present as a check to balance all the immune attacks we constantly initiate, can be equally profound. Healthy immune responses must therefore be viewed as a delicate balance, spending much of the time with one foot on the brakes and one on the gas. Hypersensitivity Reactions Allergies and asthma are examples of hypersensitivity reactions. These result from inappropriate and overly active immune responses to common innocuous environmental antigens, such as pollen, food, or animal dander. The possibility that certain substances induce increased sensitivity (hypersensitivity) rather than protection was recognized in about 1902 by Charles Richet, who attempted to immunize dogs against the toxins of a type of jellyfish. He and his colleague Paul Portier observed that dogs exposed to sublethal doses of the toxin reacted almost instantly, and fatally, to a later challenge with even minute amounts of the same toxin. Richet concluded that a successful vaccination typically results in phylaxis (protection), whereas anaphylaxis (anti-protection)—an extreme, rapid, and often lethal overreaction of the immune response to something it has encountered before—can result in certain cases in which exposure to antigen is repeated. Richet received the Nobel Prize in 1913 for his discovery of the anaphylactic response (see Table 1-2). The term is used today to describe a severe, life-threatening, allergic response. Fortunately, most hypersensitivity or allergic reactions in humans are not rapidly fatal. There are several different types of hypersensitivity reactions; some are caused by antibodies and others are the result of T-cell activity (see Chapter 15). However, most allergic or anaphylactic responses involve a type of antibody called immunoglobulin E (IgE). Binding of IgE to its specific antigen (allergen) induces the release of substances that cause irritation and inflammation, or the accumulation of cells and fluid at the site. When an allergic individual is exposed to an allergen, symptoms may include sneezing, wheezing (Figure 1-10), and difficulty in breathing (asthma); dermatitis or skin eruptions (hives); and, in more severe cases, strangulation due to constricted airways

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BOX 1-3

CLINICAL FOCUS

The Hygiene Hypothesis Worldwide, 300 million people suffer from asthma and approximately 250,000 people died from the disease in 2007 (see Chapter 15). As of 2009, in the United States alone, approximately 1 in 12 people (8.2%) are diagnosed with asthma. The most common reason for a trip to a hospital emergency room (ER) is an asthma attack, accounting for one-third of all visits. In addition to those treated in the ER, over 400,000 hospitalizations for asthma occurred in the United States in 2006, with an average stay of 3 to 4 days. In the past 25 years, the prevalence of asthma in industrialized nations has doubled. This is coupled with an overall rise in other types of allergic disease during the same time frame. What accounts for this climb in asthma and allergy in the last few decades? One idea, called the hygiene hypothesis, suggests that a decrease in human exposure to environmental microbes has had adverse effects on the human immune system. The hypothesis suggests that several categories of allergic or inflammatory disease, all disorders caused by excessive immune activation, have become more prevalent in industrialized nations thanks to diminished exposure to particular classes of microbes following the widespread use of antibiotics, immunization programs, and overall hygienic practices in those countries. This idea was first proposed by D. P. Strachan and colleagues in an article published in 1989 suggesting a link between hay fever and household hygiene. More recently, this hypothesis has been expanded to include the view by some that it may be a contributing factor in many allergic diseases, several autoimmune disorders, and, more recently, inflammatory bowel disease. What is the evidence supporting the hygiene hypothesis? The primary clinical support comes from studies that have shown a positive correlation between growing up under environmental conditions that favor microbe-rich (sometimes

called “dirty”) environments and a decreased incidence of allergy, especially asthma. To date, childhood exposure to cowsheds and farm animals, having several older siblings, attending day care early in life, or growing up in a developing nation have all been correlated with a decreased likelihood of developing allergies later in life. While viral exposures during childhood do not seem to favor protection, exposure to certain classes of bacteria and parasitic organisms may. Of late, the primary focus of attention has been on specific classes of parasitic worms (called helminthes), spawning New Age allergy therapies involving intentional exposure. This gives whole new meaning to the phrase “Go eat worms”! What are the proposed immunologic mechanisms that might underlie this link between a lack of early-life microbial exposure and allergic disease? Current dogma supporting this hypothesis posits that millions of years of coevolution of microbes and humans has favored a system in which early exposure to a broad range of common environmental bugs helps set the immune system on a path of homeostatic balance between aggression and inhibition. Proponents of this immune regulation argument, sometimes referred to as the “old friends” hypothesis, suggest that antigens present on microbial organisms that have played a longstanding role in our evolutionary history (both pathogens and harmless microbes we ingest or that make up our historical flora) may engage with the pattern recognition receptors (PPRs) present on cells of our innate immune system, driving them to warn cells involved in adaptive responses to tone it down. This hypothesis posits that without early and regular exposure of our immune cells to these old friends and their antigens, the development of “normal” immune regulatory or homeostatic responses is thrown into disarray, setting us up for an immune system poised to overreact in the future.

Animal models of disease lend some support to this hypothesis and have helped immunologists probe this line of thinking. For instance, certain animals raised in partially or totally pathogen-free environments are more prone to type 1, or insulin-dependent, diabetes, an autoimmune disease caused by immune attack of pancreatic cells (see Chapter 16). The lower the infectious burden of exposure in these mice, the greater the incidence of diabetes. Animals specifically bred to carry enhanced genetic susceptibility favoring spontaneous development of diabetes (called NOD mice, for nonobese diabetic) and treated with a variety of infectious agents can be protected from diabetes. Meanwhile, NOD mice maintained in pathogen-free housing almost uniformly develop diabetes. Much like this experimental model, susceptibility to asthma and most other allergies is known to run in families. Although all the genes linked to asthma have not yet been characterized, it is known that you have a 30% chance of developing the disease if one of your parents is a sufferer, and a 70% chance if both parents have asthma. While the jury may still be out concerning the verdict behind the hygiene hypothesis, animal and human studies clearly point to strong roles for both genes and environment in susceptibility to allergy. As data in support of this hypothesis continue to grow, the old saying concerning a dirty child—that “It’s good for their immune system”—may actually hold true! Centers for Disease Control and Prevention. 2012. CDC: Preventing Chronic Disease 9: 110054. Liu, A. H., and Murphy, J. R. 2003. Hygiene hypothesis: Fact or fiction? Journal of Allergy and Clinical Immunology 111(3):471–478. Okada, H., et al. 2010. The hygiene hypothesis for autoimmune and allergic diseases: An update. Clinical and Experimental Immunology 160:1. Sironi, M., and M. Clerici. 2010. The hygiene hypothesis: An evolutionary perspective. Microbes and Infection 12:421.

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FIGURE 1-10 Patient suffering from hay fever as a result of an allergic reaction. Such hypersensitivity reactions result from sensitization caused by previous exposure to an antigen in some individuals. In the allergic individual, histamines are released as a part of the hypersensitivity response and cause sneezing, runny nose, watery eyes, and such during each subsequent exposure to the antigen (now called an allergen) [Source: Chris Rout/Alamy.]

following extreme inflammation. A significant fraction of our health resources is expended to care for those suffering from allergies and asthma. One particularly interesting rationale to explain the unexpected rise in allergic disease is called the hygiene hypothesis and is discussed in the Clinical Focus on page 20. Autoimmune Disease Sometimes the immune system malfunctions and a breakdown in self-tolerance occurs. This could be caused by a sudden inability to distinguish between self and nonself or by a misinterpretation of a self-component as dangerous, causing an immune attack on host tissues. This condition, called autoimmunity, can result in a number of chronic debilitating diseases. The symptoms of autoimmunity differ, depending on which tissues or organs are under attack. For example, multiple sclerosis is due to an autoimmune attack on a protein in nerve sheaths in the brain and central nervous system that results in neuromuscular dysfunction. Crohn’s disease is an attack on intestinal tissues that leads to destruction of gut epithelia and poor absorption of food. One of the most common autoimmune disorders, rheumatoid arthritis, results from an immune attack on joints of the hands, feet, arms, and legs. Both genetic and environmental factors are likely involved in the development of most autoimmune diseases. However, the exact combination of genes and environmental exposures that favor the development of a particular autoimmune disease are difficult to pin down, and constitute very active areas of immunologic research. Recent discoveries in these areas and the search for improved treatments are all covered in greater detail in Chapter 16.

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Immune Deficiency In most cases, when a component of innate or adaptive immunity is absent or defective, the host suffers from some form of immunodeficiency. Some of these deficiencies produce major clinical effects, including death, while others are more minor or even difficult to detect. Immune deficiency can arise due to inherited genetic factors (called primary immunodeficiencies) or as a result of disruption/damage by chemical, physical, or biological agents (termed secondary immunodeficiencies). Both of these forms of immune deficiency are discussed in greater detail in Chapter 18. The severity of the disease resulting from immune deficiency depends on the number and type of affected immune response components. A common type of primary immunodeficiency in North America is a selective immunodeficiency in which only one type of antibody, called Immunoglobulin A is lacking; the symptoms may be an increase in certain types of infections, or the deficiency may even go unnoticed. In contrast, a more rare but much more extreme deficiency, called severe combined immunodeficiency (SCID), affects both B and T cells and basically wipes out adaptive immunity. When untreated, SCID frequently results in death from infection at an early age. By far, the most common form of secondary immunodeficiency is Acquired Immune Deficiency Syndrome (AIDS), resulting from infection with Human Immunodeficiency Virus (HIV). As discussed further in Chapter 18, humans do not effectively recognize and eradicate this virus. Instead, a state of persistent infection occurs, with HIV hiding inside the genomes of TH cells, its target cell type and the immune cell type that is critical to guiding the direction of the adaptive immune response. As the immune attack on the virus mounts, more and more of these TH cells are lost. When the disease progresses to AIDS, so many TH cells have been destroyed or otherwise rendered dysfunctional that a gradual collapse of the immune system occurs. It is estimated that at the end of 2010, more than 34 million people worldwide suffered from this disease (for more current numbers, see www.unaids.org), which if not treated can be fatal. For patients with access, certain anti-retroviral treatments can now prolong life with HIV almost indefinitely. However, there is neither a vaccine nor a cure for this disease. It is important to note that many pervasive pathogens in our environment cause no problem for healthy individuals thanks to the immunity that develops following initial exposure. However, individuals with primary or secondary deficiencies in immune function become highly susceptible to disease caused by these ubiquitous microbes. For example, the fungus Candida albicans, present nearly everywhere and a nonissue for most individuals, can cause an irritating rash and a spreading infection in the mucosal surface of the mouth and vagina in patients suffering from immune deficiency (see Figure 1-6b). The resulting rash, called thrush, can sometimes be the first sign of immune dysfunction (Figure 1-11). If left unchecked, C. albicans can spread, causing systemic candidiasis, a life-threatening condition. Such

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FIGURE 1-11 An immune deficient patient suffering from oral thrush due to opportunistic infection by Candida albicans. [Creative Commons 24 h)

Cytokinesis (20 min)

DC

FIGURE 14-14 Stages observed during the encounter between a naïve T cell and a dendritic cell. Investigators observed that T cells made transient contact with dendritic cells both early and late in a response, prior to proliferation. They described several sequential stages, depicted here. These stages may not be required—other experiments show that CD4 and CD8 T cells can form stable interactions very quickly, bypassing the first step. However, most agree that the outcome of the interaction varies with its subpopulations, for example—can provide help that activated B cells need to proliferate and differentiate into producers of high-affinity antibodies and memory cells. Each will encourage the production of different antibody isotypes, tailoring the response to the type of pathogen that initiated the activity. Activated B cells seek T-cell help at the border between the follicle and the paracortex of the lymph node. The Cyster lab pioneered dynamic imaging studies of antigen-specific B cells and generated now-classic images that revealed a B-cell/T cell choreography that was not fully anticipated by other approaches. They show B cells in the follicle moving purposefully toward the T-cell zone hours after binding antigen via the B-cell receptor (BCR) (Figure 14-15 and Accompanying Video 14-12). The investigators also showed that this migration was dependent on the chemokine receptor CCR7. When B cells bind antigen, they receive signals that up-regulate CCR7, which is not ordinarily expressed by naïve B cells. B cells can now follow chemotactic signals generated by the paracortex and migrate toward helper T cells. In other words, by inducing the up-regulation of CCR7, antigen binding redirects the B cell to the T-cell zone, where it can receive the appropriate help. The investigators captured the encounter between an activated B cell and activated helper CD4 T cell at the border between the follicle and T-cell zone (Figure 14-16 and Accompanying Video 14-13). Antigen-specific B-cell/T-cell pairs form stable connections within a day of antigen introduction and migrate together, with the B cell in full control of the antigen-specific T cell, which trails behind “helplessly.” Such behavior was not evident between T and B cells that did not share antigen specificity. The intimate and stable interactions established between T and B cells reflects the type of help that helper T cells must deliver to B cells. The cell pair forms an immunological synapse between them, with TCR, CD28, and adhesion mole-

quality—and stable interactions appear required for optimal differentiation and proliferation of effector cells. Proliferation occurs after cells have detached from the dendritic cell, although they seem to continue to make transient contacts. [Adapted from M. J. Miller, O. Safrina, I. Parker, and M. D. Cahalan (2004), Imaging the single cell dynamics of CD4⫹ T cell activation by dendritic cells in lymph nodes, Journal of Experimental Medicine 200:847–856.]

Follicle Paracortex

B cell that has encountered antigen Border between follicle and paracortex

FIGURE 14-15 AND ACCOMPANYING VIDEO 14-12 B cells travel to the border between the follicle and paracortex after being activated by antigen. Antigen-specific (BCRtransgenic) B cells (green) and nonantigen-specific (wild-type) B cells (red) were transferred together into recipient mice. Soluble antigen was injected 1 to 2 days later, and the behavior of the B cells in draining lymph nodes was imaged by two-photon microscopy. The dotted line in the static image roughly indicates the border between the B-cell zone (follicle) and T-cell zone (paracortex). An antigen-specific B cell that has been activated by antigen (1–3 hours after it was introduced) is circled, and its route can be followed in the video. It moves “purposefully” through other meandering B cells to the follicle border. (The time in hours:minutes:seconds is shown in the upper-left corner.) [Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, et al. (2005) Antigen-Engaged B Cells Undergo Chemotaxis toward the T Zone and Form Motile Conjugates with Helper T Cells. PLoS Biology 3:e150. Video S5.]

cules on the surface of T cells engaging class II MHC-peptide complexes, CD80/86 molecules, and other adhesion molecules on B cells. Into this synaptic space, the T cell delivers cytokines (e.g., IL-4) that stimulate B-cell differentiation. The synapse is also the site of stimulating interactions between CD40L on the T cell and CD40 on the B cell. CD40 signals are required for B-cell proliferation and differentiation.

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Examples of B-cell/T-cell interactions

FIGURE 14-16 AND ACCOMPANYING VIDEO 14-13 Antigen-specific B and T cells interact at the border between the follicle and paracortex. Antigen-specific B cells (red) and antigen-specific T cells (green) were transferred into a mouse that had been immunized with a soluble antigen. Eight hours after antigen was introduced, the interactions between T and B cells were observed in an isolated lymph node via two-photon microscopy. The static image shows examples of stable contacts made between T cells and B cells in the paracortex, along the border of the follicle. The video shows how the more actively migrating B cells (red) drag the interacting T cells (green) behind them as they travel. [Okada T, Miller MJ, Parker I, Krummel MF, Neighbors M, et al. (2005) Antigen-Engaged B Cells Undergo Chemotaxis toward the T Zone and Form Motile Conjugates with Helper T Cells. PLoS Biology 3: e150. Video S6.]

After receiving T-cell help, activated B cells travel to the outer edges of the follicle, following cues that are dependent on a G-protein-coupled receptor (EBI2). Here B cells continue to proliferate and, in some cases, continue to receive T-cell help. Some of these activated B cells will differentiate into plasma cells and leave the lymph node; others will return to the interior of the follicle, seeding a germinal center, where they undergo more rounds of proliferation and somatic hypermutation.

Dynamic Imaging Approaches Have Been Used to Address a Controversy about B-Cell Behavior in Germinal Centers The movements of cells within germinal centers have been visualized by several groups. Cyster and other labs revealed that germinal-center B cells were unusually motile and extended long processes—something more characteristic of DCs than lymphocytes (Figure 14-17 and Accompanying Video 14-14). Immunologists studying B cells have also used dynamic imaging studies to address a controversy about the behavior of activated B cells in the germinal center. Recall that the germinal center is divided into two major subzones based, originally, on their appearance within classically stained sections under a light microscope (see Figure 12-11b). The dark zone contains proliferating B cells and is thought to be the main site of somatic hypermutation (see Chapter 12). The light zone is replete with follicular DCs and their reticular network, which present antigen-antibody complexes and provide a scaffold for further B-cell/T-helper cell interactions

FIGURE 14-17 AND ACCOMPANYING VIDEO 14-14 Germinal-center B cells differ in their behavior from naïve B cells. In this experiment, fluorescently labeled antigen-specific B cells (green) were transferred into a mouse, which was immunized with soluble antigen 1 day later. Naïve (B and T) lymphocytes, fluorescently labeled red, were introduced 6 days later. The behaviors of antigen-activated B cells versus naïve lymphocytes were observed by two-photon imaging of isolated lymph nodes. Antigenexperienced B cells (green) occupy the germinal centers and differ dramatically in shape and activity from naïve lymphocytes (red). They extend processes that actively probe the microenvironment in ways more reminiscent of DCs. Most naïve B (and T cells) remain outside the germinal center, where they probe less actively and extend shorter processes. (Collagen fibers in the lymph node microenvironment fluoresce blue in this system.) [Allen, C.D., Okada, T., Tang, H.L., and Cyster, J.G. (2007b). Imaging of germinal center selection events during affinity maturation. Science 315:528–531. Movie S1.]

that regulate germinal center selection events. The light zone is thought to be the primary site of differentiation to plasma cells and memory B cells, as well as the site for positive selection of antigen-specific B cells that are somatically mutated in the dark zone. Positive selection is thought to occur when new B-cell specificities generated in the dark zone move to the light zone to sample antigens and test their affinities against antigens. Those that bind with high affinity received T-cell help and survived to return to the dark zone to generate even higher-affinity specificities. The highest-affinity clones arise after several cycles of somatic hypermutation in the dark zone, and antigen sampling in the light zone. Based on this model of positive selection, investigators made a simple prediction: dynamic imaging experiments should show B cells trafficking back and forth between the zones (Figure 14-18). However, the experiments showed that germinal center B cells much prefer to travel within rather than between the light and dark zones! In fact, only a few percent were found to travel between the zones every hour. In addition,

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Light zone Apoptosis

Dark zone Lower-affinity B cell Higher-affinity B cell

FIGURE 14-18 One proposed model for trafficking of germinal-center B cells between dark and light zones. Germinal-center B cells are thought to proliferate predominantly in the dark zone, where they also undergo somatic mutation. Highaffinity clones are thought to be positively selected in the light zone, where both antigen and T-cell help are available. This proposal requires cells to move from dark to light zones, perhaps even more than once. Two-photon intravital microscopy experiments surprised many when they showed that (1) B cells preferred to traffic within rather than between zones, (2) germinal-center B cells can divide in the light zone, and (3) the B cells made surprisingly brief contact with T helper cells when they did encounter them in the light zone. This recirculation model is still favored, but is currently under modification based on these and other experiments. [Adapted from A. E. Hauser, M. J. Shlomchik, and A. M. Haberman, (2007), In vivo imaging studies shed light on germinal-centre development, Nature Reviews Immunology 7:499.]

B-cell division was not simply confined to the dark zone but was also observed in the light zone. Finally, B cells made only brief contact with T-helper cells in the light zone, challenging another assumption that high-affinity B-cell clones would make stable connections with T cells that allow them to outcompete lower-affinity B-cell clones for T-cell help. No peptide

CD8ⴙ T cells arrest when they encounter antigen on DCs in the lymph node. This figure shows tracings (white) of the trajectories of antigen-specific CD8 T cells (red) interacting over a 5-minute period with DCs (green) in the lymph node of mice that had been immunized with two different concentrations of peptide antigen (109 M and 107 M) or no antigen at all. The trajectories of T cells

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Although these observations inspired new models in which B cells behave and develop independently in light and dark zones, other analyses suggested that the frequency of travel between dark and light zones is still compatible with traditional models of positive selection for high-affinity clones (see Chapter 12). B cells may simply sample antigen less frequently than once assumed and may not establish long-term contacts with T helper cells. The brevity of germinal-center B-cell interactions with T helper cells suggest that only the highest-affinity B clones, which can process and present more antigen, engage T cells effectively, even if transiently. In this case, dynamic images raised more questions than they resolved. However, they inspired novel speculation that may lead us to a more accurate understanding of the B-cell response.

CD8 T Cells Are Activated in the Lymph Node via a Multicellular Interaction Naïve CD8 T cells are the precursors of cytotoxic T cells (CTLs). Major participants in the cellular immune response, CTLs rove the body for infected cells, which they can kill very efficiently by inducing apoptosis. Like naïve CD4 T cells, naïve CD8 T cells are also activated by interacting with DCs in secondary lymphoid tissues; however, they recognize class I MHC-peptide rather than class II MHC-peptide combinations. Like B cells, they also require CD4 T-cell help to be optimally activated. The behavior of naïve and activated CD8 T cells in the lymph node has been traced using dynamic imaging techniques. The movements of those cells after antigen introduction resemble those of CD4 T cells: CD8 T cells arrest when they contact antigen on DCs and engage in both transient and stable associations. Some investigations, however, suggest that CD8 T cells form stable contacts with DCs more quickly than CD4 T cells, even at low antigen concentrations (Figure 14-19 and Accompanying Video 14-15). Like CD4 T cells, CD8 T cells will divide up to 10 times over a

10⫺9 M

FIGURE 14-19 AND ACCOMPANYING VIDEO 14-15

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10⫺7 M

interacting with antigen-exposed DCs are considerably shorter than those interacting with DCs from mice that were not immunized, indicating that the T cells arrest when they encounter antigen. [Reprinted from Beuneu, H., Lemaitre, F., Deguine, J., Moreau, H.D., Bouvier, I., Garcia, Z., Albert, M.L., and Bousso, P., Visualizing the functional diversification of CD8 T cell responses in lymph nodes. Immunity 33:412–23.]

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4- to 5-day period after antigen engagement. Mature CTLs ultimately leave the lymph node to travel to sites of infection. Dynamic imaging techniques also allowed investigators to experimentally address two important questions about T-cell activation in vivo. First, when during these stimulatory interactions does a naïve CD8 T cell start to differentiate into a functional CTL? To answer this question, investigators engineered CD8 T cells that fluoresced whenever they expressed IFN , a cytokine that is produced by differentiated CTLs. Using dynamic imaging techniques, they generated time-lapse videos of the behavior of these cells in a lymph node. Their results were unexpected. Antigenspecific (TCR-transgenic) CD8 T cells could differentiate into IFN producing cells quickly—prior to cell division (less than 24 hours after antigen encounter). The amount of IFN expressed by each activated CD8 T cell also varied widely. These findings suggest that functional differentiation of CD8 T cells begins as soon as T cells engage antigen on DCs. They also suggest that how well a cell generates IFN

depends not simply on the affinity of the TCR for antigen (which was the same for each T cell). The quality of signals delivered by the DC and/or with the quality of CD4 T-cell help are likely to play major roles. Second, how do CD4 T cells provide help, given that they cannot directly engage CD8 T cells? CD8 T cells, of course, interact with class I MHC-peptide complexes. CD4 T cells interact with class II MHC-peptide complexes, which are not expressed by CD8 cells. The simplest model for the delivery of CD4 T-cell help envisions an interaction among three cells: a single DC that presents peptides from an antigen protein in both class I and class II MHC to a CD8 T cell and a CD4 T cell, respectively (see Figure 14-11a for a schematic example of this tricellular complex). The probability that three cells with the appropriate antigen specificities and complexes could find each other in a physiological context seemed low to immunologists. However, to their surprise and delight, dynamic imaging experiments confirmed that just such an interaction occurs in a lymph node. Cellular trios (consisting of an antigenexpressing DC, and antigen-specific CD4 T and CD8 T cells) were observed by investigators 20 hours after T cells were exposed to antigen (Figure 14-20 and Accompanying Video 14-16). Further data suggest these interactions may not be as improbable as supposed. In fact, they are facilitated by chemokine interactions: DCs activated by CD4 T cells produce chemokines (CCL3, CCL4, CCL5) that specifically attract CD8 T cells that express the chemokine receptors CCR4 and CCR5.

FIGURE 14-20 AND ACCOMPANYING VIDEO 14-16 The formation of a tricellular complex in the lymph node during CD8ⴙ T-cell activation. This dynamic image and video depict a lymph node of a live, anesthetized mouse that had first been injected subcutaneously with antigen-expressing dendritic cells (blue), and subsequently injected intravenously with antigen-specific CD4 T cells (red) and CD8 T cells (green). The static image shows a stable complex that includes all three fluorescently labeled cells. The video reveals the sequence of interactions that occurred, showing that a CD4 T cell (red) and antigen-expressing dendritic cell interacts first and is then joined by CD8 T cell (green). [Reprinted with permission from Macmillan Publishers, Ltd: Castellino, F., Huang, A.Y., Altan-Bonnet, G., Stoll, S., Scheinecker, C., and Germain, R.N 2006. Chemokines enhance immunity by guiding naïve CD8 T cells to sites of CD4 T cell-dendritic cell interaction. Nature 440:890–5.]

node to perform their functions. Antibody-producing B cells (plasma cells) travel to several sites, depending on the isotype of the antibody that they are producing. Early IgM producers release antibodies from the medulla of the lymph node, many IgG producers go to the bone marrow, and IgA producers localize to mucosal-associated lymphoid tissue (MALT), particularly at the gut. Effector and memory CD4 and CD8 T cells travel to multiple organs and sites of infection, following chemokine and cytokine cues that have been generated by the innate immune response at those sites.

Activated Lymphocytes Exit the Lymph Node and Recirculate

A Summary of Our Current Understanding

Successful activation of all three lymphocyte subsets results in proliferation and differentiation into effector and memory lymphocytes. These mature cells must leave the lymph

Biochemical and dynamic imaging studies have helped to generate an overview of the timing and organization of the fundamental T, B, and DC behaviors during a primary adaptive

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OVERVIEW FIGURE

A Summary of B-Cell and T-Cell Behavior in a Lymph Node after the Introduction of Antigen T-cell response

Early hours: APC activation

Lymph node

Minutes: (Soluble) Ag entry

B-cell response

SSC Minutes to 24 h: Ag entry

Min to 6 h: B cell/ Ag interaction

HEV Follicle

~6 h: B cell migration to T cell zone

1-24 h: T cell/APC interactions

T-cell zone (paracortex)

24-96 h: T cell proliferation (and differentiation)

GC >72-96 h: Egress of effector cells Efferent lymphatics

B cell

24-48 h: B cell prolif & migration to outer follicular zone 48-96 h: development of the germinal center

Dendritic cell

CD8+ T cell CD4+ T cell

Follicular dendritic cell

Macrophage

(See text for full description.)

immune response in a lymph node (Overview Figure 14-21). Not every detail of Figure 14-21 is likely to be correct—and not every cellular player is represented. However, this graphic overview should provide a useful reference and reminder of the fundamental events that govern the development of the adaptive immune response in space and time. Briefly, naïve lymphocytes—B cells, CD4 T cells, and CD8 T cells—continually circulate through secondary lymphoid tissue, browsing for antigens that they can bind. After entering a lymph node via HEVs, lymphocytes are guided by fibrous networks as they randomly explore their microenvironments. B cells scan the surface of follicular DCs in the follicle (B-cell zone) for unprocessed antigen that they might bind. T cells scan the processed peptide-MHC complexes on the surface of DCs in the paracortex or T-cell zone. Antigen arrives in a lymph node in waves. Soluble, unprocessed, and opsonized antigen can arrive within minutes and is passed from cell to cell to the follicular dendritic cells that are scanned by naïve B cells. DCs that have processed antigen at the sites of infection arrive in the paracortex hours after infection.

When a probing lymphocyte engages an antigen, its movements slow down as it begins to commit to the interactions that induce their differentiation into effector cells and proliferation, processes that start early but continue over a 4- to 5-day period. A CD4 T cell that engages class II MHC-peptide complexes on the surfaces of DCs may differentiate into one of several types of effector helper cells. Which effector cell type it will become depends on both quantitative (affinity) and qualitative (costimulatory molecule engagements and cytokine interactions) variables. Some CD4 T cells gain the ability to help B cells to differentiate into antibody-producing cells. Some gain the ability to enhance cytotoxic cell differentiation and activity. Within hours after successfully engaging antigen in follicles with their BCRs, B cells process and present antigen peptides in their own MHC molecules and migrate to the edges of a follicle, where they seek contact with activated CD4 T cells that have differentiated into effectors that help B cells. Some differentiate directly into plasma cells; others

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reenter the follicle and establish a germinal center, where their BCR isotype and affinities are shaped by selection events and additional CD4 T-cell help. Within roughly the same period of time, CD8 T cells engage in multicellular complexes with DCs and helper CD4 T cells and start to differentiate into bona fide cytotoxic lymphocytes. The differentiation of antigen-specific lymphocytes into effector and memory helper CD4 T cells, cytotoxic CD8 T cells, and antibody-producing B cells is accompanied by proliferation, which is robust by 24 to 48 hours after antigen encounter, and can continue for 4 days and more. Fully mature effector and memory T lymphocytes leave the lymph node and travel to sites of infection to assist in the clearance of pathogen. Effector B cells also leave the lymph node and travel to several sites, including the bone marrow and the mucosal tissues, to release antibodies.

The Immune Response Contracts within 10 to 14 Days The proliferative activity of all lymphocytes ultimately declines, typically because the stimulus for activation and proliferation (pathogen) is removed. When pathogen is successfully cleared (or successfully goes into hiding from immune cells), inflammatory signals will cease. Most effector cells of the immune system have limited life spans and will ultimately die in the absence of stimulation. The life span of innate immune cells is particularly short. Neutrophils, for instance, live for little over 5 days. The contraction of effector lymphocytes, most of which also have a finite lifespan, can be accelerated by self-limiting interactions. Engagements between T cells themselves can result in cell death. All T cells express the death receptor Fas, and activated T cells express FasL. Fas-FasL interactions induce apoptosis and may help to reduce the numbers of lymphocytes at the end of a response. Regulatory T cells may also help to quell normal immune responses by releasing inhibitory cytokines (Chapter 11). By approximately 10 days after initiation of the adaptive immune response, the lymph node has quieted and returned to its antigen naïve state and structure.

Immune Cell Behavior in Peripheral Tissues Once the effector and memory cells that are generated after antigen encounter leave the lymphoid tissue, they begin to recirculate among peripheral tissues. Unlike naïve lymphocytes, effector lymphocyte populations do not reenter secondary lymphoid tissue but, instead, exhibit tissue-selective homing behavior. Antibody-producing B cells (plasma cells) travel to several sites, including the medullary region of the lymph node and the bone marrow, where they release large quantities of

antibodies. Pathogen-specific cytotoxic CD8 T cells follow chemokine cues to the sites of cellular infection, where they induce apoptosis of cells that express the class I MHCpeptide complexes that reveal their infection by intracellular pathogens. The trafficking of helper CD4 T cells varies according to effector subtypes and is still incompletely understood. Some CD4 T cells that provide signals promoting B-cell and CD8 T-cell differentiation stay in the lymph node and continue to stimulate lymphocyte differentiation. Others travel to sites of infection to enhance the ability of tissue phagocytes to clear opsonized pathogen. Some effector CD4 T cells may even complete their differentiation or change their functions at the sites of infection, responding with plasticity to the collection of signals and needs of the local response. Memory cells take up residence in both secondary lymphoid tissue (as central memory cells) and peripheral tissues throughout the body (as effector memory cells). There they share sentinel functions with residing APCs. In this section, we feature several examples of the behavior of effector lymphocytes confronting physiological antigen in peripheral tissues.

Chemokine Receptors and Integrins Regulate Homing of Effector Lymphocytes to Peripheral Tissues Not surprisingly, the trafficking of effector and memory lymphocytes to peripheral tissues is regulated by changes in the repertoire of chemokine receptors expressed on the memory or effector cell surface and the chemokines generated by innate immune cells at sites of infection. In addition, tissues display unique sets of adhesion molecules that help select for the effector subset. In general, effector cells and effector memory cells avoid redirection back to secondary lymphoid tissues because they decrease expression of L-selectin (CD62L), which prevents them from entering via HEVs. Instead, effector cells express adhesion molecules and chemokine receptors that coordinate their homing to relevant tissues. For example, a subset of memory and effector T cells that home to the intestinal mucosa expresses high levels of the integrins 47 (LPAM-1) and CD11a/CD18 (LFA-1, L2), which bind to MAdCAM and various ICAMs on intestinal lamina propria venules (Figure 14-22). Cells homing to the gut mucosa also express the chemokine receptor CCR9, which binds to CCL25 in the small intestine. IgA-secreting B cells are also recruited into gut tissue via the chemokines CCL25 and CCL28. Other memory/effector cells home preferentially to the skin because they express high levels of cutaneous lymphocyte antigen (CLA) and LFA-1, which bind to E-selectin and ICAMs on dermal venules of the skin (see Figure 14-22). The chemokines CCL17, CCL27, and CCL1 also play a role in the recruitment of skin-homing T cells.

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(a)

Skin-homing effector T cell

SS SS

E-selectin

SS

ICAM-1

MAdCAM-1

SS SS

LPAM-1

LFA-1

SS

SS

SS

SS

CLA

levels of particular homing receptors that allow them to home to endothelium in particular tertiary extralymphoid tissues. The initial interactions in homing of effector T cells to (a) mucosal and (b) skin sites are illustrated.

SS

SS

475

ceptors and vascular addressins involved in selective trafficking of naïve and effector T cells. Various subsets of effector T cells express high

L-selectin S SS S

ICAM-1

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FIGURE 14-22 Examples of homing re-

(b) Mucosal-homing effector T cell

LFA-1

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Intestinal lamina propria endothelium

Skin dermal venule endothelium

Tertiary extralymphoid tissue

Effector Lymphocytes Respond to Antigen in Multiple Tissues Dynamic imaging techniques have been used to visualize the immune response to specific pathogen, rather than noninfectious experimental antigen. Below we describe recent studies that specifically follow the behavior of T cells responding to physiologic insults in the periphery. We explore the first two in the most depth. One follows the behavior of T cells in response to infection with the protozoa that causes toxoplasmosis (Toxoplasma gondii), a pathogen that is transmitted from cats to humans (and other animals) via feces and produces an infection that is often asymptomatic. However, “Toxo” can cause harm to the fetus of a pregnant woman. The second study traces the behavior of T cells responding to—and rejecting—an allogeneic skin graft. The observations should enhance the development of therapeutic efforts to inhibit graft rejection. We close with several short, tantalizing descriptions of interactions between immune cells and physiological antigens that have been revealed by recent dynamic imaging studies. As you will see, these studies provide a powerful reminder of the influence of inflammation, which is difficult to mimic in the absence of real infection. The studies also begin to reveal immune cell behavior outside secondary lymphoid tissue. CD8 T-Cell Response to Infection by Toxoplasma gondii Investigators have recently used dynamic imaging methods to trace the behavior of the T cell specific for Toxoplasma gondii (Toxo), a protozoal parasite that infects many different tissues, including the brain. More than half of the world’s population has been infected by Toxo, which is acquired by ingesting contaminated raw meat, soil, or litter exposed to feces from infected cats. Most of us never know that we have been invaded, and some of us harbor the parasite in cysts for

long periods of time. In those with weakened immune systems, however, Toxo infection can damage the brain and eyes. The pathogen also can cross the placenta and cause disease in fetuses, whose immune systems are underdeveloped. Understanding our response to this pathogen is an important step in controlling it. In order to follow the immune response to this pathogen, investigators cleverly modified the parasite so that it expresses an antigen (an ovalbumin [OVA] peptide) that can be recognized by TCR transgenic (OT-1) CD8 T cells, which, as you now know (see Advances Box 14-1), can be more easily traced and manipulated. The Hunter lab has imaged CD8 T cells responding to this infection in both the lymph nodes and the brain. Their work shows that CD8 T cells in the lymph node respond very rapidly (within 36 hours) after intravenous injection of T. gondii, forming stable contacts with DCs that peak between 36 and 48 hours. As predicted by work described above, crawling naïve antigen-specific T cells slow down considerably (from 6–8 m/min to 4 m/ min) after encounter with DCs that have been exposed to antigen. Their movements pick up again after 48 hours, when they are actively proliferating and seem to rely on transient contacts, again as predicted by investigations with noninfectious systems. Interestingly, responding CD8 T cells and DCs appear to collect in the lymph node’s subcapsular sinus, a localization that was also seen after viral infection (Figure 14-23). Investigators speculate that this may be an active site of inflammation in the lymph node that attracts effector cells. DCs also change in appearance, becoming vacuolated—not as a result of infection, but in response to general inflammation. CD8 T cells reactive to T. gondii antigens appeared in the brain by day 3, and peaked at day 22, an increase that was coincident with peak proliferation of CD8 T cells in secondary lymphoid organs. The investigators provide evidence that the increase in T cells in the brain was due to the continual

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(a) Capsule Subcapsular sinus

Toxoplasma gondii Neutrophil

(b)

Virus Subcapsular sinus macrophage Sinus-lining cell

Virus-specific B cell

Non-specific B cell

influx of CD8 T cells and not a consequence of proliferation within the brain. T cells could be surprisingly motile when they arrived, particularly during periods when investigators determined that effector activity was highest and parasites were under control. They could also form more stationary clusters, near infected cells, consistent with the possibility that they were interacting with brain-resident APCs, which were not labeled in this experiment. Unfortunately, the ability of infiltrating T. gondii-reactive T cells to control the infection wanes over time ( 40 days), a loss of function that was associated with the up-regulation of expression of PD-1, a negative costimulatory molecule. This observation is consistent with observations that chronic infection “exhausts” lymphocytes, reducing their ability to clear pathogens. The infection altered the microenvironment of both the lymph node and the brain in several unanticipated ways. Inflammation (antigen specific and nonspecific) disrupted the reticular network of the lymph node and decreased the levels of CCL21. This disruption may have assisted the establishment of cell-cell contacts during infection, but also was likely to abrogate responses to new infections, by taking away the cues for naïve T-cell migration. In the brain, however, the infection appeared to result in the establishment of a new reticular network decorated with CCL21. This was a surprise and presumably helps organize T-cell entry and response in this tertiary tissue. T-cell interactions with this network after infection imply that this network plays a significant role in T-cell trafficking within the brain (Figure 14-24

(c)

Toxoplasma gondii T cell

Dendritic cell Infected macrophage

Infected T cell

FIGURE 14-23 Examples of immune response to pathogens within the subcapsular sinus of a lymph node. (a) Toxoplasma gondii, a protozoal parasite, can gain direct access to the subcapsular sinus and attracts swarms of neutrophils. (b) After a virus gains access, it accumulates on CD169 macrophages that line the sinus. Virus-specific B cells will then gather from the follicular side of the lining, sampling antigen from the macrophages. (c) Memory CD8 T cells also have access to the subcapsular sinus and have been shown to cluster around cells infected with T. gondii. This makes them vulnerable to further infection. [Adapted from J. L. Coombes, and E. A. Robey, E.A. 2010, Dynamic imaging of host-pathogen interactions in vivo, Nature Reviews. Immunology 10:353–364, Figure 3.]

FIGURE 14-24 AND ACCOMPANYING VIDEO 14-17 A reticular network is established at the site of infection by Toxoplasma gondii. This dynamic image was taken of brain tissue in mice that had been infected for 4 weeks with the protozoan Toxoplasma gondii, and then injected intravenously with protozoan-specific T cells (green). The image shows a fibrous network fluorescing blue, that has been established specifically in response to the infection (it is not present in uninfected tissue). The videos show T cells using the routes established by the network to migrate through the tissue. The magnified image shows T cells extending processes that interact intimately with the fibers. [Reprinted with permission from Macmillan Publishers, Ltd: Castellino, F., Huang, A.Y., Altan-Bonnet, G., Stoll, S., Scheinecker, C., and Germain, R.N, 2006. Chemokines enhance immunity by guiding naïve CD8 T cells to sites of CD4 T cell-dendritic cell interaction. Nature 440:890–5.]

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The Immune Response in Space and Time and Accompanying Video 14-17). Other studies intriguingly suggest that such reticular networks are routinely established in other tertiary tissues after infection. Host Immune Cell Response to a Skin Graft The rejection of grafts that are MHC mismatched (allografts) requires both CD4 and CD8 T-cell activity. However, the sequence of interactions that lead to rejection is not fully understood, and the role of host (recipient) versus graft (donor) APCs in mediating the activation is still disputed. Specifically, in order to be activated, naïve CD4 and CD8 T cells must interact with APCs that express the graft’s alloantigens. Are the activating APCs coming from the graft or from the host? The Bousso lab performed an elegant set of dynamic imaging experiments to address this and other questions. MHC-mismatched skin was grafted onto a mouse ear and imaged by two-photon intravital microscopy. The mismatched skin was from a mouse whose DCs fluoresced yellow (a CD11c-YFP reporter mouse), so their motions could be traced. These fluorescent DCs exited the graft and were gone within 6 days. The investigators looked for these graftderived DCs in the draining lymph nodes of the recipient mouse and found them as early as 3 days after engraftment, but they had the look of dying cells and never reappeared. The investigators then asked which cells came into the skin graft from the host mouse. To do this, they put the graft onto mice whose cells all express green fluorescent protein (GFP). They found that CD11b (myeloid) green fluorescent cells infiltrated the graft within 3 days. These cells were dominated by neutrophils at first, but over time they included more and more monocytes and DCs. Interestingly, such myeloid cells were also found in grafts from control, MHC-matched mice (isografts). The investigators took advantage of their system to look more closely at the locations of these cells and found that GFP cells infiltrated the dermis of both allografts and isografts. However, only the allografts showed evidence of GFP cells infiltrating all of the donated skin, including the outer layer (the epidermis), indicating that deep infiltration was antigen dependent. To see if the infiltrating cells carried alloantigen back to draining lymph nodes, the investigators introduced a clever twist to their system (Figure 14-25a). First they put their grafts on MHC-mismatched hosts with GFP cells and let the green, myeloid cells (neutrophils and monocytes) infiltrate the graft over a 6-day period. They then removed the graft, with its new green host cell infiltrates, and put it on another MHC-mismatched mouse, but one that did not have any GFP cells. They could then trace the fate of the green infiltrates. Three days later, they found some of these GFP cells in the T-cell zones of the draining lymph node, suggesting that infiltrating cells from the host could play a role in initiating the T-cell response to the graft. This is consistent with suggestions that host APCs cross-present the graft’s alloantigens to CD8 T cells. (Recall from Chapter 8 that is the process by which APCs pick up extracellular

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antigens and present them as class I MHC-peptide complexes to CD8 T cells.) They strengthened their case for the role of host APCs in cross-presenting alloantigen via a clever, albeit complex scheme. Essentially they grafted skin from a “strain A” class I MHC-deficient mouse onto an antigen-mismatched (“strain B”) mouse that expressed normal class I MHC (Figure 14-25b). In this system, the skin graft’s own APCs were completely unable to present antigen to class I MHC-restricted CD8 T cells; only the infiltrating cells from the recipient would be able to do so. They allowed strain B cells to infiltrate the graft, then removed it, transplanting it onto another strain B mouse, but one that was class I MHC deficient. If and only if the infiltrating (strain B) cells from the first host successfully cross-presented foreign antigen from the strain A graft would the CD8 T cells in this second recipient respond (and initiate rejection). Indeed, in vivo imaging revealed that antigen-specific CD8 T cells from this second recipient responded to the graft—confirming that host DCs that infiltrated the graft pick up and present foreign antigen to CTLs, which are responsible for graft rejection. Finally, the investigators visualized the activity of the graft-rejecting cytotoxic CD8 T cells. Dividing T cells could be found in the draining lymph node as early as two days after graft transplant. However, such cells were not found in the graft itself until day 8, and seemed to enter from the graft edges. CD8 T cells were found at the border between the dermis (deep) and epidermis (more superficial) border of the allograft and were often in proximity to dying cells, which were likely their targets. CD8 T cells were slower moving in the allografts (versus isografts), which again is consistent with indications that antigen-specific cells arrest when they meet their antigen. By day 10, when the tissue was undergoing active rejection, CD8 T cells were found throughout the graft. Dendritic Cell Contribution to Listeria Infection As you know, DCs are important for initiating an adaptive immune response against infection; however, in some cases they are responsible for maintaining infection! Listeria monocytogenes is an intracellular bacterium that resides in soil and can cause food-associated illness and fatalities (Figure 14-26; also see www.dnatube.com/video/2506/ Intracellular-Listeria-Infection for an illustrative video). In 2011, one of the worst food-borne disease outbreaks in the United States was traced to Listeria associated with cantaloupe melons. Recall that the spleen is the site of response to bloodborne pathogens: all white blood cells circulate through sinuses in the red pulp, which also is a site where DCs and monocytes can sample antigen (see Figure 2-10). White blood cells enter and scan the spleen’s version of the T-cell zone, the periarteriolar lymphoid sheath (PALS). B cells enter and scan follicles, which are very similar to those found in lymph nodes. The area that separates the red pulp from the white pulp, the marginal zone (MZ), is a relatively

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Retransplant onto B6 WT

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Skin from MHC Class I-/- mouse of Strain A

Primary transplant onto MHC Class I+/+ mouse of Strain B

Retransplant onto MHC Class I-/mouse of Strain B

dLN Cells in the graft

Cells in the lymph node

GFP cells

GFP cells CD8 T cells

FIGURE 14-25 In vivo imaging of the immune response to an antigen-mismatched skin graft. This figure shows the approaches taken by investigators to see which antigen-presenting cells—host or graft (donor) APC?—activate the CD8 T cells that will reject a skin graft. (a) Skin from a mouse of one strain (C3H) was grafted onto the ear of a mouse of an MHC-mismatched strain (B6) in which all cells expressed GFP. The cells that infiltrated the skin from this B6-GFP mouse (pseudo colored yellow) were predominantly myeloid (neutrophils, monocytes, macrophages). After time was allowed for infiltration, the investigators removed the graft and placed it on a wild-type B6 mouse whose CD8 T cells (shown in green). This allowed the investigators to see where the infiltrating cells (yellow) would go. They found the cells in the draining lymph node (dLN)

where they were interacting with CD8 T cells. These CD8 T cells ultimately distributed themselves through the graft, killing the MHCmismatched cells. (b) Skin from a strain A mouse that expressed no class I MHC was transplanted on the ear of an antigen-mismatched (strain B) mouse that expressed class I MHC. In this situation, none of the donor graft cells and only the infiltrating recipient cells could present antigen to CD8⫹ T cells. The investigators let this graft accumulate recipient cells, then retransplanted it onto an antigen-matched class I/ mouse. The T cells from this mouse responded to the strain B cells that had infiltrated the primary graft and picked up the strain A antigen.

unique microenvironment that is the first line of defense against blood-borne pathogens. It is also the site of residence of DCs and monocytes as well as a unique group of B cells (MZ B cells) that do not circulate. Dynamic imaging of fluorescently tagged Listeria bacteria showed that they arrive in the red pulp of the spleen within seconds of intravenous injection (see Figure 14-26). (During a physiologic infection, when bacteria are ingested, they probably take longer to arrive in the red pulp—either within white blood cells that they infected in the mucosa of the gut or directly from blood that they penetrated during infection.)

Within minutes, Listeria associates with and is presumably phagocytosed by the DCs in the red pulp sinuses, thereby infecting them. Listeria-specific CD8 T cells accumulate in these sinuses, forming stable contacts with the DCs that activate them. Investigators found that DCs in these sinuses recruit other innate immune cells, including monocytes, and unwittingly pass their Listeria infection to these cells (via their extensive processes, down which Listeria travel). These circulating cells, which are critical for controlling the pathogen, also become vehicles of infection that spreads throughout the body.

[Reprinted by permission from Macmillan Publishers Ltd: Celli, S., Albert, M.L., and Bousso, P., 2011. Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy, Nature Medicine 17: 744–749.]

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Tumor

Tissue

FIGURE 14-27 T cells infiltrating a tumor. Dynamic imaging of interactions of antigen-specific T cells (yellow) with tumor expressing that antigen (red) shows that some T cells infiltrate the solid tumor, but many gather in tissue borders that are not as tumor ridden. [Deguine, J., et al., 2010. Intravital Imaging Reveals Distinct Dynamics for Natural Killer and CD8 T Cells during Tumor Regression, Immunity 3: 632–644. Copyright 2010 with permission from Elsevier.]

FIGURE 14-26 Intracellular Listeria infection. Listeria (green) use the host cell actin (red) to propel themselves through the cell cytoplasm (cometlike tails of red actin can be seen trailing the green bacteria). [Courtesy Alain Charbit.]

T-Cell Response to Tumors Dynamic imaging studies have also visualized an immune reaction to experimentally generated tumors. These show that antigen-specific, activated T cells can gain access to the tumor (Figure 14-27). When they do, they migrate vigorously and exhibit effective cytolytic behavior, associating for long periods of time (6 hours or more) with tumor cells and inducing apoptosis. However, the tumors failed to regress, and T-cell response seemed limited by (1) poor access to the tissue and (2) poor antigen presentation by tumor-associated cells, not by the availability of activated CTLs or their ability to mediate cytolysis. These studies suggest that strategies to improve antigen presentation and tumor accessibility may be more effective than strategies that simply increase antigen-specific T-cell number. Regulatory T-Cell Responses Dynamic imaging has also allowed us to see when and where regulatory T cells exert their suppressive activity, and the results satisfyingly confirm predictions made from “static” experiments, suggesting that regulatory T cells can suppress immune responses in more than one way (see Chapter 9). In

a model of Type I diabetes (the result of T-cell-mediated destruction of  cells of the pancreatic islet), investigators traced the activities of fluorescently labeled antigen-specific regulatory T cells introduced prior to disease onset. They exhibited two distinct behaviors. Some prevented diabetogenic effector cells from making productive clustering contacts with cells presenting auto-antigen in the pancreatic islets, consistent with proposals that regulatory T cells can inhibit effector T-cell function by quelling the activation potential of resident APCs. Other regulatory T cells formed stable connections with effector CD8 T cells and inhibited their ability to kill pancreatic target cells, an effect associated with TGF- secretion by regulatory T cells. Memory T-Cell Responses Dynamic imaging of the trafficking of memory T and B cells is in its early stages. However, experiments have already revealed intriguing features. For instance, memory CD8 T cells appear to localize to and reside in the B-cell follicle after Listeria infection. A clever set of experiments revealed that T cells that are latecomers to the scene of an immune response in a lymph node tend to generate central memory T cells, perhaps because they do not have as much access to fully activating antigen interactions that would lead to effector cell generation. The Analyze the Data study question at the end of this chapter also describes another interesting investigation that reveals differences in the trafficking of CD4 and CD8 memory T cells.

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Dynamic imaging takes advantage of advances in microscopy to visualize the movements of cells deep in live tissue. Innate immune cells, including granulocytes and antigenpresenting cells (APCs), are the first to respond to pathogen. Innate immune cells bind pathogen via pattern recognition receptors (PRRs), which generate signals that result in the release of inflammatory signals, including chemokines and cytokines. Chemokines attract other innate immune cells to the site of infection. Neutrophils are generally the first cell type to move from the bloodstream into inflammatory sites; they can swarm around pathogens as they attack. They also produce more chemokines that attract APCs, including dendritic cells (DCs), which are the most efficient activators of naïve T cells. DCs at the site of infection engulf antigen, presenting processed peptide in class II MHC and cross-presenting it in class I MHC. They travel to draining lymph nodes via the afferent lymphatics to alert the adaptive immune system of the presence of pathogen. Antigen can enter the lymph node via multiple routes. Soluble, unprocessed antigen enters the tissue quickly and directly, or can be trapped by macrophages lining the sinuses and relayed to follicular DCs through the activity of noncognate B cells (and others). Processed antigen (in the form of MHC-peptide complexes) arrives within a few hours on the surface of DCs, which travel to the T-cell zone (paracortex). Dendritic cells extend long processes and can interact with up to 5000 T cells per hour. Lymphocytes undergo constant recirculation between the blood, lymph, lymphoid organs, and tertiary extralymphoid tissues, increasing the chances that the small number of lymphocytes specific for a given antigen (about 1 in 105 cells) will actually encounter that antigen. Cell-adhesion molecules (CAMs) and chemokine/ chemokine receptor interactions regulate the migration of leukocytes both into and within lymphoid organs and inflamed tissues. Extravasation of all white blood cells involves four steps: rolling, activation, arrest/adhesion, and transendothelial migration. Naïve B and T lymphocytes express CD62L that helps them to home secondary lymphoid organs, where they extravasate across high-endothelial venules (HEVs). Naïve lymphocytes are very motile cells in the lymph node and crawl along reticular fibers in their respective lymph node zones. B lymphocytes browse for antigen along follicular DC networks in the follicle. T lymphocytes interact with fibroblastic reticular cell networks in the paracortex, where DCs that express antigen can also be found. Their

movements are regulated in part by chemokine interactions. Naïve lymphocytes that do not encounter antigen leave the lymph node after about 12 to 18 hours and reenter circulation to probe another lymph node. ■

T lymphocytes that encounter antigen to which they bind slow down considerably and ultimately “arrest,” forming stable contacts with APCs (in the CD4 and CD8 T cells). These contacts last between 1 and 24 hours, and if productive result in proliferation. Cells can experience up to 10 divisions over the following 4 to 5 days.



B cells that encounter antigen up-regulate CCR7 and travel by chemotaxis from the center of the follicle to the T-cell zone, where they wait for T-cell help. Antigen-specific T-cell/B-cell interactions are strong and stable. The T/B cell pair moves actively at the follicular/T-cell border.





CD8 T cells quickly form stable interactions with antigenbearing DCs in the cortex. Stable tricellular complexes that include a CD8 T cell, a CD4 helper T cell, and a dendritic cell have been directly observed in dynamic images, showing that activated CD8 T cells may be specifically attracted by chemokines produced by DCs activated by CD4 T cells.



Germinal-center B cells are unusually motile and distinct in morphology (they look more like DCs). Although they tend to circulate within, rather than between, the light zone and dark zone, a small percentage traffic from dark to light zones, presumably to sample antigen and receive T-cell help. Their contact with helper T cells is surprisingly brief, a behavior that may put high-affinity B cells, which express higher densities of MHC-peptide at a competitive advantage.



Effector and memory lymphocytes leave the lymph node via portals in the medullary region of the lymph node, an event that depends on interactions between S1P1 receptors on white blood cells and S1P in their environment. They travel back into circulation via efferent lymphatics. Effector lymphocytes down-regulate CD62L, expressing a different profile of chemokine receptors that allow them to interact with inflamed vascular endothelium of tertiary tissues.



Dynamic imaging of cells responding to a protozoal parasite that infects the brain reveals that inflammation can temporarily alter the microenvironment (reticular structure) of a lymph node and can remodel the tissue that is the site of infection by establishing reticular systems for lymphocyte trafficking.



Dynamic images of cells responding to an MHC-mismatched skin graft show that the APCs of the recipient are capable of sampling and presenting alloantigen to host CD8 T cells, which ultimately distribute themselves throughout the graft and destroy it.

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R E F E R E N C E S Allen, C. D., T. Okada, and J. G. Cyster. (2007a). Germinal-center organization and cellular dynamics. Immunity 27:190–202. Allen, C. D., T. Okada, H. L. Tang, and J. G. Cyster. (2007b). Imaging of germinal center selection events during affinity maturation. Science 315:528–531. Bajénoff, M., et al. (2006). Stromal cell networks regulate lymphocyte entry, migration, and territoriality in lymph nodes. Immunity 25:989–1001.

Germain, R. N., M. J. Miller, M. L. Dustin, and M. C. Nussenzweig. (2006). Dynamic imaging of the immune system: progress, pitfalls and promise. Nature Reviews Immunology 6:497–507. Hauser, A. E., Shlomchik, M. J., and Haberman, A. M. (2007). In vivo imaging studies shed light on germinal-centre development. Nature Reviews Immunology 7:499–504. Jenkins, M. K. (2008). Imaging the immune system. Immunological Review 221:5–6.

Bajénoff, M., et al. (2007). Highways, byways and breadcrumbs: Directing lymphocyte traffic in the lymph node. Trends in Immunology 28:346–352.

John, B., et al. (2009). Dynamic imaging of CD8() T cells and dendritic cells during infection with Toxoplasma gondii. PLoS Pathogens 5:e1000505.

Beuneu, H., et al. (2010). Visualizing the functional diversification of CD8 T cell responses in lymph nodes. Immunity 33: 412–423.

Junt, T., E. Scandella, and B. Ludewig. (2008). Form follows function: Lymphoid tissue microarchitecture in antimicrobial immune defence. Nature Reviews Immunology 8:764–775.

Bousso, P. (2008). T-cell activation by dendritic cells in the lymph node: Lessons from the movies. Nature Reviews Immunology 8:675–684.

Lindquist, R. L., et al. (2004). Visualizing dendritic cell networks in vivo. Nature Immunology 5:1243–1250.

Bousso, P., and E. Robey. (2003). Dynamics of CD8 T cell priming by dendritic cells in intact lymph nodes. Nature Immunology 4: 579–585. Cahalan, M. D. (2011). Imaging transplant rejection: A new view. Nature Medicine 17:662–663. Cahalan, M. D., and I. Parker. (2005). Close encounters of the first and second kind: T-DC and T-B interactions in the lymph node. Seminars in Immunology 17:442–451. Cahalan, M. D., and I. Parker. (2008). Choreography of cell motility and interaction dynamics imaged by two-photon microscopy in lymphoid organs. Annual Review of Immunology 26:585–626.

Miller, M. J., O. Safrina, I. Parker, and M. D. Cahalan. (2004). Imaging the single cell dynamics of CD4 T cell activation by dendritic cells in lymph nodes. Journal of Experimental Medicine 200:847–856. Miller, M. J., S. H. Wei, I. Parker, and M. D. Cahalan. (2002). Two-photon imaging of lymphocyte motility and antigen response in intact lymph node. Science 296:1869–1873. Mueller, S. N., and H. D. Hickman. (2010). In vivo imaging of the T cell response to infection. Current Opinion in Immunology 22:293–298. Okada, T., et al. (2005). Antigen-engaged B cells undergo chemotaxis toward the T zone and form motile conjugates with helper T cells. PLoS Biology 3:e150.

Castellino, F., and R. N. Germain. (2006). Cooperation between CD4 and CD8 T cells: When, where, and how. Annual Review of Immunology 24:519–540.

Pereira, J. P., L. M. Kelly, and J. G. Cyster. (2010). Finding the right niche: B-cell migration in the early phases of T-dependent antibody responses. International Immunology 22:413–419.

Castellino, F., et al. (2006). Chemokines enhance immunity by guiding naïve CD8 T cells to sites of CD4 T cell-dendritic cell interaction. Nature 440:890–895.

Phan, T. G., I. Grigorova, T. Okada, and J. G. Cyster. (2007). Subcapsular encounter and complement-dependent transport of immune complexes by lymph node B cells. Nature Immunology 8:992–1000.

Catron, D. M., A. A. Itano, K. A. Pape, D. L. Mueller, and M. K. Jenkins. (2004). Visualizing the first 50 hr of the primary immune response to a soluble antigen. Immunity 21:341–347.

Schwickert, T. A. (2007). In vivo imaging of germinal centres reveals a dynamic open structure. Nature 446:83–87.

Celli, S., M. L. Albert, and P. Bousso. (2011). Visualizing the innate and adaptive immune responses underlying allograft rejection by two-photon microscopy. Nature Medicine 17:744–749.

Shwab, S. R., and J. G. Cyster. (2007). Finding a way out: Lymphocyte egress from lymphoid organs. Nature Immunology 8:1295–1301.

Celli, S., F. Lemaître, and P. Bousso. (2007). Real-time manipulation of T cell-dendritic cell interactions in vivo reveals the importance of prolonged contacts for CD4 T cell activation. Immunity 27:625–634.

Weber, C., L. Fraemohs, and E. Dejana. (2007). Adhesion molecules in vascular inflammation. Nature Reviews Immunology 7:467–477.

Coombes, J. L., and E. A. Robey. (2010). Dynamic imaging of host-pathogen interactions in vivo. Nature Reviews Immunology 10:353–364. Cyster, J. G. (2010). Shining a light on germinal center B cells. Cell 143:503–505.

Wei, S. H., et al. (2007). Ca2 signals in CD4 T cells during early contacts with antigen-bearing dendritic cells in lymph node. Journal of Immunology 179:1586–1594. Wilson, E. H., et al. (2009). Behavior of parasite-specific effector CD8 T cells in the brain and visualization of a kinesis-associated system of reticular fibers. Immunity 30:300–311.

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Useful Websites http://artnscience.us/index.html Immunologist Art Anderson has developed a lovely Web site that appreciates the importance of integrating excellent immunological information with images.

www.youtube.com/watch?v=XOeRJPIMpSs This YouTube video shows fluorescently labeled dendritic cells migrating through skin. http://multimedia.mcb.har vard.edu/media. html “The Inner Life of the Cell” is an artistic and informative animation of the intercellular and intracellular molecular events associated with leukocyte extravasation.

http://download.cell.com/immunity/mmcs/ journals/1074-7613/PIIS1074761304002389.mmc1. mov Video 14-9 Movements of DC and naïve lymphocytes in the absence of antigen.

http://download.cell.com/immunity/mmcs/ journals/1074-7613/PIIS1074761304002389.mmc2. mov Video 14-10 Movements of DC and antigen-specific

CD4 T cells and B cells after subcutaneous injection of a soluble antigen.

http://www.sciencedirect.com/science/Miami MultiMediaURL/1-s2.0-S1074761307004517/1-s2.0S1074761307004517-mmc10.mov/272197/html/ S1074761307004517/c23e22d1cf621035a8f 3f278912e7171/mmc10.mov Video 14-11 T cells arrest

Video Links

after antigen encounter.

Copy and paste these links into a web browser to view the videos. If a link does not work, try a different browser or player. Each video associated with figures in this chapter can also be found in the on-line ImmunologyPortal associated with Kuby Immunology (Chapter 14).

http://www.plosbiology.org/article/fetchSingle Representation.action?uri=info:doi/10.1371/journal. pbio.0030150.sv001 Video 14-12 B cells travel to the

http://multimedia.mcb.har vard.edu/media. html Video 14-1 Two-photon imaging of live T and B lym-

http://www.plosbiology.org/article/fetchSingle Representation.action?uri=info:doi/10.1371/journal. pbio.0030150.sv006 Video 14-13 Antigen-specific B

phocytes within a mouse lymph node.

http://download.cell.com/immunity/mmcs/ journals/1074-7613/PIIS1074761306004833.mmc3. avi Video 14-2 T cell interacting with fibroblastic reticular network.

http://download.cell.com/immunity/mmcs/ journals/1074-7613/PIIS1074761306004833.mmc6. avi Video 14-3 T lymphocytes migrate along the fibroblas-

border between the follicle and paracortex after being activated by antigen.

and T cells interact at the border between the follicle and paracortex.

http://www.sciencemag.org/content/suppl/ 2007/01/31/1136736.DC1/1136736s1.mpg Video 14-14 Germinal-center B cells differ in their behavior from naïve B cells.

http://download.cell.com/immunity/mmcs/ journals/1074-7613/PIIS1074761306004833. mmc14.avi Video 14-4 B cells migrate along follicular

http://www.sciencedirect.com/science/Miami MultiMediaURL/1-s2.0-S1074761310003213/1-s2.0S1074761310003213-mmc2.mov/272197/html/S107 4761310003213/02ec1444124cb3ef651dc939 ef632e2b/mmc2.mov Video 14-15 CD8 T cells arrest

dendritic cell networks.

when they encounter antigen on DCs in the lymph node.

http://www.nature.com/ni/journal/v5/n12/extref/ ni1139-S8.mov Video 14-5 Dendritic cells (DCs) are

http://www.nature.com/nature/journal/v440/ n7086/extref/nature04651-s9.mov Video 14-16 The

present in all lymph node microenvironments.

formation of a tricellular complex in the lymph node during CD8+ T cell activation.

tic reticular cell network.

http://www.nature.com/ni/journal/v6/n12/extref/ ni1269-S11.mov Video 14-6 Lymphocytes exit the lymph node through portals.

http://www.ncbi.nlm.nih.gov/pmc/ar ticles/ PMC2569002/bin/NIHMS71239-supplement-07. mov Video 14-7 Neutrophil swarming. http://www.nature.com/ni/journal/v8/n9/extref/ ni1494-S6.mpg Video 14-8 B cells capture antigen from macrophages in the subcapsular sinus of the lymph node.

http://www.sciencedirect.com/science/Miami MultiMediaURL/1-s2.0-S1074761309000600/1-s2.0S1074761309000600-mmc12.mov/272197/html/ S1074761309000600/8623d017a55653b6c73fb81 e2da216e5/mmc12.mov Video 14-17 A reticular network is established at the site of infection by Toxoplasma gondii.

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Q U E S T I O N S

ANALYZE THE DATA A recent paper (T. Gebhardt et al., Dif-

ferent patterns of peripheral migration by memory CD4 and CD8 T cells, 2011, Nature 477:216–219) presented the first intravital microscopy data directly comparing the behavior of memory CD4 and CD8 T cells in response to infection. Their data were surprising. Gebhardt et al.’s experimental strategy: The investigators infected the skin of mice with Herpes Simplex Virus and adoptively transferred CD4 T cells (which fluoresce green in the images and video associated with the study) specific for the virus as well as CD8 T cells (which fluoresce red), both of which expressed T-cell receptors specific for the virus. They observed the movements of these cells in the infected skin during the effector phase of the immune response (8 days after infection) as well as 5 or more weeks later when the only cells in the tissue would be memory cells. Remember that skin has two layers: an outer layer (epidermis) and an inner layer (dermis). Their results: During the effector phase, both CD4 and CD8 T cells initially moved similarly through the dermis. However, gradually, these two cell populations distributed themselves differently: CD4 T cells stayed in the dermis, CD8 T cells moved to the epidermis (!). The investigators then looked at the memory cell populations in infected skin weeks later. What they saw is depicted in the following video (memory CD8 T cells [red], memory CD4 T cells [green]): www.nature.com/ nature/journal/v477/n7363/extref/nature10339-s4.mov Your assignment: Take a look at this time-lapse video and read its legend. a. Describe what you see, identifying at least two specific

differences between CD4 and CD8 T-cell behavior. b. Propose a molecular difference that could explain one of these distinctions. c. Advance one hypothesis about the adaptive value of the difference(s) you observed. That is, what advantage (if any) may such a difference in CD4 and CD8 T-cell behavior provide an animal responding to a skin infection? EXPERIMENTAL DESIGN QUESTION You want to directly test

claims that CCR5 is important for the localization of naïve B cells to the follicles of a lymph node. You have all the reagents that you need to perform a two-photon intravital microscopy, including (1) an anti-CCR5 antibody that you know will block the interactions between CCR5 and its ligand (and can be injected), and (2) a CCR5/ mouse. Design an experiment that will definitively test these claims. Define what you will measure, and sketch one figure predicting your results.

advance a proposal. Be specific and concise. What approaches might be taken to treat this disease? 1. Which of the following are features of two-photon micros-

copy? (See Advances Box 14-1.) a. Excites fluorochromes with shorter wavelengths of light

than confocal microscopy Achieves higher resolutions than confocal microscopy Can be used to generate three-dimensional images Can be used to generate time-lapse videos Can penetrate tissue to deeper depths than confocal microscopy f. Damages tissue more seriously than confocal microscopy b. c. d. e.

2. You want to track the behavior of T cells specific for the

influenza virus in a mouse lymph node. You have a mouse whose cells all express yellow fluorescent protein (YFP). You decide to isolate T cells from this mouse and introduce them into a mouse that has been immunized with influenza, as well as into a control mouse that was given no antigen. You look at the lymph nodes of both mice, expecting to see a difference in the behavior of the cells. However, you do not see much of a difference. At first you wonder if all you read is true—perhaps T cells do not arrest when they encounter antigen! But then you realize that your experimental design was flawed. What was the problem? 3. Indicate whether each of the following statements is true or

false. If you think a statement is false, explain why. a. Chemokines are chemoattractants for lymphocytes but

not other leukocytes. b. T cells, but not B cells, express the chemokine receptor

CCR7. c. Antigen can only come into the lymph node if it is asso-

ciated with an antigen-presenting cell. d. Lymphocytes increase their motility after they engage

e. f. g. h.

dendritic cells (DCs) expressing an antigen to which they bind. T cells crawl along the follicular DC network as they scan DCs for antigen in the lymph node. Lymphocytes make use of reticular networks only in secondary lymphoid organs. Leukocyte extravasation follows this sequence: adhesion, chemokine activation, rolling, transmigration. Most secondary lymphoid organs contain high-endothelial venules (HEVs).

4. Provide an example of lymphocyte chemotaxis during an

immune response. 

CLINICAL FOCUS QUESTION Leukocyte adhesion deficiency

I (LAD I) is a rare genetic disease that results from a defect or deficiency in CD18. Patients with this condition usually do not live past childhood because they cannot fight off bacterial infections. Why? Given what you have learned in this chapter,

5. Describe where CD8 T cells and B cells receive T-cell help

within a secondary lymphoid organ. 

6. How might the behavior of an antigen-specific CD8 T cell

differ in the lymph node of a CCL3-deficient animal versus a wild-type animal?

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7. Extravasation of neutrophils and of lymphocytes occurs by

generally similar mechanisms, although some differences distinguish the two processes. a. List in order the basic steps in leukocyte extravasation. b. Which step requires chemokine activation and why? c. Naïve lymphocytes generally do not enter tissues other

than the secondary lymphoid organs. What confines them to this system? 8. Naïve T and naïve B-cell subpopulations migrate preferen-

tially into different parts of the lymph node. What is the basis for this compartmentalization? Identify both structural and molecular influences. 9. True or False? Germinal-center B cells differ in morphol-

ogy and motility from other B cells in the follicle. 10. Place a check mark next to the molecules that interact with

each other.

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______ Chemokine and L-selectin ______ E-selectin and L-selectin ______ CCL19 and CCR7 ______ ICAM and chemokine ______ Chemokine and G-protein-coupled receptor ______ BCR and MHC ______ TCR and MHC

11. Predict how a deficiency in each of the following would affect

T-cell and B-cell trafficking in a lymph node during a response to antigen. How might they affect an animal’s health? a. b. c. d.

L-selectin CCR7 CCR5 S1P1

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T

he same immune reactions that protect us from infection can also inflict a great deal of damage, not simply on a pathogen, but on our own cells and tissues. As you have learned, the immune response uses multiple strategies to reduce damage to self by turning off responses when pathogen is cleared and avoiding reactions to self antigens. However, these checks and balances can break down, leading to immune-mediated reactions that are more detrimental than protective. Some immune-mediated disorders are caused by a failure of immune tolerance. These autoimmune disorders will be discussed in Chapter 16. Others are caused by an inappropriately vigorous innate and/or adaptive response to antigens that pose little or no threat. These disorders, called hypersensitivities, will be the main focus of this chapter. Finally, some disorders are caused by a failure to turn off innate or adaptive responses, resulting in a chronic inflammatory state. We will close this chapter with a discussion of the causes and consequences of chronic inflammation, a condition that is of interest to many because of its intriguing association with the current obesity epidemic. Two French scientists, Paul Portier and Charles Richet, were the first to recognize and describe hypersensitivities. In the early twentieth century, as part of their studies of the responses of bathers in the Mediterranean to the stings of Portuguese man-o’-war jellyfish (Physalia physalis), they demonstrated that the toxic agent in the sting was a small protein. They reasoned that eliciting an antibody response that could neutralize the toxin may serve to protect the host. Therefore, they injected low doses of the toxin into dogs to elicit an immune response, and followed with a booster injection a few weeks later. However, instead of generating a protective antibody response, the unfortunate dogs responded immediately to the second injection with vomiting, diarrhea, asphyxia, and death. Richet coined the term “anaphylaxis,” derived from the Greek and translated loosely as “against protection” to describe this overreaction of the immune system, the first description of a hypersensitivity reaction. Richet was

Young girl sneezing in response to flowers. [Brand New Images/Getty Images] ■

Allergy: A Type I Hypersensitivity Reaction



Antibody-Mediated (Type II) Hypersensitivity Reactions



Immune Complex-Mediated (Type III) Hypersensitivity



Delayed-Type (Type IV) Hypersensitivity (DTH)



Chronic Inflammation

subsequently awarded the Nobel Prize in Physiology or Medicine in 1913. Since that time, immunologists have learned that there are multiple types of hypersensitivity reactions. Immediate hypersensitivity reactions result in symptoms that manifest themselves within very short time periods after the immune stimulus, like those described above. Other types of hypersensitivity reactions take hours or days to manifest themselves, and are referred to as delayed-type hypersensitivity (DTH) reactions. In general, immediate hypersensitivity reactions result from antibody-antigen reactions, whereas DTH is caused by T-cell reactions. As it became clear that different immune mechanisms give rise to distinct hypersensitivity reactions, two immunologists, P. G. H. Gell and R. R. A. Coombs, proposed a classification scheme to discriminate among the various types of hypersensitivity (see Figure 15-1). Type I hypersensitivity reactions are mediated by IgE antibodies, 485

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ADCC

Immune complex C3b

Allergen

C3b

FcεR for IgE FcεR

Allergenspecific IgE

Cytotoxic Surface cell Target antigen cell Complement C3b activation

Sensitized T cell C3b

Complement activation

Cytokines

Neutrophil Degranulation

Immune complex

(Allergy)

Tissue damage Activated macrophage

Type I Allergy and Atopy Immune mediator

IgE

C3b

Type II Antibody-mediated hypersensitivity

Type III Immune complex-mediated hypersensitivity

Type IV Delayed type hypersensitivity (DTH)

IgG or IgM

Immune complexes

T cells

Mechanism

Ag induces crosslinking of IgE bound to mast cells and basophils with release of vasoactive mediators.

Ab directed against cell surface antigens mediates cell destruction via complement activation or ADCC.

Ag-Ab complexes deposited in various tissues induce complement activation and an ensuing inflammatory response mediated by massive infiltration of neutrophils.

Sensitized T cells (TH1, TH2 and others) release cytokines that activate macrophages or TC cells which mediate direct cellular damage.

Typical manifestations

Includes systemic anaphylaxis and localized anaphylaxis such as hay fever, asthma, hives, food allergies, and eczema.

Includes blood transfusion reactions, erythroblastosis fetalis, and autoimmune hemolytic anemia.

Includes localized Arthus reaction and generalized reactions such as serum sickness, necrotizing vasculitis, glomerulonephritis, rheumatoid arthritis, and systemic lupus erythematosus.

Includes contact dermatitis, tubercular lesions, and graft rejection.

FIGURE 15-1 The four types of hypersensitivity reactions. and include many of the most common allergies to respiratory allergens, such as pollen and dust mites. Type II hypersensitivity reactions result from the binding of IgG or IgM to the surface of host cells, which are then destroyed by complement- or cell-mediated mechanisms. In type III hypersensitivity reactions, antigen-antibody complexes deposited on host cells induce complement fixation and an ensuing inflammatory response. Type lV hypersensitivity reactions result from inappropriate T-cell activation. It should be noted that, although this classification method has proven to be a useful analytical and descriptive tool, many clinical hypersensitivity disorders include molecular and cellular contributions from components belonging to more than one of these categories. The subdivisions are not as frequently evoked in real-world clinical settings as they once were. The term allergy first appeared in the medical literature in 1906, when the pediatrician Clemens von

Pirquet noted that the response to some antigens resulted in damage to the host, rather than in a protective response. Although most familiar respiratory allergies result from the generation of IgE antibodies toward the eliciting agent, and therefore are type I hypersensitivity reactions, other common reactions that are associated with allergy, such as the response to poison ivy, result from T-cell-mediated, type IV responses.

Allergy: A Type I Hypersensitivity Reaction More than half of the U.S. population (54.3%) suffers from type I hypersensitivity reactions, which encompass the most common allergic reactions, including hay fever, asthma, atopic dermatitis, and food allergies. The incidence of allergy continues to rise in the human population, and understanding

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Allergy, Hypersensitivities, and Chronic Inflammation immune mechanisms behind the response has already led to new therapies. Below we describe the molecular and cellular participants in the various type I hypersensitivities, as well as the rationale behind current treatments.

IgE Antibodies Are Responsible for Type I Hypersensitivity Type I hypersensitivity reactions (allergies) are initiated by the interaction between an IgE antibody and a multivalent antigen. Classic Experiment Box 15-1 describes the brilliant series of experiments by K. Ishizaka and T. Ishizaka in the 1960s and 1970s that led to the identification of IgE as the class of antibody responsible for allergies. In normal individuals, the level of IgE in serum is the lowest of any of the immunoglobulin classes, making further physiochemical studies of this molecule particularly difficult. It was not until the discovery of an IgE-producing myeloma by Johansson and Bennich in 1967 that extensive analyses of IgE could be undertaken.

Many Allergens Can Elicit a Type I Response Healthy individuals generate IgE antibodies only in response to parasitic infections. However, some people, referred to as atopic, are predisposed to generate IgE antibodies against common environmental antigens, such as those listed in Table 15-1. Chemical analysis revealed that most, if not all, allergens are either protein or glycoprotein in nature, with multiple antigenic sites, or epitopes, per molecule. For many years, scientists tried unsuccessfully to find any structural commonalities among molecules that induced distress in

TABLE 15-1

Common allergens associated with type I hypersensitivity

Plant pollens

Foods

Rye grass

Nuts

Ragweed

Seafood

Timothy grass

Eggs

Birch trees

Peas, Beans

Drugs

Milk

Penicillin Sulfonamides

Insect products

Local anesthetics

Bee venom

Salicylates

Wasp venom Ant venom

Mold spores

Cockroach calyx

Animal hair and dander

Dust mites

Latex Foreign serum Vaccines

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susceptible individuals, but recently several features shared by many allergens have begun to provide clues to the biological basis of their activity. First, many allergens have intrinsic enzymatic activity that affects the immune response. For example, allergen extracts from dust mites and cockroaches as well as from fungi and bacteria are relatively high in protease activity. Some of these proteases have been shown to be capable of disrupting the integrity of epithelial cell junctions, and allowing allergens to access the underlying cells and molecules of the innate and adaptive immune systems. Others, including a protease (Der p 1) produced by the dust mite, cleave and activate complement components at the mucosal surface. Still others cleave and stimulate protease-activated receptors on the surfaces of immune cells, enhancing inflammation. Thus, one factor that distinguishes allergenic from nonallergenic molecules may be the presence of enzymatic activity that affects the cells and molecules of the immune system. Second, many allergens contain potential pathogenassociated molecular patterns, or PAMPS (see Chapter 5), capable of interacting with receptors of the innate immune system, and initiating a cascade of responses leading to an allergic response. However, it is unclear why this cascade is stimulated in only a subset of individuals. Third, many allergens enter the host via mucosal tissues at very low concentrations, which tend to predispose the individual to generate TH2 responses, leading to B-cell secretion of IgE.

IgE Antibodies Act by Cross-Linking Fc␧ Receptors on the Surfaces of Innate Immune Cells IgE antibodies alone are not destructive. Instead, they cause hypersensitivity by binding to Fc receptors specific for their constant regions (Fc␧Rs). These are expressed by a variety of innate immune cells, including mast cells, basophils, and eosinophils (Chapter 2). The binding of IgE antibodies to Fc␧Rs activates these granulocytes, inducing a signaling cascade that causes cells to release the contents of intracellular granules into the blood, a process called degranulation (see Figure 15-2). The contents of granules vary from cell to cell, but typically include histamine, heparin, and proteases. Together with other mediators that are synthesized by activated granulocytes (leukotrienes, prostaglandins, chemokines, and cytokines), these mediators act on surrounding tissues and other immune cells, causing allergy symptoms. Interestingly, the serum half-life of IgE is quite short (only 2 to 3 days). However, when bound to its receptor on an innate immune cell, IgE is stable for weeks. IgE actually binds two different receptors, the high-affinity Fc␧RI, which is responsible for most of the symptoms we associate with allergy, and the lower-affinity Fc␧RII, which regulates the production of IgE by B cells, as well as its transport across cells (Chapter 13). The role of Fc␧RI in type I hypersensitivities is confirmed by experiments conducted in mice that lack an Fc␧RI ␣ chain; they are resistant to localized and systemic allergic responses, despite having normal numbers of mast cells.

