Advances in Immunology Volume 99

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Advances in

IMMUNOLOGY VOLUME

99

This page intentionally left blank

Advances in

IMMUNOLOGY VOLUME

99 Edited by

FREDERICK W. ALT Howard Hughes Medical Institute, Boston, Massachusetts, USA Associate Editors

K. FRANK AUSTEN Harvard Medical School, Boston, Massachusetts, USA

TASUKU HONJO Kyoto University, Kyoto, Japan

FRITZ MELCHERS University of Basel, Basel, Switzerland

JONATHAN W. UHR University of Texas, Dallas, Texas, USA

EMIL R. UNANUE Washington University, St. Louis, Missouri, USA

AMSTERDAM • BOSTON • HEIDELBERG • LONDON NEW YORK • OXFORD • PARIS • SAN DIEGO SAN FRANCISCO • SINGAPORE • SYDNEY • TOKYO Academic Press is an imprint of Elsevier

Academic Press is an imprint of Elsevier 32 Jamestown Road, London, NW1 7BY, UK Radarweg 29, PO Box 211, 1000 AE Amsterdam, The Netherlands Linacre House, Jordan Hill, Oxford OX2 8DP, UK 30 Corporate Drive, Suite 400, Burlington, MA 01803, USA 525 B Street, Suite 1900, San Diego, CA 92101-4495, USA Copyright # 2008 Elsevier Inc. All rights reserved No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or by any means electronic, mechanical, photocopying, recording or otherwise without the prior written permission of the publisher Permissions may be sought directly from Elsevier’s Science & Technology Rights Department in Oxford, UK: phone (+44) (0) 1865 843830; fax (+44) (0) 1865 853333; email: [email protected]. Alternatively you can submit your request online by visiting the Elsevier web site at http://elsevier.com/locate/permissions, and selecting Obtaining permission to use Elsevier material Notice No responsibility is assumed by the publisher for any injury and/ or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences, in particular, independent verification of diagnoses and drug dosages should be made ISBN: 978-0-12-374325-1 ISSN: 0065-2776 For information on all Academic Press publications visit our website at elsevierdirect.com Printed and bound in USA 08 09 10 11 10 9 8 7 6 5 4 3 2 1

CONTENTS

Contributors

ix

1. Cis-Regulatory Elements and Epigenetic Changes Control Genomic Rearrangements of the IgH Locus

1

Thomas Perlot and Frederick W. Alt Introduction The Immunoglobulin Heavy Chain Locus V(D)J Recombination During B-Cell Development Class Switch Recombination and Somatic Hypermutation IgH Rearrangements and Allelic Exclusion Accessibility Control IgH Locus Control Through Cis-Regulatory Elements Conclusions Acknowledgments References 1. 2. 3. 4. 5. 6. 7. 8.

2. DNA-PK: The Means to Justify the Ends?

2 3 4 7 8 10 16 23 23 24

33

Katheryn Meek, Van Dang, and Susan P. Lees-Miller Introduction Composition DNA Binding and Kinase Activation Structural Studies of DNA-PK Targets of DNA-PK’s Enzymatic Activity DNA-PK’s Autophosphorylation is Functionally Complex Autophosphorylation Within Two Clusters Reciprocally Regulates DNA End Access 8. Further DNA-PK Autophosphorylation is Required During NHEJ 9. Model of DNA-PK Activation 10. End Processing to Promote End Conservation 11. Does DNA-PK Regulate Dsbr Repair Pathway Choice? 12. Why is DNA-PK So Abundant? References 1. 2. 3. 4. 5. 6. 7.

34 36 37 38 39 39 40 43 44 47 49 50 52

v

vi

Contents

3. Thymic Microenvironments for T-Cell Repertoire Formation

59

Takeshi Nitta, Shigeo Murata, Tomoo Ueno, Keiji Tanaka, and Yousuke Takahama Introduction Trafficking of Developing Thymocytes Cortical Microenvironment Medullary Microenvironment Concluding Remarks Acknowledgments References 1. 2. 3. 4. 5.

4. Pathogenesis of Myocarditis and Dilated Cardiomyopathy

60 64 68 71 78 79 79

95

Daniela Cihakova and Noel R. Rose Human Myocarditis The Evidence for an Autoimmune Process in Myocarditis Mouse Models of Myocarditis Role of Proinflammatory Cytokines in Myocarditis Role of T Helper Cells in Myocarditis The Divergent Role of Macrophages in Myocarditis Conclusions/Directions for Future Research Acknowledgments References

96 100 102 104 104 107 108 109 109

5. Emergence of the Th17 Pathway and Its Role in Host Defense

115

1. 2. 3. 4. 5. 6. 7.

Darrell B. O’Quinn, Matthew T. Palmer, Yun Kyung Lee, and Casey T. Weaver Introduction Emergence of the Th17 Pathway Functions of the Th17-Derived Cytokines IL-23/IL-17-Mediated Innate Immunity Th17 Cells and Innate Immunity Th17 Cells and Acquired Immunity IL-23/IL-17-Mediated Responses in Specific Microbial Infections Closing Remarks References 1. 2. 3. 4. 5. 6. 7. 8.

6. Peptides Presented In Vivo by HLA-DR in Thyroid Autoimmunity

116 118 125 128 129 131 132 147 148

165

Laia Muixı´, In˜aki Alvarez, and Dolores Jaraquemada 1. Introduction 2. Natural Peptides: The Constitutive Ligands of MHC Molecules 3. Natural Ligands for HLA Class II in Autoimmune Thyroid Tissue

166 173 177

Contents

4. Concluding Remarks Acknowledgments References

vii

200 201 201

Index

211

Contents of Recent Volumes

217

See Color Plate Section in the back of this book

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CONTRIBUTORS

Numbers in parentheses indicate the pages on which the authors’ contributions begin.

Frederick W. Alt The Howard Hughes Medical Institute, The Children’s Hospital, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA (1) In˜aki Alvarez Immunology Unit, Institut de Biotecnologia i Biomedicina (IBB), and Department of Cell Biology, Physiology and Immunology, Universitat Auto`noma de Barcelona (UAB), Campus de Bellaterra, 08193 Barcelona, Spain (165) Daniela Cihakova Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA (95) Van Dang College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA (33) Dolores Jaraquemada Immunology Unit, Institut de Biotecnologia i Biomedicina (IBB), and Department of Cell Biology, Physiology and Immunology, Universitat Auto`noma de Barcelona (UAB), Campus de Bellaterra, 08193 Barcelona, Spain (165) Yun Kyung Lee Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA (115) Susan P. Lees-Miller Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada (33) Katheryn Meek College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan State University, East Lansing, Michigan, USA (33)

ix

x

Contributors

Laia Muixı´ Immunology Unit, Institut de Biotecnologia i Biomedicina (IBB), and Department of Cell Biology, Physiology and Immunology, Universitat Auto`noma de Barcelona (UAB), Campus de Bellaterra, 08193 Barcelona, Spain (165) Shigeo Murata Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan (59) Takeshi Nitta Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan (59) Darrell B. O’Quinn Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA (115) Matthew T. Palmer Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA (115) Thomas Perlot University of Vienna, Dr-Karl-Lueger-Ring1, Vienna, Austria; and The Howard Hughes Medical Institute, The Children’s Hospital, Immune Disease Institute, and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA (1) Noel R. Rose Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland; and W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA (95) Yousuke Takahama Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan (59) Keiji Tanaka Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan (59) Tomoo Ueno Division of Experimental Immunology, Institute for Genome Research, University of Tokushima, Tokushima 770-8503, Japan (59) Casey T. Weaver Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA (115)

CHAPTER

1 Cis-Regulatory Elements and Epigenetic Changes Control Genomic Rearrangements of the IgH Locus Thomas Perlot*,† and Frederick W. Alt*

Contents

1. 2. 3. 4.

Introduction The Immunoglobulin Heavy Chain Locus V(D)J Recombination During B-Cell Development Class Switch Recombination and Somatic Hypermutation 5. IgH Rearrangements and Allelic Exclusion 6. Accessibility Control 6.1. Germline transcripts 6.2. Spatial organization and nuclear positioning of the IgH locus 6.3. Chromatin modifications 7. IgH Locus Control Through Cis-Regulatory Elements 7.1. Promoter of DQ52 7.2. VH promoters 7.3. Intronic enhancer 7.4. 30 IgH regulatory region and I promoters 7.5. Additional potential regulatory elements 7.6. Interplay between cis-regulatory elements 8. Conclusions Acknowledgments References

2 3 4 7 8 10 10 12 13 16 16 17 18 20 21 22 23 23 24

* The Howard Hughes Medical Institute, The Children’s Hospital, Immune Disease Institute,

and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA University of Vienna, Dr-Karl-Lueger-Ring1, Vienna, Austria

{

Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00601-9

#

2008 Elsevier Inc. All rights reserved.

1

2

Abstract

Thomas Perlot and Frederick W. Alt

Immunoglobulin variable region exons are assembled from discontinuous variable (V), diversity (D), and joining (J) segments by the process of V(D)J recombination. V(D)J rearrangements of the immunoglobulin heavy chain (IgH) locus are tightly controlled in a tissue-specific, ordered and allele-specific manner by regulating accessibility of V, D, and J segments to the recombination activating gene proteins which are the specific components of the V(D)J recombinase. In this review we discuss recent advances and established models brought forward to explain the mechanisms underlying accessibility control of V(D)J recombination, including research on germline transcripts, spatial organization, and chromatin modifications of the immunoglobulin heavy chain (IgH) locus. Furthermore, we review the functions of well-described and potential new cisregulatory elements with regard to processes such as V(D)J recombination, allelic exclusion, and IgH class switch recombination.