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The Discovery and Identification of IgE as the Carrier of Allergic Hypersensitivity In a stunning series of papers published between 1966 and the mid-1970s, Kimishige Ishizaka and Teruko Ishizaka, working with a number of collaborators, identified a new class of immunoglobulins, which they called IgE antibodies, as being the major effector molecules in type 1 antibody-mediated hypersensitivity reactions. The Ishizakas built on work performed in 1921 by K. Prausnitz and H. Kustner, who injected serum from an allergic person into the skin of a nonallergic individual. When the appropriate antigen was later injected into the same site, a wheal and flare reaction (swelling and reddening) was detected. This reaction, called the P-K reaction after its originators, was the basis for the first biological assay for IgE activity. In their now classic experiments, the Ishizakas assayed for the presence of allergen-specific antibody using the wheal and flare reaction. They also employed radioimmunodiffusion, using the ability of radioactive allergen E derived from ragweed pollen to bind to and precipitate pollen-specific antibodies as an additional assay; the antibodies formed a radioactive precipitate on binding to the ragweed allergen. (Note that both the antigen and the immunoglobulin class are designated “E” in this series of experiments.) The Ishizakas reasoned that the best starting material for purifying the protein

responsible for the P-K reaction would be the serum of an allergic individual who displayed hypersensitivity to ragweed pollen E. (Serum is that component of the blood remaining after the cells and clotting components have been removed.) To purify the serum protein responsible for the allergic reaction, they took whole human serum and subjected it to ammonium sulfate precipitation (different proteins precipitate out at varying concentrations of ammonium sulfate), and ion exchange chromatography, which separates proteins on the basis of their intrinsic charge. Different fractions from the chromatography column were tested by radioimmunodiffusion for their ability to bind to radioactive antigen E from ragweed pollen. Portions of the different fractions were also injected at varying dilutions into volunteers, along with antigen, to test for a P-K reaction. Finally, each fraction was also tested semiquantitatively for the presence of IgG and IgA antibodies. The results of these experiments are shown for the serum from one of the three individuals they tested (see Table 1 below). From the results in this table, it is clear that the ability of proteins in the various fractions to induce a P-K reaction did not correlate with the amounts of either IgG or IgA antibodies, but it did correlate with the amounts of antibodies that could be detected in an immunodiffusion reaction

The High-Affinity IgE Receptor, Fc␧RI Mast cells and basophils constitutively express high levels of the high-affinity IgE receptor, Fc␧RI, which binds IgE with an exceptionally high-affinity constant of 1010M⫺1. This affinity helps overcome the difficulties associated with responding to an exceptionally low concentration of IgE in the serum (1.3 ⫻ 10⫺7 M). Eosinophils, Langerhans cells, monocytes, and platelets also express Fc␧RI, although at lower levels. Most cells express a tetrameric form of Fc␧RI, which includes an ␣ and ␤ chain and two identical disulfide-linked ␥ chains (Figure 15-3a). Monocytes and platelets express an alternative form lacking the ␤ chain. The ␣ chains of the Fc␧RI, members of the immunoglobulin superfamily, directly

with radioactive antigen E. Perhaps another antibody class was responsible for the line of immunoprecipitation on the immunodiffusion gel? The fractions containing the highest concentration of protein able to bind to allergen E were further purified using gel chromatography, which separates proteins on the basis of size and molecular shape. Again, the presence of the protein was detected on the basis of its ability to bind to radioactive antigen E and to induce a P-K reaction in the skin of a test subject who had been injected with antigen E. The resulting protein still contained small amounts of IgG and IgA antibodies, which were eliminated by mixing the fractions with antibodies directed toward those human antibody subclasses, and then removing the resultant immunoprecipitate. The Ishizakas’ final protein product was 500 to 1000 times more potent than the original serum in its ability to generate a P-K reaction, and the most active preparation generated a positive skin reaction at a dilution of 1:8000. None of its reactivities correlated with the presence of any of the other known classes of antibody, and so a new class of antibody was named, IgE, based on its ability to bind to allergens and bring about a P-K reaction. As described in Chapter 3, the level of IgE in the serum is the lowest of all the antibody classes, falling within the range

bind the IgE heavy chains, whereas the ␤ and ␥ chains are responsible for signal transduction. They contain immunoreceptor tyrosine-based activation motifs (ITAMs) (see Chapter 3) that are phosphorylated in response to IgE cross-linking. IgE-mediated signaling begins when an allergen crosslinks IgE that is bound to the surface Fc␧RI receptor (Figure 15-2). Although the specific biochemical events that follow cross-linking of the Fc␧RI receptor vary among cells and modes of stimulation, the Fc␧RI signaling cascade generally resembles that initiated by antigen receptors and growth factor receptors (Chapter 3). Briefly, IgE cross-linking induces the aggregation and migration of receptors into membrane lipid rafts, followed by phosphorylation of ITAM motifs by

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BOX 15-1 TABLE 1 Serum donor

Data from original paper identifying the immunoglobulins responsible for skin sensitization Fraction

Minimum dose for P-K reaction

Relative amount IgE

Relative amount IgG

Relative amount IgA

U

A

0.04







n

B

0.008

⫹⫹





0.26





⫹/⫺







C

⬎ 0.9

D A n

A

0.002

⫹⫹

⫹⫹



B

0.0006

⫹⫹⫹



⫹/⫺

C

0.0014

⫹⫹



⫹/⫺

D

0.005

⫹⫹





E

0.017

⫹⫹





F

0.13







Modified from original table entitled “Distribution of skin-sensitizing activity and of ␥G (IgG) and ␥A (IgA) globulin following diethylaminoethyl (DEAE) Sephadex column fractionation,” in Ishizaka, K., and T. Ishizaka, (1967), Identification of ␥E-antibodies as a carrier of reaginic activity. Journal of Immunology 99(6):1187–1198.

of 0.1 to 0.4 ␮g/ml, although atopic individuals can have as much as 10 times this concentration of IgE in their circulation. However, in 1967, Johansson and Bennich discovered an IgE-producing myeloma, which enabled a full biochemical analysis of the molecule. The structure of IgE is described in Chapter 3. In Table 1, which is modified from the original data in this classic 1967 paper, serum protein fractions from two separate donors were evaluated for the relative amounts of IgA or IgG (referred to as ␥A and ␥G, respectively, in this publication),

using rabbit antisera against the two immunoglobulin subclasses, and for the presence of the putative “IgE” using radioimmunodiffusion (as described in this chapter’s text). IgG is the most common class of immunoglobulin in serum, and IgA was included because early experiments had suggested that IgA may be the antibody responsible for the wheal and flare reaction. The “Minimum dose for P-K” column refers to the quantity of the fraction required to yield a measureable wheal and flare response. In this column, the lower the number, the higher the

associated tyrosine kinases. Adapter molecules then latch onto the phosphorylated tyrosine residues and initiate signaling cascades culminating in enzyme and/or transcription factor activation. Figure 15-4 identifies just a few of the signaling events specifically associated with mast cell activation. Fc␧RI signaling leads to mast cell and basophil (1) degranulation of vesicles containing multiple inflammatory mediators (Figure 15-5a), (2) expression of inflammatory cytokines and (3) conversion of arachidonic acid into leukotrienes and prostaglandins, two important lipid mediators of inflammation. These mediators have multiple short-term and long-term effects on tissues that will be described in more detail below (Figure 15-5b).

amount of P-K responsive antibody in the fraction (i.e., fraction B had the highest amount of the allergenic antibody). It can readily be seen that the fractions showing the strongest P-K responses (highlighted arrows: n) are also those in which the highest amounts of the so-called ␥E were measured by radio-immunodiffusion. P-K reactions did not correlate with either IgG or with IgA levels in the serum from this, or from two other donors. Ishizaka, K., and T. Ishizaka. (1967). Identification of ␥E-antibodies as a carrier of reaginic activity. Journal of Immunology 99(6):1187–1198.

The Low-Affinity IgE Receptor, Fc␧RII The other IgE receptor, designated Fc␧RII, or CD23, has a much lower affinity for IgE (Kd of 1 ⫻ 106 M⫺1) (Figure 15-3b). CD23 is structurally distinct from Fc␧RI (it is a lectin and a type II membrane protein) and exists in two isoforms that differ only slightly in the N-terminal cytoplasmic domain. CD23a is found on activated B cells, whereas CD23b is induced on various cell types by the cytokine IL-4. Both isoforms also exist as membrane-bound and soluble forms, the latter being generated by proteolysis of the surface molecule. Interestingly, CD23 binds not only to IgE, but also to the complement receptor CD21. The outcome of CD23 ligation depends on which ligands it binds to (IgE or CD21) and whether it does so as a soluble

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Allergen

CD4 Histamine, heparin, proteases

IL-4 B cell

TH2 cell

Smooth muscle cell Allergen

Small blood vessel Vasoactive amines

Fc receptor for IgE

Mucous gland

Blood platelets + Allergen

Memory cell

Plasma cell

Sensitized mast cell

Sensory nerve endings

Degranulation

Allergenspecific IgE Eosinophil

FIGURE 15-2 General mechanism underlying an immediate type I hypersensitivity reaction. Exposure to an allergen activates TH2 cells that stimulate B cells to form IgE-secreting plasma cells. The secreted IgE molecules bind to IgE-specific Fc receptors (Fc␧RI) on mast cells and blood basophils. (Many molecules of IgE with various specificities can bind to the Fc␧RI.) Second exposure to the allergen leads to cross-linking of the bound IgE, triggering the release of pharmacologically active mediators (vasoactive amines) from mast cells and basophils. The mediators cause smooth muscle contraction, increased vascular permeability, and vasodilation. (a) FcεRI: High-affinity IgE receptor

(b) FcεRII (CD23): Low-affinity IgE receptor

NH2

Ig-like domains Extracellular space

S S

Soluble CD23 α

S S S S

COOH

S S

β

γ H2N NH2 S

γ

S S

Proteolytic cleavage

S

Plasma membrane Cytoplasm COOH

ITAM

COOH

NH2 NH2 COOH COOH

FIGURE 15-3 Schematic diagrams of the high-affinity Fc␧RI and low-affinity Fc␧RII receptors that bind the Fc region of IgE. (a) Fc␧RI consists of a ␣ chain that binds IgE, a ␤ chain that participates in signaling, and two disulfide-linked ␥ chains that are the most important component in signal transduction. The ␤ and ␥ chains contain a cytoplasmic ITAM, a motif also present in the Ig␣/Ig␤ (CD79␣/␤) heterodimer of the B-cell receptor and in the T-cell receptor complex. (b) The single-chain Fc␧RII is unusual because it is a type II transmembrane protein, oriented in the membrane with its NH2-terminus directed toward the cell interior and its COOH-terminus directed toward the extracellular space.

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Ag IgE FcεRI γ γ β α Lyn

Adaptors

PKC

PLD

Degranulation

α β γ γ Lyn

MAPK NF-κB

Cytokines

PLA

Leukotrienes, prostaglandins

FIGURE 15-4 Signaling pathways initiated by IgE allergen cross-linking. By cross-linking Fc␧ receptors, IgE initiates signals that lead to mast cell degranulation, cytokine expression, and leukotriene and prostaglandin generation. The signaling cascades initiated by the Fc␧RI are similar to those initiated by antigen receptors (see Chapter 3). This figure illustrates only a few of the players in the complex signaling network. Briefly, cross-linking of Fc␧RI activates tyrosine kinases, including Lyn, which phosphorylate adaptor molecules, which organize signaling responses that lead to activation of multiple kinases, including protein kinase C (PKC) and various mitogen-activated protein kinases (MAPKs). These, in turn, activate transcription factors (e.g., NF␬B) that regulate cytokine production. They also activate lipases, including phospholipase D (PLD), which regulates degranulation, and phospholipase A (PLA), which regulates the metabolism of the leukotriene and prostaglandin precursor arachidonic acid. [K. Roth, W. M. Chen, and T. J. Lin, 2008, Positive and negative regulatory mechanisms in high-affinity IgE receptor-mediated mast cell activation, Archivum immunologiae et therapiae experimentalis 56:385–399.]

or membrane-bound molecule. For example, when soluble CD23 (sCD23) binds to CD21 on the surface of IgEsynthesizing B cells, IgE synthesis is increased. However, when membrane-bound CD23 binds to soluble IgE, further IgE synthesis is suppressed. Atopic individuals express relatively high levels of surface and soluble CD23.

IgE Receptor Signaling Is Tightly Regulated Given the powerful nature of the molecular mediators released by mast cells, basophils, and eosinophils following Fc␧RI signaling, it should come as no surprise that the responses are subject to complex systems of regulation. Below we offer just a few examples of ways in which signaling through the Fc␧RI receptor can be inhibited. Co-Clustering with Inhibitory Receptors Recall from earlier chapters that the intracellular regions of some lymphocyte receptors, including Fc␥RIIB, bear immu-

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noreceptor tyrosine inhibitory motifs (ITIMs as distinct from ITAMs) (see Figure 13-3). Cross-linking of these receptors leads to inhibition, rather than to activation of cellular responses. Interestingly, mast cells express both the activating Fc␧RI and inhibiting Fc␥RIIB. Therefore, if an allergen binds both IgG and IgE molecules, it will trigger signals through both Fc receptors. The inhibitory signal dominates. This phenomenon is part of the reason that eliciting IgG antibodies against common allergens through desensitization therapies can help atopic patients. The more anti-allergen IgG they have, the higher the probability that allergens will co-cluster Fc␧RI receptors with inhibitory Fc␥RIIB receptors. Inhibition of Downstream Signaling Molecules Because many of the reactions in the activation pathway downstream from the Fc␧RI pathways are phosphorylations, multiple phosphatases, such as SHPs, SHIPs, and PTEN, play an important role in dampening receptor signaling. In addition, the tyrosine kinase, Lyn, can play a negative as well as a positive role in Fc␧RI signaling by phosphorylating and activating the inhibitory Fc␥RIIB. Finally, Fc␧RI signaling through Lyn and Syk kinases also activates E3 ubiquitin ligases, including c-Cbl. Cbl ubiquitinylates Lyn and Syk, as well as Fc␧RI itself, triggering their degradation. Thus, Fc␧RI activity contributes to its own demise.

Innate Immune Cells Produce Molecules Responsible for Type I Hypersensitivity Symptoms The molecules released in response to Fc␧RI cross-linking by mast cells, basophils, and eosinophils are responsible for the clinical manifestations of type I hypersensitivity. These inflammatory mediators act on local tissues as well as on populations of secondary effector cells, including other eosinophils, neutrophils, T lymphocytes, monocytes, and platelets. When generated in response to parasitic infection, these mediators initiate beneficial defense processes, including vasodilation and increased vascular permeability, which brings an influx of plasma and inflammatory cells to attack the pathogen. They also inflict direct damage on the parasite. In contrast, mediator release induced by allergens results in unnecessary increases in vascular permeability and inflammation that lead to tissue damage with little benefit. The molecular mediators can be classified as either primary or secondary (Table 15-2). Primary mediators are preformed and stored in granules prior to cell activation, whereas secondary mediators are either synthesized after target-cell activation or released by the breakdown of membrane phospholipids during the degranulation process. The most significant primary mediators are histamine, proteases, eosinophil chemotactic factor (ECF), neutrophil chemotactic factor (NCF), and heparin. Secondary mediators include platelet-activating factor (PAF), leukotrienes, prostaglandins, bradykinins, and various cytokines and chemokines.

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(a)

(b) Cytoplasmic granules

Mast cell activation

Cytokines, chemokines, growth factors, lipid mediators, granule-associated mediators

Cellular targets

Stromal and muscle cells

Nerves

Tissue changes and remodeling

Vascular endothelial cells

Acute changes in function

Days-weeks

FIGURE 15-5 Mast cell activity. (a) A mast cell in the process of degranulating (colorized EM image of mast cell membrane). (b) Mast cell mediators and their effects. Different stimuli activate mast cells to secrete different amounts and/or types of products. Activated mast cells immediately release preformed, granuleassociated inflammatory mediators (including histamine, proteases, and heparin) and are induced to generate lipid mediators (such as leukotrienes and prostaglandins), chemokines, cytokines, and growth factors (some of which can also be packaged in granules).

Hematopoietic cells

Epithelial cells

Inflammation Hours-days

These mediators act on different cell types, and have both acute and chronic effects. When produced over long periods of time, mast cell mediators have a significant influence on tissue structure by enhancing proliferation of fibroblasts and epithelial cells, increasing production and deposition of collagen and other connective tissue proteins, stimulating the generation of blood vessels, and more. [(a) http://t3.gstatic.com/images?q⫽tbn:ANd9GcQh3-PP1n1mbEiIsiHmciOqeniEL5D-iMw3HTWlbVZsQ-TW0QPbQ. (b) Figure 1 in Galli, S. J., and S. Nakae. (2003). Mast Cells to the Defense. Nature Immunology 4:1160–1162.]

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TABLE 15-2 Principal mediators involved in type I hypersensitivity. Mediator

Effects Primary

Histamine, heparin

Increased vascular permeability; smooth muscle contraction

Serotonin (rodents)

Increased vascular permeability; smooth muscle contraction

Eosinophil chemotactic factor (ECF-A)

Eosinophil chemotaxis

Neutrophil chemotactic factor (NCF-A)

Neutrophil chemotaxis

Proteases (tryptase, chymase)

Bronchial mucus secretion; degradation of blood vessel basement membrane; generation of complement split products

Platelet-activating factor

Platelet aggregation and degranulation; contraction of pulmonary smooth muscles

Leukotrienes (slow reactive substance of anaphylaxis, SRS-A)

Increased vascular permeability; contraction of pulmonary smooth muscles

Prostaglandins

Vasodilation; contraction of pulmonary smooth muscles; platelet aggregation

Bradykinin

Increased vascular permeability; smooth muscle contraction

Secondary

Cytokines IL-1 and TNF-␣

Systemic anaphylaxis; increased expression of adhesion molecules on venular endothelial cells

IL-4 and IL-13

Increased IgE production

IL-3, IL-5, IL-6, IL-10, TGF-␤, and GM-CSF

Various effects (see text)

The varying manifestations of type I hypersensitivity in different tissues and species reflect variations in the primary and secondary mediators present. Below we briefly describe the main biological effects of several key mediators. Histamine Histamine, which is formed by decarboxylation of the amino acid histidine, is a major component of mast-cell granules, accounting for about 10% of granule weight. Its biological effects are observed within minutes of mast-cell activation. Once released from mast cells, histamine binds one of four different histamine receptors, designated H1, H2, H3, and H4. These receptors have different tissue distributions and mediate different effects on histamine binding. Serotonin is also present in the mast cells of rodents and has effects similar to histamine. Most of the biologic effects of histamine in allergic reactions are mediated by the binding of histamine to H1 receptors. This binding induces contraction of intestinal and bronchial smooth muscles, increased permeability of venules (small veins), and increased mucous secretion. Interaction of histamine with H2 receptors increases vasopermeability (due to contraction of endothelial cells) and vasodilation (by relaxing the smooth muscle of blood vessels), stimulates exocrine glands, and increases the release of acid in the stomach. Binding of histamine to H2 receptors on mast cells and basophils suppresses degranulation; thus, histamine ultimately exerts negative feedback on the further release of mediators.

Leukotrienes and Prostaglandins As secondary mediators, the leukotrienes and prostaglandins are not formed until the mast cell undergoes degranulation and phospholipase signaling initiates the enzymatic breakdown of phospholipids in the plasma membrane. An ensuing enzymatic cascade generates the prostaglandins and the leukotrienes. In a type I hypersensitivity-mediated asthmatic response, the initial contraction of human bronchial and tracheal smooth muscles is at first mediated by histamine; however, within 30 to 60 seconds, further contraction is signaled by leukotrienes and prostaglandins. Active at nanomole levels, the leukotrienes are approximately 1000 times more effective at mediating bronchoconstriction than is histamine, and they are also more potent stimulators of vascular permeability and mucus secretion. In humans, the leukotrienes are thought to contribute significantly to the prolonged bronchospasm and buildup of mucus seen in asthmatics. Cytokines and Chemokines Adding to the complexity of the type I reaction is the variety of cytokines released from mast cells and basophils. Mast cells, basophils, and eosinophils secrete several interleukins, including IL-4, IL-5, IL-8, IL-13, GM-CSF, and TNF-␣. These cytokines alter the local microenvironment and lead to the recruitment of inflammatory cells such as neutrophils and eosinophils. For instance, IL-4 and IL-13 stimulate a TH2 response and thus increase IgE production by B cells.

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IL-5 is especially important in the recruitment and activation of eosinophils. The high concentrations of TNF-␣ secreted by mast cells may contribute to shock in systemic anaphylaxis. IL-8 acts as a chemotactic factor, and attracts further neutrophils, basophils, and various subsets of T cells to the site of the hypersensitivity response. GM-CSF stimulates the production and activation of myeloid cells, including granulocytes and macrophages.

Type I Hypersensitivities Are Characterized by Both Early and Late Responses Type I hypersensitivity responses are divided into an immediate early response and one or more late phase responses (Figure 15-6). The early response occurs within minutes of allergen exposure and, as described above, results from the release of histamine, leukotrienes, and prostaglandins from local mast cells. However, hours after the immediate phase of a type I hypersensitivity reaction begins to subside, mediators released during the course of the reaction induce localized inflammation, called the late-phase reaction. Cytokines released from mast cells, particularly TNF-␣ and IL-1, increase the expression of cell adhesion molecules on venular endothelial cells, thus facilitating the influx of neutrophils, eosinophils, and TH2 cells that characterizes this phase of the response. Eosinophils play a principal role in the late-phase reaction. Eosinophil chemotactic factor, released by mast cells during the initial reaction, attracts large numbers of eosinophils to the affected site. Cytokines released at the site, including IL-3, IL-5, and GM-CSF, contribute to the growth and differentiation of these cells, which are then activated by binding of antibody-coated antigen. This leads to degranulation and further release of inflammatory mediators that contribute to the extensive tissue damage typical of the latephase reaction. Neutrophils, another major participant in late-phase reactions, are attracted to the site of an ongoing type I reaction by neutrophil chemotactic factor released from degranulating mast cells. Once activated, the neutrophils release their granule contents, including lytic enzymes, platelet-activating factor, and leukotrienes. Recently, a third phase of type I hypersensitivity has been described in models of type I hypersensitivity reactions in the skin. This third phase starts around 3 days post antigen challenge and peaks at day 4. It is also characterized by massive eosinophil infiltration but, in contrast to the second phase, requires the presence of basophils. As shown in Figure 15-7, which illustrates an example of the late-phase responses in the mouse ear, cytokines and proteases released from basophils act on tissue-resident cells such as fibroblasts. These fibroblasts then secrete chemokines that are responsible for the recruitment of larger numbers of eosinophils and neutrophils to the skin lesion. Subsequent degranulation of the eosinophils and neutrophils adds to the considerable tissue damage at the site of the initial allergen contact. These experiments illustrate

how multiple granulocyte subsets can cooperate in the induction of chronic allergic inflammation.

There Are Several Categories of Type I Hypersensitivity Reactions The clinical manifestations of type I reactions can range from life-threatening conditions, such as systemic anaphylaxis and severe asthma, to localized reactions, such as hay fever and eczema. The nature of the clinical symptoms depends on the route by which the allergen enters the body, as well as on its concentration and on the prior allergen exposure of the host. In this section, we describe the pathology of the various type I hypersensitivity reactions. Systemic Anaphylaxis Systemic anaphylaxis is a shocklike and often fatal state that occurs within minutes of exposure to an allergen. It is usually initiated by an allergen introduced directly into the bloodstream or absorbed from the gut or skin. Symptoms include labored respiration, a precipitous drop in blood pressure leading to anaphylactic shock, followed by contraction of smooth muscles leading to defecation, urination, and bronchiolar constriction. This leads to asphyxiation, which can lead to death within 2 to 4 minutes of exposure to the allergen. These symptoms are all due to rapid antibody-mediated degranulation of mast cells and the systemic effects of their contents. A wide range of antigens has been shown to trigger this reaction in susceptible humans, including the venom from bee, wasp, hornet, and ant stings; drugs such as penicillin, insulin, and antitoxins; and foods such as seafood and nuts. If not treated quickly, these reactions can be fatal. Epinephrine, the drug of choice for treating systemic anaphylactic reactions, counteracts the effects of mediators such as histamine and the leukotrienes, relaxing the smooth muscles of the airways and reducing vascular permeability. Epinephrine also improves cardiac output, which is necessary to prevent vascular collapse during an anaphylactic reaction. Localized Hypersensitivity Reactions In localized hypersensitivity reactions (atopy), the pathology is limited to a specific target tissue or organ, and often occurs at the epithelial surfaces first exposed to allergens. Atopic allergies include a wide range of IgE-mediated disorders, such as allergic rhinitis (hay fever), asthma, atopic dermatitis (eczema), and food allergies. The most common atopic disorder, affecting almost 50% of the U.S. population, is allergic rhinitis or hay fever. Symptoms result from the inhalation of common airborne allergens (pollens, dust, viral antigens), which react with IgE molecules bound to sensitized mast cells in the conjunctivae and nasal mucosa. Cross-linking of IgE receptors induces the release of histamine and heparin from mast cells, which then cause vasodilation, increased capillary permeability, and production of exudates in the eyes and respiratory tract. Tearing, runny nose, sneezing, and coughing are the main symptoms.

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LATE RESPONSE

EARLY RESPONSE

IL-4

Mast cell

PAF

TH 2 IL-4

Histamine Leukotrienes Prostaglandins

APC

IL-5 ECF NCF TNF-α Leukotrienes

Vasodilation

Recruitment of inflammatory cells

Mucus secretion

Inflammatory cells (eosinophils; neutrophils)

Bronchoconstriction

Mucous glands

Epithelial injury Early response

Late response

Eosinophils

Blood vessel

Thickened basement membrane Curschmann's spirals

EARLY RESPONSE (minutes)

LATE RESPONSE (hours)

Histamine Prostaglandins Leukotrienes

IL-4, TNF-α, LTC4 PAF, IL-5, ECF IL-4, IL-5

Vasodilation Bronchoconstriction Mucus secretion

Increased endothelial cell adhesion Leukocyte migration Leukocyte activation

FIGURE 15-6 The early and late inflammatory responses in asthma. The immune cells involved in the early and late responses are represented at the top. The effects of various mediators on an airway, represented in cross-section, are illustrated in the center and also described in the text.

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Epidermis Dermis

3

2

Cytokines

4

Chemokines

Other factors

5 Blood vessel

1

IgE FcεRI

Basophils

FIGURE 15-7 Multiple innate immune cells are involved in the chronic type 1 hypersensitivity response in a mouse ear-swelling model. Late in a type I response, basophils migrate into the dermis of the ear (1) and are activated by IgE/antigen complexes (2). They release cytokines and other inflammatory mediators

Alternatively, an asthma attack can be induced by exercise or cold, apparently independently of allergen stimulation (intrinsic asthma). Like hay fever, allergic asthma is triggered by activation and degranulation of mast cells, with subsequent release of inflammatory mediators, but instead of occurring in the nasal mucosa, the reaction develops deeper in the lower respiratory tract. Contraction of the bronchial smooth muscles, mucus secretion, and swelling of the tissues surrounding the airway all contribute to bronchial constriction and airway obstruction. Asthmatic patients may be genetically predisposed to atopic responses. Some, for instance, have abnormal levels of receptors for neuropeptides (substance P) that contract smooth muscles and decreased expression of receptors for vasoactive intestinal peptide, which relaxes smooth muscles. Atopic dermatitis (allergic eczema) is an inflammatory disease of skin and another example of an atopic condition. It is observed most frequently in young children, often developing during infancy. Serum IgE levels are usually elevated. The affected individual develops erythematous (red) skin eruptions that fill with pus if there is an accompanying bacterial infection. Unlike a DTH reaction, which involves TH1 cells (see below), the skin lesions in atopic dermatitis contain TH2 cells and an increased number of eosinophils. Food Allergies: A Common Type of Atopy on the Rise Food allergies, whose incidence is on the rise, are another common type of atopy. In children, food allergies account

Eosinophils

Neutrophils

(3) that stimulate stromal cells (e.g., fibroblasts) to release chemokines (4), which attract other granulocytes, including eosinophils and neutrophils to the tissue, contributing to a chronic inflammation and tissue damage. [Adapted from H. Karasuyama et al., 2011, Nonredundant roles of basophils in immunity, Annual Review of Immunology 29:45–69.]

for more anaphylactic responses than any other type of allergy. They are highest in frequency among infants and toddlers (6%–8%) and decrease slightly with maturity. Approximately 4% of adults display reproducible allergic reactions to food. The most common food allergens for children are found in cow’s milk, eggs, peanuts, tree nuts, soy, wheat, fish, and shellfish. Among adults, nuts, fish, and shellfish are the predominant culprits. Most major food allergens are water-soluble glycoproteins that are relatively stable to heat, acid, and proteases and, therefore, digest slowly. Some food allergens (e.g., the major glycoprotein in peanuts, Ara h 1) are also capable of acting directly as an adjuvant and promoting a TH2 response and IgE production in susceptible individuals. Allergen cross-linking of IgE on mast cells along the upper or lower gastrointestinal tract can induce localized smooth muscle contraction and vasodilation and thus such symptoms as vomiting or diarrhea. Mast-cell degranulation along the gut can increase the permeability of mucous membranes, so that the allergen enters the bloodstream. Basophils also play a significant role in acute food allergy symptoms. Several hypotheses have been advanced to explain why some individuals become sensitive to antigens that are well tolerated by the rest of the population. One possibility is that a temporary viral or bacterial infection may lead to a shortlived increase in the permeability of the gut surface, allowing increased absorption of allergenic antigens and sensitization. Alternatively, sensitization may occur via the respiratory

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TABLE 15-3 Immune basis for some food allergies Disorder

Symptoms

Common trigger

Notes about mechanism

Hives (urticaria)

Wheal and flare swellings triggered by ingestion or skin contact

Multiple foods

Oral allergy

Itchiness, swelling of mouth

Fruits, vegetables

Due to sensitization by inhaled pollens, producing IgE that cross-reacts with food proteins

Asthma, rhinitis

Respiratory distress

Inhalation of aerosolized food proteins

Mast-cell mediated

Anaphylaxis

Rapid, multiorgan inflammation that can result in cardiovascular failure

Peanuts, tree nuts, fish, shellfish, milk, etc.

Exercise-induced anaphylaxis

As above, but occurs when one exercises after eating trigger foods

Wheat, shellfish, celery (may be due to changes in gut absorption associated with exercise)

Atopic dermatitis

Rash (often in children)

Egg, milk, wheat, soy, etc.

May be skin T cell mediated

Gastrointestinal inflammation

Pain, weight loss, edema, and/or obstruction

Multiple foods

Eosinophil mediated

Most often seen in infants: diarrhea, poor growth, and/or bloody stools

Cow’s milk (directly or via breast milk), soy, grains

TNF-␣ mediated

IgE mediated (acute)

IgE and cell mediated (chronic)

Cell mediated (chronic) Intestinal inflammation brought about by dietary protein (e.g., enterocolitis, proctitis)

Adapted from S. H. Sicherer and H. A. Sampson, 2009, Food allergy, Annual Review of Medicine 60:261–277.

route or via absorption of allergens through the skin. This is thought to be the case for one type of allergic reaction to apple proteins. Exposure to birch pollen can induce respiratory type I hypersensitivity, and the IgE that is generated cross-reacts to a protein from apples, leading to a severe digestive allergic response. Finally, various dietary conditions may bias an individual’s responses in the direction of TH2 generation. These include reduced dietary antioxidants, altered fat consumption, and over- or under-provision of Vitamin D. Table 15-3 lists various immune mechanisms that play a role in food allergy. Note that although most are IgE mediated, some are mediated by T cells. The efficiency of the gut barrier improves with maturity, and the food allergies of many infants resolve without treatment as they grow, even though allergen-specific IgE can still be detected in their blood. However, resolution is not always achieved, and in some cases the continuation of the allergic state reflects a reduced frequency of regulatory T cells in allergic versus nonallergic individuals. Depending on where the allergen is deposited, patients with atopic dermatitis and food hypersensitivity can also exhibit other symptoms. For example, some individuals

develop asthmatic attacks after ingesting certain foods. Others develop atopic urticaria, commonly known as hives, when a food allergen is carried to sensitized mast cells in the skin, causing swollen (edematous), erythematous eruptions.

There Is a Genetic Basis for Type I Hypersensitivity The susceptibility of individuals to atopic responses has a strong genetic component that has been mapped to several possible loci by candidate gene association studies, genomewide linkage analyses, and genome expression studies (see Clinical Focus Box 15-2). As might be expected from the pathogenesis of allergy and asthma, many of the associated gene loci encode proteins involved in the generation and regulation of immune responsiveness (e.g., innate immune receptors, cytokines and chemokines and their receptors, MHC proteins) as well as with airway remodeling (e.g., growth factors and proteolytic enzymes). Other proteins that have been implicated in the hereditary predisposition to allergy and asthma include transcription factors and proteins that regulate epigenetic modifications.

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CLINICAL FOCUS

The Genetics of Asthma and Allergy It has long been appreciated that a predisposition to asthma and allergic responses runs strongly in families, suggesting the presence of an hereditary component. In addition, twin studies in humans and mice have implicated both environmental and epigenetic, as well as genetic, factors in determining the susceptibility of an individual to hypersensitivity responses. With this degree of complexity, it is not surprising that the identification of the genes involved in controlling an individual’s vulnerability to hypersensitivity responses has proven to be a difficult task. However, since the late 1980s, the geneticist’s toolkit has markedly expanded with the availability of genome wide sequence information in addition to libraries of single nucleotide polymorphisms (SNPs). These tools, along with more classical genetic approaches have been used to map hypersensitivity susceptibility genes. One approach to determining which genes are associated with a particular pathological state is to use knowledge of the disease to develop and then genetically test an hypothesis regarding potential candidate genes (i.e. “educated guesses”). For example, we know that asthma is associated with high numbers of differentiated TH2 cells, and high levels of IL-4 expression introduce a bias in the differentiation of activated CD4 T cells toward the TH2 state. We can therefore hypothesize that asthma sufferers may

exhibit polymorphisms in structural or regulatory regions of the IL-4 gene, leading to unusually high levels of IL-4 production. Using this theoretical framework, geneticists selected a region on human chromosome 5, 5q31-33, for detailed analysis. This region contains a cluster of cytokine genes, among which are the genes for IL-3, IL-4, IL-5, IL-9, and IL-13, as well as the gene encoding granulocytemacrophage colony stimulating factor. Careful study of this region revealed a polymorphism associated with the predisposition to asthma that maps to the promoter region of IL-4—a confirmation of the hypothesis advanced above. In addition, two alleles of IL-9 associated with a predisposition to atopy were also identified. A second approach starts with a statistically based search for genes associated with particular disease states and is referred to as a genome wide association survey (GWAS). The genomes of individuals who do and those who do not have the disease in question are mapped with respect to the presence of SNPs. Statistically significant association of disease with a particular polymorphism then provides the motivation for detailed sequence analysis in the region of the SNP, and a search for likely candidate genes. Cloning of genes begins in the region identified by the candidate SNP and then proceeds by a sequential search of contiguous

Diagnostic Tests and Treatments Are Available for Type I Hypersensitivity Reactions Type I hypersensitivity is commonly assessed by skin testing, an inexpensive and relatively safe diagnostic approach that allows screening of a wide range of antigens at once. Small amounts of potential allergens are introduced at specific skin sites (e.g., the forearm or back), either by intradermal injection or by dropping onto a site of a superficial scratch. Thirty minutes later, the sites are reexamined. Redness and swelling

sequences until a gene of interest is identified. This technique is referred to as positional cloning, and several genes important in asthma and atopy have been identified in this way. Figure 1 illustrates some of the products of genes already identified as having polymorphisms relevant to the development of asthma or atopy. However, this investigation is far from complete. Sometimes the same SNP has been shown to have different effects in various racial or ethnic populations, illustrating the complexity of such disease-associated genetic studies.

(the result of local mast cell degranulation) indicate an allergic response (Figure 15-8). Less commonly, physicians may elect to measure the serum levels of either total or allergenspecific IgE using ELISA or Western blot technologies (see Chapter 20). Treatment of type I hypersensitivity reactions always begins with measures to avoid the causative agents. However, no one can avoid contact with aeroallergens such as pollen, and a number of immunological and pharmaceutical interventions are now available.

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BOX 15-2 TLR

Prostaglandin E2 receptor

Microbial product

Pollen Defensin-β1

Epithelial cell

CC16

STAT6 Dendritic cell

IL-4Rα

STAT3

IL-4Rα TLR

IL-13 Soluble mediators IL-5

TREG cell

MHC class II Antigen

CCL5

GATA3 STAT6

IL-12β T-Bet

Precursor TH cell

FcεRIB

IL-4 IL-13

IL-10 TGF-β1

TCR

TH1 cell

Eosinophil

IL-5Rα

IL-4 IL-13

IgE Allergic Inflammation

STAT6

TH2 cell

B cell IL-4 IL-13

FcεRIB FcεRIB

Basophil

Mast cell

FIGURE 1 Products of genes that exhibit polymorphisms associated with predisposition to allergy. The genes coding for the products shown have been discovered by multiple genome wide survey techniques. They can be divided into three broad categories. One group of genes codes for proteins that trigger an immune response. These include pattern recognition receptors (TLR2, TLR4, TLR6), pollen receptors (Prostaglandin E2 receptor), and inhibitory cytokines (IL-10, TGF␤), as well as genes coding for proteins that regulate antigen presentation (MHC class II). Another group of genes codes for proteins that regulate TH2 differentiation and innate immune cell responses. These include polarizing cytokines (IL-4, IL-12), signaling and transcriptional regulators (STAT6, T-bet GATA3), Fc␧ receptors, effector cytokines (IL-4, IL-5, and IL-13), and cytokine receptors (IL-4R, IL-5R, IL-13R). Another group of genes is expressed by epithelial cells and smooth muscle cells and codes for proteins that regulate the tissue response. These include chemokines (CCL6), defensins (Defensin ␤2), and other signaling molecules. [Vercelli, D. Discovering susceptibility genes for asthma and allergy. Nature Reviews. Immunology 8:169–182.]

Hyposensitization For many years, physicians have been treating allergic patients with repeated exposure (via ingestion or injection) to increasing doses of allergens, in a regimen termed hyposensitization or immunotherapy. This mode of treatment attacks the disease mechanism of the allergic individual at the source and, when it works, is by far the most effective way to manage allergies. Hyposensitization can reduce or even eliminate symptoms for months or years after the desensitization course is complete.

How does hyposensitization work? Two main mechanisms have been proposed (Figure 15-9). Repeated exposure to low doses of allergen may induce an increase in regulatory T cells producing the immunosuppressive cytokines TGF-␤ and/or IL-10 (a form of tolerance). It may also induce an increase in noninflammatory IgG (specifically IgG4) antibodies specific for the allergens (desensitization). These antibodies either competitively inhibit IgE binding or induce co-clustering of antigen with inhibitory Fc receptors as described above. Regardless of mechanism, hyposensitization

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Histamine

Negative control

Feather

Plane pollen

Cat

Dog

Horse

Sheep wool APC

Birch pollen

Grass pollen

Daisy pollen

Alternaria (mould)

Naïve CD4+ T cell

TGF-β IL-10

FoxP3

FIGURE 15-8 Skin testing for hypersensitivity. This photograph shows an example of a skin test for a variety of antigens. These were introduced by superficial injection and read after 30 minutes. The positive control for a response is histamine; the negative control is typically just saline. This individual is clearly atopic; the skin test reveals responses to multiple animal and plant allergens. [Southern Illinois University/Getty Images]

TREG cell Desensitization Increased IgG4 Decreased IgE Decreased basophil reactivity Decreased mast cell reactivity

Tolerance Increased FoxP3+ TREGs Increased IFN-γ, IL-10, TNF-α

FIGURE 15-9 Mechanisms underlying hyposensitization results in a reduction of allergen-specific TH2 cells, and a concomitant decrease in eosinophils, basophils, mast cells, and neutrophils in the target organs. Although often strikingly successful, hyposensitization does not work in every individual for every allergen. Patients whose disease is refractory to hyposensitization, or who choose not to use it, can try other therapeutic strategies that have taken advantage of our growing knowledge of mechanisms behind mast cell degranulation and the activity of hypersensitivity mediators. Antihistamines, Leukotriene Antagonists, and Inhalation Corticosteroids For many years now, antihistamines have been the most useful drugs for the treatment of allergic rhinitis. These drugs inhibit histamine activity by binding and blocking histamine receptors on target cells. The H1 receptors are blocked by the first-generation antihistamines such as diphenhydramine and chlorpheniramine, which are quite effective in controlling the symptoms of allergic rhinitis. Unfortunately, since they are capable of crossing the blood-brain barrier, they also act on H1 receptors in the nervous system and have multiple side effects. Because these first-generation drugs bind to muscarinic acetylcholine receptors, they can also induce dry mouth, urinary retention, constipation, slow heartbeat, and sedation. Second-generation antihistamines such as fexofenadine, loratidine, and desloratidine were developed in the early 1980s, and exhibit significantly less cross-reactivity with muscarinic receptors. Leukotriene antagonists, specifically montelukast, have also been used to treat type I hypersensitivities and are com-

treatment for type I allergy. This figure illustrates two major mechanisms that are likely to contribute to successful hyposensitization treatment (immunotherapy). Repeated injection or ingestion of low doses of antigen may lead to immune tolerance via the induction of regulatory T cells that quell the immune response to the allergen. Alternatively or in addition, it may induce the generation of IgG antibodies (specifically IgG4), which either compete with IgE for binding to antigen or induce co-clustering of Fc␧RI with inhibitory Fc␥RII receptors (see chapter text). This inhibits basophil and mast cell activity, reducing symptoms (desensitization). [A. M. Scurlock, B. P. Vickery, J. O’B. Hourihane, and A. W. Burks, 2010, Pediatric food allergy and mucosal tolerance, Mucosal Immunology 3(4):345–354, www.nature.com/mi/journal/ v3/n4/fig_tab/mi201021f1.html.]

parable in effectiveness to antihistamines. Finally, inhalation therapy with low-dose corticosteroids reduces inflammation by inhibiting innate immune cell activity and has been used successfully to reduce frequency and severity of asthma attacks. Immunotherapeutics Therapeutic anti-IgE antibodies have been developed; one such antibody, omalizumab, has been approved by the FDA and is available as a pharmacological agent. Omalizumab binds the Fc region of IgE and interferes with IgE-Fc␧R interactions. This reagent has been used to treat both allergic rhinitis and allergic asthma. However, for the treatment of allergic rhinitis, omalizumab is no more effective than secondgeneration antihistamines and is rarely prescribed because of its higher cost. Other monoclonal reagents are also being evaluated for their clinical value.

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Allergy, Hypersensitivities, and Chronic Inflammation Other Medications Used to Control Allergic Asthma In addition to prescribing therapies that focus on inhibiting the molecular and cellular causes of type I hypersensitivities, physicians prescribe drugs that alleviate the symptoms. In particular, drugs that enhance production of the second messenger cAMP help to counter the bronchoconstriction of asthma and the degranulation of mast cells. Epinephrine, or epinephrine agonists (like albuterol), do this by binding to their G protein-coupled receptors, which generate signals that generate cAMP (see Chapter 3). Theophylline, another commonly used drug in the treatment of asthma, does this by antagonizing phosphodiesterase (PDE), which normally breaks down cAMP.

The Hygiene Hypothesis Has Been Advanced to Explain Increases in Allergy Incidence Asthma incidence has increased dramatically in the developed world over the past two decades. This observation supported suggestions that reduction in air quality associated with industrialization played a role in respiratory hypersensitivity. Indeed, the incidence and severity of asthma among those growing up in inner cities is significantly higher. However, it became clear that air quality, although very important, was not the only factor contributing to the increase in asthma incidence. Another contributing cause was suggested by surprising studies from Europe, the United States, Australia, and New Zealand demonstrating that children exposed to a farm environment either prenatally or neonatally were significantly less likely to suffer from hay fever, atopic dermatitis, asthma, and wheezing compared with the control population. In addition, exposure of a pregnant mother or baby to barns and stables, farm animals, hay and grain products, and/or unprocessed cow’s milk all resulted in a decreased tendency to develop type I hypersensitivity later in life. Exposure of children to day care situations and older siblings also correlated with a reduction in asthma incidence. What all these conditions have in common is early exposure to pathogens and potential allergens. Intrigued by this commonality, investigators advanced the hygiene hypothesis, which proposed that exposure to some pathogens during infancy and youth benefits individuals by stimulating immune responses and establishing a healthy balance of T-cell subset activities so that no one response dominates. For instance, the immune system of newborn babies may be biased in the TH2 direction by the uterine environment. TH1-TH2 balance may be restored by the occurrence of infections in the developing neonate. However, in the sanitary conditions promoted by Western medicine, the neonatal immune system may not have the exposure to infections that would otherwise reorient it to generate TH1-type responses. Evidence that pathogen exposure induces NK-mediated interferon ␥ secretion, which biases the responses of the

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subject away from the TH2 direction and thus away from antibody production that contributes to asthma and other allergies, also supports this view. Other investigations, however, focus on the possibility that exposure to viral, bacterial, and parasitic pathogens helps establish the broad array of regulatory T cells that is critical for moderating normal immune responses and quelling autoimmune reactions. The hygiene hypothesis has been advanced to explain increases in the incidence of all allergic responses (e.g., food allergies) as well as increases in the frequency of people suffering from autoimmune diseases (Chapter 16). Studies testing various predictions of the hypothesis are ongoing, and it continues to be modified in its particulars. Understanding the cellular and molecular players that contribute to allergy is clearly important in evaluating the data from these studies.

Antibody-Mediated (Type II) Hypersensitivity Reactions Type II hypersensitivity reactions involve antibody-mediated destruction of cells by immunoglobulins of heavy chain classes other than IgE. Antibody bound to a cell-surface antigen can induce death of the antibody-bound cell by three distinct mechanisms (see Chapters 6 and 13). First, certain immunoglobulin subclasses can activate the complement system, creating pores in the membrane of a foreign cell. Secondly, antibodies can mediate cell destruction by antibodydependent cell-mediated cytotoxicity (ADCC), in which cytotoxic cells bearing Fc receptors bind to the Fc region of antibodies on target cells and promote killing of the cells. Finally, antibody bound to a foreign cell also can serve as an opsonin, enabling phagocytic cells with Fc or C3b receptors to bind and phagocytose the antibody-coated cell. In this section, we examine three examples of type II hypersensitivity reactions. Certain autoimmune diseases involve autoantibody-mediated cellular destruction by type II mechanisms and will be described in Chapter 16.

Transfusion Reactions Are an Example of Type II Hypersensitivity Several proteins and glycoproteins on the membrane of red blood cells are encoded by genes with several allelic forms. An individual with a particular allele of a blood-group antigen can recognize other allelic forms in transfused blood as foreign, and mount an antibody response. Blood types are referred to as A, B, or O, and the antigens that are associated with the blood types are identified as A, B, and H, respectively. Interestingly, the blood type antigens (ABH) are carbohydrates, rather than proteins. This was demonstrated by simple experiments in which the addition of high concentrations of particular simple sugars was shown to inhibit antibody binding to red blood cells bearing particular types of red

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(a)

Galactose

Lipid or protein

N–Acetylglucosamine

Fucose

H antigen N–Acetylgalactosamine

A antigen

Galactose

B antigen

(b) Genotype

Blood–group phenotype

Antigens on erythrocytes (agglutinins)

Serum antibodies (isohemagglutinins)

AA or AO BB or BO AB OO

A B AB O

A B A and B H

Anti–B Anti–A None Anti–A and anti–B

FIGURE 15-10 ABO (ABH) blood groups. (a) Structure of terminal sugars, which constitute the distinguishing epitopes of the A, B, and H blood antigens. All individuals express the H antigen, but not all individuals express the A or B antigens. The blood group of those who express neither A or B antigens (but, like all people express the H antigen) is referred to as O. (b) ABO genotypes, corresponding phenotypes, agglutinins (antigens), and isohemagglutinins (antibodies that react to nonhost antigens).

blood cell antigens. These inhibition reactions revealed that antibodies directed to group A antigens predominantly bound to N-acetyl glucosamine residues, those to group B antigens bound to galactose residues, and those directed toward the so-called H antigens bound to fucose residues (Figure 15-10a). Note that the H antigen is present in all blood types. The A, B, and H antigens are synthesized by a series of enzymatic reactions catalyzed by glycosyltransferases. The final step of the biosynthesis of the A and B antigens is catalyzed by A and B transferases, encoded by alleles A and B at the ABO genetic locus. Although initially detected on the surface of red blood cells, antigens of the ABO blood type system also occur on the surface of other cells as well as in bodily secretions. Antibodies directed toward ABH antigens are termed isohemagglutinins. Figure 15-10b shows the pattern of blood cell antigens and expressed isohemagglutinins normally found within the human population. Most adults possess IgM antibodies to those members of the ABH family they do not express. This is because common microorganisms express carbohydrate antigens very similar in structure to the carbohydrates of the ABH system and induce a B-cell response. B cells generating antibodies specific for the ABH antigens expressed by the host, however, undergo negative selection. For example, an individual with blood type A recognizes B-like epitopes on microorganisms and produces isohem-

agglutinins to the B-like epitopes. This same individual does not respond to A-like epitopes on the same microorganisms because they have been tolerized to self-A epitopes. If a type A individual is transfused with blood containing type B cells, a transfusion reaction occurs in which the preexisting anti-B isohemagglutinins bind to the B blood cells and mediate their destruction by means of complement-mediated lysis. Individuals with blood type O express only the H antigen. Although they can donate blood to anyone, they have antibodies that will react to both A-type or B-type blood. Their anti-A- and anti-B-producing B cells were never exposed to A or B antigens and therefore were never deleted. The clinical manifestations of transfusion reactions result from massive intravascular hemolysis of the transfused red blood cells by antibody plus complement. These manifestations may be either immediate or delayed. Reactions that begin immediately are most commonly associated with ABO blood-group incompatibilities, which lead to complementmediated lysis triggered by the IgM isohemagglutinins. Within hours, free hemoglobin can be detected in the plasma; it is filtered through the kidneys, resulting in hemoglobinuria. As the hemoglobin is degraded, the porphyrin component is metabolized to bilirubin, which at high levels is toxic to the organism. Typical symptoms of bilirubinemia include fever, chills, nausea, clotting within blood vessels, pain in the lower back, and hemoglobin in the urine. Treatment involves prompt termination of the transfusion and

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Allergy, Hypersensitivities, and Chronic Inflammation maintenance of urine flow with a diuretic, because the accumulation of hemoglobin in the kidney can cause acute tubular necrosis. Antibodies to other blood-group antigens such as Rh factor (see below) may result from repeated blood transfusions because minor allelic differences in these antigens can stimulate antibody production. These antibodies are usually of the IgG class. These incompatibilities typically result in delayed hemolytic transfusion reactions that develop between 2 and 6 days after transfusion. Because IgG is less effective than IgM in activating complement, complementmediated lysis of the transfused red blood cells is incomplete. Free hemoglobin is usually not detected in the plasma or urine in these reactions. Rather, many of the transfused cells are destroyed at extravascular sites by agglutination, opsonization, and subsequent phagocytosis by macrophages. Symptoms include fever, increased bilirubin, mild jaundice, and anemia.

Hemolytic Disease of the Newborn Is Caused by Type II Reactions Hemolytic disease of the newborn develops when maternal IgG antibodies specific for fetal blood-group antigens cross the placenta and destroy fetal red blood cells. The consequences of such transfer can be minor, serious, or lethal. Severe hemolytic disease of the newborn, called erythroblastosis fetalis, most commonly develops when the mother and fetus express different alleles of the Rhesus (Rh) antigen. Although there are actually five alleles of the Rh antigen, expression of the D allele elicits the strongest immune response. We therefore designate individuals bearing the D allele of the Rh antigen as Rh⫹. An Rh⫺ mother fertilized by an Rh⫹ father is in danger of developing a response to the Rh antigen and rejecting an Rh⫹ fetus. During pregnancy, fetal red blood cells are separated from the mother’s circulation by a layer of cells in the placenta called the trophoblast. During her first pregnancy with an Rh⫹ fetus, an Rh⫺ woman is usually not exposed to enough fetal red blood cells to activate her Rh-specific B cells. However, at the time of delivery, separation of the placenta from the uterine wall allows larger amounts of fetal umbilical cord blood to enter the mother’s circulation. These fetal red blood cells stimulate Rh-specific B cells to mount an immune response, resulting in the production of Rh-specific plasma cells and memory B cells in the mother. The secreted IgM antibody clears the Rh⫹ fetal red cells from the mother’s circulation, but memory cells remain, a threat to any subsequent pregnancy with an Rh⫹ fetus. Importantly, since IgM antibodies do not pass through the placenta, IgM anti-Rh antigens are no threat to the fetus. Activation of IgG-secreting memory cells in a subsequent pregnancy results in the formation of IgG anti-Rh antibodies, which, however, can cross the placenta and damage the fetal red blood cells (Figure 15-11). Mild to severe anemia can develop in the fetus, sometimes with fatal

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consequences. In addition, conversion of hemoglobin to bilirubin can present an additional threat to the newborn because the lipid-soluble bilirubin may accumulate in the brain and cause brain damage. Because the blood-brain barrier is not complete until after birth, very young babies can suffer fatal brain damage from bilirubin. Fortunately, bilirubin is rapidly broken down on exposure of the skin to ultraviolet (UV) light, and babies who display the telltale jaundiced appearance that signifies high levels of blood bilirubin are treated by exposure to UV light in their cribs (Figure 15-12). Hemolytic disease of the newborn caused by Rh incompatibility in a second or later pregnancy can be almost entirely prevented by administering antibodies against the Rh antigen to the mother at around 28 weeks of pregnancy and within 24 to 48 hours after the first delivery. Anti-Rh antibodies are also administered to pregnant women after amniocentesis. These antibodies, marketed as Rhogam, bind to any fetal red blood cells that may have entered the mother’s circulation and facilitate their clearance before B-cell activation and ensuing memory-cell production can take place. In a subsequent pregnancy with an Rh⫹ fetus, a mother who has been treated with Rhogam is unlikely to produce IgG anti-Rh antibodies; thus, the fetus is protected from the damage that would occur when these antibodies cross the placenta. The development of hemolytic disease of the newborn caused by Rh incompatibility can be detected by testing maternal serum at intervals during pregnancy for antibodies to the Rh antigen. A rise in the titer of these antibodies as pregnancy progresses indicates that the mother has been exposed to Rh antigens and is producing increasing amounts of antibody. Treatment depends on the severity of the reaction. For a severe reaction, the fetus can be given an intrauterine blood-exchange transfusion to replace fetal Rh⫹ red blood cells with Rh⫺ cells. These transfusions are given every 10 to 21 days until delivery. In less severe cases, a blood-exchange transfusion is not given until after birth, primarily to remove bilirubin; the infant is also exposed to low levels of UV light to break down the bilirubin and prevent cerebral damage. The mother can also be treated during the pregnancy by plasmapheresis. In this procedure, a cell separation machine is used to separate the mother’s blood into two fractions: cells and plasma. The plasma containing the anti-Rh antibody is discarded, and the cells are reinfused into the mother in an albumin or fresh plasma solution. The majority of cases (65%) of hemolytic disease of the newborn, however, are caused by ABO blood-group incompatibility between the mother and fetus and are not severe. Type A or B fetuses carried by type O mothers most commonly develop these reactions. A type O mother can develop IgG antibodies to the A or B blood-group antigens through exposure to fetal blood-group A or B antigens in successive pregnancies. Usually, the fetal anemia resulting from this incompatibility is mild; the major clinical manifestation is a

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DEVELOPMENT OF ERYTHROBLASTOSIS FETALIS (WITHOUT RHOGAM) Placenta

PREVENTION (WITH RHOGAM)

Plasma cells

Mother

Maternal circulation

Mother (treated with Rhogam)

B cell Anti-Rh IgM

RBCs with Rh antigen 1st Pregnancy

Delivery

Rh-specific B cell

Rhogam

Memory cell

Prevents B-cell activation and memory cell formation

Memory cell

Plasma cells IgG

2nd Pregnancy

IgG anti-Rh Ab crosses placenta and attacks fetal RBCs causing erythroblastosis fetalis

FIGURE 15-11 Destruction of Rh positive red blood cells during erythroblastosis fetalis of the newborn. Development of erythroblastosis fetalis (hemolytic disease of the newborn) is caused when an Rh⫺ mother carries an Rh⫹ fetus (left). The effect of treatment with anti-Rh antibody, or Rhogam, is shown on the right.

slight elevation of bilirubin, with jaundice. Exposure of the infant to low levels of UV light is often enough to break down the bilirubin and avoid cerebral damage. In severe cases, transfusion may be required.

Hemolytic Anemia Can Be Drug Induced

FIGURE 15-12 Ultraviolet light is used to treat bilirubinemia of the newborn. [Stephanie Clarke/123RF]

Certain antibiotics (e.g., penicillin, cephalosporins, and streptomycin), as well as other well-known drugs (including ibuprofen and naproxen), can adsorb nonspecifically to proteins on red blood cell membranes, forming a drugprotein complex. In some patients, such drug-protein complexes induce formation of antibodies. These antibodies then bind to the adsorbed drug on red blood cells, inducing complement-mediated lysis and thus progressive anemia. When the drug is withdrawn, the hemolytic anemia disappears. Penicillin is notable in that it can induce all four types of hypersensitivity with various clinical manifestations (Table 15-4).

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TABLE 15-4

Penicillin-induced hypersensitive reactions

Type of reaction

Antibody or lymphocytes induced

Clinical manifestations

I

IgE

Urticaria, systemic anaphylaxis

II

IgM, IgG

Hemolytic anemia

III

IgG

Serum sickness, glomerulonephritis

IV

TH1 cells

Contact dermatitis

Immune Complex-Mediated (Type III) Hypersensitivity The reaction of antibody with antigen generates immune complexes. Generally, these complexes facilitate the clearance of antigen by phagocytic cells and red blood cells. In some cases, however, the presence of large numbers and networks of immune complexes can lead to tissue-damaging type III hypersensitivity reactions. The magnitude of the reaction depends on the number and size of immune complexes, their distribution within the body, and the ability of the phagocyte system to clear the complexes and thus minimize the tissue damage. The deposition of these complexes initiates a reaction that results in the recruitment of complement components and neutrophils to the site, with resultant tissue injury.

Immune Complexes Can Damage Various Tissues The formation of antigen-antibody complexes occurs as a normal part of an adaptive immune response. It is usually followed by Fc receptor-mediated recognition of the complexes by phagocytes, which engulf and destroy them and/or by complement activation resulting in the lysis of the cells on which the immune complexes are found. However, under certain conditions, immune complexes are inefficiently cleared and may be deposited in the blood vessels or tissues, setting the stage for a type III hypersensitivity response. These conditions include (1) the presence of antigens capable of generating particularly extensive antigen-antibody lattices, (2) a high intrinsic affinity of antigens for particular tissues, (3) the presence of highly charged antigens (which can affect immune complex engulfment) and (4) a compromised phagocytic system. All have been associated with the initiation of a type III response. Immune complexes bind to mast cells, neutrophils, and macrophages via Fc receptors, triggering the release of vasoactive mediators and inflammatory cytokines, which

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interact with the capillary epithelium and increase the permeability of the blood vessel walls. Immune complexes then move through the capillary walls and into the tissues where they are deposited and set up a localized inflammatory response. Complement fixation results in the production of the anaphylatoxin chemokines C3a and C5a, which attract more neutrophils and macrophages. These in turn are further activated by immune complexes binding to their Fc receptors to secrete proinflammatory chemokines and cytokines, prostaglandins, and proteases. Proteases digest the basement membrane proteins collagen, elastin, and cartilage. Tissue damage is further mediated by oxygen free radicals released by the activated neutrophils. In addition, immune complexes interact with platelets and induce the formation of tiny clots. Complex deposition in the tissues can give rise to symptoms such as fever, urticaria (rashes), joint pain, lymph node enlargement, and protein in the urine. The resulting inflammatory lesion is referred to as vasculitis if it occurs in a blood vessel, glomerulonephritis if it occurs in the kidney, or arthritis if it occurs in the joints.

Immune Complex-Mediated Hypersensitivity Can Resolve Spontaneously If immune complex-mediated disease is induced by a single large bolus of antigen that is then gradually cleared, it can resolve spontaneously. Spontaneous recovery is seen, for example, when glomerulonephritis is initiated following a streptococcal infection. Streptococcal antigen-antibody complexes bind to the basement membrane of the kidney and set up a type III response, which resolves as the bacterial load is eliminated. Similarly, patients being treated by injections of passive antibody can develop immune responses to the foreign antibody and generate large immune complexes. This was seen initially during the use of horse antibodies in the treatment of diphtheria in the early 1900s. On repeated injections with the horse antibodies, patients developed a syndrome known as serum sickness, which resolved as soon as the antibodies were withdrawn. Serum sickness is an example of a systemic form of immune complex disease, which resulted in arthritis, skin rash, and fever. A more modern manifestation of the same problem occurs in patients who receive therapeutic mouse-derived monoclonal antibodies designed to treat previously intractable cancers. After several such treatments, some patients generate their own antibodies against the foreign monoclonals and develop serum sickness–like symptoms. We know now that the injection of the mouse antibodies caused a generalized type III reaction, and in many cases the therapeutic antibodies were actually cleared before they could reach their pathogenic target. To avoid this response, current therapeutic antibodies are genetically engineered to remove most of the foreign epitopes (they are humanized).

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Examples of diseases resulting from type III hypersensitivity reactions.

Autoimmune diseases Systemic lupus erythematosus Rheumatoid arthritis Multiple sclerosis Drug reactions Allergies to penicillin and sulfonamides Infectious diseases Poststreptococcal glomerulonephritis Meningitis Hepatitis Mononucleosis Malaria Trypanosomiasis

Autoantigens Can be Involved in Immune Complex-Mediated Reactions If the antigen in the immune complex is an autoantigen, it cannot be eliminated and type III hypersensitivity reactions cannot be easily resolved. In such situations, chronic type III responses develop. For example, in systemic lupus erythematosus, persistent antibody responses to autoantigens are an identifying feature of the disease, and complexes are deposited in the joints, kidneys, and skin of patients. Examples of diseases resulting from type III hypersensitivity reactions are found in Table 15-5.

Arthus Reactions Are Localized Type III Hypersensitivity Reactions One example of a localized type III hypersensitivity reaction has been used extensively as an experimental tool. If an animal or human subject is injected intradermally with an antigen to which large amounts of circulating antibodies exist (or have been recently introduced by intravenous injections), antigen will diffuse into the walls of local blood vessels and large immune complexes will precipitate close to the injection site. This initiates an inflammatory reaction that peaks approximately 4 to 10 hours post injection and is known as an Arthus reaction. Inflammation at the site of an Arthus reaction is characterized by swelling and localized bleeding, followed by fibrin deposition (Figure 15-13). A sensitive individual may react to an insect bite with a rapid, localized type I reaction, which can be followed, some 4 to 10 hours later, by the development of a typical Arthus reaction, characterized by pronounced erythema and edema. Intrapulmonary Arthus-type reactions induced by bacterial

FIGURE 15-13 An Arthus reaction. This photograph shows an Arthus reaction on a thigh of a 72-year-old woman. This occurred at the site of injection of a chemotherapeutic drug, 3 to 4 hours after the patient received a second injection (15 days after the first). This response was accompanied by fever and significant discomfort. [From P. Boura et al., 2006, Eosinophilic cellulitis (Wells’ syndrome) as a cutaneous reaction to the administration of adalimumab, Annals of the Rheumatic Diseases 65:839–840. doi:10.1136/ard.2005.044685.]

spores, fungi, or dried fecal proteins can also cause pneumonitis or alveolitis. These reactions are known by a variety of common names reflecting the source of the antigen. For example, farmer’s lung develops after inhalation of actinomycetes from moldy hay, and pigeon fancier’s disease results from inhalation of a serum protein in dust derived from dried pigeon feces.

Delayed-Type (Type IV) Hypersensitivity (DTH) Type lV hypersensitivity, commonly referred to as DelayedType Hypersensitivity (DTH), is the only hypersensitivity category that is purely cell mediated rather than antibody mediated. In 1890, Robert Koch observed that individuals infected with Mycobacterium tuberculosis developed a localized inflammatory response when injected intradermally (in the skin) with a filtrate derived from a mycobacterial culture. He therefore named this localized skin reaction a tuberculin reaction. Later, as it became apparent that a variety of other antigens could induce this cellular response (Table 15-6), its name was changed to delayed-type, or type IV, hypersensitivity. The hallmarks of a type IV reaction are its initiation by T cells (as distinct from antibodies), the delay required for

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TABLE 15-6

Intracellular pathogens and contact antigens that induce delayed-type (type IV) hypersensitivity

Intracellular bacteria

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Mycobacterium leprae

Variola (smallpox)

Brucella abortus

Measles virus

507

(a) Sensitization phase

Intracellular bacteria TH1 cells (generally)

Intracellular viruses

Mycobacterium tuberculosis

APC CD4+ TH

Listeria monocytogenes Intracellular fungi

|

Contact antigens

Pneumocystis carinii

Picrylchloride

Candida albicans

Hair dyes

Histoplasma capsulatum

Nickel salts

Cryptococcus neoformans

Poison ivy Poison oak

Antigen-presenting cells: Macrophages Langerhans cells

DTH-mediating cells: CD4+ TH1 generally and TH17,TH2, and CD8+ cells occasionally

(b) Effector phase

Intracellular parasites Leishmania sp.

Secreted IFN-γ

the reaction to develop, and the recruitment of macrophages (as opposed to neutrophils or eosinophils) as the primary cellular component of the infiltrate that surrounds the site of inflammation. The most common type IV hypersensitivity is the contact dermatitis that occurs after exposure to Toxicodendron species, which include poison ivy, poison oak, and poison sumac. This is a significant public health problem. Approximately 50% to 70% of the U.S. adult population is clinically sensitive to exposure to Toxicodendron; only 10% to 15% of the population is tolerant. Some responses can be severe and require hospitalization.

The Initiation of a Type IV DTH Response Involves Sensitization by Antigen A DTH response begins with an initial sensitization by antigen, followed by a period of at least 1 to 2 weeks during which antigen-specific T cells are activated and clonally expanded (Figure 15-14a). A variety of antigen-presenting cells (APCs) are involved in the induction of a DTH response, including Langerhans cells (dendritic cells found in the epidermis) and macrophages. These cells pick up antigen that enters through the skin and transport it to regional lymph nodes, where T cells are activated. In some species, including humans, the vascular endothelial cells express class II MHC molecules and can also function as APCs in the development of the DTH response. Generally, the T cells activated during the sensitization phase of a traditional DTH response are CD4⫹, primarily of the TH1 subtypes. However, recent studies indicate that TH17, TH2, and CD8⫹ cells can also play a role.

Membrane TNF-β Resting Sensitized macrophage TH1 TH1 secretions: Cytokines: IFN-γ, LT-α (TNF-β), IL-2, IL-3, GM-CSF, MIF Chemokines: IL-8/CXCL8, MCP-1/CCL2

Class II MHC

TNF receptor

Activated macrophage Effects of macrophage activation: ↑ Class II MHC molecules ↑ TNF receptors ↑ Oxygen radicals ↑ Nitric oxide

FIGURE 15-14 The DTH response. (a) In the sensitization phase after initial contact with antigen (e.g., peptides derived from intracellular bacteria), TH cells proliferate and differentiate into TH1 cells. Cytokines secreted by these T cells are indicated by the black balls. (b) In the effector phase after subsequent exposure of sensitized TH cells to antigen, TH1 cells secrete a variety of cytokines and chemokines. These factors attract and activate macrophages and other nonspecific inflammatory cells. Activated macrophages are more effective in presenting antigen, thus perpetuating the DTH response, and function as the primary effector cells in this reaction. Other helper T-cell subsets are now thought to participate in DTH (TH2 and TH17) and CD8⫹ T cells also contribute.

The Effector Phase of a Classical DTH Response Is Induced by Second Exposure to a Sensitizing Antigen A second exposure to the sensitizing antigen induces the effector phase of the DTH response (see Figure 15-14b). In

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the effector phase, T cells are stimulated to secrete a variety of cytokines, including interferon-␥ (IFN-␥) and Lymphotoxin-␣ (TNF-␤), which recruit and activate macrophages and other inflammatory cells. A DTH response normally does not become apparent until an average of 24 hours after the second contact with the antigen and generally peaks 48 to 72 hours after this stimulus. The delayed onset of this response reflects the time required for the cytokines to induce localized influxes of macrophages and their activation. Once a DTH response begins, a complex interplay of nonspecific cells and mediators is set in motion that can result in extensive amplification of the response. By the time the DTH response is fully developed, only about 5% of the participating cells are antigen-specific TH1 cells; the remainder are macrophages and other innate immune cells. TH1 cells are important initiators of DTH, but the principal effector cells of the DTH response are activated macrophages. Cytokines elaborated by helper T cells, including IFN-␥ and Lymphotoxin-␣, induce blood monocytes to adhere to vascular endothelial cells, migrate from the blood into the surrounding tissues, and differentiate into activated macrophages. As described in Chapter 2, activated macrophages exhibit enhanced phagocytosis and an increased ability to kill microorganisms. They produce cytokines, including TNF-␣ and IL-1␤, that recruit more monocytes and neutrophils, and enhance the activity of TH1 cells, amplifying the response. The heightened phagocytic activity and the buildup of lytic enzymes from macrophages in the area of infection lead to nonspecific destruction of cells and thus of any intracellular pathogens, such as Mycobacteria. Usually, any presented pathogens are cleared rapidly with little tissue damage. However, in some cases, and especially if the antigen is not easily cleared, a prolonged DTH response can develop, which becomes destructive to the host, causing a visible granulomatous reaction. Granulomas develop when continuous activation of macrophages induces them to adhere closely to one another. Under these conditions, macrophages assume an epithelioid shape and sometimes fuse to form multinucleated giant cells (Figure 15-15a). These giant cells displace the normal tissue cells, forming palpable nodules, and releasing high concentrations of lytic enzymes, which destroy surrounding tissue. The granulomatous response can damage blood vessels and lead to extensive tissue necrosis. The response to Mycobacterium tuberculosis illustrates the double-edged nature of the DTH response. Immunity to this intracellular bacterium involves a DTH response in which activated macrophages wall off the organism in the lung and contain it within a granuloma-type lesion called a tubercle (see Figure 15-15b). Often, however, the release of concentrated lytic enzymes from the activated macrophages within the tubercles damages the very lung tissue that the immune response aims to preserve.

(a)

TH1 cell

Multinucleated giant cell Epithelioid cell

Intracellular bacteria

Activated macrophage

(b)

FIGURE 15-15 A prolonged DTH response can lead to formation of a granuloma, a nodule-like mass. (a) Lytic enzymes released from activated macrophages in a granuloma can cause extensive tissue damage. (b) Stained section of a granuloma associated with tuberculosis. [Biophoto Associates/Getty Images]

observing whether a characteristic skin lesion develops days later at the injection site. A positive skin-test reaction indicates that the individual has a population of sensitized TH1 cells specific for the test antigen. For example, to determine whether an individual has been exposed to M. tuberculosis, PPD, a protein derived from the cell wall of this mycobacterium, is injected intradermally. Development of a red, slightly swollen, firm lesion at the site between 48 and 72 hours later indicates previous exposure. Note, however, that a positive test does not allow one to conclude whether the exposure was due to a pathogenic form of M. tuberculosis or to a vaccine antigen, which is used in some parts of the world.

The DTH Reaction Can be Detected by a Skin Test

Contact Dermatitis Is a Type IV Hypersensitivity Response

The presence of a DTH reaction can be measured experimentally by injecting antigen intradermally into an animal and

Contact dermatitis is one common manifestation of a type IV hypersensitivity. The simplest form of contact dermatitis

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Allergy, Hypersensitivities, and Chronic Inflammation occurs when a reactive chemical compound binds to skin proteins and these modified proteins are presented to T cells in the context of the appropriate MHC antigens. The reactive chemical may be a pharmaceutical, a component of a cosmetic or a hair dye, an industrial chemical such as formaldehyde or turpentine, an artificial hapten such as fluoro-dinitrobenzene, a metal ion such as nickel, or the active allergen from poison ivy. Sometimes, however, the antigen is presented to T cells via a less familiar route. Some chemicals are first metabolized by the body to form a reactive metabolite, which, in turn, binds to proteins presented by the MHC. Some investigators propose that other molecules have the capacity to bind directly to the T-cell receptor and directly stimulate an effector or memory T cell. These ideas are new and still being evaluated. What seems clear, however, is that many small molecules and drugs can attach to cell-surface proteins of T cells as well as of APCs and give rise to the hypersensitivity reactions characteristic of contact dermatitis. The classical DTH response to Mycobacterium antigens described above is mediated by CD4⫹, TH1 T cells. However, we now know that T-cell hypersensitivity reactions directed toward other antigens are mediated by a variety of T-cell subtypes and cytokines. The severe dermatitis associated with some type IV hypersensitivities to drugs, for example, is caused by CD8⫹ T cells and NK cells. These cytotoxic cells induce death of keratinocytes and sloughing of the skin or mucus membrane. Diseases in which this mechanism is active include erythema multiforme, Stevens-Johnson syndrome, and toxic epidermal necrolysis; they can be fatal. The

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allergen in these cases can be associated with drugs as common as the nonsteroidal anti-inflammatory medication ibuprofen. The incidence of such complications is higher in males than in females and usually occurs in young adults. At present the best way to avoid a DTH response it to avoid the causative antigen. Once hypersensitivity has developed, topical or oral corticosteroids can be used to suppress the destructive immune response.

Chronic Inflammation Regardless of whether an immune response is activated appropriately or inappropriately, a full-blown immune response makes one feel ill, largely because of the activity of inflammatory mediators released by innate immune cells. In most cases, the misery subsides when the insult (antigen, allergen, or toxin) is removed. However, in some circumstances, an inflammatory stimulus persists, generating a chronic inflammatory response that has systemic effects, which have been associated most directly with Type 2 diabetes. Recent studies also raise a possibility that chronic inflammation exacerbates heart disease, kidney disease, Alzheimer’s, and cancer (Figure 15-16).

Infections Can Cause Chronic Inflammation Chronic inflammatory conditions have a variety of causes, some of which are still being identified (Figure 15-16). Some are the result of infections that persist because

Non-infectious causes: • Obesity • Tissue damage • Heart disease and atherosclerosis

Infectious causes: • Unresolved infection • Intestinal microbes

Chronic inflammation IL-6, TNFα, IL-1β, IL-18

Tissue changes: • Cell death • Scarring (fibrosis) • Blood vessel growth (angiogenesis) • Cell proliferation

Metabolic changes: • Impaired insulin signaling

Type 2 diabetes

Other systemic disease: • Organ failure (kidney, heart, liver) • Cancer • Alzheimer’s

FIGURE 15-16 Causes and consequences of chronic inflammation. Chronic inflammation has infectious and noninfectious causes, including obesity. Chronic inflammatory conditions, regardless of cause, have common systemic consequences, some of which are related to the effects of inflammatory mediators on metabolism

(Type 2 diabetes) and some of which are related to the effects of inflammatory mediators on tissue organization and cell proliferation (e.g., cancer). Other disorders have also been associated with chronic inflammation although the mechanisms behind the association may be indirect and are still being studied.

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pathogen has continual access to the body. For instance, gum disease and unhealed wounds make a body vulnerable to continual microbe invasion and immune stimulation. Gut pathogens can also contribute. Although our commensal bacteria play an important role in dampening our reaction to microbes that we ingest, this protective mechanism can fail, and gut microbes can contribute to a chronic inflammatory state. Some chronic inflammatory conditions are caused by pathogens that evade the immune system and remain active in the body, inspiring continual low-level inflammatory reactions. Fungi and mycobacteria are two examples of pathogens that are not always successfully cleared and have the ability to continually stimulate immune cells that release inflammatory molecules and cytokines.

They appear to have the capacity to bind toll-like receptors on adipocytes, initiating a signaling cascade analogous to that experienced by innate immune cells that recognize pathogen. Obesity is now recognized as a major, if not the major, cause of chronic inflammation, which, as you will see below, has severe consequences. Interestingly, approximately 6% of individuals who are considered obese by weight do not generate inflammatory cytokines and show few signs of metabolic dysfunction. The basis for the ability of these individuals to tolerate excess fat is an area of active investigation.

There Are Noninfectious Causes of Chronic Inflammation

The specific consequences of chronic inflammation vary with the tissue of origin as well as the sex, age, and health status of the individual. However, given that those who suffer from chronic inflammation all exhibit increased circulation of inflammatory mediators, including the classical proinflammatory cytokine trio (IL-1, IL-6, and TNF-␣), it is not surprising that many suffer similar systemic disorders (Figure 15-16).

Interestingly, pathogens are not the only causes of chronic inflammation. Physical damage to tissue also releases molecules (damage-associated molecular patterns, or DAMPs) that induce the secretion of inflammatory cytokines. If tissue damage is not resolved because of continual mechanical interference, for instance, the inflammatory stimulus persists. Tumors, autoimmune disorders, atherosclerosis, and heart disease, each of which results in tissue damage that stimulates immune responses, are other examples of disorders that can cause chronic inflammation. The biomedical community was startled, however, to discover that one of the most common noninfectious causes of chronic inflammation today is obesity, a condition that did not, at first, suggest a relationship to inflammation.

Obesity Is Associated with Chronic Inflammation Obesity has long been associated with a constellation of metabolic and systemic disorders, including Type 2 diabetes. The biological mechanisms responsible for these associations are still being investigated. However, recent work suggests that many of the systemic effects of obesity are mediated by inflammation. What does fat have to do with inflammation? It turns out that the immune system is not the only source of inflammatory cytokines. Visceral adipocytes, fat cells that surround organs (as opposed to subcutaneous adipocytes, which are located under the skin), are very active, responsive cells. Not only do they generate hormones such as leptin that regulate metabolism, but they also secrete a variety of proinflammatory mediators, including TNF-␣ and IL-6. What triggers this release? Some studies suggest that intracellular stress responses associated with excessive lipid buildup induce signals that enhance production of cytokines and inflammatory mediators. Free fatty acids, a common consequence of obesity, may also play a role.

Chronic Inflammation Can Cause Systemic Disease

Chronic Inflammation and Insulin Resistance Type 2 diabetes is one of the most common consequences of chronic inflammation. Diabetes results from a failure in insulin signaling, a failure that leads to general metabolic dysfunction. Type 1 diabetes (Chapter 16) is caused by the autoimmune-mediated destruction of pancreatic islet cells that make insulin. Type 2 diabetes, however, is caused by a failure of cells to respond to insulin, a state known as insulin resistance. What does inflammation have to do with insulin resistance? Inflammatory cytokines, particularly TNF-␣ and IL-6, induce signaling cascades that inhibit the ability of the insulin receptor to function. This interference is in large part due to the cytokines’ ability to activate JNK, a MAPK that is often associated with stress and inflammatory responses. JNK can phosphorylate and inactivate IRS-1, a key downstream mediator of insulin receptor signaling. This observation also helps explain the long-recognized association between obesity and Type 2 diabetes (metabolic syndrome). Inflammatory cytokines released by visceral adipocytes in response to excess lipid induce signals that inhibit insulin signaling, leading to insulin resistance, a primary cause of Type 2 diabetes. This perspective is directly supported by studies in mouse models where obesity was uncoupled from inflammation. Wild-type mice fed a highfat diet became obese and developed Type 2 diabetes. Mice that lack JNK also became obese on the same diet, but did not develop diabetes. (See Clinical Focus Box 15-3 for a more complete discussion of the association between obesity, inflammation, and diabetes.)