1. INTRODUCTION An individual clone of mature B-cells expresses immunoglobulin (Ig) molecules as an antigen receptor. The typical subunit of an Ig molecule consists of two identical heavy chains (HC) and two identical light chains (LC). The N-terminal region of these chains contains the highly variable antigen binding site; whereas the C-terminal part is called constant region (C region). The C region of the IgH chain (CH) determines the effector functions of antibodies, which are the secreted form of Ig molecules. Immunoglobulin (Ig) and T-cell receptor (TCR) variable region exons are assembled from large arrays of V (variable), D (diversity), and J ( joining) gene segments during the development, respectively, of B and T lymphocytes. Once a functional immunoglobulin chain is expressed, allelic exclusion operates through a feedback mechanism to prevent further rearrangements of Ig heavy (IgH) and Ig light (IgL) chain genes. V(D)J recombination is mediated by a common recombinase complex that includes the recombination-activating gene products RAG1 and RAG2, which harbor endonuclease activity that introduces DNA double-strand breaks (DSBs) at V, D, and J segments. The V(D)J reaction is completed by the ubiquitously expressed nonhomologous end-joining (NHEJ) factors that join the broken V, D, and J segments together. Still, Ig loci are only fully assembled in B lineage cells and TCR loci are only assembled in T lineage cells. Within a lineage, different loci are rearranged in a specific order. For example, IgH locus variable region exons are assembled before those of Ig light chains (IgL), and within the IgH locus D to JH recombination precedes VH to DJH recombination. Given

Genomic Rearrangements of the IgH Locus

3

such locus-specific regulation and a common V(D)J recombinase, accessibility of the different loci to the common V(D)J recombinase must underlie the cell-type and stage-dependent assembly of the different IgH and TCR gene families ( Jung et al., 2006). Activation of mature B-cells can alter their IgH loci through a separate form of genomic rearrangement which is termed IgH class switch recombination (CSR). CSR allows B-cells to express IgH chains with different constant regions which can change the effector functions of antibodies without altering variable region specificity. CSR is initiated by activationinduced cytosine deaminase (AID), the activity of which ultimately leads to DSBs in regions upstream of CH genes which are then joined by NHEJ or other end-joining pathways to complete the CSR reaction (Chaudhuri et al., 2007). Ig and TCR loci contain a number of cis-regulatory elements which regulate V(D)J rearrangements, IgH CSR, and Ig gene expression at various levels. In this chapter, we will focus on the impact of cis-regulatory elements on genetic and epigenetic regulation of recombination events within the IgH locus.

2. THE IMMUNOGLOBULIN HEAVY CHAIN LOCUS The murine IgH locus is a complex genomic region, spanning about 3 Mb close to the telomere of the long arm on chromosome 12. The IgH locus comprises arrays of V, D, and J segments upstream of several constant region exons (Fig. 1.1A). Different mouse strains carry varying numbers of VH and D elements. Some 150 VH segments are distributed over 2.5 Mb in the 50 part of the IgH locus and are classified in 16 VH gene families defined by sequence similarities ( Johnston et al., 2006). These VH gene families are partially interspersed with one another but, depending on position, can be divided into proximal (30 part of the VH cluster, close to IgH–D region, for example, VH7183), intermediate (e.g., VHS107), and distal (50 part of the VH cluster, distant from IgH–D region, for example, VHJ558) families. 30 of the VH elements, separated by 90 kb, lie 10–15 D segments (Retter et al., 2007; Ye, 2004) followed by 4 JH elements. Because of the uniform transcriptional orientation of V, D, and J segments, V(D)J recombination events at the IgH locus result in deletion of the intervening sequence. The 30 part of the IgH locus contains a series of sets of different constant (C) region exons Cm, Cd, Cg3, Cg1, Cg2b, Cg2a, Ce, Ca, which will be referred to as ‘‘CH genes’’ (Fig. 1.1B). A large number of cis-regulatory elements were identified throughout the IgH locus. The intronic enhancer, Em, is located in the intron between JH4 and the CH exons (Fig. 1.1); the 30 IgH regulatory region (IgH 30 RR) consists of several DNase hypersensitive sites and is located at the very

4

Thomas Perlot and Frederick W. Alt

VH7183

5⬘RR? B

DH

VD RR? ImP

CH

PDQ52 Em

Ig 1P AAA

VHDJH Em

JH

Cm Cd

AAA

Cg 3

Cg 1

IgH 3⬘RR

Cg 2b Cg 2a

Ce

Ca

HS3B HS4 HS5 HS6 HS7

VHS107

HS1,2

VHJ558

HS3A

A

IgH 3⬘RR

FIGURE 1.1 Schematic depiction of the murine IgH locus. (A) VH, DH, JH gene segments and CH exons are shown as rectangles, known and potential regulatory elements as ovals. The VH families VHJ558, VHS107, and VH7183 are depicted as examples for distal, intermediate, and proximal VH families, respectively. The cis-regulatory elements PDQ52 (promoter of DQ52), Em (intronic enhancer), and IgH 30 RR (IgH 30 regulatory region) are depicted. The potential regulatory elements 50 RR (50 regulatory region) and VD RR (VH–DH intergenic regulatory region) are depicted with a question mark. Drawing not to scale. (B) The 30 part of the IgH locus. An assembled VHDJH exon is shown as a white rectangle, CH genes as squares, Em and individual DNaseI hypersensitive sites within the IgH 30 RR are depicted as black ovals, switch regions as white circles. I promoters are located upstream of every switch region (Chaudhuri et al., 2007; Lennon and Perry, 1985; Lutzker and Alt, 1988), only m and g1 I promoters (ImP, Ig1P) are depicted. Transcripts from I promoters get spliced and polyadenylated. Switch regions also get transcribed in the antisense orientation (Apel et al., 1992; Julius et al., 1988, Morrison et al., 1998; Perlot et al., 2008). Concomitant transcription from ImP and, for example, Ig1P can target AID to m and g1 switch regions and thereby initiate CSR to Cg1.

30 end of the IgH locus (Fig. 1.1). Transcriptional promoters are present upstream of every VH segment (Fig. 1.2B), upstream of DH segments (Fig. 1.2A and C), and upstream of CH genes (Fig. 1.2A). In addition, antisense transcripts from less well-defined promoters were described in the VH, DH, JH regions, and upstream of CH genes. Section 7 of this chapter contains a detailed discussion of these IgH cis-regulatory elements.

3. V(D)J RECOMBINATION DURING B-CELL DEVELOPMENT The IgH locus V(D)J exon is assembled at the pro-B-cell stage leading to the production of m IgH heavy chains via splicing of the VHDJH exon onto the adjacent Cm constant region exons. Functional mHC and surrogate Ig light chain proteins form a complex that is expressed on the surface of pre-B-cells and is known as the pre-B-cell receptor (pre-BCR)

5

Genomic Rearrangements of the IgH Locus

A PDQ52 DQ52

DH

JH1 JH2 JH3 JH4

AAA AAA

Em CH

m0 Im

DH-JH antisense germline transcripts B

VH sense germline transcripts AAA

VHP

VH

L

VHP

AAA

VH

L

VHP

AAA

VH

L

or VH antisense germline transcripts C PDH

DH

DJH JH4

AAA AAA

Em CH

Dm Im

DH antisense germline transcript D

mHCVHP

L

VH

VHDJH

JH4

L VHDJH antisense transcript

Em

Sm

AAA mRNA AAA

CH

Im

Sm antisense transcript

FIGURE 1.2 Transcripts within the IgH locus. VH, DH, JH gene segments and CH exons are shown as rectangles, enhancer and promoter elements as ovals. 12 bp and 23 bp RSSs are depicted as black and white triangles, respectively. Drawings not to scale. (A) The IgH locus in germline configuration is transcribed from the promoter of DQ52 (PDQ52) to produce the m0 transcript (Alessandrini and Desiderio, 1991), and from within the Em enhancer to generate the Im transcript (Lennon and Perry, 1985; Su and Kadesch, 1990), both of which are getting spliced and polyadenylated (Kottmann et al., 1994, Su and Kadesch, 1990). DH and JH elements are transcribed in the antisense orientation (Bolland et al., 2007; Chakraborty et al., 2007), suggested start sites (dashed arrows) are located around PDQ52 (Chakraborty et al., 2007) and Em (Bolland et al., 2007). Sites of transcriptional termination of DH–JH antisense germline transcripts are unknown. (B) Unrearranged VH segments are transcribed in the sense orientation from the individual VH promoters (VHP) (Yancopoulos and Alt, 1985). The intron between the leader (L) and the VH exon (VH) is spliced out, and the VH sense germline transcript gets polyadenylated (Yancopoulos and Alt, 1985). The VH segments and VH intergenic regions can also get transcribed in the antisense orientation (Bolland et al., 2004). Start and termination sites of VH antisense germline transcripts are unknown. Therefore, individual antisense transcripts could comprise one VH segment and its adjacent regions or multiple VH segments including intergenic regions, shown as short and long solid arrows, respectively. (C) Upon D to JH recombination, the assembled DJH exon gets transcribed from the DH promoter (PDH) and spliced to the Cm exons to generate the Dm transcript (Alessandrini and