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BOX 15-3

CLINICAL FOCUS

Type 2 Diabetes, Obesity, and Inflammation As late as the 1960s, scientists and physicians searching for information on Type 2 diabetes, obesity, or inflammation would have looked in separate chapters of physiology or pathology books. Obesity was viewed as a problem of poor nutritional management or resources, psychology, or (in the case of rare hormonal disorders) endocrinology. Type 2 diabetes was known to be a result of the inability to effectively use insulin, resulting in high blood glucose levels (hyperglycemia) and was, therefore, seen solely as the province of endocrinologists. Inflammation had been understood for close to a thousand years to be a result of immune system activity. However, research conducted over the past few decades has resulted in a dramatic shift in our thinking regarding the centrality of inflammatory responses in many human pathologies, including Type 2 diabetes. Furthermore, knowledge about how inflammatory mediators work and the cells from which they are released has helped us to understand the linkages between obesity and Type 2 diabetes. Here we will explore the intersecting biological pathways that connect obesity, inflammation and Type 2 diabetes. Obesity and Type 2 diabetes currently represent a public health problem of stunning proportions in the United States. As of summer 2011, 33.8% of adults in the United States are obese (defined as having a body mass index [BMI] of 30 or above), and childhood obesity rates are rising rapidly, with 17% of children and adolescents aged from 2 to 19 years falling into this category. Nor is the problem restricted to the United States. The World Health Organization reports that 300 million adults are obese and as many as 1 billion are reported to be overweight worldwide. But why should this be a problem, and what does it have to do with immunology? In Type 1 diabetes, the insulin-producing ␤ cells of the islets of Langerhans are

subject to autoimmune attack and are destroyed. However, in Type 2 diabetes, patients experience a state of insulin resistance, in which the body still makes insulin, but the responses to it are dulled and the amount of insulin in the circulation is unable to do its job of driving dietary sugar out of the bloodstream and into the waiting cells. The first indication that Type 2 diabetes may result from, or at least be exacerbated by, inflammatory signals came almost a hundred years ago, when it was discovered that patients receiving salicylate (aspirin) for inflammatory conditions showed an increase in insulin sensitivity. Studies published in the early years of the twenty-first century further demonstrated that patients suffering from a variety of infectious diseases, including hepatitis C and HIV, as well as those with autoimmune diseases such as rheumatoid arthritis, displayed insulin resistance. These diseases share the common feature of inducing an active inflammatory response. In each case, the insulin resistance was improved upon treatment with antiinflammatory drugs. What do we know about the mechanism by which inflammation results in insulin resistance? Insulin signals a cell to import glucose by binding to a cell surface receptor that is a member of the receptor tyrosine kinase (RTK) family. When insulin binds to the receptor on the external surface of the cell, a signal is transmitted to the intracellular part of the receptor, activating the intrinsic tyrosine kinase activity of the receptor (see Chapter 3). The two halves of the insulin-bound dimeric receptor phosphorylate one another on tyrosine residues, and these residues then act as docking sites for other proteins in the signaling cascade. A set of six proteins termed the Insulin Receptor Substrate (IRS) proteins are among the early, pivotally important substrates of the insulin receptor, and they bind to it through their phosphotyrosine

binding domains. The IRS proteins are then phosphorylated by the RTKs and subsequently act as adapter molecules for transmitting the insulin signal to downstream molecules such as the kinases PI3 kinase and Fyn, and the docking proteins Grb2 and SHP2. However, if the IRS proteins are phosphorylated on serine residues by IRS serine/threonine kinases, their signaling capacity is inhibited (see Figure 1). Inflammatory cytokines, such as IL-6, IL-1␤ and TNF-␣, bind to cell-surface receptors and signal the activation of kinases, including JNK, which phosphorylate IRS-1 on serine residues, inhibiting its activity. Thus, inflammatory cytokines act to inhibit the insulin signal, leading to insulin resistance. However, what is the source of the excess inflammatory cytokines released in individuals with Type 2 diabetes? Patients with Type 2 diabetes are frequently (although not always) obese, and so investigators began to explore the relationships between obesity and the generation of inflammatory cytokines. Mice fed high-fat diets, or those with a genetic predisposition to obesity, were found to develop chronically elevated levels of inflammatory mediators, such as TNF-␣, IL-1␤, and IL-6, and increased local concentrations of chemokines, such as CCL2, which draw immune cells, particularly macrophages, into adipose tissue. Investigators therefore advanced the hypothesis that the nutrients themselves might be activating signaling pathways leading to the release of these mediators. But with what receptors are the nutrients interacting? One clue has come from genetically modified mice. Animals in which the gene encoding the TLR4 receptor has been eliminated are protected from the insulin resistance engendered by eating a highfat diet. This suggests that the Toll receptors may be recognizing the excess nutrients and initiating the inflammatory (continued)

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Box 15-3

(continued) Fatty Acids TNF-α IL-6

IR

TLR

IRS Tyr-P Phosphorylation on Tyrosine residues enhances IRS signaling

IL-6R

TNFR

Ser-P Phosphorylation on Serine residues inhibits IRS signaling JNK and other kinases

Glucose transport Lipid and sugar metabolism

FIGURE 1 Signaling events that link obesity to insulin resistance. Several factors trigger signaling cascades that interfere with insulin receptor (IR) signaling cascades. Inflammatory cytokines, including TNF-␣ and IL-6, are produced by adipocytes themselves, as well as immune cells. These trigger signaling events in multiple cells that activate kinases, including JNK, which inactivate IRS by phosphorylating it at a serine residue. Thus, insulin signaling is impaired. JNK can also be activated (and insulin signaling inactivated) by the interaction between toll like receptors and free fatty acids, which are increased in obesity. [Based on Gray, S., and J. K. Kim. (2011, October). New insights into insulin resistance in the diabetic heart. Trends in Endocrinology and Metabolism 22(10):394–403.]

response. Indeed, TLR4 and TLR2 have both been shown to be responsive to high levels of free fatty acids. A second observation which implicates the TLR4 receptor in the sensing of a nutrient-rich environment is that serum lipopolysaccharide (LPS) in mice is increased after feeding. Perhaps

intestinal permeability is increased immediately after feeding, which allows the introduction of inflammatory molecules such as LPS directly into the circulation. In an animal fed a normal diet, this inflammation is short-lived. However, it is possible that in animals living in the nutrient-rich

Chronic Inflammation and Susceptibility to Other Diseases As part of the normal healing process, inflammatory cytokines also enhance blood vessel flow and blood vessel formation (angiogenesis), induce proliferation and activation of fibroblasts and immune cells, and regulate death of infected or damaged cells. Together, these events induce tissue remodeling that gives immune cells better access to pathogens and scarring that heals wounds. However, continual stimulation of this healing process has deleterious consequences. Overstimulation of fibroblasts leads to excessive tissue scarring (fibrosis), which can physically and severely impair organ function. Continual stimulation of cell proliferation enhances the probability of mutations

environment characteristic of obesity, intestinal permeability remains relatively high long after a meal is completed and thus the animal is chronically exposed to low levels of inflammatory signals. If Toll receptors are signaling the release of inflammatory cytokines, what is the nature of the cell that carries these Toll receptors? A significant number of studies have demonstrated the presence of macrophages and mast cells in adipose (fat) tissue, suggesting that these two cell types are at least in part responsible for the detection of excess nutrients and the development of inflammatory signals in adipose tissues. The roles of macrophages and mast cells in the development of obesity-associated inflammation are supported by the finding that depletion of cells bearing the CD11c marker (macrophages, dendritic cells, and neutrophils) increases insulin sensitivity. In addition, it is possible that macrophages, newly recruited into expanding adipose tissue, may differentiate into a more potent proinflammatory phenotype than macrophages normally resident in lean adipose tissue. Improved insulin sensitivity in adipose tissue was also found upon depletion of mast cells, NKT cells, and CD8⫹ T cells, whereas increased levels of CD4⫹ and TREG cells are associated with higher levels of insulin sensitivity. TREG cells associated with adipose tissue secrete large amounts of

and may contribute to tumor formation or growth. Enhancement of blood vessel formation can also enhance survival of cells in solid tumors. Although focusing on similarities between acute and chronic inflammation is helpful in understanding some of the consequences described, it is also important not to oversimplify the relationship. For instance, although neutrophil infiltration is a cardinal feature of acute inflammation, monocytes, macrophages, and lymphocytes accumulate during chronic inflammation. Fibroblasts associated with chronic inflammation secrete distinct cytokines and may represent distinct lineages. These differences and others underscore the importance of thoughtfully considering and customizing approaches to ameliorate inflammatory conditions.

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BOX 15-3 IL-10, which acts, in part, to reduce the inflammatory response. Finally, recent work shows that some adipocytes (fat cells) themselves are capable of generating inflammatory cytokines, including TNF-␣ and IL-6. Adipocytes also express Toll-like receptors that trigger cytokine release. Excess internal lipid also appears to stimulate internal stress responses by adipocytes that enhance cytokine production. So it appears that macrophages, mast cells, NKT cells, CD8 T cells, and adipocytes all play a part in the release of proinflammatory mediators in adipose tissues. Another stimulus for the secretion of proinflammatory cytokines from adipose tissue-associated cells may be hypoxia, which develops in rapidly expanding adipose tissue. If an animal fed a high-fat diet puts on weight fairly rapidly, the newly developing vasculature (blood supply) is not always able to keep pace and the tissue may be temporarily starved of oxygen. This is known as a state of ischemia, and ischemic tissues recruit macrophages. Once present in ischemic tissues, macrophages are induced to express proinflammatory proteins, as well as angiogenic proteins, which aid in signaling the development of an increased blood supply. Adipocytes may also contribute to this response directly. Once an animal starts down the road to obesity, its problems can become self-

perpetuating. Adipocytes that are full of fat will tend to leak free fatty acids into the circulation, and these in turn induce further inflammation. Indeed, free fatty acids and cellular stress have also been shown to be additional triggers for IRS protein kinases. In addition, high levels of proinflammatory cytokines block the formation of new adipocytes and reduce the secretion of adiponectin, an important regulator of adipocyte production. As the obese adipocyte expands, it approaches its mechanical limit. Cellular responses to mechanical stress, or death, lead to the release of cytokines and additional fatty acids into the circulation. Furthermore, the swollen adipocyte also undergoes endoplasmic reticulum (ER) stress, in which an unusually high number of unfolded proteins accumulates in the ER. This, in turn, provokes the release of inflammatory cytokines and chemokines. However, the problems of the Type 2 diabetic are not confined just to the adipocytes. In the liver, normally the site of glucose homeostasis, increased levels of inflammatory cytokines also help to induce insulin resistance. Gluconeogenesis (the formation of new glucose) is normally inhibited by insulin, but under inflammatory conditions gluconeogenesis is no longer suppressed and the high blood glucose levels characteristic of the Type 2 diabetic are further increased. In the pancreas, the site of insulin produc-

tion, the high blood glucose levels initially induce hyperproliferation of the pancreatic ␤ cells, but eventually apoptosis of the insulin-producing cells occurs, further exacerbating the state of high blood glucose. In the brain of animals fed a high-fat diet, inflammatory pathways are also activated in the hypothalamus, leading to resistance to the effects of both insulin and leptin, a hormone that normally signals satiety, thus setting up a positive feedback loop: the fatter the animal, the more it needs to eat to achieve satiety. In summary, current research suggests that obese animals exhibit a state of chronic inflammation resulting from the release of nutrient-stimulated inflammatory mediators by adipocytes, themselves, as well as macrophages and mast cells. These inflammatory mediators in turn act on adipocytes and other cells to reduce their sensitivity to insulin, leading to the syndrome we now know as Type 2 diabetes. Inflammation stimulated by nutrient excess is chronic and is associated with a reduced metabolic rate. Some have suggested that it be distinguished from the acute inflammatory state set up by infectious stimuli by using the term metaflammation. Clinical research has already demonstrated some success in using anti-inflammatory medications such as salicylate homologs and IL-1 receptor antagonists in the treatment of insulin resistance.

S U M M A R Y S U M M A R Y ■

Hypersensitivities are immune disorders caused by an inappropriate response to antigens that are not pathogens.





Hypersensitivities are classically divided into four categories (types I–IV) that differ by the immune molecules and cells that cause them and the way they induce damage.





The hygiene hypothesis has been advanced to explain increases in asthma and allergy incidence in the developed world and proposes that early exposure to pathogens inhibits allergy by balancing the representation and polarization of regulatory and helper T cell subsets.





Allergy is a type I hypersensitivity reaction that is mediated by IgE antibodies. IgE antibodies bind to antigen via their variable regions and to one of two types of Fc receptors via their constant regions. Mast cells, basophils, and to a lesser extent eosinophils express Fc␧RI, and are the main mediators of allergy symptoms. Cross-linking of Fc␧RI receptors by allergen/IgE complexes initiates multiple signaling cascades that resemble those initiated by antigen receptors.

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Mast cells, basophils, and eosinophils that are stimulated by Fc␧ cross-linking release their granular contents (including histamine, proteases) in a process called degranulation. They also generate and secrete inflammatory cytokines and lipid inflammatory molecules (leukotrienes and prostaglandins). Degranulation releases both nonprotein (histamine) and protein (protease) molecules that induce rapid inflammatory responses (e.g., vasodilation and edema, smooth muscle constriction). Mast cell activity can also be down-regulated by inhibitory signals, including phosphatase activity, inhibitory FcR signaling, and ubiquitinylation and degradation of signaling molecules. Allergy symptoms vary depending on where the IgE response occurs and whether it is local or systemic. Asthma, atopic dermatitis, and food allergies are examples of local allergic responses. Anaphylaxis refers to a systemic IgE response and can be caused by systemic (e.g., intravenous) introduction of the same allergen that induces local responses. Individuals predisposed to allergic responses are referred to as atopic. Both genetic and environmental factors contribute to allergy susceptibility. Why some antigens induce allergy and others do not is still not fully understood. Some antigens that cause allergy appear to have intrinsic protease activity. Skin tests are an effective way to diagnose allergies. Allergies can be treated by hyposensitization, which increases IgG responses to allergens, in turn inhibiting IgE activity. They are also treated with pharmacological inhibitors of inflammation, including antihistamines, leukotriene inhibitors, and corticosteroids. Type II hypersensitivities are caused by IgG and IgM antibodies binding to an antigen on red blood cells and inducing cell destruction by recruiting complement or by ADCC. Transfusion reactions are caused by antibodies that bind to A, B, or H carbohydrate antigens, which are expressed on the surface of red blood cells. Individuals with different blood types (A, B, or O) express different carbohydrate antigens. They are tolerant to their own antigens, but generate antibodies against the antigen (A or B) that they do not express. All individuals express antigen H, so no antibodies are generated to this carbohydrate. Hemolytic disease of the newborn is caused by maternal antibody reaction to the Rh antigen, which can happen if mother is Rh⫺ and father is Rh⫹. Drug-induced hemolytic anemia is caused by antibody responses to red blood cells that have bound drug molecules or metabolites.



Immune complexes of antibody and antigen can cause type III hypersensitivities when they cannot be cleared by phagocytes. This may be due to peculiarities of the antigen itself, or disorders in phagocytic machinery.



Uncleared immune complexes can induce degranulation of mast cells and inflammation, and can be deposited in tissues and capillary beds where they induce more innate immune activity, blood vessel inflammation (vasculitis), and tissue damage.



Arthus reactions are examples of immune complex (type III) hypersensitivity reactions and can be induced by insect bites, as well as inhalation of fungal or animal protein. They are characterized by local and sometimes severe inflammation of blood vessels.



Delayed-type hypersensitivity (type IV hypersensitivity) is cell mediated, not antibody mediated. Examples include contact dermatitis caused by poison ivy, as well as the tuberculin reaction. DTH responses are responsible for granulomas associated with tuberculosis.



DTH requires T cells to be sensitized to antigen. Subsequent reexposure to antigen results in cytokine generation, inflammation, and the recruitment of macrophages, which produce DTH symptoms 2 to 4 days after reexposure.



TH1 cells are classically associated with DTH, but other helper cell subsets have also been implicated recently.



Chronic inflammation is a pathological condition characterized by persistent, increased expression of inflammatory cytokines.



Chronic inflammation has infectious and noninfectious origins. It can be caused by an infection that is not fully resolved as well as by chronic tissue damage brought about by wounds, tumors, autoimmune disease, or organ disease. Obesity is now recognized as one of the most common causes of chronic inflammation.



Obesity can result in chronic inflammation in part because visceral fat cells (adipocytes) can be stimulated to produce inflammatory cytokines directly.



Chronic inflammation, regardless of cause, increases the susceptibility of individuals to several systemic diseases, including Type 2 diabetes.



Inflammatory cytokines associated with chronic inflammation contribute to insulin resistance (Type 2 diabetes) by interfering with the activity of enzymes (e.g., JNK) downstream of the insulin receptor.



Cytokines produced during chronic inflammation can also induce tissue scarring (leading to organ dysfunction) as well as cell proliferation and angiogenesis (which may contribute to tumor development).

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R E F E R E N C E S Abramson, J., and I. Pecht. (2007). Regulation of the mast cell response to the type 1 Fc epsilon receptor. Immunological Reviews 217: 231–254. Acharya, M., et al. (2010). CD23/FcepsilonRII: molecular multitasking. Clinical and Experimental Immunology 162:12–23. Adam, J., W. J. Pichler, and D. Yerly. (2011). Delayed drug hypersensitivity: Models of T-cell stimulation. British Journal of Clinical Pharmacology 71:701–707. Boura-Halfon, S., and Y. Zick. (2009). Phosphorylation of IRS proteins, insulin action, and insulin resistance. American Journal of Physiology—Endocrinology and Metabolism 296:E581–591. Cavani, A., and A. De Luca. Allergic contact dermatitis: Novel mechanisms and therapeutic perspectives. Current Drug Metabolism 11:228–233. Donath, M. Y., and S. E. Shoelson. (2011). Type 2 diabetes as an inflammatory disease. Nature Reviews Immunology 11:98–107. Galli, S. J., S. Nakae, and M. Tsai. (2005). Mast cells in the development of adaptive immune responses. Nature Immunology 6:135–142. Gerull, R., M. Nelle, and T. Schaible. (2011). Toxic epidermal necrolysis and Stevens-Johnson syndrome: A review. Critical Care Medicine 39:1521–1532. Gilfillan, A. M., and J. Rivera. (2009). The tyrosine kinase network regulating mast cell activation. Immunological Reviews 228:149–169. Gladman, A. C. (2006). Toxicodendron dermatitis: Poison ivy, oak, and sumac. Wilderness and Environmental Medicine 17:120–128. Gregor, M. F., and G. S. Hotamisligil. (2011). Inflammatory mechanisms in obesity. Annual Review of Immunology 29:415–445. Holgate, S. T., R. Djukanovic, T. Casale, T., and J. Bousquet. (2005). Anti-immunoglobulin E treatment with omalizumab in allergic diseases: An update on anti-inflammatory activity and clinical efficacy. Clinical & Experimental Allergy 35:408–416. Hotamisligil, G. S. (2010). Endoplasmic reticulum stress and the inflammatory basis of metabolic disease. Cell 140:900–917. Karasuyama, H., K. Mukai, K. Obata, Y. Tsujimura, and T. Wada. (2011). Nonredundant roles of basophils in immunity. Annual Review of Immunology 29:45–69. Madore, A. M., and C. Laprise. (2010). Immunological and genetic aspects of asthma and allergy. Journal of Asthma and Allergy 3:107–121. Minai-Fleminger, Y., and F. Levi-Schaffer. (2009). Mast cells and eosinophils: The two key effector cells in allergic inflammation. Inflammation Research 58:631–638. Mullane, K. (2011). Asthma translational medicine: Report card. Biochemical Pharmacology 82:567–585.

Nizet, V., and M. E. Rothenberg. (2008). Mitochondrial missile defense. Nature Medicine 14:910–912. Roediger, B., and W. Weninger. (2011). How nickel turns on innate immune cells. Immunology and Cell Biology 89:1–2. Rosenwasser, L. J. (2011). Mechanisms of IgE Inflammation. Current Allergy and Asthma Reports 11:178–183. Rothenberg, M. E., and S. P. Hogan. (2006). The eosinophil. Annual Review of Immunology 24:147–174. Sicherer, S. H., and H. A. Sampson. (2009). Food allergy: Recent advances in pathophysiology and treatment. Annual Review of Medicine 60:261–277. Vercelli, D. (2008). Discovering susceptibility genes for asthma and allergy. Nature Reviews Immunology 8:169–182. Wan, Y. I., et al. (2011). A genome-wide association study to identify genetic determinants of atopy in subjects from the United Kingdom. Journal of Allergy and Clinical Immunology 127:223–231, 231 e1–3. Yamamoto, F. (2004). Review: ABO blood group system—ABH oligosaccharide antigens, anti-A and anti-B, A and B glycosyltransferases, and ABO genes. Immunohematology 20:3–22.

Useful Web Sites https//chriskresser.com/how-inflammation-makesyou-fat-and-diabetic-and-vice-versa This is an interesting and credible series of commentaries by Chris Kresser who did not go to medical school, but graduated from an alternative medicine program and is open about his interest in examining the assumptions that underlie medical practices. His online articles on obesity and inflammation are informed and clearly written.

www.youtube.com/watch?v=IGDXNHMwcVs An above-average and roughly accurate YouTube animation on the cells and molecules involved in type I hypersensitivity (allergy). www.youtube.com/watch?v ⫽ y3bOgdvV_M&feature⫽related is another YouTube animation about type I hypersensitivity that is clear, if overly simplified. faculty.ccbcmd.edu/courses/bio141/lecguide/unit5/ hypersensitivity/type3/typeiii.html An animation from Dr. Gary Kaiser’s Microbiology website (Community College of Baltimore County) that helps one visualize type III (immune-complex mediated) hypersensitivity and how it induces vasculitis.

www.youtube.com/watch?v=wfmsFfC8WIM A YouTube mini-lecture on delayed-type hypersensitivity (focusing on the immunology behind poison ivy). Includes some good pictures of poison ivy lesions.

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Q U E S T I O N S

1. You have in your possession a number of mouse strains,

each of which lacks a specific gene (gene knockout animals). How might the type I hypersensitivity response of each knockout strain (a–e) differ from a wild-type mouse? How might the type II hypersensitivity response differ? Explain your answer. a. Mouse is unable to generate a ␧ heavy chain. b. Mouse is unable to generate a high-affinity Fc␧RI

receptor. c. Mouse is unable to generate a low-affinity Fc␧RII receptor. d. Mouse is deficient in the ability to generate the complement attack complex. e. Mouse is unable to express CD21. 2. What is the difference between primary and secondary

pharmacological mediators in the type I hypersensitivity response? Name two of each. 3. How does histamine suppress its own release? 4. Immunotherapy of type I hypersensitivity responses is

aimed at raising the levels of IgG antibodies specific for allergens. Describe one mechanism by which allergenspecific IgG can dampen down the IgE response to an allergen. ⫹

5. A mother has an Rh blood type and the father has an Rh



blood type. Under these circumstances, the family pediatrician is not worried about the possibility of a type II hypersensitivity reaction. However, if the converse is true, and the mother is Rh⫺ and the father Rh⫹, the pediatrician does worry and asks the obstetrician to inject the mother with antibodies toward the end of her first pregnancy. Explain his reasoning in both cases. 6. A mother has an Rh





and the father an Rh blood type. The first baby born to the parents was Rh⫹. However, the parents elect for the mother not to receive Rhogam. Are all

future babies of this couple at risk for type II hypersensitivity reactions? Why?/Why not? 7. Define type III hypersensitivity, illustrating the initiating

cells and molecules, the cells and molecules that bring about the pathological effects and indicating two triggers for this type of response. 8. Indicate which type(s) of hypersensitivity reaction (I–IV)

apply to the following characteristics. Each characteristic can apply to one, or more than one, type. Is an important defense against intracellular pathogens. Can be induced by penicillin. Involves histamine as an important mediator. Can be induced by poison oak in sensitive individuals. Can lead to symptoms of asthma. Occurs as a result of mismatched blood transfusion. Systemic form of reaction is treated with epinephrine. Can be induced by pollens and certain foods in sensitive individuals. i. May involve cell destruction by antibody-dependent cell-mediated cytotoxicity. j. One form of clinical manifestation is prevented by Rhogam. k. Localized form characterized by wheal-and-fl are reaction. a. b. c. d. e. f. g. h.

9. As described in the text, a small group of obese individuals

(6% or so) do not suffer from the chronic inflammatory state characteristic of most forms of obesity. a. These individuals do not develop Type 2 diabetes. Why

not? Provide a specific, molecular answer. b. Some have suggested that these individuals express

genetic polymorphisms that make them less susceptible to obesity-generated inflammation. Describe one possible genetic polymorphism that could uncouple obesity from inflammation.

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arly in the twentieth century, Paul Ehrlich realized that the immune system could go awry. Instead of reacting only against foreign antigens, it could focus its attack on the host. This condition, which he termed horror autotoxicus, can result in a clinical syndrome generically referred to as autoimmunity. This inappropriate response of the immune system, directing humoral and/or T-cell-mediated immune activity against self components, is the cause of autoimmune diseases such as rheumatoid arthritis (RA), multiple sclerosis (MS), systemic lupus erythematosus (SLE, or lupus) and certain types of diabetes. Autoimmune reactions can cause serious damage to cells and organs, sometimes with fatal consequences. In some cases the damage to self cells or organs is caused by antibodies; in other cases, T cells are the culprit. Simply stated, autoimmunity results from some failure of the host’s immune system to distinguish self from nonself, causing destruction of self cells and organs. Although on the rise, autoimmunity is still rare, suggesting that mechanisms to protect an individual from this sort of anti-self immune attack must exist, and they do. This process and the mechanisms that control it are collectively termed tolerance, or self-tolerance. Establishing self-tolerance is complicated, involving both the elimination of immune cells that can react against self-antigens and active inhibition of immune responses against self proteins. Our understanding of the mechanisms that control self-tolerance have really blossomed in the last decade, giving rise to new ways of understanding and treating autoimmune disease. When self-tolerance processes are working correctly, host tissues should remain undisturbed by the immune system and only foreign invaders should be attacked. As we know from Chapter 9 and will address further below, the mechanisms that maintain self-tolerance do so by establishing what is “us,” making the “them” clearer. These elaborate mechanisms that maintain self-tolerance also cause rejection of any transplanted tissues or cells that carry new proteins, as occurs whenever the donor is not genetically identical to the recipient. Transplantation refers to the act of transferring cells, tissues, or organs

Characteristic butterfly rash in an SLE patient. [From L. Steinman, 1993, Scientific American 269(3):80.] ■

Establishment and Maintenance of Tolerance



Autoimmunity



Transplantation Immunology

from one site to another—typically from one individual to another. Transfers between two sites on the same individual (e.g., skin) or between identical twins, although not free of complication, are more likely to survive. Many diseases can be cured by implantation of a healthy organ, tissue, or cells (a graft). The development of surgical techniques that allow this has removed one barrier to successful transplantation, but others remain. A worldwide shortage of organs for transplantation leaves tens of thousands of individuals waiting for a transplant, sometimes for many years. And yet, the most formidable barrier to making transplantation a routine medical treatment is the immune system. In this chapter, we first describe our current understanding of the general mechanisms that establish and maintain tolerance to self antigens. When these mechanisms fail, autoimmunity, the second major topic of this chapter, can result. Several common human autoimmune diseases resulting from failures of these mechanisms are described. These can be divided into two broad categories: organ specific and multiorgan (systemic) autoimmune diseases. Such diseases affect 5% to 8% of 517

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the human population; they are often chronic and debilitating, necessitating prolonged medical intervention. Several experimental animal models used to study autoimmunity, as well as therapies for treating autoimmune disease, are also discussed. In the final part of this chapter, we turn to the topic of transplantation, or situations in which self-tolerance can work against us. We begin by discussing the immunologic processes governing graft rejection, followed by current therapeutic modalities for suppressing these responses. We end with some of the most commonly transplanted tissues and the potential future role of xenotransplantation (cross-species grafts) in clinical medicine.

Establishment and Maintenance of Tolerance The term tolerance applies to the many layers of protection imposed by the immune system to prevent the reaction of its cells and antibodies against host components. In other words, individuals should not typically respond aggressively against their own antigens, although they will respond to pathogens or even cells from another individual of the same species. Until fairly recently, this was thought to be mediated by the elimination of cells that can react against self antigens, yielding a state of unresponsiveness to self. Contemporary studies of tolerance provide evidence for a much more active role of immune cells in the selective inhibition of responses to self antigens. Rather than ignore self proteins, the immune system protects them. For instance, the discovery and characterization of regulatory T cells, which in fact recognize self proteins, have revolutionized the field of tolerance and autoimmunity, not to mention transplanation. In the first step of this process, a phenomenon termed central tolerance deletes T- or B-cell clones before the cells are allowed to mature if they possess receptors that recognize self antigens with high affinity (see Chapter 9). Central tolerance occurs in the primary lymphoid organs: the bone marrow for B cells and the thymus for T cells (Figure 16-1a). Because central tolerance is not perfect and some self-reactive lymphocytes find their way into the periphery and secondary lymphoid tissues, additional safeguards limit their activity. These backup precautions include peripheral tolerance, which renders some self-reactive lymphocytes in secondary lymphoid tissues inactive and generates others that actively inhibit immune responses against self (Figure 16-1b). The possibility of damage from self-reactive lymphocytes is further limited by the life span of activated lymphocytes, which is regulated by programs that induce cell death (apoptosis) following receipt of specific signals. The mechanisms mediating peripheral tolerance vary. Under normal circumstances, encounter of mature lymphocytes with an antigen leads to stimulation of the immune response. However, presenting the antigen in some alterna-

tive form, time, or location may instead lead to tolerance. Antigens that induce tolerance are called tolerogens rather than immunogens. Here, context is important; the same chemical compound can be both an immunogen and a tolerogen, depending on how and where it is presented to the immune system. For instance, an antigen presented to T cells without appropriate costimulation results in a form of tolerance known as anergy (unresponsiveness to antigenic stimulus), whereas the same antigen presented with costimulatory molecules can become a potent immunogen. When some antigens are introduced orally, tolerance can be the result, whereas the same antigen given as an intradermal or subcutaneous injection can be immunogenic. In other instances, mucosally administered antigens provide protective immunity, such as in the case of Sabin’s oral polio vaccine. However, there is one general truth: tolerogens are antigen specific. The inactivation of an immune response does not result in general immune suppression, but rather is specific for the tolerogenic antigen. In adults, most encounters with foreign antigen lead to an immune response aimed at eradication. This is not true in the fetus, where, due to the immature state of the immune system, exposure to antigens frequently results in tolerance. Other than fetal exposure, factors that promote tolerance rather than stimulation of the immune system by a given antigen include the following: • High doses of antigen • Long-term persistence of antigen in the host • Intravenous or oral introduction • Absence of adjuvants (compounds that enhance the immune response to antigen) • Low levels of costimulation • Presentation of antigen by immature or unactivated antigen-presenting cells (APCs) In the 1960s, researchers believed that all self-reactive lymphocytes were eliminated during their development in the bone marrow and thymus. The conventional wisdom suggested that failure to eliminate these lymphocytes led to autoimmune consequences. More recent experimental evidence has countered that belief. Healthy individuals have been shown to possess mature, recirculating, self-reactive lymphocytes. Since the presence of these self-reactive lymphocytes in the periphery does not predict or inevitably result in autoimmune reactions, their activity must be regulated in healthy individuals through other mechanisms. Mechanisms that maintain tolerance include the induction of cell death or cell anergy in lymphocytes and limitations on the activity of selfreactive cells by regulatory processes (see Figure 16-1b). Cell death plays an important role in establishing and maintaining both central and peripheral tolerance. This is evidenced by the development of systemic autoimmune diseases in mice with naturally occurring mutations of either the death receptor, Fas, or Fas ligand (FasL). As discussed in Chapter 9,

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Lymphoid precursor

Newly emerged (immature) clones of lymphocytes

Exposure to self antigens during development

Bone marrow (B cells) or thymus (T cells) as depicted here

Maturation of clones not specific for self antigens present in primary lymphoid organs

Deletion of lymphocytes specific for self antigens present in generative organs

Maturation of clones that suppress self antigen recognition (regulatory cells)

(b) Peripheral tolerance Mature lymphocytes

Bind self antigen Bind foreign antigen Regulation

Apoptosis Anergy

Activation of effector cells against foreign antigen

Peripheral tolerance: deletion, anergy or regulation of lymphocytes that can recognize self antigens in peripheral tissues

FIGURE 16-1 Central and peripheral tolerance. (a) Central tolerance is established by deletion of lymphocytes in primary lymphoid organs (thymus for T cells and bone marrow for B cells) if they possess receptors that can react with self antigens or by the emergence of regulatory T cells that can inhibit self-reactive cells. (b) Peripheral tolerance involves deleting, rendering anergic or actively suppressing escaped lymphocytes that possess receptors that react with self antigens. This process occurs in secondary lymphoid organs.

activated T cells express increased levels of Fas and FasL. In both B and T cells, engagement of Fas by FasL induces a rapid apoptotic death known as activation-induced cell death (AICD). Mice that carry inactivating mutations in Fas (lpr/lpr) or FasL (gld/gld) are not able to engage the AICD pathway and develop autoimmune disease early in life (see below).

Antigen Sequestration Is One Means to Protect Self Antigens from Attack In addition to the various mechanisms of central and peripheral tolerance, an effective means to avoid selfreactivity is sequestration or compartmentation of antigens.

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For example, the anterior chamber and lens of the eye are considered sequestered sites, without lymphatic drainage and possessing tissue-specific privileged antigens that are normally isolated from interaction with immune cells. This sequestration allows these antigens to avoid encounter with reactive lymphocytes under normal circumstances; if the antigen is not exposed to immune cells, there is little possibility of reactivity. However, one possible consequence of sequestration is that the antigen is never encountered by developing lymphocytes, and thus active tolerance to the sequestered antigen is not established. If barriers between immune cells and the sequestered antigens are breached (by trauma, for example), the newly exposed antigen may be seen as foreign because it was not previously encountered. Trauma to one eye that allows entry of immune cells can result in inflammation in that eye, leading to tissue destruction and impaired vision. In these cases, the other eye may also become inflamed due to the sudden entry of clones of these recently activated immune cells recognizing newly discovered tissue-specific antigens. This is not to suggest that a lack of exposure to the immune system is the only factor that mediates immune privilege. A locally immunosuppressive microenvironment in tissues conventionally considered immune privileged, such as the eye and central nervous system (CNS), in addition to other active mechanisms, is believed to bias the immune response toward tolerance in these locations. We discuss further some of these pathways in the following sections, as well as in Chapters 9 and 10.

Central Tolerance Limits Development of Autoreactive T and B Cells One mechanism strongly influencing central tolerance is the deletion during early stages of maturation of lymphocyte clones that have the potential to react with self components later (see Figure 16-1a). Consider the mechanisms that generate diversity in T-cell or B-cell receptors. As discussed in Chapter 7, the genetic rearrangements that give rise to a functional T-cell receptor (TCR) or immunoglobulin (Ig) occur through a process whereby any V-region gene segment can associate with any D or J gene segment. This means that the generation of variable regions that react with self antigens is almost inevitable. If this were allowed to occur frequently, such TCR or Ig receptors could produce mature functional T or B cells that recognize self antigens, and autoimmune disease would ensue. Although our understanding of the precise molecular mechanisms mediating central tolerance in T and B cells is not complete, we do know that these cells undergo a developmentally regulated event called negative selection. This results in the induction of death in some, but not all, cells that carry potentially autoreactive TCR or Ig receptors. A classic experiment by C. C. Goodnow and colleagues, described in Chapter 10, mated transgenic mice expressing hen egg white lysozyme (HEL) with mice expressing a trans-

genic immunoglobulin specific for HEL to demonstrate that self-reactive lymphocytes are removed or inactivated after encounter with self antigen. David Nemazee and colleagues showed that some developing B cells can undergo receptor editing. In this process, the antigen-specific V region is “edited” or switched for a different V-region gene segment via V(D)J recombination, sometimes producing a less autoreactive receptor with an affinity for self antigens below a critical threshold that would lead to disease, allowing the cell to survive. As mentioned in Chapters 9 and 10, the central tolerance processes of negative selection and receptor editing work to eliminate many autoreactive T cells in the thymus and autoreactive B cells in the bone marrow. Receptor editing, as well as clonal deletion or apoptosis, are now recognized as mechanisms that lead to central tolerance in developing B cells (see Chapter 10). By similar mechanisms, T cells developing in the thymus that have a high affinity for self antigen are deleted, primarily through the induction of apoptosis (see Chapter 9). More recently, it has been discovered that some of these self-reactive T cells in the thymus may be spared, and that these cells may function in the periphery as antigen-specific regulatory cells working to dampen immune responses to the antigens that they recognize (see below and Chapter 9).

Peripheral Tolerance Regulates Autoreactive Cells in the Circulation Numerous studies have shown that central tolerance is not a foolproof process; all possible self-reactive lymphocytes are not deleted. In fact, it is now clear that lymphocytes with specificity for self antigens are not uncommon in the periphery. Two factors contribute to this: (1) not all self antigens are expressed in the central lymphoid organs where negative selection occurs, and (2) there is a threshold requirement for affinity to self antigens before clonal deletion is triggered, allowing some weakly self-reactive clones to survive the weeding-out process. Just like central tolerance, the mechanisms that control peripheral tolerance have been demonstrated by a variety of experimental strategies. As we saw in Chapter 11, in order for T cells to become activated, the TCR must bind antigen presented by self-MHC (major histocompatibility complex molecules) (signal 1), while at the same time the T cell must undergo costimulatory engagement (signal 2). Early experiments by Marc Jenkins and colleagues showed that when CD4 T-cell clones are stimulated in vitro through the TCR alone, without costimulation, they become anergic. Subsequent data showed that the interaction between CD28 on the T cell and CD80/86 (B7) on the APC provided the costimulatory signal required for T-cell activation. This led to a careful examination of costimulation, revealing the existence of other molecules that could bind to CD80/86 and the discovery of a related molecule, called CTLA-4. This molecule inhibits rather than stimulates T-cell activation upon binding CD80/86. We now appreciate that many such molecules

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Tolerance, Autoimmunity, and Transplantation deliver supplementary signals during T-cell activation, and the group of molecules that regulate T-cell behavior are now often referred to as immunomodulatory, to cover both costimulatory and inhibitory behavior. CTLA-4 expression is induced only after T cells are activated, providing a mechanism to dampen T-cell activity and regulate the immune response. Mice lacking CTLA-4 display massive proliferation of lymphocytes and widespread autoimmune disease, suggesting an essential role for this molecule in maintaining peripheral tolerance. Peripheral tolerance in B cells appears to follow a similar set of rules. For instance, experiments with transgenic mice have demonstrated that when mature B cells encounter most soluble antigens in the absence of T-cell help, they become anergic and never migrate to germinal centers. In this way, maintenance of T-cell tolerance to self antigens enforces B-cell tolerance to the same antigens. In T cells, a third mechanism for maintaining tolerance, in addition to T-cell anergy and apoptosis, is through the activity of regulatory T cells (TREG cells). Acting in secondary lymphoid tissues and at sites of inflammation, TREG cells recognize specific self antigens, and sometimes foreign antigens, via TCR interactions. However, they down-regulate immune processes when they engage with these antigens in the periphery. These cells can be generated both naturally, in the thymus (nTREG cells, Figure 16-2), and after induction by antigen in the periphery (iTREG cells; see below). In fact, many of the circulating T cells with specificity for self antigens may be such regulatory cells. Some scientists postulate a division of labor, with nTREG cells specializing in regulating responses against self antigen to inhibit autoimmune disease and iTREG cells controlling reactions against benign foreign antigens at mucosal surfaces, where the immune system comes in constant contact with the outside world (e.g., gut commensals or respiratory allergens). In a very recent study, specifically blocking iTREG but not nTREG cell development at the maternal-fetal interface correlated with a drop in the number of regulatory cells specific for paternal antigens and an increase in fetal death, suggesting that these cells may also be involved in regulating the immune response to fetal alloantigens and may influence pregnancy outcomes. The existence of immune inhibitory T cells was first proposed in the early 1970s by scientists who identified this activity within the CD8 subset and called these cells CD8 suppressor T cells. However, a lack of available reagents and expertise for isolating, propagating, and characterizing these cells meant that many decades passed without sufficient supporting evidence of their existence. During this time, the idea of naturally occurring immunosuppressive cells fell out of favor, only to be resurrected 25 years later by S. Sakaguchi and colleagues with the characterization of a subset of CD4 T cells with immunosuppressive abilities. Regulatory cells are currently the focus of much research, where subsets of CD8 and CD4 T cells, as well as certain types of APCs, have been found to possess these immunedampening capabilities.

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Thymus Thymocyte

Low affinity for self antigen High affinity for self antigen

Intermediate affinity for self antigen

Apoptosis Up-regulation of FoxP3

T cell

nTREG cells suppress reaction to self antigens

nTREG cell

FIGURE 16-2 T regulatory (TREG) cells generated from thymocytes during negative selection in the thymus can inhibit self-reactive T cells in the periphery. During T cell development in the thymus, thymocytes with high affinity for self antigens undergo apoptosis, while those with low affinity are positively selected and released. Thymocytes with intermediate affinity for antigens encountered in the thymus up-regulate the transcription factor FoxP3 and become natural nTREG cells, which are released into the periphery and serve to keep self-reactive T-cell responses in check. [M. Kronenberg and A. Rudensky. 2005. Regulation of immunity by selfreactive T cells. Nature. 435:598–604.]

Regulatory CD4 T Cells As discussed in Chapter 9, TREG cells were most recently characterized as a unique subset of CD4 T cells that express high levels of the IL-2R  chain (CD25), the low-affinity receptor for this cytokine. Naturally occurring nTREG cells arise from a subset of T cells expressing receptors with intermediate affinity for self antigens in the thymus (see Figure 16-2). Certain of these cells up-regulate the transcription factor FoxP3 and then develop into cells that migrate out of the thymus and are capable of suppressing reactions to self antigens. Evidence that CD4 TREG cells can control the immune response to self antigens has now been demonstrated in many experimental settings. In experiments in nonobese diabetic (NOD) mice and BioBreeding (BB) rats, two strains prone to the development of autoimmune-based diabetes, the onset of diabetes was delayed when these animals were injected with normal CD4 T cells from histocompatible donors. Further characterization of the CD4 T cells of nondisease-prone mice revealed that a subset expressing

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high levels of CD25 was responsible for the suppression of diabetes. This population was further characterized using transgenic reporter mice expressing a green fluorescent protein (GFP) fused with the transcription factor FoxP3 (FoxP3GFP mice). The GFP T cells but not the GFP T cells from these mice could be used to transfer the immune-suppressive activity, identifying this transcription factor as a major regulator controlling the development of these cells. CD4 TREG cells have also been found to suppress responses to some nonself antigens. For example, these cells may control allergic responses against innocuous environmental substances and/or responses to the commensal microbes that make up the normal gut flora. In mice experimentally manipulated to lack CD4 TREG cells (approximately 5–10% of their peripheral CD4 T-cell population), inflammatory bowel disease is common. In strains of mice that are genetically resistant to the induction of the autoimmune disease experimental autoimmune encephalomyelitis (EAE; a murine model of MS), depletion of this CD4 T cell subset renders the mice susceptible to disease, suggesting that these regulatory cells play a role in suppressing autoimmunity. The pathway by which some CD4 T cells develop regulatory functions in the thymus is still unclear, despite intense investigation. During development, engagement of the TCR with self antigens can result in either death of the developing thymocyte by negative selection or generation of this regulatory capacity (nTREGs). The difference between these fates may be due to other intercellular interactions (i.e., the binding of CD28 with CD80/86 or CD40 with CD40L) or the presence of certain cytokines. The importance of FoxP3, which appears to be both essential and sufficient for the induction of immunosuppressive function, can be seen in humans inheriting a mutated form of this X-linked gene, which causes a multiorgan autoimmune disease (see Autoimmunity below and Chapter 18). Naïve T cells that have escaped to the periphery also can be induced to express FoxP3 and acquire regulatory function (iTREG cells). Factors that favor the development of iTREG cells include the presence of certain cytokines during antigenic stimulation, chronic low-dose antigen exposure, and lack of costimulation or the presentation of antigen by immature dendritic cells (DCs). For example, there is significant evidence that iTREG cells and other regulators of immunity are present in the gut-associated lymphoid tissue (GALT). These cells are continuously exposed to gut microbes and food-borne antigens, which themselves may play a significant role in regulating immunity (see Clinical Focus Box 16-1). The microenvironment of the GALT is rich in lymphoid tissue-derived transforming growth factor beta (TGF-), which is believed to encourage the development of iTREG cells. There is also some evidence that T cells can switch types, meaning that in certain circumstances TREG cells (immunosuppressive) can acquire effector function (immune activating), and vice versa. The cues that determine this switch are not known, although the amount of IL-2 and other key cytokines in the microenvironment may be a contributing factor.

TREG cell

CTLA-4 MHC class II

CD80/86

Dendritic cell

Tryptophan IDO

Immunosuppressive microenvironment Kynurenin

Inhibition of IL-6

Inhibition of TNF-α

TNF-α

IL-6

FIGURE 16-3 CTLA-4 mediated inhibition of APCs by TREG cells. One of the proposed mechanisms used by TREG cells to inhibit APCs involves signaling through the CD80/CD86 (B7) receptor. In the APC, this engagement results in decreased expression of CD80/86, activation of indoleamine-2,3-dioxygenase (IDO; an enzyme that converts tryptophan to kynurenin, creating an immunoinhibitory microenvironment), and changes in transcription leading to decreased expression of IL-6 and TNF-. [Adapted from Wing K, Sakaguchi S. 2010. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nature Immunology 11:7–13]

In studying the mechanisms by which TREG cells inhibit immune responses, both contact-dependent and contactindependent processes have been observed. CD4 TREG cells have been shown to kill APCs or effector T cells directly, by means of granzyme and perforin. TREG cells may also modulate the function of other cells responding to antigen by surface receptor engagement. One prime example of this is the expression of CTLA-4. TREG cells express high levels of this immune inhibitory receptor. As shown in Figure 16-3, interaction of CTLA-4 on TREG cells with CD80/86 on an APC can lead to inhibition of APC function, including reduced expression of costimulatory molecules and proinflammatory cytokines, such as IL-6 and tumor necrosis factor- (TNF-). At the same time, these targeted APCs begin to express soluble factors that inhibit local immune cells, including indoleamine-2,3-dioxygenase (IDO). TREG cells themselves also secrete immune inhibitory cytokines, such as IL-10, TGF-, and IL-35, suppressing the activity of other nearby T cells and APCs. Finally, because TREG cells express only the low-affinity IL-2R (CD25) but not the  or  subunits, which are required for signal

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CLINICAL FOCUS

It Takes Guts to Be Tolerant In the past decade, a significant appreciation has developed for the gastrointestinal tract and the host’s microflora in regulating the immune response. Specifically, the commensal organisms that comprise the gut microbiota appear to work actively at inducing tolerance to themselves. Maybe even more important, the gut and the organisms that reside there seem to play key roles in maintaining a level of systemic homeostasis that allows for the development of tolerance to self and that sets the stage for effective identification and elimination of pathogens (see also Clinical Focus Box 1-3). Formerly thought to be “ignored” by immune cells, host microbiota are now known to participate in a two-way communication with the immune system. This cross-talk results in advantages for both the host and the microbe. For the microbe, this interaction drives immune tolerance to the bugs, allowing them to continue to thrive in their home. For the host, there appear to be multiple advantages to immune health, depending somewhat on the composition of the microbe(s) in question. For instance, germ-free mice have been found to harbor defects in both humoral and adaptive immunity, and in some strains there is an increased susceptibility to the development of autoimmunity. In one recent study looking at the early stages of human rheumatoid arthritis, significantly less of specific bacterial species were found in the intestinal flora of afflicted individuals. Collectively, these observations in humans and in animal models suggest that communication between commensal microbes and the host immune system may influence the induction or severity of some autoimmune diseases. The antibody responses of germ-free mice to exogenous antigens are depressed, as are responses to Conconavalin A (Con A),

a strong T-cell mitogen. Germ-free animals were found to exhibit a TH2 cytokine bias in response to antigenic challenge, which could be restored to balance by colonization with just a single microbe, specifically Bacteroides fragilis. Further, this restored immune balance was most strongly associated with microbial expression of one particular molecule: the surface-expressed polysaccharide A. This strongly suggests that single microorganisms, and in some cases maybe even single molecules expressed by these microbes, can have systemic effects on the balance of immunity in the host. This increased awareness of gutassociated immune regulation has spawned several interesting hypotheses related to tolerance. One, called the microflora or altered microbiota hypothesis, posits that changes in gut microflora due to dietary modifications and/or increased antibiotic use have disrupted normal microbially mediated pathways important for regulating immune tolerance. There is already a clear link between intestinal microbiota and health of the gut, including a role for certain microbes in regulating inflammatory syndromes of the bowel. Further evidence for this comes from transplantation studies, where individuals treated with immune ablation therapy and high-dose antibiotics show overcolonization with certain, sometimes pathogenic, microbes. These individuals frequently manifest correlating immune defects, which can be reversed by directly manipulating the gut microflora. But how do our commensal microbes influence the immune balance? Some posit that intestinal epithelial cells (IECs) and mucosal dendritic cells (DCs), which express several key innate receptors, including Toll-like receptors and NOD-like receptors (TLRs and NLRs), may be involved.

transduction, they can act as a sponge, absorbing this growthand survival-promoting cytokine and further discouraging expansion of local immunostimulatory effector T cells. This pathway of inhibition is believed to be quite antigen specific; TREG cells inhibit APCs presenting their cognate

Mucosal DCs in the gut are known to sample the contents of the intestinal lumen. These cells could be another connection between commensal microbes and the maintenance of tolerance. In a study by R. Medzhitov and colleagues, mice engineered to lack the MyD88 gene, important for signaling through these innate receptors, were more susceptible to intestinal injury and autoimmunity. This suggests that signaling through these receptors can in some instances induce tolerance rather than an inflammatory response. This could be explained by the delivery of tolerogenic and homeostatic signals by as yet undefined antigens carried by the gut microbiota. Experiments to identify the specific ligands and receptors important for this cross-talk between microflora and the host immune system are ongoing. In case you were thinking of taking this role of the gut in immune balance with a grain of salt, you might want to think again. Sodium chloride may be a new addition to the list of dietary contributors to immune pathway development. In vitro studies have shown that the addition of sodium chloride to cultures of CD4 T cells can drive development of TH17 cells. Expansion of this cell type has been linked to the development of certain autoimmune diseases. It now appears that “We are what we eat” extends to the immune system, which may have a discriminating palate of its own.

Rakoff-Nahoum S, Paglino J, Eslami-Varzaneth F, Edberg S and Medzhitov R. 2004. Recognition of commensal microflora by toll-like receptors is required for intestinal homeostasis. Cell 118:229–41. Round J, O’Connell R and Masmanian S. 2010. Coordination of tolerogenic immune responses by the commensal microbiota. Journal of Autoimmunity. 34:220–225.

antigen or effector T cells that share their same antigen specificity and not T cells with a different specificity. However, again taking advantage of FoxP3-GFP mice bred with TCR transgenic animals, it was shown that it is possible for FoxP3 T cells to inhibit T cells recognizing other antigens,

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T regulatory cell (specificity for A) T effector cell (specificity for A)

T effector cell (specificity for B) TREG

CD4+

CD4+

TCR Peptide MHC class II Peptide A

Peptide B

Antigen presenting cell

Linked suppression

FIGURE 16-4 Linked suppression mediated by TREG cells. In cases where a single antigen-presenting cell (APC) engages simultaneously with T cells of different specificity, inhibitory signals meant for one can be transmitted to both and lead to a “spreading” of immune suppression to include other antigens. [R. I. . Lechler, O. A. Garden, and L. A. Turka, 2003, The complementary roles of deletion and regulation in transplantation tolerance, Nature Reviews Immunology 3:147–158]

as occurs when both the TREG cell and the “bystander” T cell recognizing another antigen interact with the same APC. The result is inhibition of the APC, via both contact-dependent and -independent pathways, as well as inhibition of the bystander T cell through soluble inhibitory factors and decommissioning of the APC (Figure 16-4). This simultaneous processing and presentation of different antigens might happen naturally in vivo when the antigens in question are parts of the same pathogen, although based on the findings in these experimental systems this was not required. This phenomenon, termed linked suppression, has now been seen in multiple experimental systems and may represent another way that TREG cells support local self-tolerance in tissues lacking any pathogen-induced danger signals. Regulatory CD8 T Cells Although an immunosuppressive role for CD8 T cells was suggested as early as 1970, it took over 35 years to confirm and partially characterize this cell population. Although much work is yet to be done, the fact that CD8 TREG cells can play a role in inhibiting responses to self antigens is now fairly well established. For instance, adoptive transfer of a subset of CD8 T cells can induce tolerance to heart transplants in recipient rats and can protect mice from the auto-

immune disease EAE. Although the specific phenotype/s of these cells and the mechanisms they use are still under debate, some central themes have begun to coalesce. First, unlike in the case of CD4 TREG cells, the contribution to this population from thymic selection (nTREG cells) is likely very small. In studies using TCR ovalbumin-specific transgenic mice where all the T cells are specific for this nonself antigen, no FoxP3-expressing CD8 nTREG cells were identified. In other experimental systems, only rare CD8 T cells emigrating from the thymus with this natural regulatory phenotype could be detected. However, in the presence of antigen and TGF-, CD8 regulatory T cells expressing FoxP3 can be induced (iTREG). In fact, the plasticity of this phenotype may be quite significant. Some hypothesize that almost any naïve T cell (CD4 or CD8) presented with antigen and the right cocktail of cytokines along with lack of costimulation will develop into an iTREG cell, with the potential to revert back to immune stimulatory later! The phenotype of these cells is also complicated. Several phenotypic markers have been associated with separate CD8 T-cell populations that suppress immune responses. These include CD8 (as opposed to the more common  and  chains), both high- and low-affinity receptors for IL-2 (CD25 and CD122, respectively) and dendritic cell markers (CD11c), as well as others. In many cases, but not all, the master transcriptional regulator FoxP3 is also present. As with their CD4 counterparts, CD8 TREG cells likely use a range of mechanisms to inhibit other cells from responding to antigen. Whether this is mediated by separate populations of cells or by cells with the potential for many methods of inhibition is still unclear. Like with CD4 TREG cells, three main pathways seem to exist: APC lysis, inhibition of APC function, and regulation of effector T cells that share cognate antigen with the CD8 TREG cell. The hypothesis that regulatory CD8, as well as some CD4, T cells work primarily by “decommissioning” specific APCs has received much attention of late. For example, CD8 T cells can efficiently use conventional cytolysis to kill APCs presenting a self antigen, leading to a reduced frequency of presentation of this autoantigen and therefore fewer effector T cells (both CD4 and CD8) activated against this self molecule. Alternatively, regulatory cells may signal APCs to reduce their costimulatory potential, with a similar net effect. A reduction in CD80/86 expression on the APC may be even more efficient in favoring immunosuppression; it bars an APC from stimulating T cells and, at the same time, encourages further production of iTREG cells upon interaction with the APC (see Figure 16-3). In fact, a reduction in the expression of various costimulatory molecules, but not MHC class I or class II, has been seen in several experimental systems designed to study TREG cells. Some mouse CD8 TREG cells, like their CD4 counterparts, recognize antigen using conventional MHC molecules. However, the most well-characterized CD8 TREG cells are restricted to the nonclassical MHC class I molecules: Qa-1 in mice and HLA-E in humans. These nonclassical MHC

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Tolerance, Autoimmunity, and Transplantation molecules play key roles in presenting lipid antigens (see Chapter 8). Studies using mice engineered to lack Qa-1restricted CD8 TREG cells showed that these animals develop aggressive autoimmune reactions against self antigens, suggesting that this population is involved in regulating CD4 T-cell responses to self antigens to maintain peripheral tolerance. In various in vitro experimental systems, CD8 iTREG cells have been found to express inhibitory cytokines, including IL-10 and TGF-, although whether these cells are actually using any of these cytokines in vivo to suppress immune responses is still an open question.

Autoimmunity Simply stated, autoimmune disease is caused by failure of the tolerance processes described above to protect the host from the action of self-reactive lymphocytes. These diseases result

TABLE 16-1

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from the destruction of self proteins, cells, and organs by auto-antibodies or self-reactive T cells. For example, autoantibodies are the major offender in Hashimoto’s thyroiditis, in which antibodies reactive with thyroid-specific antigens cause severe tissue destruction. On the other hand, many autoimmune diseases are characterized by tissue destruction mediated directly by T cells. A well-known example is rheumatoid arthritis (RA), in which self-reactive T cells attack the tissue in joints, causing an inflammatory response that results in swelling and tissue destruction. Other examples of T-cell-mediated autoimmune disease include insulindependent or Type 1 diabetes mellitus (T1DM) and multiple sclerosis (MS). Table 16-1 lists several of the more common autoimmune disorders, as well as their primary immune mediators. Autoimmune disease is estimated to affect between 3% and 8% of individuals in the industrialized world, making this a rising problem in terms of morbidity and mortality

Some autoimmune diseases in humans

Disease

Self antigen/Target gene

Immune effector

ORGAN-SPECIFIC AUTOIMMUNE DISEASES

Addison’s disease

525

Adrenal cells

Auto-antibodies

Autoimmune hemolytic anemia

RBC membrane proteins

Auto-antibodies

Goodpasture’s syndrome

Renal and lung basement membranes

Auto-antibodies

Graves’ disease

Thyroid-stimulating hormone receptor

Auto-antibody (stimulating)

Hashimoto’s thyroiditis

Thyroid proteins and cells

TH1 cells, auto-antibodies

Idiopathic thrombocytopenia purpura

Platelet membrane proteins

Auto-antibodies

Type 1 diabetes mellitus

Pancreatic beta cells

TH1 cells, auto-antibodies

Myasthenia gravis

Acetylcholine receptors

Auto-antibody (blocking)

Myocardial infarction

Heart

Auto-antibodies

Pernicious anemia

Gastric parietal cells; intrinsic factor

Auto-antibody

Poststreptococcal glomerulonephritis

Kidney

Antigen-antibody complexes

Spontaneous infertility

Sperm

Auto-antibodies

SYSTEMIC AUTOIMMUNE DISEASES

Ankylosing spondylitis

Vertebrae

Immune complexes

Multiple sclerosis

Brain or white matter

TH1 cells and TC cells, auto-antibodies

Rheumatoid arthritis

Connective tissue, IgG

Auto-antibodies, immune complexes

Scleroderma

Nuclei, heart, lungs, gastrointestinal tract, kidney

Auto-antibodies

Sjögren’s syndrome

Salivary gland, liver, kidney, thyroid

Auto-antibodies

Systemic lupus erythematosus (SLE)

DNA, nuclear protein, RBC and platelet membranes

Auto-antibodies, immune complexes

Immune dysregulation polyendocrinopathy enteropathy X-linked (IPEX)

Multiorgan, loss of FoxP3 gene

Missing regulatory T cells

Autoimmune polyendocrinopathycandidiasis-ectodermal dystrophy (APECED)

Multiorgan, loss of aire gene

Defective central tolerance

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Experimental animal models of autoimmune diseases

Animal model

Possible human disease counterpart

Inducing antigen

Disease transferred by T cells

SPONTANEOUS AUTOIMMUNE DISEASES

Nonobese diabetic (NOD) mouse

Insulin-dependent diabetes mellitus (1)

Unknown

Yes

(NZB X NZW) F1 mouse

Systemic lupus erythematosus (SLE)

Unknown

Yes

Obese-strain chicken

Hashimoto’s thyroiditis

Thyroglobulin

Yes

EXPERIMENTALLY INDUCED AUTOIMMUNE DISEASES*

Experimental autoimmune myasthenia gravis (EAMG)

Myasthenia gravis

Acetylcholine receptor

Yes

Experimental autoimmune encephalomyelitis (EAE)

Multiple sclerosis (MS)

Myelin basic protein (MBP); proteolipid protein (PLP)

Yes

Autoimmune arthritis (AA)

Rheumatoid arthritis (RA)

M. tuberculosis (proteoglycans)

Yes

Experimental autoimmune thyroiditis (EAT)

Hashimoto’s thyroiditis

Thyroglobulin

Yes

* These diseases can be induced by injecting appropriate animals with the indicated antigen in complete Freund’s adjuvant. Except for autoimmune arthritis, the antigens used correspond to the self antigens associated with the human disease counterpart. Rheumatoid arthritis involves reaction to proteoglycans, which are self antigens associated with connective tissue.

around the globe. These diseases are often categorized as either organ-specific or systemic, depending on whether they affect a single organ or multiple systems in the body. Another method of grouping involves the immune component that does the bulk of the damage: T cells versus antibodies. In this section, we describe several examples of both organ-specific and systemic autoimmune disease. In each case, we discuss the antigenic target (when known), the causative process (either cellular or humoral), and the resulting symptoms. When available, examples of animal models used to study these disorders are also considered (Table 16-2). Finally, we touch on the factors believed to be involved in induction or control of autoimmunity, and the treatments for these conditions.

Some Autoimmune Diseases Target Specific Organs Autoimmune diseases are caused by immune stimulatory lymphocytes or antibodies that recognize self components, resulting in cellular lysis and/or an inflammatory response in the affected organ. Gradually, the damaged cellular structure is replaced by connective tissue (fibrosis), and the function of the organ declines. In an organ-specific autoimmune disease, the immune response is usually directed to a target antigen unique to a single organ or gland, so that the manifestations are largely limited to that organ. The cells of the target organs may be damaged directly by humoral or cellmediated effector mechanisms. Alternatively, anti-self anti-

bodies may overstimulate or block the normal function of the target organ. Hashimoto’s Thyroiditis In Hashimoto’s thyroiditis, an individual produces autoantibodies and sensitized TH1 cells specific for thyroid antigens. This disease is much more common in women, often striking in middle-age (see Clinical Focus Box 16-2). Antibodies are formed to a number of thyroid proteins, including thyroglobulin and thyroid peroxidase, both of which are involved in the uptake of iodine. Binding of the auto-antibodies to these proteins interferes with iodine uptake, leading to decreased thyroid function and hypothyroidism (decreased production of thyroid hormones). The resulting delayed-type hypersensitivity (DTH) response is characterized by an intense infiltration of the thyroid gland by lymphocytes, macrophages, and plasma cells, which form lymphocytic follicles and germinal centers. (See Chapter 15 for a description of the DTH response.) The ensuing inflammatory response causes a goiter, or visible enlargement of the thyroid gland, a physiological response to hypothyroidism. Type 1 Diabetes Mellitus Type 1 diabetes mellitus (T1DM) or insulin-dependent diabetes, affects almost 2 in 1000 children in the U.S.; roughly double the incidence observed just 20 years ago. It is seen mostly in youth under the age of 14 and is less common than Type 2, or non-insulin dependent diabetes mellitus. T1DM is caused by an autoimmune attack against insulin-producing

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impeded vascular flow), renal failure, and blindness. If untreated, death can result. The most common therapy for T1DM is daily administration of insulin. Although this is helpful, sporadic doses are not the same as metabolically regulated, continuous, and controlled release of the hormone. Unfortunately, diabetes can remain undetected for many years, allowing irreparable loss of pancreatic tissue to occur before treatment begins. One of the best-studied animal models of this disease is the NOD mouse, which spontaneously develops a form of diabetes that resembles human T1DM. This disorder also involves lymphocytic infiltration of the pancreas and destruction of beta cells, and carries a strong association with certain MHC alleles. Disease is mediated by bonemarrow-derived cells; normal mice reconstituted with an injection of bone marrow cells from NOD mice will develop diabetes, and healthy NOD mice that have not yet developed disease can be spared by reconstitution with bone marrow cells from normal mice. NOD mice housed in germ-free environments show a higher incidence of diabetes compared to those in normal housing, suggesting that microbes may influence the development of autoimmune disease. In genome-wide scans, over 20 insulin-dependent diabetes (Idd) loci associated with disease susceptibility have been identified, including at least one member of the TNF receptor family.

(a)

(b)

FIGURE 16-5 Photomicrographs of an islet of Langerhans in (a) pancreas from a normal mouse and (b) pancreas from a mouse with a disease resembling insulin-dependent diabetes mellitus. Note the lymphocyte infiltration into the islet (insulitis) in (b). [From M. A. Atkinson and N. K. Maclaren, 1990, Scientific American 263:1, 62.] [© 2007 W. H. Freeman and Company.]

cells (beta cells) scattered throughout the pancreas, which results in decreased production of insulin and consequently increased levels of blood glucose. The attack begins with cytotoxic T lymphocyte (CTL) infiltration and activation of macrophages, frequently referred to as insulitis (Figure 16-5b), followed by cytokine release and the production of autoantibodies, which leads to a cell-mediated DTH response. The subsequent beta-cell destruction is thought to be mediated by cytokines released during the DTH response and by lytic enzymes released from the activated macrophages. Autoantibodies specific for beta cells may contribute to cell destruction by facilitating either antibody-mediated complement lysis or antibody-dependent cell-mediated cytotoxicity (ADCC). The abnormalities in glucose metabolism associated with T1DM result in serious metabolic problems that include ketoacidosis (accumulation of ketone, a breakdown product from fat) and increased urine production. The late stages of the disease are often characterized by atherosclerotic vascular lesions (which cause gangrene of the extremities due to

Myasthenia Gravis Myasthenia gravis is the classic example of an autoimmune disease mediated by blocking antibodies. A patient with this disease produces auto-antibodies that bind the acetylcholine receptors (AchRs) on the motor end plates of muscles, blocking the normal binding of acetylcholine and inducing complement-mediated lysis of the cells. The result is a progressive weakening of the skeletal muscles (Figure 16-6). Ultimately, the antibodies cause the destruction of the cells bearing the receptors. The early signs of this disease include drooping eyelids and inability to retract the corners of the mouth. Without treatment, progressive weakening of the muscles can lead to severe impairment of eating as well as problems with movement. However, with appropriate treatment, this disease can be managed quite well and afflicted individuals can lead a normal life. Treatments are aimed at increasing acetylcholine levels (e.g., using cholinesterase inhibitors), decreasing antibody production (using corticosteroids or other immunosuppressants), and/or removing antibodies (using plasmapheresis). One of the first experimentally induced autoimmune disease animal models was discovered serendipitously in 1973, when rabbits immunized with AChRs purified from electric eels suddenly became paralyzed. (The original aim was to generate monoclonal antibodies for research use.) These rabbits developed antibodies against the foreign AChR that cross-reacted with their own AChRs. These autoantibodies then blocked muscle stimulation by Ach at the synapse and led to progressive muscle weakness. Within a

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Why Are Women More Susceptible Than Men to Autoimmunity? Gender Differences in Autoimmune Disease Of the nearly 50 million individuals in the United States believed to be living with autoimmune disease, approximately 78% are women. As shown in the following table, female-biased predisposition to autoimmunity is more apparent in some diseases than others. For example, the female-to-male ratio of individuals who suffer from diseases such as multiple sclerosis (MS) or rheumatoid arthritis (RA) is approximately two or three females to one male. There are nine women for every one man afflicted with systemic lupus erythematosus (SLE). However, these statistics do not tell the entire story. In some diseases, such as MS, the severity can be worse in men than in women. That women are more susceptible to autoimmune disease has been recognized for many years. The reasons are not entirely understood, although recent advances are helping to clarify this difference. Although it may seem unlikely, considerable evidence suggests significant gender differences in immune responses. In general, females tend to mount more vigorous humoral and cellular immune responses. Immune cell activation, cytokine secretion after infection, numbers of circulating CD4 T cells and mitogenic

responses are all higher in women than men. Immunization studies conducted in mice and humans show that females produce a higher titer of antibodies than males; this is true during both primary and secondary responses. In transplantation, women also suffer from a higher rate of graft rejection. As one might guess, this enhanced immunity in females means that males, in general, are slightly more prone to infections. The prevailing view is that sex hormone differences between men and women account for at least part of the observed gender difference in the rates of autoimmunity. Some of this evidence comes from observations made in SLE, where young women of child-bearing age are at greatest risk for the disease. Lupus flares during pregnancy (a high estrogen state) and increased rates of remission following menopause (a low estrogen state) also point to sex hormones as potential regulators of this autoimmune disease. The general consensus is that estrogens, the more female-specific hormones, are associated with enhanced immunity whereas androgens, or malebased hormones, are associated with its suppression.

In mice, whose gender differences are easier to study, a large body of literature documents gender differences in immune responses. Female mice are much more likely than male mice to develop TH1 responses and, in infections for which proinflammatory TH1 responses are beneficial, are more likely to be resistant to the infection. An excellent example is infection by viruses such as vesicular stomatitis virus (VSV), herpes simplex virus (HSV), and Theiler’s murine encephalomyelitis virus (TMEV). Clearance of these viruses is enhanced by TH1 responses. In other cases, however, a pro-inflammatory response can be deleterious. For example, a TH1 response to lymphocytic choriomeningitis virus (LCMV) correlates with more severe disease and significant pathology. Thus, female mice are more likely to succumb to infection with LCMV. The importance of gender in LCMV infection is underscored by experiments demonstrating that castrated male mice behave immunologically like females and are more likely to experience autoimmune disease than uncastrated males. Why this dichotomy between the sexes? One hypothesis posits that this increased risk of autoimmunity in women

BLOCKING AUTO-ANTIBODIES (Myasthenia gravis)

Nerve

Nerve

Acetylcholine

AChR

Auto-antibody to AChR Muscle cell

Muscle activation

Muscle activation inhibited

FIGURE 16-6 In myasthenia gravis, binding of auto-antibodies to the acetylcholine receptor (AChR; right) blocks the normal binding of acetylcholine (burgundy dots) and subsequent muscle activation (left). In addition, the anti-AChR auto-antibody activates complement, which damages the muscle end plate; the number of acetylcholine receptors declines as the disease progresses. [© 2013 W. H. Freeman and Company.]

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Box 16-2 is a by-product of the evolutionary role of women as bearers of children. Pregnancy may give us a clue as to how sex plays a role in regulating immune response. During pregnancy, it is critical that the mother tolerate the fetus, which is a type of foreign semi-allograft. This makes it very likely, maybe even crucial for successful implantation and fetal development, that the female immune system undergoes important modifications during pregnancy. Recall that females normally tend to mount more TH1-like responses than TH2 responses. During pregnancy, however, women mount more TH2-like responses. It is thought that pregnancy-associated levels of sex steroids may promote an antiinflammatory environment. In this regard, it is notable that diseases enhanced by TH2like responses, such as SLE, which has a strong antibody-mediated component, can be exacerbated during pregnancy, whereas diseases that involve inflammatory responses, such as RA and MS, sometimes are ameliorated in pregnant women. Another effect of pregnancy is the presence of fetal cells in the maternal circulation, creating a state called microchimerism. Fetal cells can persist in the maternal circulation for decades. These long-lived fetal cells may play a role in the development of autoimmune disease. Furthermore, the exchange of cells during pregnancy is bidirectional (cells of the mother may also

Gender prevalence ratios for selected autoimmune diseasesa

TABLE 1

Autoimmune disease

Ratio (female/male)

Hashimoto’s thyroiditis/hypothyroidism

50:1

Systemic lupus erythematosus

9:1

Sjögren’s syndrome

9:1–20:1

Graves’ disease/hyperthyroidism

7:1

Rheumatoid arthritis

3:1–4:1

Scleroderma

3:1–4:1

Myasthenia gravis

2:1–3:1

Multiple sclerosis

2:1

Type 1 diabetes mellitus

1:1–2:1

Ulcerative colitis

1:1

Autoimmune myocarditis

1:1.2

a

Modified from Gleicher and Barad, 2007.

appear in the fetal circulatory system), so that the presence of the mother’s cells in the male circulation could also be a contributing factor in autoimmune disease. Although some studies have linked the presence of microchimerism with certain autoimmune syndromes, other studies have contradicted these findings, casting

year, this animal model, called experimental autoimmune myasthenia gravis (EAMG), led to the discovery that autoantibodies to the AChR were also the cause of myasthenia gravis in humans.

Some Autoimmune Diseases Are Systemic In systemic autoimmune diseases, the immune response is directed toward a broad range of target antigens and involves a number of organs and tissues. These diseases reflect a general defect in immune regulation that results in hyperactive T cells and/or B cells. Tissue damage is typically widespread, both from cell-mediated immune responses and from direct cellular damage caused by auto-antibodies or by accumulation of immune complexes.

some doubt on this as a significant mode of induction of autoimmunity. Zandman-Goddard G, Peeva E and Shoenfeld Y 2007. Gender and autoimmunity. Autoimmunity Reviews, 6(6): 366–72. Gleicher N and Barad DH. 2007. Gender as risk factor for autoimmune diseases. Journal of Autoimmunity, 28 (1): 1–6.

Systemic Lupus Erythematosus One of the best examples of a systemic autoimmune disease is systemic lupus erythematosus (SLE). Like several of the other autoimmune syndromes, this disease is more common in women, with approximately a 9:1 ratio (see Clinical Focus Box 16-2). Onset of symptoms typically appears between 20 and 40 years of age and is more frequent in African American and Hispanic women than in Caucasians, for unknown reasons. In identical twins where one suffers from SLE, the other has up to a 60% chance of developing SLE, suggesting a genetic component. However, although close relatives of an SLE patient are 25 times more likely to contract the disease, still only 2% of these individuals ever develop SLE. Affected individuals may produce auto-antibodies to a vast array of tissue antigens, such as DNA, histones, RBCs,

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FIGURE 16-8 Diagnostic test for anti-nuclear antibodies FIGURE 16-7 Characteristic “butterfly” rash over the cheeks of a woman with systemic lupus erythematosus. [From L. Steinman,1993, Scientific American 269(3):80.]

platelets, leukocytes, and clotting factors. Signs and symptoms include fever, weakness, arthritis, skin rashes (Figure 16-7), and kidney dysfunction. Auto-antibodies specific for RBCs and platelets can lead to complement-mediated lysis, resulting in hemolytic anemia and thrombocytopenia, respectively. When immune complexes of auto-antibodies with various nuclear antigens are deposited along the walls of small blood vessels, a type III hypersensitivity reaction develops. The complexes activate the complement system and generate membrane-attack complexes and complement fragments (C3a and C5a) that damage the wall of the blood vessel, resulting in vasculitis and glomerulonephritis. In severe cases, excessive complement activation produces elevated serum levels of certain complement fragments, leading to neutrophil aggregation and attachment to the vascular endothelium. Over time, the number of circulating neutrophils declines (neutropenia) and occlusions of various small blood vessels develop (vasculitis), which can lead to widespread tissue damage. Laboratory diagnosis of SLE involves detection of antinuclear antibodies directed against double-stranded or singlestranded DNA, nucleoprotein, histones, and nucleolar RNA. Indirect immunofluorescent staining with serum from SLE patients produces characteristic nuclear-staining patterns (Figure 16-8). New Zealand Black (NZB) mice and F1 hybrids of NZB x New Zealand White (NZW) mice spontaneously develop autoimmune diseases that closely resemble SLE. NZB mice develop autoimmune hemolytic anemia between 2 and 4 months of age, at which time various auto-antibodies can be detected, including antibodies to erythrocytes, nuclear proteins, DNA, and T lymphocytes. F1 hybrids develop glomerulonephritis from immune-complex deposits in the kidney and die prematurely. As in human SLE, the incidence of autoimmunity in F1 hybrids is greater in females.

using serum from an SLE patient. Serum dilutions from a patient are mixed with cells attached to a glass slide. Fluorescently labeled secondary antibodies directed against human antibodies are then added and reveal staining of the nucleus under a fluorescence microscope. [Courtesy ORGENTEC Diagnostika GmbH ]

Multiple Sclerosis Multiple sclerosis (MS) is the most common cause of neurologic disability associated with disease in Western countries. MS occurs in women two to three times more frequently than men (see Clinical Focus Box 16-2) and, like SLE, frequently develops during childbearing years (approximately 20–40 years of age). Individuals with this disease produce autoreactive T cells that participate in the formation of inflammatory lesions along the myelin sheath of nerve fibers in the brain and spinal cord. Since myelin functions to insulate the nerve fibers, a breakdown in the myelin sheath leads to numerous neurologic dysfunctions, ranging from numbness in the limbs to paralysis or loss of vision. Epidemiological studies indicate that MS is most common in the Northern Hemisphere and, interestingly, in the United States. Populations north of the 37th parallel have a markedly higher prevalence of MS than those south of this latitude. Individuals born south of the 37th parallel who move north before age 15 assume the higher relative risk. These provocative data suggest that an environmental component early in life affects the risk of contracting MS. Genetic influences are also important. The average person in the United States has about one chance in 1000 of developing MS; this increases to 1 in 50 to 100 for children or siblings of people with MS, and to 1 in 3 for an identical twin. The cause of MS is not well understood. Infection by certain viruses, such as Epstein-Barr virus (EBV), may predispose a person to MS. Some viruses can cause demyelinating diseases, but the data linking viruses to MS are not definitive. EAE, one of the best-studied animal models of autoimmune disease, is mediated solely by T cells. It can be induced in a variety of species by immunization with

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Tolerance, Autoimmunity, and Transplantation myelin basic protein (MBP) or proteolipid protein (PLP)— both components of the myelin sheaths surrounding neurons in the CNS—in complete Freund’s adjuvant. Within 2 to 3 weeks, the animals develop cellular infiltration of the CNS, resulting in demyelination and paralysis. Most of the animals die, but others have milder symptoms, and some develop a chronic form of the disease that resembles relapsing and remitting MS in humans. Those that recover are resistant to the development of disease from a subsequent challenge with MBP and adjuvant. In these experiments, exposure of immature T cells to self antigens that normally are not present in the thymus presumably led to tolerance to these antigens. EAE was also prevented in susceptible rats by prior injection of MBP directly into the thymus. MBP is normally sequestered from the immune system by the blood-brain barrier, but in EAE the immune system is exposed to it under nonphysiologic conditions. EAE in small mammals does provide a system for testing treatments for human MS. Rheumatoid Arthritis Rheumatoid arthritis (RA) is a fairly common autoimmune disorder, most often diagnosed between the ages of 40 to 60 and more frequently seen in women. The major symptom is chronic inflammation of the joints (Figure 16-9), although the hematologic, cardiovascular, and respiratory systems are also frequently affected. Many individuals with RA produce a group of auto-antibodies called rheumatoid factors that are reactive with determinants in the Fc region of IgG—in other words, antibodies specific for antibodies! The classic rheumatoid factor is an IgM antibody that binds to normal circulating IgG, forming IgM-IgG complexes that are deposited in the joints. These immune complexes can activate the complement cascade, resulting in a type III hypersensitivity reaction, which leads to chronic inflammation of the joints. Treatments for RA include nonspecific drugs aimed at reducing inflammation, such as nonsteroidal anti-inflammatory drugs (NSAIDs) and corticosteroids.

FIGURE 16-9 Swollen hand joints in a woman with rheumatoid arthritis. [James Stevenson/Photo Researchers]

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More disease-specific immune modifiers have also been introduced, including antibodies that block TNF- and IL-6 (see treatment below).

Both Intrinsic and Extrinsic Factors Can Favor Susceptibility to Autoimmune Disease Overzealous immune activation can lead to autoimmunity, although suboptimal immune stimulation results in insufficiency that can allow microbes to take control. But what tips the balance toward a break in tolerance and the development of autoimmunity? Experiments with germ-free mouse models, discordance data in identical twins, and epidemiologic studies of geographic associations all suggest roles for both the environment and genes in susceptibility to the development of autoimmunity. Environmental Factors Favoring the Development of Autoimmune Disease As mentioned earlier, some autoimmune syndromes are more common in certain geographic locations or in particular climates. This suggests a link between environmental exposures (some of which may be microbial) or lifestyle factors, such as diet, in the development of autoimmune disease. For instance, we now know that cross-talk between gut microflora and the systemic immune system may help regulate peripheral tolerance, which could impact the development of autoimmune disease. Animals maintained in germ-free environments often show heightened susceptibility to autoimmune disease compared to their germ-laden counterparts. How commensal microbes influence tolerance and autoimmunity is an active area of research (see Clinical Focus Box 16-1). Infections may also influence the induction of autoimmunity. For example, tissue pathology following infection may result in the release of sequestered self antigens that are presented in a way that fosters immune activation rather than tolerance induction. Likewise, the molecular structures of certain microbes may share chemical features with self components, resulting in the activation of immune cells with cross-reactive potential. The Role of Genes in Susceptibility to Autoimmunity Certain alleles within the MHC have been linked to several different autoimmune disorders. The strongest association between an HLA allele and autoimmunity is seen in ankylosing spondylitis, an inflammatory disease of vertebral joints. Individuals who express HLA-B27 are 90 times more likely to develop ankylosing spondylitis than individuals with a different HLA allele at this locus. This does not imply causation; not all individuals who express HLA-B27 experience this syndrome, suggesting that the relationship between MHC alleles and development of autoimmune disease is multifaceted. Interestingly, unlike many other autoimmune diseases, 90% of the cases of ankylosing spondylitis are seen in males.

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Some non-MHC inherited genetic mutations can have causative effects on the development of autoimmunity. Not surprisingly, these tend to play prominent roles in immune regulation. Inactivating mutations in two immune-related genes, aire and FoxP3, result in forms of immune deficiency that impact central and peripheral tolerance, respectively (see above, as well as Chapters 9 and 18). A mutant form of aire has been shown to cause autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED), a systemic disease resulting from defective deletion or inactivation of autoreactive T cells in the thymus. The cause of another human autoimmune disorder—immune dysregulation, polyendocrinopathy, enteropathy, and X-linked syndrome (IPEX) has been mapped to mutations in the FoxP3 gene. The product of this gene is required for the formation of many, but not all, regulatory T cells, suggesting that this disease is caused by an inability to generate the TREG cells needed to maintain peripheral tolerance. Many other genes with more subtle or even cumulative effects on susceptibility to autoimmunity have also been discovered. Not surprisingly, most of these play some role in the immune response. Genes for cytokines and their receptors, antigen processing and presentation, c-type lectin receptors, signaling pathways, adhesion molecules, and costimulatory or inhibitory receptors have all been linked to specific autoimmune diseases (Table 16-3). In many instances, multiple genes (sometimes with compounding environmental factors) may be required in order to predispose an individual to a particular autoimmune disease. In some cases, a single gene can heighten susceptibility to multiple different autoimmune disorders. For instance, a mutant form of PTPN22, a tyrosine phosphatase, results in reduced

TCR signaling capacity and has been linked to T1DM, RA, and SLE. It is believed that attenuation of TCR signaling during positive and negative selection may be what predisposes carriers of this allele to autoimmunity. The Role of Certain T Helper Cell Types in Autoimmunity In both organ-specific and systemic autoimmunity, CD4 rather than CD8 T cells have been linked to disease pathogenesis. However, the T helper (TH) cell type or set of cytokines most closely associated with autoimmunity depends somewhat on the model system or human disease in question. As we know from Chapter 11, the antigen, the type of APC to make first encounter, and the surface receptors used during this engagement set the stage for the transition from innate to adaptive immunity. In this transition, the cytokine milieu will help determine which subsets of a TH cell will predominate. The induction of autoimmunity is likewise a complex process, where even experimental models of the same human disease can be induced by different means, making outcomes in each case difficult to correlate. Nevertheless, a few themes have emerged from both human and animal studies of autoimmune disease. Much of the initial data collected from various studies of autoimmune disease supported a role for autoreactive TH1 cells and IFN-. For example, IFN- levels in the CNS of mice with EAE correlate with the severity of disease, and treatment with this cytokine exacerbates MS in humans. Likewise, adoptive transfer of IFN--producing CD4 TH cells from mice with EAE can induce disease in naïve hosts. On the flip side, elimination of IFN- using either neutralizing antibodies or removal of the gene does not protect animals from EAE; in fact, it worsens the symptoms.

TABLE 16-3

Examples of genetic associations with autoimmune disease

Disease

C-type lectin

Cytokines, their receptors and regulators

Type 1 diabetes

CLEC16A

IL-2R

Rheumatoid arthritis (RA)

DCIR

STAT4

REL, C5-TRAFI

IL-1A, IL-23R

KIR complex

Ankylosing spondylitis (AS) Multiple sclerosis (MS)

CLEC16A

Systemic lupus erythematosus (SLE) Crohn’s disease

CLEC16A

Innate immune response

IL-2RA, IL-7R

Adhesion and costimulation

Antigen processing and presentation

CTLA4

VNTR-Ins PTPN22

CD40

PTPN22, MHC2TA ERAP1

CD40

STAT4, IRF5

TNFAIP3

TNFSF4

PTPN22

IL-23R

NOD2, NCF4

TNFSF15

PTPN2

Source: R. Thomas, 2010, The balancing act of autoimmunity: Central and peripheral tolerance versus infection control, International Reviews of Immunology 29:211, Table 1 with modifications.

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TABLE 16-4

Common pro-inflammatory environmental factors in autoimmune diseases

Group

Examples

Disease association examples

Infection

Viral

Type I diabetes

Bacterial

Reiter’s syndrome

Fungal

Mucocutaneous candidiasis (APECED)

Smoking

Rheumatoid arthritis

Fabric dyes

Scleroderma

Iodine

Thyroiditis

Psychological

Multiple sclerosis, Systemic lupus erythematosus (SLE)

Oxidative, metabolic

Rheumatoid arthritis

Ultraviolet light

SLE

Endoplasmic reticulum

Ulcerative colitis

Gluten

Celiac disease

Breastfeeding cessation

Type I diabetes

Gastric bypass

Spondyloarthropathy

Toxins

Stress

Food

Source: R. Thomas, 2010. The balancing act of autoimmunity: Central and peripheral tolerance versus infection control. International Reviews of Immunology 29:211, Table 2.]

These conflicting results led to the study of other cytokines or T cell types that may be involved in the induction of autoimmunity, especially those connected to IFN-. Recall from Chapter 11 that IL-12 and IL-23, which can be produced by APCs during activation, encourage the production of other cytokines, such as either IFN- or IL-17, favoring T-cell development along TH1 or TH17 pathways, respectively. Studies have shown that mice engineered to lack the gene for the p40 subunit of IL-12, which happens to be shared with IL-23, are protected from EAE. This protection is due to inhibition of IL-23, a cytokine required to sustain TH17 cells. Mice lacking IL-17A are less susceptible to both EAE and collagen-induced arthritis, a model for human RA. In follow-up studies of patients with RA and psoriasis, elevated IL-17 expression has been found at the site of inflammation, and increased serum levels of IL-17 and IL-23 have been observed in patients with SLE. Collectively, these findings support the notion that TH17 cells may be an important driver of some autoimmune diseases.

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Several Possible Mechanisms Have Been Proposed for the Induction of Autoimmunity In addition to genetic and environmental predisposing factors, autoimmunity likely develops from a number of different events. Disease may be induced by certain genetic mutations, the release of sequestered antigens, overstimulation of antigen-specific receptors, and stochastic events. In most cases, a combination of these is the cause. Another issue is the sex difference in autoimmune susceptibility, with diseases such as Hashimoto’s thyroiditis, SLE, MS, and RA preferentially affecting women. Factors that may account for this, such as hormonal differences between the sexes and the potential effects of fetal cells in the maternal circulation following pregnancy, are discussed in the Clinical Focus Box 16-2. As a result of random V(D)J recombination, over half of all antigen-specific receptors recognize self proteins. Not all of these are deleted during negative selection (see Chapter 9). Potentially self-reactive T and B cells found in the periphery are normally held in check by anergic or regulatory mechanisms, such as TREG cells. However, exposure to carcinogens or infectious agents that favor DNA damage or polyclonal activation can potentially interfere with this regulation and/or lead to the expansion and survival of rare T- or B-cell clones with autoimmune potential (Table 16-4). Genes that, when mutated, could favor expansion include those encoding antigen receptors, signaling molecules, costimulatory or inhibitory molecules, apoptosis regulators, or growth factors (see Table 16-3). Gram-negative bacteria, cytomegalovirus, and EBV are all known polyclonal activators, inducing the proliferation of numerous clones of B cells that express IgM in the absence of T-cell help. If B cells reactive to self antigens are activated by this mechanism, autoantibodies can appear. A role for particular microbial agents in autoimmunity was postulated for several reasons beyond their potential for DNA damage or polyclonal activation. As discussed earlier, some autoimmune syndromes are associated with certain geographic regions, and immigrants to an area can acquire enhanced susceptibility to the disorder associated with that region. This, coupled with the fact that a number of viruses and bacteria possess antigenic determinants that are similar or even identical to normal host-cell components, led to a hypothesis known as molecular mimicry. This proposes that some pathogens express protein epitopes resembling self components in either conformation or primary sequence. For instance, rheumatic fever, a disease caused by autoimmune destruction of heart muscle cells, can develop after a Group A Streptococcus infection. In this case, antibodies to streptococcal antigens have been shown to cross-react with the heart muscle proteins, resulting in immune complex deposition and complement activation, a type II hypersensitivity reaction (see Chapter 15). In one study, 600 different monoclonal antibodies specific for 11 different viruses were evaluated for their reactivity with

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normal tissue antigens. More than 3% of the virus-specific antibodies tested also bound to normal tissue, suggesting that molecular similarity between foreign and host antigens may be fairly common. In these cases, susceptibility may also be influenced by the MHC haplotype of the individual, since certain class I and class II MHC molecules may be more effective than others in presenting the homologous peptide for T-cell activation. Release of sequestered antigens is another proposed mechanism of autoimmune initiation, one that may in some cases also be connected with environmental exposures. The induction of self-tolerance in T cells results from exposure of immature thymocytes to self antigens in the thymus, followed by clonal deletion or inactivation of any self-reactive cells (see Chapter 9). Tissue antigens that are not expressed in the thymus will not engage with developing T cells and will thus not induce self-tolerance. Trauma to tissues following either an accident or an infection can release these sequestered antigens into the circulation. For instance, the release of heart muscle antigens following myocardial infarction (heart attack) can lead to the formation of auto-antibodies that target healthy heart muscle cells. Studies involving injection of normally sequestered antigens directly into the thymus of susceptible animals support this proposed mechanism: injection of CNS myelin proteins or pancreatic beta cells can inhibit the development of EAE or diabetes, respectively. In these experiments, exposure of immature T cells to self antigens normally not present in the thymus presumably led to central and possibly also peripheral tolerance to these antigens. It is worth reiterating that, although certain events may be associated with the development of autoimmunity, a complex combination of genotype and environmental factors likely influences the balance of self-tolerance versus development of autoimmune disease.

Autoimmune Diseases Can Be Treated by General or Pathway-Specific Immunosuppression Ideally, treatment for autoimmune diseases should reduce only the autoimmune response, leaving the rest of the immune system intact. However, implementing this strategy has proven difficult. The current therapies to treat autoimmune disease fall into two categories: broad spectrum immunosuppressive treatments and more recent mechanism- or cell-type-specific strategies (Table 16-5). Broad-Spectrum Therapies Most first-generation therapies for autoimmune diseases are not cures but merely palliatives, reducing symptoms to provide the patient with an acceptable quality of life. For example, most general immunosuppressive treatments (e.g., corticosteroids, azathioprine, and cyclophosphamide) are strong anti-inflammatory drugs that suppress lymphocytes

by inhibiting their proliferation or by killing these rapidly dividing cells. Side effects of these drugs include general cytotoxicity, an increased risk of uncontrolled infection, and the development of cancer. In some autoimmune diseases, removal of a specific organ or set of toxic compounds can alleviate symptoms. Patients with myasthenia gravis often have thymic abnormalities (e.g., thymic hyperplasia or thymomas), in which case thymectomy can increase the likelihood of remission. Plasmapheresis may also provide significant if short-term benefit for diseases involving antigen-antibody complexes (e.g., myasthenia gravis, SLE, and RA), where removal of a patient’s plasma antibodies temporarily eliminates these complexes. Strategies That Target Specific Cell Types When antibodies and/or immune complexes are heavily involved in autoimmune pathology, strategies aimed at B cells can improve clinical symptoms. For example, a monoclonal antibody against the B-cell-specific antigen CD20 (Rituximab) depletes a subset of B cells and provides short-term benefit for RA. However, most cell-type specific agents used to treat autoimmune disorders target T cells or their products because these cells are either directly pathogenic or provide help to autoreactive B cells. The first anti-T-cell antibodies used to treat autoimmune disease targeted the CD3 molecule and were designed to deplete T cells without signaling through this receptor. Although somewhat effective in the treatment of T1DM, this method still induced broad-spectrum immune suppression. Anti-CD4 antibodies successfully reversed MS and arthritis in animal models, although human trials of this treatment have shown no efficacy. A possible reason for this failure is that anti-CD4 may interfere with the activity of CD4CD25 regulatory T cells, a cell type we now know is key to the regulation of tolerance. With this in mind and with the discovery of the TH17 subset, scientists are beginning to target specific T helper cell pathways. In several mouse models of autoimmunity, including MS, T1DM, SLE, and IPEX, the transfer of TREG cells can clearly inhibit disease pathogenesis. The greatest difficulty with translating this from mouse to human is in selecting a population of TREG cells, as FoxP3 in humans does not correlate well with immunosuppressive activity. Therefore, most of the emphasis in the clinical applications of this approach is currently directed toward mimicking TREG-like mechanisms of suppression (e.g., using IL-10) or inhibiting TH17mediated effects (e.g., using IL-17 or IL-23 blocking antibodies). Therapies That Block Steps in the Inflammatory Process Since chronic inflammation is a hallmark of debilitating autoimmune disease, steps in the inflammatory process are potential targets for intervention. Drugs that block TNF-, one of the early mediators in the inflammatory process, are

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TABLE 16-5

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Drugs currently approved by the FDA or undergoing clinical trials to treat autoimmune disease or suppress the immune response, arranged according to mechanism of action

Name

Brand name

Mechanism of action

Target disease

T- OR B-CELL DEPLETING AGENTS

Lymphocyte immune globulin (horse), anti-thymocyte globulin (rabbit)

Atgam (horse), Thymoglobulin (rabbit)

Depleting horse/rabbit polyclonal anti-thymocyte antibody

Renal transplant; aplastic anemia

Muronomab (OKT3)

Orthoclone OKT3

Mouse anti-human CD3 mAb

Acute transplant rejection; graft-versus-host disease (GVHD)

Human anti-CD4 mAb, partially depleting

Rheumatoid arthritis (RA)

Chimeric anti-CD20 mAb

RA

Zanolimumab (HuMax-CD4)

Rituximab (IDEC-C2B8)

Rituzan

TARGETING TRAFFICKING/ADHESION

Fingolimod (FTY720)

S1P receptor agonist

Relapsing/remitting multiple sclerosis (MS); renal transplant

TARGETING TCR SIGNALING

Cyclosporine A

Gengraf, Neoral, Sandimmune

Calcineurin inhibitor

Transplant; severe active RA; severe plaque psoriasis

Tacrolimus (FK506)

Prograf (systemic), Protopic (topical)

Calcineurin inhibitor

Transplant; moderate-severe atopic dermatitis; ulcerative colitis (UC); RA; myasthenia gravis; GVHD

TARGETING COSTIMULATORY AND ACCESSORY MOLECULES

Abatacept (BMS-188667)

Orencia

Belatacept (BMS-224818, LEA29Y)

Fc fusion protein with extracellular domain of CTLA-4, blocks CD28-CD80/86 interaction

RA; lupus nephritis; inflammatory bowel disease (IBD); juvenile idiopathic arthritis (JIA)

Same as Abatacept, higher affinity

Transplant

TARGETING CY TOKINES/CY TOKINE SIGNALING

Sirolimus

Rapamune

mTOR inhibitor

Renal transplant, GVHD

Source: Scott M. Steward-Tharp, Yun-Jeong Song, Richard M. Siegel, John J. O’Shea, 2010, New insights into T cell biology and T celldirected therapy for autoimmunity, inflammation, and immunosuppression. Annals of the New York Academy of Sciences 1183:123, Table 1 with modifications.

widely used to treat RA, psoriasis, and Crohn’s disease. An IL-1 receptor antagonist is approved for treatment of RA, as are antibodies directed against the IL-6 receptor and IL-15. Other anti-inflammatory, cytokine-based experimental treatments for autoimmunity include targeting the IL-2 receptor (CD25 and CD122), IL-1, and IFNs. More broadly, the class

of drugs designated as statins, used by millions to reduce cholesterol levels, have been found to lower serum levels of C-reactive protein (an acute-phase protein and indicator of inflammation), reduce levels of pro-inflammatory cytokines, and decrease expression of adhesion molecules on endothelial cells. Clinical trials of statins for treatment of RA and MS

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have shown encouraging results. The addition of such drugs with prior FDA approval and extensive safety testing is a tremendous advantage, considering that 95% of agents fail human trials due to safety concerns. Compounds that block the chemokine or adhesion molecule signals controlling lymphocyte movement into sites of inflammation can also thwart autoimmune processes. The most well-characterized inhibitor of cell trafficking is FTY720. This compound is an analog of sphingosine 1-phosphate (S1P), which is involved in the migration of lymphocytes into the blood and lymph. By acting as a receptor antagonist, it inhibits egress of all subsets of T cells, resulting in a reduction of up to 85% in circulating blood lymphocytes. Importantly, FTY720, which has been effective in treating MS, is also reported to inhibit TH1 and TH17 cells, and to enhance TREG cell activity. Strategies That Interfere with Costimulation T cells require both antigenic stimulation via the TCR (signal 1) and costimulation (signal 2) in order to become fully activated (see Chapter 11). Without costimulation, T cells undergo apoptosis, become anergic, or are induced as immune inhibitors. Therefore, one way to control T-cell activation would be to regulate costimulation. To this end, a fusion protein was generated consisting of the extracellular domain of CTLA-4 combined with the human IgG1 constant region. CTLA-4 binds to its CD80/86 partner with an affinity that is approximately 20 times greater than that of CD28. This therapeutic fusion protein, called abatacept (Orencia) and approved for the treatment of RA, has also been studied with limited success in patients with MS, T1DM, SLE, and inflammatory bowel disease. This drug blocks CD80/86 on APCs from engaging with CD28 on T cells. Antigen-Specific Immunotherapy The holy grail of immunotherapy to treat autoimmune disease is a strategy that specifically targets the autoreactive cells, sparing all other leukocytes. In an ideal world, clinical therapies that induce tolerance to the auto-antigen could reverse the course of autoimmune disease. However, even in cases when the auto-antigen is known, as with T1DM and MS, there is a risk of exacerbating the disease by introduction of the tolerogen, as was seen in some of the early trials. Nonetheless, glatiramer acetate (GA), a polymer of four basic amino acids found commonly in MBP and approved for treating MS, selectively increased the number of TREG cells and modulated the function of APCs, suggesting that this strategy may also be effective for the treatment of other autoimmune disorders. This drug is the only FDA-approved antigen-specific treatment currently available for an autoimmune disease, although others are in the pipeline. BHT3021, a DNA-based product aimed at tolerizing the immune system to proinsulin, if approved, would be the first antigenbased treatment for type 1 diabetes.

Transplantation Immunology Alexis Carrel reported the first systematic study of transplantation in 1908; he interchanged both kidneys in a series of cats, some of which maintained urinary output for up to 25 days. Although all the cats eventually died, the experiment established that a transplanted organ could carry out its normal function in the recipient. The first human kidney transplant, attempted in 1935 by a Russian surgeon, failed because a mismatch of blood types between donor and recipient caused almost immediate rejection of the kidney. We now know that this rapid immune response, termed hyperacute rejection, is mediated by preformed antibodies (described below). Finally, in 1954 a team in Boston headed by Joseph Murray performed the first successful human kidney transplant between identical twins, followed 3 years later by the first transplant between nonidentical individuals. Today, the transfer of various organs and tissues between individuals is performed with ever-increasing frequency and rates of success, at least for their short-term survival. Although a supply of organs is provided by accident victims and, in some cases, living donors, many more people are in need of transplants than can be accommodated with available organs. According to the U.S Department of Health and Human Services, as of December 2012 over 116,000 individuals in the United States are on the waiting list for an organ transplant (see http://optn.transplant.hrsa.gov for real-time data). The majority of those on the list (over 75%) require a kidney, for which the median waiting period ranges from 3 to 5 years. Immunosuppressive agents can delay or prevent rejection of transplanted organs, but they have side effects. New treatments that promise longer transplant survival and more specific tolerance to the graft without suppressing other immune function are under development. This section describes the mechanisms underlying graft rejection, procedures that are presently used to prolong graft survival, and the current status of transplantation as a clinical tool.

Graft Rejection Occurs Based on Immunologic Principles The degree and type of immune response to a transplant varies with the type and source of the grafted tissue. The following terms denote different types of transplants: • Autograft is self tissue transferred from one body site to another in the same individual. Examples include transferring healthy skin to a burned area in burn patients and using healthy blood vessels to replace blocked coronary arteries. • Isograft is tissue transferred between genetically identical individuals. This occurs in inbred strains of mice or identical human twins, when the donor and recipient are syngeneic.

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(a) Autograft acceptance

(b) First-set rejection

Grafted epidermis

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(c) Second-set rejection

Grafted epidermis

Grafted epidermis

Blood vessels

Days 3–7: Revascularization

Days 3–7: Revascularization

Days 3–4: Cellular infiltration

Mediators

Days 7–10: Healing

Days 7–10: Cellular infiltration

Days 5–6: Thrombosis and necrosis

Blood clots Neutrophils Necrotic tissue

Days 12–14: Resolution

Days 10–14: Thrombosis and necrosis

Necrotic tissue Blood clots

Damaged blood vessels

FIGURE 16-10 Schematic diagrams of the process of graft acceptance and rejection. (a) Acceptance of an autograft is completed within 12 to 14 days. (b) First-set rejection of an allograft begins 7 to 10 days after grafting, with full rejection occurring by 10 to 14 days. (c) Second-set rejection of an allograft begins within 3 to 4 days, with full rejection by 5 to 6 days. The cellular infiltrate that invades an allograft (b, c) contains lymphocytes, phagocytes, and other inflammatory cells. [© 2013 W. H. Freeman and Company.] • Allograft is tissue transferred between genetically different members of the same species. In mice this means transferring tissue from one strain to another, and in humans this occurs in transplants in which the donor and recipient are not genetically identical (the majority of cases). • Xenograft is tissue transferred between different species (e.g., the graft of a baboon heart into a human). Because of significant shortages of donated organs, raising animals for the specific purpose of serving as organ donors for humans is under serious consideration. Autografts and isografts are usually accepted, owing to the genetic identity between donor and recipient (Figure 16-10a). Because an allograft is genetically dissimilar to the host and

therefore expresses unique antigens, it is often recognized as foreign by the immune system and is therefore rejected. Obviously, xenografts exhibit the greatest genetic and antigenic disparity, engendering a vigorous graft rejection response. Specificity and Memory in Allograft Rejection The rate of allograft rejection varies according to the tissue involved; skin grafts are generally rejected faster than other tissues, such as kidney or heart. Despite these time differences, the immune response culminating in graft rejection always displays the attributes of specificity and memory. If an inbred mouse of strain A is grafted with skin from strain B, primary graft rejection, known as first-set rejection, occurs (Figure 16-10b). The skin first becomes revascularized between days 3 and 7. As the reaction develops, the

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First-set rejection

First skin graft, strain A

Necrosis 14 days

Second-set rejection

Second skin graft, strain A Time

Necrosis

6 days

Naïve strain = B mouse

FIGURE 16-11 Experimental demonstration that

Splenic T cells

Second-set rejection First skin graft, strain A

T cells can transfer allograft rejection. When T cells derived from an allograft-primed mouse are transferred to an unprimed syngeneic mouse, the recipient mounts a second-set rejection to an initial allograft from the original allogeneic strain. [© 2013 W. H. Freeman and Company.]

Necrosis

6 days Naïve strain = B mouse

tions resulted in more pronounced graft rejection. These data are supported by human studies showing CD4 and CD8 T cells infiltrating human kidney allografts. The role of DCs in rejection or tolerance of an allograft is the subject of increasing interest due to their immunostimulatory capacity and their role in the induction of tolerance. As discussed in Chapter 8, DCs can present exogenous antigens in the context of class I MHC molecules via cross-presentation, giving CD8 T cells the opportunity to recognize alloantigens as part of the rejection process. In mice, inhibition of DCs can aid graft acceptance (presumably by interfering with the presentation of donor antigens), although pretreatment with donor DCs can promote survival of both heart and pancreas transplants (possibly by inducing tolerance to donor antigens).

Role of T Cells in Graft Rejection Using adoptive transfer studies in the early 1950s, Avrion Mitchison showed that donor lymphocytes but not serum antibody could transfer allograft rejection responses. Later studies further defined these as T cells. For instance, nude mice, which lack a thymus and consequently lack functional T cells, were found to be incapable of allograft rejection; these mice even accept xenografts. In other studies, T cells derived from an allograft-primed mouse were shown to transfer second-set allograft rejection to an unprimed syngeneic recipient as long as that recipient was grafted with the same allogeneic tissue (Figure 16-11). Analysis of the T-cell subpopulations involved in allograft rejection has implicated both CD4 and CD8 populations. In one study, mice were injected with monoclonal antibodies to deplete one or both types of T cells and then the rate of graft rejection was measured. As shown in Figure 16-12, removal of the CD8 population alone had no effect on graft survival, and the graft was rejected at the same rate as in control mice (15 days). Removal of the CD4 T-cell population alone prolonged graft survival from 15 days to 30 days. However, removal of both CD4 and CD8 T cells resulted in long-term survival (up to 60 days) of the allografts. This study indicated that both CD4 and CD8 T cells participated in rejection and that the collaboration of the two subpopula-

Antigenic Profiles and Transplantation Tolerance Tissues that share sufficient antigenic similarity, allowing transfer without immunologic rejection, are said to be histocompatible. This is the case when the transfer occurs Surviving grafts, %

vascularized transplant becomes infiltrated with inflammatory cells. There is decreased vascularization of the transplanted tissue by 7 to 10 days, visible necrosis by 10 days, and complete rejection by 12 to 14 days. Immunologic memory is demonstrated when a second strain-B graft is transferred to a previously engrafted strain-A mouse. In this case, the anti-graft reaction develops more quickly, with complete rejection occurring within 5 to 6 days. This secondary response is called second-set rejection (Figure 16-10c). Specificity can be demonstrated by grafting skin from an unrelated mouse of strain C at the same time as the second strain-B graft. Rejection of the strain-C graft proceeds according to the slower, first-set rejection kinetics, whereas the strain-B graft is rejected in an accelerated second-set fashion.

100

50

0

Anti– CD8

Control

15

Anti–CD4

30

Anti–CD4 and anti–CD8 60

Time after grafting, days

FIGURE 16-12 The role of CD4ⴙ and CD8ⴙ T cells in allograft rejection is demonstrated by the curves showing survival times of skin grafts between mice mismatched at the MHC. Animals in which the CD8 T cells were removed by treatment with an anti-CD8 monoclonal antibody (red) showed little difference from untreated control mice (black). Treatment with monoclonal anti-CD4 antibody (blue) improved graft survival significantly, and treatment with both anti-CD4 and anti-CD8 antibody prolonged graft survival most dramatically (green). [Cobbold SP, Martin G, Qin S, Waldmann H. 1986. Monoclonal antibodies to promote marrow engraftment and tissue graft tolerance. Nature 323:164–166.]

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Tolerance, Autoimmunity, and Transplantation between identical twins. Tissues that display significant antigenic differences are histoincompatible and typically induce an immune response that leads to tissue rejection. Of course, there are many shades of gray in the degree of histocompatibility between a donor and recipient. The antigens involved are encoded by more than 40 different loci, but the loci responsible for the most vigorous allograft rejection reactions are located within the MHC. The organization of the MHC—called the H-2 complex in mice and the HLA complex in humans—was described in detail in Chapter 8 (see Figures 8-7 and 8-8). Because the genes in the MHC locus are closely linked, they are usually inherited as a complete set from each parent, called a haplotype. In inbred strains of mice, offspring inherit the same haplotype from each parent, meaning they are homozygous at the MHC locus. When mice from two different inbred strains are mated, the F1 progeny each inherit one maternal and one paternal haplotype (see Figure 8-9). These heterozygous F1 offspring express the MHC type from both parents (b/k, in Figure 8-10), which means they are tolerant to the alleles from both haplotypes and can accept grafts from either parent. However, neither of the parental strains can accept grafts from the F1 offspring because each parent lacks one of the F1 haplotypes and will therefore reject these MHC antigens. In outbred populations, there is a high degree of heterozygosity at most loci, including the MHC. In matings between members of an outbred species, there is only a 25% chance that any two offspring will inherit identical MHC haplotypes unless the parents share one or more haplotypes. Therefore, for purposes of organ or bone marrow grafts, it can be assumed that there is a 25% chance of MHC identity between any two siblings. With parent-to-child grafts, the donor and recipient will always have one haplotype in common (50% match), which is why these grafts are so common. However, in this case, the donor and recipient are still nearly always mismatched for all or most alleles inherited from the other parent, providing a target for the immune system. Role of Blood Group and MHC Antigens in Graft Tolerance The most intense graft rejection reactions are due to differences between donor and recipient in ABO blood-group and MHC antigens. The blood-group antigens are expressed on RBCs, epithelial cells, and endothelial cells, requiring the donor and recipient to first be screened for ABO compatibility. If the recipient carries antibodies to any of these antigens, the transplanted tissue will induce rapid antibody-mediated lysis of the incompatible donor cells. For this reason, most transplants are conducted between individuals with a matching ABO blood group. Next, the MHC compatibility between potential donors and a recipient is determined. The first choice is usually parents or first-order siblings with at least a partial MHC match, followed by other family members and even friends. Given our current success with immunosuppression and immune

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tolerance induction protocols, solid organ transplants between individuals with even a total HLA mismatch can be successful. The most rigorous testing is conducted in bone marrow transplants, where at least partial HLA matching is crucial. Several tests can be used to determine the HLA compatibility, and the choice is somewhat dependent on the organ or tissue in question. Molecular assays using sequence-specific primers to establish which HLA alleles are expressed by the recipient and potential donors (called tissue typing) has become more common in recent years, especially in bone marrow transplantation. Molecular assays provide greater specificity and higher resolution than assays that characterize MHC molecules serologically, using antigen-antibody interactions alone, which was standard practice in the past. The presence of any preformed antibodies against potential donor HLA alloantigens must also be evaluated in the recipient. We generate antibodies against nonself HLA proteins for a number of reasons, but transplant recipients who have received prior allografts are especially likely to possess them. Testing for this is called cross-matching, and is the most important level of compatibility testing that occurs prior to solid organ transfer; a positive cross-match means that the recipient has antibodies against HLA proteins carried by the donor. The most common method used today is the Luminex assay, which employs fluorochrome-labeled microbeads impregnated with specific HLA proteins (Figure 16-13). Each HLA protein is associated with a fluorochrome of a different intensity. These HLA-impregnated beads are mixed with recipient serum, allowing clinicians to determine more precisely which donor specific anti-HLA antibodies are present HLA antigen Luminex bead

Recipient serum anti-HLA antibody

PE-labeled secondary antibody (anti-IgG)

FIGURE 16-13 The Luminex cross-matching assay. Microbeads impregnated with fluorochromes of different intensity each carry a different HLA protein. Recipient serum is incubated with these beads, and any antibody binding is detected using phycoerythrin (PE)-labeled secondary anti-human immunoglobulin. Laser excitation and detection are used to determine the fluorochrome intensity of bound beads, and therefore the associated HLA molecule(s) with which the serum reacts. [B. D. Tait, 2009, Luminex technology for HLA antibody detection in organ transplantation. Nephrology 14:247–254, with modifications.]

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in the recipient prior to transplantation. The importance of careful cross-matching was shown in a seminal 1969 study, where up to 80% of kidney transplant patients with a positive cross-match experienced almost immediate transplant rejection, while only 5% of patients with a negative cross-match had this outcome. The MHC makeup of donor and host is not the sole factor determining tissue acceptance. Even when MHC antigens are identical, the transplanted tissue can be rejected because of differences at various other loci, including the minor histocompatibility locus. As described in Chapter 8, the major histocompatibility antigens are recognized directly by TH and TC cells, a phenomenon termed alloreactivity. In contrast, minor histocompatibility antigens are recognized only when peptide fragments are presented in the context of selfMHC molecules. Rejection based on only minor histocompatibility differences is usually less vigorous but can still lead to graft rejection. Therefore, even in cases of HLA-identical matches, some degree of immune suppression is usually still required. The Sensitization Stage of Graft Rejection Graft rejection occurs in stages and can be caused by both humoral and cell-mediated immune responses to alloantigens (primarily, MHC molecules) expressed on cells of the graft. Antibody-mediated, DTH, and cell-mediated cytotoxicity reactions have all been implicated, but the latter are primarily credited with orchestrating this response. As we discuss later, immediate hyperacute rejection is caused primarily by preexisting anti-HLA antibodies. When graft rejection occurs in the absence of this preexisting immunity, it can be divided into two stages: (1) a sensitization phase, which occurs shortly after transplantation when antigenreactive lymphocytes of the recipient proliferate in response to alloantigens on the graft, and (2) a later effector stage, in which immune destruction of the graft takes place. During the sensitization phase, CD4 and CD8 T cells recognize alloantigens expressed on cells of the foreign graft and proliferate in response. Both major and minor histocompatibility alloantigens can be recognized. In general, the response to minor histocompatibility antigens is weak, although the combined response to several minor differences can be quite vigorous. The response to major histocompatibility antigens involves recognition of either the donor MHC molecule directly (direct presentation) or recognition of peptides from donor HLA in the cleft of the recipient’s own MHC molecules (indirect presentation). A host TH cell becomes activated when it interacts with an APC that both expresses an appropriate antigenic-ligand/ MHC-molecule complex and provides the requisite costimulatory signal. Because DCs are found in most tissues and constitutively express high levels of class II MHC molecules, activated allogeneic donor DCs can mediate direct presentation in grafts or in the draining lymph node, to which they can sometimes migrate. APCs of host origin can also migrate into a graft and endocytose the foreign alloantigens (both

major and minor histocompatibility molecules), where they become activated and present alloantigens indirectly as processed peptides bound to self-MHC molecules. The crosspresentation ability of DCs (see Chapter 8) also allows them to present endocytic antigens in the context of class I MHC molecules to CD8 T cells, which can then participate in allograft rejection. In addition to DCs, other cell types have been implicated in alloantigen presentation and immune activation leading to graft rejection, including Langerhans cells and endothelial cells lining the blood vessels. Both of these cell types express class I and class II MHC antigens. However, as we know from Chapter 11, T cells that respond to antigen via the TCR in the absence of costimulation or danger signals can become tolerant. This may help to explain a long-standing clinical observation: transfusion of donor blood into a graft recipient prior to transplantation can facilitate acceptance of a subsequent graft from that donor. This suggests that exposure to donor cells in this nonactivating context induced tolerance to donor alloantigens. Newer immunomodulation protocols based on this observation, as well as related experimental studies, are currently underway to design techniques to effectively induce donorspecific tolerance prior to engraftment. Effector Stage of Graft Rejection A variety of mechanisms participate in the effector stage of allograft rejection (Figure 16-14). The most common are cellmediated reactions; less common mechanisms (except during hyperacute rejection) are antibody-mediated complement lysis and destruction by ADCC. The hallmark of graft rejection involving cell-mediated reactions is an influx of immune cells into the graft. Among these are T cells, especially CD4, and APCs, often macrophages. Histologically, the infiltration in many cases resembles that seen during a DTH response, in which cytokines produced by T cells promote inflammatory cell infiltration (see Figure 15-14). Although probably less important, recognition by host CD8 T cells of either foreign class I alloantigens on the graft or alloantigenic peptides cross-presented in the context of class I MHC by DCs can lead to CTL-mediated killing. In each of these effector mechanisms, cytokines secreted by TH cells play a central role. For example, IL-2 and IFN- produced by TH1 cells have been shown to be important mediators of graft rejection. These two cytokines promote T-cell proliferation (including CTLs), DTH responses, and the synthesis of IgG by B cells, with resulting complement activation. A number of cytokines that encourage the expression of MHC class I and class II molecules (e.g., the interferons and TNFs) increase during graft rejection episodes, inducing a variety of cell types within the graft to increase surface expression of these proteins. Many of the cytokines most closely associated with TH2 and TH17 cells have also been implicated in graft rejection. Elevations in IL-4, -5, and -13, responsible for B-cell activation and eosinophil accumulation in allografts, and in IL-17, have all been linked to transplant rejection. Recent studies showing that

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APC

TH cell

IL–2, IL–4, IL–5, IL–6

IL–2

TDTH

Activated macrophage

IL–2

CD8+ TC

CD4+ TC

B cell

IFN–γ Lymphotoxin–α

Cytotoxicity



MHC expression

CD8+ CTL

Lytic enzymes

NK cell or macrophage

Complement Membrane damage

CD4+ CTL Lysis

Class I MHC alloantigen

Class II MHC alloantigen

ADCC Fc receptor

Graft

FIGURE 16-14 Effector mechanisms involved in allograft rejection. The generation or activity of various effector cells depends directly or indirectly on cytokines (blue) secreted by activated TH cells. ADCC  antibody-dependent cell-mediated cytotoxicity. [© 2013 W. H. Freeman and Company.]

neutralization of IL-17 could extend the survival of cardiac allografts in the mouse have generated much interest in this cytokine and in the role of TH17 cells in graft rejection. Finally, antibody-mediated rejection (AMR), although less frequent, is still a major issue in clinical transplantation. These antibodies are often directed against donor HLA molecules or endothelial antigens. The hallmarks of this response, which is dependent on T-cell maintenance of these alloreactive B cells, are the activation of complement and deposition of C4d, especially among endothelial cells lining graft capillaries. AMR, although most associated with the earliest stages of rejection, can occur at any time during the clinical course of allograft rejection.

Graft Rejection Follows a Predictable Clinical Course Graft rejection reactions, although somewhat variable in their time courses depending on the type of tissue transferred and the immune response involved, follow a fairly predictable course. Hyperacute rejection reactions typically occur within the first 24 hours after transplantation, acute rejection reactions usually begin in the first few weeks after transplantation, and chronic rejection reactions can occur from months to years after transplantation. Careful crossmatching can avoid most cases of hyperacute rejection. Current immunosuppressive agents have greatly advanced our

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2

Antibodies bind to antigens of renal capillaries and activate complement (C– )

1 Preexisting host antibodies are carried to kidney graft

C

C

Capillary endothelial walls

C

Kidney graft 3

Complement split products attract neutrophils, which release lytic enzymes

Enzymes

4

FIGURE 16-15 Steps in the hyperacute rejection of

Neutrophil lytic enzymes destroy endothelial cells; platelets adhere to injured tissue, causing vascular blockage

Platelets

a kidney graft. [© 2013 W. H. Freeman and Company.]

ability to reduce instances of acute rejection, although chronic rejection still remains a major problem.

isting antibodies that cross-react with common antigens of the donor species that are not present in the recipient species.

Hyperacute Rejection by Preexisting Antibodies In rare instances, a transplant is rejected so quickly that the grafted tissue never becomes vascularized. These hyperacute reactions are caused by preexisting host serum antibodies specific for antigens of the graft and have the greatest impact in highly vascularized grafts (such as kidney and heart). Preexisting recipient antibodies bind to HLA antigens on the endothelial cells of the graft. These antigen-antibody complexes activate the complement system and result in an intense accumulation of neutrophils. The ensuing inflammatory reaction causes endothelial damage and obstructing blood clots within the capillaries, preventing vascularization of the graft (Figure 16-15). Several mechanisms can account for the presence of preexisting antibodies specific for allogeneic MHC antigens. These include repeated blood transfusions that induced antibodies to MHC antigens expressed on allogeneic WBCs in the blood; repeated pregnancies, in which women develop antibodies against paternal alloantigens of the fetus; exposure to infectious agents, which can elicit MHC cross-reactive antibodies; or a previous transplant, which induced high levels of antibodies to the allogeneic MHC antigens present in that graft. In some cases, preexisting antibodies specific for blood-group antigens may also be present and can mediate hyperacute rejection. However, with careful cross-matching and ABO blood-group typing, many instances of hyperacute rejection can be avoided. Xenotransplants (see below) are also often rejected in a hyperacute manner because of preex-

Acute Rejection Mediated by T-Cell Responses Cell-mediated allograft rejection manifests as an acute rejection of the graft beginning about 7 to 10 days after transplantation (see Figure 16-10b). Histopathologic examination reveals a massive infiltration of macrophages and lymphocytes at the site of tissue destruction, suggestive of TH-cell activation and proliferation. Acute graft rejection occurs by the mechanisms described for the effector stage of graft rejection (see Figure 16-14). Acute AMR may also be involved during this stage; it is the suggested cause of 20% to 30% of acute rejection cases. Chronic Rejection Phase Chronic rejection reactions develop months or years after acute rejection reactions have subsided. The mechanisms include both humoral and cell-mediated responses by the recipient. Although immunosuppressive drugs and advanced tissue-typing techniques have dramatically increased survival of allografts during the first years, little progress has been made in long-term survival. In data collected in the United States as of 2008, 1-year kidney graft survival rates approached 97%. However, even in cases of a living donor—the most ideal scenario—10-year survival rates were only 60% (based on procedures performed in 2000). Immunosuppressive drugs usually do little to manage chronic rejection, which not infrequently necessitates another transplant. In up to 60% of cases in which there is some form of chronic allograft dysfunction, antidonor antibodies can be found in the recipient, suggesting that in addition to cell-mediated responses, AMR may also be involved in chronic rejection.

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Allogeneic transplantation always requires some degree of immunosuppression if the transplant is to survive. Most immunosuppressive treatments are nonspecific, resulting in generalized suppression of responses to all antigens, not just those of the allograft. This places the recipient at increased risk of infection and cancer. In fact, infection is the most common cause of transplant-related death. Many immunosuppressive measures slow the proliferation of activated lymphocytes, thus affecting any rapidly dividing nonimmune cells (e.g., gut epithelial cells or bone marrow hematopoietic stem cells), and leading to serious or even life-threatening complications. Patients on long-term immunosuppressive therapy are also at increased risk of hypertension and metabolic bone disease. Fine-tuning of their immunosuppressive cocktail, and eventual weaning off these drugs, is an ongoing process for most, if not all, transplant recipients. Total Lymphoid Irradiation to Eliminate Lymphocytes Because lymphocytes are extremely sensitive to x-rays, x-irradiation can be used to eliminate them in the transplant recipient just before grafting. Although not a part of most immunosuppressive regimens, this is often used in bone marrow transplantation or to treat graft-versus-host disease (GVHD), in which the graft rejects the host. In total lymphoid irradiation, the recipient receives multiple x-ray exposures to the thymus, spleen, and lymph nodes before the transplant, and the recipient is engrafted in this immunosuppressed state. Because the bone marrow is not x-irradiated, lymphoid stem cells proliferate and renew the population of recirculating lymphocytes. These newly formed lymphocytes appear to be more likely to become tolerant to the antigens of the graft. Generalized Immunosuppressive Therapy In 1959, Robert Schwartz and William Dameshek reported that treatment with 6-mercaptopurine suppressed immune responses in animal models. Joseph Murray and colleagues then screened a number of its chemical analogues for use in human transplantation. One, azathioprine, when used in combination with corticosteroids, dramatically increased survival of allografts. Murray received a Nobel prize in 1991 for this clinical advance, and the developers of the drug, Gertrude Elion and George Hitchings, received the Nobel prize in 1987. Azathioprine (Imuran) is a potent mitotic inhibitor often given just before and after transplantation to diminish both B- and T-cell proliferation. Other mitotic inhibitors that are sometimes used in conjunction with immunosuppressive agents are cyclophosphamide and methotrexate. Cyclophosphamide is an alkylating agent that inserts into the DNA helix and becomes cross-linked, leading to disruption of the DNA chain. It is especially effective against rapidly dividing cells and is therefore sometimes given at the time of grafting to block T-cell proliferation. Methotrexate acts as a folic-acid antagonist

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to block purine biosynthesis. Because mitotic inhibitors act on all rapidly dividing cells, they cause significant side effects, especially affecting the gut and liver, in addition to their target, bone marrow-derived cells. Most often, these mitotic inhibitors are combined with immunosuppressive drugs such as corticosteroids (e.g., prednisone and dexamethasone). These potent anti-inflammatory agents exert their effects at many levels of the immune response and therefore help prevent acute episodes of graft rejection. More specific immune suppression became possible with the development of several fungal metabolites, including cyclosporin A (CsA), FK506 (tacrolimus), and rapamycin (also known as sirolimus). Although chemically unrelated, these exert similar effects, blocking the activation and proliferation of resting T cells. Some of these also prevent transcription of several genes encoding important T-cell activation molecules, such as IL-2 and the high-affinity IL-2 receptor (IL-2R). By inhibiting TH-cell proliferation and cytokine expression, these drugs reduce the subsequent activation of various effector populations involved in graft rejection, making them a mainstay in heart, liver, kidney, and bone marrow transplantation. In one study of 209 kidney transplants from deceased donors, the 1-year survival rate was 80% among recipients receiving CsA and 64% among those receiving other immunosuppressive treatments. Similar results have been obtained with liver transplants (Figure 16-16). Despite these impressive results, CsA does have some side effects, most notably toxicity to the kidneys. FK506 and rapamycin are 10 to 100 times more potent immunosuppressants than CsA and therefore can be administered at lower doses and with fewer side effects. 100 90 80 70 Survival, %

Immunosuppressive Therapy Can Be Either General or Target-Specific

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60 50 40 30 20 10 0 1

3 6 12 24 Time after transplantation, months

36

FIGURE 16-16 Comparison of the survival rates of liver transplants following azathioprine versus cyclosporin A treatment. Transplant survival rates are shown over a 3-year period for 84 liver transplant patients immunosuppressed using a combination of azathioprine plus corticosteroids (black) compared with another 55 patients treated with cyclosporin A plus corticosteroids (blue). [Sabesin SM, Williams JW. 1987. Current status of liver transplantation. Hospital Practice 22:75–86.]

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Specific Immunosuppressive Therapy The ideal immunosuppressant would be antigen-specific, inhibiting the immune response to the alloantigens present in the graft while preserving the recipient’s ability to respond to other foreign antigens. Although this goal has not yet been achieved, several more targeted immunosuppressive agents have been developed. Most involve the use of monoclonal antibodies (mAbs) or soluble ligands that bind specific cell-surface molecules. One limitation of most first-generation mAbs came from their origin in animals. Recipients of these frequently developed an immune response to the nonhuman epitopes, rapidly clearing the mAbs from the body. This limitation has been overcome by the construction of humanized mAbs and mouse-human chimeric antibodies (see Chapter 20). Many different mAbs have been tested in transplantation settings, and the majority work by either depleting the recipient of a particular cell population or by blocking a key step in immune signaling. Antithymocyte globulin (ATG), prepared from animals exposed to human lymphocytes, can be used to deplete lymphocytes in recipients prior to transplantation, but has significant side effects. A more subset-specific strategy uses a mAb to the CD3 molecule of the TCR, called OKT3, and rapidly depletes mature T cells from the circulation. This depletion appears to be caused by binding of antibody-coated T cells to Fc receptors on phagocytic cells, which then phagocytose and clear the T cells from the circulation. In a further refinement of this strategy, a cytotoxic agent such as diphtheria toxin is coupled with the mAb. Antibody-bound cells then internalize the toxin and die. Another technique uses mAbs specific for the high-affinity IL-2 receptor CD25 (Basiliximab). Since this receptor is expressed only on activated T cells, this treatment specifically blocks proliferation of T cells activated in (a)

response to the alloantigens of the graft. However, since TREG cells also express CD25 and may aid in alloantigen tolerance, this strategy may have drawbacks. More recently, a mAb against CD20 (Rituximab) has been used to deplete mature B cells and is aimed at suppressing AMR responses. Finally, in cases of bone marrow transplantation, mAbs against T-cellspecific markers have been used to pretreat the donor’s bone marrow to destroy immunocompetent T cells that may react with the recipient tissues, causing GVHD (described below). Because cytokines appear to play an important role in allograft rejection, these compounds can also be specifically targeted. Animal studies have explored the use of mAbs specific for the cytokines implicated in transplant rejection, particularly TNF-, IFN-, and IL-2. In mice, anti-TNF- mAbs prolong bone marrow transplants and reduce the incidence of GVHD. Antibodies to IFN- and to IL-2 have each been reported in some cases to prolong cardiac transplants in rats. As described in Chapter 11, TH-cell activation requires a costimulatory signal in addition to the signal mediated by the TCR. The interaction between CD80/86 on the membrane of APCs and the CD28 or CTLA-4 molecule on T cells provides one such signal (see Figure 11-3). Without this costimulatory signal, antigen-activated T cells become anergic (see Figure 11-4). CD28 is expressed on both resting and activated T cells, while CTLA-4 is expressed only on activated T cells and binds CD80/86 with a 20-fold-higher affinity. In mice, D. J. Lenschow, J. A. Bluestone, and colleagues demonstrated prolonged graft survival by blocking CD80/86 signaling with a soluble fusion protein consisting of the extracellular domain of CTLA-4 fused to human IgG1 heavy chain (called CTLA4Ig). This new drug, belatacept, was shown to induce anergy in T cells directed against the grafted tissue and has been (b)

CD28

CD80/86

T cell

APC

CTLA-4Ig

T cells that recognize graft antigens become activated

T cells that recognize graft antigens lack costimulation and become anergic

Graft rejected

Graft survives

FIGURE 16-17 Blocking costimulatory signals at the time of transplantation can cause anergy instead of activation of the T cells reactive against the graft. T-cell activation requires both the interaction of the TCR with its ligand and the reaction of costimulatory receptors with their ligands (a). In (b), contact between one of the costimulatory receptors, CD28 on the T cell,

and its ligand, CD80/86 on the APC, is blocked by reaction of CD80/86 with the soluble ligand CTLA-4Ig. The CTLA-4 is coupled to an Ig H chain, which slows its clearance from the circulation. This process specifically suppresses graft rejection without inhibiting the immune response to other antigens. [© 2013 W. H. Freeman and Company.]

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CTLA-4Ig CD40

⫺ ⫺

CD40L

Anti-CD3 CD28 mAb Anti-CD40L ⫺ Costimulation TH cell

CTLA-4 ⫺ CTLA-4Ig

⫺ Anti-CD25 mAb

IL-2R␣ (CD25)

PLCγ

Cyclosporin A ⫺ Calcineurin FK506

Nucleotide synthesis



JAK3 ⫺ JAK3 inhibitor

Cell cycle

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antigens of the donor in a manner that causes immune tolerance rather than sensitization. One unique example of the latter, involving fetal exposure to foreign antigens, occurs in species where nonidentical twins share a placenta during fetal development (see Classic Experiments Box 16-3).

CD80/86

TCR

|

⫺ Rapamycin

Azathioprine Cyclophosphamide Mycophenolate mofetil Methotrexate

FIGURE 16-18 Sites of action for various agents used in clinical transplantation. [© 2013 W. H. Freeman and Company.]

approved by the FDA for prevention of organ rejection in adult kidney transplant patients (Figure 16-17). Some of the treatments used to suppress transplant rejection in clinical settings are summarized in Figure 16-18, along with their sites of action.

Immune Tolerance to Allografts Is Favored in Certain Instances Sometimes, an allograft may be accepted with little or no use of immunosuppressive drugs. Obviously, with tissues that lack alloantigens (e.g., cartilage or heart valves), no immunologic barrier to transplantation exists. Acceptance of an allograft can be favored in one of two situations: when cells or tissue are grafted to a so-called privileged site that is sequestered from immune system surveillance, or when a state of tolerance has been induced biologically, usually by previous exposure to the

Cells and Cytokines Associated with Graft Tolerance There is now significant evidence that FoxP3-expressing TREG cells play a role in transplantation tolerance. In clinical operational tolerance, where the graft survives despite the removal of all immunosuppressive therapy, there is an increase in the number of TREG cells in the circulation and in the graft. These cells are believed to inhibit alloreactive cells using a combination of direct contact and expression of immunosuppressive cytokines, such as TGF-, IL-10, and IL-35. To date, difficulties identifying and isolating this population of T cells have limited their use as a treatment for inducing transplant tolerance. However, strategies that use existing or induced TREG cells to limit graft rejection, especially in GVHD, are an active area of research. Immunologically Privileged Sites An allograft placed in an immunologically privileged site, or an area without significant immune cell access (e.g., the anterior chamber of the eye, cornea, uterus, testes, and brain), is less likely to experience rejection. Each of these sites is characterized by an absence of lymphatic vessels, and sometimes also blood vessels. Consequently, the alloantigens of the graft are not able to sensitize the recipient’s lymphocytes, and the graft has an increased likelihood of acceptance even when HLA antigens are not matched. The privileged status of the cornea has allowed corneal transplants to be highly successful. Ironically, the successful transplantation of allogeneic pancreatic islet cells into the thymus in a rat model of diabetes suggests that the thymus may also be a unique type of immunologically privileged site. Immunologically privileged sites fail to induce an immune response because they are effectively sequestered from the cells of the immune system. This suggests the possibility of physically sequestering grafted cells. In one study, pancreatic islet cells were encapsulated in semipermeable membranes and then transplanted into diabetic mice. The islet cells survived and produced insulin. The transplanted cells were not rejected, because the recipient’s immune cells could not penetrate the membrane. This novel transplant method may have application for treatment of human diabetics. Inducing Transplantation Tolerance Methods for inducing tolerance to allow acceptance of allografts have been studied extensively in animal models, and some of the discoveries have now been applied to humans. The current favorite involves the induction of a state of mixed hematopoietic chimerism, where donor and recipient hematopoietic cells coexist in the host prior to transplantation. The seed for this strategy originated from

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CLASSIC EXPERIMENT

Early Life Exposure to Antigens Favors Tolerance Induction In 1945, Ray Owen, an immunologist working at the California Institute of Technology, reported a novel observation in cattle. His discovery would advance our understanding of immune tolerance and provide grist for many transplant immunologists who followed in his footsteps. He noticed that nonidentical or dizygotic cattle twins retained the ability to accept cells or tissue from their genetically distinct sibling throughout their lives. This was not true for the nonidentical twins of other mammalian species that did not share a placenta in utero. In cattle, the shared placenta allowed free blood circulation from one twin to the other throughout the embryonic and fetal period. Although the twins may have inherited distinct paternal and maternal antigens, they did not recognize those of their placental partner as foreign and could therefore later accept grafts from them. He hypothesized that exposure to the alloantigens of their placental sibling during this early stage of life somehow

induced a lifelong state of immune tolerance to these antigens; in other words, these alloantigens were treated as self. In 1953, Owen’s observations were extended in a seminal paper by Rupert Billingham, Leslie Brent, and Peter Medawar. They showed that inoculation of fetal mice with cells from a genetically distinct donor mouse strain led to subsequent acceptance of skin grafts from donor mice of the same strain. This and other work led to the hypothesis that fetal development is an immunologically privileged period, during which exposure to an antigen induces tolerance to that antigen later in life. Peter Medawar shared the 1960 Nobel Prize in Physiology or Medicine with Sir Frank MacFarlane Burnet, for their shared work in the discoveries that led to our understanding of acquired immunological tolerance. Although no experimental data are available to demonstrate such specific tolerance in humans, there is anecdotal evidence. For example, transplants in very

animal studies and observations in humans. For instance, transplant recipients who underwent total myeloablative therapy followed by donor bone marrow transfer prior to receiving a solid organ from the same donor displayed enhanced tolerance for the solid organ graft. A modified protocol involving a less intense non-myeloablative procedure followed by bone marrow transfer resulted in mixed chimerism that, even when quite transient, was still associated with improved graft outcomes. The mechanism for this induction of tolerance is still unclear; both central deletion of alloreactive T cells and an enhancement of immune suppression by TREG cells are hypothesized.

Some Organs Are More Amenable to Clinical Transplantation Than Others For a number of illnesses, a transplant is the only means of therapy. Figure 16-19 summarizes the major organ and cell transplants being performed today. Certain combinations of organs, such as heart and lung or kidney and pancreas, are being transplanted simultaneously with increasing frequency. Since the first kidney transplant was performed in the 1950s, it is estimated that over 500,000 kidneys have been

young children show a slightly higher success rate than those in older individuals, suggesting that early life exposure to antigens may bias toward tolerance induction in humans as well. There are also clinical examples in adults where allografts mismatched at a single HLA locus are accepted with little or no immune suppression. When this mismatched antigen happens to be expressed by the transplant recipient’s mother, it is possible that perinatal exposure to this maternal antigen induced subsequent tolerance to the alloantigen. Because human maternal cells do not normally cross the placental barrier, the mechanism for such specific tolerance to noninherited maternal antigens is unknown. Billingham, R. E., L. Brent, P. B. Medawar. (1953). ‘Actively acquired tolerance’ of foreign cells. Nature 172:603–606. Owen, R. D. (1945). Immunogenetic consequences of vascular anastomoses between bovine twins. Science 102:400–401.

transplanted worldwide. The next most frequently transplanted solid organ is the liver, followed by the heart, the lung, and the pancreas. In 2011, over 28,000 solid organ transplants were performed in the United States, in addition to more than 46,000 corneal tissue grafts. Although the clinical outcomes have improved considerably in the past few years, major obstacles still exist. Immunosuppressive drugs greatly increase the short-term survival of the transplant, but medical problems arise from their use and chronic rejection remains a lingering problem. The need for additional transplants after rejection exacerbates the shortage of organs that is a major obstacle to the widespread use of transplantation. Research on artificial organs continues, but there are no reports of universal long-term successes. This makes the idea of looking outside our species more compelling to some (see Clinical Focus Box 16-4). The frequency with which a given organ or tissue is transplanted depends on a number of factors, including alternative treatment options, organ availability, and the level of difficulty of the procedure. Several factors contribute to the kidney being the most commonly transplanted organ. Many common diseases (e.g., diabetes) result in kidney failure that can be alleviated by transplantation. Because kidneys come in pairs and we can survive with only one, this organ is

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Transplants Performed in 2011

Cornea: 46196, cadaver Skin grafts: Mostly autologous, number n/a

Blood: An estimated 15 million units of RBCs

Lung: 1821-cadaver, 1-living Heart and Lung: 27-cadaver

Pancreas: 28-cadaver Kidney and Pancreas: 795-cadaver

Heart: 2322-cadaver

Kidney: 11043-cadaver, 5771-living

Liver: 6095-cadaver, 24-living

Hematopoietic Stem Cell Transfer (bone marrow or cord blood): >20,000-living donations

FIGURE 16-19 Transplantation routinely conducted in clinical practice. For the solid organs, the number of transplants performed in the United States in 2011 is indicated. Estimates are included for other transplants if available. [© 2013 W. H. Freeman and Company.]

available from living as well as deceased donors. In most transplant situations, transfer from a living donor affords enhanced chances of graft survival. Surgical procedures for kidney transfer are also simpler than for the liver or heart. Because many transplants of this type have been conducted for many years, patient-care procedures and effective immunosuppressive regimens are well established. Matching of blood and histocompatibility groups presents no special problems, and transplants can even be conducted across significant mismatches. This contrasts with bone marrow transplants, which must be at least partially matched. The major problems faced by patients waiting for a kidney are the shortage of organs and the increasing number of sensitized recipients. The latter results from rejection of a first transplant, which leaves the recipient sensitized to the alloantigens in that graft. As with all nonidentical transplants, it is typically necessary to maintain kidney transplant patients on some form of immunosuppression for their entire lives. Unfortunately, this gives rise to complications, including risks of cancer and infection, as well as other side effects such as hypertension and metabolic bone disease.

After the kidney, bone marrow is the most frequent transplant. This procedure is increasingly used to treat hematologic diseases, including leukemia, lymphoma, and immunodeficiencies, especially severe combined immunodeficiency (SCID; see Chapter 18). Although the supply of bone marrow, which is a renewing resource, is less of a problem than is the supply of kidneys, finding a matched donor is a major obstacle. However, current tissue typing techniques can quickly identify donors with at least partial HLA matches. Bone marrow transplant recipients are typically immunologically suppressed before transfer, making graft rejection rare. However, the presence of foreign immunocompetent cells means that GVHD is a real risk, although the use of immunosuppressive drugs and pretreatment to deplete T cells from the graft have improved outcomes. Perhaps the most dramatic forms of transplantation involve the transfer of heart, lung, or both: situations where the recipient must be kept alive via artificial means during surgery. The human heart can remain viable for a limited time in ice-cold buffer solutions, which delay tissue damage. However,

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BOX 16-4

CLINICAL FOCUS

Is There a Clinical Future for Xenotransplantation? Unless organ

donations increase drastically, most of the over 116,000 U.S. patients still on the waiting list for a transplant at the end of 2012 will not receive one. In fact, fewer than 30,000 donor organs will become available in time to save these waiting patients. The disparity in numbers of individuals on a waiting list for a transplant and the number of available organs grows every year, making it increasingly unlikely that human organs can fill this need. One solution to this shortfall is to utilize animal organs, a process called xenotransplantation. Clinical attempts at using nonhuman primates as donors led to some short-term success, including function of a baboon liver for 70 days and a chimpanzee kidney for 9 months in human recipients. Although there are advantages in the phylogenetic similarity between primate species, the use of nonhuman primates as organ donors has several important disadvantages. This similarity carries with it elevated risk in terms of the transfer of pathogenic viruses, not to mention the impracticalities and ethical concerns that arise in the use of these close cousins. The use of pigs to supply organs for humans has been under serious consideration for many years. Pigs breed rapidly, have large litters, can be housed in pathogen-free environments, and share considerable anatomic and physiologic similarity with humans. In fact, pigs have served as donors of cardiac valves for humans for years. However, balancing the advantages of pig donors are several serious difficulties. For example, if a pig kidney were implanted into a human by techniques standard for human transplants, it would likely fail in a rapid and dramatic fashion due to hyperacute rejection. This antibody-mediated rejection is due to the presence on the pig

cells (and cells of most mammals except humans and the highest nonhuman primates) of a disaccharide antigen called galactosyl--1,3-galactose (Gal1,3Gal). The presence of this antigen on many microorganisms means that nearly everyone has been exposed and has formed antibodies against it. The preexisting antibodies crossreact with pig cells, which are then lysed rapidly by complement. The absence of human regulators of complement activity on the pig cells, including human decay accelerating factor (DAF) and human membrane cofactor protein (MCP), intensifies the complement lysis cycle (see Chapter 6 for descriptions of DAF and MCP). How can this major obstacle be circumvented? Strategies for absorbing the antibodies from the circulation on solid supports and the use of soluble gal-gal disaccharides to block antibody reactions were both tested. A more elegant solution involved genetically engineered pigs in which the gene for the enzyme responsible for the addition of Gal1,3Gal to pig proteins was knocked out. These galactosyl transferase gene knockout (GalT-KO) pigs have been used as heart or kidney donors for baboons in experimental systems. K. Kuwaki and colleagues transplanted GalT-KO pig hearts into baboons immunosuppressed with antithymocyte globulin and an antiCD154 monoclonal antibody (the CD40 ligand found mostly on T cells) and then maintained with commonly used immunosuppressive drugs. The mean survival time was 92 days, and one GalT-KO pig heart transplant survived in a baboon for 179 days. K. Yamada and coworkers demonstrated kidney function in recipients of GalT-KO pig kidneys for up to 83 days using a regimen of simultaneous thymus transplant in an attempt to establish tolerance in

eventually lack of oxygen (ischemia) and the resulting deprivation of ATP lead to irreversible organ death. The surgical methods for implanting a heart have been available since the first heart transplant was carried out in 1964 in South Africa by Dr. Christian Barnard. Today, the 1-year survival rate for

the baboon recipients. Although these studies were not conclusive, some promising results have encouraged further exploration of the use of pigs for xenotransplantation in a clinical setting. Even if all issues of antigenic difference were resolved, additional concerns remain for those considering pigs as a source of transplanted tissue. Pig endogenous retroviruses introduced into humans as a result of xenotransplantation could cause significant disease. Opponents of xenotransplantation raise the specter of another HIV-type epidemic resulting from human infection by a new animal retrovirus. Continuing work on development of pigs free of endogenous pig retroviruses could reduce the possibility of this bleak outcome. Will we see the use of pig kidneys in humans in the near future? The increasing demand for organs is driving the commercial development of colonies of pigs suitable for such purposes. Although kidneys are the most sought-after organ at present, other organs and cells from the specially bred and engineered animals will find use if they are proven to be safe and effective. A statement issued in 2000 from the American Society of Transplantation and the American Society of Transplant Surgeons endorses the use of xenotransplants if certain conditions are met, including the demonstration of feasibility in a nonhuman primate model, proven benefit to the patient, and lack of infectious disease risk. Although certain barriers remain to the clinical use of xenotransplants, serious efforts are in motion to overcome these difficulties. Ekser B and Cooper D. 2010. Overcoming barriers to xenotransplantation: prospects for the future. Expert Reviews in Clinical Immunology, 6(2):219–230.

heart transplants has climbed to greater than 80%. Brain-dead accident victims with an intact circulatory system and a functioning heart are the typical source of these organs. HLA matching is often not possible because of the limited supply of these organs and the urgency of the procedure.

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Tolerance, Autoimmunity, and Transplantation The liver is important because it clears and detoxifies substances in the body. Malfunction of this organ can be caused by viral diseases (e.g., hepatitis) or exposure to harmful chemicals (e.g., chronic alcoholism), although most liver transplants are actually performed to correct congenital abnormalities. This organ has a complicated circulatory network, posing some unique technical challenges. However, its large size also presents opportunity; the liver from a single donor can often be split and given to at least two different recipients. One of the more common diseases in the United States is diabetes mellitus. This disease is caused by malfunction of insulin-producing islet cells in the pancreas. Newer protocols that avoid whole-organ transfer involve harvesting donor islet cells and perfusing them into the recipient’s liver, where they become permanently established in the liver sinusoids. Initial results indicate that 53% of recipients are insulin independent after such a transplant, some for up to 2 years. Several factors favor survival of functioning pancreatic cells, the most important being the condition of the islet cells used for implantation.

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Most skin transplants are conducted with autologous tissue. However, after severe burns, foreign skin thawed from frozen deposits may also be used. These grafts generally act as biologic dressings because the cellular elements are no longer viable and the graft does not grow in the new host. True allogeneic skin grafting using fresh viable donor skin has been undertaken, but rejection is a significant issue that must be managed with aggressive immunosuppressive therapy, which unfortunately also increases the already high infection risk. This list of commonly transplanted tissue is in no way comprehensive, and will surely expand with time. Improved procedures for inducing tolerance and controlling rejection, along with any advances in future organ availability, would add significantly to this list. For instance, the recent use of intracerebral neural cell grafts has restored function in victims of Parkinson’s disease. In studies conducted thus far, the source of neural donor cells was human embryos; the possibility of using those from other animal species is being tested. Likewise, the transfer of composite tissues (e.g., whole digit, limb, and even facial transplants) is still relatively rare and extremely complicated, but advances are being made.

S U M M A R Y ■











A major task of the immune system is to distinguish self from nonself. Failure to do so results in immune attacks against cells and organs of the host with the possible onset of autoimmune disease. Mechanisms to prevent self-reactivity (i.e., tolerance) operate at several levels. Central tolerance serves to delete self-reactive T or B lymphocytes; peripheral tolerance inactivates or regulates self-reactive lymphocytes that survive the initial screening process. Human autoimmune diseases can be divided into organspecific and systemic diseases. The organ-specific diseases involve an autoimmune response directed primarily against a single organ or gland. The systemic diseases are directed against a broad spectrum of tissues. There are both spontaneous and experimental animal models for autoimmune diseases. Spontaneous autoimmune diseases result from genetic defects, whereas experimental animal models have been developed by immunizing animals with self antigens in the presence of adjuvant. There is evidence for genetic and environmental influences on autoimmunity. In particular, certain alleles of MHC have been strongly linked to autoimmunity. In addition, defects in many different genes involved in immunity can predispose individuals to autoimmune disease. However, environmental factors, including microflora and infection, can also have impacts on autoimmune susceptibility. CD4 rather than CD8 T cells are most associated with autoimmunity. There is evidence for both TH1 and TH17 cells in the development of autoimmunity, depending on the disease in question.







A variety of mechanisms have been proposed for induction of autoimmunity, including release of sequestered antigens, molecular mimicry, and polyclonal stimulation of lymphocytes. Evidence exists for each of these mechanisms, reflecting the many different pathways leading to autoimmune reactions. Current therapies for autoimmune diseases include treatment with generally immunosuppressive drugs as well as treatments that inhibit specific cell types or pathways, such as B cells, T cells, adhesion molecules, costimulation, and TH17 cells. Strategies aimed at enhancement of TREG cells, induction of tolerance, and antigen-specific targeting are also under development. Graft rejection is an immunologic response displaying the attributes of specificity, memory, and self-nonself recognition. There are three major types of rejection reactions: ■

Hyperacute rejection, mediated by preexisting host antibodies against graft antigens



Acute graft rejection, in which TH cells and/or CTLs mediate tissue damage



Chronic rejection, which involves both cellular and humoral immune components



The immune response to tissue antigens encoded within the major histocompatibility complex is the strongest force in rejection.



The match between a recipient and potential graft donors is assessed by typing blood-group antigens and MHC antigens, and evaluating existing anti-donor antibodies (cross-matching).

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The process of graft rejection can be divided into a sensitization stage, in which T cells are stimulated, and an effector stage, in which they attack the graft.



In most clinical situations, graft rejection is suppressed by nonspecific immunosuppressive agents or by total lymphoid x-irradiation. Experimental approaches using monoclonal antibodies offer the possibility of more specific immunosuppression. These antibodies may act by: ■ Depleting certain populations of reactive cells ■ Blocking TCR engagement or interfering with costimulation ■ Inhibiting the trafficking of certain cell types ■ Interfering with specific cytokine signaling





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Certain sites in the body are immunologically privileged, including the cornea of the eye, brain, testes, and uterus,









and transplants in these sites may not be rejected despite genetic mismatch between donor and recipient. Specific tolerance to alloantigens can be induced by exposure to these antigens in utero or as a neonate. In some cases, prior exposure of adults to alloantigens in the form of hematopoietic cells, creating a state of mixed chimerism, can favor later success of grafts expressing these same alloantigens. Of all the organs or cell types amenable to transplantation, kidney transplants are the most common, and this organ is in the greatest demand. A major complication in bone marrow transplantation is GVHD, mediated by the lymphocytes contained within the donor marrow that target the recipient’s cells. The critical shortage of organs available for transplantation may be solved in the future by using organs from nonhuman species (xenotransplants).

R E F E R E N C E S Abdelnoor, A. M., et al. 2009. Influence of HLA disparity, immunosuppressive regimen used, and type of kidney allograft on production of anti-HLA class-I antibodies after transplant and occurrence of rejection. Immunopharmacology and Immunotoxicology 31(1):83–87. Anderson, M. S., et al. 2005. The cellular mechanism of Aire control of T cell tolerance. Immunity 23:227. Costa, V. S., T. C. Mattana, and M. E. da Silva. 2010. Unregulated IL-23/IL-17 immune response in autoimmune diseases. Diabetes Research and Clinical Practice 88(3):222–226. Chinen, J., and R. H. Buckley. 2010. Transplantation immunology: Solid organ and bone marrow. Journal of Allergy and Clinical Immunology 125(2 Suppl 2):S324-–335. Damsker, J. M., A. . Hansen, and R. R. 2010. Th1 and Th17 cells: Adversaries and collaborators. Annals of the New York Academy of Sciences 1183:211–221. Gorantla, V. S., et al. 2010. T regulatory cells and transplantation tolerance. Transplantation Reviews (Orlando) 24(3):147–159. Hafler, D. A., et al. 2005. Multiple sclerosis. Immunological Reviews 204:208. Hogquist, K. A., T. A. Baldwin, and S. C. Jameson. 2005. Central tolerance: Learning self-control in the thymus. Nature Reviews Immunology 5:772. Issa, F., A. Schiopu, and K. J. Wood. 2010. Role of T cells in graft rejection and transplantation tolerance. Expert Review of Clinical Immunology 6(1):155–169. 

Kapp, J. A., and R. P. Bucy. 2008. CD8 suppressor T cells resurrected. Hum Immunol 69(11):715–720. Kunz, M., and S. M. Ibrahim. 2009. Cytokines and cytokine profiles in human autoimmune diseases and animal models of autoimmunity. Mediators of Inflammation 2009:979258.

Lu, L., and H. Cantor. 2008. Generation and regulation of CD8() regulatory T cells. Cellular and Molecular Immunology 5(6):401–406. Pomié, C., I. Ménager-Marcq, and J. P. van Meerwijk. 2008. Murine CD8 regulatory T lymphocytes: The new era. Human Immunology 69(11):708–714. Ricordi, C., and T. B. Strom. 2004. Clinical islet transplantation: Advances and immunological challenges. Nature Reviews. Immunology 4:259. Rioux, J. D., and A. K. Abbas. 2005. Paths to understanding the genetic basis of autoimmune disease. Nature 435:584. Round, J. L., R. M. O’Connell, and S. K. Mazmanian. 2010. Coordination of tolerogenic immune responses by the commensal microbiota. Journal of Autoimmunity 34(3):J220–J225. Sakaguchi, S. 2004. Naturally arising CD4 regulatory T cells for immunologic self-tolerance and negative control of immune responses. Annual Review of Immunology 22:531. Sayegh, M. H., and C. B. Carpenter. 2004. Transplantation 50 years later—progress, challenges, and promises. New England Journal of Medicine 351:26. Steward-Tharp, S. M., Y. J. Song, R. M. Siegel, and J. J. O’Shea. 2010. New insights into T cell biology and T cell-directed therapy for autoimmunity, inflammation, and immunosuppression. Annals of the New York Academy of Sciences 1183:123–148. Tait, B. D. 2009. Solid phase assays for HLA antibody detection in clinical transplantation. Current Opinion in Immunology 21(5):573–577. Thomas, R. 2010. The balancing act of autoimmunity: Central and peripheral tolerance versus infection control. International Reviews of Immunology 29(2):211–233.

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Tolerance, Autoimmunity, and Transplantation Turka, L. A., and R. I. Lechler. 2009. Towards the identification of biomarkers of transplantation tolerance. Nature Reviews Immunology 9(7):521–526. Turka, L. A., K. Wood, and J. A. Bluestone. 2010. Bringing transplantation tolerance into the clinic: Lessons from the ITN and RISET for the Establishment of Tolerance consortia. Current Opinion in Organ Transplantation 15(4):441–448. Veldhoen, M. 2009. The role of T helper subsets in autoimmunity and allergy. Current Opinion in Immunology 21(6):606–611. von Boehmer, H., and F. Melchers 2010. Checkpoints in lymphocyte development and autoimmune disease. Nature Immunology 11(1):14–20. Waldmann, H., and S. Cobbold. 2004. Exploiting tolerance processes in transplantation. Science 305:209. Wing, K., and S. Sakaguchi. 2010. Regulatory T cells exert checks and balances on self tolerance and autoimmunity. Nature Immunology 11(1):7–13.

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www.niddk.nih.gov Home page for the National Institute of Diabetes and Digestive and Kidney Diseases. This site contains an exhaustive list of links to other diabetes healthrelated sites. www.unos.org The United Network for Organ Sharing site has information concerning solid-organ transplantation for patients, families, doctors, and teachers, as well as up-todate numbers on waiting patients. www.marrow.org The National Marrow Donor Program website contains information about all aspects of bone marrow transplantation. http://optn.transplant.hrsa.gov/data The Organ Procurement and Transplantation Network site is run by the U.S. Department of Health and Human Services. It maintains real-time numbers on waiting patients, as well as data on organ transplants in the United States.

www.who.int/transplantation/knowledgebase/en

www.lupus.org/index.html The site for the Lupus Foundation of America contains valuable information for patients and family members as well as current information about research in this area.

www.niams.nih.gov Home page for the National Institute of Arthritis and Musculoskeletal and Skin Diseases. This site contains links to other arthritis sites.

The World Health Organization runs this site as a clearinghouse of information relating to organ, tissue, and cell donation and transplantation worldwide.

www.immunetolerance.org This website, run by the U.S.-based Immune Tolerance Network, is aimed at translating basic research findings in tolerance induction into therapy for autoimmunity, allergy, and transplantation.

Q U E S T I O N S

CLINICAL FOCUS QUESTION What are some of the possible rea-

sons why females are more susceptible to autoimmune diseases than males? 1. Explain why all self-reactive lymphocytes are not elimi-

nated in the thymus or bone marrow. How are the surviving self reactors prevented from harming the host? 2. Why is tolerance critical to the normal functioning of the

immune system? 3. What is the importance of receptor editing to B-cell

tolerance? 4. For each of the following autoimmune diseases (a–j), select

the most appropriate characteristic (1–10) listed below. Disease a. _____ b. _____ c. _____ d. _____ e. _____ f. _____ g. _____

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www.srtr.org The Scientific Registry of Transplant Recipients is a national database of transplantation statistics.

Useful Websites

S T U D Y

|

Experimental autoimmune encephalitis (EAE) Graves’ disease Systemic lupus erythematosus (SLE) Type 1 diabetes mellitus (T1DM) Rheumatoid arthritis (RA) Hashimoto’s thyroiditis Experimental autoimmune myasthenia gravis (EAMG)

h. _____ i. _____ j. _____

Myasthenia gravis Multiple sclerosis (MS) Autoimmune hemolytic anemia

Characteristics (1) Auto-antibodies to acetylcholine receptor (2) TH1-cell reaction to thyroid antigens (3) Auto-antibodies to RBC antigens (4) T-cell response to myelin (5) Induced by injection of myelin basic protein (MBP) plus complete Freund’s adjuvant (6) Auto-antibody to IgG (7) Auto-antibodies to DNA and DNA-associated protein (8) Auto-antibodies to receptor for thyroid-stimulating hormone (9) Induced by injection of acetylcholine receptors (10) TH1-cell response to pancreatic beta cells 5. Experimental autoimmune encephalitis (EAE) has proved

to be a useful animal model of autoimmune disorders. a. Describe how this animal model is made. b. What is unusual about the animals that recover from EAE? c. How has this animal model indicated a role for T cells

in the development of autoimmunity?

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6. Molecular mimicry is one mechanism proposed to account

d. All allografts between individuals with identical HLA

for the development of autoimmunity. How has induction of EAE with myelin basic protein contributed to the understanding of molecular mimicry in autoimmune disease?

e. Cytokines produced by host TH cells activated in response

7. Describe at least three different mechanisms by which a

13. Indicate whether a skin graft from each donor to each

localized viral infection might contribute to the development of an organ-specific autoimmune disease.

recipient listed in the following table would result in rejection (R) or acceptance (A). If you believe a rejection reaction would occur, indicate whether it would be a first-set rejection (FSR), occurring in 12 to 14 days, or a second-set rejection (SSR), occurring in 5 to 6 days. All the mouse strains listed have different H-2 haplotypes.