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(Cobb et al., 2006). Signaling through the pre-BCR induces proliferation, signals cessation of further VH to DJH rearrangements at the IgH locus (i.e., allelic exclusion, see below), and promotes the onset of IgL variable region exon (VLJL) assembly. Thus, expression of the pre-BCR represents an important checkpoint at the pro- to pre-B-cell transition (Ma˚rtensson et al., 2007). Subsequently, Igk and Igl LC variable regions are assembled during the pre-B-cell stage. Expression of a functional Igk or Igl LC along with mHC forms a complete Ig molecule which is expressed on the cell surface of the resulting immature B-cells (Gorman and Alt, 1998). Immature B-cells migrate to the periphery where mature naı¨ve B-cells can be activated and undergo further modification of their IgH locus including IgH CSR and somatic hypermutation (SHM) (see below). All V, D, and J segments are flanked by recombination signal sequences (RSSs) that consist of a conserved palindromic heptamer and a conserved AT-rich nonamer separated by a less conserved 12 bp or 23 bp spacer (Sakano et al., 1980). The RAG1/2 endonuclease recognizes and binds a pair of RSSs with different spacer lengths in the context of the 12/23 rule (Early et al., 1980; Sakano et al., 1980), which allows for efficient V(D)J recombination only between gene segments flanked by 12 bp and 23 bp RSSs (Fugmann et al., 2000). The 12/23 restriction provides some direction for which Ig gene segments can be assembled. For example, IgH D segments are flanked with 12 bp RSSs on both sides; whereas VH and JH segments are flanked with 23 bp RSSs, thus preventing direct VH–JH joining. In the TCRb locus, however, direct Vb to Jb joints would be allowed according to the 12/23 rule but are denied by ‘‘beyond 12/23’’ restrictions (Bassing et al., 2000). Differential composition of RSSs implement ‘‘beyond 12/23’’ restriction at the nicking and pairing step of V(D)J recombination (Drejer-Teel et al., 2007; Jung et al., 2003).

Desiderio, 1991; Reth and Alt, 1984), which in one reading frame encodes for a short mHC molecule (Reth and Alt, 1984). DH antisense germline transcription is present throughout the remaining unrearranged DH segments (Chakraborty et al., 2007). Suggested origin of DH antisense germline transcripts is the region around the promoter of the recombined DH segment (depicted as PDH) (Chakraborty et al., 2007), transcriptional termination sites are unknown. (D) Upon VH to DJH recombination, the promoter of the rearranged VH segment (depicted as VHP) drives expression of mRNA encoding for the mHC. In addition to Im sense transcription, the Sm switch region is also transcribed in the antisense orientation (Perlot et al., 2008) from promoters residing within Sm (Apel et al., 1992; Morrison et al., 1998), the transcriptional termination site of the Sm antisense transcript is unknown. The assembled VHDJH exon and the adjacent JH region are transcribed in the antisense orientation potentially from start sites within the JH cluster (dashed arrow) (Perlot et al., 2008), the transcriptional termination site of the VHDJH antisense transcript is unknown. Upstream unrearranged VH segments are transcriptionally silenced upon assembly of a functional VHDJH exon (Bolland et al., 2004; Yancopoulos and Alt, 1985).

Genomic Rearrangements of the IgH Locus

7

RAG cutting precisely between RSSs and variable region gene segments results in the formation of blunt RSS ends, and the formation of coding ends (CE) of the V, D, or J segments as closed hairpins. Coding joints (CJs) are formed through a joining reaction mediated by members of the NHEJ repair pathway. In this reaction, Ku proteins bind the free CEs and recruit DNA-dependent protein kinase catalytic subunit (DNAPKcs), which activates the endonuclease activity of Artemis to open the hairpins. Subsequently, ends are joined by the XRCC4/DNA ligaseIV complex (Rooney et al., 2004). In contrast, the blunt SEs are precisely ligated to each other by NHEJ. Tight regulation of V(D)J recombination is imperative to ensure proper lymphocyte development and genomic integrity. While V(D)J recombination is of enormous advantage in order to efficiently combat infections, erroneous V(D)J recombination can have adverse consequences including chromosomal translocations, which can contribute to neoplastic transformation and the development of leukemias and lymphomas.

4. CLASS SWITCH RECOMBINATION AND SOMATIC HYPERMUTATION After a VHDJH variable exon is assembled upstream of the C region exons, a promoter 50 of the rearranged VH segment drives expression of m and d HC molecules in mature B-cells. Upon antigen encounter and activation, a B-cell can switch to expression of downstream CH genes to generate antibodies with the same variable region specificity but a different CH effector function by CSR. Repetitive switch (S) regions are located upstream of every CH gene except Cd. Introduction of DSBs in Sm and a downstream switch region can result in joining of the two switch regions, deletion of the intervening sequence, and consequently expression of a downstream CH gene with the same variable region exon. AID is absolutely required for CSR. It appears to function by deaminating cytosines to uracils within the substrate S region DNA with the resulting mismatches somehow being processed into DSBs by cooption of normal repair pathways (Di Noia and Neuberger, 2007). As AID is a singlestrand DNA-specific cytosine deaminase, its activity on duplex S region DNA is targeted by transcription (Chaudhuri et al., 2007). In this context, switch regions can be transcribed from an I (intervening) promoter located upstream of each S region, which allows for AID targeting to specific transcribed S regions (Fig. 1.2B). In addition to these sense germline transcripts, antisense transcripts were described in several S regions (Apel et al., 1992; Julius et al., 1988; Morrison et al., 1998; Perlot et al., 2008). AID initiated S region DSBs are joined by NHEJ or an alternative endjoining pathway to complete CSR.

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Thomas Perlot and Frederick W. Alt

AID is also required for SHM, a process during which the variable region exon gets mutated at a relatively high frequency in activated B-cells. SHM is initiated by transcription-dependent targeting of AID to assembled variable regions followed by error prone repair of the resulting mismatches (Di Noia and Neuberger, 2007). Through affinity maturation, B-cell clones producing higher affinity antibodies are selected and an efficient adaptive immune response is elicited.

5. IgH REARRANGEMENTS AND ALLELIC EXCLUSION Expression of RAG1 and RAG2 is absolutely required for V(D)J recombination. In the hematopoietic lineage, RAG activity can first be demonstrated in common lymphoid progenitor (CLP) cells, which are precursor cells that can develop into B-cells, T-cells, natural killer (NK) cells, and dendritic cells (DC) (Borghesi et al., 2004). Together with the detection of D to JH rearrangements in non-B-cell lymphoid lineages (Borghesi et al., 2004; Born et al., 1988; Kurosawa et al., 1981), expression of RAG in CLPs suggests that the first IgH rearrangement step can occur, at least at low level, in CLPs. Thus, the IgH D to JH recombination step is not absolutely restricted to the B-lineage, in contrast to VH to DJH rearrangements which normally occur only in B-cells. Efficient D to JH rearrangement on both IgH alleles takes place after B lineage commitment in the pro-B-cell stage (Alt et al., 1984). Once DJH segments are formed, one of the upstream VH elements can join to form a complete VHDJH exon. In the murine IgH locus, proximal VH segments are preferentially rearranged compared to distal VH elements throughout ontogeny (Malynn et al., 1990; Yancopoulos et al., 1984). However, peripheral B-cells do not show this preference as selection alters the B-cell repertoire (Yancopoulos et al., 1988). Both D to JH and VH to DJH recombination take place at the pro-B-cell stage, however, in an ordered manner such that D to JH rearrangement nearly always occurs before VH to DJH rearrangement (Alt et al., 1984). In this regard, VH to DJH recombination is the step that is regulated in the context of allelic exclusion to ensure expression of only one functional HC. Successful VH to DJH recombination and expression of a mHC from one IgH allele prevents a second DJH allele from undergoing VH to DJH rearrangement ( Jung et al., 2006). Considering the junctional diversity generated during V(D)J recombination, only one out of three VHDJH exons will be in frame with the downstream Cm exons; whereas two out of three will be out of frame and therefore unable to express a functional mHC (Mostoslavsky et al., 2004). The percentage of functional recombination events is further decreased by usage of VH pseudogenes containing stop codons, frameshifts, defective splice sites, or lacking an ATG