8. Monoclonal antibodies have been administered for therapy

in various autoimmune animal models. Which monoclonal antibodies have been used, and what is the rationale for these approaches?

haplotypes will be accepted. to alloantigens play a major role in graft rejection.

9. Indicate whether each of the following statements is true or

false. If you think a statement is false, explain why. a. TH1 cells have been associated with development of

autoimmunity. b. Immunization of mice with IL-12 prevents induction of

EAE by injection of MBP plus adjuvant. c. The presence of the HLA B27 allele is diagnostic for ankylosing spondylitis, an autoimmune disease affecting the vertebrae. d. A defect in the gene encoding Fas can reduce programmed cell death by apoptosis. 10. For each of the following autoimmune disorders (a–d),

indicate which of the following treatments (1–5) may be appropriate: Disease a. Hashimoto’s thyroiditis b. Systemic lupus erythematosus c. Graves’ disease d. Myasthenia gravis Treatment (1) Cyclosporin A (2) Thymectomy (3) Plasmapheresis (4) Kidney transplant (5) Thyroid hormones 11. Which of the following are examples of mechanisms for the

development of autoimmunity? For each possibility, give an example. a. b. c. d. e.

Polyclonal B-cell activation Tissue damage Viral infection Increased expression of TCR molecules Increased expression of class II MHC molecules

12. Indicate whether each of the following statements is true or

false. If you think a statement is false, explain why. a. Acute rejection is mediated by preexisting host antibod-

ies specific for antigens on the grafted tissue.

Donor

Recipient

BALB/c

C3H

BALB/c

Rat

BALB/c

Nude mouse

BALB/c

C3H, had previous BALB/c graft

BALB/c

C3H, had previous C57BL/6 graft

BALB/c

BALB/c

BALB/c

(BALB/c x C3H)F1

BALB/c

(C3H x C57BL/6)F1

(BALB/c x C3H)F1

BALB/c

(BALB/c x C3H)F1

BALB/c, had previous F1 graft

14. Graft-versus-host disease (GVHD) frequently develops

after certain types of transplantations. a. Briefly outline the mechanisms involved in GVHD. b. Under what conditions is GVHD likely to occur? c. Some researchers have found that GVHD can be dimin-

ished by prior treatment of the graft with monoclonal antibody plus complement or with monoclonal antibody conjugated with toxins. List at least two cell-surface antigens to which monoclonal antibodies could be prepared and used for this purpose, and give the rationale for your choices. 15. What is the biologic basis for attempting to use soluble

CTLA-4Ig or anti-CD40L to block allograft rejection? Why might this be better than treating a graft recipient with CsA or FK506? 16. Immediately after transplantation, a patient is often given

extra strong doses of anti-rejection drugs and then allowed to taper off as time passes. Describe the effects of the commonly used anti-rejection drugs azathioprine, cyclosporine A, FK506, and rapamycin. Why is it possible to decrease the use of some of these drugs at some point after transplantation?

b. Second-set rejection is a manifestation of immunologic

CLINICAL FOCUS QUESTION What features would be desir-

memory. c. Host dendritic cells can migrate into grafted tissue and act as APCs.

able in an ideal animal donor for xenotransplantation? How would you test your model prior to doing clinical trials in humans?

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S

urviving infectious disease outbreaks was one of the primary drivers for our earliest forays into the study of immunology. This led to the development and use of rudimentary vaccines even before we understood how a vaccine could induce protective immunity (see Chapter 1). Since those early vaccination trials of Edward Jenner and Louis Pasteur, vaccines have been developed for many diseases that were once major afflictions of humankind. For example, the incidence of diphtheria, measles, mumps, pertussis (whooping cough), rubella (German measles), poliomyelitis, and tetanus, which once collectively claimed the lives of millions, has declined dramatically as vaccination has become more common. Clearly, vaccination is a cost-effective weapon for disease prevention, and yet the need for safe and effective vaccines for many life-threatening infectious diseases remains. These and other public health concerns led to the development of agencies to help organize the accumulating data concerning infectious disease, such as the World Health Organization (WHO) and the U.S.based Centers for Disease Control and Prevention (CDC). These organizations monitor public health and disease, guide health care policy discussions, respond to sudden infectious disease outbreaks, and report regularly on their findings. Although the local and international expenditures on these practices are questioned at times, there is no doubt that these and present-day biomedical advances have led us to an age in which rapid and often effective response to sudden infectious disease outbreaks is commonplace. It has also allowed us to better appreciate the conditions and policies that can limit outbreaks of infectious disease. Although vaccination or naturally acquired protective immunity can provide critical defense against many pathogens, infectious diseases still cause the death of millions each year. Although the number varies greatly by region, about 25% of deaths worldwide are associated with communicable diseases, which kill an estimated 11 million to 12 million people each year (Figure 17-1). Sanitation, antibiotics, and vaccination have reduced the impact of infectious disease, but infections still account

The bacteria Listeria monocytogenes polymerizing host cell actin into comet tails. [Courtesy Matteo Bonazzi, PhD, Edith Gouin, and Pascale Cossart] ■

The Importance of Barriers to Infection and the Innate Response



Viral Infections



Bacterial Infections



Parasitic Infections



Fungal Infections



Emerging and Re-emerging Infectious Diseases



Vaccines

for almost half of the leading causes of death in the developing world, especially among the very young. Adding to the endemic infectious disease burden most heavily borne by the developing world, new diseases are emerging and others are resurfacing. Influenza and West Nile virus (WNV) strains prevalent in birds have adapted to cause human infection. Previously rare infections by certain bacteria or fungi are increasing because of the rise in the numbers of individuals with impaired immunity, primarily due to the prevalence of human immunodeficiency virus (HIV)-induced acquired immunodeficiency syndrome (AIDS). Increasing antibiotic resistance in existing pathogens, such as Staphylococcus aureus and Mycobacterium tuberculosis, has some infections 553

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Cardiovascular conditions, 17.3 million

Infectious diseases

Annual deaths (million)

Respiratory infections Diarrheal diseases HIV/AIDS Tuberculosis Malaria Vaccine-preventable childhood diseases Meningitis Hepatitis B and C Tropical parasitic diseases STDs (other than HIV) Dengue Leprosy

3.53 2.46 1.78 1.34 0.83

Infectious diseases, 11.2 million

All other causes of death

Perinatal conditions, 2.6 million Asthma and chronic obstructive pulmonary diseases, 4.2 million

Neoplastic diseases, 7.8 million Injuries, 5.1 million

0.45 0.34 0.20 0.14 0.12 0.02 0.01

FIGURE 17-1 Infectious diseases are among the leading causes of death worldwide. The 11.2 million deaths attributable annually directly to infections are broken down by category in this table. [based on WHO 2008 global burdens of disease estimates]

spreading at an alarming rate in developing as well as in industrialized countries. In certain instances, a common infectious agent has become associated with a new disease. Such is the case for the recently identified disease necrotizing fasciitis, caused by the so-called flesh-eating strain of Streptococcus pyogenes, a bacterium most commonly associated with the now rare disease scarlet fever. In this chapter, the concepts of immunity described throughout the text are applied to selected infectious diseases caused by the four main types of pathogens (viruses, bacteria, fungi, and parasites). We focus on particular infectious diseases that affect large numbers of people, that illustrate specific immune concepts, and that use novel strategies to subvert the immune response, as well as some diseases that have warranted recent headlines. The chapter concludes with a section on vaccines, divided by the type of vaccine design being applied and including examples of specific pathogens that have been successfully targeted using these strategies.

The Importance of Barriers to Infection and the Innate Response First and foremost, in order for a pathogen to establish an infection in a susceptible host, it must breach physical and chemical barriers. One of the first and most important of these barriers consists of the epithelial surfaces of the skin and the lining of the gut. The difficulty of penetrating these surfaces ensures that most pathogens never gain productive entry into the host. In addition, the epithelia produce chemicals that are useful in preventing infection. The secretion of gastric enzymes by specialized epithelial cells lowers the pH of the stomach and upper gastrointestinal tract, and other

specialized cells in the gut produce antibacterial peptides. In addition, normal commensal flora present at mucosal surfaces (the gastrointestinal, genitourinary, and respiratory tracts) can competitively inhibit the binding of pathogens to host cells. When pathogen dose and virulence are minimal, these barriers can often block productive infection altogether. Interventions that introduce barriers to infection in intermediate hosts can be used as an indirect strategy to disrupt the cycle of infectious disease in humans. For example, many pathogens make use of arthropod vectors, such as the mosquito, for parts of their life cycle and as vehicles for transmission to humans. Very recent studies in Dengue virus, transmitted by the bite of an infected mosquito and the cause of an often fatal hemorrhagic fever in humans, suggest that it may be possible to engineer mosquitoes that are resistant to infection with the virus. When these engineered mosquitoes were released into the wild, they began to supplant the wild-type, virus-susceptible mosquito population, suggesting that they may have the potential to break the cycle of transmission. This and other exciting new avenues of research that target animal disease vectors could advance infectious disease eradication without the requirement to intervene with the human immune response. Of course, this strategy is not a possibility with most infectious diseases, for which there is no animal vector. When the basic human barriers to infection are breached, more directed innate immune responses come into play at or near the site of infection. These early responses are often tailored to the type of pathogen, using molecular pattern recognition receptors (see Chapter 5). Some bacteria produce endotoxins such as lipopolysaccharide (LPS), which stimulate macrophages or endothelial cells to produce cytokines, such as IL-1, IL-6, and TNF-␣. These cytokines can activate nearby innate cells, encouraging phagocytosis of the bacteria. The cell walls of many gram-positive bacteria contain a peptidoglycan that activates the alternative complement pathway, leading to opsonization and phagocytosis or lysis (see Chapter 6).

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Mechanisms of humoral and cell-mediated immune responses to viruses

Response type

Effector molecule or cell

Activity

Humoral

Antibody (especially secretory IgA)

Blocks binding of virus to host cells, thus preventing infection or reinfection

IgG, IgM, and IgA antibody

Blocks fusion of viral envelope with host cell’s plasma membrane

IgG and IgM antibody

Enhances phagocytosis of viral particles (opsonization)

Cell mediated

IgM antibody

Agglutinates viral particles

Complement activated by IgG or IgM antibody

Mediates opsonization by C3b and lysis of enveloped viral particles by membrane-attack complex

IFN-␭ secreted by TH or TC cells

Has direct antiviral activity

Cytotoxic T lymphocytes (CTLs)

Kill virus-infected self cells

NK cells and macrophages

Kill virus-infected cells by antibody-dependent cell-mediated cytotoxicity (ADCC)

Viruses commonly induce the production of interferons, which can inhibit viral replication by inducing an antiviral response in neighboring cells. Viruses are also controlled by natural killer (NK) cells, which frequently form the first line of defense in these infections (see Chapter 5). In many cases, these innate responses can lead to the resolution of infection. Cells responding via innate immunity at the infection site receive signals that help coordinate the subsequently more specific adaptive immune response. During this very pathogen-specific stage of the immune response, final eradication of the foreign invader often occurs, typically leaving a memory response capable of halting secondary infections. However, just as adaptive immunity in vertebrates has evolved over many millennia, pathogens have evolved a variety of strategies to escape destruction by the adaptive immune response. Some pathogens reduce their own antigenicity either by growing within host cells, where they are sequestered from immune attack, or by shedding their membrane antigens. Other pathogen strategies include camouflage (expressing molecules with amino acid sequences similar to those of host cell membrane molecules or acquiring a covering of host membrane molecules); suppressing the immune response selectively or directing it toward a pathway that is ineffective at fighting the infection; and continual variation in surface antigens. Examples of each of these strategies are included in the following sections.

Viral Infections Viruses are small segments of nucleic acid with a protein or lipoprotein coat that require host resources for their replication. Typically, a virus enters a cell via a cell-surface receptor for which it has affinity and preempts cell biosynthetic machinery to replicate all components of itself, including its genome. This genome replication step is often error prone, generating numerous mutations. Because large numbers of new viral

particles (virions) are produced in a replication cycle, many different mutants with individual survival advantages can be selected for the ability to propagate most effectively in the host. A virus is more likely to thrive if it does not kill its host, as sustained coexistence in the host favors the survival and spread of the virus. However, the mutability of the viral genome sometimes gives rise to lethal variants that do not conform to this state of equilibrium with their host. If such mutants cause the early death of their host, survival of the virus requires that it spread to new hosts rapidly. Among the other survival strategies available to viruses is a long latency period before severe illness, during which time the host may pass the virus to others unknowingly, as in the case of HIV. One additional strategy used by viruses is facile transmission, such as with influenza and the smallpox virus, where infection is efficiently transferred during even a short acute illness. The life cycle of some viruses pathogenic for humans, such as WNV, may also include nonhuman hosts, providing them with additional reservoirs. A number of specific immune effector mechanisms, together with nonspecific defense mechanisms, prevent or eliminate most viral infections (Table 17-1). Passage across the mucosa of the respiratory, genitourinary, or gastrointestinal tracts accounts for most instances of viral transmission. Entrance of the virus may also occur through broken skin, usually as a result of an insect bite or puncture wound. The outcome of this infection depends on how effectively the host’s defensive mechanisms resist the offensive tactics of the virus. The innate immune response to viral infection primarily begins with the recognition of pathogen associated molecular patterns (PAMPs) and leads to the generation of antiviral effectors. For example, double-stranded RNA (dsRNA) molecules and other virus-specific structures are detected by one of several PAMP receptors, inducing the expression of type I interferons (IFN-␣ and IFN-␤), the assembly of intracellular inflammasome complexes, and the activation of NK cells.

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Type I interferons can induce an antiviral response or resistance to viral replication by binding to the IFN-␣/-␤ receptor, thereby activating the JAK-STAT pathway and the production of new transcripts, one of which encodes an enzyme that leads to viral RNA degradation (see Figure 5-16). IFN-␣/-␤ binding also induces dsRNA-dependent protein kinase (PKR), which leads to inactivation of protein synthesis, thus blocking viral replication in infected cells. The binding of type I interferon to NK cells induces lytic activity, making them very effective in killing virally infected cells. This activity is enhanced by IL-12, a cytokine that is produced by dendritic cells very early in the response to viral infection.

vate NK cells, which play an important role in host defense and lysis of infected cells during the first days of many viral infections, until a specific CTL response develops. In most viral infections, specific CTL activity arises within 3 to 4 days after infection, peaks by 7 to 10 days, and then declines. Within 7 to 10 days of primary infection, most virions have been eliminated, paralleling the development of CTLs. CTLs specific for the virus eliminate virus-infected self cells and thus eliminate potential sources of new virus. Virus-specific CTLs confer protection against that virus in nonimmune recipients following adoptive transfer. Transfer of a CTL clone specific for influenza virus strain X protects mice against strain X but not against influenza virus strain Y.

Many Viruses Are Neutralized by Antibodies Antibodies specific for viral surface antigens are often crucial in containing the spread of a virus during acute infection and in protecting against reinfection. Antibodies are particularly effective if they are localized at the site of viral entry into the body and if they bind to key viral surface structures, interfering with their ability to attach to host cells. For example, influenza virus binds to sialic acid residues in cell membrane glycoproteins and glycolipids, rhinovirus binds to intercellular adhesion molecules (ICAMs), and EpsteinBarr virus (EBV) binds to type 2 complement receptors on B cells. The advantage of the attenuated oral polio vaccine, discussed later in this chapter, is that it induces production of secretory IgA, which effectively blocks attachment of poliovirus to epithelial cells lining the gastrointestinal tract. Viral neutralization by antibody sometimes involves mechanisms that operate after viral attachment to host cells. For example, antibodies may block viral penetration by binding to epitopes that are necessary to mediate fusion of the viral envelope with the plasma membrane. If the induced antibody is of a complement-activating isotype, lysis of enveloped virions can ensue. Antibody or complement can also agglutinate viral particles and function as an opsonizing agent to facilitate Fcor C3b-receptor-mediated phagocytosis of the free virions.

Cell-Mediated Immunity Is Important for Viral Control and Clearance Although antibodies have an important role in containing the spread of a virus in the acute phases of infection, they cannot eliminate established infection once the viral genome is integrated into host chromosomal DNA. Once such an infection is established, cell-mediated immune mechanisms are most important in host defense. In general, both CD8⫹ TC cells and CD4⫹ TH1 cells are required components of the cell-mediated antiviral defense. Activated TH1 cells produce a number of cytokines, including IL-2, IFN-␥, and tumor necrosis factor-␣ (TNF-␣), which defend against viruses either directly or indirectly. IFN-␥ acts directly by inducing an antiviral state in nearby cells. IL-2 acts indirectly by assisting the development of cytotoxic T lymphocyte (CTL) precursors into an effector population. Both IL-2 and IFN-␥ acti-

Viruses Employ Several Different Strategies to Evade Host Defense Mechanisms Despite their restricted genome size, a number of viruses encode proteins that interfere with innate and adaptive levels of host defense. Presumably, the advantage of such proteins is that they enable viruses to replicate more effectively amid host antiviral defenses. As described above, the induction of type I interferon is a major innate defense against viral infection, but some viruses have developed strategies to evade the action of IFN-␣/-␤. These include hepatitis C virus, which has been shown to overcome the antiviral effect of the interferons by blocking or inhibiting the action of PKR (see Figure 5-16). Another mechanism for evading host responses is inhibition of antigen presentation by infected host cells. Herpes simplex virus (HSV) produces an immediate-early protein (synthesized shortly after viral replication) that very effectively inhibits the human transporter molecule needed for antigen processing (TAP; see Figure 8-17). Inhibition of TAP blocks antigen delivery to class I MHC molecules in HSV-infected cells, thus preventing presentation of viral antigen to CD8⫹ T cells. This results in the trapping of empty class I MHC molecules in the endoplasmic reticulum and effectively shuts down a CD8⫹ T-cell response to HSV-infected cells. Likewise, adenoviruses and cytomegalovirus (CMV) use distinct molecular mechanisms to reduce the surface expression of class I MHC molecules, again inhibiting antigen presentation to CD8⫹ T cells. Other viruses, such as measles virus and HIV, reduce levels of class II MHC molecules on the surface, thus blocking the function of antigen-specific antiviral helper T cells. Complement activation is another of the antibodymediated destruction pathways of viruses, resulting in opsonization and elimination of the virus by phagocytic cells. A number of viruses, such as vaccinia virus, evade complementmediated destruction by secreting a protein that binds to the C4b complement component, inhibiting the classical complement pathway. HSV also makes a glycoprotein component that binds to the C3b complement component, inhibiting both the classical and alternative pathways. A number of viruses escape immune attack by constantly changing their surface antigens. The influenza virus is a prime example, as discussed below. Antigenic variation

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Infectious Diseases and Vaccines among rhinoviruses, the causative agent of the common cold, is responsible for our inability to produce an effective vaccine for colds. Nowhere is antigenic variation greater than in HIV, the causative agent of AIDS, estimated to accumulate mutations 65 times faster than the influenza virus. A section of Chapter 18 is dedicated to HIV and AIDS. Viruses such as EBV, CMV, and HIV cause generalized or specific immunosuppression. In some cases, immunosuppression is caused by direct viral infection of lymphocytes or macrophages. The virus can then either directly destroy the immune cells by cytolytic mechanisms or alter their function. In other cases, immunosuppression is the result of a cytokine imbalance or diversion of the immune responses toward pathways less effective at virus eradication. For instance, EBV, the cause of mononucleosis, produces a protein that is homologous to IL-10; like IL-10, this protein

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suppresses cytokine production by the TH1 subset, resulting in an immunosuppressed state.

Influenza Has Been Responsible for Some of the Worst Pandemics in History The influenza virus infects the upper respiratory tract and major central airways in humans, horses, birds, pigs, and even seals. Between 1918 and 1919, the largest influenza pandemic (worldwide epidemic) in recent history occurred, killing between 20 million and 50 million people. The sequence of this virus has recently been reconstructed, leading to much controversy about its publication (Clinical Focus Box 17-1). Two other less major pandemics occurred in the twentieth century, caused by influenza strains that were new or had not circulated in the recent past, leaving most people with little immunity to them.

BOX 17-1

CLINICAL FOCUS

The 1918 Pandemic Influenza Virus: Should It Publish or Perish? The most virulent and devastating of the pandemic strains of influenza virus in recent history was seen in 1918 and 1919. Worldwide deaths from that socalled “Spanish flu” strain may have reached 50 million in less than 1 year, compared with the roughly 10,000 to 15,000 who die yearly from nonpandemic strains. Approximately 675,000 of the victims of Spanish flu were located the United States, with certain areas, such as Alaska and the Pacific Islands, losing more than half of their population during the outbreak. Mortality rates for the 1918 pandemic flu were surprisingly high, especially among young and healthy individuals, reaching 2.5% in infected individuals compared to less than 0.1% during other flu epidemics. Most of these deaths were the result of a virulent pneumonia, which felled some patients in as little as 5 days. Thanks to present-day molecular techniques and chance, the recent reconstruction and sequencing of the virus that caused the 1918 pandemic became possible. After several failed attempts, a research team led by Jeffrey Tautenberger published the final genetic sequences of the deadly 1918 flu virus. Their results were made possible following isolation of viral RNA from Spanish flu victims using formalin-fixed lung autopsy samples and

tissue collected from an Inuit woman who was buried in permafrost in Alaska. Analysis of the sequence revealed that this highly virulent strain was derived from an avian virus and differed significantly from other human influenza A strains, making it the most “bird-like” of the influenza strains ever isolated from humans. The reconstructed virus sequence became the object of intense study, as well as controversy. Thanks to a cDNA reconstruction of live virus, scientists were able to study the virulence factors at play in this deadly strain. In mouse studies, they found that the reconstructed virus spread rapidly in the respiratory tract and produced high numbers of progeny, causing pervasive damage in the lungs. Using recombinant virus strains, they found that three polymerase genes and the HA gene appeared to account for the high lethality of the virus; replacing the polymerase genes significantly reduced the virulence of the strain while a new HA gene completely blocked its ability to kill the host. Word of the imminent publication of the sequence of the 1918 influenza caused a scientific and public controversy. On the one hand, many virologists, molecular biologists, and epidemiologists were eager to glimpse this highly virulent sequence for clues to what determinants might play

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a role in lethality and what measures could be taken to avoid this in the future. On the other hand, some feared that this sequence might be used for evil rather than for good, leading to a potential do-ityourself recombinant reconstructionist frenzy, culminating in a weaponized version of the influenza virus. In the end, the sequences were published and follow-up studies led to the conclusion that the 1918 virus is sensitive to the seasonal flu vaccine and even treatable with available anti-flu drugs; this may have calmed many fears. Nevertheless, controversy persists. Recently, the National Science Advisory Board for Biosecurity (NSABB), a U.S.-based body that advises the community about research concerning agents deemed a national security threat, recommended against the release of data related to the current avian H5N1 strain. These data would reveal the mutations behind the virus’s transmissibility to humans. After 8 months of deliberations, Science magazine published a special open-access issue in June 2012 describing this work and related policy issues, placing another one in the publish-rather-than-perish column. T. M. Tumpey et al. 2005. The 1918 Flu Virus Is Resurrected. Science 310:77–80; and P. Palese. 2012. Don’t Censor Life-Saving Science. Nature 481:115.

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(b)

Hemagglutinin

Nucleocapsid

Matrix protein

Neuraminidase

Lipid bilayer

NS1, NS2

(a)

M1, M2 NA NP HA PA PB1 PB2

0

10 20 30 40 50 Nanometers

FIGURE 17-2 Influenza virus. (a) Electron micrograph of influenza virus reveals roughly spherical viral particles enclosed in a lipid bilayer with protruding hemagglutinin and neuraminidase glycoprotein spikes. (b) Schematic representation of influenza structure. The envelope is covered with neuraminidase and hemagglutinin spikes. Inside is an inner layer of matrix protein surrounding the nucleocap-

Properties of the Influenza Virus Influenza virions are surrounded by an outer envelope, a lipid bilayer derived from the plasma membrane of the infected cell, plus various virus-specific proteins. Imbedded in this envelope are two key viral glycoproteins, hemagglutinin (HA) and neuraminidase (NA) (Figure 17-2a). HA trimers are responsible for the attachment of the virus to host cells, binding to the sialic acid groups on host-cell glycoproteins and glycolipids. NA is an enzyme that cleaves N-acetylneuraminic (sialic) acid from nascent viral glycoproteins and host-cell membrane glycoproteins, facilitating viral budding from the infected host cell. Thus these two structures are essential for viral attachment and for exit of new virus from infected cells—so important in fact that we track new strains of influenza based on their antigenic subtypes of HA and NA (e.g., H1N1 versus H5N1 virus). Within the envelope, an inner layer of matrix protein surrounds the nucleocapsid, which consists of eight different strands of single-stranded RNA (ssRNA) associated with protein and RNA polymerase (Figure 17-2b). Each RNA strand encodes one or more different influenza proteins. There are three basic types of influenza (A, B, and C), each differing in the makeup of its nuclear and matrix proteins. Type A is the most common and is responsible for the

sid, which consists of eight ssRNA molecules associated with nucleoprotein. The eight RNA strands encode 10 proteins: PB1, PB2, PA, HA (hemagglutinin), NP (nucleoprotein), NA (neuraminidase), M1, M2, NS1, and NS2. [Source: (a) Courtesy of G. Murti, Department of Virology, St. Jude Children’s Research Hospital, Memphis, Tenn.]

major human pandemics. Influenza virus strains are tracked yearly by the Centers for Disease Control and Prevention (CDC) and the World Health Organization (WHO). According to WHO nomenclature, each virus strain is defined by its animal host of origin (specified if other than human), geographical origin, strain number, year of isolation, and the antigenic structures of HA and NA. For example, A/Sw/ Iowa/15/30 (H1N1) designates strain-A isolate 15 that arose in swine in Iowa in 1930 and has antigenic subtypes 1 for both HA and NA (refer to Table 17-2, pandemic strains). Variation in Epidemic Influenza Strains To date, there are 13 different antigenic subtypes for HAs and 9 for NAs. Antigenic variation in HA and NA is generated by two different mechanisms: antigenic drift and antigenic shift. Antigenic drift involves a series of spontaneous point mutations that occur gradually, resulting in minor changes in HA and NA over time. Antigenic shift results in the sudden emergence of a new subtype of influenza, where the structures of HA and/or NA are considerably different from that of the virus present in a preceding year. The immune response contributes to the emergence of these antigenically distinct influenza strains. In a typical year, the predominant virus strain undergoes antigenic

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N

(a)

|

HA

Human influenza

559 Swine influenza

Virus

Host cell Antigenic drift Person A, subsequent years

Person A, year 1

Secondary host cell Antigenic shift

FIGURE 17-3 Two mechanisms generate variations in

Mutated virus

influenza surface antigens. (a) In antigenic drift, the accumulation of point mutations eventually yields a variant protein that is no longer recognized by antibody to the original antigen. (b) Antigenic shift may occur by reassortment of an entire ssRNA between human and animal virions infecting the same cell. For clarity, only the HA surface antigens of the virus are shown in part b. Only two of the eight RNA strands are depicted.

TABLE 17-2

Species Human

Some influenza A strains and their hemagglutinin (H) and neuraminidase (N) subtype Virus strain designation

Antigenic subtype

A/Puerto Rico/8/34

H0N1

A/Fort Monmouth/1/47

H1N1

A/Singapore/1/57

H2N2

A/Hong Kong/1/68

H3N2

A/USSR/80/77

H1N1

A/Brazil/11/78

H1N1

A/Bangkok/1/79

H3N2

A/Taiwan/1/86

H1N1

A/Shanghai/16/89

H3N2

A/Johannesburg/33/95

H3N2

A/Wuhan/359/95

H3N2

A/Texas/36/95

H1N1

A/Hong Kong/156/97

H5N1

A/California/04/2009

H1N1

Swine

A/Sw/Iowa/15/30

H1N1

A/Sw/Taiwan/70

H3N2

Horse (equine)

A/Eq/Prague/1/56

H7N7

Bird

A/Eq/Miami/1/63

H3N8

A/Fowl/Dutch/27

H7N7

A/Tern/South America/61

H5N3

A/Turkey/Ontario/68

H8N4

A/Chicken/Hong Kong/258/97

H5N1

Human cell

drift, generating minor antigenic variants. As individuals infected with influenza mount an effective immune response, they will eliminate that strain. However, the accumulation of point mutations sufficiently alters the antigenicity of some variants so that they are able to escape immune elimination (Figure 17-3a) and become a new variant of influenza that is transmitted to others, causing another local epidemic cycle. The role of antibody in such immunologic selection can be demonstrated in the laboratory by mixing an influenza strain with a monoclonal antibody specific for that strain and then culturing the virus in cells. The antibody neutralizes all unaltered viral particles, and only those viral particles with mutations resulting in altered antigenicity escape neutralization and are able to continue the infection. Within a short time in culture, a new influenza strain emerges, just as it does in nature. In this way, influenza evolves during a typical flu season, such that the dominant strains at the start and end of the season are antigenically distinct. This is why we are offered a new flu vaccine each year. The vaccine formulation is based on carefully constructed models tracking the dominant variant(s) from the end of the previous season. And our guesses are not always 100% accurate, making some years’ influenza vaccinations more effective than others. Episodes of antigenic shift are thought to occur through a different mechanism. The primary mechanism is genetic

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(a) Primary response

Naïve B cell binds to the pathogen

Memory B cell binds to the pathogen

Naïve B cell binds to the pathogen

FcR





Naïve B cell activation, antibody production, and pathogen eradication

(b)

Antigen epitope specificity



Negative signal prevents activation

Memory B cell activation, antibody production, and pathogen eradication

First infection

Second infection

Third infection

Fourth infection

A

A

A

F

B

B

E

E

C Viral strain

D

D

D

Primary response

Memory response

Memory response

Primary response

A B C D E F

FIGURE 17-4 The presence of preformed antibody inhibits primary responses to a pathogen. (a) During a primary response, naïve B cells are activated and produce antibodies specific to epitopes on the pathogen. During a secondary response to a variant of that pathogen, memory B cells specific to epitopes encountered in the past will be reactivated and help to eradicate the pathogen. The Fc regions of antibodies bound to the surface of the pathogen will bind to the FcRs on naïve B cells and inhibit them from responding, even to new epitopes on the pathogen. (b) This inhibition of primary responses

reassortment between influenza virions from humans and those from various animals (Figure 17-3b). The fact that the influenza genome contains eight separate strands of ssRNA makes possible the reassortment of individual RNA strands of human and animal virions within a secondary (nonhuman) host cell infected with both viruses—in other words, shuffling of the DNA segments derived from the animal and human strains. Both pigs and birds can harbor human influenza A viruses, as well as their own and maybe those of other species, making them perfect conduits for in vivo genetic reassortment between human influenza A

against unique epitopes on pathogens that elicit memory cell responses is called original antigenic sin. No immune response is mounted to each new epitope during subsequent exposures to the pathogen until the pathogen expresses a significant number of unique epitopes and memory cells can no longer eradicate the organism. In this case, a new primary response is mounted and disease symptoms are severe until a new adaptive response has been established, resetting original antigenic sin. [Source: Adapted from P. Parham, 2009, The Immune System, New York: Garland Science. a: Fig. 10.23; b: Fig. 10.25.]

viruses and the potential sources for strains that have been coined “swine flu” or “bird or avian flu.” As one might imagine, the contribution of viral proteins from the viruses of these animals is frequently “new” to humans who will have little or no immunity, sparking pandemics. Original Antigenic Sin and Susceptibility to Influenza Immunologic memory is an amazing and beautiful thing. For pathogens that don’t change much from one encounter to the next, it can provide us with lifelong protection. However, preformed immunity can come with caveats for pathogens

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Infectious Diseases and Vaccines that have evolved to vary their antigenic structure. During secondary encounters with a pathogen that bears a strong molecular resemblance to a pathogen seen in the past (i.e., we have developed adaptive immunity to some of the epitopes before), memory cells specific for previously encountered epitopes are engaged rapidly and efficiently. As long as these cells and their products, like antibodies, can dispatch the pathogen efficiently, there is no need to mount a primary response to any new epitopes carried by that pathogen. In fact, the presence of antibodies attached to a pathogen, either as residuals from a recent infection or produced by reactivation of memory B cells, will divert naïve B cells from responding (Figure 17-4a). This occurs when the Fc region of the pathogen-associated antibody binds with Fc receptors on naïve B cells, inducing anergy. In other words, if there is a way to take care of an infection with memory, this will be the default pathway. This concept is referred to as original antigenic sin, or the tendency to focus an immune attack on those structures that were present during the original, or primary, encounter with a pathogen and for which we have established memory. This means that our immune systems effectively ignore the subtle changes occurring each year in pathogens that drift antigenically, like influenza virus (Figure 17-4b). Once the organism has drifted sufficiently that there are only “new” epitopes, or insufficient numbers of key epitopes to effectively dispatch with existing memory cells, a new primary response is mounted. In such a year, we experience a bad case of the flu. Since we all begin our journeys of original antigenic sin at different times and in response to different antigenic variants, we don’t typically all get a bad case of the flu at the same time; the exceptions are pandemic influenza years (see Clinical Focus Box 17-1).

Bacterial Infections Immunity to bacterial infections is achieved by means of antibody unless the bacterium is capable of intracellular growth, in which case delayed-type hypersensitivity (DTH) has an important role. Bacteria enter the body either through a number of natural routes (e.g., the respiratory, gastrointestinal, and genitourinary tracts) or through normally inaccessible routes opened up by breaks in mucous membranes or skin. Depending on the number of organisms entering and their virulence, different levels of host defense are enlisted. If the inoculum size and the virulence are both low, then localized tissue phagocytes may be able to eliminate the bacteria via nonspecific innate defenses. Larger inocula or organisms with greater virulence tend to induce antigen-specific adaptive immune responses. In some bacterial infections, disease symptoms are caused not by the pathogen itself but by the immune response. As described in Chapter 4, pathogen-stimulated overproduction of cytokines leads to the symptoms associated with

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bacterial septic shock, food poisoning, and toxic shock syndrome. For instance, cell wall endotoxins of some gramnegative bacteria activate macrophages, resulting in release of high levels of IL-1 and TNF-␣, which can cause septic shock. In staphylococcal food poisoning and toxic shock syndrome, exotoxins produced by the pathogens function as superantigens, which can activate all T cells that express T-cell receptors with a particular V␤ domain (see Table 11-2). The resulting systemic production of cytokines by activated TH cells is overwhelming, causing many of the symptoms of these diseases.

Immune Responses to Extracellular and Intracellular Bacteria Can Differ Infection by extracellular bacteria induces production of antibodies, which are ordinarily secreted by plasma cells in regional lymph nodes and the submucosa of the respiratory and gastrointestinal tracts. The humoral immune response is the main protective response against extracellular bacteria. The antibodies act in several ways to protect the host from the invading organisms, including removal of the bacteria and inactivation of bacterial toxins (Figure 17-5). Extracellular bacteria can be pathogenic because they induce a localized inflammatory response or because they produce toxins. The toxins—endotoxin or exotoxin—can be cytotoxic but also may cause pathogenesis in other ways. An excellent example of this is the toxin produced by diphtheria, which blocks protein synthesis. Endotoxins, such as LPS, are generally components of bacterial cell walls, whereas exotoxins, such as diphtheria toxin, are secreted by the bacteria. Antibody that binds to accessible antigens on the surface of a bacterium can, together with the C3b component of complement, act as an opsonin that increases phagocytosis and thus clearance of the bacterium. In the case of some bacteria—notably, the gram-negative organisms—complement activation can lead directly to lysis of the organism. Antibodymediated activation of the complement system can also induce localized production of immune effector molecules that help to develop an amplified and more effective inflammatory response. For example, the complement fragments C3a and C5a act as anaphylatoxins, inducing local mast-cell degranulation and thus vasodilation and the extravasation of lymphocytes and neutrophils from the blood into tissue spaces (see Figure 17-5). Other complement split products serve as chemotactic factors for neutrophils and macrophages, thereby contributing to the buildup of phagocytic cells at the site of infection. Antibody to a bacterial toxin may bind to the toxin and neutralize it; the antibody-toxin complexes are then cleared by phagocytic cells in the same manner as any other antigen-antibody complex. Although innate immunity is not very effective against intracellular bacterial pathogens, intracellular bacteria can activate NK cells, which in turn provide an early defense against these organisms. Intracellular bacterial infections tend

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OVERVIEW FIGURE

Antibody-Mediated Mechanisms for Combating Infection by Extracellular Bacteria

Bacteria

Toxin 1 Toxin neutralization

Complement activation C3b

C3b 2 Complement–mediated lysis

C3b

C3b

C3b

C3b 3 Opsonization and phagocytosis

4 Anaphylatoxins mediate mast-cell degranulation

Macrophage

5 Chemotaxis

C3a, C5a

Mast cell Mediators

Extravasation

Neutrophil

Lymphocyte Macrophage

(1) Antibody neutralizes bacterial toxins. (2) Complement activation on bacterial surfaces leads to complement-mediated lysis of bacteria. (3) Antibody and the complement split product C3b bind to bacteria, serving as opsonins to increase phagocytosis. (4) C3a and C5a, generated by antibody-initiated complement activation, induce local mast-cell degranulation, releasing substances that mediate vasodilation and extravasation of lymphocytes and neutrophils. (5) Other complement products are chemotactic for neutrophils and macrophages.

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Host immune responses to bacterial infection and bacterial evasion mechanisms

Infection process

Host defense

Bacterial evasion mechanisms

Attachment to host cells

Blockage of attachment by secretory IgA antibodies

Secretion of proteases that cleave secretory IgA dimers (Neisseria meningitidis, N. gonorrhoeae, Haemophilus influenzae) Antigenic variation in attachment structures (pili of N. gonorrhoeae)

Proliferation

Phagocytosis (Ab- and C3b-mediated opsonization)

Production of surface structures (polysaccharide capsule, M protein, fibrin coat) that inhibit phagocytic cells Mechanisms for surviving within phagocytic cells Induction of apoptosis in macrophages (Shigella flexneri)

Complement-mediated lysis and localized inflammatory response

Generalized resistance of gram-positive bacteria to complementmediated lysis Insertion of membrane-attack complex prevented by long side chain in cell-wall LPS (some gram-negative bacteria)

Invasion of host tissues

Ab-mediated agglutination

Secretion of elastase that inactivates C3a and C5a (Pseudomonas)

Toxin-induced damage to host cells

Neutralization of toxin by antibody

Secretion of hyaluronidase, which enhances bacterial invasiveness

to induce a cell-mediated immune response, specifically DTH. In this response, cytokines secreted by CD4⫹ T cells are important—most notably IFN-␥, which activates macrophages to kill ingested pathogens more effectively.

Bacteria Can Evade Host Defense Mechanisms at Several Different Stages There are four primary steps in bacterial infection: 1. Attachment to host cells 2. Proliferation

shortened half-life in mucous secretions and are not able to agglutinate microorganisms. Some bacteria evade the antibody responses of the host by changing their surface antigens. In Neisseria gonorrhoeae, for example, pilin (the protein component of the pili) has a highly variable structure, generated by gene rearrangements of its coding sequence. The pilin locus consists of 1 or 2 expressed genes and 10 to 20 silent genes. Each gene is arranged into six regions called minicassettes. Pilin variation is generated by a process of gene conversion, in which one or more minicassettes from the silent genes replace a minicassette of the expression gene. This process generates enormous antigenic

3. Invasion of host tissue 4. Toxin-induced damage to host cells Host-defense mechanisms act at each of these steps, and many bacteria have evolved ways to circumvent some of them (Table 17-3). Some bacteria express molecules that enhance their ability to attach to host cells. A number of gram-negative bacteria, for example, have pili (long hairlike projections), which enable them to attach to the membrane of the intestinal or genitourinary tract (Figure 17-6). Other bacteria, such as Bordetella pertussis, the cause of whooping cough, secrete adhesion molecules that attach to both the bacterium and the ciliated epithelial cells of the upper respiratory tract. Secretory IgA antibodies specific for such bacterial structures can block bacterial attachment to mucosal epithelial cells and are the main host defense against bacterial attachment. However, some bacteria, such as the species of Neisseria that cause gonorrhea and meningitis, evade the IgA response by secreting proteases that cleave secretory IgA at the hinge region; the resulting Fab and Fc fragments have a

P

FIGURE

17-6 Electron micrograph of Neisseria gonorrhoeae attaching to urethral epithelial cells. Pili (P) extend from the gonococcal surface and mediate the attachment.

[Source: M. E. Ward and P. J. Watt, Adherence of Neisseria gonorrhoeae to urethral mucosal cells: an electron microscope study of human gonorrhea. 1972, Journal of Infectious Disease 126:601.]

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variation, which may contribute to the pathogenicity of N. gonorrhoeae by increasing the likelihood that expressed pili will go undetected by antibody, allowing them to bind firmly to epithelial cells and avoid neutralization by IgA. Bacteria may also possess surface structures that inhibit phagocytosis. A classic example is Streptococcus pneumoniae, whose polysaccharide capsule prevents phagocytosis very effectively. The 84 serotypes of S. pneumoniae differ from one another by distinct capsular polysaccharides, and during infection the host produces antibody against the infecting serotype. This antibody protects against reinfection with the same serotype but will not protect against infection by a different serotype. In this way, genetic variants of S. pneumoniae can cause disease many times in the same individual. On other bacteria, such as Streptococcus pyogenes, a surface protein projection called the M protein inhibits phagocytosis, a key step in bacterial removal. Some pathogenic staphylococci are able to assemble a protective coat from host blood proteins. These bacteria secrete a coagulase enzyme that precipitates a fibrin coat around them, shielding them from phagocytic cells. Mechanisms for interfering with the complement system help other bacteria survive. In some gram-negative bacteria, for example, long side chains on the lipid A moiety of the cell wall core polysaccharide help to resist complementmediated lysis. Pseudomonas secretes an enzyme, elastase, that inactivates both the C3a and C5a anaphylatoxins, thereby diminishing the localized inflammatory reaction. A number of bacteria escape host-defense mechanisms through their ability to survive within phagocytic cells. Bacteria such as Listeria monocytogenes escape from the phagolysosome to the cytoplasm, a favorable environment for their growth. Other bacteria, such as members of the Mycobacterium genus, block lysosomal fusion with the phagolysosome or resist the oxidative attack that typically takes place within the phagolysosome.

Tuberculosis Is Primarily Controlled by CD4⫹ T Cells Until recently, tuberculosis, caused by Mycobacterium tuberculosis, was the leading cause of death in the world from a single infectious agent. Today, M. tuberculosis and HIV vie for the lead in deaths due to an infectious agent, with an increasing number of individuals infected by both. Roughly one-third of the world’s population is infected with M. tuberculosis. Although tuberculosis was believed to be eliminated as a public health problem in the United States, the disease re-emerged in the early 1990s, particularly in areas where HIV-infection levels are high. This disease is still the leading killer of individuals with AIDS. M. tuberculosis spreads easily, and pulmonary infection usually results from inhalation of small droplets of respiratory secretions containing a few bacilli. The inhaled bacilli are ingested by alveolar macrophages in the lung and are able to survive and multiply intracellularly by inhibiting forma-

TH1 cell Activated macrophages Macrophage with bacilli

Caseous center Bacilli

Activated macrophages

FIGURE 17-7 A tubercle formed in pulmonary tuberculosis.

tion of phagolysosomes. When the infected macrophages lyse, large numbers of bacilli are released. The most common clinical pattern of infection with M. tuberculosis, seen in 90% of infected individuals, is pulmonary tuberculosis. In this pattern, CD4⫹ T cells are activated within 2 to 6 weeks after infection and secrete cytokines that induce the infiltration of large numbers of activated macrophages. These cells wall off the organism inside a granuloma called a tubercle (Figure 17-7), a cluster of small lymphocytes surrounding infected macrophages. The localized concentrations of lysosomal enzymes in these granulomas can cause extensive tissue necrosis. The massive activation of macrophages that occurs within tubercles often results in the concentrated release of lytic enzymes. These enzymes destroy nearby healthy cells, resulting in circular regions of necrotic tissue, which eventually form a lesion with a caseous (cheeselike) consistency. As these lesions heal, they become calcified and are readily visible on x-rays of the lungs as a defined shadow. Much of the tissue damage seen with M. tuberculosis is thus actually due to pathology associated with the cell-mediated immune response. Because the activated macrophages suppress proliferation of the phagocytosed bacilli, infection is contained. Cytokines produced by CD4⫹ T cells (TH1 subset) play an important role in the response by activating macrophages so that they are able to kill the bacilli or inhibit their growth. The role of IFN-␥ in the immune response to mycobacteria has been demonstrated with knockout mice lacking IFN-␥. These mice died when they were infected with an attenuated strain of mycobacteria, whereas IFN-␥⫹ wild-type mice survived.

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Infectious Diseases and Vaccines The CD4⫹ T-cell-mediated immune response mounted by the majority of people exposed to M. tuberculosis controls the infection and later protects against reinfection. However, in about 10% of infected individuals, the disease progresses to chronic pulmonary tuberculosis or to extrapulmonary tuberculosis. This progression may occur years after the primary infection. In this clinical pattern, accumulation of large concentrations of mycobacterial antigens within tubercles leads to chronic CD4⫹ T-cell activation and ensuing macrophage activation, with high concentrations of lytic enzymes causing the necrotic caseous lesions to liquefy into a rich medium that allows the tubercle bacilli to proliferate extracellularly. Eventually the lesions rupture, and the bacilli disseminate in the lung and/or are spread through the blood and lymphatic vessels. Tuberculosis has traditionally been treated for long periods of time with several different antibiotics, sometimes in combination. The intracellular growth of M. tuberculosis makes it difficult for drugs to reach the bacilli, necessitating up to 9 months of daily treatment. Recent clinical trials showed a slightly more effective combination of antibiotics that can be taken less frequently and for a shorter period. Infected individuals with latent tuberculosis were more likely to complete this course of antibiotics, reducing the chances of disease spread. At present, the only vaccine for M. tuberculosis is an attenuated strain of M. bovis called Bacille Calmette-Guérin (BCG). This vaccine is fairly effective against extrapulmonary tuberculosis but less so against the more common pulmonary tuberculosis; in some cases, BCG vaccination has even increased the risk of infection. Moreover, after BCG vaccination the tuberculin skin test cannot be used as an effective monitor of exposure to wild-type M. tuberculosis. Because of these drawbacks, this vaccine is not used in the United States but is used in several other countries. However, the alarming increase in multidrug-resistant strains has stimulated renewed efforts to develop a more effective tuberculosis vaccine.

Diphtheria Can Be Controlled by Immunization with Inactivated Toxoid Diphtheria is the classic example of a bacterial disease caused by a secreted exotoxin. Immunity to Corynebacterium diphtheriae, the causative agent, can be induced by immunization with an inactivated form of the toxin, known as a toxoid. Natural infection with C. diphtheriae occurs only in humans, and is spread by respiratory droplets. The organism colonizes the nasopharyngeal tract and causes little tissue damage, with only a mild inflammatory reaction. Virulence is due to its potent exotoxin, which destroys the underlying tissue and results in heart, liver, and kidney damage, as well as to suffocation following formation of a tough fibrous membrane in the respiratory tract. Interestingly, the exotoxin is encoded not by the bacteria but by the tox gene carried by a bacterial virus (called phage ␤). The toxin inhib-

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its protein synthesis and is extremely potent: a single molecule has been shown to kill a cell. Immunization with the toxoid-based vaccine caused a rapid decrease in the number of cases of diphtheria, although sporadic outbreaks have occurred in areas where vaccination coverage is allowed to lapse. Diphtheria toxoid is administered in a vaccine combination with Bordetella pertussis (the cause of whooping cough) and tetanus toxoid (called DPT, for diphtheria, pertussis, and tetanus). DPT or DTaP, is given to children beginning at 6 to 8 weeks of age as part of the normal course of childhood immunizations (see below). Immunization with the toxoid induces the production of antibodies (antitoxin), which can bind to the toxin and neutralize its activity. Because antitoxin levels decline slowly over time, booster shots are recommended at 10-year intervals to maintain antitoxin levels within the protective range.

Parasitic Infections The term parasite encompasses a vast number of protozoan and helminthic organisms (worms). The diversity of the parasitic universe makes it difficult to generalize, but a major difference between these types of parasites is that the protozoans are unicellular eukaryotes that usually live and multiply within host cells for at least part of their life cycle, whereas helminths are multicellular organisms that can be quite large and have the ability to live and reproduce outside their human host. Most clinically relevant protozoan parasites also require an intermediate host for a portion of their life cycle and for transmission to human hosts. Parasites can evade the immune system, allowing them to chronically infect their human hosts and exact a lifelong toll. Malaria, African sleeping sickness, Chagas’s disease, leishmaniasis, and toxoplasmosis are among the most common parasitic diseases. Experimental systems, especially mouse models of infection, have defined how immunity to certain parasites is achieved, but the diversity of parasites and the complexity of the infections they cause preclude easy generalizations.

Protozoan Parasites Account for Huge Worldwide Disease Burdens Infections caused by parasites account for an enormous disease burden worldwide, especially in tropical or subtropical regions and developing countries where sanitation and living conditions are poor. The presence of sewage-tainted water promotes parasite spread and transmission to humans. Likewise, many of the protozoan parasites spend part of their time in arthropod hosts, such as mosquitoes, flies, or ticks, which serve as an essential microenvironment for completion of their life cycle and as a vector for transmission to humans. Targeting these blood-feeding vectors can therefore be quite effective in interrupting protozoan propagation and reducing transmission rates.

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The type and effectiveness of immune response to protozoan infection depends in part on the location of the parasite within the host and the life cycle stage of the parasite. Many protozoans spend part of their time free within the bloodstream; humoral antibody is most effective during these stages. At other stages they may grow intracellularly, making cell-mediated immune reactions the most effective host defense. In the development of vaccines for protozoan diseases, the life cycle stages of these pathogens and the branch of the immune response that is most likely to confer protection must be carefully considered. Malaria Malaria is the number-one parasitic cause of death worldwide. Half of the world’s population lives in a malaria endemic zone, and nearly 10% of the world population is infected by the causative agent of malaria: one of several species of the genus Plasmodium, of which P. falciparum is the most virulent. The alarming development of multiple-drug resistance in Plasmodium and the increased pesticide resistance of the Anopheles mosquito, the arthropod vector of Plasmodium, underscore the importance of developing new strategies to hinder the spread of malaria. Plasmodium has an extremely complex life cycle. Female Anopheles mosquitoes serve as the vector and host for part of the parasite’s life cycle. (Because male Anopheles mosquitoes feed on plant juices, they do not transmit Plasmodium.) Human infection begins when sporozoites, one of the life cycle stages of Plasmodium, enter the bloodstream as an infected female mosquito takes a blood meal (Figure 17-8). Sporozoites are long, slender cells that are covered by a 45-kDa protein called circumsporozoite (CS) antigen, which mediates their adhesion to hepatocytes. The binding site on the CS antigen is a conserved region that has a high degree of sequence homology with known human cell adhesion molecules. In hepatocytes, the parasites differentiate into merozoites, which infect red blood cells, initiating the major symptoms and pathology of malaria. Eventually some of the merozoites differentiate into male and female gametocytes, which may be ingested by a female Anopheles mosquito during a blood meal from an infected individual. Within the mosquito’s gut, the male and female gametocytes differentiate into gametes that fuse to form a zygote, which multiplies and differentiates into sporozoites within the mosquito’s salivary gland, initiating the cycle again. The symptoms of malaria are recurrent chills, fever, and sweating that peak roughly every 48 hours, when successive generations of merozoites are released from infected red blood cells. An infected individual eventually becomes weak and anemic. The merozoites can block capillaries, causing intense headaches, renal failure, heart failure, or cerebral damage (called cerebral malaria), often with fatal consequences. Some malaria symptoms may be caused by excessive production of cytokines, a hypothesis stemming from the observation that cancer patients treated with recombinant TNF-␣ developed symptoms that mimicked malaria. The connection

Sporozoites

Liver

Merozoites

RBC In mosquito gut

Gametocytes

FIGURE 17-8 The life cycle of Plasmodium. Sporozoites enter the bloodstream when an infected mosquito takes a blood meal. The sporozoites migrate to the liver, where they multiply, transforming liver hepatocytes into giant multinucleate schizonts, which release thousands of merozoites into the bloodstream. The merozoites infect red blood cells, which eventually rupture, releasing more merozoites. Eventually some of the merozoites differentiate into male and female gametocytes, which are ingested by a mosquito and differentiate into gametes in the mosquito’s gut. The gametes fuse to form a zygote that differentiates to the sporozoite stage within the salivary gland of the mosquito.

between TNF-␣ and malaria symptoms was studied by infecting mice with a mouse-specific strain of Plasmodium, which causes rapid death by cerebral malaria. Injection of these mice with antibodies to TNF-␣ prevented this rapid death. In regions where malaria is endemic, the immune response to Plasmodium infection is poor. Children younger than 14 years old mount the weakest immune response and consequently are most likely to develop malaria. In some regions, the childhood mortality rate for malaria reaches 50%. Even in adults, the degree of immunity is far from complete. Most people living in endemic regions have lifelong low-level Plasmodium infections.

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Infectious Diseases and Vaccines A number of factors may contribute to these low levels of immune response to Plasmodium. The maturational changes allow the organism to keep changing its surface molecules, resulting in continual changes in the antigens seen by the immune system. The intracellular phases of the life cycle reduce the degree of immune activation generated by the pathogen and allow the organism to multiply shielded from attack. The most accessible stage, the sporozoite, circulates in the blood for such a short time before infecting hepatocytes (approx. 30 minutes) that effective immune activation is unlikely to occur. Even when an antibody response does develop to sporozoites, Plasmodium overcomes that response by sloughing off the CS surface antigens, thus rendering the antibodies ineffective. The development of drug resistance by Plasmodium has complicated drug treatment choices for malaria, making the search for a vaccine very important. African Sleeping Sickness Two species of African trypanosomes cause African sleeping sickness, a chronic, debilitating disease transmitted to humans and cattle by the bite of the tsetse fly. In the bloodstream, the trypanosome, a flagellated protozoan, differentiates into a long, slender form that continues to divide every 4 to 6 hours. The disease progresses from an early, systemic stage in which trypanosomes multiply in the blood to a neurologic stage in which the parasite infects cells of the central nervous system, leading to meningoencephalitis and eventual loss of consciousness—thus the name. The surface of the Trypanosoma parasite is covered with a variable surface glycoprotein (VSG). Several unusual genetic processes generate extensive variation in these surface structures, enabling the organism to escape immunologic clearance. An individual trypanosome carries a large repertoire of VSG genes, each encoding a different VSG primary sequence, but expresses only a single VSG gene at a time. Trypanosoma brucei, for example, carries more than 1000 VSG genes in its genome. Activation of a VSG gene results in duplication of the gene and its transposition to a transcriptionally active expression site (ES) at the telomeric end of specific chromosomes (Figure 17-9a). Activation of a new VSG gene displaces the previous gene from the telomeric ES, like placing a new DVD in the reader. Trypanosomes have multiple transcriptionally active ES sites, so that a number of VSG genes can potentially be expressed; unknown control mechanisms limit expression to a single VSG expression site at any time. As parasite numbers increase after infection, an effective humoral response develops to the VSG covering the surface of the parasite. These antibodies eliminate most of the parasites from the bloodstream, both by complement-mediated lysis and by opsonization and subsequent phagocytosis. However, about 1% of the organisms bear an antigenically different VSG because of transposition of that VSG gene into the ES. These parasites escape the initial antibody response, begin to proliferate in the bloodstream, and go on to populate

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the next wave of parasitemia in the host. The successive waves of parasitemia reflect a unique mechanism of antigenic shift by which the trypanosomes evade the immune response to their surface antigens. Each new variant that arises in the course of a single infection escapes the humoral antibodies generated in response to the preceding variant, and so waves of parasitemia recur (Figure 17-9b). The new variants arise not by clonal outgrowth from a single escape variant cell, but from the expansion of multiple cells that have activated the same VSG gene in the current wave of parasitic growth. It is not known how this process is coordinated. This continual shifting of surface epitopes has made vaccine development extremely difficult. Leishmaniasis The protozoan parasite Leishmania major illustrates how powerfully different host responses can impact disease outcome, leading to either clearance of the parasite or death from the infection. Leishmania is a flagellated protozoan that lives in the phagosomes of macrophages and is transmitted by sandflies. It usually results in one of two syndromes: a localized cutaneous lesion that is generally painless and self-resolving, or a systemic form of the disease, called visceral leishmaniasis, which is nearly always fatal without treatment. Resistance to leishmaniasis correlates well with the production of IFN-␥ and the development of a TH1 response. Strains of mice that are naturally resistant to Leishmania develop a TH1 response and produce IFN-␥ upon infection. If IFN-␥ production or signaling is blocked in these strains, the mice become highly susceptible to Leishmania-induced fatality. However, a few strains of mice, such as BALB/c, are naturally susceptible to Leishmania-induced death. BALB/c animals mount a TH2-type response to Leishmania infection, producing high levels of IL-4 and essentially no IFN-␥. Studies have shown that a small subset of CD4⫹ T cells in the susceptible animals recognize a particular epitope on L. major, and produces high levels of IL-4 early in the response to the parasite, skewing the response towards a TH2-dominated pathway. Understanding how different T-helper responses affect the outcome of infections could contribute to the rational design of effective treatments and vaccines against this and other pathogens.

A Variety of Diseases Are Caused by Parasitic Worms (Helminths) Parasitic worms, or helminths, are responsible for a wide variety of diseases in humans and animals. The adult forms are large, multicellular organisms that can often be seen with the naked eye. Parasitic worms are frequently categorized based on their structure and site of infection: nematodes (roundworms), cestodes (tapeworms), and trematodes (flukes). Most enter their animal hosts through the intestinal tract; helminth eggs can contaminate food, water, feces, and soil. Although helminths are exclusively extracellular and therefore more accessible to the immune system than protozoans, most

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OVERVIEW FIGURE

Successive Waves of Parasitemia after Infection with Trypanosoma Result from Antigenic Shifts in the Parasite’s Variant Surface Glycoprotein (VSG) (a) 5′

VSG1

VSG2

VSG3

VSG4

VSGn

VSG1

Expression site VSG1 3′

VSG1 VSG1

Duplication and translocation to expression site

5′

VSG1

VSG2

VSG3

VSG4

VSGn

VSG2

Expression site VSG2 3′

VSG3

Expression site 5′

VSG1

VSG2

VSG3

VSG4

VSGn

VSG2

VSG2

Duplication and translocation to expression site

VSG3

3′ VSG3

VSG3 (b) Antibodies to variant 3 Antibodies to variant 2 Antibodies to variant 1

Millions of trypanosomes per milliliter of blood

1.5 Variant 2

Variant 3

Variant 1 1.0

0.5

0 25

26

27 28 29 Approximate time after tsetse fly bite, weeks

30

(a) Antigenic shifts in trypanosomes occur by the duplication of gene segments encoding variant VSG molecules and their translocation to an expression site located close to the telomere. (b) Antibodies develop against each variant as the numbers of these parasites rise, but each new variant that arises is unaffected by the humoral antibodies induced by the previous variant. [Source: Part (b) adapted from J. Donelson, 1988, The Biology of Parasitism, New York: Alan R. Liss.]

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Infectious Diseases and Vaccines infected individuals carry few parasites. Unlike protozoan parasites, helminths do not multiply within their hosts. Thus, the immune response is not strongly engaged, and the level of immunity generated can be very poor. More than 300 million people are infected with Schistosoma, which causes the chronic, debilitating, and sometimes-fatal disease schistosomiasis. Infection occurs through contact with free-swimming infectious larvae that are released from an infected snail and bore into the skin, frequently while individuals wade through contaminated water. As they mature, they migrate in the body, with the final site of infection varying by species. The females produce eggs, some of which are excreted and infect more snails. Most symptoms of schistosomiasis are initiated by the eggs, which invade tissues and cause hemorrhage. A chronic state can develop in which the unexcreted eggs induce cell-mediated DTH reactions, resulting in large granulomas that can obstruct the venous blood flow to the liver or bladder. An immune response does develop to the schistosomes, but it is usually not sufficient to eliminate the adult worms. Instead, the worms survive for up to 20 years, causing prolonged morbidity. Adult schistosome worms have several unique mechanisms that protect them from immune defenses. These include decreasing the expression of antigens on their outer membrane and enclosing themselves in a glycolipid-and-glycoprotein coat derived from the host, masking the presence of their own antigens. Among the antigens observed on the adult worm are the host’s own ABO blood-group and histocompatibility antigens! The immune response is, of course, diminished by this covering made of the host’s self antigens, which must contribute to the lifelong persistence of these organisms. The major contributors to protective immunity against schistosomiasis are controversial. The immune response to infection with S. mansoni is dominated by TH-2-like mediators, with high titers of antischistosome IgE antibodies, localized increases in degranulating mast cells, and an influx of eosinophils (Figure 17-10, top). These cells can then bind the antibody-coated parasite using their Fc receptors for IgE or IgG, inducing degranulation and death to the parasite via antibody-dependent cell-mediated cytotoxicity (ADCC; see Figure 13-14). One eosinophil mediator, called basic protein, has been found to be particularly toxic to helminths. However, immunization studies in mice suggest that a TH1 response, characterized by IFN-␥ and macrophage accumulation, may actually be more effective for inducing protective immunity (Figure 17-10, bottom). In fact, inbred strains of mice with deficiencies in mast cells or IgE can still develop protective immunity to S. mansoni following vaccination. Based on these observations, it has been suggested that the ability to induce an ineffective TH2-like response may have evolved in schistosomes as a clever defense mechanism to ensure that IL-10 and other TH-1 inhibitors are induced in order to block initiation of a more effective TH1-dominated pathway.

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Fungal Infections Fungi are a diverse and ubiquitous group of organisms that occupy many niches and also perform services for humans, including the fermentation of bread, cheese, wine, and beer, as well as the production of penicillin. As many as a million species of fungi are known to exist; only about 400 are potential agents of human disease. Infections may result from introduction of exogenous organisms due to injury or inhalation, or from endogenous organisms such as the commensals present in the gut and on the skin. Fungal diseases, or mycoses, are classified based on the following criteria: • Site of infection—superficial, cutaneous, subcutaneous, or deep and systemic • Route of acquisition—exogenous or endogenous • Virulence—primary or opportunistic These categories (summarized in Table 17-4) are not mutually exclusive. For example, an infection such as coccidiomycosis may progress from a cutaneous lesion to a systemic infection of the lungs. Cutaneous infections include attacks on skin, hair, and nails; examples are ringworm, athlete’s foot, and jock itch. Subcutaneous infections are normally introduced by trauma and accompanied by inflammation; if inflammation is chronic, extensive tissue damage may ensue. Deep mycoses involve the lungs, the central nervous system, bones, and the abdominal viscera. These infections can occur through ingestion, inhalation, or inoculation into the bloodstream. A very rare and deadly outbreak of fungal meningitis in 2012 was linked to Exserohilium rostratum, a fungal contaminant in a preparation of corticosteroids used for epidural injections. Virulence can be divided into primary, indicating the rare agents with high pathogenicity, and opportunistic, denoting weakly virulent agents that primarily infect individuals with compromised immunity. Most fungal infections of healthy individuals are resolved rapidly, with few clinical signs. The most commonly encountered and best-studied human fungal pathogens are Cryptococcus neoformans, Aspergillus fumigatus, Coccidioides immitis, Histoplasma capsulatum, and Blastomyces dermatitidis. Diseases caused by these fungi are named for the agent; for example, C. neoformans causes cryptococcosis and B. dermatitidis causes blastomycosis. In each case, infection with these environmental agents is aided by predisposing conditions that include AIDS, immunosuppressive drug treatment, and malnutrition.

Innate Immunity controls Most Fungal Infections The barriers of innate immunity control most fungi. Commensal organisms also help control the growth of potential pathogens, as demonstrated by long-term treatment with broad-spectrum antibiotics, which destroy normal mucosal bacterial flora and often lead to oral or vulvovaginal infection

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OVERVIEW FIGURE

Overview of the Immune Response Generated against Schistosoma mansoni IgE

Inflammation Mast cell Mediators NCF

Plasma cell

ECF

PAF PAF

C3a C5a

Platelets

C Eosinophil B a si c p rot

Neutrophil C3b

ein

Adult worm C C3a C5a

C3b

Macrophage IFN-γ

T H1

Mast cell Chemotaxis Mediators

The response includes an IgE humoral component (top) and a cell-mediated component involving CD4⫹ T cells (bottom). C ⫽ complement; ECF ⫽ eosinophil chemotactic factor; NCF ⫽ neutrophil chemotactic factor; PAF ⫽ platelet-activating factor.

with Candida albicans, an opportunistic agent. Phagocytosis by neutrophils is a strong defense against most fungi, and so people with neutropenia (low neutrophil count) are generally more susceptible to fungal disease. Resolution of infection in normal, healthy individuals is often rapid and initiated by recognition of common fungal

cell wall PAMPs. The three most medically relevant cell wall components include ␤-glucans (polymers of glucose), mannans (long chains of mannose), and chitin (a polymer of N-acetylglucosamine). The importance of certain pattern recognition receptors (PRRs) in resolving fungal infection has been demonstrated by the increased susceptibility to

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Classification of fungal diseases

Site of infection

Superficial Cutaneous Subcutaneous Deep or systemic

Epidermis, no inflammation Skin, hair, nails Wounds, usually inflammatory Lungs, abdominal viscera, bones, CNS

Route of acquisition

Exogenous Endogenous

Environmental, airborne, cutaneous, or percutaneous Latent reactivation, commensal organism

Virulence

Primary Opportunistic

Inherently virulent, infects healthy host Low virulence, infects immunocompromised host

mycoses seen in individuals with defects in these components. For instance, certain molecular variants of dectin 1, a C-type lectin receptor (see Chapter 5), are associated with chronic mucocutaneous candidiasis. Toll-like receptors 2, 4 and 9, as well as complement receptor 3 (CR3), are also involved in the innate response to fungi. In sum, recognition of these cell wall components leads to the activation of complement (via both alternative and lectin pathways) along with the induction of phagocytosis and destruction of fungal cells. The key role of CR3, which recognizes complement deposited on the ␤-glucans of fungal cells, was confirmed by the fact that mortality from experimental infections of mice with Cryptococcus increased after an antibody to CR3 was administered. Like other microbes, fungi have evolved mechanisms to evade the innate immune response. These include production of a capsule, as in the case of C. neoformans, which blocks PRR binding. Another evasion strategy employed by this organism involves fungi-induced expulsion from macrophages that does not kill host cells and therefore avoids inflammation.

Immunity against Fungal Pathogens Can Be Acquired A convincing demonstration of acquired immunity against fungal infection is the protection against subsequent attacks following an infection. This protection is not always obvious for fungal disease because primary infection often goes unnoticed. However, positive skin reactivity to fungal antigens is a good indicator of prior infection and the presence of memory responses. For instance, a granulomatous inflammation controls spread of C. neoformans and H. capsulatum, indicating the presence of acquired cell-mediated immunity. However, the organism may remain in a latent state within the granuloma, reactivating only if the host becomes immunosuppressed. The presence of antibodies is another sign of prior exposure and lasting immunity, and antibodies against C. neoformans are commonly found in healthy subjects. However, probably the most convincing argument for preexisting immunity against fungal pathogens comes from the fre-

quency of normally rare fungal diseases in patients with compromised immunity. AIDS patients suffer increased incidences of mucosal candidiasis, histoplasmosis, coccidiomycosis, and cryptococcosis. These observations in T-cellcompromised AIDS patients and data showing that B-cell–deficient mice have no increased susceptibility to fungal disease indicate that cell-mediated mechanisms of immunity likely control most fungal pathogens. The study of immunity to fungal pathogens has become more pressing with the advent of AIDS and the increase in individuals receiving immunosuppressive drugs for other conditions. This has led to the observation that strong TH1 responses and the production of IFN-␥, important for optimal macrophage activation, are most commonly associated with protection against fungi. Conversely, TH2 and TREG cell responses, or their products, are associated with susceptibility to mycoses. This is apparent in patients displaying distinct T helper responses to coccidioidomycosis, where TH1 immune activity is associated with a mild, asymptomatic infection and TH2 responses result in a severe and often relapsing form of the disease. Although the role for other cell types is less certain, recently a regulatory role for TH17 cells in controlling adaptive immunity against fungi has been postulated, where these cells are hypothesized to help support TH1- and discourage TH2-cell activation.

Emerging and Re-emerging Infectious Diseases At least yearly, it seems, we hear about a new virus or bacterium arising, accompanied by severe illness or death. Newly described human pathogens are referred to as emerging pathogens. Examples include HIV, SARS, WNV, the widely publicized Ebola virus, and Legionella pneumophila, the bacterial causative agent for Legionnaires’ disease. These often appear to come from nowhere and, as far as we know, are caused by new human pathogens. On the other hand, reemerging pathogens are those that were formerly rare or largely eradicated but suddenly begin to infect a widening number of individuals. The re-emergence of these diseases is

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The Immune System in Health and Disease Multidrug-resistant tuberculosis

Lyme disease

Cryptosporidiosis

Cryptosporidiosis Vancomycin-resistant S. aureus Cyclosporiasis

vCJD West Nile virus

Diphtheria

SARS Vancomycinresistant S. aureus

Typhoid fever Cholera

E. coli O157:H7

E. coli O157:H7

H5N1 influenza

Human monkeypox

Drug-resistant malaria Vancomycinresistant S. aureus

Whitewater arroyo virus

Nipah virus

Hantavirus pulmonary syndrome

Hendra virus

H1N1 influenza

Enterovirus 71 Hepatitis C

Dengue

Rift Valley fever Cholera Yellow fever Hantavirus pulmonary syndrome

HIV Human monkeypox

Lassa fever Yellow fever Drug-resistant malaria Cholera

Plague

Ebola

hemorrhagic fever Marburg hemorrhagic fever

FIGURE 17-11 Examples of emerging and re-emerging diseases showing points of origin. Red represents newly emerging diseases, black re-emerging diseases. [Source: Adapted from A. S. Fauci, 2001, Infectious diseases: Considerations for the 21st Century, Clinical Infectious Diseases 32:675.]

not surprising if we consider that some microbes can adapt to a range of environments, and that many microbes can exist in dormant states or can survive in intermediate hosts. Examples of many of the emerging and re-emerging infectious diseases are shown in Figure 17-11.

Some Noteworthy New Infectious Diseases Have Appeared Recently Ebola was first recognized after an outbreak in Africa in 1976. The disease received a great deal of attention because of the severity and rapid progression to death after the onset of symptoms. By 1977, the causative virus had been isolated and classified as a filovirus, a type of RNA virus that includes the similarly deadly Marburg virus, a close relative of Ebola. The most pathogenic strain, Ebola-Zaire, causes a particularly severe hemorrhagic fever, killing roughly 50% to 90% of those infected, often within days of the onset of symptoms. Although the risk of death is very high after infection, it is fairly easy to control the spread of the virus by isolating infected individuals. This is an example of a pathogen that likely resides in some wild animals, which helps it to remain

in the environment for long periods of time and only infect humans when some form of contact is made. Although the animal reservoir for Ebola has not been found, fruit bats are the most likely candidate. Legionnaires’ disease is a virulent pneumonia first reported in attendees at an American Legion convention in Philadelphia in 1976. Of the 221 afflicted, 34 died. The organism causing the disease turned out to be a bacterium, later named Legionella pneumophila. It proliferates in cool, damp areas, including the condensing units of large commercial air-conditioning systems. Infection can spread when the air-conditioning system emits an aerosol containing the bacteria. Improved design of air-conditioning and plumbing systems has greatly reduced the danger from this disease. In November 2002, an unexplained atypical pneumonia was seen in the Guandong province of China, proving resistant to any treatment. A physician who had cared for some of these patients traveled to Hong Kong and infected guests in his hotel, who then seeded a multinational outbreak that lasted until May 2003. By the time the disease, called severe acute respiratory syndrome (SARS), was contained, 8096 cases had been reported, with 774 deaths. A rapid response

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(b) Virulent SARS virus

Civet SARS virus

Mild-virulence SARS virus

Nonbinding

SARS spike protein Weak binding

Amino acid substitution

FIGURE 17-12 The coronavirus that caused the outbreak of severe acute respiratory syndrome, or SARS. (a) The virus is studded with spikes that in cross-section give it the appearance of a crown, hence the name coronavirus. (b) The human receptor for the SARS virus is angiotensin-converting enzyme 2 (ACE2). The spike protein from the highly virulent form of the virus binds with high affinity to this receptor; the variant of this virus found in the civet cat

by the biomedical community identified the etiologic agent as a coronavirus, so named because the spike proteins emanating from these viruses give them a crownlike appearance (Figure 17-12a). This virus was soon traced to the civet cat after the earliest cases of SARS were found to occur in animal vendors. Human coronaviruses had been known for many years, primarily as the cause of a mild form of the common cold, but this newly emerged variant had not been seen previously. X-ray crystallography showed that a mutated civet SARS virus spike protein could bind to a human cell surface protein, human angiotensin-converting enzyme type 2 (ACE2), 1000 times more tightly than did the original virus spike protein, answering the question of how this virus jumped from the civet cat to humans (Figure 17-12b). Animal models for SARS showed that antibodies to the outer spike protein could thwart replication of the virus, leading to the later development of an intranasal vaccine that could induce a strong local humoral response as well as cell-mediated immunity. West Nile virus (WNV), first identified in Uganda in 1937, was not seen outside Africa or western Asia until 1999, when it showed up in the New York City area. By 2006, it had been reported in all but six states in the continental United States. WNV is a flavivirus that replicates very well in certain species of birds and is carried by mosquitoes from infected birds to so-called dead-end hosts such as horses and humans. Transmission between humans via mosquitos is inefficient because the titer of virus in human blood is low and the amount of blood transferred by the insect bite is small and does not contain sufficient virus to cause infection. WNV may, however, be transferred from human to human by blood transfusion and may be passed from

Human receptor for the SARS virus: angiotensinconverting enzyme 2 (ACE2)

and in a version that causes mild human disease have two different amino acid substitutions in the spike protein. The civet cat SARS virus does not bind to the human receptor, and the version that causes mild disease in humans binds 1000 times more weakly than the most virulent form. [Source: Part a from Dr. Linda Stannard, UCT/Photo Researchers; part b adapted from K. V. Holmes, 2005, Structural biology. Adaptation of SARS coronavirus to humans. Science 309:1822.]

infected pregnant mothers to their newborns. It is only a health hazard in individuals with compromised immune function, in whom it can cross the blood-brain barrier and cause life-threatening encephalitis or meningitis. The primary public health control measure is education of the public about mosquito control. Why are these new diseases emerging and others reemerging around the globe? One reason may be the crowding of the world’s poorest populations into ever smaller areas within huge cities. Another factor is the great increase in international travel: an individual can become infected on one continent and then spread the disease on another continent all in the same day. The mass distribution and importation of food can also expose large populations to potentially contaminated food. Indiscriminate use of antibiotics in humans and in veterinary applications also fosters resistance in pathogens to the drugs commonly used against them. Finally, changes in climate and weather patterns, along with the extension of human populations into the previously unbroached domains of animals, can introduce new pathogens into human populations.

Diseases May Re-emerge for Various Reasons Tuberculosis is a re-emerging disease now receiving considerable attention. Twenty years ago, public health officials were convinced that tuberculosis would soon disappear as a major health consideration in the United States. A series of events conspired to interrupt the trend, including the AIDS epidemic and other conditions that result in immunosuppressed individuals, allowing Mycobacterium strains to gain a foothold and evolve resistance to the conventional battery

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of antibiotics. These individuals can then pass on newly emerged, antibiotic-resistant strains of M. tuberculosis to others. Despite the disappointment of not eradicating this disease in the United States, rates of tuberculosis have been declining annually, with a record decline of more than 10% in 2009. However, incidence rates worldwide are falling at less than 1% annually, despite multiple efforts to tackle tuberculosis globally. Laxity in adherence to vaccination programs can also lead to re-emergence of diseases that were nearly eradicated. For example, diphtheria began to re-emerge in parts of the former Soviet Union in 1994, where it had almost vanished thanks to European vaccination programs. By 1995, over 50,000 cases were reported and thousands died. The social upheaval and instability that came with the breakup of the Soviet Union was almost certainly a major factor in the reemergence of this disease, due to lapses in vaccination and other public health measures. Even in the United States, an increasing trend in some regions to delay or opt out of childhood vaccination has led to sporadic local outbreaks in previously rare childhood diseases such as measles and whooping cough. The WHO and the CDC both actively monitor new infections, and work closely to detect and identify new infectious agents and key re-emerging pathogens. Their collective data help to provide up-to-date information for travelers to parts of the world where such emerging and re-emerging infectious agents may pose a risk.

Vaccines Preventative vaccines have led to the control or elimination of many infectious diseases that once claimed millions of lives. Since October 1997, not a single naturally acquired smallpox case has been reported anywhere. On the heels of the global victory over smallpox, the program to eradicate polio went into overdrive. Led by the WHO and several large philanthropic donors, numbers of polio cases were reduced worldwide by over 95% by the year 2000. Alas, cultural and religious resistance to vaccination led to polio’s reappearance in recent years, although many vaccination programs are now back on track. Worldwide vaccination campaigns can also be credited with the control of at least 10 other major infectious diseases (measles, mumps, rubella, typhoid, tetanus, diphtheria, pertussis, influenza, yellow fever, and rabies), many of which previously affected mostly babies and young children. Still, a crying need remains for vaccines against many other diseases, including malaria, tuberculosis, and AIDS. More work is needed for existing vaccines as well: to improve safety and efficacy of some, or to lower the cost and simplify the delivery of existing vaccines so that they can reach those who need them most, especially in developing countries. Based on WHO data, millions of infants still die due to diseases that could be prevented by existing vaccines. Development of effective new vaccines is a long, complicated, and costly process, rarely reaching the stage of years-

long clinical trials. Many vaccine candidates that were successful in laboratory and animal studies fail to prevent disease in humans, have unacceptable side effects, or may even worsen the disease they were meant to prevent. Stringent testing is an absolute necessity, because approved vaccines will be given to large numbers of well people. Clear information for consumers about adverse side effects, even those that occur at very low frequency, must be made available and carefully balanced against the potential benefit of protection by the vaccine. Vaccine development begins with basic research. An appreciation of the differences in epitopes recognized by T and B cells has enabled immunologists to design vaccine candidates to maximize activation of both cellular and humoral immune responses. As differences in antigenprocessing pathways became evident, design strategies and additives can be employed to activate specific, desired immune pathways and to maximize antigen presentation. Targeting strategies to elicit protection at mucosal surfaces, the most common site of infection, are also underway. Finally, techniques like genetic engineering are being employed to develop vaccines that maximize the immune response to selected epitopes and that simplify delivery. Here we describe current vaccine strategies, some vaccines presently in use, and new strategies that may lead to future vaccines.