Genomic Rearrangements of the IgH Locus

9

translation start site, by stop codons in DH segments as well as through selection against certain reading frames of DJH joins (Gu et al., 1991). As a result, a substantial fraction of developing B-cells will not be able to generate a functional mHC from either IgH allele and will undergo apoptosis (Rajewsky, 1996). If a nonfunctional VH to DJH rearrangement occurs on the first allele, the second DJH allele can still undergo VH to DJH rearrangement (Alt et al., 1984). Allelic exclusion of VH to DJH rearrangement is mediated by feedback regulation; a functional mHC together with surrogate light chains are assembled to a pre-BCR, which signals the cessation of further VH to DJH rearrangements (Alt et al., 1984; Jung et al., 2006). In this regard, endogenous IgH rearrangements are largely inhibited by the expression of a preassembled membrane-bound mHC transgene (Nussenzweig et al., 1988). Likewise, allelic exclusion was broken by targeted deletion of the mHC transmembrane exons (Kitamura and Rajewsky, 1992), by lack of a functional pre-BCR (Lo¨ffert et al., 1996), and by combined deletion of the downstream pre-BCR signaling molecules Syk and ZAP-70 (Schweighoffer et al., 2003). The combined data from these studies strongly support a feedback-mediated mechanism for allelic exclusion that is mediated by signaling through a functional mHC in the pre-BCR signaling complex. The complete chain of events that leads to cessation of VH to DJH rearrangements and implementation of allelic exclusion is still elusive. However, it was shown that the onset of allelic exclusion after successful VH to DJH recombination is accompanied by the transient downregulation of RAG (Grawunder et al., 1995), decontraction of the IgH locus (Rolda´n et al., 2005), and loss of accessibility correlates such as VH germline transcripts and marks of active chromatin (see below). It has been estimated that only 1 in 10,000 wild-type B-lymphocytes actually escape allelic exclusion and express a functional mHC from both IgH alleles (Barreto and Cumano, 2000). Feedback regulation can explain cessation of VH to DJH rearrangement but would be ineffective if both IgH alleles would rearrange simultaneously. Therefore, it was suggested that the V(D)J recombination machinery targets one allele at a time (Alt et al., 1980). Supportive of this hypothesis was the observation that all Ig loci as well as the TCRb locus undergo asynchronous replication (Mostoslavsky et al., 2001; Norio et al., 2005). At the allelically excluded Igk locus it is thought that asynchronous replication facilitates allelespecific chromatin changes (Mostoslavsky et al., 1998) that lead to the early replicating allele rearranging first (Mostoslavsky et al., 2001). A similar mechanism for VH to DJH recombination, the allelically excluded IgH rearrangement step, was speculated, but has not yet been demonstrated. Thus, asynchronous replication could conceivably play a role in the initiation phase of allelic exclusion. However, it does not

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provide an explanation for the maintenance of allelic exclusion during subsequent B-cell stages, which prevents further IgH rearrangements in the presence of RAG, which must be affected by feedback mechanisms that influence accessibility.

6. ACCESSIBILITY CONTROL The accessibility hypothesis was proposed to explain how a single common V(D)J recombinase can target the different Ig and TCR loci in a lineage- and stage-specific manner (Yancopoulos and Alt, 1985). For example, Ig variable region exons are only fully assembled in B-cells while TCR variable region exons are only rearranged in T-cells. Similarly, IgH loci are rearranged during the pro-B-cell stage and not in pre-B-cells where IgL variable region assembly occurs. The accessibility hypothesis was first proposed based on the finding that germline VH gene segments are transcribed in pro-B-cells but not in subsequent B-cell stages, with germline VH transcription providing a potential correlate of accessibility (Yancopoulos and Alt, 1985). This hypothesis was proven by experiments that showed transfected TCR gene substrates could be rearranged by proB lines that do not rearrange endogenous TCR gene segments, first demonstrating a common V(D)J recombinase (Yancoupouls, 1986). This conclusion was confirmed and extended by other studies (Krangel, 2003; Stanhope-Baker et al., 1996). However, the precise mechanisms that mediate differential accessibility of Ig and TCR gene segments to V(D)J recombination are still not clear. Over the decades, several correlates of accessibility have been defined and a general picture is beginning to emerge as to how accessibility control might be regulated and implemented. Among the known correlates of accessibility are germline transcripts, chromatin modifications, DNase hypersensitivity, spatial organization, and positioning of Ig and TCR loci in the interphase nucleus.

6.1. Germline transcripts Germline transcription is the production of transcripts from V, D, or J segments and adjacent regions before they undergo rearrangement (Fig. 1.2). Sense germline transcripts starting from promoters upstream of V, D, and J segments have been described in all Ig and TCR loci (Hesslein and Schatz, 2001), and their stage-specific expression patterns strongly correlate with accessibility of these transcribed elements (e.g., Yancopoulos and Alt, 1985). The precise role of sense germline transcripts is still not understood and has been debated (Krangel, 2003). Recent studies support the notion that active transcription mediates chromatin changes that render the transcribed regions accessible to the recombinase

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(Sen and Oltz, 2006). However, it has been debated whether germline transcripts are the cause or the effect of these chromatin changes, and neither possibility has been unequivocally proven or disproved. On one hand, the levels of germline transcripts exhibit a positive correlation with rearrangement efficiency (Sun and Storb, 2001), which could suggest that the process of transcription itself could promote RAG targeting. However, others have shown that the correlation between individual rearrangements and germline transcription is not absolute (Angelin-Duclos and Calame, 1998; Sikes et al., 2002). The IgH locus in germline configuration is transcribed from the promoter of DQ52 (PDQ52), the 30 most DH segment, toward Cm, thereby producing the so-called m0 transcript (Fig. 1.2A). After D to JH rearrangement, the recombined DJH element is transcribed (Fig. 1.2C) (Alessandrini and Desiderio, 1991; Reth and Alt, 1984); and at the same time, unrearranged VH segments are transcribed from their promoters (Fig. 1.2B). Germline VH transcription appears to be silenced upon a productive rearrangement (Corcoran, 2005; Yancopoulos et al., 1985). More recently, antisense transcripts have been found to occur throughout the VH cluster (Fig. 1.2B) (Bolland et al., 2004), in the DH region (Fig. 1.2A and C) (Bolland et al., 2007; Chakraborty et al., 2007), and in the JH region (Fig. 1.2A) (Bolland et al., 2007; Perlot et al., 2008). VH antisense transcripts appear to be biallelic, and it has been argued that such transcripts are large and span several VH segments and the adjacent intergenic regions; but formal proof of their initiation sites is still lacking. VH antisense transcription was shown to initiate during D to JH recombination, and to be rapidly downregulated after VH to DJH recombination (Bolland et al., 2004). DH antisense transcripts were detected in RAGdeficient pro-B-cells as well as on the D–JH rearranged allele of B-cell lines with a functionally assembled IgH gene (Chakraborty et al., 2007). DH antisense transcripts have been suggested to originate from the 30 most DH (Chakraborty et al., 2007) or from the JH region (Bolland et al., 2007). The functional significance of antisense transcription in the context of V(D)J recombination has not been fully elucidated. It has been postulated that antisense transcription promotes an active chromatin state rendering the locus more accessible (Bolland et al., 2004), based on the observed correlation between antisense VH germline transcription and active VH to DJH recombination (Bolland et al., 2004). Similar conclusions were reached based on the observation of reduced antisense DH transcripts and reduced D to JH rearrangements in mice lacking the intronic enhancer, Em (Afshar et al., 2006; Bolland et al., 2007). Conversely, others have raised the possibility that antisense transcripts, at least in the DSP DH segments, could pair with low levels of postulated germline sense transcripts and elicit RNA interference-mediated transcriptional gene silencing (Chakraborty et al., 2007; Koralov et al., 2008). It should be noted that true germline sense

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transcripts have not been identified as yet in the germline DH segments, but their level may be as low as those originally identified in the S. pombe centromeric repeats and may only be revealed in an RNAi-deficient background (Volpe et al., 2002).

6.2. Spatial organization and nuclear positioning of the IgH locus The spatial organization of the Ig and TCR loci was analyzed by threedimensional fluorescence in situ hybridization (3D FISH), in which nuclear organization remains preserved. Several groups showed that before undergoing rearrangement, the IgH locus moves from its default position at the nuclear periphery to a more central compartment (Fuxa et al., 2004; Kosak et al., 2002), going along with the observation that the nuclear periphery has a repressive effect on transcription (Andrulis et al., 1998; Baxter et al., 2002; Reddy et al., 2008) and, therefore, might keep the IgH locus in an inaccessible state. These observations are consistent with the peripheral location of the IgH locus in thymocytes which have only low levels of D to JH and no VH to DJH rearrangements (Fuxa et al., 2004; Kosak et al., 2002; Kurosawa et al., 1981). The centrally located IgH locus in pro-B-cells can undergo D to JH rearrangement; however, for rearrangements of the distant VH elements, long-range contraction and looping of the IgH locus ( Jhunjhunwala et al., 2008) seems to be crucial as lack of IgH locus contraction in Pax5-deficient pro-B-cells does not allow for rearrangements of intermediate and distal VH families (Fuxa et al., 2004; Sayegh et al., 2005). After successful rearrangement, the IgH locus decontracts and, thereby, has been proposed to impede further VH to DJH rearrangements by increasing the distance between VH elements and the DJH region (Rolda´n et al., 2005). Therefore, it seems that one aspect of VH to DJH recombination accessibility might be influenced by spatial arrangement of the IgH locus within the nucleus. In B lineage stages subsequent to the pro-B stage, one IgH allele is positioned in close proximity to centromeric heterochromatin (Rolda´n et al., 2005; Skok et al., 2001). This finding was interpreted as the monoallelic silencing of the nonproductive IgH allele, because transcriptionally silent genes have been shown to associate with centromeric heterochromatin (Brown et al., 1997). However, the VH cluster gets silenced on both alleles in the context of germline transcription, and considering the fact that rearrangements from both IgH alleles, productive and nonproductive in either DJH or VHDJH configuration, are expressed in all B-cell stages that were examined (Daly et al., 2007; Fukita et al., 1998; Ono and Nose, 2007), monoallelic silencing might be either a short-time transient phenomenon or recruitment to centromeric heterochromatin might have other implications in the process of allelic exclusion.

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Studies of interactions between IgH and Igk alleles demonstrated coordinated patterns of action of Ig loci during B-cell development. Interactions between IgH and Igk, predominantly in pre-B-cells, were demonstrated to reposition the interacting IgH allele to centromeric heterochromatin and induce IgH locus decontraction (Hewitt et al., 2008). Therefore, this interchromosomal interaction could play a role in IgH allelic exclusion and the transition from accessible IgH alleles to accessible Igk alleles.

6.3. Chromatin modifications Eukaryotic DNA is packaged into nucleosomes in which genomic DNA is wrapped around histone octamers. The N-terminal ends of histones, called histone tails, can be marked by diverse modifications (e.g., acetylation, methylation, phosphorylation, ubiquitination, and others). The ‘‘histone code’’ ( Jenuwein and Allis, 2001) translates patterns of histone modifications into repression or activation of chromatin. An extensive effort has been made to investigate the effects of histone modifications and also various other chromatin attributes such as DNA methylation, DNase sensitivity, and nucleosome remodeling on accessibility of Ig and TCR loci with hopes of shedding light on the epigenetic regulation of V(D)J recombination. Posttranslational modifications of N-terminal histone tails can affect genome regulation in several ways. Histone modifications can directly affect chromatin structure, for example, through a change in charge. In this context, histone acetylation can loosen the association between DNA and the histone core or can alter higher order chromatin packaging. Alternatively chromatin modifications can disrupt or provide binding sites for chromatin remodeling complexes or other effector molecules. Prominent examples of such specialized binding domains are bromodomains specifically binding acetylated lysines, and chromodomains binding to dimethylated lysine 9 on histone 3 (Kouzarides and Berger, 2007). Many studies showed that marks of active chromatin correlate with V(D)J rearrangements. For example, acetylated lysine 9 on histone 3 (H3K9ac), hyperacetylated histone 4, and dimethylated lysine 4 on histone 3 (H3K4me2) are active chromatin marks (Kouzarides and Berger, 2007). They are present in the D–JH region peaking around the 50 most D segment, DFL16.1, and over the JH elements (Chakraborty et al., 2007; Morshead et al., 2003) in early pro-B-cells that are poised to undergo D to JH rearrangements. However, they are almost absent in thymocytes (Chakraborty et al., 2007). Following D to JH recombination, the proximal VH elements become hyperacetylated and, thereafter, in a manner that is dependent on IL-7R signaling and on its downstream effector STAT5 (signal transducer and activator of transcription 5), the distal VH segments

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become hyperacetylated (Bertolino et al., 2005; Chowdhury and Sen, 2001). Acetylation patterns seem to be narrowly confined to the VH segment, its promoter, and RSS ( Johnson et al., 2003). Histone hyperacetylation is lost after productive VH to DJH recombination, thereby contributing to rendering the VH cluster inaccessible in pre-B-cells (Chowdhury and Sen, 2003). Notably, an engineered locus that actively recruits an H3K9 methyltransferase shows downregulation of germline transcripts and impaired V(D)J recombination (Osipovich et al., 2004). H3K9me2 is absent in the D–JH region of pro-B-cells and present in thymocytes (Chakraborty et al., 2007), and removal of H3K9me2 from the VH region before VH to DJH recombination is dependent on Pax5 ( Johnson et al., 2004), a transcription factor essential for B-cell commitment (Busslinger, 2004). In agreement with this data, loss of Pax5 leads to an inability to rearrange distal VH gene families (Hesslein et al., 2003; Nutt et al., 1997). The antagonistic Polycomb (PcG) and Trithorax (trxG) groups of protein complexes establish and propagate a silenced or active chromatin state, respectively (Ringrose and Paro, 2004). Curiously, targeted deletion of the PcG protein Ezh2, an H3K27 methyltransferase, inhibits rearrangements of the distal VHJ558 family without affecting germline transcription (Su et al., 2003). H3K27 methylation was reported to be a mark of inactive chromatin (Kouzarides and Berger, 2007); therefore, it remains to be determined whether the results observed in the Ezh2 knockout are direct or indirect effects. Recent studies reported that the PhD finger domain of RAG2 specifically binds to trimethylated H3K4 (Liu et al., 2007; Matthews et al., 2007), a histone modification associated with transcriptional start regions (Pokholok et al., 2005) also shown to be present in accessible IgH regions (Liu et al., 2007). Mutation of the conserved tryptophan residue W453 within the PhD finger domain of RAG2 abrogates RAG2 binding to H3K4me3 and impairs V(D)J recombination of chromosomal and extrachromosomal substrates (Liu et al., 2007; Matthews et al., 2007). However, removal of the entire RAG2 noncore region, including the PhD domain, only leads to a partial impairment of V(D)J recombination (Liang et al., 2002). These seemingly contradicting data have been suggested to reflect the presence of an inhibitory function within the noncore region of RAG-2, which is relieved upon binding to H3K4me3, or can be circumvented by deleting the entire noncore region (Liu et al., 2007; Matthews et al., 2007). These recent studies provide the first direct link between epigenetic control of V(D)J rearrangement and RAG recombinase accessibility. Chromatin remodeling complexes can change the composition, structure, or position of nucleosomes within chromatin. These changes are noncovalent and are dependent on ATP hydrolysis (Martens and Winston, 2003). The SWI/SNF chromatin remodeling complex contains

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a bromodomain that allows it to efficiently bind acetylated chromatin and mobilize nucleosomes or change nucleosome structure (Martens and Winston, 2003). In this regard, it was shown that unmodified or even hyperacetylated nucleosomes located directly on RSSs are inhibitory to RAG cleavage in vitro (Golding et al., 1999) and that addition of SWI/SNF improved substrate cleavage (Kwon et al., 2000). RSSs strongly attract nucleosomes and, thus, may implement some aspect of accessibility control (Baumann et al., 2003). Moreover, nucleosome positioning appears pivotal for V(D)J recombination in vivo (Cherry and Baltimore, 1999). Further supporting the importance of SWI/SNF complexes in V(D)J recombination, BRG1 (the ATPase subunit of SWI/SNF) was found to associate at Ig and TCR loci within hyperacetylated chromatin regions (Morshead et al., 2003). Functional targeting of BRG1 to a TCRb minilocus lacking the essential Db promoter rescued V(D)J recombination of this substrate (Osipovich et al., 2007), substantiating the role of SWI/SNF complexes in V(D)J recombination and suggesting a role for transcriptional promoters in recruitment of chromatin remodeling complexes. Another readout to assess chromatin structure is the DNase sensitivity assay. Less tightly packed chromatin- or nucleosome-free DNA is more sensitive to DNase or restriction enzyme digestion than heterochromatin regions. While cis-acting elements such as promoters and enhancers can be devoid of nucleosomes and, therefore, are DNase hypersensitive, accessible chromatin of Ig and TCR loci shows general DNase sensitivity (Yancopoulos et al., 1986). In this context, the region between DQ52 and Em is DNase sensitive before D to JH rearrangement and JH RSSs show enhanced sensitivity and seem to be nucleosome free (Mae¨s et al., 2006). The VH region becomes nuclease sensitive before VH to DJH rearrangement and reverts to a refractory state after successful VH to DJH recombination (Chowdhury and Sen, 2003). Cytosines in mammalian DNA can be methylated in CpG dinucleotides. Generally, cytosine methylation corresponds to silenced genes (Stein et al., 1982; Vardimon et al., 1982) or silent regions throughout the genome; whereas promoter regions of expressed genes are found in an unmethylated state. Cytosine methylation can act by inhibiting regulatory proteins from binding to DNA (Watt and Molloy, 1988), or by recruiting methyl-CpG binding proteins which in turn can interact with HDACs to enforce a silent chromatin state ( Jaenisch and Bird, 2003). In this regard, methylated V(D)J recombination substrates are refractory to active rearrangement (Cherry and Baltimore, 1999; Hsieh and Lieber, 1992); in particular, methylated RSSs can abolish RAG cleavage and V(D)J recombination (Whitehurst et al., 2000). Demethylation alone, however, is not sufficient to initiate V(D)J recombination (Cherry et al., 2000). The DH–JH cluster gets demethylated before the onset of D to JH recombination (Mae¨s et al., 2001; Storb and Arp, 1983) characteristic of an accessible state. In this

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context, the JCk region gets monoallelically demethylated and this demethylated allele undergoes rearrangement first (Mostoslavsky et al., 1998). The second allele stays in a repressive environment and somehow can get demethylated, if the rearrangement on the first Igk allele is nonproductive (Goldmit and Bergman, 2004). More extensive studies on DNA methylation of the IgH locus could potentially help to elucidate aspects of accessibility control within this locus.