Protective Immunity Can Be Achieved by Active or Passive Immunization Immunization is the process of eliciting a long-lived state of protective immunity against a disease-causing pathogen. Exposure to the live pathogen followed by recovery is one route to immunization. Vaccination, or intentional exposure to forms of a pathogen that do not cause disease (a vaccine), is another. In an ideal world, both engage antigen-specific lymphocytes and result in the generation of memory cells, providing long-lived protection. However, vaccination does not ensure immunity, and a state of immune protection can be achieved by means other than infection or vaccination. For example, the transfer of antibodies from mother to fetus or the injection of antiserum against a pathogen both provide immune protection. Without the development of memory B or T cells specific to the organism, however, this state of immunity is only temporary. Thus, vaccination is an event, whereas immunization (the development of a protective memory response) is a potential outcome of that event. Here we describe current use of passive and active immunization techniques. Passive Immunization by Delivery of Preformed Antibody Edward Jenner and Louis Pasteur are recognized as the pioneers of vaccination, or early attempts to induce active immunity, but similar recognition is due to Emil von Behring and Hidesaburo Kitasato for their contributions to passive

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Common agents used for passive immunization

Disease

Agent

Black widow spider bite

Horse antivenin

Botulism

Horse antitoxin

Cytomegalovirus

Human polyclonal Ab

Diphtheria

Horse antitoxin

Hepatitis A and B

Pooled human immunoglobulin

Measles

Pooled human immunoglobulin

Rabies

Human or horse polyclonal Ab

Respiratory disease

Monoclonal anti-RSV*

Snake bite

Horse antivenin

Tetanus

Pooled human immunoglobulin or horse antitoxin

Varicella zoster virus

Human polyclonal Ab

*

Respiratory syncytial virus

Source: Adapted from A. Casadevall, 1999, Passive antibody therapies: progress and continuing challenges. Clinical Immunology 93:5.

immunity. These investigators were the first to show that immunity elicited in one animal can be transferred to another by injecting serum from the first. Passive immunization, in which preformed antibodies are transferred to a recipient, occurs naturally when maternal IgG crosses the placenta to the developing fetus. Maternal antibodies to diphtheria, tetanus, streptococci, rubeola, rubella, mumps, and poliovirus all afford passively acquired protection to the developing fetus. Later, maternal antibodies present in breast milk can also provide passive immunity to the infant in the form of maternally produced IgA. Passive immunization can also be achieved by injecting a recipient with preformed antibodies, called antiserum, from immune individuals. Before vaccines and antibiotics became available, passive immunization was the only effective therapy for some otherwise fatal diseases, such as diphtheria, providing much needed humoral defense (see Chapter 1, Clinical Focus Box 1-2). Currently, several conditions still warrant the use of passive immunization, including the following: • Immune deficiency, especially congenital or acquired B-cell defects • Toxin or venom exposure with immediate threat to life • Exposure to pathogens that can cause death faster than an effective immune response can develop Babies born with congenital immune deficiencies are frequently treated with passive immunization, as are chil-

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dren experiencing acute respiratory failure caused by respiratory syncytial virus (RSV). Passive immunity is used in unvaccinated individuals exposed to the organisms that cause botulism, tetanus, diphtheria, hepatitis, measles, and rabies (Table 17-5), or to protect travelers and health-care workers who expect exposure to potential pathogens for which they lack protective immunity. Antiserum provides protection against poisonous snake and insect bites. In all these instances, it is important to remember that passive immunization does not activate the host’s immune response. Therefore, it generates no memory response and protection is transient. Although passive immunization may be effective, it should be used with caution because certain risks are associated with the injection of preformed antibody. If the antibody was produced in another species, such as a horse (one of the most common animal sources), the recipient can mount a strong response to the isotypic determinants of the foreign antibody, or the parts of the antibody that are unique to the horse species. This anti-isotype response can cause serious complications. Some individuals will produce IgE antibody specific for horse-specific determinants. High levels of these IgE-horse antibody immune complexes can induce pervasive mast-cell degranulation, leading to systemic anaphylaxis (see Chapter 15). Other individuals produce IgG or IgM antibodies specific for the foreign antibody, which form complement-activating immune complexes. The deposition of these complexes in the tissues can lead to type III hypersensitivity reactions. Even when human antiserum, or gamma globulin, is used, the recipient can generate an anti-allotype response to the human immunoglobulin (recognition of within species antigenic differences), although its intensity is usually much less than that of an anti-isotype response. Active Immunization to Induce Immunity and Memory Whereas the aim of passive immunization is transient protection or alleviation of an existing condition, the goal of active immunization is to trigger the adaptive immune response in a way that will elicit protective immunity and immunologic memory. When active immunization is successful, a subsequent exposure to the pathogenic agent elicits a secondary immune response that successfully eliminates the pathogen or prevents disease mediated by its products. Active immunization can be achieved by natural infection with a microorganism, or it can be acquired artificially by administration of a vaccine. In active immunization, as the name implies, the immune system plays an active role— proliferation of antigen-reactive T and B cells is induced and results in the formation of protective memory cells. This is the primary goal of vaccination. Active immunization with various types of vaccines has played an important role in the reduction of deaths from infectious diseases, especially among children. In the United States, vaccination of children begins at birth. The American

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Recommended childhood immunization schedule in the United States, 2012 AGE

Vaccine

Birth

Hepatitis B

Hep B

1 mo

2 mo

4 mo

6 mo

9 mo

Hep B

12 mo

15 mo

19–23 mo

2–3 yr

4–6 yr

Hep B

Rotavirus

RV

RV

RV

Diphtheria, tetanus, pertussis

DTaP

DTaP

DTaP

Haemophilus influenzae type b

Hib

Hib

Hib

Hib

Pneumococcal

PCV

PCV

PCV

PCV

Inactivated poliovirus

IPV

IPV

DTaP

DTaP

PPSV

IPV

IPV Influenza (yearly)

Influenza Measles, mumps, rubella

MMR

MMR

Varicella

Varicella Hepatitis A

18 mo

(Two doses at least 6 months apart)

Meningococcal

Varicella Dose 1

HepA series MCV4

Recommendations in effect as of 12/23/11. Any dose not administered at the recommended age should be administered at a subsequent visit, when indicated and feasible. A combination vaccine is generally preferred over separate injections of equivalent component vaccines. MCV4 and PPSV ranges are recommended for certain high risk groups. See website for further details. Source: Modified version of 2012 American Academy of Pediatrics recommendations, found on CDC chart, www.cdc.gov/vaccines/schedules/index.html.

Academy of Pediatrics sets nationwide recommendations (updated in 2012) for childhood immunizations in this country, as outlined in Table 17-6. The program includes the following vaccines for children from birth to age 6: • Hepatitis B vaccine (HepB) • Diphtheria-tetanus-(acellular) pertussis (DTaP) combined vaccine • Haemophilus influenzae type b (Hib) vaccine to prevent bacterial meningitis and pneumonia • Inactivated (Salk) polio vaccine (IPV) • Measles-mumps-rubella (MMR) combined vaccine • Varicella zoster vaccine for chickenpox • Meningococcal vaccine (MCV4) against Neisseria meningitidis • Pneumococcal conjugate vaccine (PCV) or pneumococcal polysaccharide vaccine (PPSV) against Streptococcus pneumoniae • Influenza virus vaccine (seasonal flu) • Hepatitis A vaccine (HepA) • Rotavirus (RV)

New recommendations as of February 2012 for young adults add males ages 11 to 12, to the individuals recommended to receive vaccination against sexually-transmitted human papillomavirus (HPV), the primary cause of cervical cancer in women. Since 2006 this vaccine has been recommended for females, starting at age 11 to 12 (see Clinical Focus Box 19-1). As indicated in Table 17-6, children typically require boosters (repeated inoculations) at appropriately timed intervals to achieve protective immunity. In the first months of life, the reason for this may be persistence of circulating maternal antibodies in the young infant. For example, passively acquired maternal antibodies bind to epitopes on the DTaP vaccine and block adequate activation of the immune system; therefore, this vaccine must be given several times after the maternal antibody has been cleared from an infant’s circulation in order to achieve adequate immunity. Passively acquired maternal antibody also interferes with the effectiveness of the measles vaccine; for this reason, the MMR vaccine is not given before 12 to 15 months of age. In developing countries, however, the measles vaccine is administered at 9 months, even though maternal antibodies are still present, because 30% to 50% of young children in these countries contract the disease before 15 months of age. Multiple immunizations with the polio vaccine are required to ensure that an adequate immune response is generated to each of the three strains of poliovirus that make up the vaccine.

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Infectious Diseases and Vaccines The widespread use of vaccines for common, life-threatening diseases in the United States has led to a dramatic decrease in the incidence of these diseases. As long as these immunization programs are maintained, especially in young children, the incidence of these diseases typically remains low. However, the occurrence or even the suggestion of possible side effects to a vaccine, as well as general trends toward reduced vaccination in children, can cause a drop in vaccination rates that leads to re-emergence of that disease. For example, rare but significant side effects from the original pertussis attenuated bacterial vaccine included seizures, encephalitis, brain damage, and even death. Decreased usage of the vaccine led to an increase in the incidence of whooping cough, before the development of an acellular pertussis vaccine (the aP in DTaP), as effective as the older vaccine but with fewer side effects, became available in 1991. However, recent trends toward altered vaccination schedules or refusal to vaccinate children may be fueling outbreaks of this disease. There were 18,000 cases and several deaths in U.S. children in just the first part of 2012, putting that year on track to be the worst in the past five decades. Despite the safety record of this vaccine and the frightening rise in this potentially deadly childhood disease, some parents still elect not to vaccinate their children (see Chapter 1, Clinical Focus Box 1-1). Recommendations for vaccination of adults depend on the risk group. Vaccines for meningitis, pneumonia, and influenza are often given to groups living in close quarters (e.g., military recruits and incoming college students) or to individuals with reduced immunity (e.g., the elderly). Depending on their destination, international travelers are also routinely immunized against endemic diseases such as cholera, yellow fever, plague, typhoid, hepatitis, meningitis,

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typhus, and polio. Immunization against the deadly disease anthrax had been reserved for workers coming into close contact with infected animals or animal products. Concerns about the potential use of anthrax spores by terrorists or in biological warfare has widened use of the vaccine to military personnel and civilians in areas believed to be at risk. Vaccination is not 100% effective. With any vaccine, a small percentage of recipients will respond poorly and therefore will not be adequately protected. Th is is not a serious problem if the majority of the population is immune to an infectious agent, significantly reducing the pathogen reservoir. In this case, the chance of a susceptible individual contacting an infected individual is very low. This phenomenon is known as herd immunity. The appearance of measles epidemics among college students and preschool-age children in the United States resulted partly from an overall decrease in vaccinations, which had lowered the herd immunity of the population (Figure 17-13). Among preschool-age children, 88% of those who developed measles were unvaccinated. Most of the college students who contracted measles had been vaccinated as children but only once. The failure of the single vaccination to protect them may have resulted from the lingering presence of passively acquired maternal antibodies (disappearing at 12 to 15 months of age), which reduced their overall response to the vaccine. This prompted the revised recommendation that children receive two MMR immunizations: one at 12 to 15 months of age and the second at 4 to 6 years. The CDC has called attention to the decline in vaccination rates and herd immunity among U.S. children. For example, based on data collected by California health

1000

800 Vaccine licensed

700 600

Number of cases, in thousands

15

900 Number of cases, in thousands

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58

60

62 64

66 68 70 Year

72 74

76

78

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82 84

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FIGURE 17-13 Introduction of the measles vaccine in 1962 led to a dramatic decrease in the annual incidence of this disease in the United States. Occasional outbreaks of measles in the 1980s (inset) occurred mainly among unvaccinated young children and among college students; most of the latter had been vaccinated but only once, when they were very young. [Source: Data from Centers for Disease Control and Prevention.]

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officials, pockets of vaccine noncompliance, mostly due to fear of adverse consequences, have likely decreased local herd immunity, resulting in outbreaks. Such a decrease portends serious consequences, as illustrated by recent events in the newly independent states of the former Soviet Union. By the mid-1990s, a diphtheria epidemic was raging in many regions of these new countries, linked to a decrease in herd immunity resulting from decreased vaccination rates after the breakup of the Soviet Union. Mass immunization programs now control this infectious disease.

There Are Several Vaccine Strategies, Each with Unique Advantages and Challenges Three key factors must be kept in mind in the development of a successful vaccine: the vaccine must be safe, it must be effective in preventing infection, and the strategy should be reasonably achievable given the population in question. Population considerations can include geographical locale, access to the target group (which may require several vaccinations), complicating co-infections or nutritional states, and, of course, cost. Critical for success is the branch of the immune system that is activated, and therefore vaccine designers must recognize the important differences between activation of the humoral and cell-mediated branches, or divergent pathways within these branches. Protection must also reach the relevant site of infection; mucosal surfaces are the prime candidates. An additional factor is the development of long-term immunologic memory. For example, a vaccine that induces a protective primary response may fail to induce the formation of memory cells, leaving the host unprotected after the primary response subsides. Before vaccines can progress from the laboratory bench to the bedside, they must go through rigorous testing in animals and humans. Most vaccines in development never progress beyond animal testing. The type of testing depends on what animal model systems are available, but frequently involves rodents and/or nonhuman primates. When these animal studies prove fruitful, follow-up clinical trials in humans can be initiated. Phase I clinical trials assess human safety; a small number of volunteers are monitored closely for adverse side effects. Only once this hurdle has been successfully passed can a trial move on to phase II, where effectiveness against the pathogen in question is evaluated. In this case, development of a measurable immune response to the immunogen is tested. However, even a positive result at this juncture does not necessarily mean that a state of protective immunity has been achieved or that long-term memory is established, and many vaccines fail at this stage. Phase III clinical trials are run in expanded volunteer populations, where protection against “the real thing” is the desired outcome, and where safety, the evaluation of several measurable immune markers, and the incidence of wild-type infections with the relevant pathogen are all monitored carefully over time. The relative significance of memory cells in immunity depends, in part, on the incubation period of the pathogen.

For influenza virus, which has a very short incubation period (1 or 2 days), disease symptoms are already underway by the time memory cells are reactivated. Effective protection against influenza therefore depends on maintaining high levels of neutralizing antibody by repeated immunizations; those at highest risk are immunized each year, usually at the start of the flu season. For pathogens with a longer incubation period, the presence of detectable neutralizing antibody at the time of infection is not necessary. The poliovirus, for example, requires more than 3 days to begin to infect the central nervous system. An incubation period of this length gives the memory B cells time to respond by producing high levels of serum antibody. Thus, the vaccine for polio is designed to induce high levels of immunologic memory. After immunization with the Salk vaccine (an inactivated form, see below), serum antibody levels peak within 2 weeks and then decline. However, the memory response continues to climb, reaching maximal levels at 6 months post vaccine and persisting for years. If an immunized individual is later exposed to the poliovirus, these memory cells will respond by differentiating into plasma cells that produce high levels of serum antibody, which defend the individual from the effects of the virus. In the remainder of this section, various approaches to the design of vaccines—both currently used vaccines and experimental ones—are described, with an examination of the ability of the vaccines to induce humoral and cellmediated immunity and the production of memory cells. As Table 17-7 indicates, the common vaccines currently in use consist of live but attenuated organisms, inactivated (killed) bacterial cells or viral particles, as well as protein or carbohydrate fragments (subunits) of the target organism. Several new types of vaccine candidate provide potential advantages in protection, production, or delivery to those in need. The primary characteristics and some advantages and disadvantages of the different types of vaccines are included in the following discussions. Live, Attenuated Vaccines In some cases, microorganisms can be attenuated or disabled so that they lose their ability to cause significant disease (pathogenicity) but retain their capacity for transient growth within an inoculated host. Some agents are naturally attenuated by virtue of their inability to cause disease in a given host, although they can immunize these individuals. The first vaccine used by Jenner is of this type: vaccinia virus (cowpox) inoculation of humans confers immunity to smallpox but does not cause smallpox. Attenuation can often be achieved by growing a pathogenic bacterium or virus for prolonged periods under abnormal culture conditions. This selects mutants that are better suited for growth in the abnormal culture conditions than in the natural host. For example, an attenuated strain of Mycobacterium bovis called Bacillus Calmette-Guérin (BCG) was developed by growing M. bovis on a medium containing increasing concentrations of bile. After 13 years, this strain had adapted to growth in strong bile and had become sufficiently attenuated that it was suitable as

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Classification of common vaccines for humans

Vaccine type

Diseases

Advantages

Disadvantages

WHOLE ORGANISMS

Live attenuated

Measles Mumps Polio (Sabin vaccine) Rotavirus Rubella Tuberculosis Varicella Yellow fever

Strong immune response; often lifelong immunity with few doses

Requires refrigerated storage; may mutate to virulent form

Inactivated or killed

Cholera Influenza Hepatitis A Plague Polio (Salk vaccine) Rabies

Stable; safer than live vaccines; refrigerated storage not required

Weaker immune response than live vaccines; booster shots usually required

PURIFIED MACROMOLECULES

Toxoid (inactivated exotoxin)

Diphtheria Tetanus

Immune system becomes primed to recognize bacterial toxins

Subunit (inactivated exotoxin)

Hepatitis B Pertussis Streptococcal pneumonia

Specific antigens lower the chance of adverse reactions

Conjugate

Haemophilus influenzae type B Streptococcal pneumonia

Primes infant immune systems to recognize certain bacteria

Difficult to develop

OTHER

DNA

In clinical testing

Strong humoral and cellular immune response; relatively inexpensive to manufacture

Not yet available

Recombinant vector

In clinical testing

Mimics natural infection, resulting in strong immune response

Not yet available

a vaccine for tuberculosis. Due to variable effectiveness and difficulties in follow-up monitoring, BCG is not used in the United States. The Sabin form of the polio vaccine and the measles vaccine both consist of attenuated viral strains. Attenuated vaccines have advantages and disadvantages. Because of their capacity for transient growth, such vaccines provide prolonged immune system exposure to the individual epitopes on the attenuated organisms and more closely mimic the growth patterns of the “real” pathogen, resulting in increased immunogenicity and efficient production of memory cells. Thus, these vaccines often require only a single immunization, a major advantage in developing coun-

tries, where studies show that a significant number of individuals fail to return for boosters. The ability of many attenuated vaccines to replicate within host cells makes them particularly suitable for inducing cell-mediated responses. The oral polio vaccine (OPV) designed by Albert Sabin, consisting of three attenuated strains of poliovirus, is administered orally to children. The attenuated viruses colonize the intestine and induce production of secretory IgA, an important defense against naturally acquired poliovirus. The vaccine also induces IgM and IgG classes of antibody and ultimately protective immunity to all three strains of virulent poliovirus. Unlike most other attenuated vaccines, OPV

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Reported polio cases 1988

0 cases 1–10 cases More than 10 cases No report

2011

0 cases 1–10 cases More than 10 cases No report

FIGURE 17-14 Progress toward the worldwide eradication of polio. Comparison of polio infection numbers for 1988 with those for 2011 show considerable progress in most parts of the world, although some areas in Africa and Asia have shown recent increases. Some experts question whether the use of live attenuated oral polio vaccine (OPV) will cause reversion to pathogenic forms at a rate sufficiently high to prevent total eradication of this once-prevalent crippling disease. [Source: Data from World Health Organization.]

requires boosters, because the three strains of attenuated poliovirus interfere with each other’s replication in the intestine. With the first immunization, one strain will predominate in its growth, inducing immunity to that strain. With the second immunization, the immunity generated by the previous immunization will limit the growth of the previously predominant strain in the vaccine, enabling one of the two remaining strains to colonize the intestines and induce immunity. Finally, with the third immunization, immunity to all three strains is achieved. A major disadvantage of attenuated vaccines is that these live forms may mutate and revert to virulent forms in vivo, resulting in paralytic disease in the vaccinated individual and serving as a source of pathogen transmission. The rate of reversion of the OPV is about 1 case in 2.4 million doses of vaccine. This reversion can also allow patho-

genic forms of the virus to find their way into the water supply, especially in areas where sanitation is not rigorous or wastewater must be recycled. This possibility has led to the exclusive use of the inactivated polio vaccine in this country (see Table 17-6). The projected eradication of paralytic polio (Figure 17-14) may be impossible as long as OPV is used anywhere in the world. The alternative inactivated polio vaccine created by Jonas Salk will likely be substituted as the number of cases decreases, although there are problems with delivery in developing countries. The ultimate goal is to achieve a polio-free world in which no vaccine is needed. Attenuated vaccines also may be associated with complications similar to those seen in the natural disease. A small percentage of recipients of the measles vaccine, for example, develop postvaccine encephalitis or other complications,

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Infectious Diseases and Vaccines although the risk of vaccine-related complications is still much lower than risks from infection. An independent study showed that 75 million doses of measles vaccine were given between 1970 and 1993, with 48 cases of vaccine-related encephalopathy (approx. 1 per 1.5 million). This low incidence compared with the rate of encephalopathy associated with infection argues for the efficacy of the vaccine. An even more convincing argument for vaccination is the high death rate associated with measles infection, even in developed countries. In addition to culturing methods, genetic engineering provides a way to attenuate a virus irreversibly, by selectively removing genes that are necessary for virulence or for growth in the vaccinee. This has been done with a herpesvirus vaccine for pigs, in which the thymidine kinase gene was removed. Because thymidine kinase is required for the virus to grow in certain types of cells (e.g., neurons), removal of this gene rendered the virus incapable of causing disease. A live, attenuated vaccine against influenza was developed recently under the name FluMist. The virus was grown at lower-than-normal temperatures until a coldadapted strain resulted that is unable to grow at human body temperature of 37ºC. This live attenuated virus is administered intranasally and causes a transient infection in the upper respiratory tract, sufficient to induce a strong immune response. The virus cannot spread beyond the upper respiratory tract because of its inability to grow at the elevated temperatures of the inner body. Because of the ease of administration and induction of good mucosal immunity, cold-adapted, nasally administered flu vaccines are on the rise. Inactivated or “Killed” Vaccines Another common means to make a pathogen safe for use in a vaccine is by treatment with heat or chemicals. This kills the pathogen, making it incapable of replication, but still allows it to induce an immune response to at least some of the antigens contained within the organism. It is critically important to maintain the structure of epitopes on surface antigens during inactivation. Heat inactivation is often unsatisfactory because it causes extensive denaturation of proteins; thus, any epitopes that depend on higher orders of protein structure are likely to be altered significantly. Chemical inactivation with formaldehyde or various alkylating agents has been successful. The Salk polio vaccine is produced by formaldehyde inactivation of the poliovirus. Although live attenuated vaccines generally require only one dose to induce long-lasting immunity, killed vaccines often require repeated boosters to achieve a protective immune status. Because they do not replicate in the host, killed vaccines typically induce a predominantly humoral antibody response and are less effective than attenuated vaccines in inducing cell-mediated immunity or in eliciting a secretory IgA response, key components of an ideal protective and mucosally based response.

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Even though the pathogens they contain are killed, inactivated whole-organism vaccines still carry certain risks. A serious complication with the first Salk vaccines arose when formaldehyde failed to kill all the virus in two vaccine lots, leading to paralytic polio in a high percentage of recipients. Risk is also encountered in the manufacture of the inactivated vaccines. Large quantities of the infectious agent must be handled prior to inactivation, and those exposed to the process are at risk of infection. However, in general, the safety of inactivated vaccines is greater than that of live attenuated vaccines. Inactivated vaccines are commonly used against both viral and bacterial diseases, including the classic yearly flu vaccine and vaccines for hepatitis A and cholera. In addition to their relative safety, their advantages include stability and ease of storage and transport. Subunit Vaccines Many of the risks associated with attenuated or killed wholeorganism vaccines can be avoided with a strategy that uses only specific, purified macromolecules derived from the pathogen. The three most common applications of this strategy, referred to as a subunit vaccine, are inactivated exotoxins or toxoids, capsular polysaccharides or surface glycoproteins, and key recombinant protein antigens (see Table 17-7). One limitation of some subunit vaccines, especially polysaccharide vaccines, is their inability to activate TH cells. Instead, they activate B cells in a thymus-independent type 2 (TI-2) manner, resulting in IgM production but little class switching, no affinity maturation, and little, if any, development of memory cells. However, vaccines that conjugate a polysaccharide antigen to a protein carrier can alleviate this problem by inducing TH cell responses (see below). Some bacterial pathogens, including those that cause diphtheria and tetanus, produce exotoxins that account for all or most of the disease symptoms resulting from infection. Diphtheria and tetanus vaccines have been made by purifying the bacterial exotoxin and then inactivating it with formaldehyde to form a toxoid. Vaccination with the toxoid induces antitoxoid antibodies, which are capable of binding to the toxin and neutralizing its effects. Conditions for the production of toxoid vaccines must be closely controlled and balanced to avoid excessive modification of the epitope structure while also accomplishing complete detoxification. As discussed previously, passive immunity can also be used to provide temporary protection in unvaccinated individuals exposed to organisms that produce these exotoxins, although no long-term protection is achieved in this case. The virulence of some pathogenic bacteria depends primarily on the antiphagocytic properties of their hydrophilic polysaccharide capsule. Coating the capsule with antibodies and/or complement greatly increases the ability of macrophages and neutrophils to phagocytose such pathogens. These findings provide the rationale for vaccines consisting of purified capsular polysaccharides. The current vaccine

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for Streptococcus pneumoniae (the organism which causes pneumococcal pneumonia) consists of 13 antigenically distinct capsular polysaccharides (PCV13). The vaccine induces formation of opsonizing antibodies and is now on the list of vaccines recommended for all infants (see Table 17-6). The vaccine for Neisseria meningitidis, a common cause of bacterial meningitis, also consists of purified capsular polysaccharides. Some viruses carry surface glycoproteins (e.g., an envelope protein from HIV-1) that have been tested for use in antiviral vaccines, with little success. However, glycoprotein-D from HSV-2 has been shown to prevent genital herpes in clinical trials of some vaccines, suggesting that this may be a viable approach for some antiviral vaccines as well. Theoretically, the gene encoding any immunogenic protein can be cloned and expressed in cultured cells using recombinant DNA technology, and this technique has been applied widely in the design of many types of subunit vaccines. For example, the safest way to produce sufficient quantities of the purified toxins that go into the generation of toxoid vaccines involves cloning the exotoxin genes from pathogenic organisms into easily cultured host cells. A number of genes encoding surface antigens from viral, bacterial, and protozoan pathogens have also been successfully cloned into cellular expression systems for use in vaccine development. The first such recombinant antigen vaccine approved for human use is the hepatitis B vaccine, developed by cloning the gene for the major hepatitis B surface antigen (HBsAg) and expressing it in yeast cells. The recombinant yeast cells are grown in large fermenters, allowing HBsAg to accumulate in the cells. The yeast cells are harvested and disrupted, releasing the recombinant HBsAg, which is then purified by conventional biochemical techniques. Recombinant hepatitis B vaccine induces the production of protective antibodies and holds much worldwide promise for protecting against this human pathogen. Recombinant Vector Vaccines Recall that live attenuated vaccines prolong antigen delivery and encourage cell-mediated responses, but have the disadvantage that they can sometimes revert to pathogenic forms. Recombinant vectors maintain the advantages of live attenuated vaccines while avoiding this major disadvantage. Individual genes that encode key antigens of especially virulent pathogens can be introduced into attenuated viruses or bacteria. The attenuated organism serves as a vector, replicating within the vaccinated host and expressing the gene product of the pathogen. However, since most of the genome of the pathogen is missing, reversion potential is virtually eliminated. Recombinant vector vaccines have been prepared utilizing existing licensed live, attenuated vaccines and adding to them genes encoding antigens present on newly emerging pathogens. Such chimeric virus vaccines can be more quickly tested and approved than an entirely new product. A very recent example of this is the yellow

fever vaccine that was engineered to express antigens of WNV. A number of organisms have been used as the vector in such preparations, including vaccinia virus, the canarypox virus, attenuated poliovirus, adenoviruses, attenuated strains of Salmonella, the BCG strain of Mycobacterium bovis, and certain strains of Streptococcus that normally exist in the oral cavity. Vaccinia virus, the attenuated vaccine used to eradicate smallpox, has been widely employed as a vector for the design of new vaccines. This large, complex virus, with a genome of about 200 genes, can be engineered to carry several dozen foreign genes without impairing its capacity to infect host cells and replicate. The procedure for producing a vaccinia vector that carries a foreign gene from another pathogen is outlined in Figure 17-15. The genetically engineered vaccinia expresses high levels of the inserted gene product, which can then serve as a potent immunogen in an inoculated host. Like the smallpox vaccine, genetically engineered vaccinia vector vaccines can be administered simply by scratching the skin, causing a localized infection in host cells. If the foreign gene product expressed by the vaccinia vector is a viral envelope protein, it is inserted into the membrane of the infected host cell, inducing development of cell-mediated as well as antibodymediated immunity. Other attenuated vectors may prove to be safer than vaccinia in vaccine preparations. A relative of vaccinia, the canarypox virus, is also large and easily engineered to carry multiple genes. Unlike vaccinia, it does not appear to be virulent, even in individuals with severe immune suppression. Another possible vector is an attenuated strain of Salmonella typhimurium, which has been engineered with genes from the bacterium that causes cholera. The advantage of this vector for use in vaccines is that Salmonella infects cells of the mucosal lining of the gut and therefore will induce secretory IgA production. Similar strategies are underway for organisms that enter via oral or respiratory routes, targeting bacteria that are normal fl ora at these sites as vectors for the addition of pathogen-specific genes. Eliciting immunity at the mucosal surface could provide excellent protection at the portal of entry for many common infectious agents, such as cholera and gonorrhea. DNA Vaccines A more recent vaccination strategy, called a DNA vaccine, utilizes plasmid DNA encoding antigenic proteins that are injected directly into the muscle of the recipient. This strategy relies on the host cells to take up the DNA and produce the immunogenic protein in vivo, thus directing the antigen through endogenous MHC class I presentation pathways, helping to activate better CTL responses. The DNA appears either to integrate into the chromosomal DNA or to be maintained for long periods in an episomal form, and is often taken up by dendritic cells or muscle cells in the injection area. Since muscle cells express low levels

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Vaccinia promoter

Restriction-enzyme cleavage site

DNA encoding Ag from pathogen

TK gene

TK gene Plasmid

Cleavage and ligation

Vaccinia promoter

Gene from pathogen TK gene

TK gene Recombinant plasmid

Vaccinia virus Transfection

Infection Tissue culture cells

Homologous recombination

BRdU selection

Recombinant vaccinia vector vaccine

FIGURE 17-15 Production of vaccine using a recombinant vaccinia vector. (top) The gene that encodes the desired antigen (orange) is inserted into a plasmid vector adjacent to a vaccinia promoter (pink) and flanked on either side by the vaccinia thymidine kinase (TK) gene (green). (bottom) When tissue culture cells are incubated simultaneously with vaccinia virus and the recombinant plasmid, the antigen gene and promoter are inserted into the vaccinia virus genome by homologous recombination at the site of the nonessential TK gene, resulting in a TK− recombinant virus. Cells containing the recombinant vaccinia virus are selected by addition of bromodeoxyuridine (BrdU), which kills TK⫹ cells.

of class I MHC molecules and do not express costimulatory molecules, delivery to local dendritic cells may be crucial to the development of antigenic responses to DNA vaccines. Tests in animal models have shown that DNA vaccines are able to induce protective immunity against a number of

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pathogens, including influenza and rabies viruses. The addition of a follow-up booster shot with protein antigen (called a DNA prime and protein boost strategy), or inclusion of supplementary DNA sequences in the vector, may enhance the immune response. One sequence that has been added to some vaccines is the common CpG DNA motif found in some pathogens; recall that this sequence is the ligand for TLR9 (see Chapter 5). DNA vaccines offer some potential advantages over many of the existing vaccine approaches. Since the encoded protein is expressed in the host in its natural form—there is no denaturation or modification—the immune response is directed to the antigen exactly as it is expressed by the pathogen, inducing both humoral and cell-mediated immunity. To stimulate both arms of the adaptive immune response with non-DNA vaccines normally requires immunization with a live attenuated preparation, which incurs additional risk. DNA vaccines also induce prolonged expression of the antigen, enhancing the induction of immunological memory. Finally, DNA vaccines present important practical advantages (see Table 17-7). No refrigeration of the plasmid DNA is required, eliminating longterm storage challenges. In addition, the same plasmid vector can be custom tailored to insert DNA encoding a variety of proteins, which allows the simultaneous manufacture of a variety of DNA vaccines for different pathogens, saving time and money. An improved method for administering DNA vaccines entails coating gold microscopic beads with the plasmid DNA and delivery of the coated particles through the skin into the underlying muscle with an air gun (called a gene gun). This allows rapid delivery of vaccine to large populations without the need for huge numbers of needles and syringes, improving both safety and cost. Human trials are underway with several different DNA vaccines, including those for malaria, HIV, influenza, Ebola, and herpesvirus, along with several vaccines aimed at cancer therapy (Table 17-8). Although there are currently no licensed human DNA vaccines, three such vaccines have been licensed for veterinary use, including a WNV vaccine that is protective in horses. This vaccine has been tested in humans; after three doses, most volunteers demonstrated titers of neutralizing antibody similar to those seen in horses, as well as CD8⫹ and CD4⫹ T cell responses against the virus. Since the widespread development of DNA vaccines for use in humans is still in its early stages, the risks associated with the use of this strategy are still largely unknown.

Conjugate or Multivalent Vaccines Can Improve Immunogenicity and Outcome One significant drawback to the techniques that do not utilize a live vaccine strategy is that they may induce weak or limited adaptive responses. To address this, schemes have been developed that employ the fusing of a highly immunogenic

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Diseases for which DNA vaccines have entered clinical trials

Infectious diseases

Cancer

Human immunodeficiency virus

B-cell lymphoma

Influenza

Prostate cancer

(Seasonal, Pandemic)

Polysaccharide coating of bacterium

Toxoid

Melanoma Breast cancer

Malaria

Ovarian cancer

Hepatitis B virus

Cervical cancer

Severe acute respiratory syndrome Marburg hemorrhagic fever

Hepatocellular cancer

Ebola virus

Bladder cancer

Human papillomavirus

Lung cancer

West Nile virus

Sarcoma

Dengue fever

Renal cell cancer

Herpes simplex virus Measles Malaria Source: M. Liu 2011. DNA vaccines: an historical perspective and view to the future. Immunological Reviews, 239:62–84.

protein to a weak vaccine immunogen (a conjugate) or mixing in extraneous proteins (multivalent) to enhance or supplement immunity to the pathogen. The vaccine against Haemophilus influenzae type b (Hib), a major cause of bacterial meningitis and infection-induced deafness in children, is a conjugate formulation included in the recommended childhood regimen (see Table 17-6). It consists of type b capsular polysaccharide covalently linked to a protein carrier, tetanus toxoid (Figure 17-16). Introduction of the conjugate Hib vaccines has resulted in a rapid decline in Hib cases in the United States and other countries that have introduced this vaccine. The polysaccharide-protein conjugate is considerably more immunogenic than the polysaccharide alone; and because it activates TH cells, it enables class switching from IgM to IgG. Although this type of vaccine can induce memory B cells, it cannot induce memory T cells specific for the pathogen. In the case of the Hib vaccine, it appears that the memory B cells can be activated to some degree in the absence of a population of memory TH cells, thus accounting for its efficacy. Like Hib, the MCV4 vaccine uses a similar strategy and is another

Linked toxoid and polysaccharide to be used in conjugate vaccine

FIGURE 17-16 A conjugate vaccine protects against Haemophilus influenzae type b (Hib). The vaccine is prepared by conjugating the surface polysaccharide of Hib to a protein molecule, making the vaccine more immunogenic than either alone.

example of a conjugate vaccine that protects young children from meningitis (see Table 17-6). It is a multivalent vaccine consisting of individual capsular Neisseria polysaccharide antigens joined to the highly immunogenic diphtheria toxoid protein. In a recent study, immunization with ␤-glucan isolated from brown alga and conjugated to diphtheria toxoid raised antibodies in mice and rats that protected against challenge with both Aspergillus fumigatus and Candida albicans. The protection was transferred by serum or vaginal fluid from the immunized animals, indicating that the immunity is antibody based. Infections with fungal pathogens are a serious problem for immunocompromised individuals. The availability of immunization or antibody treatment could circumvent problems with toxicity of antifungal drugs and the emergence of resistant strains, an issue that is especially important in hospital settings. Since subunit polysaccharide or protein vaccines tend to induce humoral but not cell-mediated responses, a method is needed for constructing vaccines that contain both immunodominant B-cell and T-cell epitopes. Furthermore, if a CTL response is desired, the vaccine must be delivered intracellularly so that the peptides can be processed and presented via class I MHC molecules. One innovative means of producing a multivalent vaccine that can deliver many copies of the antigen into cells is to incorporate antigens into protein micelles, lipid vesicles (called liposomes), or immunostimulating complexes (Figure 17-17a). Mixing proteins in detergent and then removing the detergent forms micelles. The individual proteins orient themselves with their hydrophilic residues toward the aqueous environment and the hydrophobic

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Phospholipid bilayer

Micelle Liposome ISCOM

Antigen

(b) ISCOM delivery of antigen into cell

TAP ER ISCOM

Proteasome

FIGURE 17-17 Multivalent subunit vaccines. (a) Protein micelles, liposomes, and immunostimulating complexes (ISCOMs) can all be prepared from detergent-extracted antigens or antigenic peptides. In micelles and liposomes, the hydrophilic residues of the antigen molecules are oriented outward. In ISCOMs, the long fattyacid tails of the external detergent layer are adjacent to the hydrophobic residues of the centrally located antigen molecules. (b) ISCOMs and liposomes can deliver agents inside cells, so they mimic endogenous antigens. Subsequent processing by the endogenous pathway and presentation with class I MHC molecules induces a cell-mediated response. ER = endoplasmic reticulum; TAP = transporter associated with antigen processing.

residues at the center to exclude their interaction with the aqueous environment. Protein-containing liposomes are prepared by mixing the proteins with a suspension of phospholipids under conditions that form lipid bilayer vesicles; the proteins are incorporated into the bilayer with the hydrophilic residues exposed. Immunostimulating complexes (ISCOMs) are lipid carriers prepared by mixing protein with detergent and a glycoside called Quil A, an adjuvant. Membrane proteins from various pathogens, including influenza virus, measles virus, hepatitis B virus, and HIV, have been incorporated into micelles, liposomes, and ISCOMs and are being assessed as potential vaccines. In addition to their increased immunogenicity, liposomes and ISCOMs appear to fuse with the plasma membrane to deliver the antigen intracellularly, where it can be processed by the endogenous pathway, leading to CTL responses (Figure 17-17b).

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Adjuvants Are Included to Enhance the Immune Response to a Vaccine In an ideal case, vaccines mimic most of the key immunologic events that occur during a natural infection, eliciting strong and comprehensive immune responses but without the risks associated with live agents. A discussion of vaccines would be incomplete without mentioning the importance of adjuvants, substances that are added to vaccine preparations to enhance the immune response to the antigen or pathogen with which they are mixed. This is especially important when the vaccine preparation is a pathogen subunit or other nonliving form of the organism, where immunogenicity can be quite low. These additives mixed with the vaccines, such as those described above, can both enhance the immune response and help with delivery of the vaccine to the immune system. For almost 80 years, the only adjuvant used in human vaccines was aluminum salts (called alum), a fairly good enhancer of TH2 responses but a weaker stimulator of TH1 pathways. Alum is mixed in an emulsion with the immunogen and is primarily thought to work by creating slow release delivery of the immunogen at the injection site, which helps in sustained stimulation of the immune response. It may also help to recruit APCs and encourage the formation of large antigen complexes that are more likely to be phagocytosed by these cells. In recent years, two new adjuvants have been licensed for use in human vaccines. One, MF59, is an oil-in-water emulsion that, like alum, is believed to aid in slow antigen delivery. The other, AS04, contains alum plus a TLR4 agonist. TLR4 is a PRR for bacterial lipopolysaccharides (LPS), and signaling by this adjuvant ultimately encourages TH1 responses. AS04 is currently used in vaccines against HPV and HSV-2. All of these adjuvants have been found to enhance the production of antibodies as compared to unadjuvanted vaccine preparations. Some creative uses of existing adjuvants and next generation adjuvants or immune modulators are also under development. These include compounds designed to stimulate certain PRRs—specifically, several TLRs and at least one NOD-like receptor. Although adjuvants have improved humoral immunity, few if any actually enhance cellular immunity, especially CD8⫹ T-cell responses. Recently, a combination of two already licensed adjuvant components was used in mice treated with an influenza virus peptide and was found to generate protective CD8⫹ T-cell memory responses. This strategy is attractive, as it utilizes adjuvants with an already established track record of safety and efficacy in humans. One very recent and particularly novel strategy utilizes a subunit vaccine followed by a chemokine, aimed at eliciting protective immunity and then moving the immune cells to the relevant mucosal surface (see Advances Box 17-2).

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BOX 17-2

ADVANCES

A Prime and Pull Vaccine Strategy for Preventing Sexually Transmitted Diseases Most

pathogens breach the physical barriers of the body at mucosal surfaces, such as the gastrointestinal, respiratory, or genital tracts. Therefore, some form of protective immunity stationed at these portals would be the most effective means of interrupting infection by such pathogens. However, until recently the nuances of the specialized secondary lymphoid structures that serve these surfaces (collectively referred to as mucosal associated lymphoid tissues, or MALT) and the unique mechanisms of establishing and maintaining adaptive immune memory at these structures have not been fully appreciated. Traditional vaccination strategies, such as subcutaneous injection of antigen, often fail to elicit protective IgA responses at mucosal sites but instead drive the development of serum IgG, which is less protective against most mucosally encountered pathogens. Positioning protective immunity at mucosal sites of entry is being studied in the design of vaccines to protect against sexually transmitted infections (STIs). According to the WHO, there are more than 30 different sexually acquired pathogens, including viral, bacterial, and parasitic organisms. It is estimated that up to 1 million people become infected with an STI every day! STIs are one of the top five reasons for individuals to seek health care in developing countries, and 30% to 40%

of female infertility is linked to postinfection damage of the fallopian tubes. The most common STIs are chlamydia, gonorrhea, syphilis, hepatitis B, and HIV. Worldwide, these STIs take their greatest toll on young women of childbearing age. Based on anatomy, women are more likely to become infected following unprotected sex with a carrier, whereas men are more likely to transmit these infections through semen. Although many of these infections are treatable, an absence of or delay in symptoms means that many unwitting carriers transmit infection to their partners. Even low levels of genital infection can increase the likelihood of transmission of new STIs, including HIV. Establishing and maintaining memory cells that home to the genital tract and can protect against STIs is difficult. In experimental systems, intranasal or intravaginal immunogen delivery has been shown to elicit antigen-specific T- and B-cell responses that traffic to the female genital tract, where protective IgA can also be found. However, as in the case of HIV, the ideal vaccine would recruit antigen-specific CD8⫹ T cells and IgAsecreting B cells to the genital tract, but not activated CD4⫹ T cells. The latter are potential targets for new infections and could therefore inadvertently enhance transmission rates in vaccinees. Akiko Iwasaki’s group at Yale University has recently applied a new vaccine strat-

egy called “prime and pull” in a mouse model of genital herpes. They used a conventional subcutaneous injection of attenuated HSV-2 to prime systemic T cell responses in mice, followed by a topical application of the chemokine CXCL9 to the vaginal canal of the mice. This chemokine resulted in the specific recruitment of effector T cells with a memory phenotype to the mucosal tissues of the vagina: the “pull.” Mice treated with this prime and pull strategy showed a significant increase in survival after challenge with live herpesvirus compared with mice that received only the priming vaccine injection. If this scheme proves safe and effective in human trials, the pull arm of this approach could theoretically be appended to conventional vaccines already approved for use in humans, shortening the lengthy pipeline for most “new” vaccines. Likewise, the chemotactic agent could be modified to recruit different target cells, or applied to other mucosal tissues, reeling in the memory cells elicited from existing vaccines directly to the sites most in need of protection.

H. Shin and A. Iwasaki. 2012. A vaccine strategy that protects against genital herpes by establishing local memory T cells. Nature 491: 463–467. A. Iwasaki. 2010. Antiviral immune responses in the genital tract: Clues for vaccines. Nature Reviews Immunology 10: 699–711.

S U M M A R Y ■

Physical barriers, such as the skin and mucosal secretions, can block infection. Innate immune responses form the initial defense against pathogens that breach these barriers. These include the nonspecific production of complement components, phagocytic cells, and certain cytokines that are elicited in response to local infection by various pathogens.





The immune response to viral infections involves both humoral and cell-mediated components. Some viruses mutate rapidly and and/or have acquired specific mechanisms to evade parts of the innate or adaptive immune response to them. The immune response to extracellular bacterial infections is generally mediated by antibody. Antibody can activate

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complement-mediated lysis of the bacterium, neutralize toxins, and serve as an opsonin to increase phagocytosis. Host defense against intracellular bacteria depends largely on CD4⫹ T-cell-mediated responses. Parasites are a very broad infectious disease category. Both humoral and cell-mediated immune responses have been implicated in immunity to protozoan infections. In general, humoral antibody is effective against blood-borne stages of the protozoan life cycle, but once protozoans have infected host cells, cell-mediated immunity is necessary. Protozoans escape the immune response through several evasion strategies. Helminths are large parasites that normally do not multiply within human hosts. Only low levels of immunity are induced to these organisms, partly because so few organisms are carried by an affected individual or exposed to the immune system. Helminths can form chronic infections and generally are attacked by antibody-mediated defenses. Fungal diseases, or mycoses, are rarely severe in normal, healthy individuals but pose a greater problem for those with immunodeficiency. Both innate immunity and adaptive immunity control infection by fungi. Emerging and re-emerging pathogens include some newly described organisms and others previously thought to have been controlled by public health practices. Factors leading to the emergence of such pathogens include increased travel, poor sanitation, and intense crowding of some populations. A state of immunity can be induced by passive or active immunization. Short-term passive immunization is induced by the transfer of preformed antibodies. Natural infection or vaccination can induce active immunization and lead to long-term immunity.

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Five types of vaccines are currently used or under experimental consideration in humans: live, attenuated (avirulent) microorganisms; inactivated (killed) microorganisms; purified macromolecules (subunits); viral vectors carrying recombinant genes (live, recombinant); and DNA vaccines.



Realizing the optimum benefit of vaccines will require cheaper manufacture and improved delivery methods for existing vaccines. Live vaccines (either attenuated or viral vectors) have the advantage of inducing both humoral and cell-mediated immunity, and can produce more effective overall protective immunity. However, live, attenuated vaccines carry the risk of reversion, which is not an issue with recombinant forms. Isolated protein components of pathogens expressed in cell culture can be used to create effective vaccines, especially when the toxic effects of the pathogen are due to discrete protein products.











Polysaccharide and other less immunogenic vaccines may be conjugated to more immunogenic proteins to enhance or maximize the immune response. Introduction of plasmid DNA encoding protein antigens from a pathogen can be used to induce both humoral and cell-mediated responses. Such DNA vaccines for several diseases are presently in human clinical trials. Adjuvants are substances added to vaccine preparations that help aid in delivery of the vaccine to the immune system and that enhance responses. A new generation of adjuvants are being developed that target specific pattern recognition receptors (PRRs) or that modify responses in ways that may help to direct the immune response toward more protective pathways.

R E F E R E N C E S Alcami, A., and U. H. Koszinowski. 2000. Viral mechanisms of immune evasion. Trends in Microbiology 8:410–418.

Kaufmann, S. H., A. Sher, and R. Ahmed, eds. 2002. Immunology of Infectious Diseases. Washington, DC: ASM Press.

Bachmann, M. F., and G. T. Jennings. 2010. Vaccine delivery: A matter of size, geometry, kinetics and molecular patterns. Nature Reviews Immunology 11:787–796.

Knodler, L. A., J. Celli, and B. B. Finlay. 2001. Pathogenic trickery: Deception of host cell processes. Nature Reviews Molecular Cell Biology 2:578–588.

Bloom, B. R., ed. 1994. Tuberculosis: Pathogenesis, Protection and Control. Washington, DC: ASM Press.

Li, F., et al. 2005. Structure of SARS coronavirus spike receptor-binding domain complexed with receptor. Science 309:1864–1868.

Borst, P., et al. 1998. Control of VSG gene expression sites in Trypanosoma brucei. Molecular and Biochemical Parasitology 91:67–76. Coffman, R. L., A. Sher, and R. A Seder. 2010. Vaccine adjuvants: Putting innate immunity to work. Immunity 33(4):492–503. Iwasaki, A. 2010. Antiviral immune responses in the genital tract: Clues for vaccines. Nature Reviews Immunology 10(10):699–711.

Liu, M. A. 2011.DNA vaccines: An historical perspective and view to the future. Immunology Reviews 239(1):62–84. Lorenzo, M. E., H. L. Ploegh, and R. S. Tirabassi. 2001. Viral immune evasion strategies and the underlying cell biology. Seminars in Immunology 13:1–9. Merrell, D. S., and S. Falkow. 2004. Frontal and stealth attack strategies in microbial pathogenesis. Nature 430:250–256.

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Macleod, M. K., et al. 2011. Vaccine adjuvants aluminum and monophosphoryl lipid A provide distinct signals to generate protective cytotoxic memory CD8 T cells. Proceedings of the National Academy of Sciences USA 108(19):7914–7919. Morens, D. M., G. K. Folkers, and A. S. Fauci. 2008. Emerging infections: A perpetual challenge. Lancet Infectious Diseases 8(11):710–719. Romani, L. 2011. Immunity to fungal infections. Nature Reviews Immunology 11(4):275–288. Schofield, L., and G. E. Grau. 2005. Immunological processes in malaria pathogenesis. Nature Reviews Immunology 5:722–735. Skowronski, D. M., et al. 2005. Severe acute respiratory syndrome. Annual Review of Medicine 56:357. Torosantucci, A., et al. 2005. A novel glyco-conjugate vaccine against fungal pathogens. Journal of Experimental Medicine 202:597–606. Yang, Y.-Z., et al. 2004. A DNA vaccine induces SARS coronavirus neutralization and protective immunity in mice. Nature 248:561.

Useful Websites

Institutes of Health that sponsors research in infectious diseases, and its Web site provides a number of links to other relevant sites.

www.who.int This is the home page of the World Health Organization, the international organization that monitors infectious diseases worldwide. www.cdc.gov The Centers for Disease Control and Prevention (CDC) is a United States government agency that tracks infectious disease outbreaks and vaccine research in the United States. www.upmc-biosecurity.org The University of Pittsburgh Center for Biosecurity Web site provides information about select agents and emerging diseases that may pose a security threat.

www.gavialliance.org The Global Alliance for Vaccines and Immunization (GAVI) is a source of information about vaccines in developing countries and worldwide efforts at disease eradication. This site contains links to major international vaccine information sites. www.ecbt.org Every Child by Two offers useful informa-

www.niaid.nih.gov The National Institute of Allergy and Infectious Diseases is the institute within the National

S T U D Y

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tion on childhood vaccination, including recommended immunization schedules.

Q U E S T I O N S

INFECTIOUS DISEASES CLINICAL FOCUS QUESTION 1. The effect of the MHC on the immune response to peptides

of the influenza virus nucleoprotein was studied in H-2b mice that had been previously immunized with live influenza virions. The CTL activity of primed lymphocytes was determined by in vitro CML assays using H-2k fibroblasts as target

Target cell (H-2k fibroblast)

Test antigen

CTL activity of influenzaprimed H-2b lymphocytes (% lysis)

(A) Untransfected

Live influenza

0

(B) Transfected with class I Db

Live influenza

60

(C) Transfected with class I Db

Nucleoprotein peptide 365–380

50

(D) Transfected with class I Db

Nucleoprotein peptide 50–63

2

(E) Transfected with class I Kb

Nucleoprotein peptide 365–380

0.5

(F) Transfected with class I Kb

Nucleoprotein peptide 50–63

1

cells. The target cells had been transfected with different H-2b class I MHC genes and were infected either with live influenza or incubated with nucleoprotein synthetic peptides. The results of these assays are shown in the following table. a. Why was there no killing of the target cells in system A

even though the target cells were infected with live influenza? b. Why was a CTL response generated to the nucleoprotein in system C, even though it is an internal viral protein? c. Why was there a good CTL response in system C to peptide 365–380, whereas there was no response in system D to peptide 50–63? d. If you were going to develop a synthetic peptide vaccine for influenza in humans, how would these results obtained in mice influence your design of a vaccine? 2. Describe the nonspecific defenses that operate when a

disease-producing microorganism first enters the body. 3. Describe the various specific defense mechanisms that the

immune system employs to combat various pathogens. 4. What is the role of the humoral response in immunity to

influenza? 5. Describe the unique mechanisms each of the following patho-

gens has for escaping the immune response: (a) African trypanosomes, (b) Plasmodium species, and (c) influenza virus. 6. M. F. Good and co-workers analyzed the effect of MHC hap-

lotype on the antibody response to a malarial circumsporozoite

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Infectious Diseases and Vaccines (CS) peptide antigen in several recombinant congenic mouse strains. Their results are shown in the table below:

H-2 alleles Strain

K

IA

IE

S

D

Antibody response to CS peptide

B10.BR

k

k

k

k

k

⬍1

B10.A (4R)

k

k

b

b

b

⬍1

B10.HTT

s

s

k

k

d

⬍1

B10.A (5R)

b

b

k

d

d

67

B10

b

b

b

b

b

73

B10.MBR

b

k

k

k

q

⬍1

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Secreting proteases to inactivate antibodies Having low virulence Developing resistance to complement-mediated lysis Allowing point mutations in surface epitopes, resulting in antigenic drift g. Increasing phagocytic activity of macrophages c. d. e. f.

11. Which of the following is a characteristic of the inflamma-

tory response against extracellular bacterial infections?

Source: Adapted from M. F. Good et al., 1988, The T cell response to the malaria circumsporozoite protein: an immunological approach to vaccine development. Annual Review of Immunology 6:663–688.

a. b. c. d. e. f.

Increased levels of IgE Activation of self-reactive CD8⫹ T cells Activation of complement Swelling caused by release of vasodilators Degranulation of tissue mast cells Phagocytosis by macrophages

12. Your mother may have scolded you for running around

outside without shoes. This is sound advice because of the mode of transmission of the helminth Schistosoma mansoni, the causative agent of schistosomiasis. a. If you disobeyed your mother and contracted this

a. Based on the results of this study, which MHC molecule(s)

serve(s) as restriction element(s) for this peptide antigen? b. Since antigen recognition by B cells is not MHC

restricted, why is the humoral antibody response influenced by the MHC haplotype? 7. Fill in the blanks in the following statements. a. The current vaccine for tuberculosis consists of an

attenuated strain of M. bovis called ______. b. Variation in influenza surface proteins is generated by

______ and ______. c. Variation in pilin, which is expressed by many gram-

negative bacteria, is generated by the process of ______. d. The mycobacteria causing tuberculosis are walled off in

e. f. g. h.

granulomatous lesions called ______, which contain a small number of ______ and many ______. The diphtheria vaccine is a formaldehyde-treated preparation of the exotoxin, called a ______. A major contribution to nonspecific host defense against viruses is provided by ______ and ______. The primary host defense against viral and bacterial attachment to epithelial surfaces is ______. Two cytokines of particular importance in the response to infection with M. tuberculosis are ______, which stimulates development of TH1 cells, and ______, which promotes activation of macrophages.

8. Despite the fact that there are no licensed vaccines for them,

life-threatening fungal infections are not a problem for the general population. Why? Who may be at risk for them? 9. Discuss the factors that contribute to the emergence of new

pathogens or the re-emergence of pathogens previously thought to be controlled in human populations. 10. Which of the following are strategies used by pathogens to

evade the immune system? For each correct choice, give a specific example. a. Changing the antigens expressed on their surfaces b. Going dormant in host cells

parasite, what cells of your immune system would fight the infection? b. If your doctor administered a cytokine to drive the immune response, which would be a good choice, and how would this supplement alter maturation of plasma cells to produce a more helpful class of antibody? 13. Usually, the influenza virus changes its structure very slightly

from one year to the next. However, although we are being exposed to these “modified” influenza strains every year, we do not always come down with the flu, even when the virus successfully breaches physical barriers. Sometimes, we do get a really bad case of the flu, despite the fact that we presumably have memory cells left from an earlier primary response to influenza. Aside from higher doses of virus and the possibility of a particularly pathogenic strain, why is it that some years we get very sick and other years we do not? For example, I might get a bad case of the flu while you experience no disease, and yet we are both being exposed to the exact same virus strain that year. What is happening here? Assume that you are not receiving the yearly influenza vaccine.

VACCINES CLINICAL FOCUS QUESTION A connection between the new

pneumococcus vaccine and a relatively rare form of arthritis has been reported. What data would you need to validate this report? How would you proceed to evaluate this possible connection? 1. Indicate whether each of the following statements is true or

false. If you think a statement is false, explain why. a. Transplacental transfer of maternal IgG antibodies against

measles confers short-term immunity on the fetus. b. Attenuated vaccines are more likely to induce cell-

mediated immunity than killed vaccines are. c. One disadvantage of DNA vaccines is that they don’t

generate significant immunologic memory. d. Macromolecules generally contain a large number of

potential epitopes. e. A DNA vaccine only induces a response to a single

epitope.

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2. What are the advantages and disadvantages of using live

attenuated organisms as vaccines? 3. A young girl who had never been immunized to tetanus

stepped on a rusty nail and got a deep puncture wound. The doctor cleaned out the wound and gave the child an injection of tetanus antitoxin. a. Why was antitoxin given instead of a booster shot of

tetanus toxoid? b. If the girl receives no further treatment and steps on a rusty nail again 3 years later, will she be immune to tetanus? 4. What are the advantages of the Sabin polio vaccine com-

pared with the Salk vaccine? Why is the Sabin vaccine no longer recommended for use in the United States?

treatment (gammaglobulin or antiserum) against the poisonous snake venom. You recover from your snakebite and return home for some TLC. One year later during an environmental studies field trip, you are bitten once again by the same type of snake. Please answer the following questions: a. Since you fully recovered from the first snakebite, are

you protected from the effects of the poison this second time (i.e., did you develop adaptive immunity)? b. Immunologically, what occurred the first time you were bitten and treated for the bite? c. Compared to the first snakebite, are you more sensitive, less sensitive, or equally sensitive to the venom from the second bite?

5. Why doesn’t the live attenuated influenza vaccine (Flu-

Mist) cause respiratory infection? 6. In an attempt to develop a synthetic peptide vaccine, you

have analyzed the amino acid sequence of a protein antigen for (a) hydrophobic peptides and (b) strongly hydrophilic peptides. How might peptides of each type be used as a vaccine to induce different immune responses? 7. Explain the phenomenon of herd immunity. How does it

relate to the appearance of certain epidemics? 8. You have identified a bacterial protein antigen that confers

protective immunity to a pathogenic bacterium and have cloned the gene that encodes it. The choices are either to express the protein in yeast and use this recombinant protein as a vaccine or to use the gene for the protein to prepare a DNA vaccine. Which approach would you take and why? 9. Explain the relationship between the incubation period of

a pathogen and the approach needed to achieve effective active immunization. 10. List the three types of purified macromolecules that are

currently used as vaccines. 11. Some parents choose not to vaccinate their infants. Rea-

sons include religion, allergic reactions, fear that the infant will develop the disease the vaccine is raised against, and, recently, a fear, unsupported by research, that vaccines can cause autism. What would be the consequence if a significant proportion of the population was not vaccinated against childhood diseases such as measles or pertussis?

ANALYZE THE DATA T. W. Kim and coworkers (Enhancing

DNA vaccine potency by combining a strategy to prolong dendritic cell life with intracellular targeting strategies. Journal of Immunology 171:2970, 2003) investigated methods to enhance the immune response against human papillomavirus (HPV)16 E7 antigen. Groups of mice were vaccinated with the following antigens incorporated in DNA vaccine constructs: • ⫹ HPV E7 antigen • ⫹ E7 ⫹ heat-shock protein 70 (HSP70) • ⫹ E7 ⫹ calreticulin • ⫹ E7 ⫹ Sorting signal of lysosome-associated membrane protein 1 (Sig/LAMP-1) A second array of mice received the same DNA vaccines and was coadministered an additional DNA construct incorporating the anti-apoptosis gene Bcl-xL. To test the efficacy of these DNA vaccine constructs in inducing a host response, spleen cells from vaccinated mice were harvested 7 days after injection, the cells were incubated overnight in vitro with MHC class I– restricted E7 peptide (aa 49–57), and then the cells were stained for both CD8 and IFN-␥ (part (a) of the figure on the following page). In another experiment Kim and group determined how effective their vaccines were if mice lacked CD4⫹ T cells (part (b), shown on the following page). a. Which DNA vaccine(s) is (are) the most effective in

b.

12. For each of the following diseases or conditions, indicate

what type of vaccination is used: a. b. c. d. e. f. g.

Polio Chickenpox Tetanus Hepatitis B Cholera Measles Mumps

1. Inactivated 2. Attenuated 3. Inactivated exotoxin 4. Purified macromolecule

13. While on a backpacking trip you are bitten by a poison-

ous snake. The medevac comes to airlift you to the nearest hospital, where you receive human immunoglobulin

c. d.

e.

inducing an immune response against papillomavirus E7 antigen? Explain your answer. Propose a hypothesis to explain why expressing calreticulin in the vaccine construct was effective in inducing CD8⫹ T cells. Propose a mechanism to explain the data in part (a) of the figure. If you were told that the ⫹E7 ⫹Sig/LAMP-l construct is the only one that targets antigen to the class II MHC processing pathway, propose a hypothesis to explain why antigen that would target MHC II molecules enhances a CD8⫹ T-cell response. Why do you think a special signal was necessary to target antigen to MHC II? What four variables contribute to the E7-specific CD8⫹ T lymphocyte response in vitro as measured in part (b) of the figure?

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(a)

(b)

Amount of CD8+ expressed (surface) Amount of IFN- expressed (intracellular)

Coadministered with BCL-xL DNA construct

3

3

Control (empty vector)

5

58

Number of E7-specific IFN- CD8 T cells /3  105 splenocytes

Infectious Diseases and Vaccines

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Bcl-xL

2000

591

With peptide Without peptide

1500 1000 Bcl-xL

500 0

Wild-type mice

CD4 knockout mice

+E7

E7-specific CD8⫹ T lymphocyte response in CD4 knockout mice vaccinated with ⫹E7 ⫹Sig/LAMP-1 DNA construct, with or without ⫹Bcl-xL DNA construct. 501

1143

1578

3687

167

2039

+E7 + HSP70

+E7 +calreticulin

+E7 + Sig/LAMP-1

Intracellular cytokine staining followed by flow cytometry analysis to determine the E7 -specific CD8⫹ T-cell response in mice vaccinated with DNA vaccines using intracellular targeting strategies. A DNA construct including the anti-apoptosis gene Bcl-xL was coadministered to the group on the right. The number at top right in each graph is the number of cells represented in the top right quadrant.

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18 Immunodeficiency Disorders

L

ike any complex multicomponent system, the immune system can be subject to failures of some or all of its parts. These failures can have dire consequences. When the system loses its sense of self and begins to attack the host’s own cells, the result is autoimmunity, described in Chapter 16. When the system errs by failing to protect the host from diseasecausing agents, the result is immunodeficiency, the subject of this chapter. Immunodeficiency resulting from an inherited genetic or developmental defect in the immune system is called a primary immunodeficiency. In such a condition, the defect is present at birth, although it may not manifest until later in life. These diseases can be caused by defects in virtually any gene involved in immune development or function, innate or adaptive, humoral or cell mediated, plus genes not previously associated with immunity. As one can imagine, the nature of the component(s) that fail(s) determines the degree and type of the immune defect; some immunodeficiency disorders are relatively minor, requiring little or no treatment, although others can be life threatening and necessitate major intervention. Secondary immunodeficiency, also known as acquired immunodeficiency, is the loss of immune function that results from exposure to an external agent, often an infection. Although several external factors can affect immune function, by far the most well-known secondary immunodeficiency is acquired immunodeficiency syndrome (AIDS), which results from infection with the human immunodeficiency virus (HIV). A global summary of the AIDS epidemic conducted by the Joint United Nations Programme on HIV/AIDS (UNAIDS) shows that by the end of 2011 (the most recent data available) over 34 million people were living with HIV and 2.5 million new infections occurred in just that year (330,000 of them in children under age 15 years). In 2011, AIDS killed approximately 1.7 million people. The good news is that, largely thanks to increased access to antiretroviral drugs, this was roughly a 24% decrease in the rate of AIDS-related deaths as compared to just 6 years earlier. People with AIDS, like individuals

Interaction between dendritic cell and T cell indicating passage of HIV-1 (green dots) between the cells. [Courtesy of Thomas J. Hope, Northwestern University.] ■

Primary Immunodeficiencies



Secondary Immunodeficiencies

with severe inherited immunodeficiency, are at risk of opportunistic infections, caused by microorganisms that healthy individuals can easily eradicate but that cause disease and even death in those with significantly impaired immune function. The first part of this chapter describes the most common primary immunodeficiencies, examines progress in identifying new defects that can lead to these types of disorders, and considers approaches to their study and treatment. The rest of the chapter describes acquired immunodeficiency, with a focus on HIV infection and AIDS, along with the current status of therapeutic and prevention strategies for combating this often fatal disorder.

Primary Immunodeficiencies To date, over 150 different types of primary, or inherited, immunodeficiency have been identified. Theoretically, any component important to immune function that is defective can lead to some form of immunodeficiency. Collectively, primary immunodeficiency disorders (PIDs) have helped immunologists to appreciate the importance of specific cellular events or proteins that are required for proper immune system function. Most of these disorders are monogenic, or 593

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Cellular (T cell), 10%

Phagocytic, 18%

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Humoral (B cell), 50%

Combined (B and T cell), 20%

FIGURE 18-1 Distribution of primary immunodeficiencies by type. Primary immunodeficiency can involve either innate processes (phagocytosis, complement, or other defects) or the adaptive immune response (humoral, cellular, or both). Of these categories, adaptive immune disruptions are the most common, with antibody defects making up the largest portion of these. [Song et al., 2011, Clinical and Molecular Allergy 9:10. doi:10.1186/1476-7961-9-10]

caused by defects in a single gene, and are extremely rare. Primary immunodeficiency diseases vary in severity from mild to nearly fatal. They can be loosely categorized as affecting either innate immunity or adaptive responses, and are often grouped by the specific components of the immune system most affected (Figure 18-1). The most common forms of primary immunodeficiency, and frequently the least severe, are those that impair one or more antibody isotype. However, due to the complex interconnections of the immune response, defects in one pathway can also manifest in other arms of the immune response, and different gene defects can produce the same phenotype, making strict categorization complicated. The cellular consequences of a particular gene disruption depend on the specific immune system component involved and the severity of the disruption (Figure 18-2). Defects that interrupt early hematopoietic cell development affect everything downstream of this step, as is the case for reticular dysgenesis, a disease in which all hematopoietic cell survival is impaired. Defects in more highly differentiated downstream compartments of the immune system, such as in selective immunoglobulin deficiencies, have consequences that tend to be more specific and usually less severe. In some cases, the loss of a gene not specifically associated with immunity has been found to have undue influence on cells of the hematopoietic lineage, such as the lymphoid cell destruction seen in adenosine deaminase deficiency (ADA), which disables both B and T cells, leading to a form of severe combined immunodeficiency. Decreased production of phagocytes, such as neutrophils, or the inhibition of phagocytic processes typically manifest as increased susceptibility to bacterial or fungal infections, as seen in defects that affect various cells within the myeloid lineage (Figure 18-2,

orange). In general, defects in the T-cell components of the immune system tend to have a greater overall impact on the immune response than genetic mutations that affect only B cells or innate responses. This is due to the pivotal role of T cells in directing downstream immune events, and occurs because defects in this cell type often affect both humoral and cell-mediated responses. Immunodeficiency disorders can also stem from developmental defects that alter a specific organ. This is most commonly seen in sufferers of DiGeorge syndrome, where T-cell development is hindered by a congenital defect that blocks growth of the thymus. Since many B-cell responses require T-cell help, most of the adaptive immune response is compromised in patients who suffer the complete form of the disease in which little or no thymic tissue is present, even though B cells are intact. Finally, a more recent category of immunodeficiency syndrome has come to light, illustrating the importance of immune regulation, or “the brakes” of the immune system. APECED and IPEX are both immunodeficiency disorders that result in overactive immune responses, or autoimmunity, due to the dysregulation of self-reactive T cells. Some of the most well-characterized primary immunodeficiency disorders with known genetic causes are listed in Table 18-1, along with the specific gene defect and resulting immune impairment. The nature of the immune defect will determine which groups of pathogens are most challenging to individuals who inherit these immunodeficiency disorders (Table 18-2). Inherited defects that impair B cells, resulting in depressed expression of one or more of the antibody classes, are typically characterized by recurring bacterial infections. These symptoms are similar to those exhibited by some of the individuals who inherit mutations in genes that encode complement components. Phagocytes are so important for the removal of fungi and bacteria that individuals with disruptions of phagocytic function suffer from more of these types of infections. Finally, the pivotal role of the T cell in orchestrating the direction of the immune response means that disruptions to the performance of this cell type can have wide-ranging effects, including depressed antibody production, dysregulation of cytokine expression, and impaired cellular cytotoxicity. In some instances, such as when T- and B-cell responses to self are not properly regulated, autoimmunity can become the primary symptom. The first part of this section on primary immunodeficiency diseases looks at defects within adaptive immunity, starting with the most extreme cases, characterized by defects to T cells, B cells, or both. This is followed by a discussion of disruptions to innate responses, including cells of the myeloid lineage, receptors important for innate immunity, and complement defects. The autoimmune consequences stemming from dysregulation of the immune system are also described. Finally, we look at the current treatment options available to affected individuals and the use of animal models of primary immunodeficiency in basic immunology research.

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OVERVIEW FIGURE

Primary Immunodeficiencies Result from Congenital Defects in Specific Cell Types Reticular dysgenesis

Stem cell

Myeloid progenitor cell

Chronic granulomatous disease

Neutrophil

Leukocyte-adhesion deficiency

Severe combined immunodeficiency (SCID)

Lymphoid progenitor cell

Monocyte

Pre–B cell

Pre–T cell

Severe combined immunodeficiency

X-linked agammaglobulinemia

APECED DiGeorge syndrome

Thymus Bare- lymphocyte syndrome

IPEX Mature B cell Common variable hypogammaglobulinemia

Mature T cell Wiskott-Aldrich syndrome X- linked hyper- IgM syndrome

Selective immunoglobulin deficiency

Plasma cell

Memory B cell

Orange ⫽ phagocytic deficiencies, green ⫽ humoral deficiencies, red ⫽ cell-mediated deficiencies, pink ⫽ regulatory cell deficiencies, and purple ⫽ combined immunodeficiencies, or defects that affect more than one cell lineage. APECED ⫽ autoimmune polyendocrinopathy and ectodermal dystrophy. IPEX ⫽ immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome.

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Some primary human immunodeficiency diseases and underlying genetic defects

Immunodeficiency disease Severe combined immunodeficiency (SCID)

Specific defect

Impaired function

RAG1/RAG2 deficiency

No TCR or Ig gene rearrangement

Inheritance mode* AR

ADA deficiency PNP deficiency

f

Toxic metabolite in T and B cells

e

AR AR

JAK-3 deficiency IL-2R␥ deficiency

f

Defective signals from IL-2, -4, -7, -9, -15, -21

e

AR XL

ZAP-70 deficiency

Defective signal from TCR

AR

Bare-lymphocyte syndrome (BLS)

Defect in class II MHC gene promoter

No class II MHC molecules

AR

Wiskott-Aldrich syndrome (WAS)

Cytoskeletal protein (WASP)

Defective T-cells and platelets

XL

Mendelian susceptibility to mycobacterial diseases (MSMD)

IFN-␥R IL-12/IL-12R STAT1

Impaired immunity to mycobacteria

AR or AD

DiGeorge syndrome

Thymic aplasia

T-cell development

AD

Gammaglobulinemias

X-linked agammaglobulinemia

Bruton’s tyrosine kinase (Btk); no mature B cells

XL

X-linked hyper-IgM syndrome

Defective CD40 ligand

XL

Common variable immunodeficiency

Low IgG, IgA; variable IgM

Complex

Selective IgA deficiency

Low or no IgA

Complex

phox

XL AR

Chronic granulomatous disease

gp91

Chediak-Higashi syndrome

Defective intracellular transport protein (LYST)

Inability to lyse bacteria

AR

Leukocyte adhesion defect

Defective integrin ␤2 (CD18)

Leukocyte extravasation

AR

Autoimmune polyendocrinopathy and ectodermal dystrophy (APECED)

AIRE defect

T-cell tolerance

AR

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome

FoxP3 defect

Absence of TREG cells

XL

phox

p67

phox

, p47

phox

, p22

f

No oxidative burst for phagocytic killing

e

* AR ⫽ autosomal recessive; AD ⫽ autosomal dominant; XL⫽ X linked; “Complex” inheritance modes include conditions for which precise genetic data are not available and that may involve several interacting loci.

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Patterns of infection and illness associated with primary immunodeficiency diseases Disease OPPORTUNISTIC INFECTIONS

Disorder Antibody

Sinopulmonary (pyogenic bacteria) Gastrointestinal (enterovirus, giardia)

Cell-mediated immunity

Pneumonia (pyogenic bacteria, Pneumocystis carinii, viruses)

OTHER SYMPTOMS

Autoimmune disease (autoantibodies, inflammatory bowel disease)

Gastrointestinal (viruses), mycoses of skin and mucous membranes (fungi) Complement

Sepsis and other blood-borne infections (streptococci, pneumococci, neisseria)

Phagocytosis

Skin abscesses, reticuloendothelial infections (staphylococci, enteric bacteria, fungi, mycobacteria)

Regulatory T cells

N/A

Autoimmune disease (systemic lupus erythematosus, glomerulonephritis)

Autoimmune disease

Source: Adapted from H. M. Lederman, 2000, The clinical presentation of primary immunodeficiency diseases, Clinical Focus on Primary Immune Deficiencies. Towson, MD: Immune Deficiency Foundation 2(1):1.

Combined Immunodeficiencies Disrupt Adaptive Immunity Among the most severe forms of inherited immunodeficiency are a group of disorders termed combined immunodeficiences (CIDs): diseases resulting from an absence of T cells or significantly impaired T-cell function, combined with some disruption of antibody responses. Defects within the T-cell compartment generally also affect the humoral system because TH cells are typically required for complete B-cell activation, antibody production, and isotype switching. Therefore, some depression in the level of one or more antibody isotypes and an associated increase in susceptibility to bacterial infection are common with CIDs. T-cell impairment can lead to a reduction in both delayed-type hypersensitivity responses and cell-mediated cytotoxicity, resulting in increased susceptibility to almost all types of infectious agents, but especially viruses, protozoa, and fungi. For instance, infections with species of Mycobacteria are common in CID patients, reflecting the importance of T cells in eliminating intracellular pathogens. Likewise, viruses that are otherwise rarely pathogenic (such as cytomegalovirus or even live, attenuated measles vaccine) may be life threatening for individuals with CIDs. The following section first discusses the most severe CIDs, such as when there is an absence of both T and B cells, followed by less severe forms of the disease, in which more minor disruptions to particular components of the T- and B-cell compartments are observed. Severe Combined Immunodeficiency (SCID) The most extreme forms of CID make up a family of disorders termed severe combined immunodeficiency (SCID).

These stem from genetic defects that lead to a virtual or absolute lack of functional T cells in the periphery. As a general rule, these defects target steps that occur early in T-cell development or that affect the stem cells that feed the lymphoid lineage. The four general categories of events that have been found to result in SCID include the following: 1.

Defective cytokine signaling in T-cell precursors, caused by mutations in certain cytokines, cytokine receptors, or regulatory molecules that control their expression

2.

Premature death of the lymphoid lineage due to accumulation of toxic metabolites, caused by defects in the purine metabolism pathways

3.

Defective V(D)J rearrangement in developing lymphocytes, caused by mutations in the genes for RAG1 and RAG2, or other proteins involved in the rearrangement process

4.

Disruptions in pre-TCR or TCR signaling during development, caused by mutations in tyrosine kinases, adapter molecules, downstream messengers, or transcription factors involved in TCR signaling

Depending on the underlying genetic defect, an individual with SCID may have a loss of only T cells (T⫺B⫹) or both T and B cells (T⫺B⫺). In either case, both cellular and humoral immunity are either severely depressed or absent. Clinically, SCID is characterized by a very low number of circulating lymphocytes and a failure to mount immune responses mediated by T cells. In many cases, the thymus will not fully develop without a sufficient number of T cells, and the few circulating T cells present in some SCID patients

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often do not respond to stimulation by mitogens, indicating that they cannot proliferate in response to antigens. In many cases, myeloid and erythroid cells (red-blood-cell precursors) appear normal in number and function, indicating that only lymphoid cells are affected. Infants born with SCID experience severe recurrent infections that, without early, aggressive treatment, can quickly prove fatal. Although both the T and B lineages may be affected, the initial manifestation in these infants is typically infection by fungi or viruses that are normally dealt with by cellular immune responses. This is because antibody deficits can be masked in the first few months of life by the presence of passive antibodies derived from transplacental circulation or breast milk. Infants with SCID often suffer from chronic diarrhea, recurrent respiratory infections, and a general failure to thrive. The life span of these children can be prolonged by preventing contact with all potentially harmful microorganisms—for example, by confinement in a sterile atmosphere. However, extraordinary effort is required to prevent contact with all opportunistic microorganisms; any object, including food, that comes in contact with the sequestered SCID patient must first be sterilized. Such isolation is feasible only as a temporary measure, pending replacement therapy treatments and/or bone marrow transplantation (more on these below). The immune system is so compromised in SCID patients that common microbes and even live-attenuated vaccines can cause persistent infection and life-threatening disease. For this reason, it is important to diagnose SCID early, especially prior to the administration of live vaccines, such as the rotavirus vaccine, which is recommended at 2 months of age (see Chapter 17). A screening test for SCID has been developed that utilizes the standard blood samples collected from neonates via heel or finger pricks. This rapid polymerase chain reaction (PCR)-based assay looks for evidence of gene recombination as in excised DNA from the TCR or BCR locus, called T-cell receptor excision circles (TRECs) and ␬-deleting recombination excision circles (KRECs). In 2010, recommendations to screen every newborn for SCID were approved. To date, approximately half of the babies born in the United States receive standard newborn screening for SCID, before live vaccines are administered and when the implementation of aggressive therapy is most beneficial. Deficiency in cytokine signaling is at the root of the most common forms of SCID, and defects in the gene encoding the common gamma (␥) chain of the IL-2 receptor (IL2RG; see Figure 4-8) are the most frequent culprits. This particular form of immunodeficiency is often referred to as X-linked SCID (or SCIDX1) because the affected gene is located on the X chromosome, and the disorder is thus more common in males. Defects in this chain impede signaling not only through IL-2R but also through receptors for IL-4, -7, -9, -15, and -21, all of which use this chain in their structures. This leads to widespread defects in B-, T-, and NK-cell development. Although this chain was first identified as a part of the IL-2 receptor, impaired IL-7 signaling is likely the source

Hematopoietic stem cell (HSC)

Common lymphoid precursor (CLP)

Impaired IL-15R signaling (NK cell development)

Blocked RAG1/2 or Artemis expression (no BCR or TCR)

Blocked IL-7R signaling (T and B cell development) Natural killer (NK) cell

T cell progenitor

B cell progenitor

FIGURE 18-3 Defects in lymphocyte development and signaling can lead to severe combined immunodeficiency (SCID). SCID may result from defects in the recombination-activating genes (RAG1 and RAG2) or the DNA excision-repair pathway (e.g., Artemis) required for synthesis of the functional immunoglobulins and T-cell receptors in developing lymphocytes. Likewise, defects in the common ␥ chain of receptors for IL-2, -4, -7, -9, and -15, required for the hematopoietic development of lymphocytes, or JAK-3, which transduces these signals (not shown), can also lead to SCID. of both T-and B-cell developmental defects, while lack of IL-15 signaling is believed to account for the block to NK cells (Figure 18-3). Deficiency in the kinase JAK-3, which associates with the cytoplasmic region of the common gamma (␥) chain, can produce a phenotype similar to X-linked SCID, as this enzyme is required for the intracellular signaling cascade utilized by all of these cytokine receptors (see Chapter 4). Defects in the pathways involved in the recombination events that produce immunoglobulin and T-cell receptors highlight the importance of early signaling through these receptors for lymphocyte survival. Mutations in the recombinase activating genes (RAG1 and RAG2) and genes encoding proteins involved in the DNA excision-repair pathways employed during gene rearrangement (e.g., Artemis) can also lead to SCID (see Figure 18-3). In these cases, production of antigen-specific receptors is blocked at the pre-T- and pre-B-cell receptor stages of development, leading to a virtual absence of functioning T and B cells, while leaving the numbers and function of NK cells largely intact (see Clinical Focus Box 7-3). Another relatively common defect resulting in SCID is adenosine deaminase (ADA) deficiency. Adenosine deaminase catalyzes conversion of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. Its deficiency results in

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Immunodeficiency Disorders the intracellular accumulation of toxic adenosine metabolites, which interferes with purine metabolism and DNA synthesis. This housekeeping enzyme is found in all cells, so these toxic compounds also produce neurologic and metabolic symptoms, including deafness, behavioral problems, and liver damage. Defects in T, B, and NK cells are due to toxic metabolite-induced apoptosis of lymphoid precursors in primary lymphoid organs. Deficiency in another purine salvage pathway enzyme, purine nucleoside phosphorylase (PNP), produces a similar phenotype via much the same mechanism. In some instances, the genetic defects associated with SCID lead to perturbations in hematopoiesis. In reticular dysgenesis (RD), the initial stages of hematopoietic cell development are blocked by defects in the adenylate kinase 2 gene (AK2), favoring apoptosis of myeloid and lymphoid precursors and resulting in severe reductions in circulating leukocytes (see Figure 18-2). The resulting general failure leads to impairment of both innate and adaptive immunity, resulting in susceptibility to infection by all types of microorganisms. Without aggressive treatment, babies with this very rare form of SCID usually die in early infancy from uncontrolled infection. MHC Defects That Can Resemble SCID A failure to express MHC molecules can lead to general failures of immunity that resemble SCID without directly impacting lymphocytes themselves. For example, without class II MHC molecules, positive selection of CD4⫹ T cells in the thymus is impaired, and with it, peripheral T helper cell responses are impaired. This type of immunodeficiency is called bare-lymphocyte syndrome and is the topic of Clinical Focus Box 8-4. The important and ubiquitous role of class I MHC molecules is highlighted in patients with defective class I expression. This rare immunodeficiency disorder can be caused by mutations in the TAP genes, which are vital to antigen processing and presentation by class I MHC molecules (see Figure 8-17). This defect, which typically allows for some residual expression of class I molecules, results in impaired positive selection of CD8⫹ T cells, depressed cellmediated immunity, and heightened susceptibility to viral infection. Developmental Defects of the Thymus Some immunodeficiency syndromes affecting T cells are grounded in failure of the thymus to undergo normal development. These thymic malfunctions can have subtle to profound outcomes on T-cell function, depending on the nature of the defect. DiGeorge syndrome (DGS), also called velocardiofacial syndrome, is one example. This disorder typically results from various deletions in a region on chromosome 22 containing up to 50 genes, with the T-box transcription factor (TBX1) thought to be most influential. This transcription factor is highly expressed during particular stages of embryonic development, when facial structures,

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FIGURE 18-4 A child with DiGeorge syndrome showing characteristic dysplasia of ears and mouth and abnormally wide distance between the eyes. [R. Kretschmer et al., 1968, New England Journal of Medicine 279:1295; photograph courtesy of F. S. Rosen.]

heart, thyroid, parathyroid, and thymus tissues are forming (Figure 18-4). For this reason, the syndrome is sometimes also called the third and fourth pharyngeal pouch syndrome. Not surprisingly, DGS patients present with symptoms of immunodeficiency, hypoparathyroidism, and congenital heart anomalies, where the latter are typically the most critical. Although most DGS sufferers show some degree of immunodeficiency, the degree varies widely. In very rare cases of complete DGS, where no thymic tissue develops, severe depression of T-cell numbers and poor antibody responses due to lack of T-cell help leave patients susceptible to all types of opportunistic pathogens. Thymic transplantation and passive antibody treatment can be of value to these individuals, although severe heart disease can limit longterm survival even when the immune defects are corrected. In the majority of DGS patients, in which some residual thymic tissue develops and functional T cells are found in the periphery, treatments to avoid bacterial infection, such as antibiotics, are often sufficient to compensate for the immune defects. Wiskott-Aldrich Syndrome (WAS) Although SCID is caused by genetic defects that result in the loss of T cells or major T-cell impairment, a number of other CIDs can result from less severe disruptions to T-cell function. The defect in patients suffering from Wiskott-Aldrich syndrome (WAS) occurs in an X-linked gene named for this disease (WASP), which encodes a cytoskeletal protein highly

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expressed in hematopoietic cells (see Table 18-1). The WAS protein (WASP) is required for assembly and reorganization of actin filaments in cells of the hematopoietic lineage, events critical to proper immune synapse formation and intracellular signaling. Clinical manifestations, which usually appear early in the first year of life, vary widely and severity depends on the specific mutation, but eczema and thrombocytopenia (low platelet counts and smaller than normal platelets, which can result in near fatal bleeding) are both common. Humoral defects, including lower than normal levels of IgM, as well as impaired cell-mediated immunity, are also common features. WAS patients often experience recurrent bacterial infections, especially by encapsulated strains such as S. pneumoniae, H. influenzae type b (Hib), and S. aureus. As the disease develops, autoimmunity and B-cell malignancy are not uncommon, suggesting that regulatory T-cell functions are also impaired. Mild forms of the disease can be treated by targeting the symptoms—transfusions for bleeding and passive antibodies or antibiotics for bacterial infections—but severe cases and long-term corrective measures require hematopoietic stem cell transfer. Hyper IgM Syndrome An inherited deficiency in CD40 ligand (CD40L or CD154) leads to impaired communication between T cells and antigen-presenting cells (APCs), highlighting the role of this surface molecule in this costimulatory process. In this X-linked disorder, TH cells fail to express functional CD40L on their plasma membrane, which typically interacts with the CD40 molecule present on B cells and dendritic cells (DCs). This costimulatory engagement is required for APC activation, and its absence in B cells interferes with class switching, B-cell responses to T-dependent antigens, and the production of memory cells (Figure 18-5). The B-cell response to T-independent antigens, however, is unaffected, accounting for the presence of IgM antibodies in these patients, which range from normal to high levels and give the disorder its common name, hyper IgM syndrome (HIM). Without class switching or hypermutation, patients make very low levels of all other antibody isotypes and fail to produce germinal centers during a humoral response, which highlights the role of the CD40-CD40L interaction in the

generation of these structures. Because CD40-CD40L interactions are also required for DC maturation and IL-12 secretion, defects in this pathway result in increased susceptibility to intracellular pathogens. Affected children therefore suffer from a range of recurrent infections, especially in the respiratory tract. Although this form of immunodeficiency results in alterations in antibody production and presents with symptoms similar to HIM variants seen in the next section on antibody deficiencies, it is classified as a CID. This is because the underlying deficiency is present in T cells, leading to a secondary defect in B-cell activation. Several other recessively inherited variants of HIM syndrome have been linked to downstream events, such as mutations in one of the enzymes involved in class switching, with the net result of depressed production of all antibody isotypes except IgM. Hyper IgE Syndrome (Job Syndrome) Another primary immunodeficiency is characterized by skin abscesses, recurrent pneumonia, eczema, and elevated levels of IgE, accompanied by facial abnormalities and bone fragility. This multisystem disorder, known as hyper IgE syndrome (HIE), is most frequently caused by an autosomal dominant mutation in the STAT3 gene. This gene is involved in the intracellular signaling cascade induced by IL-6 and TGF-␤ receptor ligation, and is important for TH17 cell differentiation (see Figure 11-11). Its absence is thought to lead to dysregulation of TH pathway development and may be the reason for overproduction of IgE. Patients with Job syndrome have lower-than-normal levels of circulating TH17 cells, and naïve cells isolated from these individuals are not capable of producing IL-17 or IL-22 in response to antigenic stimulation. Depressed TH17 responses, which are important for clearance of fungal and extracellular bacterial infections, explain the susceptibility of these patients to C. albicans and S. aureus. STAT3 defects also inhibit IL-10 signaling and the development of regulatory T cells, which is evident in the reduction of induced TREG cells in these patients. Although STAT3 is involved in the signal transduction of many cytokines and therefore could play a role in the elevation of IgE in these patients, no clear mechanism for this has been defined. FIGURE 18-5 Defects in components

Defect in CD40L (HIM) CD40L

CD40 Class II MHC Ig class switching

TCR CD4

IgM T cell

CD28

CD80/86

B cell

of APC-T cell interactions can give rise to primary immunodeficiency. Defects in CD40/CD40L costimulation between T cells and APCs lead to a block in APC maturation. In B cells, this manifests as a defect in class switching, leading to elevated levels of IgM and no other isotypes (called hyperIgM syndrome, or HIM). In DCs, this blocks maturation and the secretion of costimulatory cytokines, such as IL-12, which are important for T-cell differentiation.

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B-Cell Immunodeficiencies Exhibit Depressed Production of One or More Antibody Isotypes Immunodeficiency disorders caused by B-cell defects make up a diverse spectrum of diseases ranging from the complete absence of mature recirculating B cells, plasma cells, and immunoglobulin, to the selective absence of only certain classes of immunoglobulins. Patients with inherited B-cell defects are usually subject to recurrent bacterial infections but display normal immunity to most viral and fungal infections because the T-cell branch of the immune system is largely unaffected. In patients with these types of immunodeficiencies, the most common infections are caused by encapsulated bacteria such as staphylococci, streptococci, and pneumococci, because antibody is critical for the opsonization and clearance of these organisms. Although the underlying defects have been identified for some of these conditions, several of the more common deficiencies, such as common variable immunodeficiency and selective IgA deficiency, appear to involve multiple genes and a continuum of phenotypes. X-Linked Agammaglobulinemia X-linked agammaglobulinemia (X-LA), or Bruton’s hypogammaglobulinemia, is characterized by extremely low IgG levels and by the absence of other immunoglobulin classes. Babies born with this disorder have virtually no peripheral B cells (⬍ 1%) and suffer from recurrent bacterial infections. X-LA is caused by a defect in Bruton’s tyrosine kinase (Btk), which is required for signal transduction through the BCR (see Figure 3-28 and Clinical Focus Box 3-2). Without functional Btk, B-cell development in the bone marrow is arrested at the pro-B- to pre-B-cell stage, and the B lymphocytes in these patients remain in the pre-B stage, with heavy chains rearranged but light chains in their germ-line configuration. Present-day use of antibiotics and replacement therapy in the form of passively administered antibodies can make this disease quite manageable. Common Variable Immunodeficiency Disorders The defects underlying the complex group of diseases belonging to this category are more different than they are similar. However, sufferers of common variable immunodeficiency disorders (CVIDs) do share recurrent infection resulting from immunodeficiency, marked by reduction in the levels of one or more antibody isotype and impaired B-cell responses to antigen, all with no other known cause. This condition can manifest in childhood or later in life, when it is sometimes called late-onset hypogammaglobulinemia or, incorrectly, acquired hypogammaglobulinemia. Respiratory tract infection by common bacterial strains is the most common symptom, and can be controlled by administration of immunoglobulin. Most cases of CVID have undefined genetic causes, and most patients have normal numbers of B cells, suggesting that B-cell development

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is not the underlying defect in most cases. Reflecting the diversity of this set of diseases, inheritance can follow autosomal recessive or autosomal dominant patterns, although most cases are sporadic. Several different proteins involving various steps of the B-cell activation cascade have been implicated in recent years. Selective IgA Deficiency A number of immunodeficiency states are characterized by significantly lowered amounts of specific immunoglobulin isotypes. Of these, IgA deficiency is by far the most common, affecting approximately 1 in 700. Individuals with selective IgA deficiency typically exhibit normal levels of other antibody isotypes and may enjoy a full life span, troubled only by a greater-than-normal susceptibility to infections of the respiratory and genitourinary tracts, the primary sites of IgA secretion. Family-association studies have shown that IgA deficiency sometimes occurs in the same families as CVID, suggesting some overlap in causation. The spectrum of clinical symptoms of IgA deficiency is broad; most of those affected are asymptomatic (up to 70%), whereas others may suffer from an assortment of serious complications. Problems such as intestinal malabsorption, allergic disease, and autoimmune disorders can be associated with low IgA levels. The reasons for this variability in the clinical profile are not clear but may relate to the ability of some, but not all, patients to substitute IgM for IgA as a mucosal antibody. The defect in IgA deficiency is related to the inability of IgAexpressing B cells to undergo normal differentiation to the plasma-cell stage. A gene or genes outside of the immunoglobulin gene complex is suspected of being responsible for this fairly common syndrome.

Disruptions to Innate Components May Also Impact Adaptive Responses Most innate immune defects are caused by problems in the myeloid-cell lineage or in complement (see Figure 18-2). Most of these defects result in depressed numbers of phagocytic cells or defects in the phagocytic process that are manifested by recurrent microbial infection of greater or lesser severity. The phagocytic processes may be faulty at several stages, including cell motility, adherence to and phagocytosis of organisms, and intracellular killing by macrophages. Leukocyte Adhesion Deficiency As described in Chapter 3, cell-surface molecules belonging to the integrin family of proteins function as adhesion molecules and are required to facilitate cellular interaction. Three of these, LFA-1, Mac-1, and gp150/95 (CD11a, b, and c, respectively), have a common ␤ chain (CD18) and are variably present on different monocytic cells; CD11a is also expressed on B cells. An immunodeficiency related to dysfunction of the adhesion molecules is rooted in a defect

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localized to the common ␤ chain and affects expression of all three of the molecules that use this chain. This defect, called leukocyte adhesion deficiency (LAD), causes susceptibility to infection with both gram-positive and gram-negative bacteria as well as various fungi. Impairment of adhesion of leukocytes to vascular endothelium limits recruitment of cells to sites of inflammation. Viral immunity is somewhat impaired, as would be predicted from the defective T–B-cell cooperation arising from the adhesion defect. LAD varies in its severity; some affected individuals die within a few years, whereas others survive into their forties. The reason for the variable disease phenotype in this disorder is not known. Chronic Granulomatous Disease Chronic granulomatous disease (CGD) is the prototype of immunodeficiency that impacts phagocytic function and arises in at least two distinct forms: an X-linked form in about 70% of patients and an autosomal recessive form found in the rest. This group of disorders is rooted in a defect in the nicotinamide adenine dinucleotide phosphate (NADPH) oxidative pathway by which phagocytes generate superoxide radicals and other reactive compounds that kill phagocytosed pathogens. For this reason, CGD patients suffer from infection by bacterial and fungal pathogens, as well as excessive inflammatory responses that lead to the formation of granulomas (a small mass of inflamed tissue). Genetic causes have been mapped to several missing or defective phagosome oxidase (phox) proteins that participate in this pathway (see Table 18-1). Standard treatment includes the use of antibiotics and antifungal compounds to control infection. Of late, the addition of IFN-␥ to this regimen has been shown to improve CGD symptoms in both humans and animal models. Although the mechanism for this is still debated, in vitro studies have shown that IFN-␥ treatment induces TNF-␣ and the production of nitric oxide (NO, another oxidative mediator) and enhances the uptake of inflammation-inducing apoptotic cells, which could play a role in inhibiting the formation of granulomas during inflammation in these patients. Chediak-Higashi Syndrome This rare autosomal recessive disease is an example of a lysosomal storage and transport disorder. Chediak-Higashi syndrome (CHS) is characterized by recurrent bacterial infections as well as defects in blood clotting, pigmentation, and neurologic function. Immunodeficiency hallmarks include neutropenia (depressed numbers of neutrophils) as well as impairments in T cells, NK cells, and granulocytes. CHS is associated with oculocutaneous albinism, or lightcolored skin, hair, and eyes, accompanied by photosensitivity. The underlying cause has been mapped to mutations in the lysosomal trafficking regulator (LYST) gene that cause defects in the LYST protein, which is important for transport of proteins into lysosomes as well as for controlling lysosome size, movement, and function. Disruption to this and related

organelles, such as the melanosomes of skin cells (melanocytes), results in enlarged organelles and defective transport functions. Affected phagocytes produce giant granules, a diagnostic hallmark, but are unable to kill engulfed pathogens, and melanocytes fail to transport melanin (responsible for pigmentation). Similar enlarged lysosome-like structures in platelets and nerve cells are also thought to interfere with blood clotting and neurologic function, respectively. Exocytosis pathways are likewise affected, which could account for the defects in killing seen in TC cells and NK cells, as well as impaired chemotactic responses. Without early antimicrobial therapy followed by bone marrow transplant, patients often die due to opportunistic infection before reaching 10 years of age. However, no therapies are currently available to treat the defects in other cells, so even when immune function is restored, neurologic and other complications continue to progress. Mendelian Susceptibility to Mycobacterial Diseases Recently, a set of immunodeficiency disorders has been grouped into a mixed-cell category based on the shared characteristic of single gene (Mendelian) inheritance of susceptibility to mycobacterial diseases (MSMD). Discovery of the underlying defects in MSMD highlights the connections between innate and adaptive immunity, as well as the key role played by IFN-␥ in fighting infection by mycobacteria, intracellular organisms that can cause tuberculosis and leprosy. During natural mycobacterial infection, macrophages in the lung or DCs in the draining lymph node recognize these bacteria through pattern recognition receptors (PRRs), such as TLR2 and TLR4, which trigger migration to lymph nodes followed by APC activation and differentiation. In the presence of strong costimulation, such as engagement of CD40 on the APC with CD40L on the T cell, these activated APCs produce significant amounts of IL-12 and IL-23, which can bind to their receptors on TH cells and NK cells, respectively. This leads to production of cytokines such as IFN-␥, IL-17, and TNF-␣. In a positive feedback loop, TH cells in this environment differentiate into TH1-type cells, further producers of IFN-␥. Upon binding to the IFN-␥R on APCs, this cytokine induces a signaling cascade, involving Janus kinases and STAT1, which results in enhanced phagocytosis and optimal phago-lysosomal fusion, effectively killing engulfed bacteria. This story of mycobacterial infection makes the defective genes or proteins now implicated in MSMD no great surprise (Figure 18-6). To date, six genes within the IFN-␥/ IL-12/IL-23 pathways have been linked to MSMD, including those encoding IL-12, IL-12R, IFN-␥R (both chains), STAT1, and a kinase downstream of IL-12 signaling (TYK2). Another gene linked to MSMD, called NEMO, controls the behavior of the signal transduction molecule NF-␬B (see Figure 3-17), which can affect CD40-dependent induction of IL-12. However, most mutations in NEMO lead to more widespread immune defects and susceptibility patterns than are seen in typical MSMD patients. The specific gene and type of mutation

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IL-12/23 Mycobacteria

IL-12/23R Phagocytosis

TYK2 Changes in gene expression leading to enhanced phagocytosis JAK1

JAK2 STAT4

Changes in gene expression leading to activation and differentiation

STAT1 JAK2 IFN-γR

Phagocyte

T cell IFN-γ

FIGURE 18-6 Genetic defects resulting in Mendelian susceptibility to mycobacterial diseases (MSMD). Many primary immunodeficiencies associated with increased susceptibility to mycobacterial infection are associated with defects in either the IFN-␥ pathway (e.g., IFN-␥R or the related STAT1 signaling molecule) or IL-12/23 signaling pathway (e.g., IL-12, IL-12R, and the associated TYK2 signaling molecule). These pathways are particularly important for clearing intravesicular infections. [Cottle, L. E., Mendelian susceptibility to mycobacterial disease, Clinical Genetics 2011:79, 17–22, with modifications.]

determine which other pathogens, if any, also pose a risk to these patients and influence prognosis as well as treatment options.

Complement Deficiencies Are Relatively Common Immunodeficiency diseases resulting from defects in the complement system, which has innate as well as adaptive triggers, are described in Chapter 6. Depending on the specific component that is defective, these immunodeficiencies can manifest as a generalized failure to activate complement (e.g., C4 defects) or failures of discrete pathways or functions (e.g., alternative pathway activation). Most complement deficiencies are associated with increased susceptibility to bacterial infections and/or immunecomplex diseases. For example, deficiency in properdin, which stabilizes the C3 convertase in the alternative complement pathway, is caused by a defect in a gene located on the X chromosome and is specifically associated with increased risk of infection with species of Neisseria. These types of bacterial infection are also more common in those with defects in the late components of complement, including C5–C9. Defects in mannose-binding lectin (MBL) result in increased susceptibility to a variety of infections by bacterial or fungal agents. Recall from Chapter 6 that MBL is a key initiator of the complement attack on many pathogens and is an important component of the innate immune response to many organisms.

Immunodeficiency That Disrupts Immune Regulation Can Manifest as Autoimmunity In addition to recognizing and eliminating foreign antigens, the adaptive immune system must learn to recognize self

MHC proteins and to be proactive in suppressing reactions to self antigens in the host. These processes are carried out by the induction of tolerance in the thymus and by the surveillance activities of regulatory T cells (TREG cells; see Chapters 9 and 16). Disruptions to genes involved in these immune regulatory or homeostatic processes, although caused by inborn immunodeficiencies, actually manifest as immune overactivity, or autoimmunity (see below and Chapter 16). Autoimmune Polyendocrinopathy and Ectodermal Dystrophy Individuals with a defect in the autoimmune regulatory gene AIRE, discussed in detail in Chapter 9, suffer from a disease called autoimmune polyendocrinopathy and ectodermal dystrophy (APECED). The AIRE protein is expressed in medullary epithelial cells of the thymus, where it acts as a transcription factor to control expression of a whole host of tissue-restricted antigens. Proper expression of these peripheral proteins in the thymus facilitates the negative selection of potentially autoreactive T cells before they can exit into the circulation. It appears that depressed expression of AIRE in these individuals results in reduced levels of tissue-specific antigens in thymic epithelial cells, allowing the escape of autoreactive T cells into the periphery, where they precipitate organ-specific autoimmunity. APECED patients experience inhibition of endocrine function, including hypoadrenalism, hypoparathyroidism, and hypothyroidism, along with chronic candidiasis. Autoimmune responses against antigens present in these endocrine organs, as well as the adrenal cortex, gonads, and pancreatic ␤-cells, are observed in these individuals. Although autoantibodies to these tissues are also observed, these may result from the tissue destruction mediated by pathogenic T cells.

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Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) Syndrome Although many T cells with the ability to recognize self antigens are destroyed in the thymus during negative selection, one class of CD4⫹ T cells with regulatory capabilities survive and actively inhibit reactions to these self antigens. The development and function of these TREG cells are controlled by a master regulator and transcription factor, called FoxP3 (see Chapters 9 and 16). Patients with immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome have inherited a mutated FoxP3 gene and lack expression of this protein, leading to a near absence of TREG cells. Without these regulatory cells in the periphery, autoreactive T cells that have escaped central tolerance in the thymus go unchecked, leading to systemic autoimmune disease. Affected infants exhibit immune destruction of the bowel, pancreas, thyroid, and skin, and they often die in the first 2 years of life due to sepsis and failure to thrive.

Immunodeficiency Disorders Are Treated by Replacement Therapy Although there are no sure-fire cures for immunodeficiency disorders, there are several treatment possibilities. In addition to the use of antimicrobial agents, and the drastic option of total isolation from exposure to any opportunistic pathogen, treatment options for immunodeficiencies include the following:

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For disorders that impair antibody production, the classic course of treatment is administration of the missing protein immunoglobulin. Pooled human gammaglobulin given either intravenously or subcutaneously protects against recurrent infection in many types of immunodeficiency. Maintenance of reasonably high levels of serum immunoglobulin (5 mg/ml serum) will prevent most common infections in the agammaglobulinemic patient. Advances in the preparation of human monoclonal antibodies and in the ability to genetically engineer chimeric antibodies with mouse V regions and human-derived C regions make it possible to prepare antibodies specific for important pathogens (see Chapter 20). Advances in molecular biology make it possible to clone the genes that encode other immunologically important proteins, such as cytokines, and to express these genes in vitro, using bacterial or eukaryotic expression systems. The availability of such proteins allows new modes of therapy in which immunologically important proteins may be replaced or their concentrations increased in the patient. For example, the delivery of recombinant IFN-␥ has proven effective for patients with CGD, and recombinant

adenosine deaminase has been successfully administered to ADA-deficient SCID patients. Cell replacement as therapy for some immunodeficiencies has been made possible by progress in bone marrow, or hematopoietic stem cell (HSC), transplantation (see Chapter 16) and is the primary potential long-term cure for SCID patients. Transfer of HSCs from an immunocompetent donor allows development of a functional immune system (see Clinical Focus Box 2-2). Success rates of greater than 90% have been reported for those who are fortunate enough to have an human leukocyte antigen (HLA)-identical donor. In cases where there is partial HLA matching, treatment in the first few months of life has the best prognosis for relatively long-lasting results. These procedures can also be relatively successful with SCID infants when haploidentical (complete match of one HLA gene set or haplotype) donor marrow is used. In this case, T cells are depleted to avoid graft versus host disease, and CD34⫹ stem cells are enriched before introducing the donor bone marrow into the recipient. A variation of bone marrow transplantation is the injection of parental CD34⫹ cells in utero when the birth of an infant with SCID is expected. If a single gene defect has been identified, as in adenosine deaminase or IL-2R␥ defects, replacement of the defective gene, or gene therapy, may be a treatment option. During the last 20 years, several clinical tests of gene therapy for these two types of SCID have been undertaken, with mixed results. In these trials, CD34⫹ HSCs are first isolated from the bone marrow or umbilical cord blood of HLA-identical or haploidentical donors. These cells are transduced with the corrected gene and then introduced into the patient, in some cases after myeloablation conditioning, a pre-treatment that destroys existing leukocytes, “making space” for engraftment of the new cells. As of 2012, we are now more than two decades out from these initial trials. In general, gene therapy for the IL-2R␥ defect has been slightly more successful than have similar treatments for ADA deficiency, yielding immunodeficiency correction rates of approximately 85% versus 70%, respectively. This is likely due to the presence of ADA defects in cell types other than leukocytes. Although these therapies have allowed a majority of the treated individuals to recover significant immune function, there have been some setbacks. In 5 of the first 20 cases of gene therapy for X-linked SCID, insertion of the vector used to transfer the corrected gene led to mutagenesis and development of leukemia. Most of these cases were successfully resolved; however, one individual died from this cancer, effectively halting additional trials. Studies are currently underway to redesign the vectors used, to make them safer, and to specifically direct integration into inactive regions of the genome.

Animal Models of Immunodeficiency Have Been Used to Study Basic Immune Function There is a good reason PIDs are sometimes called nature’s experiments. Many of the molecular details of how the

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Immunodeficiency Disorders immune system works have come from discovering and studying the broken parts. More important, observations made in humans with PIDs and in animal models of immunodeficiency have taught us what questions to ask. Does the immune system play a role in surveillance for cancer? (The answer is yes, and it’s not always a good one; see Chapter 19.) How can a defect in the IL-2R lead to a total lack of B cells as well as T cells? How can mutation of a single gene related to immunity cause immunologic attack of a whole range of different self proteins? (See above for answers to both.) And the list goes on. Experimental animals with spontaneous or engineered primary immunodeficiencies have provided fertile ground for manipulating and studying basic immune processes. By comparing the phenotypes of animals with and without these blocks in certain components of the immune system, scientists have been able to tease out many details of normal immune processes. The two most widely used animal models of primary immunodeficiency are the athymic, or nude, mouse and the SCID mouse. However, development of other genetically altered animals in which a single target immune gene is knocked out or mutated has also yielded valuable information about the role of these genes in combating infection, and has highlighted some unexpected connections between the immune system and other systems in the body. Nude (Athymic) Mice A genetic trait designated nu (now called Foxn1nu), which is controlled by a recessive gene on chromosome 11, was discovered in 1962 by Norman Roy Grist. Mice homozygous for this trait (nu/nu, or nude mice) are hairless and have a vestigial thymus (Figure 18-7). Heterozygotic nu/wt littermates have hair and a normal thymus. We now know that the mutated gene FOXN1 encodes a transcription factor, mainly expressed in the thymus and skin epithelial cells, that plays a role in cell differentiation and survival, suggesting that the hair loss and immunodeficiency may be caused by the same defect. Like humans born with severe immunodeficiency, these mice do not survive for long without intervention, and 50% or more die within the first 2 weeks after birth from opportunistic infection if housed under standard conditions. When these animals are to be used for experimental purposes, precautions include the use of sterilized food, water, cages, and bedding. The cages are protected from dust by placing them in a laminar flow rack or by using cage-fitted air filters.

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Nude mice have now been studied for many years and have been developed into a tool for biomedical research. For example, because these mice can permanently tolerate both allografts and xenografts (tissue from another species), they have a number of practical experimental uses in the study of transplantation and cancer. Hybridomas (immortalized B cells) or solid tumors from any origin can be grown in the nude mouse, allowing their propagation and the evaluation of new tumor imaging techniques or pharmacological treatments for cancer in these animals. The SCID Mouse In 1983, Melvin and Gayle Bosma and their colleagues described an autosomal recessive mutation in mice that gave rise to a severe deficiency in mature lymphocytes. They designated the trait SCID because of its similarity to human severe combined immunodeficiency. The SCID mouse was shown to have early B- and T-lineage cells but a virtual absence of lymphoid cells in the thymus, spleen, lymph nodes, and gut tissue, the usual locations of functional T and B cells. Precursor cells in the SCID mouse appeared to be unable to differentiate into mature functional B and T lymphocytes. Inbred mouse lines carrying this defect, which have now been propagated and studied in great detail, neither make antibody nor carry out delayed-type hypersensitivity or graft rejection reactions. Lacking much of their adaptive response, they succumb to infection early in life if not kept in extremely pathogen-free environments. Hematopoietic cells other than lymphocytes develop normally in the SCID mouse; red blood cells, monocytes, and granulocytes are present and functional. Like humans, SCID mice may be rendered immunologically competent by transplantation of stem cells from a matched donor. The mutation was discovered in a gene called protein kinase, DNA activated, catalytic polypeptide (PRKDC), which was later shown to participate in the double-stranded DNA break-repair pathway important for antigen-specific receptor gene recombination in B and T cells. This defect is a leaky mutation: a certain number of SCID mice do produce immunoglobulin, and about half of these mice can also reject skin allografts, suggesting components of both humoral and adaptive immunity are present. This leaky phenotype has somewhat limited the widespread use of these mice in research laboratories. However, their ability, like the nude mouse, to accept engrafted tissue from any species has FIGURE 18-7 A nude mouse (Foxn1nu/ Foxn1nu). This defect leads to absence of a thymus or a vestigial thymus and cell-mediated immunodeficiency. [Courtesy of the Jackson Laboratory, Bar Harbor, Maine.]

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led to the development of chimeric mice reconstituted with a humanized immune system (called hu-SCID). These human cells can develop in a normal fashion and, as a result, hu-SCID mice contain T cells, B cells, and immunoglobulin of human origin. In one important application, these mice can be infected with HIV-1, a pathogen that does not infect mouse cells. This provides an animal model in which to test therapeutic or prophylactic strategies against HIV infection. RAG Knockout Mice The potential utility of a mouse model that lacks adaptive immunity, or certain components of adaptive responses, led to the engineering of mice with more targeted mutations. Arguably, the most widely used have been mice with deletions in one of the recombination-activating enzymes, RAG1 and RAG2, responsible for the rearrangement of immunoglobulin or T-cell receptor genes. Since both enzymes are required for recombination, the phenotypes of the two are almost identical, although absence of RAG2 blocks B-cell and T-cell development at an earlier stage and more completely. Unlike nude or SCID mice, RAG2 knockout mice exhibit “tight” defects in both B-cell and T-cell compartments; precursor cells cannot rearrange the genes for antigen-specific receptors or proceed along a normal developmental path, and thus both B and T cells are absent. With a SCID phenotype, RAG knockout mice can be used as an alternative to nude or conventional SCID mice. Their applications include experimental cancer and infectious disease research, as well as more targeted investigations of immune gene function. RAG knockout mice can be the background strain for the production of transgenic mice carrying specific rearranged T-cell or B-cell receptor genes. For example, since these loci have already rearranged, T-cell receptor transgenes will not require the RAG enzymes, and can develop “normally” in the thymus, allowing immunologists to study the events that occur during positive and negative selection while observing the behavior of a million or more T cells of the same clonotype. Although the degree to which this represents truly typical in vivo development of a T cell is questionable, this model has been widely used to ask and to answer many important questions related to MHC restriction and tolerance.

Secondary Immunodeficiencies As described above, a variety of defects in the immune system can give rise to immunodeficiency. In addition to the inherited primary immunodeficiencies, there are also acquired (secondary) immunodeficiencies. Although AIDS resulting from HIV infection is the most well known of these, other factors, such as drug treatment, metabolic disease, or malnutrition, can also impact immune function and lead to secondary deficiencies. As in primary immunodeficiency, symptoms include heightened susceptibility to common infectious agents

and sometimes opportunistic infections. The effect depends on the degree of immune suppression and inherent host susceptibility factors, but can range from no clinical symptoms to almost complete collapse of the immune system, as in HIVinduced AIDS. In most cases, withdrawal of the external condition causing the deficiency can result in restoration of immune function. The first part of this section will cover secondary immunodeficiency due to some non-HIV causes, and the remainder will deal with AIDS. One secondary immunodeficiency that has been recognized for some time but has an unknown cause is acquired hypogammaglobulinemia. This condition is sometimes confused with CVID, a condition that shows genetic predisposition (see above). Symptoms include recurrent infection, and the condition typically manifests in young adults who have very low but detectable levels of total immunoglobulin with normal T-cell numbers and function. However, some cases do involve T-cell defects, which may grow more severe as the disease progresses. The disease is generally treated by immunoglobulin therapy, allowing patients to live a relatively normal life. Unlike the similar primary deficiencies described above, there is no evidence for genetic transmission of this disease. Mothers with acquired hypogammaglobulinemia deliver normal infants. However, at birth these infants will be deficient in circulating immunoglobulin due to the lack of IgG in maternal circulation that can be passively transferred to the infant. Another form of secondary immunodeficiency, agentinduced immunodeficiency, results from exposure to any of a number of environmental agents that induce an immunosuppressed state. These could be immunosuppressive drugs used to combat autoimmune diseases such as rheumatoid arthritis or the corticosteroids commonly used during transplantation procedures to blunt the attack of the immune system on donor organs. The mechanism of action of these immunosuppressive agents varies, as do the defects in immune function, although T cells are a common target. As described in Chapter 16, recent efforts have been made to use more specific means of inducing tolerance to allografts to circumvent the unwanted side effects of general immunosuppression. In addition, cytotoxic drugs or radiation treatments given to treat various forms of cancer, as well as accidental radiation exposure, frequently damage rapidly dividing cells in the body, including those of the immune system, inducing a state of temporary immunodeficiency as an unwanted consequence. Patients undergoing such therapy must be monitored closely and treated with antibiotics or immunoglobulin if infection appears. Extremes of age are also natural factors in immune function. The very young and elderly suffer from impairments to immune function not typically seen during the remainder of the life span. Neonates, and especially premature babies, can be very susceptible to infection, with degree of prematurity linked to the degree of immune dysfunction. Although all the basic immune components are in place in full-term, healthy newborns, the complete range of innate and adaptive

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Immunodeficiency Disorders immune functions take some time to mature. Along with presence of passive maternal antibody for about the first 6 months of life, this is part of the reason for a gradual vaccination program against the common childhood infectious diseases that peak around 1 year of age (see Chapter 17). In later life, individuals again experience an increasing risk of infection, especially by bacteria and viruses, as well as more malignancies. Cell-mediated immunity is generally depressed, and although there are increased numbers of memory B cells and circulating IgG, the diversity of the B-cell repertoire is diminished. The single most common cause of acquired immunodeficiency, even dwarfing the number of individuals worldwide affected by AIDS, is severe malnutrition, affecting both innate and adaptive immunity. Sustained periods with very low protein-calorie diets (hypoproteinemia) are associated with depression in T-cell numbers and function, although deleterious B cell effects may take longer to appear. The reason for this is unclear, although some evidence suggests a bias toward anti-inflammatory immune pathways (e.g., IL-10 and TREG cells) when protein is scarce. In addition to protein, an insufficiency in micronutrients, such as zinc and ascorbic acid, likely contributes to the general immunodeficiency and increased susceptibility to opportunistic infection that occurs with malnutrition. This can be further complicated by stress and infection, both of which may contribute to diarrhea, further reducing nutrient absorption in the gut. Deficiency in vitamin D, required for calcium uptake and bone health, has also been linked to an inhibition in the ability of macrophages to act against intracellular pathogens, such as M. tuberculosis, endemic in many regions of the world where people are at greatest risk of malnutrition. Severe malnourishment thus ranks as one of the most preventable causes of poor immune function in otherwise healthy individuals, and when combined with chronic infection (as with HIV/AIDS, tuberculosis, or cholera) can be all the more deadly.

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cell-mediated immune responses and a significant decrease in the subpopulation of T cells that carry the CD4 marker (T helper cells). When epidemiologists examined the background of the first patients with this new syndrome, they found that the majority were homosexual males. In those early days before we knew the cause or transmission route, and as the number of AIDS cases climbed throughout the world, people thought to be at highest risk for AIDS were homosexual males, promiscuous heterosexual individuals of either sex and their partners, intravenous drug users, people who received blood or blood products prior to 1985, and infants born to HIV-infected mothers. We now know that all these initial patients had intimate contact with an HIVinfected individual or exposure to HIV-tainted blood. Since its discovery in the early 1980s, AIDS has increased to epidemic proportions throughout the world. As of December 2011, approximately 34 million people were living with HIV infection, 1.3 million in the United States. Although reporting of AIDS cases is mandatory in the United States, many states do not require reporting of cases of HIV infection that have not yet progressed to AIDS, making the count of HIV-infected individuals an estimate. The demographic profile of new HIV infections is evolving in the United States, where racial and ethnic minorities, especially men, are being disproportionately affected (Figure 18-8). The toll of HIV/AIDS in the United States is dwarfed by figures for other parts of the world. The global distribution of those afflicted with HIV is shown in Figure 18-9. In subSaharan Africa, the region most affected, an estimated 23.5 million people were living with HIV at the end of 2010, and another 4 million were in South and Southeast Asia. Epidemiologic statistics estimate that more than 24 million people worldwide have died from AIDS since the beginning 120 103.9

In recent years, all other forms of immunodeficiency have been overshadowed by an epidemic of severe immunodeficiency caused by the infectious agent called human immunodeficiency virus (HIV). HIV causes acquired immunodeficiency syndrome (AIDS) and was first recognized as opportunistic infections in a cluster of individuals on both coasts of the United States in June 1981. This group of patients displayed unusual infections, including the opportunistic fungal pathogen Pneumocystis carinii, which causes P. carinii pneumonia (PCP) in people with immunodeficiency. Previously, these infections were limited primarily to individuals taking immunosuppressive drugs. In addition to PCP, some of those early patients had Kaposi’s sarcoma, an extremely rare skin tumor, as well as other, rarely encountered opportunistic infections. More complete evaluation showed that all patients had a common marked deficiency in

White Hispanic/Latino Black

100 Rate per 100,000

HIV/AIDS Has Claimed Millions of Lives Worldwide

607

80 60

20

39.7

39.9

40

15.9

11.8 2.6

0

Male

Female

FIGURE 18-8 Rate of new HIV-1 infections in the United States in 2009, sorted by race/ethnicity and gender. Recent demographic data suggest a disproportionate and widening increase in the number of new HIV-1 infections among blacks and Hispanics as compared to whites, especially among men. [Centers for Disease Control, www.cdc.gov/hiv/resources.]

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GLOBAL TOTALS • People living with HIV/AIDS (as of December 2011) = 34 million • Estimated number of deaths from AIDS in 2011 = 1.7 million • New HIV infections in 2011 = 2.5 million

East Europe/Central Asia 1.4 million Western Central Europe 900,000 North America 1.4 million

North Africa/Mideast 300,000

Caribbean 230,000

East Asia 830,000

South/Southeast Asia 4 million Oceania 53,000

Latin America 1.4 million

Sub-Saharan Africa 23.5 million

Australia/New Zealand 24,700

FIGURE 18-9 The global AIDS epidemic. As of 2011, approximately 34 million people worldwide were living with HIV; most of them were in sub-Saharan Africa and Southeast Asia. Although the rate of new infections is decreasing, 2.5 million people are estimated to have contracted HIV in 2011. [UNAIDS Report on the Global AIDS Epidemic (2012), www.unaids.org/globalreport/global_report.htm.]

of the epidemic, leaving millions of children orphaned. Despite a better understanding of how HIV is transmitted, estimates indicate the occurrence of 2.5 million new HIV infections in 2011, amounting to almost 7,000 new infections each day! Now the good news: rates of new HIV infections decreased by 20% worldwide in 2011 compared to 2001, and access to lifesaving drugs has significantly expanded. These gains are attributable partly to the United Nations Declaration of Commitment on HIV/AIDS, signed in 2001, which has paved the way for stepped-up prevention and education programs around the world as well as expanded drug access programs. Of course this has also led to climbing numbers of individuals living with AIDS, as the period of time from onset of AIDS to opportunistic infection lengthens. Yet, there is still no indication of an end to the epidemic.

The Retrovirus HIV-1 Is the Causative Agent of AIDS Within a few years after recognition of AIDS as an infectious disease, the causative agent, now known as HIV-1, was discovered and characterized in the laboratories of Luc

Montagnier in Paris and Robert Gallo in Bethesda, Maryland (Figure 18-10). About 2 years later, the infectious agent was found to be a retrovirus of the lentivirus genus, which display long incubation periods (lente is Latin for “slow”). Retroviruses carry their genetic information in the form of RNA, and when the virus enters a cell this RNA is reversetranscribed (RNA to DNA, rather than the other way around) by a virally encoded enzyme, reverse transcriptase (RT). This copy of DNA, which is called a provirus, is integrated into the cell genome and is replicated along with the cell DNA. When the provirus is expressed to form new virions (viral particles), the cell lyses. Alternatively, the provirus may remain latent in the cell until some regulatory signal starts the expression process. The discovery of a retrovirus as the cause of HIV was novel, since at the time only one other human retrovirus, human T-cell lymphotropic virus I (HTLV-I), had been identified. Although comparisons of their genomic sequences revealed that HIV-1 is not a close relative of HTLV-I, similarities in overall characteristics led to use of the name HTLV-III for the AIDS virus in early reports. About 5 years after the discovery of HIV-1, a close retroviral cousin, HIV-2, was isolated from some AIDS sufferers in

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OVERVIEW FIGURE

Structure of HIV gp120

(a)

(b)

gp41

p32 integrase p17 (matrix) p24 (capsid) ssRNA

p10 protease

MHC proteins

Reverse transcriptase (p64)

(a) Cross-sectional schematic diagram of HIV. Each virion carries 72 glycoprotein projections composed of gp120 and gp41: gp41 is a transmembrane molecule that crosses the lipid bilayer of the viral envelope, gp120 is associated with gp41, and together they interact with the target receptor (CD4) and coreceptor (CXCR4 or CCR5) on host cells. The viral envelope derives from the host cell and contains some host-cell membrane proteins, including class I and class II MHC molecules. Within the envelope is the viral matrix (p17) and the core, or nucleocapsid (p24). The HIV genome consists of two

Africa. Unlike HIV-1, its prevalence is mostly limited to areas of Western Africa, and disease progresses much more slowly, if at all. In Guinea-Bissau, where HIV-2 is most common, up to 8% of the population may be persistently infected and yet most of these individuals experience a nearly normal lifespan. There is some hope that scientists can gain a better understanding of HIV-1 from the study of the more benign cohabitation of HIV-2 and its human host. Viruses related to HIV-1 have been found in nonhuman primates, and some of these are believed to be the original source of HIV-1 and -2 in humans. These viruses, variants of simian immunodeficiency virus (SIV), can cause immunodeficiency disease in certain infected monkeys. Typically, SIV strains cause no disease in their natural hosts but produce immunodeficiency similar to AIDS when injected into another species. For example, the virus from African green monkeys (SIVagm) is present in a high percentage of normal, healthy African green monkeys in the wild. However, when SIVagm is injected into macaques, it causes a severe and often lethal immunodeficiency. HIV-1 is believed to have evolved from a strain of SIV that jumped the species barrier from African chimpanzees to humans, although HIV-2 is thought to have arisen from a separate but similar transfer from SIV-

copies of single-stranded RNA (ssRNA), which are associated with two molecules of reverse transcriptase (p64) plus p10, a protease, and p32, an integrase. (b) Electron micrograph of HIV virions magnified 200,000 times. The glycoprotein projections are faintly visible as “knobs” extending from the periphery of each virion. [Part a adapted from B. M. Peterlin and P. A. Luciw, 1988, AIDS 2:S29; part b from a micrograph by Hans Geldenblom of the Robert Koch Institute (Berlin), in R. C. Gallo and L. Montagnier, 1988, Scientific American 259(6):41.]

infected sooty mangabeys. Both of these events are believed to have occurred some time during the twentieth century, making this a relatively new pathogen for the human population. A number of other animal retroviruses more or less similar to HIV-1 have been reported. These include the feline immunodeficiency virus and bovine immunodeficiency virus (FIV and BIV, respectively) and the mouse leukemia virus. Study of these animal viruses has yielded information concerning the general nature of retrovirus action and pathways to the induction of immunodeficiency. Because HIV does not replicate in typical laboratory animals, model systems to study it are few. Only the chimpanzee supports infection with HIV-1 at a level sufficient to be useful in vaccine trials, but infected chimpanzees rarely develop AIDS, which limits the value of this model in the study of viral pathogenesis. In addition, the number of chimpanzees available for such studies is low, and both the expense and the ethical issues involved preclude widespread use of this infection model. The SCID mouse (see above) reconstituted with human lymphoid tissue for infection with HIV-1 has been useful for certain studies of HIV-1 infection, especially in the development of drugs to combat viral replication.

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CLINICAL FOCUS

Prevention of Infant HIV Infection by Anti-Retroviral Treatment It is estimated that almost 700,000 infants became infected with HIV through mother-to-child transmission in 2005 (just prior to the widespread implementation of standard prophylactic treatment for HIV-infected mothers). The majority of these infections resulted from transmission of virus from HIV-infected mothers during childbirth or by transfer of virus from milk during breast-feeding. The incidence of maternally acquired infection can be reduced by treatment of the infected mother with a course of zidovudine or azidothymidine (AZT, a nucleoside analog reverse-transcriptase inhibitor [NRTI]) for several months prior to delivery and treatment of her infant for 6 weeks after birth. This treatment regimen is standard practice in the United States. However, the majority of worldwide HIV infection in infants occurs in sub-Saharan Africa and other less-developed areas, where the cost and timing of the zidovudine regimen render it an impractical solution to the problem of maternalinfant HIV transmission. A 1999 clinical trial of the anti-retroviral drug nevirapine (Viramune, an NRTI) brought hope for a practical way to combat HIV transmission at birth in less-than-ideal conditions of clinical care. The trial took place at Mulago Hospital in Kampala,

FIGURE 1 Mural showing mother and child on an outside wall of Mulago Hospital Complex in Kampala, Uganda, site of the clinical trial demonstrating that maternal-infant HIV-1 transmission at birth was greatly reduced by nevirapine. [Courtesy of Thomas Quinn, Johns Hopkins University.]

Uganda, and enrolled 645 mothers who tested positive for HIV infection (Figure 1). About half of the mothers were given a single dose of nevirapine at the onset of labor, and their infants were given a single dose one day after birth. The dose and timing were dictated by the customary rapid

HIV-1 Is Spread by Intimate Contact with Infected Body Fluids Although some of the details involving the mechanism by which HIV-1 infects an individual are still incomplete, the big picture of transmission routes has been resolved. Epidemiological data indicate that the most common means of transmission include vaginal and anal intercourse, receipt of infected blood or blood products, and passage from HIVinfected mothers to their infants. Before routine tests for HIV-1 were in place, patients who received blood transfusions and hemophiliacs who received blood products were at risk for HIV-1 infection. Exposure to infected blood accounts for the high incidence of AIDS among intravenous drug users, who often share hypodermic needles. Infants born to

discharge at the hospital. The control arm of the study involved a more extensive course of zidovudine, but local conditions did not allow the full course typically administered to infected mothers in the United States. The subjects were followed for at least 18 months. The infected mothers breast-fed

mothers who are infected with HIV-1 are at high risk of infection; without prophylaxis, over 25% of these newborns may become infected with the virus. However, elective Cesarean section delivery and antiretroviral treatment programs for HIV⫹ pregnant women and their newborns are making a real dent in these numbers (see Clinical Focus Box 18-1). In the worldwide epidemic, it is estimated that approximately 75% of the cases of HIV transmission are attributable to sexual contact. The presence of other sexually transmitted diseases (STDs) increases the likelihood of transmission, and in situations where STDs flourish, such as unregulated prostitution, these infections probably represent a powerful cofactor for sexual transmission of HIV-1. The open lesions and activated inflammatory cells (some of which may express receptors for HIV) associated with STDs favor the

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BOX 18-1 99% of the infants in this study, so the test measures a considerable interval during which the infants were exposed to risk of infection at birth and while nursing. The infants were tested for HIV-1 infection at several times after birth up to 18 months of age using an RNA PCR test and at later times for HIV-1 antibody (after maternal antibody would no longer interfere with the test). The overall rate of infection for infants born to untreated mothers is estimated to be about 37%. In the United States, when the full course of zidovudine is used, the rate drops to 20%. The highly encouraging results of the Uganda study revealed infection in only 13.5% of the babies in the nevirapine group when tested at 16 weeks of age. Of those given a short course of zidovudine, 22.1% were infected at this age compared to 40.2% in a small placebo group. In the 18-month follow-up of the test group, 15.7% of those given nevirapine were infected, whereas 25.8% given zidovudine were positive for HIV-1. These promising results led to World Health Organization (WHO) recommendations that this nevirapine regimen be used in all instances where mother-tochild transmission of HIV was a danger. But what about the risks associated with breast-feeding? In 2007, it was estimated that up to 200,000 infants become infected with HIV annually through breast

milk. In resource-limited countries, poor early nutrition and susceptibility to disease are key factors in infant death rates; breast-feeding significantly reduces these risks. Therefore, a follow-up investigation called the Breastfeeding, Antiretrovirals, and Nutrition (BAN) study was conducted in Lilongwe, Malawi, in 2006 to 2008. This investigation employed a dose of nevirapine for the mother and child at birth, followed by 7 days of treatment of the pair with two NRTIs. The 2369 mother-infant pairs were then randomized to one of three post-delivery prophylaxis groups: maternal treatment, infant treatment, or placebo control. Mothers in the maternal regimen received a triple-drug cocktail for 28 weeks post delivery, including two NRTIs and either an NNRTI or a protease inhibitor. The babies in the infant treatment group received nevirapine daily for the same period of time, while initial participants in the control population received no additional treatment (in the latter part of the study, no new pairs were enrolled in this group). All mothers were encouraged to breast-feed for 6 months and wean before 7 months. Comparing the rate of HIV transmission that occurred during breast-feeding (from weeks 2–28) in the control population to the treatment groups, results demonstrated a 53% protective effect for the maternal regimen and a 74% protective effect in the infant treatment group.

transfer and attachment of the virus during intercourse. Estimates of transmission rates per exposure vary widely and depend on many factors, such as the presence of STDs and number of virions. However, male-to-female transfer between discordant couples during vaginal intercourse is approximately twice as risky to the female as to the male, based solely on anatomical considerations, and receptive partners in anal intercourse are even more at risk. Data from studies in India and in Africa indicate that men who are circumcised are at significantly lower risk of acquiring HIV-1 via sexual contact, possibly because foreskin provides a source of cells that can become infected or harbor the virus. However, this did not work in reverse: circumcised males were equally likely to transmit HIV-1 to their sexual partners. No similarly protective effect of circumcision was

Collectively, the conclusions from these efficacy and safety trials have led to new worldwide recommendations for protecting newborns from maternally transmitted HIV. In 2009, the WHO revised its recommendations to encourage breast-feeding for at least 12 months, based on studies in Africa showing increased infant mortality rates in babies weaned as early as 6 months. The hope is that with an extended nursing period that includes nevirapine prophylaxis, both the danger of death from disease or malnutrition and the risk of HIV transmission can be significantly reduced, even in severely resource-limited settings. As mentioned above, these studies were designed to conform to the reality of maternal health care in less developed nations. Nevirapine has significant advantages in this regard, including stability of the drug at room temperature, reasonable cost, and relatively few side effects. The dose of nevirapine administered to the mother and infant at birth costs about 200 times less than the zidovudine regimen used in the United States to block HIV transmission at birth. In fact, the treatment is sufficiently inexpensive as to suggest that it may be cost-effective to treat all mothers and infants at the time of delivery in areas where the rates of infection are high and the nevirapine treatment costs are less than the tests used to determine HIV infection.

seen for other STDs, including herpes simplex type 2, syphilis, or gonorrhea. Identifying the initial events that take place during HIV transmission is logistically and ethically challenging, as we know that immediate antiviral treatment significantly diminishes the odds of infection. Nonetheless, hypotheses concerning the most likely sequence of events have been pieced together based on observations in humans and animals, including in vitro studies using explanted human tissue and in vivo studies in macaques, a nonhuman primate. Based on these observations, we believe that free virus and virusinfected cells, which can both be found in vaginal secretions and semen, contribute to infection. Direct infection of the many activated but resting memory CD4⫹ T cells present within the vaginal mucosa is likely the primary initial source

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FIGURE 18-11 Interaction between dendritic cell and T cell, indicating passage of HIV-1 (green dots) between the cells. Note that particles cluster at the interface between the large dendritic cell and the smaller T cell. [Courtesy of Thomas J. Hope, North-

Although the female genital tract is a relatively robust barrier to most infectious agents (with the adaptive immune response taking over from there), emerging evidence based on viral sequence analysis suggests that a single HIV-1 virion may be responsible for all or most of the systemic infection in many male-to-female transfers. Because transmission of HIV-1 infection requires direct contact with infected blood, milk, semen, or vaginal fluid, preventive measures can be taken to block these events. Scientific researchers and medical professionals who take reasonable precautions, which include avoiding exposure of broken skin or mucosal membranes with fluids from their patients, significantly decrease their chances of becoming infected. When exposure does occur, rapid administration of anti-HIV treatment can often prevent systemic infection. The use of condoms when having sex with individuals of unknown infection status also significantly reduces chances of infection. One factor contributing to the spread of HIV is the long period after infection during which no clinical signs may appear but during which the infected individual may infect others. Thus, universal use of precautionary measures is important whenever infection status is uncertain.

western University.]

In Vitro Studies Have Revealed the Structure and Life Cycle of HIV-1

of infection in the female genital tract (the most studied location). In macaques, a foothold or attachment can be established in as little as 30 to 60 minutes, and high numbers of infected CD4⫹ cells are seen within 1 day of exposure. Replication of HIV-1 in the vaginal mucosa was also shown to help activate local CD4⫹ T cells, providing yet more targets for the virus and creating a vicious cycle. Likewise, the inflammation associated with STDs is thought to enhance the number of TH cells and their susceptibility to infection. Viral transcytosis through epithelial cells, or endocytic transport from the luminal to the basal surface of the cell, is another possible route. Langerhans cells (LCs), a type of intraepithelial DC with long processes that reach close to the vaginal lumen, have also been shown to take up virus, although they may not become infected. These and other DCs may transport intact infectious virus, possibly for days, within endocytic compartments, and can transfer this virus to CD4⫹ T cells (Figure 18-11). The role of macrophages in these early events, as transporters or targets of infection, is somewhat controversial but not suspected to be a major contributor. Finally, free virus can squeeze between epithelial cells or gain access through microabrasions, eventually encountering susceptible cells or afferent lymphatic vessels. Whether free or cell-associated, the virus then migrates through the submucosa to the draining lymph node, where the adaptive immune response can be initiated. However, once there, further viral spread is facilitated, some through cell-to-cell hand-off via infectious synapses, as many more cells with the proper surface receptors are encountered.

The HIV-1 genome and encoded proteins have been fairly well characterized, and the functions of most of these proteins are known (Figure 18-12). HIV-1 carries three structural genes (gag, pol, and env) and six regulatory or accessory genes (tat, rev, nef, vif, vpr, and vpu). The structural genes and the proteins they encode were the first to be sequenced and meticulously characterized. The gag gene encodes several proteins, including the capsid and matrix, which enclose the viral genome and associated proteins. The pol gene codes for the three main enzymes (in addition to those supplied by the host cell) that are required for the viral life cycle: protease, integrase, and reverse transcriptase. In fact, the protease enzyme is required to process precursor proteins of many of the other viral peptides. As we will see shortly, these uniquely viral enzymes are some of the main targets for therapeutic intervention. The final structural gene, env, is the source of the surface proteins gp120 and gp41, involved in attachment of the virus to the CD4⫹ viral receptor and its coreceptor, either CXCR4 or CCR5. The regulatory genes expressed by HIV-1, which took longer to characterize, encode functions such as modulating CD4 and class I MHC expression, inactivating host proteins that interfere with viral transcription, and facilitating intracellular viral transport. Much has been learned about the life cycle of HIV-1 from in vitro studies, where cultured human T cells have been used to map out virus attachment and post-attachment intracellular events (Figure 18-13a). HIV-1 infects cells that carry the CD4 antigen on their surface; in addition to TH cells, these can include monocytes and macrophages, as well as other cells expressing CD4. This preference for CD4⫹ cells

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(a)

Kbp 0

1

2

3

4

5

6 vif

gag

7

8

9

vpu

pol

env

5'LTR

3'LTR vpr

rev tat

(b)

613

Gene

Protein product

gag

53-kDa precursor p17 p24 p9 p7

env

160-kDa precursor gp41 gp120

pol

Precursor p64 p51 p10 p32

nef

Function of encoded proteins Nucleocapsid proteins Forms outer core-protein layer (matrix) Forms inner core-protein layer (capsid) Is component of nucleoid core Binds directly to genomic RNA Envelope glycoproteins Is transmembrane protein associated with gp120 and required for fusion Protrudes from envelope and binds CD4 Enzymes Has reverse transcriptase and RNase activity Has reverse transcriptase activity Is protease that cleaves gag precursor Is integrase Regulatory proteins

tat

p14

Strongly activates transcription of proviral DNA

rev

p19

Allows export of unspliced and singly spliced mRNAs from nucleus Auxiliary proteins

nef

p27

Down-regulates host-cell class I MHC and CD4

vpu

p16

Is required for efficient viral assembly and budding. Promotes extracellular release of viral particles, degrades CD4 in ER

vif

p23

Promotes maturation and infectivity of viral particle

vpr

p15

Promotes nuclear localization of preintegration complex, inhibits cell division

FIGURE 18-12 Genetic organization of HIV-1 (a) and functions of encoded proteins (b). The three major genes—gag, pol, and env—encode polypeptide precursors that are cleaved to yield the nucleocapsid core proteins, enzymes required for replication, and to envelop core proteins. Of the remaining six genes, three (tat, rev, and nef) encode regulatory proteins that play a major role in

controlling expression, two (vif and vpu) encode proteins required for virion maturation, and one (vpr) encodes a weak transcriptional activator. The 5⬘ long terminal repeat (LTR) contains sequences to which various regulatory proteins bind. The organization of the HIV-2 and SIV genomes is very similar except that the vpu gene is replaced by vpx in both of them.

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OVERVIEW FIGURE

HIV Infection of Target Cells and Activation of Provirus (a) Infection of target cell 1

(b) Activation of provirus CXCR4 or CCR5 2

CD4 3

ssRNA 4

2

Reverse transcriptase

Provirus 5

LTR 7

2

3a 3b

RNA-DNA hybrid LTR

1

mRNAs

Precursors

6

ssRNA Proteins 4 4

HIV dsDNA 5a 5b

1 HIV gp120 binds to CD4 on target cell.

1 Transcription factors stimulate transcription of proviral DNA into genomic ssRNA and, after processing, several mRNAs.

2 HIV gp41 binds to a chemokine receptor (CXCR4 or CCR5) and fuses with the target cell membrane.

2 Viral RNA is exported to cytoplasm.

3 Nucleocapsid containing viral genome and enzymes enters cells.

3a Host-cell ribosomes catalyze synthesis of viral precursor proteins.

4 Viral genome and enzymes are released following removal of core proteins.

3b Viral protease cleaves precursors into viral proteins.

5 Viral reverse transcriptase catalyzes reverse transcription of ssRNA, forming RNA-DNA hybrids. 6 Original RNA template is partially degraded by ribonuclease H, followed by synthesis of second DNA strand to yield HIV dsDNA. 7 The viral dsDNA is then translocated to the nucleus and integrated into the host chromosomal DNA by the viral integrase enzyme.

4 HIV ssRNA and proteins assemble beneath the host-cell membrane, into which gp41 and gp120 are inserted. 5a The membrane buds out, forming the viral envelope. 5b Released viral particles complete maturation; incorporated precursor proteins are cleaved by viral protease present in viral particles.

(a) Following entry of HIV into cells and formation of dsDNA, integration of the viral DNA into the host-cell genome creates the provirus. (b) The provirus remains latent until events in the infected cell trigger its activation, leading to formation and release of viral particles.

is due to a high-affinity interaction between gp120 and the CD4 molecule on the host cell. However, this interaction alone is not sufficient for viral entry and productive infection. Expression of another cell-surface molecule, called a coreceptor, is required for HIV-1 access to the cell. Each of the two known coreceptors for HIV-1, CCR5 and CXCR4, belongs to a separate class of molecule known as a chemokine receptor. The role of chemokine receptors in the body is to bind their natural ligands, chemokines, which are chemotactic messengers driving the movement of leukocytes (see Chapter 4). The infection of a T cell is assisted by the CXCR4 coreceptor, while the analogous CCR5 seems to be the pre-

ferred coreceptor for viral entry into monocytes and macrophages, and is now a target for antiviral intervention. After HIV-1 has entered a cell, the RNA genome of the virus is reverse-transcribed and a cDNA copy integrates into the host genome. The integrated provirus is transcribed, and the various viral RNA messages are spliced and translated into proteins that, along with a complete new copy of the RNA genome, are used to form new viral particles (Figure 18-13b). These initial viral proteins are cleaved by the virally encoded protease into the forms that make up the nuclear capsid in a mature infectious viral particle. Virus expression leads to newly formed virions that bud from the surface of the infected cell, often

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CCR5

SDF-1

615 RANTES, MIP-1α, MIP-1β

CD4

CD4

T cell

Monocyte

FIGURE 18-15 CXCR4 and CCR5 serve as coreceptors for HIV infection. Although CD4 binds to the envelope glycoprotein of HIV-1, a second coreceptor is necessary for entry and infection. T-cell–tropic strains of HIV-1 use the coreceptor CXCR4, whereas the macrophage-tropic strains use CCR5. Both are receptors for chemokines, and their normal ligands (SDF-1, RANTES, and MIP) can block HIV infection of the cell.

FIGURE 18-14 Once the HIV provirus has been activated, buds representing newly formed viral particles can be observed on the surface of an infected T cell. The extensive cell damage resulting from budding and release of virions leads to the death of infected cells. [Courtesy of R. C. Gallo, 1988, HIV—The cause of AIDS: An overview on its biology, mechanisms of disease induction, and our attempts to control it. Journal of Acquired Immune Deficiency Syndromes 1:521.]

causing cell lysis (Figure 18-14). However, HIV-1 can also become latent, or remain unexpressed, for long periods of time in an infected cell. This period of dormancy makes the task of finding these latently infected cells especially difficult for the immune response to HIV-1; latent infection is believed to aid in the establishment of HIV reservoirs, or safe havens, where both drug therapy and antiviral immunity can have little impact. Studies of the viral envelope protein gp120 identified a region called the V3 loop, which plays a role in the choice of receptors used by the virus. It is clear from these studies that a single amino acid difference in this region of gp120 may be sufficient to determine which receptor is used. Moreover, a mutation in the CCR5 gene that occurs with varying frequency, depending on ethnic background, imparts nearly total resistance to infection with the strains of HIV-1 that are most commonly encountered in sexual exposure. Individuals who are homozygous for this mutation express no CCR5 on the surface of their cells, making them impervious to viral strains that require this coreceptor but, remarkably, otherwise apparently unperturbed by loss of this chemokine receptor. This has led to some recent stories, and new hopes, of HIV elimination. For instance, one HIV-infected patient who received a set of bone marrow transplants (to treat leukemia) from a donor who lacked the CCR5 coreceptor protein may, or may not, be virus free. Confirmation and long-term follow-up of this finding, as well as development

of other techniques to exploit this coreceptor block as a method of virus elimination, are currently underway. The discovery that CXCR4 and CCR5 serve as coreceptors for HIV-1 on T cells and macrophages, respectively, explained why some strains of HIV-1 preferentially infect T cells (T-tropic strains), whereas others prefer macrophages (M-tropic strains). T-tropic strains use the CXCR4 coreceptor, whereas M-tropic strains use CCR5 (Figure 18-15). This also helped to explain some observed roles of chemokines in virus replication. It was known from in vitro studies that certain chemokines, such as RANTES, had a negative effect on virus replication. CCR5 and CXCR4 cannot bind simultaneously to HIV-1 and to their natural chemokine ligands. Competition for the receptor between the virus and the natural chemokine ligand can thus block viral entry into the host cell. Early enthusiasm for the use of these chemokines as antiviral agents was dampened when significant RANTES expression was observed in some HIV-1 infected individuals who progress to disease, with no obvious antiviral effect. Despite this, an antagonist of CCR5 has recently been approved for use as a therapeutic inhibitor of HIV spread (see below).

Infection with HIV-1 Leads to Gradual Impairment of Immune Function Isolation of HIV-1 and its growth in culture allowed purification of viral proteins and the development of tests for infection with the virus. The most commonly used test is an ELISA (see Chapter 20) to detect the presence of antibodies directed against proteins of HIV-1, especially the gag p24 protein (see Figures 18-10a and 18-12b), one of the most immunogenic of the HIV proteins. These antibodies generally appear in the serum of infected individuals within 6 to 12 weeks after exposure, but can take up to 6 months to appear. When antibodies appear in the blood, the individual is said to have seroconverted or to be seropositive for HIV-1. Positive p24 ELISA results are then confirmed using the more specific Western blot technique, which detects the presence of antibodies against several HIV-1 proteins.

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Seroconversion Acute phase

Asymptomatic phase +

CD4+ T-cell count in blood (cells/mm3)

Death

Anti-HIV antibody + + +

AIDS +

+ 106

1,000 800

105 CD4 T cells

600 104 400 HIV viral load 200 0

0

6 12 Weeks

1 2 3 4 5 6 7 8 9 10 11 Years

103

102

Viral load in blood (HIV RNA copies/ml plasma)

616

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FIGURE 18-16 The typical course of an HIV infection. Soon after infection, viral RNA is detectable in the serum. However, HIV infection is most commonly detected by the presence of anti-HIV antibodies after seroconversion, which normally occurs within a few months after infection. Clinical symptoms indicative of AIDS generally do not appear for up to 8 years after infection, but this interval is variable, especially when antiretroviral therapy is used. The onset of clinical AIDS is usually signaled by a decrease in T-cell numbers and a sharp increase in viral load. [Adapted from A. Fauci et al., 1996, Immunopathogenic mechanisms of HIV infection. Annals of Internal Medicine 124:654.]

Although the precise course of HIV-1 infection and disease onset varies considerably in different patients, a general scheme for the progression to AIDS can be outlined (Figure 18-16). First, there is the acute, or primary, stage of infection. This is the period immediately after infection, where there are often no detectable anti-HIV-1 antibodies. Estimates vary, but some reports find that more than half of the individuals undergoing primary infection experience flu-like symptoms, including fever, lymphadenopathy (swollen lymph nodes), and malaise approximately 2 to 4 weeks after exposure. During this acute phase, HIV-1 infection is spreading and the viral load (number of virions) in the blood as well as in other body fluids can be quite high, elevating the risk of transfer to others. For unknown reasons, seroconversion, or the appearance of antibody against HIV antigens, can take months to develop. This stage is followed by an asymptomatic period during which there is a gradual decline in CD4⫹ T cells but usually no outward symptoms of disease. This is driven by an immune response involving both antibody and cytotoxic CD8⫹ T lymphocytes that keeps viral replication in check and drives down the viral load. The length of this asymptomatic window varies greatly and is likely due to a combination of host and viral factors. Although the infected individual

normally has no clinical signs of disease at this stage, viral replication continues, CD4⫹ cell levels gradually fall, and viral load in the circulation can be measured by PCR assays for viral RNA. These measurements of viral load have assumed a major role in the determination of the patient’s status and prognosis. Even when the level of virus in the circulation is stable, large amounts of virus are produced in infected CD4⫹ T cells; as many as 109 virions are released every day and continually infect and destroy additional host T cells. Despite this high rate of replication, the virus is kept in check by the immune system throughout the asymptomatic phase of infection, and the level of virus in circulation from about 6 months after infection (the set point) can be a predictor of the course of disease. Low levels of virus in this period correlate with a longer asymptomatic period and opportunistic pathogen-free window. Without treatment, most HIV-1 infected patients eventually progress to AIDS, where opportunistic infection is the hallmark. Diagnosis of AIDS occurs only once four criteria have been met: evidence of infection with HIV-1 (presence of antibodies or viral RNA in blood), greatly diminished numbers of CD4⫹ T cells (⬍ 200 cells/␮l of blood), impaired or absent delayed-type hypersensitivity reactions, and the occurrence of opportunistic infections (Table 18-3). The first overt indication of AIDS is often opportunistic infection with the fungus Candida albicans, which causes the appearance of sores in the mouth (thrush) and, in women, a vulvovaginal yeast infection that does not respond to treatment. A persistent hacking cough caused by P. carinii infection of the lungs is another early indicator. A rise in the level of circulating HIV-1 in the plasma (viremia) and a concomitant drop in the number of CD4⫹ T cells generally precedes this first appearance of symptoms. Late-stage AIDS patients generally succumb to tuberculosis, pneumonia, severe wasting diarrhea, or various malignancies. Without treatment, the time between acquisition of the virus and death from the immunodeficiency averages 9 to 11 years.

Active Research Investigates the Mechanism of Progression to AIDS Of intense interest to immunologists are the events that take place between the initial encounter with HIV-1 and the takeover and collapse of the host immune system. Understanding how the immune system holds HIV-1 in check during the asymptomatic phase could aid in the design of effective therapeutic and preventive strategies. For this reason, the handful of HIV-1 infected individuals who remain asymptomatic for very long periods without treatment (long-term nonprogressors), estimated to be ⬍ 2% of HIV-1⫹ individuals in the United States, are the subject of intense study. Another group of heavily studied individuals consists of those in high-risk groups, such as many prostitutes. From the study of virus-immune system interactions in the handful of individuals in these high-risk groups who remain

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TABLE 18-3 Stage*

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617

Stage definition for HIV infection among adults and adolescents CD4 T-cell count

CD4 T-cell percentage

Clinical evidence

1

⭓ 500/␮L

or

⬎ 29%

and

No AIDS-defining condition

2

200–499/␮L

or

14–28%

and

No AIDS-defining condition

⬍ 200/␮L

or

⬍ 14%

or

Presence of AIDS-defining condition

3 (AIDS)

AIDS-DEFINING CONDITIONS:

• Candidiasis of bronchi, trachea, or lungs • Candidiasis of esophagus • Cervical cancer, invasive • Coccidioidomycosis, disseminated or extrapulmonary • Cryptococcosis, extrapulmonary • Cryptosporidiosis, chronic intestinal (⬎ 1 month duration) • Cytomegalovirus disease (other than liver, spleen, or nodes) • Cytomegalovirus retinitis (with loss of vision) • Encephalopathy, HIV related • Herpes simplex: chronic ulcers (⬎ 1 month duration) or bronchitis, pneumonitis, or esophagitis • Histoplasmosis, disseminated or extrapulmonary • Isosporiasis, chronic intestinal (⬎ 1 month duration) • Kaposi’s sarcoma • Lymphoid interstitial pneumonia or pulmonary lymphoid hyperplasia complex • Lymphoma, Burkitt (or equivalent term) • Lymphoma, immunoblastic (or equivalent term) • Lymphoma, primary, of brain • Mycobacterium avium complex or Mycobacterium kansasii, disseminated or extrapulmonary • Mycobacterium tuberculosis of any site, pulmonary, disseminated, or extrapulmonary • Mycobacterium, other species or unidentified species, disseminated or extrapulmonary • Pneumocystis jirovecii pneumonia • Pneumonia, recurrent • Progressive multifocal leukoencephalopathy • Salmonella septicemia, recurrent • Toxoplasmosis of brain • Wasting syndrome attributed to HIV * All require laboratory confirmation of HIV infection. Source: AIDS-Defining Conditions, 2008, Centers for Disease Control, www.cdc.gov/mmwr/preview/mmwrhtml/rr5710a2.htm.

seronegative despite known and repeated exposure, we hope to gain clues to mechanisms of control or possibly even protection. In addition to the discovery of CCR5 deletion (see above), several interesting findings have emerged from studying high-risk populations, including the presence of strong CD8⫹ T-cell responses against HIV in many of these individuals, as well as HLA-associated influences on disease susceptibility.

Although the viral load in plasma remains fairly stable throughout the period of chronic HIV infection, examination of the lymph nodes and gastrointestinal (GI) tract tissue reveals a different picture. Fragments of nodes obtained by biopsy from infected subjects show high levels of infected cells at all stages of infection; in many cases, the structure of the lymph node is completely destroyed by virus long before plasma viral load increases above the

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(c)

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% T cells expressing CD4

(e) 100

HIV⫺

80

HIV⫹

60 40 20 0

GI tract

FIGURE 18-17 Endoscopic and histologic evidence for depletion of CD4 T cells in the GI tract of AIDS patients. Panels (a) and (c) show the intestinal tract of a normal individual and a stained section from a biopsy of the same area (terminal ileum) with obvious large lymphoid aggregates (arrows, a) and CD4⫹ T cells stained with antibody (brown color, c). Similar analysis of samples from an HIV⫹ patient in the acute stage of infection in panels (b) and

steady-state level. In fact, data from 2004 show that the gut may be the main site of HIV-1 replication and CD4⫹ T-cell depletion. Work from the laboratories of Ashley Haase and Daniel Douek indicates a dramatic depletion of lymphoid tissue and specifically CD4⫹ T cells from the GI tract during HIV infection, starting as early as the acute stages of infection (Figure 18-17). Subsequent investigations of the association between the GI tract and HIV have suggested that TH17 cells, which express both the CCR5 and CXCR4 coreceptors, are the primary targets of infection and destruction. These TH17 cells are thought to play an important role in homeostatic regulation of the innate and adaptive responses to microbial flora in the gut. Destruction of these cells and disruption of the integrity of the mucosal barrier in the GI tract may allow for the translocation of microbial products across the epithelial lining, explaining some of the rampant immune stimulation that is characteristic of HIV infection. In a deadly feedback loop, this immune stimulation generates yet more activated CD4⫹ cells, the favored targets for HIV infection and replication. The severe decrease in CD4⫹ T cells is a clinical hallmark of AIDS, and several explanations have been advanced for it. In early studies, direct viral infection and destruction of CD4⫹ T cells was discounted as the primary cause, because the large numbers of circulating HIV-infected T cells predicted by the model were not found. More recent studies indicate that it is difficult to find the infected cells

Peripheral blood

Lymph nodes

(d) indicate absence of normal lymphoid tissue and sparse staining for CD4⫹ T cells. (e) Comparison of CD4⫹ T-cell numbers in samples from GI tract, peripheral blood (PB), and lymph nodes of AIDS-positive and -negative individuals. [From J. M. Brenchley et al. 2004, CD4⫹ T cell depletion during all stages of HIV disease occurs predominantly in the gastrointestinal tract. Journal of Experimental Medicine 200:749.]

because they are so rapidly killed by HIV (the half-life of an actively infected CD4⫹ T cell is less than 1.5 days) and because most of the infected cells may localize to the GI tract. Smaller numbers of CD4⫹ T cells become infected but do not actively replicate virus. These latently infected cells persist for long periods, and the integrated proviral DNA replicates in cell division along with cell DNA. Studies in which viral load is decreased by antiretroviral therapy show a concurrent increase in CD4⫹ T-cell numbers in the peripheral blood and eventually in the gut. Apoptosis due to nonspecific immune activation and bystander effects of free virus or infected cells acting on uninfected cells have also been postulated to play a role in HIV-induced lymphopenia. Although depletion of CD4⫹ T cells is the primary focus of follow-up testing in HIV-infected individuals, other immunologic consequences involving both adaptive and innate immune functions can be observed during the progression to AIDS. These include a decrease or absence of delayed-type hypersensitivity to antigens for which the individual normally reacts, decreased serum immunoglobulins (especially IgG and IgA), and impaired cellular responses to antigens. Generally, the HIV-infected individual loses the ability to mount T-cell responses in a predictable sequence: responses to specific recall antigens (e.g., influenza virus) are lost first, then response to alloantigens declines, and finally mitogenic responses to stimuli disappear. Innate responses are also impacted, including NK and dendritic cell functions.

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Immunologic abnormalities associated with HIV infection

Stage of infection

Typical abnormalities observed LYMPH NODE STRUCTURE

Early

Infection and destruction of dendritic cells; some structural disruption, especially to gastrointestinal tractassociated lymphoid tissues

Late

Extensive damage and tissue necrosis; loss of follicular dendritic cells and germinal centers; inability to trap antigens or support activation of T and B cells T HELPER (T H ) CELLS

Early

Depletion of CD4⫹ T cells, especially in the gut (TH17 main targets); loss of in vitro proliferative response to specific antigen

Late

Further decrease in TH-cell numbers and corresponding helper activities; no response to T-cell mitogens or alloantigens ANTIBODY PRODUCTION

Early

Enhanced nonspecific IgG and IgA production but reduced IgM synthesis

Late

No proliferation of B cells specific for HIV-1: no detectable anti-HIV antibodies in some patients; increased numbers of B cells with low CD21 expression and enhanced Ig secretion CY TOKINE PRODUCTION

Early

Increased levels of some cytokines

Late

Shift in cytokine production from TH1 subset to TH2 subset DELAYED-TYPE HYPERSENSITIVITY

Early

Highly significant reduction in proliferative capacity of TH1 cells and reduction in skin-test reactivity

Late

Elimination of DTH response; complete absence of skin-test reactivity T CY TOTOXIC (T C ) CELLS

Early

Normal reactivity

Late

Reduction but not elimination of CTL activity due to impaired ability to generate CTLs from TC cells

Table 18-4 lists some immune abnormalities common to HIV/AIDS. Individuals infected with HIV-1 often display dysfunction of the central and peripheral nervous systems, especially in the later stages of infection. Viral sequences have been detected by HIV-1 probes in the brains of children and adults with AIDS, suggesting that viral replication occurs there. Quantitative comparison of specimens from brain, lymph node, spleen, and lung of AIDS patients with progressive encephalopathy indicated that the brain was heavily infected. A frequent complication in later stages of HIV infection is AIDS dementia, a neurological syndrome characterized by abnormalities in cognition, motor performance, and behavior. It remains unknown whether or not AIDS dementia and other pathological effects observed in

the central nervous systems of infected individuals are a direct effect of HIV-1 on the brain, a consequence of immune responses to the virus, or a result of infection by opportunistic agents.

Therapeutic Agents Inhibit Retrovirus Replication Development of a vaccine to prevent the spread of AIDS is the highest priority for immunologists. Meanwhile, drugs that can reverse the effects of HIV-1 have greatly improved the outlook for infected individuals. There are several strategies for the development of effective antiviral drugs that take advantage of the life cycle of HIV (Figure 18-18). The key to success for such therapies is that they must be

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Reverse transcription

ssRNA Reverse transcriptase

Provirus

Inhibit integrase

Activation Integration

Transcription

Translation

2

mRNAs CD4 Cleavage Proteins

Precursors

Inhibit fusion

ssRNA

Assembly CCR5 HIV 1

Budding

5

Inhibit protease

Chemokine receptor antagonists

specific for HIV-1 and interfere minimally with normal cell processes. Thus far, antiviral agents targeting five separate steps in the viral life cycle have proven effective. The first success came with drugs that interfere with the reverse transcription of viral RNA to cDNA ( 3 in

TABLE 18-5

Categories of HIV-1 drugs in clinical use

Category

FDA approval date*

Nucleoside/nucleotide analogues

1987

Nonnucleoside reverse transcriptase inhibitors

1996

Protease inhibitors

1995

Fusion/attachment inhibitors

2003

Chemokine coreceptor antagonists

2007

Integrase inhibitors

2007

*Year of first FDA approval for a drug to treat HIV-1 infection in that drug category. Source: Antiretroviral Drug Profiles. (2012). HIV InSite, University of California, San Francisco. http://hivinsite.ucsf.edu/InSite?page⫽ar-drugs

FIGURE 18-18 Stages in the viral replication cycle that provide targets for therapeutic antiretroviral drugs. The first licensed drugs with anti-HIV activity interfered with reverse transcription of viral RNA to cDNA (3), followed by drugs which blocked the viral protease that cleaves precursor proteins into the peptides needed to assemble new virions (5). In the past decade, newer drugs have come on the market that interfere with other steps in the viral life cycle, such as HIV coreceptor attachment (1) or fusion to the cell membrane (2), as well as the viral integrase necessary for insertion of proviral DNA into the host cell chromosome (4).