7. IgH LOCUS CONTROL THROUGH CIS-REGULATORY ELEMENTS A formidable number of cis-regulatory elements have been identified throughout the IgH locus (Fig. 1.1). Enhancers are located in the JH–Cm intronic region and at the very 30 end of the locus. Promoters are found 50 of VH and D segments as well as 50 of most CH genes. Cis-elements in the IgH locus not only govern gene expression, but also play crucial roles in accessibility control in all its above-mentioned aspects and also control CSR. An extensive effort has been made to elucidate the many roles of these transcription elements. Ongoing research also aims at identifying missing regulatory elements and elucidating their role in IgH locus control.

7.1. Promoter of DQ52 DQ52 is the 30 most D segment. Its promoter becomes active before D to JH rearrangement to generate the m0 transcript (Fig. 1.2A) (Alessandrini and Desiderio, 1991; Kottmann et al., 1994; Schlissel et al., 1991a). This transcript runs all the way through the Cm exons, which get spliced to the JH1 splice donor site (Schlissel et al., 1991b). The same promoter region also gives rise to a low-level antisense transcript (Chakraborty et al., 2007). It has been suggested that the repetitive nature of the DH region in combination with bidirectional transcription can elicit RNA interference-mediated transcriptional gene silencing that would lead to the observed inactive chromatin state of the DSP elements. However, as mentioned above, only antisense and no sense transcripts have been detected thus far in the germline DH region (Chakraborty et al., 2007). Every DH element upstream of DQ52 has a bidirectional promoter which, upon D to JH rearrangement, potentially through approximation to the Em enhancer, gets activated to generate an antisense transcript and a sense transcript (Fig. 1.2C) (Alessandrini and Desiderio, 1991; Chakraborty et al., 2007). The sense transcript gets spliced in a way that the rearranged DJH segment is joined to the Cm exons. In one reading frame, this mRNA can encode for a shorter version of the mHC (Reth and

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Alt, 1984), which can inhibit subsequent VH to DJH rearrangements (Gu et al., 1991; Lo¨ffert et al., 1996; Malynn et al., 2002). Targeted deletion of the DQ52 promoter, which has both promoter and enhancer activity (Kottmann et al., 1994), in mice had no major impact on D to JH rearrangement, other than a slight shift in JH usage (Afshar et al., 2006; Nitschke et al., 2001). However, in these studies, m0-like transcripts were still evident, suggesting that activity of the heterogeneous promoter of DQ52 was not entirely abrogated. In a different study, the intronic Em enhancer was replaced with a phosphoglycerate kinase promoter–neomycin resistance gene cassette (PGK-NeoR), which resulted in complete absence of m0 transcripts and complete inhibition of D to JH rearrangement (Perlot et al., 2005). In this regard, targeted deletion of an analogous promoter element in the TCRb locus, the promoter of Db1, led to diminished germline transcripts from this promoter and reduced Db1 rearrangements (Whitehurst et al., 1999), demonstrating an accessibility control function for this element in Db–Jb recombination.

7.2. VH promoters Every VH element has its own promoter that initiates VH germline transcripts before VH to DJH rearrangement (Fig. 1.2B), as well as transcripts of the assembled VHDJH exon after rearrangement (Fig. 1.2D). Most VH promoters can generate a germline transcript, in which a leader exon gets spliced to a VH exon (Fig. 1.2B). The transcript gets polyadenylated and contains an open reading frame (Yancopoulos and Alt, 1985); however, no VH protein or its function has thus far been demonstrated. The most conserved element across VH promoters is the octamer ATGCAAAT (Parslow et al., 1984). This sequence element has been shown to be necessary for VH transcription (Mason et al., 1985), and it binds the ubiquitously expressed Oct-1 and the B-cell-specific Oct-2, both POU family transcription factors. Most but not all VH promoters contain a TATA box, an initiator (Inr) element (Buchanan et al., 1997), a heptamer, and a pyrimidine stretch (Eaton and Calame, 1987). Additionally, binding sites for a number of mostly B-lineage-specific transcription factors and chromatin remodeling complexes have been identified in VH promoter regions ( Johnston et al., 2006). Germline transcripts from unrearranged VH promoters are generated upon D to JH rearrangement in pro-B-cells, and downregulated after completed VH to DJH recombination and assembly and expression of a functional VHDJH exon (Bolland et al., 2004; Hardy et al., 1991). The promoter of a recombined VH element stays active throughout B-cell development, and cell line experiments showed that the first upstream unrearranged VH segment can also be continuously expressed at reduced levels (Wang and Calame, 1985). Promoter activity of a functionally

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rearranged VH element was shown to be partially dependent on the 30 regulatory region (Pinaud et al., 2001). Thus, VH promoters might fulfill a dual role: to help confer accessibility to germline VH segments and to drive expression of the assembled heavy chain gene.

7.3. Intronic enhancer The IgH intronic enhancer (Em) was the first cellular (as opposed to viral) eukaryotic enhancer element described (Alt et al., 1982; Banerji et al., 1983; Gillies et al., 1983). Em comprises a 220 bp enhancer core (cEm) and two flanking matrix attachment regions (MARs). Targeted deletion of both MARs shows that they are dispensable for efficient V(D)J recombination within the IgH locus (Sakai et al., 1999). Deletion of Em in B-cells (Chen et al., 1993; Sakai et al., 1999; Serwe and Sablitzky, 1993) and in the germline of mice (Afshar et al., 2006; Perlot et al., 2005) led to reduced D to JH rearrangement and severely impaired VH to DJH rearrangement. The residual V(D)J recombination activity in the IgH locus implies that activation of IgH rearrangements may also involve one or more additional enhancer type elements. One candidate for such a compensating element is the promoter/enhancer region PDQ52, which was speculated to promote D to JH recombination (Alessandrini and Desiderio, 1991). However, deletion of PDQ52 along with Em did not show increased impairment above that seen with deletion of Em alone (Afshar et al., 2006). However, since the deletion of PDQ52 appeared to be incomplete, this element cannot yet be ruled out as having redundant functions with Em in conferring accessibility to the DH–JH region. Another candidate for cooperative function with Em is the 30 IgH regulatory region, but the double knockout of Em and the IgH 30 RR has not been generated. By analogy, deletion of the intronic Igk enhancer (iEk) reduces Vk to Jk rearrangements (Xu et al., 1996); whereas a double knockout of iEk and the 30 Ek enhancer completely blocks recombination of the Igk locus (Inlay et al., 2002). The iEk and 30 Ek in the Igk locus are the enhancer elements corresponding to the position of Em and IgH 30 RR in the IgH locus. It has been puzzling why in Em knockout mice the VH to DJH step is more severely impaired than the D to JH step, even though the Em enhancer has no obvious effect on germline transcripts of intermediate and distal VH families (Perlot et al., 2005). One explanation could be significant underestimation of D to JH impairment in Em knockout mice. Initial very low levels of D to JH rearrangements could limit the crucial DJH substrates for subsequent VH to DJH rearrangements and, therefore, result in the observed strong reduction of VH to DJH recombination. After a productive rearrangement, feedback regulation inhibits further VH to DJH recombination, but does not block further D to JH rearrangements

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(Reth et al., 1987). Therefore, D to JH recombination might ‘‘catch up’’ over the course of B-cell development and mask a stronger impairment. Notably, replacement of Em with a PGK-NeoR cassette (Chen et al., 1993; Perlot et al., 2005; Sakai et al., 1999) or introduction of PGK-NeoR cassette just 50 of Em (Chen et al., 1993; Delpy et al., 2002) results in a much more severe impairment or a complete block of V(D)J recombination and concomitant complete loss of m0 transcripts (Perlot et al., 2005). This phenomenon could be explained by a promoter competition/insulating mechanism. In such a scenario, the PGK-NeoR gene and its promoter might compete with PDQ52 for activity from a downstream cis-element such as the IgH 30 RR, which is known to act over long distances (Pinaud et al., 2001). Similar promoter competition for the IgH 30 RR has been observed between I promoters and the PGK-NeoR cassette introduced in the CH region (see below). Alternatively, the PGK-NeoR cassette could induce local chromatin changes that impede m0 germline transcription and accessibility of D and JH segments. Extensive studies revealed an array of binding sites for B-lineagespecific transcription factors and also for ubiquitously expressed proteins within the Em enhancer and the flanking MARs (Calame and Sen, 2004). The unique combination of these factors is likely to mediate the enhancer’s predominant activity in pro-B-cells (Inlay et al., 2006). In this context, replacement of iEk with Em leads to premature Igk rearrangement in proB-cells and absence of Igk rearrangements in pre-B-cells, the stage when LC rearrangement normally takes place (Inlay et al., 2006), corroborating the pro-B-cell specificity of Em. Em was suggested to play a role in regulating antisense transcripts through the JH and DH region (Afshar et al., 2006; Bolland et al., 2007), and additionally a promoter region within Em was identified that gives rise to the Im transcript (Lennon and Perry, 1985; Su and Kadesch, 1990). Starting at Em, this transcript extends through the m switch region and Cm. Transcription of switch regions was shown to be necessary for CSR, probably for targeting AID, and in this regard, deletion of Em leads to reduced Im transcript levels and reduced CSR (Bottaro et al., 1998; Perlot et al., 2005). Deletion of Em has no obvious effect on somatic hypermutation of VHDJH exons in mature B-cells (Perlot et al., 2005). An open question is how the activity of AID is specifically targeted to regions within Ig loci. It was speculated that cis-regulatory elements could determine this specificity, but neither cEm nor the IgH 30 RR, alone (Morvan et al., 2003), seems to have a crucial role in targeting SHM to the IgH locus VHDJH segments. While an absolute requirement of cEm or the IgH 30 RR for SHM can be excluded, there is a possibility that smaller defects of SHM in these mutants are masked by selection processes during affinity maturation. Also, a combined function of cEm and the IgH 30 RR in promoting or targeting SHM is another possibility that needs to be tested.