Figure 18-18); several drugs that use two possible mechanisms of action operate at this step. The second generation of drugs inhibits the viral protease ( 5 in Figure 18-18) required to cleave precursor proteins into the units needed for construction of new mature virions. This was followed by development of an inhibitor of the viral gp41 that blocks fusion of virus with the host cell membrane ( 2 in Figure 18-18), inhibiting new infection of cells. The two newest antiviral agents on the market target attachment of the virus to the cell by competition for access to the CCR5 chemokine coreceptor used by the virus ( 1 in Figure 18-18) or interfere with integrase ( 4 in Figure 18-18) required for insertion of the viral genome into host cell DNA. Table 18-5 lists the currently available categories of anti-HIV therapies, along with the year in which they were first approved for use. It should be stressed that the development of any drug to the point at which it can be used for patients is a long and arduous procedure. The drugs that pass the rigorous tests for safety and efficacy represent a small fraction of those that receive initial consideration. There are two possible strategies for developing pharmaceutical agents that can interfere with reverse transcription: nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs). The prototype and earliest (approved in 1987) of the drugs that interferes with reverse transcription was zidovudine, or azidothymidine (AZT), in the NRTI class.

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Immunodeficiency Disorders The introduction of AZT, a nucleoside analogue and competitive inhibitor of the enzyme, into the growing cDNA chain of the retrovirus causes termination of the chain. AZT is effective in some but not all patients, and its efficacy is further limited because long-term use has adverse side effects and because resistant viral mutants develop in treated patients. The administered AZT is used not only by the HIV-1 reverse transcriptase (RT) but also by human DNA polymerase. The incorporation of AZT into the DNA of host cells kills them. Precursors of red blood cells and other rapidly dividing cells are especially sensitive to AZT, resulting in anemia and other side effects. NNRTI drugs inhibit the action of RT by binding to a different site on the enzyme. These noncompetitive inhibitors of RT have less of an adverse effect on host proteins, and therefore fewer side effects. However, they are still susceptible to the development of resistance as the virus mutates, and for this reason are typically only used in combination with other anti-HIV drugs that have different targets. Nevirapine, licensed in 1996, was the first such compound designed to treat HIV, but since then many more such drugs have come on the market. A separate class of drugs, called protease inhibitors, target the HIV-1 protease that cleaves viral protein precursors into the peptides required for packaging into virions in the final stages of HIV replication. The first of the protease inhibitors, saquinavir, came to market in the mid1990s, but many more have emerged since then. They are most commonly used today as a part of a multidrug cocktail designated as highly active antiretroviral therapy (HAART). In most cases, this combines the use of three or more anti-HIV drugs from different classes. The combination strategy appears to overcome the ability of the virus to rapidly produce mutants that are drug resistant. In many cases, HAART has lowered plasma viral load to levels that are not detectable by current methods and has improved the health of AIDS patients to the point that they can again function at a normal level. The decrease in the number of AIDS deaths in the United States in recent years is attributed to this advance in therapy. Despite the optimism engendered by success with HAART, present drawbacks include the need for consistent adherence to this regimen, lest viral drug-resistant mutants be favored in the patient. In addition, some patient may experience serious side effects that become too severe to allow the use of HAART. This said, access to multiple drugs within most of the drug categories in the United States makes substitution possible. The success of HAART has led researchers to wonder whether it might be possible to eradicate all virus from an infected individual and thus actually cure AIDS. Most AIDS experts are not convinced that this is feasible, mainly because of the persistence of latently infected CD4⫹ T cells and macrophages, which can serve as a reservoir of infectious virus if and when the provirus becomes reactivated.

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Even with a viral load beneath the level of detection by PCR assays, the immune system may not recover sufficiently to clear virus should it begin to replicate in response to some activation signal. In addition, virus may persist in sites, such as the brain, that are not readily penetrated by the antiretroviral drugs, even though the virus in circulation is undetectable. The use of immune modulators, such as recombinant IL-2 and IL-7 as adjuvants to HAART, is being examined as a strategy to help reconstitute the immune system and restore normal immune function, with mixed results. Some forms of antiviral therapy may also work to prevent HIV infection in individuals at high risk. Based on studies completed in 2011 and 2012, when both partners in an HIV discordant couple (where one is infected and the other is not) were given a cocktail of two NRTIs, rates of sexual transmission of HIV dropped by 60% to 75%. Cost considerations aside, this pre-exposure prophylaxis (or PrEP) treatment was approved by the FDA in 2012 as a preventive measure for healthy, HIV-negative individuals at high risk of infection.

A Vaccine May Be the Only Way to Stop the HIV/AIDS Epidemic The AIDS epidemic continues to rage despite the advances in therapeutic approaches outlined above. The expense of HAART (roughly $1000/month in the United States), the strict adherence required to avoid development of resistance, and the possibility of side effects preclude universal application. At present, it appears that the best option to stop the spread of AIDS is a safe, effective vaccine that prevents infection and/or progression to disease. The cause of AIDS was discovered over 25 years ago. Why don’t we have an AIDS vaccine by now? The best way to approach an answer to this question is to examine the specific challenges presented by HIV-1. HIV mutates rapidly, creating a moving target for both the immune response and any vaccine design. This means that there are many possible variants of the virus to contend with in any one geographical location, not even considering many different countries. We know from HIV seropositive individuals that the development of humoral immunity during a natural infection, even the presence of neutralizing antibodies, does not necessarily inhibit viral spread. This means that a strong humoral response alone is unlikely to block infection. Despite years of study, data from long-term nonprogressor and exposed seronegative individuals have shed little light on the immune correlates of protection from disease or infection. Without clear goalposts, it is difficult to know which types of cellular immune response will be most effective, or when we have reached enough of a response to protect against a natural infection. In addition, good animal models for testing these fledgling vaccines are limited and expensive.

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Major HIV-1 vaccine trials

Vaccine design

Study name

Status

Result

Purified protein (gp120)

VAX003, VAX004

Completed in 2003

No protection

Recombinant adenovirus vector (gag/pol/nef)

HVTN 502 STEP HVTN 503 Phambili

Terminated in 2007

No protection

Recombinant canarypox vector (env/gag/protease) ⫹ env gp120 protein boost

RV 144

Completed in 2009

30% reduction

DNA vaccine - 6-plasmids (env/gag/pol/nef) ⫹ Recombinant adenovirus vector boost (same genes)

HVTN 505

Began in 2009

N/A (in progress)

HVTN ⫽ HIV vaccine trials network Source: Adapted from D. H. Barouch and B. Korber. 2010. HIV-1 vaccine development after STEP. Annual Reviews in Medicine 61:153.

Still, valuable lessons have been learned from over a decade of HIV vaccine initiatives, both from studies in nonhuman primates and from clinical trials in humans (Table 18-6). The earliest of these trials in humans aimed at eliciting humoral immunity to neutralize incoming virus. This approach used purified envelope protein from HIV (specifically, gp120) as an immunogen. These studies, completed in the United States and Thailand in 2003, showed weak neutralizing antibody responses and no protection from infection. This was followed by a new wave of vaccine design aimed at eliciting cellular immunity to the virus using recombinant viral vectors that would better mimic natural infection. The initial vector chosen was adenovirus serotype 5 (Ad5), carrying recombinant DNA derived from the gag, pol, and nef genes of HIV-1. Ad5 is derived from a naturally occurring human virus to which between 30% and 80% of individuals (depending on geographic location) have previously been exposed, appearing to make this a safe vector choice. These trials included two stages of vaccination: an initial priming dose followed by a vaccine boost using the same vector. Initially conducted in the Americas, the Caribbean, and Australia, these human trials began in 2003 but were halted prematurely in 2007, midway through the trial period, because they did not demonstrate the desired reduction in viral load for those volunteers who became infected after receiving the vaccine. More important, the rate of infection appeared to be higher in the vaccinated group of the trial than in the placebo control population, especially among individuals who had pretrial immune responses to adenovirus. This was a resounding blow to the AIDS research community and sent many vaccine design teams back to the drawing board. These disappointing results have led to new thinking in terms of targets for the next wave of HIV-1 vaccine design:

vaccines that elicit both humoral and cellular immunity. One trial using this strategy has recently been completed. It used a prime-boost combination with a recombinant DNA vector containing HIV-1 genes (the prime) followed by peptides derived from the virus (the boost). The vector chosen was canarypox, a virus that does not replicate in humans and that was engineered to carry DNA from the env, gag, and the portion of pol that encodes the protease of HIV-1. The protocol also included a boost with a companion vaccine consisting of an engineered version of HIV-1 gp120 protein. Results from this study were modest but promising, demonstrating an approximately 30% reduction in infection rates among the vaccine treated group compared to the matched placebo control population. Although statistically significant, these results are still far from the near 100% protection rate that is the aim for all vaccines against infectious disease. Nevertheless, this is one of the first really promising steps forward in HIV vaccine research. In 2009, trials were launched for a new DNA vaccine containing six plasmids representing multiple clades of env genes plus single clade gag, pol, and nef (prime), followed by an adenoviral vector containing the same genes (boost). Results from this trial, which expanded its scope in 2011, are still pending. It is clear that development of an HIV vaccine is not a simple exercise in classic vaccinology. More research is needed to understand how this viral attack against the immune system can be thwarted. Although much has been written about the subject and large-scale initiatives are proposed, the path to an effective vaccine is not obvious. It is certain only that all data must be carefully analyzed and that all possible means of creating immunity must be tested. This is one of the greatest public health challenges of our time. An intense and cooperative effort will be required to devise, test, and deliver a safe and effective vaccine for AIDS.

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S U M M A R Y ■



















Immunodeficiency results from the failure of one or more components of the immune system. Primary immunodeficiencies are based on genetic defects present at birth; secondary or acquired immunodeficiencies arise from a variety of external causes. Immunodeficiencies may be classified by the cell types involved and may affect either the lymphoid- or the myeloid-cell lineage, or both. Combined immunodeficiencies (CIDs) disrupt adaptive immunity by interfering with both T-cell and B-cell responses. Severe combined immunodeficiency, or SCID, is the most extreme form of CID and arises from a lack of functional T cells, which also manifests as no T-cell help for B-cell responses. Genetic failures of thymic development and major histocompatibility (MHC) expression can lead to CID because of the disruption of T-cell development. B-cell immunodeficiencies, which are associated with susceptibility to bacterial infection, can range from single isotype defects to total disruptions of humoral immunity. Selective immunoglobulin deficiencies are the less-severe form of B-cell deficiency and result from defects in more highly differentiated cell types. Myeloid immunodeficiencies cause impaired phagocytic function. Affected individuals suffer from increased susceptibility to bacterial infection. Complement deficiencies are relatively common and vary in their clinical impact, but are generally associated with increased susceptibility to bacterial infection. Immunodeficiencies that disrupt immune regulation can lead to overactive immune responses that manifest as autoimmune syndromes. Immunodeficiency disorders can be treated by replacement of defective or missing proteins, cells, or genes.

Administration of human immunoglobulin is a common treatment, especially for those disorders that primarily disrupt antibody responses. ■

Animal models for immunodeficiency include nude and SCID mice. Targeted gene knockout mice provide a means to study the role of specific genes in immune function.



Secondary or acquired immunodeficiency can result from immunosuppressive drugs, infection, and malnutrition; the most well-known form of this is AIDS, caused by human immunodeficiency virus-1 (HIV-1), which is a retrovirus.



HIV-1 infection is spread by contact with infected body fluids such as occurs during sex, intravenous drug use, and from mother to infant during childbirth or breastfeeding.



The HIV-1 genome contains three structural and six regulatory genes that encode the proteins necessary for infection and propagation in host cells.



HIV-1 uses the host CD4 molecule as well as a chemokine coreceptor (CCR5 or CXCR4) to attach to and fuse with host cells.



Infection with HIV-1 results in gradual and severe impairment of immune function, marked by depletion of CD4⫹ T cells, especially in the gut, and can result in death from opportunistic infection.



Treatment of HIV infection with antiretroviral drugs that target specific steps in the viral life cycle, especially in combination, can lower the viral load and provide relief from some symptoms of infection. However, no cures for HIV are available.



Efforts to develop a vaccine for HIV-1 have been only modestly successful. The millions of new infections occurring yearly emphasize the need for an effective vaccine.

R E F E R E N C E S Belizário, J. E. 2009. Immunodeficient mouse models: An overview. The Open Immunology Journal 2:79–85. Brenchley, J. M., and D. C. Douek. 2008. HIV infection and the gastrointestinal immune system. Mucosal Immunity 1(1):23. Buckley, R. H. 2004. Molecular defects in human severe combined immunodeficiency and approaches to immune reconstitution. Annual Review of Immunology 22:625. Calvazzano-Calvo, M., et al. 2005. Gene therapy for severe combined immunodeficiency. Annual Review of Medicine 56:585.

Chasela, C. S., et al. 2010. Maternal or infant antiretroviral drugs to reduce HIV-1 transmission. The New England Journal of Medicine 362(24):2271. Chinen, J., and W. T. Shearer. 2009. Secondary immunodeficiencies, including HIV infection. Journal of Allergy and Clinical Immunology 125(2):S195. Corey, L., et al. 2009. Post STEP modifications for research on HIV vaccines. AIDS 23(1):3. Douek, D. C., et al. 2009. Emerging concepts in the immunopathogenesis of AIDS. Annual Reviews in Medicine 60:471.

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Fischer, A. 2007. Human primary immunodeficiency diseases. Immunity 27:835. Fischer, A., et al. 2010. 20 years of gene therapy for SCID. Nature Immunology 11(6):457. Fried A. J., and F. A. Bonilla. 2009. Pathogenesis, diagnosis, and management of primary antibody deficiencies and infections. Clinical Microbiology Reviews 22(3):396. Granich, R., et al., 2010. Highly active antiretroviral treatment for prevention of HIV transmission. Journal of the International AIDS Society 13:1. Hladik, F., and T. J. Hope. 2009. HIV infection of the genital mucosa in women. Current HIV/AIDS Reports 6:20–28. Hui-Qi, Q., S. P. Fisher-Hoch, and J. B. McCormick. 2011. Molecular immunity to mycobacteria: Knowledge from the mutation and phenotype spectrum analysis of Mendelian susceptibility to mycobacterial diseases. International Journal of Infectious Diseases 15(5):e305–e313.

Piacentini, L., et al. 2008. Genetic correlates of protection against HIV infection: The ally within. Journal of Internal Medicine 265:110. Rerks-ngarm, S., et al., 2009. Vaccination with ALVAC and AIDSVAX to prevent HIV-1 infection in Thailand. The New England Journal of Medicine 361(23):2209.

Useful Websites www.niaid.nih.gov/topics/immunedeficiency/ The National Institute of Allergy and Infectious Diseases (NIAID) maintains a Web site for learning more about primary immunodeficiency diseases, their treatment, and current research.

www.niaid.nih.gov/topics/hivaids/research/ vaccines Another NIAID Web page that includes information about AIDS vaccines and links to documents about vaccines in general.

Isgrò, A., et al. 2005. Bone marrow clonogenic capability, cytokine production, and thymic output in patients with common variable immunodeficiency. Journal of Immunology 174(8):5074–5081.

www.scid.net This Web site contains links to periodicals and databases with information about SCID.

Jackson, J. B., et al. 2003. Intrapartum and neonatal singledose nevirapine compared to zidovudine for prevention of mother-to-child transmission of HIV-1 in Kampala, Uganda: 18-month follow-up of the HIVNET 012 randomized trial. Lancet 362:859.

site is maintained by the University of California, San Francisco, one of many centers of research in this field.

Moore, J. P., et al. 2004. The CCR5 and CXCR4 coreceptors— central to understanding the transmission and pathogenesis of human immunodeficiency virus type I infection. AIDS Research and Human Retroviruses 20:111. Moraes-Vasconcelos, D., et al. 2008. Primary immune deficiency disorders presenting as autoimmune diseases: IPEX and APECED. Journal of Clinical Immunology 28 (Suppl 1): S11. Notarangelo, L. D. 2010. Primary immunodeficiences. Journal of Allergy and Clinical Immunology 125(2):S182. Ochs, H. D., et al. 2009. TH17 cells and regulatory T cells in primary immunodeficiency diseases. Journal of Allergy and Clinical Immunology 123(5):977.

S T U D Y

http://hivinsite.ucsf.edu This HIV/AIDS information

www.unaids.org/en Information about the national and global AIDS epidemic can be accessed from this site. http://hiv-web.lanl.gov This Web site, maintained by the Los Alamos National Laboratory, contains all available sequence data on HIV and SIV along with up-to-date reviews on topics of current interest to AIDS research. www.aidsinfo.nih.gov This site, maintained by the National Institutes of Health, contains information and national guidelines on the treatment and prevention of AIDS.

http://clinicaltrials.gov This National Institutes of Health-sponsored Web site is a registry of all private and government funded clinical trials in the United States and worldwide.

Q U E S T I O N S

CLINICAL FOCUS QUESTION The spread of HIV/AIDS from in-

fected mothers to infants can be reduced by single-dose regimens of the reverse transcriptase inhibitor nevirapine. What would you want to know before giving this drug to all mothers and infants (without checking infection status) at delivery in areas of high endemic infection? 1. Indicate whether each of the following statements is true or

false. If you think a statement is false, explain why. a. Complete DiGeorge syndrome is a congenital birth

defect resulting in absence of the thymus. b. X-linked agammaglobulinemia (XLA) is a combined B-cell and T-cell immunodeficiency disease.

c. The hallmark of a phagocytic deficiency is increased

susceptibility to viral infections. d. In chronic granulomatous disease, the underlying

defect is in phagosome oxidase or an associated protein. e. Injections of immunoglobulins are given to treat indi-

viduals with X-linked agammaglobulinemia. f. Multiple defects have been identified in human SCID. g. Mice with the SCID defect lack functional B and T lym-

phocytes. h. Mice with SCID-like phenotype can be produced by

knockout of RAG genes. i. Children born with SCID often manifest with increased

infection by encapsulated bacteria in the first months of life.

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j. Failure to express class II MHC molecules in bare-

10. Immunologists have studied the defect in SCID mice in an

lymphocyte syndrome affects cell-mediated immunity only.

effort to understand the molecular basis for severe combined immunodeficiency in humans. In both SCID mice and humans with this disorder, mature B and T cells fail to develop.

2. For each of the following immunodeficiency disorders,

indicate which treatment would be appropriate.

a. In what way do rearranged Ig heavy-chain genes in

Immunodeficiency

b. In SCID mice, rearrangement of ␬ light-chain DNA is

a. b. c. d. e. f.

SCID mice differ from those in normal mice?

Chronic granulomatous disease ADA-deficient SCID X-linked agammaglobulinemia DiGeorge syndrome IL-2R-deficient SCID Common variable immunodeficiency

not seen. Explain why.

c. If you introduced a rearranged, functional ␮ heavy-

chain gene into progenitor B cells of SCID mice, would the ␬ light-chain DNA undergo a normal rearrangement? Explain your answer. 11. Indicate whether each of the following statements is true or

Treatment

false. If you think a statement is false, explain why.

1. 2. 3. 4. 5.

a. HIV-1 and HIV-2 are both believed to have evolved fol-

Full bone marrow transplantation Pooled human gamma globulin Recombinant IFN-␥ Recombinant adenosine deaminase Thymus transplant in an infant

3. Patients with X-linked hyper-IgM syndrome express nor-

mal genes for other antibody subtypes but fail to produce IgG, lgA, or IgE. Explain how the defect in this syndrome accounts for the lack of other antibody isotypes.

b. c. d. e.

4. Patients with DiGeorge syndrome are born with either no

f.

thymus or a severely defective thymus. In the severe form, the patient cannot develop mature helper, cytotoxic, or regulatory T cells. If an adult suffers loss of the thymus due to accident or injury, little or no T-cell deficiency is seen. Explain this discrepancy.

g.

5. Infants born with SCID experience severe recurrent infec-

h.

lowing the species jump of SIV from chimpanzees to humans. HIV-1 causes immune suppression in both humans and chimpanzees. SIV is endemic in the African green monkey. The anti-HIV drugs zidovudine and saquinavir both act on the same point in the viral replication cycle. T-cell activation increases transcription of the HIV proviral genome. HIV-infected patients meet the criteria for AIDS diagnosis as soon as their CD4⫹ T-cell count drops below 500 cells/␮l. The polymerase chain reaction is a sensitive test used to detect antibodies to HIV. If HAART is successful, viral load will typically decrease.

12. Various mechanisms have been proposed to account for

tions. The initial manifestation in these infants is typically fungal or viral infections, and only rarely bacterial ones. Why are bacterial infections less of an issue in these newborns?

the decrease in the numbers of CD4⫹ T cells in HIVinfected individuals. What seems to be the most likely reason for depletion of CD4⫹ T cells?

6. In B-cell immunodeficiencies, infections by bacteria are

13. Would you expect the viral load in the blood of HIV-

common ailments. However, not all types of bacteria prove equally problematic, and encapsulated bacteria, such as staphylococci, are often the most problematic. Why is this type of bacteria such a problem for individuals who inherit this type of immunodeficiency? 7. Granulocytes from patients with leukocyte adhesion defi-

ciency (LAD) express greatly reduced amounts of CD11a, b, and c adhesion molecules. a. What is the nature of the defect that results in decreased

expression of these adhesion molecules in LAD patients? b. What is the normal function of the integrin molecule

LFA-1? Give specific examples.

infected individuals in the early years of the asymptomatic phase of HIV-1 infection to vary significantly (assuming no drug treatment)? What about CD4⫹ T-cell counts? Why? 14. If viral load begins to increase in the blood of an HIV-

infected individual and the level of CD4⫹ T cells decrease, what would this indicate about the infection?

15. Clinicians often monitor the level of skin-test reactivity, or

delayed-type hypersensitivity, to commonly encountered infectious agents in HIV-infected individuals. Why do you think these immune reactions are monitored, and what change might you expect to see in skin-test reactivity with progression into AIDS?

8. How can an inherited defect in the IL-2 receptor cause the

16. Certain chemokines have been shown to suppress infec-

demise of developing B cells, as well as T cells, if B cells do not possess receptors for IL-2 signaling?

tion of cells by HIV, and proinflammatory cytokines enhance cell infection. What is the explanation for this?

9. Primary immunodeficiency results from a defective com-

17. Treatments with combinations of anti-HIV drugs (HAART)

ponent of the immune response. Typically, this manifests as failure to fight off one or more types of pathogen. Very occasionally, immunodeficiency results in autoimmune syndromes, where the immune response attacks self tissues. Explain how this might occur.

have reduced virus levels significantly in some treated patients and can delay the onset of AIDS. If an AIDS patient becomes free of opportunistic infection and has no detectable virus in the circulation, can that person be considered cured?

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18. Suppose you are a physician who has two HIV-infected

als with CVID. They looked at the T-cell phenotypes of CVID patients (as shown in the following table) as well as some of the cytokines made by these individuals (see accompanying graphs).

patients. Patient B. W. has a bacterial infection of the skin (S. aureus), and patient L. S. has a mycobacterial infection. The CD4⫹ T-cell counts of both patients are about 250 cells/␮l. Would you diagnose either patient or both of them as having AIDS?

a. What is the impact of CVID on T helper cells? b. Naïve CTLs require IL-2 production from T helper cells

19. ANALYZE THE DATA. Common variable immunodefi-

in order to become activated (see Chapter 14). How might CVID impact the generation of CTLs? c. True or false? CVID inhibits cytokine production. Explain your answer. Speculate on the physiological impact of the cytokine pattern of CVID patients. d. Would you predict an effect of CVID on the humoral immune response?

ciency (CVID) causes low concentrations of serum Igs and leads to frequent bacterial infections in the respiratory and gastrointestinal tracts. People with CVID also have an increased prevalence of autoimmune disorders and cancers. Isgrò and colleagues (Journal of Immunology, 2005, 174:5074) examined the bone marrow of several individu-

Patients

CD4⫹

% cells/␮l

Naïve CD4



1

2

5

6

7

8

9

10

11

CVID

Control (n  10)

47

36

28

28

27

19

19

32

57

34

47.5

296

234

278

1652

257

361

289

248

982

351

%

4

cells/␮l Activated CD4⫹

14

12

2

10

52

12

16

72

66

30

29

40

30

20

31

519

2

5

22

3

9

8

12

9

8

37

%

11.6

7.9

1.5

12

61

50

23

29

35

22

15

25

385

38

30

57

44

56

47

45

21

39

20

195

247

298

3363

420

1065

714

348

362

414

404

%

25

30

43.9

20

13

15

22

58

cells/␮l

49

74

131

143

45

54

88

233

cells/␮l Naïve CD8

4

6

% ⫹

25.8

31

cells/␮l CD8⫹

6.8

7

16.9

235

12.9

71

138

(a) 100 IL-2 pg/mL

pg/mL

80 60 40

Control

20 CVID 0

180 160 140 120 100 80 60 40 20 0

TNF-␣

CVID Control

(b) 100

Control

80

60 40

CVID

pg/mL

pg/mL

100

IL-2

80

0

0

72 Hours

96

CVID

40 20

48

TNF-␣

60

20 24

1024

Control 24

48

72 Hours

96

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Cancer and the Immune System

A

s the death toll from infectious disease has declined in the Western world, cancer has become the second leading cause of death, topped only by heart disease. Current estimates project that half of all men and one in three women in the United States will develop cancer at some point in their lifetimes, and that one in five will die from it. From an immunologic perspective, cancer cells can be viewed as altered self cells that have escaped normal growth-regulating mechanisms. This chapter examines the unique properties of cancer cells, giving particular attention to those properties that can be recognized by the immune system. We then describe the immune responses that develop against cancer cells, as well as the methods by which cancers manage to evade those responses. The final section surveys current clinical and experimental immunotherapies for cancer. In most organs and tissues of a mature animal, a balance is maintained between cell renewal and cell death. The various types of mature cells in the body have a given life span; as these cells die, new cells are generated by the proliferation and differentiation of various types of stem cells. This cell growth and proliferation are essential for wound healing and homeostasis. Under normal circumstances in the adult, the production of new cells is regulated so that the number of any particular type of cell remains fairly constant. Occasionally, however, cells arise that no longer respond to normal growth control mechanisms; these cells proliferate in an unregulated manner, giving rise to cancer. In the following sections, we first cover common terminology related to cancer, and then discuss the pathways that can lead to this uncontrolled cell growth.

Terminology and Common Types of Cancer Cells that give rise to clones of cells that can expand in an uncontrolled manner will produce a tumor or neoplasm. A tumor that is not capable of indefinite growth and does not invade the healthy surrounding tissue extensively is said to

A cluster of prostate cancer cells, stained to visulize the nuclei (green), Golgi apparatus (pink), and actin filaments (purple). [Nancy Kedersha/Getty Images] ■

Terminology and Common Types of Cancer



Malignant Transformation of Cells



Tumor Antigens



The Immune Response to Cancer



Cancer Immunotherapy

be benign. A tumor that continues to grow and becomes progressively more invasive is called malignant; the term cancer refers specifically to a malignant tumor. In addition to uncontrolled growth, malignant tumors exhibit metastasis, whereby small clusters of cancerous cells dislodge from the original tumor, invade the blood or lymphatic vessels, and are carried to other distant tissues, where they take up residence and continue to proliferate. In this way, a primary tumor at one site can give rise to a secondary tumor at another site (Figure 19-1). Malignant tumors or cancers are classified according to the embryonic origin of the tissue from which the tumor is derived. Most (80–90%) are carcinomas, tumors that arise from epithelial origins such as skin, gut, or the epithelial lining of internal organs and glands. Skin cancers and the majority of cancers of the colon, breast, prostate, and lung are carcinomas. The leukemias, lymphomas, and myelomas are malignant tumors of hematopoietic cells derived from the bone marrow and account for about 9% of cancer incidence 627

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19-1

OVERVIEW FIGURE

Tumor Growth and Metastasis (a)

Initially modified tumor cell

(c)

Invasive tumor cells

Basal lamina

Blood vessel

(b)

Mass of tumor cells (localized benign tumor)

(d)

Tumor cells invade blood vessels, allowing metastasis to occur

(a) A single cell develops altered growth properties at a tissue site. (b) The altered cell proliferates, forming a mass of localized tumor cells, or a benign tumor. (c) The tumor cells become progressively more invasive, spreading to the underlying basal lamina. The tumor is now classified as malignant. (d) The malignant tumor metastasizes by generating small clusters of cancer cells that dislodge from the tumor and are carried by the blood or lymph to other sites in the body. [Adapted from J. Darnell et al., 1990, Molecular Cell Biology, 2nd ed., Scientific American Books.]

in the United States. Leukemias proliferate as single detached cells, whereas lymphomas and myelomas tend to grow as tumor masses. Sarcomas, which arise less frequently (around 1% of the incidence of cancer in the United States), are derived from mesodermal connective tissues, such as bone, fat, and cartilage. Historically, the leukemias were classified as acute or chronic according to the clinical progression of the disease. The acute leukemias appeared suddenly and progressed rapidly, whereas the chronic leukemias were much less aggressive and developed slowly as mild, barely symptomatic diseases. These clinical distinctions apply to untreated leukemias; with current treatments, the acute leukemias often have a good prognosis, and permanent remission is possible. Now the major distinction between acute and chronic leukemias is the maturity of the cell involved. Acute leukemias tend to arise in less mature cells, whereas chronic leukemias arise in mature cells, although each can arise from lymphoid

or myeloid lineages. The acute leukemias include acute lymphocytic leukemia (ALL) and acute myelogenous leukemia (AML). These diseases can develop at any age and have a rapid onset. The chronic leukemias include chronic lymphocytic leukemia (CLL) and chronic myelogenous leukemia (CML), which develop more slowly and are seen primarily in adults.

Malignant Transformation of Cells Much has been learned about cancer from in vitro studies of primary cells. Treatment of normal cultured cells with specific chemical agents, irradiation, and certain viruses can alter their morphology and growth properties. In some cases, this process leads to unregulated growth and produces cells capable of growing as tumors when they are injected into animals. Such cells are said to have undergone transformation, or

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Cancer and the Immune System malignant transformation, and they often exhibit properties in vitro similar to those of cancer cells that form in vivo. For example, they have decreased requirements for the survival factors required by most cells (such as growth factors and serum), are no longer anchorage dependent, and grow in a density-independent fashion. Moreover, both cancer cells and transformed cells can be subcultured in vitro indefinitely; that is, for all practical purposes, they are immortal. Because of the similar properties of cancer cells and transformed cells, the process of malignant transformation has been studied extensively as a model of cancer induction.

DNA Alterations Can Induce Malignant Transformation Transformation can be induced by various chemical substances (such as formaldehyde, DDT, and some pesticides), physical agents (e.g., asbestos), and ionizing radiation; all are linked to DNA mutations. For this reason, these agents are commonly referred to as carcinogens. The International Agency for Research on Cancer (IARC) tracks the most common causes of cancer in humans and maintains lists of potential cancer-causing agents (see Useful Web Sites). Infection with certain viruses, most of which share the property of integrating into the host cell genome and disrupting chromosomal DNA, can also lead to transformation. Table 19-1 lists the most common viruses associated with cancer. Although the activity of each of these agents is associated with cancer initiation, thanks to the variety of DNA repair mechanisms present in our cells, exposure to carcinogens does not always lead to cancer. Instead, a confluence of factors must occur before enough changes to normal cellular genes occurs to

TABLE 19-1

Common human infectious carcinogens

Viral Agent

Type

Cancer

HTLV-1 Human T-cell leukemia virus -1

RNA

Adult T-cell leukemia or lymphoma

HHV-8 Human herpesvirus-8

DNA

Kaposi’s sarcoma (especially in HIV⫹)

HPV Human papillomavirus

DNA

Cervical carcinoma

HBV and HCV Hepatitis B and C viruses

DNA

Liver carcinoma

EBV Epstein-Barr virus

DNA

Burkitt’s lymphoma and nasopharyngeal carcinoma

Sources: The National Institute for Occupational Safety and Health (NIOSH) and the Centers for Disease Control and Prevention (CDC). [http://www.cdc. gov/niosh/topics/cancer/]

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induce malignant transformation, as we discuss further in the following sections.

The Discovery of Oncogenes Paved the Way for Our Understanding of Cancer Induction The discovery that disruption of normal cellular genes can lead to cancer came from studies done with cancer-causing viruses. In 1916, Peyton Rous, working at the Rockefeller Institute, showed that cells cultured with a particular retrovirus underwent malignant transformation. This virus was later named Rous sarcoma virus. Fifty years after his initial results, Rous was awarded the Nobel Prize in Physiology or Medicine for his discoveries, which led to the term oncogene, from the Greek word ónkos (which means “mass” or “tumor”). This term was originally used to describe the unique genetic material present in certain viruses that promotes malignant transformation of infected cells. In 1971, Howard Temin suggested that oncogenes might not be unique to transforming viruses but might also be found in normal cells. Temin proposed that a virus might acquire a normal growth-promoting gene from the genome of a cell it infects. He called these normal cellular genes proto-oncogenes to distinguish them from the cancerpromoting sequences carried by some viruses (viral oncogenes, or v-onc). The following year, R. J. Huebner and G. J. Todaro proposed that mutations or genetic rearrangements of in situ proto-oncogenes by carcinogens or viruses might alter the normally regulated function of these genes, converting them into cancer inducers, called cellular oncogenes (c-onc; Figure 19-2). Considerable evidence supporting this hypothesis accumulated in subsequent years. For example, some malignantly transformed cells contain multiple copies of cellular oncogenes, leading to increases in oncogene products. Such amplification of cellular oncogenes has been observed in cells from various types of human cancers. In the mid-1970s, J. Michael Bishop and Harold E. Varmus at the University of California, San Francisco, again used the Rous retrovirus to establish the origins of these viral oncogenes. Retroviruses have RNA genomes that must be reverse-transcribed into DNA during their life cycle, followed by integration of this DNA into the host-cell chromosome. Retroviruses therefore make intimate contact with the genomes of their host cell, especially those genes that happen to become their neighbors following chromosomal integration. In elegant studies that led to another Nobel Prize in 1989, Bishop and Varmus demonstrated that the oncogene carried by Rous sarcoma virus (later called v-src) was in fact just a version of a normal growth-promoting cellular gene (c-src) found throughout many species and probably acquired previously from a host cell during viral replication. By randomly incorporating this normal cellular gene into its own genome, the Rous sarcoma virus acquired the ability to induce malignant transformation in the cells it infected, giving it a selective advantage. The evidence that oncogenes alone could induce malignant transformation

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Normal cells

Transformed cells Viral oncogenes Transforming virus

Proto-oncogenes

Normal expression

Essential growthcontrolling proteins Growth factors Growth factor receptors Signal transducers Intranuclear factors Regulators of apoptosis

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Carcinogens and genetic predisposition Cellular oncogenes Altered expression 1 Qualitatively altered, hyperactive proteins 2 Quantitative alterations (gene amplification or translocation) resulting in increased or decreased levels of products

FIGURE 19-2 The relationship of proto-oncogenes to oncogenes. The process of malignant transformation can result from infection with transforming viruses, which carry oncogene sequences, or via carcinogen-induced conversion of a wild-type protooncogene into a cellular oncogene, sometimes compounded by genetic predisposition in the host cell. Cellular oncogenes arise due to DNA changes that alter expression of proto-oncogenes, including DNA mutations that result in the production of qualitatively different gene products (1), as well as DNA amplification or chromosomal translocation, leading to increased or decreased expression of these gene products (2).

came again from studies of the v-src oncogene; when this oncogene was cloned and transfected into normal cells in culture, the cells underwent malignant transformation.

Genes Associated with Cancer Control Cell Proliferation and Survival Normal tissues maintain homeostasis through a tightly regulated process of cell proliferation balanced by cell death. An imbalance at either end of the scale encourages development of a cancerous state. The genes involved in these homeostatic processes work by producing proteins that either encourage or discourage cellular proliferation and survival. Not surprisingly, it is the disruption of these same growth-regulating genes that accounts for most, if not all, forms of cancer. The activities of these proteins can occur anywhere in the pathway, from signaling events at the surface of the cell, to intracellular signal transduction processes and nuclear events.

Normal cellular genes that are associated with the formation of cancer fall into three major categories based on their activities: oncogenes, tumor-suppressor genes, and genes involved in programmed cell death, or apoptosis (Table 19-2). As discussed earlier, oncogenes are involved in cell growth-promoting processes. On the flip side, tumorsuppressor genes play the opposite role in homeostasis, dampening cellular growth and proliferation. Unlike oncogenes, which become the villain when their activity is enhanced, tumor-suppressor genes, also known as antioncogenes, become involved in cancer induction when they fail. Finally, many of the genes involved in apoptosis have also been associated with cancer, as these genes either enforce or inhibit cell death signals. Recently, several unifying hallmarks have been proposed that help to characterize the transition of non-neoplastic cells to a cancerous state. These include sustained proliferation, subversion of negative growth regulators, and resistance to cell death—controlled, respectively, by the activity of oncogenes, tumor-suppressor genes, and genes that regulate apoptosis. Other proposed hallmarks of cancer include replicative immortality, the growth of new blood vessels (also called angiogenesis), and the potential to invade other tissues (metastasis). Two underlying factors that help to enable the development of these hallmarks—genetic instability and inflammation—are also now recognized contributors to tumorigenesis. In the last several years, the significance of specific cells and chemical signals associated with the immune response in both suppressing and encouraging tumor development has been documented. Many of these will be discussed further in the section later in this chapter entitled “The Immune Response to Cancer.” Cancer-Promoting Activity of Oncogenes The proteins encoded by a particular oncogene and its corresponding proto-oncogene have a very similar function. Sequence comparisons of viral and cellular oncogenes reveal that they are highly conserved in evolution. Although most cellular oncogenes consist of the typical series of exons and introns, their viral counterparts consist of uninterrupted coding sequences, suggesting that the virus acquired the oncogene through an intermediate RNA transcript from which the intron sequences were removed during RNA processing. The actual coding sequences of viral oncogenes and the corresponding proto-oncogenes exhibit a high degree of homology. In some cases, a single point mutation is all that distinguishes a viral oncogene from the corresponding proto-oncogene. In many cases, the conversion of a proto-oncogene into an oncogene accompanies a mere change in the level of expression of a normal growthcontrolling protein. One category of proto-oncogenes and their viral counterparts encodes growth factors or growth factor receptors. Included among these are sis, which encodes a form of platelet-derived growth factor, and fms, erbB, and neu, which encode growth factor receptors (see Table 19-2). In normal

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Functional classification of cancer-associated genes

Type/name

Nature of gene product CATEGORY I: GENES THAT INDUCE CELLULAR PROLIFERATION

Growth factors sis

A form of platelet-derived growth factor (PDGF)

Growth factor receptors fms erbB neu erbA

Receptor for colony-stimulating factor 1 (CSF-1) Receptor for epidermal growth factor (EGF) Protein (HER2) related to EGF receptor Receptor for thyroid hormone

Signal transducers src abl Ha-ras N-ras K-ras

Tyrosine kinase Tyrosine kinase GTP-binding protein with GTPase activity GTP-binding protein with GTPase activity GTP-binding protein with GTPase activity

Transcription factors jun fos myc

Component of transcription factor AP1 Component of transcription factor AP1 DNA-binding protein CATEGORY II: TUMOR SUPRESSOR GENES, INHIBITORS OF CELLULAR PROLIFERATION*

Rb

Suppressor of retinoblastoma

TP53

Nuclear phosphoprotein that inhibits formation of small-cell lung cancer and colon cancers

DCC

Suppressor of colon carcinoma

APC

Suppressor of adenomatous polyposis

NF1

Suppressor of neurofibromatosis

WT1

Suppressor of Wilm’s tumor CATEGORY III: GENES THAT REGULATE PROGRAMMED CELL DEATH

bcl-2

Suppressor of apoptosis

Bcl-xL

Suppressor of apoptosis

Bax

Inducer of apoptosis

Bim

Inducer of apoptosis

Puma

Inducer of apoptosis

*

The activity of the normal products of the category II genes inhibits progression of the cell cycle. Loss of a gene or its inactivation by mutation in an indicated tumorsuppressor gene is associated with development of the indicated cancers.

cells, the expression of growth factors and their receptors is carefully regulated. Usually, one population of cells secretes a growth factor that acts on another population of cells carrying the receptor for the factor, thus stimulating proliferation of the second population. Inappropriate expression of either a growth factor or its receptor can result in uncon-

trolled proliferation. In breast cancer, increased synthesis of the growth factor receptor encoded by c-neu has been linked with a poor prognosis. Other oncogenes in this category encode products that function in signal transduction pathways or as transcription factors. The src and abl oncogenes encode tyrosine

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kinases, and the ras oncogene encodes a GTP-binding protein (see Table 19-2). The products of these genes act as signal transducers. The myc, jun, and fos oncogenes all encode transcription factors. Overactivity of any of these oncogenes may result in unregulated proliferation. Some transformations arise due to chromosomal translocations that bring proto-oncogenes under the control of other genomic regions. For instance, a number of B- and T-cell leukemias and lymphomas arise from translocations involving immunoglobulin or T-cell receptor loci, very transcriptionally active regions in these cells. One of the best characterized is the translocation of c-myc from its position on chromosome 8 to the immunoglobulin heavy-chain enhancer region on chromosome 14, accounting for 75% of Burkitt’s lymphoma cases (Figure 19-3). In the remaining patients with Burkitt’s lymphoma, c-myc remains on chromosome 8 but the ␬ or ␥ light-chain genes are translocated to c-myc on the tip of chromosome 8. As a result of these translocations, synthesis of the c-Myc protein, which functions as a transcription factor controlling the behavior of many genes involved in cell growth and proliferation,

increases and allows unregulated cellular growth. The consequences of enhanced and constitutive myc expression in lymphoid cells have been investigated in transgenic mice. In one study, mice were engineered to express a transgene consisting of all three c-myc exons and the immunoglobulin heavy-chain enhancer. Of 15 transgenic pups born, 13 developed lymphomas of the B-cell lineage within a few months of birth. Cellular transformation has also been associated with mutations in proto-oncogenes. This may be a major mechanism by which chemical carcinogens or x-irradiation convert a proto-oncogene into a cancer-inducing oncogene. For instance, single-point mutations in c-ras, which encodes a GTPase, have been detected in carcinomas of the bladder, colon, and lung (see Table 19-2). Alterations that result in overactivity of the ras oncogene, part of the epidermal growth factor (EGF) receptor signaling pathway, are seen in up to 30% of all human cancers and nearly 90% of all pancreatic cancers. Viral integration into the host-cell genome may in itself serve to convert a proto-oncogene into a transforming oncogene. For

(a) Burkitt’s lymphoma

c–myc 8

(b) Promoter

JH

D

14

CH VH

8 q–

14 q+

Switch region Sμ

5′ L

VH

CH c–myc

3′

Rearranged Ig heavy–chain gene on chromosome 14

3′

Translocated c–myc gene in some Burkitt’s lymphomas

3′

Translocated c–myc gene in other Burkitt’s lymphomas

Enhancer

VH JH

Cμ exons

(c) Sμ

5′ 3

2

1

Enhancer Cμ exons

c–myc exons (d)

5′ 3

2



c–myc exons

FIGURE 19-3 Chromosomal translocations resulting in Burkitt’s lymphoma. (a) Most cases of Burkitt’s lymphoma arise following a chromosomal translocation that moves part of chromosome 8, containing the c-myc gene, to the Ig heavy-chain locus on chromosome 14, shown in more detail in (b). (c) In some cases, the

Cμ exons

entire c-myc gene is inserted near the Ig heavy-chain enhancer. (d) In other cases, only the coding exons (2 and 3) of c-myc are inserted at the ␮ switch site. The proto-oncogene myc encodes a transcription factor involved in the regulation of many genes with roles in cell growth, proliferation, and apoptosis.

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Cancer and the Immune System example, avian leukosis virus (ALV) is a retrovirus that does not carry any viral oncogenes, yet it is able to transform B cells into lymphomas. This particular retrovirus has been shown to integrate within the c-myc proto-oncogene, resulting in increased synthesis of c-Myc. Some DNA viruses have also been associated with malignant transformation, such as certain serotypes of the human papillomavirus (HPV), which are linked with cervical cancer (see Table 19-1). Tumor-Suppressor Genes A second category of cancer-associated genes—called tumor-suppressor genes, or anti-oncogenes—encodes proteins that inhibit excessive cell proliferation. In their normal state, tumor-suppressor genes prevent cells from progressing through the cell cycle inappropriately, functioning like brakes. A release of this inhibition is what can lead to cancer induction. The prototype of this category of oncogenes is Rb, the retinoblastoma gene (see Table 19-2). Hereditary retinoblastoma is a rare childhood cancer in which tumors develop from neural precursor cells in the immature retina. The affected child inherits a mutated Rb allele; later, somatic inactivation of the remaining Rb allele is what leads to tumor growth. Unlike oncogenes where a single allele alteration can lead to unregulated growth, tumor-suppressor genes require a “two-hit” disabling sequence, as the one functional allele is enough to suppress the development of cancer. Probably the single most frequent genetic abnormality in human cancer, found in 60% of all tumors, is a mutation in the TP53 gene. This tumor-suppressor gene encodes p53, a nuclear phosphoprotein with multiple cellular roles, including involvement in growth arrest, DNA repair, and apoptosis. Over 90% of small-cell lung cancers and over 50% of breast and colon cancers have been shown to be associated with mutations in TP53. The Role of Apoptotic Genes A third category of cancer-associated genes is sequences involved in programmed cell death, or apoptosis. These genes can encode proteins that either induce or block apoptosis. Pro-apoptotic genes act like tumor suppressors, normally inhibiting cell survival, whereas anti-apoptotic genes behave more like oncogenes, promoting cell survival. Thus, a failure of the former or overactivity of the latter can encourage neoplastic transformation of cells. Included in this category are genes such as bcl-2, an antiapoptosis gene (see Table 19-2). This oncogene was originally discovered because of its association with B-cell follicular lymphoma. Since its discovery, bcl-2 has been shown to play an important role in regulating cell survival during hematopoiesis and in the survival of selected B cells and T cells during maturation. Interestingly, the EpsteinBarr virus (which can cause infectious mononucleosis) contains a gene that has sequence homology to bcl-2 and may act in a similar manner to suppress apoptosis.

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Malignant Transformation Involves Multiple Steps The development from a normal cell to a cancerous cell is typically a multistep process of clonal evolution driven by a series of somatic mutations. These mutations progressively convert the cell from normal growth to a precancerous state and finally a cancerous state, where all barriers designed to contain cell growth have been surmounted. Induction of malignant transformation appears to involve at least two distinct phases: initiation and promotion. Initiation involves changes in the genome but does not, in itself, lead to malignant transformation. Malignant transformation can occur during the rampant cell division that follows the initiation phase, and results from the accumulation of new DNA alterations, typically affecting proto-oncogenes, tumorsuppressor genes or apoptotic genes, that lead to truly unregulated cellular growth. The presence of myriad chromosomal abnormalities in precancerous and cancerous cells lends support to the role of multiple mutations in the development of cancer. This has been demonstrated in human colon cancer, which typically progresses in a series of well-defined morphologic stages (Figure 19-4). Colon cancer begins as small, benign tumors called adenomas in the colorectal epithelium. These precancerous tumors grow, gradually displaying increasing levels of intracellular disorganization until they acquire the malignant phenotype (Figure 19-4b). The morphologic stages of colon cancer have been correlated with a sequence of gene changes (Figure 19-4a) involving inactivation or loss of three tumor-suppressor genes (APC, DCC, and TP53) and activation of one cellular proliferation oncogene (K-ras). Studies with transgenic mice also support the role of multiple steps in the induction of cancer. Transgenic mice expressing high levels of Bcl-2, a protein encoded by the anti-apoptotic gene bcl-2, develop a population of small resting B cells (derived from secondary lymphoid follicles) that have greatly extended life spans. Gradually, these transgenic mice develop lymphomas. Analysis of lymphomas from these mice has shown that approximately half have a c-myc translocation (a proto-oncogene) to the immunoglobulin H-chain locus. The synergism of Myc and Bcl-2 is highlighted in double-transgenic mice produced by mating the bcl-2⫹ transgenic mice with myc⫹ transgenic mice. These mice develop leukemia very rapidly. The role of DNA mutations and the progressive genomic changes that can lead to the induction of cancer are clearly illustrated by diseases such as xeroderma pigmentosum (XP). This rare disorder is inherited in an autosomal recessive manner and is caused by defects in any one of a number of genes involved in normal DNA repair, collectively known as the nucleotide excision repair (NER) pathway. Individuals with this disease are unable to repair ultraviolet (UV)induced mutations, which occur naturally with even moderate exposure to sunlight and are typically repaired via the NER pathway. A buildup of unrepaired DNA in the

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(a)

Chromosomal site

5q

12p

18q

17p

Alteration

Loss

Activation

Loss

Loss

Gene

APC

DCC

TP53

Normal epithelium

K-ras DNA hypomethylation

Hyperproliferative epithelium

Early adenoma

Other alterations

Intermediate adenoma

Late adenoma

Carcinoma

Metastasis

(b)

Normal

APC

Early adenoma

K-ras

Intermediate adenoma

FIGURE 19-4 Model of sequential genetic alterations leading to metastatic colon cancer. Each of the stages indicated in (a) is morphologically distinct, as illustrated in (b), allowing researchers to determine the sequence of genetic alterations. In this sequence, benign colorectal polyps progress to carcinoma following mutations resulting in the inactivation or loss of three tumor-suppressor

cutaneous cells of young children with XP leads to random genomic alterations, including to genes involved in regulating normal cell growth and division. This leads to unregulated growth of some cells, allowing further DNA mutations to occur and promoting the development of neoplasms, such as malignant melanoma or squamous cell carcinoma, the most common forms of skin cancer in XP patients. The mean age of skin cancer in children with XP is age 8, as compared to 60⫹ years of age in the general population. Figure 19-5 shows a child with the early skin manifestations common to XP. Accumulating data from several recent studies suggest that within a growing tumor, not all cells have equal potential for unlimited growth. Clinical studies of at least three different types of tumors, including those originating in the gut, brain, and skin, all suggest that a subset of cells within a tumor may be the true engines of tumor growth. This subset (called cancer stem cells) displays true unlimited regenera-

DCC

Late adenoma

TP53

Carcinoma

genes (APC, DCC, and TP53) and the activation of one oncogene linked to cellular proliferation (K-ras). [(a) Adapted from B. Vogelstein and K. W. Kinzler, 1993, The multistep nature of cancer, Trends in Genetics 9:138. (b) Adapted from P. Rizk and N. Barker, 2012, Gut stem cells in tissue renewal and disease: Methods, markers, and myths, Systems Biology and Medicine 4:5, 475–496.]

tive potential, and is the major producer of the other cancer cells, populating the mass of the tumor by sharing this unrestrained ability to divide. If these new discoveries prove valid and can be applied more broadly, future therapies will undoubtedly require that we hone in on these rare cancer stem cells, cutting off the source of tumor cell expansion and, potentially, providing hope for cancer eradication.

Tumor Antigens Neoplastic cells are self cells and thus most of the antigens associated with them are subject to the tolerance-inducing processes that maintain homeostasis and inhibit the development of autoimmunity. However, unique or inappropriately expressed antigens can be found in many tumors and are frequently detected by the immune system. Some of these antigens may be the products of oncogenes, where

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Examples of common tumor antigens

TABLE 19-3

Category

|

Antigen/s

Associated cancer types

Tumor-Specific Antigens (TSAs)

Viral

HPV: L1, E6, E7

Cervical carcinoma

HBV: HBsAg

Hepatocellular carcinoma

SV40: Tag

Malignant pleural mesothelioma (cancer of the lung lining)

Tumor-Associated Antigens (TAAs) MUC1

Breast, ovarian

MUC13/CA-125

Ovarian

HER-2/neu

Breast, melanoma, ovarian, gastric, pancreatic

MAGE

Melanoma

PSMA

Prostate

TPD52

Prostate, breast, ovarian

CEA

Colon

Gp100

Melanoma

Overexpression

FIGURE 19-5 Xeroderma pigmentosum. This rare autosomal-recessive inherited disorder arises from mutations to one of several genes involved in DNA repair. This disorder is characterized by extreme skin sensitivity to ultraviolet light, abnormal skin pigmentation, and a high frequency of skin cancers, especially on sun-exposed skin of the face, neck and arms. [CID/ISM/Phototake]

there is no qualitative difference between the oncogene and proto-oncogene products; instead, it is merely increased levels of the oncogene product that can be recognized by the immune system. Most tumor antigens give rise to peptides that are recognized by the immune system following presentation by self major histocompatibility complex (MHC) molecules. In fact, many of these antigens have been identified by their ability to induce the proliferation of antigen-specific cytotoxic T lymphocytes (CTLs) or helper T cells. To date, tumor antigens recognized by human T cells fall into one of four groups based on their source:

Differentiation AFP Stage Tyrosinase

Hepatocellular carcinoma Melanoma

PSA

Prostate

PAP

Prostate

Abbreviations: SV40, simian virus 40; L, late gene; E, early gene; HBsAg, hepatitis B surface antigen; Tag, large tumor antigen; MUC, mucin; MAGE, melanomaassociated antigen; HER/neu, human epidermal receptor/neurological; PSMA, prostate-specific membrane antigen; TP, tumor protein; PSA, prostate-specific antigen; PAP, prostatic acid phosphatase. Source: Adapted from Table 1 in J. F. Aldrich, et al., 2010. Vaccines and immunotherapeutics for the treatment of malignant disease, Clinical and Developmental Immunology.

• Antigens encoded by genes exclusively expressed by tumors (e.g., viral genes) • Antigens encoded by variant forms of normal genes that are altered by mutation • Antigens normally expressed only at certain stages of development • Antigens that are overexpressed in particular tumors Table 19-3 lists several categories of common antigens associated with tumors. As one can imagine, many clinical

research studies aim to utilize these antigens as diagnostic or prognostic indicators, as well as therapeutic targets for tumor elimination. There are two main types of tumor antigens, categorized by their uniqueness: tumor-specific antigens (TSAs) and tumor-associated antigens (TAAs). Originally these were designated as transplantation antigens (TSTAs and TATAs), stemming from studies in which these antigens were discovered by transplanting them into recipient animals, inducing a rejection immune response.

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Normal cell

Self peptide

Self peptide Class I MHC Class I MHC Altered self peptide

Oncofetal peptide

Mutation generates a new peptide presented by class I MHC (TSA)

Overexpression of normal protein (TAA)

Inappropriate expression of embryonic gene (TAA)

FIGURE 19-6 Different mechanisms generate tumorspecific antigens (TSAs) and tumor-associated antigens (TAAs). TSAs are unique to tumor cells, and can result from DNA mutations that lead to expression of altered self proteins or from expression of viral antigens (not shown) in transformed cells. TAAs

Tumor-Specific Antigens Are Unique to Tumor Cells TSAs are unique proteins that may result from mutations in tumor cells that generate altered proteins and, therefore, new antigens. Cytosolic processing of these proteins then gives rise to novel peptides that are presented with class I MHC molecules (Figure 19-6), inducing a cell-mediated response by tumor-specific CTLs. TSAs have been identified on tumors induced with chemical or physical carcinogens, as well as on some virally induced tumors. Demonstrating the presence of TSAs on spontaneously occurring or chemically induced tumors is particularly difficult. These antigens can be quite diverse and are only identified by their ability to induce T-cell-mediated rejection. The immune response to such tumors typically eliminates all of the tumor cells bearing sufficient numbers of these unique antigens, and thus selects for cells bearing few or none. Nonetheless, experimental methods have been developed to facilitate the characterization of TSAs, which have been shown to differ from normal cellular proteins by as little as a single amino acid. Further characterization of TSAs has demonstrated that many of these antigens are not cell membrane proteins; rather, they are short peptides derived from cytosolic proteins that have been processed and presented together with class I MHC molecules. In contrast to chemically induced tumors, virally induced tumors express tumor antigens shared by all tumors induced

are more common and represent normal cellular proteins displaying unique expression patterns, such as an embryonic protein expressed in the adult or overexpression of self proteins. Both types of tumor antigens can be detected by the immune system following presentation in class I MHC.

by the same virus, making their characterization simpler. For example, when syngeneic mice are injected with killed cells from a particular polyoma virus-induced tumor, the recipients are protected against subsequent challenge with live cells from any polyoma-induced tumors. Likewise, when lymphocytes are transferred from mice with a virus-induced tumor into normal syngeneic recipients, the recipients reject subsequent transplants of all syngeneic tumors induced by the same virus, suggesting that lymphocytes recognize and kill cells expressing a virally derived TSA. In some cases, the presence of virus-specific tumor antigens is an indicator of neoplastic transformation. In humans, Burkitt’s lymphoma cells have been shown to express a nuclear antigen of the Epstein-Barr virus that may indeed be a tumorspecific antigen for this type of tumor. HPV E6 and E7 proteins are found in more than 80% of invasive cervical cancers, and they provide the clearest example of a virally encoded tumor antigen. In fact, the first clinically approved cancer vaccine is one used to prevent infection with HPV and block the emergence of cervical cancer (see Clinical Focus Box 19-1).

Tumor-Associated Antigens Are Normal Cellular Proteins with Unique Expression Patterns In contrast to TSAs, TAAs are not unique to the cancer. Instead, these represent normal cellular proteins typically

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BOX 19-1

CLINICAL FOCUS

A Vaccine to Prevent Cervical Cancer, and More Globally, cervical cancer is the third leading cause of death among women, and second only to breast cancer in terms of cancer deaths in women. Over 500,000 women each year develop cervical cancer (80% in developing countries), and approximately 275,000 women die from the disease annually. Periodic cervical examination (using the Papanicolaou test, or Pap smear) to detect abnormal cervical cells significantly reduces the risk for women. However, a health-care program that includes regular Pap smears is commonly beyond the means of the less advantaged and is largely unavailable in many developing countries. Human papillomavirus (HPV), the most common sexually transmitted infection, is implicated in over 99% of cervical cancers. HPV is also associated with most cases of vaginal, vulval, anal, penile, and oropharyngeal (head and neck) cancers, as well as genital warts. Among the hundred-plus genotypes of HPV, approximately 40 are associated with genital or oral infections. Two of these, types 16 and 18, account for more than 70% of all instances of cervical cancer, whereas types 6 and 11 are most often involved in HPV-associated genital warts. It is estimated that the majority of sexually active men and women become infected with HPV at some point in their lives. A study of female college students at the University of Washington published in 2003 showed that after 5 years, more than 60% of study participants (all of whom were HPV negative when enrolled in the study) became infected. Most infections are resolved without disease; it is persistent infection leading to cervical or anal intraepithelial neoplasia that is associated with high cancer risk. Preventing cervical cancer, therefore, appears to be a matter of preventing HPV infection. Gardasil (manufactured by Merck), the first vaccine ever approved for the prevention of cancer, was licensed in 2006 for the prevention of infection with HPV and potential development of cervical cancer or genital warts. This quadrivalent formulation targets HPV types 6, 11, 16, and 18. Three years later, GlaxoSmithKline received a

license for Cervarix, a vaccine to prevent cervical cancer that targets only HPV types 16 and 18. These vaccines are between 95% and 99% effective in preventing infection by HPV. Conclusive evidence that this will translate into significantly reduced rates of cervical cancer in women, which can take many years to develop, will not be available until long-term follow-up studies have been completed. In June 2006, the federal Advisory Committee on Immunization Practices (ACIP) recommended routine HPV vaccination for girls ages 11 to 12, and catch-up immunizations for females ages 13 to 26 who have not already received the vaccine. Although the committee did not recommend routine immunization for boys at that time, it did suggest that Gardasil be made available to males ages 9 to 26. As of 2007, 25% of 13- to 17-year-old girls in the United States reported receiving at least one dose of this vaccine. In 2011, this number rose to 53% in girls, still far short of targeted numbers (~80%) and significantly lower than the rates of compliance for most other routine childhood vaccines (somewhere around 90%, depending on the age of the child). In 2011, boys ages 11 to 13 were added to the ACIP list of recommendations for routine HPV vaccination. The hope is that this will curb the rising tide of anal and oropharyngeal cancers among men, but also cut back the infection cycle and impact rates of cervical cancer in women. However, HPV vaccination rates among young men remained at only 8% at the end of 2011. The idea was that, with Gardasil in particular, the ability to reduce the incidence of unsightly genital warts might provide added incentive for male vaccination. With a safe and effective vaccine against a common and deadly cancer available for several years, why are the rates of immunization in young people still so low? The answer depends somewhat on the country in question, as well as social and economic factors. Especially in developing countries, cost and ease of use are major barriers. Development is currently underway for a second generation of HPV vaccines, which

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are more cost effective, easier to administer and produce longer-lived immunity to a broader range of HPV genotypes. Controversy based on social and ethical issues, as well as misinformation, are also high on the list of reasons why HPV vaccination rates are believed to remain so low. In U.S.-based studies of factors that influence decisions to vaccinate adolescents against HPV, mother’s attitudes, physician recommendations, and misunderstandings in all groups were highlighted. For instance, since HPV is a sexually transmitted infection, most parents prefer to consider this an issue for “the future,” assuming that their children are not sexually active and that there is plenty of time before a vaccine for a sexually transmitted disease should be considered. In fact, based on the Centers for Disease Control and Prevention (CDC) surveillance data from 2011, 47% of high school students in the U.S. have had sexual intercourse; greater than 6% beginning before the age of 13. The HPV vaccine regimen, which involves three intramuscular injections administered over a 6-month period, is most effective when completed prior to exposure, and produces the most robust immune response in 11- to 12-year-olds, the target population. Physician recommendations are also key to making a dent in the rates of HPV vaccination. In a 2011 study, women 19 to 26 years old were asked about whether they had received an HPV vaccine. In the group that had received a provider recommendation, 85% were immunized, compared with only 5% among women who did not receive a physician recommendation. More public and professional information concerning the advantages of this vaccine before the onset of sexual activity, as well as the lifetime risk of disease caused by HPV, may help drive down the cycle of infection and worldwide deaths due to this sexually transmitted killer. Winer RL, Lee SK, Hughes JP, Adam DE, Kiviat NB, Koutsky LA.Genital human papillomavirus infection: incidence and risk factors in a cohort of female university students. Am J Epidemiol. 2003 Feb 1;157(3):218–26. CDC. Youth risk behavior surveillance—United States, 2011. MMWR 2012;61(SS-4).

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expressed only during specific developmental stages, such as in the fetus, or at extremely low levels in normal conditions, but which are upregulated in tumor cells (see Figure 19-6). Those derived from mutation-induced reactivation of certain fetal or embryonic genes, called oncofetal tumor antigens, normally only appear early in embryonic development, before the immune system acquires immunocompetence. When transformation of cells causes them to appear at later stages of development on neoplastic cells of the adult, they are recognized as nonself and induce an immunologic response. Two well-studied oncofetal antigens are alpha-fetoprotein (AFP) and carcinoembryonic antigen (CEA). AFP, the most abundant fetal protein, drops from milligram levels in fetal serum to between 5 ng/ml and 50 ng/ml after birth. Elevated levels of this glycoprotein can also be found in women, especially during the early stages of pregnancy. Significantly elevated AFP levels in nonpregnant adults are not uncommon in ovarian, testicular, and liver cancers, where serum levels above 300 ng/ml can be indicative of small lesions even in asymptomatic individuals. Monitoring of these levels can help clinicians to make prognoses and to evaluate treatment efficacy, especially in liver cancer. CEA is another oncofetal membrane glycoprotein found on gastrointestinal and liver cells of 2- to 6-month-old fetuses. Approximately 90% of patients with advanced colorectal cancer and 50% of patients with early colorectal cancer have increased levels of CEA in their serum; some patients with other types of cancer also exhibit increased CEA levels. However, because AFP and CEA can be found in trace amounts in some normal adults and in some noncancerous disease states, the presence of these oncofetal antigens is not necessarily diagnostic of tumors but can still be used to monitor tumor growth. If, for example, a patient has had surgery to remove a colorectal carcinoma, CEA levels are monitored after surgery; an increase in the CEA level is an indication that tumor growth has resumed. In addition to embryonic antigens, the category of TAAs also includes the products of some oncogenes, such as several growth factors and growth factor receptors. These proteins, although transcribed in the adult, are normally tightly regulated and expressed only at low levels. For instance, a variety of tumor cells express the epidermal growth factor (EGF) receptor at levels 100 times greater than in normal cells. Another, melanotransferrin, designated p97, has fibroblast growth factor-like activities. Whereas normal cells express fewer than 8,000 molecules of p97 per cell, melanoma cells express 50,000 to 500,000 molecules per cell. The gene that encodes p97 has been cloned, and a recombinant vaccinia virus vaccine has been prepared that carries the cloned gene. When this vaccine was injected into mice, it induced both humoral and cell-mediated immune responses, which protected the mice against live melanoma cells expressing the p97 antigen. Targeting such oncogene products has yielded some significant clinical success, such as in the case of breast cancers overexpressing HER2 (discussed further in the final section of this chapter).

The Immune Response to Cancer As mentioned earlier, cells have multiple built-in (or intrinsic) mechanisms to prevent cancer. One example is the NER pathway of DNA repair, which encourages cell senescence (permanent cell cycle arrest) or even apoptosis at the first signs of unregulated growth. However, in the event that this system fails, control mechanisms external to the cell can kick in. At their most basic, these cell-extrinsic mechanisms involve environmental signals that instruct a cell to activate internal pathways leading to growth arrest or apoptosis, to prevent cancer cell spread. For example, when epithelialcell/extracellular-matrix associations are disrupted due to malignant transformation, death signals are triggered that block proliferation and spread of these contact dependent cells. Thus these extracellular attachments serve as inhibitors of cell death, which when broken set off a safety mechanism promoting apoptosis. If unregulated growth continues, identification and rejection of tumor cells by components of the immune system may help salvage homeostasis. Although several key immune cell types and effector molecules that participate in this response have been identified in recent years, much still remains to be learned about natural mechanisms of anti-tumor immunity and how best to induce these in clinical settings. For over a century, the controversy over whether and how the immune system participates in cancer recognition and destruction has raged. However, data collected in the past couple of decades from both animal models and clinical studies has clearly defined a role for the immune response in tumor cell identification and eradication. To date, there are three proposed mechanisms by which the immune system is thought to control cancer: • By destroying viruses that are known to transform cells • By eliminating pathogens and reducing pro-tumor inflammation • By actively identifying and eliminating cancerous cells This final mechanism, involving tumor cell identification and eradication, is termed immunosurveillance. It posits that the immune system continually monitors for and destroys neoplastic cells. Evidence from animal models as well as immune deficiency disorders and induced immune suppression in humans supports this hypothesis. For instance, AIDS patients and transplant recipients on immunosuppressive drugs have a much higher incidence of several types of cancer than do individuals with fully competent immune systems. However, recent data also point to potential pro-tumor influences of the immune response on cancer. For instance, chronic inflammation and immune-mediated selection for malignant cells may actually contribute to cancer cell spread and survival. Contemporary studies of immunity to cancer have now generated a more nuanced hypothesis of immune involvement in neoplastic regulation. This model is called

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immunoediting; it incorporates observations of both tumor-inhibiting and tumor-enhancing processes mediated by the immune system. In the following section we describe this revised view of the role of the immune response in cancer.

immune system in cancer eradication or survival during cancer immunoediting.

Immunoediting Both Protects Against and Promotes Tumor Growth

As early as the 1860s, Rudolph Virchow observed that leukocytes infiltrate the site of a solid tumor, and he proposed a link between inflammation and cancer induction. However, as infiltrating leukocytes can be found in both cancers that progress and those that resolve, the role of inflammation in immunity to cancer was obscure. More recent scientific advances have allowed a more detailed analysis of the location and type of cells involved, leading to the identification of key indicators of cancer regression in certain types of tumors. With this newfound knowledge, advances are being made in cancer diagnostic tools, prognostic indicators, and targets for clinical intervention. Much of the research aimed at probing the relationship between the immune system and cancer has employed mouse model systems. Many or even most mice rendered immune deficient via targeted gene knockouts or neutralizing antibodies display some increased incidence of cancer, be it spontaneous or carcinogen induced. Likewise, spontaneous or induced tumors in immune-competent mice have allowed scientists to model human cancer induction and test hypotheses concerning specific cell types and immune pathways in cancer eradication. Although many of the elements of both innate and adaptive immunity can be linked in some way to tumor-cell recognition and destruction, certain components appear to play key roles in immune-mediated cancer control. Studies in mice and humans have led to the awareness that there are both “good” and “bad” forms of inflammation in the response to cancer (e.g. Figure 19-7, bottom right). On the good or anti-tumor side are innate responses dominated by immune-activating macrophages (called M1), cross-presenting dendritic cells (DCs; see Chapter 8), and NK cells. These cells and the cytokines they produce help elicit strong TH1 and CTL responses, which are associated with a good prognosis and tumor regression. Conversely, the immune cell infiltrates found in tumors that are more likely to progress and metastasize include anti-inflammatory macrophages (M2) and myeloid-derived suppressor cells (MDSCs). Concomitantly, adaptive responses to cancer dominated by the TH2 pathway (and in some cases, also TH17 or Treg cells) are associated with poorer clinical outcomes and reduced survival times. In the following sections, we discuss in greater detail our developing awareness of both the positive and negative relationships of some of these specific pathways to cancer immunity.

In the mid-1990s, research in animal models of cancer suggested that natural immunity could eliminate tumors. Armed with this understanding, researchers identified some of the key cell types and effector molecules involved. Experimental studies showed that mice lacking intact T-cell compartments or interferon (IFN)-␥ signaling pathways were more susceptible to chemically induced or transplanted tumors, respectively. Likewise, RAG2 knockout mice, which fail to generate T, B, or natural killer T (NKT) cells, were more likely to spontaneously develop cancer as they aged and were more susceptible to chemical carcinogens. However, the real surprise came when scientists used the same chemical carcinogen to induce tumors in both wildtype and RAG2 knockout mice, then adoptively transferred these tumors into syngeneic wild-type recipients. (See Chapters 12 and 20 for further discussion of adoptive transfer.) All of the tumors coming from wild-type animals grew aggressively in their new hosts, whereas up to 40% of the tumors taken from immunodeficient mice were rejected by recipients. This suggested that tumors growing in immunedeficient environments are more immunogenic than those arising in an immunocompetent environment. These observations led to the idea that the immune system exerts a dynamic influence on cancer, inhibiting tumor cells but also sculpting them in a Darwinian process of selection: those that survive are better able to outwit the immune response and have a survival advantage. The three currently proposed phases to the immunoediting hypothesis are elimination, equilibrium, and escape (Figure 19-7). The first phase, elimination, is the traditional view of the immune system as a major player in the identification and destruction of newly formed cancer cells. Equilibrium is the proposed second phase, characterized by a state of balance between destruction and survival of a small number of neoplastic cells. Ample clinical evidence now suggests that phase 2 can continue for up to decades after the emergence of a tumor. However, identifying residual transformed cells and targeting them during this window is challenging. Escape is the final phase of cancer progression, when the most aggressive and least immunogenic of the residual tumor cells begin to thrive and spread. Most basic research studies have focused on the role of the immune system in the elimination phase, where both innate and adaptive processes identify and target transformed cells for destruction, sometimes setting the stage for what will occur during the equilibrium and escape phases. The following sections discuss our current understanding of the role of the

Key Immunologic Pathways Mediating Tumor Eradication Have Been Identified

Innate Inhibitors of Cancer Identified over 35 years ago, natural killer (NK) cells were among the first cell type to be recognized for their inherent ability to destroy tumor cells, from which their name derives. Mice rendered NK cell deficient, either by gene knockout or via neutralizing antibodies, show an increased

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OVERVIEW FIGURE

The Three Stages of Cancer Immunoediting Healthy tissue Malignant transformation– carcinogens, viral infection, chronic inflammation, genetic predisposition

Intrinsic tumor suppression (e.g., repair, apoptosis)

Tumor antigen

NKG2D ligands

Transformed cells

Phase 1: Elimination (cancer immunosurveillance) CD8⫹ T cell

NKT cell

Phase 2: Equilibrium (cancer persistence/dormancy)

NK cell

CD8⫹ T cell

CD4⫹ T cell

IL-12

Phase 3: Escape (cancer progression) CD8⫹ T cell

CD4⫹ T cell

NK cell M1 Mφ

Anti-tumor microenvironment, including IL-12 and IFN-γ

IFN-γ Gradual progression

CD4⫹ M1 Mφ T cell

Innate and adaptive immunity (including IFN-α/β, IFN-γ, IL-12)

Genetic instability and immunoselection (i.e., editing)

CTLA-4 M2 Mφ

Protection (i.e., extrinsic tumor suppression)

PD-L1 MDSC

CTLA-4

CD8⫹ T cell

Pro-tumor microenvironment, including TGF-β, IDO, IL-10 and chronic inflammation

TREG

Cancer immunoediting

Recognition and targeting of tumor cells by the immune system is believed to occur in three phases. Phase I, Elimination: cancer cells are recognized by the immune system via their tumor antigens and targeted for destruction. In the process, some cells acquire mutations that allow them to resist immune destruction. Phase II, Equilibrium: low levels of abnormal cells persist, but their proliferation and spread are held in check by the adaptive immune response. Phase III, Escape: further mutation in the surviving tumor cells leads

incidence of lymphomas and sarcomas. The importance of NK cells in tumor immunity is highlighted by the mutant mouse strain called beige and by Chediak-Higashi syndrome in humans. In both, a genetic defect causes marked impairment of NK cells, and in each case an associated increase is present in certain types of cancer.

to the capacity for immortal growth and metastasis. Over time, inhibitory immune responses begin to dominate and immune activity shifts from anti- to pro-tumor. Tan cells are normal; pink to red cells represent progressive development of decreased immunogenicity in tumor cells. Abbreviations: M⌽, macrophage; MDSC, myeloid-derived suppressor cells; PD-L1, programmed death ligand 1. [Modified from M. D. Vesely, et al., 2011, Natural innate and adaptive immunity to cancer, Annual Review of Immunology 29:235–271.]

NK cell recognition mechanisms use a series of surface receptors that respond to a balance of activating and inhibiting signals delivered by self cells (see Chapter 13). Since many transforming viruses can induce the downregulation of MHC expression, detecting “missing self ” is likely at least one of the ways in which NK cells participate in tumor-cell

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Cancer and the Immune System identification and eradication. However, inducing signals in the form of molecular expressions of danger can also engage NK cell-activating receptors (e.g., NKG2D) delivering what is now referred to as an “altered” or “induced-self ” signal (see Figure 19-7). Various forms of cellular stress, including viral infection, heat shock, UV radiation, and other agents that induce DNA damage can trigger expression of the ligands for these activating NK cell receptors. Using activating signals induced by DNA damage pathways, NK cells may thus be able to distinguish cancerous or precancerous cells from healthy neighboring cells. Once engaged, these cells use cytolytic granules that include such compounds as perforin to target their killing machinery at cells expressing these activating ligands. In fact, deficiency in perforin, a cytolytic compound used by both NK cells and CTLs to kill target cells, is linked to increased cancer susceptibility. Indirectly, NK cells may also participate in cancer eradication by secreting IFN-␥, a potent anticancer cytokine that encourages DCs to stimulate strong CTL responses in vitro (see adaptive responses below). Numerous observations indicate that activated macrophages also play a significant role in the immune response to tumors. For example, macrophages are often observed to cluster around tumors, and the presence of proinflammatory macrophages, such as type M1, is correlated with tumor regression. Like NK cells, macrophages are not MHC restricted and express Fc receptors, enabling them to bind to antibody on tumor cells and to mediate antibody-dependent cell-mediated cytotoxicity (ADCC; discussed further below). The anti-tumor activity of activated macrophages is likely mediated by lytic enzymes, as well as reactive oxygen and nitrogen intermediates. In addition, activated macrophages secrete a cytokine called tumor necrosis factor alpha (TNF␣) that has potent anti-tumor activity. More recently, a previously unsuspected role for the eosinophil in cancer immunity has also come to light. Mice engineered to lack eotaxin or CCL11, two chemoattractants for eosinophils, or IL-5, a stimulatory cytokine for this cell type, were all found to be more susceptible to carcinogeninduced cancers than wild-type mice. In addition, IL-5 transgenic animals, which display higher levels of circulating eosinophils, are more resistant to chemically induced sarcomas. Adaptive Cell Types Involved in Cancer Eradication In experimental animals, tumor antigens induce humoral and cell-mediated immune responses that lead to the destruction of transformed cells expressing these proteins. Animals that lack either ␣␤ or ␥␦ T cells are more susceptible to a number of induced and spontaneous tumors. Several tumors have been shown to induce CTLs that recognize tumor antigens presented by class I MHC on these neoplastic cells. In fact, strong anti-tumor CTL activity correlates significantly with tumor remission and is primarily credited with maintaining the equilibrium stage of cancer detection, or a state of immune-mediated neoplastic cell dormancy (see

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Figure 19-7). Evidence for this comes from studies of mice treated with low-dose carcinogens. In the fraction of animals that do not develop cancer, rendering the animals immunodeficient can result in the sudden onset of cancer. Importantly, blocking NK-cell responses at this stage does not result in eruption of occult cancer, but blocking CD8⫹ T-cell responses or IFN-␥ does, highlighting the importance of adaptive immunity during this stage of cancer. The pressures of adaptive immunity on cancerous cells during this relatively long stage are believed to sculpt tumors (thus the immunoediting name), driving the selective survival of neoplastic cells that are less immunogenic and have accumulated mutations most favorable for immune evasion. One example of this is increased expression of PD-L1 on tumor cells, which binds to PD-1 on CTLs and inhibits engagement (see Figure 19-7). In clinical studies of cancer, the frequency of tumorinfiltrating lymphocytes (TILs)—a combination of T cells, NKT cells, and NK cells—correlates with a prognosis of cancer regression. For instance, in one seminal study of ovarian cancer, 38% of women with high numbers of TILs compared with 4.5% of women with low numbers of TILs survived more than 5 years past diagnosis. However, beyond numbers, the type of infiltrating cells may be even more crucial and in some cases can have more prognostic power than clinical cancer staging. In general, a high frequency of CD8⫹ T cells, and sometimes a high ratio of CTL to TREG cells is associated with enhanced survival. However, the story with TREG cells is complicated; several studies have observed a positive impact of this cell type on anticancer immune responses (discussed further in the following sections). B cells respond to tumor-specific antigens by generating anti-tumor antibodies that can foster tumor-cell recognition and lysis. Using their Fc receptors, NK cells and macrophages again participate in this response, mediating ADCC (see Chapter 13). However, some anti-tumor antibodies serve a more detrimental role, blocking CTL access to tumor-specific antigens and enhancing survival of the cancerous cells. For this reason, a clear positive or negative role for B cells in cancer immunity is less obvious. The Role of Cytokines in Cancer Immunity Animal models in which cytokines or cytokine response pathways are eliminated have helped us to identify the role of specific cytokines in tumor-cell eradication. IFN-␥ and the regulatory components of this pathway are clearly important in cance