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7.4. 30 IgH regulatory region and I promoters I promoters are located upstream of all switch regions (Chaudhuri et al., 2007; Lennon and Perry, 1985; Lutzker and Alt, 1988). Transcripts initiating from I promoters are processed in such a way that an I (intervening) exon, located immediately downstream of the I promoter, is spliced to the associated CH exons. In this process, the intronic region including the S region is spliced out and the transcript gets polyadenylated. However, these transcripts appear ‘‘sterile,’’ as they do not contain an open reading frame and could not be shown to encode for a protein (Chaudhuri et al., 2007). Active transcription from I promoters is necessary for CSR as only transcribed S regions can become AID targets during CSR. In this context, deletion of I promoters abrogates efficient CSR to the associated CH genes; while replacement of I promoters with a constitutively active promoter directs CSR to the associated CH gene (Manis et al., 2002). Transcription from different I promoters prior to CSR can be induced upon stimulation with different activators or cytokines. Corresponding surface receptors for these molecules and their associated downstream signaling pathways affect different combinations of activating or repressive response elements within I promoter regions, which leads to CSR to different IgH isotypes under different stimulation conditions (Stavnezer, 2000). Most I promoters do not appear to act in isolation as efficient transcription from them also requires the IgH 30 RR (Pinaud et al., 2001) and physical interaction between the IgH 30 RR and specific I promoters has been implicated (Wuerffel et al., 2007). The IgH 30 RR is located downstream of Ca at the very 30 end of the IgH locus (Fig. 1.2B). This regulatory region consists of a number of DNaseI hypersensitive sites scattered over 35 kb (Dariavach et al., 1991; Garrett et al., 2005; Lieberson et al., 1991; Matthias and Baltimore, 1993; Pettersson et al., 1990); up until now, none of them was shown to play a role in V(D)J recombination but more studies are needed (Khamlichi et al., 2000; Pinaud et al., 2001). The most striking function, control of IgH CSR, has been assigned to HS3b, HS4 within the IgH 30 RR. Targeted deletions in mice revealed severely reduced CSR to most IgH isotypes and reduced germline transcription from I promoters through the corresponding S regions (Pinaud et al., 2001), a process required for CSR (Jung et al., 1993; Zhang et al., 1993). Deletion of the more 50 DNaseI hypersensitive sites within the IgH 30 RR HS3a and HS1,2 had no effect on CSR (Manis et al., 1998); however, replacement of HS3a or HS1,2 with a PGK-NeoR cassette resulted in a similar defect as in the HS3b, HS4 deletion (Cogne´ et al., 1994, Manis et al., 1998). The latter observations suggested a potential promoter competition/insulation between I promoters and the PGK-NeoR cassette for signals from within the IgH 30 RR. This hypothesis was strengthened by insertion of a PGK-NeoR cassette at the Ig2b promoter or the Ce gene,

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respectively. In both cases, germline transcription and class switching to CH genes 30 of the inserted PGK-NeoR cassette was unaffected; while germline transcription and class switching to CH genes 50 of the inserted PGK-NeoR cassette was impaired (Seidl et al., 1999). These results suggest that the inserted PGK-NeoR cassette can interfere with the long-range control effect of IgH 30 RR on CSR in a position-dependent manner. The IgH 30 RR is necessary for efficient expression of the rearranged HC from the promoter upstream of the assembled VHDJH exon (Pinaud et al., 2001), whereas the much more proximal Em enhancer is not required for HC expression (Perlot et al., 2005). Because it can influence expression of rearranged VHDJH segments, the IgH 30 RR can function over a distance of at least 200 kb. Such long-range activity may be important for activating oncogenes translocated into the upstream portions of the CH locus in lymphomas. Not all of the seven described hypersensitivity sites in the spacious 30 regulatory region have been knocked out yet, therefore, other potential functions still remain to be discovered. Apart from the abovementioned effects on germline transcription, CSR, and IgH expression, it has been speculated that parts of the 30 regulatory region might have a role in long-range chromatin organization. Finally, activity of the Ig1 promoter does not appear to be dependent on the IgH 30 RR; suggesting that it carries sufficient regulatory elements itself or that there are other long-range IgH locus elements that function in CSR to be defined.

7.5. Additional potential regulatory elements Several laboratories suggested that the IgH locus can be associated with the nuclear periphery via its 50 region (Kosak et al., 2002; Yang et al., 2005). The 50 end of the IgH locus does not get deleted in the course of V(D)J recombination and as such is an attractive location for a missing regulatory element that controls processes such as accessibility control of the distal VH genes, positioning of the IgH locus, or feedback regulation. In fact, 30 kb upstream of the most distal VH element an array of DNaseI hypersensitive sites has been identified (Pawlitzky et al., 2006). One of these sites, HS1, was reported to be pro-B-cell specific and potentially contain binding sites for the transcription factors PU.1, Pax5, and E2A. However, preliminary knockout experiments, in which HS1 was deleted, showed no effect on the IgH locus, as targeted alleles could still undergo efficient V(D)J recombination including all VH gene families. Furthermore, allelic exclusion was unaffected (Perlot, Pawlitzky, Brodeur, and Alt, unpublished data). Other potential functions of these sites, including acting as a boundary area as was suggested by DNA modifications confined to one side of 50 IgH hypersensitive sites (Reddy et al., 2008), are still being tested. Another area that was speculated to harbor a regulatory element is the 90 kb region between the VH and the DH clusters. This region could

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contain an element that ensures the ordered rearrangement of the D to JH and VH to DJH steps, such as a boundary element that influences activation of separate IgH locus domains. Moreover, the VH to DH intergenic region is deleted on a productively rearranged allele but remains in place on a DJH rearranged allele, suggesting an element might reside in this region that is responsible for shutting down the incompletely rearranged allele in the context of allelic exclusion. Potential support for such an element came from placement of a VH segment into the DH region, which resulted in breaking of lineage specificity, ordered rearrangement, and allelic exclusion of the introduced VH segment (Bates et al., 2007). Preliminary studies in which this intergenic region has been deleted have provided direct support for the notion that this region contains elements important for regulation of lineage specificity of VH to DJH rearrangement (Giallourakis, Franklin, and Alt, unpublished data).

7.6. Interplay between cis-regulatory elements The transition from an inactive to an active chromatin state of the IgH locus is in part governed by Em. The intronic enhancer plays an important role in placing active chromatin marks throughout the DH–JH region (Chakraborty, Perlot, Subrahmanyam, Alt, and Sen, unpublished data). In addition, Em promotes transcripts from PDQ52 and supports formation of the DNaseI hypersensitive site at this promoter element (Perlot et al., 2005; Chakraborty, Perlot, Subrahmanyam, Alt, and Sen, unpublished data). These data argue in favor of a direct interaction between Em and PDQ52 reminiscent of a corresponding promoter/enhancer holocomplex described in the TCRb locus (Oestreich et al., 2006). The fact that Em is not absolutely required for stimulation of PDQ52 suggests partial compensation by another cis-element. It was speculated that the IgH 30 RR can take over this role of interaction with PDQ52 and of activating the DH–JH region, because its ability to function over a long range to activate I region promoters and to influence expression of the promoter of a rearranged VHDJH segment (Pinaud et al., 2001). No single cis-regulatory element has thus far been identified that is responsible for the bulk of VH germline transcription. It seems to be likely that VH promoters can be activated in trans by B-lineage and stagespecific factors. VH antisense transcripts might be a prerequisite of VH sense germline transcripts by initializing an active chromatin state and accessibility of the VH locus (Bolland et al., 2004). Start sites of these transcripts still remain elusive, which exacerbates the manipulation of such transcripts and a direct proof for this hypothesis. After rearrangement of a complete VHDJH exon, the assembled VHDJH–Cm gene is transcribed from the VH promoter 50 of the rearranged VH. Transcription from the rearranged VH promoter is mainly supported

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by the IgH 30 RR (Pinaud et al., 2001) suggesting direct interaction of the two cis-elements. In this regard, the ability of the IgH 30 RR to form a direct complex with a region around Em and downstream I promoters was demonstrated during CSR (Wuerffel et al., 2007). However, cEm does not appear to be involved in that interaction, since deletion of that element does not affect complex formation (Wuerffel et al., 2007). As mentioned above, the interaction of the IgH 30 RR with I region promoters likely underlies the cooperation of these two elements in I region transcription and regulation of IgH CSR.

8. CONCLUSIONS Antigen receptor genes are assembled by the process of V(D)J recombination in a developmentally controlled manner. Differential accessibility at Ig and TCR loci is regulated at least in part by cooperative action of cisregulatory elements. Tremendous progress has been made in identifying and elucidating the multiple layers of control of the IgH locus during V(D)J recombination; however, many important questions remain unanswered and new questions are emerging. Among these are processes involved in ordered IgH rearrangements, asynchronous VH to DJH rearrangements, enforcement of feedback regulation, the precise relevance and impact of chromosome positioning and movements, the role of antisense transcription throughout the IgH locus, and chromatin modifications. Likewise, a remarkable amount of progress has been made in elucidating the role of cis-acting elements in the regulation of IgH CSR, but again there are still many unanswered questions including precisely how these elements function to specifically target AID to S regions and the precise mechanisms by which the IgH 30 RR and I region promoters elements cooperate in response to external stimuli to specifically activate CSR to particular CH genes. To fully understand the genetic and epigenetic regulation of the IgH locus, all involved cis-regulatory elements and trans acting factors need to be identified and analyzed. More work also will need to be done to understand how these factors influence regulation at the level of chromatin structure and spatial organization. Understanding the mechanisms governing the IgH locus, a model system for gene expression and epigenetic regulation will also advance our understanding of various other unsolved biological problems.

ACKNOWLEDGMENTS We thank Cosmas Giallourakis and John Manis for critically reviewing the manuscript and for discussions. T. P. received a Boehringer Ingelheim Fonds PhD scholarship. This work was supported by National Institutes of Health Grants PO1CA092625-05 and 2PO1AI031541-15 (to F.W.A.). F.W.A. is an investigator of the Howard Hughes Medical Institute.

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Parslow, T. G., Blair, D. L., Murphy, W. J., and Granner, D. K. (1984). Structure of the 5 ends of immunoglobulin genes: A novel conserved sequence. Proc. Natl. Acad. Sci. USA 81(9), 2650–2654. Pawlitzky, I., Angeles, C. V., Siegel, A. M., Stanton, M. L., Riblet, R., and Brodeur, P. H. (2006). Identification of a candidate regulatory element within the 5 flanking region of the mouse Igh locus defined by pro-B cell-specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J. Immunol. 176(11), 6839–6851. Perlot, T., Alt, F. W., Bassing, C. H., Suh, H., and Pinaud, E. (2005). Elucidation of IgH intronic enhancer functions via germ-line deletion. Proc. Natl. Acad. Sci. USA 102(40), 14362–14367. Perlot, T., Li, G., and Alt, F. W. (2008). Antisense transcripts from immunoglobulin heavychain locus V(D)J and switch regions. Proc. Natl. Acad. Sci. USA 105(10), 3843–3848. Pettersson, S., Cook, G. P., Bru¨ggemann, M., Williams, G. T., and Neuberger, M. S. (1990). A second B cell-specific enhancer 30 of the immunoglobulin heavy-chain locus. Nature 344 (6262), 165–168. Pinaud, E., Khamlichi, A. A., Le Morvan, C., Drouet, M., Nalesso, V., Le Bert, M., and Cogne´, M. (2001). Localization of the 30 IgH locus elements that effect long-distance regulation of class switch recombination. Immunity 15(2), 187–199. Pokholok, D. K., Harbison, C. T., Levine, S., Cole, M., Hannett, N. M., Lee, T. I., Bell, G. W., Walker, K., Rolfe, P. A., Herbolsheimer, E., Zeitlinger, J., Lewitter, F., et al. (2005). Genome-wide map of nucleosome acetylation and methylation in yeast. Cell 122(4), 517–527. Rajewsky, K. (1996). Clonal selection and learning in the antibody system. Nature 381(6585), 751–758. Reddy, K. L., Zullo, J. M., Bertolino, E., and Singh, H. (2008). Transcriptional repression mediated by repositioning of genes to the nuclear lamina. Nature. Reth, M. G., and Alt, F. W. (1984). Novel immunoglobulin heavy chains are produced from DJH gene segment rearrangements in lymphoid cells. Nature 312(5993), 418–423. Reth, M., Petrac, E., Wiese, P., Lobel, L., and Alt, F. W. (1987). Activation of V kappa gene rearrangement in pre-B cells follows the expression of membrane-bound immunoglobulin heavy chains. EMBO J. 6(11), 3299–3305. Retter, I., Chevillard, C., Scharfe, M., Conrad, A., Hafner, M., Im, T. H., Ludewig, M., Nordsiek, G., Severitt, S., Thies, S., Mauhar, A., Blo¨cker, H., et al. (2007). Sequence and characterization of the Ig heavy chain constant and partial variable region of the mouse strain 129S1. J. Immunol. 179(4), 2419–2427. Ringrose, L., and Paro, R. (2004). Epigenetic regulation of cellular memory by the Polycomb and Trithorax group proteins. Annu. Rev. Genet. 38, 413–443. Rolda´n, E., Fuxa, M., Chong, W., Martinez, D., Novatchkova, M., Busslinger, M., and Skok, J. A. (2005). Locus ‘decontraction’ and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat. Immunol. 6(1), 31–41. Rooney, S., Chaudhuri, J., and Alt, F. W. (2004). The role of the non-homologous end-joining pathway in lymphocyte development. Immunol. Rev. 200, 115–131. Sakai, E., Bottaro, A., Davidson, L., Sleckman, B. P., and Alt, F. W. (1999). Recombination and transcription of the endogenous Ig heavy chain locus is effected by the Ig heavy chain intronic enhancer core region in the absence of the matrix attachment regions. Proc. Natl. Acad. Sci. USA 96(4), 1526–1531. Sakano, H., Maki, R., Kurosawa, Y., Roeder, W., and Tonegawa, S. (1980). Two types of somatic recombination are necessary for the generation of complete immunoglobulin heavy-chain genes. Nature 286(5774), 676–683. Sayegh, C., Jhunjhunwala, S., Riblet, R., and Murre, C. (2005). Visualization of looping involving the immunoglobulin heavy-chain locus in developing B cells. Genes Dev. 19 (3), 322–327.

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CHAPTER

2 DNA-PK: The Means to Justify the Ends? Katheryn Meek,* Van Dang,* and Susan P. Lees-Miller†

Contents

1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12.

Abstract

Introduction Composition DNA Binding and Kinase Activation Structural Studies of DNA-PK Targets of DNA-PK’s Enzymatic Activity DNA-PK’s Autophosphorylation is Functionally Complex Autophosphorylation Within Two Clusters Reciprocally Regulates DNA End Access Further DNA-PK Autophosphorylation is Required During NHEJ Model of DNA-PK Activation End Processing to Promote End Conservation Does DNA-PK Regulate Dsbr Repair Pathway Choice? Why is DNA-PK So Abundant? References

34 36 37 38 39 39 40 43 44 47 49 50 52

The DNA-dependent protein kinase (DNA-PK) is central to the process of nonhomologous end joining because it recognizes and then binds double strand breaks initiating repair. It has long been appreciated that DNA-PK protects DNA ends to promote end joining. Here we review recent work from our laboratories and others demonstrating that DNA-PK can regulate end access both

* College of Veterinary Medicine and Department of Pathobiology and Diagnostic Investigation, Michigan

State University, East Lansing, Michigan, USA Departments of Biochemistry and Molecular Biology and Oncology, University of Calgary, Calgary, Alberta, Canada

{

Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00602-0

#

2008 Elsevier Inc. All rights reserved.

33

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positively and negatively. This is accomplished via distinct autophosphorylation events that result in opposing effects on DNA end access. Additional autophosphorylations that are both physically and functionally distinct serve to regulate kinase activity and complex dissociation. Finally, DNA-PK both positively and negatively regulates DNA end access to repair via the homologous recombination pathway. This has particularly important implications in human cells because of DNA-PK’s cellular abundance.

1. INTRODUCTION DNA is the blueprint for all living organisms; accordingly, all organisms have evolved numerous mechanisms to ensure maintenance of an exact copy of their genomes for propagation. In somatic cells of multicellular organisms, genomic maintenance is critical to the organism to ensure cellular viability and prevent oncogenesis. Given its importance to life, it is somewhat surprising that evolution has allowed DNA to be so labile, being quite sensitive to various forms of damage including oxidation, hydrolysis, and methylation. Efficient DNA repair systems have evolved to repair base damage (base excision repair, BER), nucleotide damage (nucleotide excision repair, NER), single strand breaks (single strand break repair, SSBR), and double strand breaks (double strand break repair, DSBR). Double strand DNA breaks (DSBs) are perhaps the most lethal form of DNA damage. Two major DNA repair pathways [nonhomologous end joining (NHEJ) and homologous recombination (HR)] repair DSBs in all eukaryotes. NHEJ (the primary pathway in higher eukaryotes) is active throughout the cell cycle (Critchlow and Jackson, 1998; Lieber, 1999; Pastink et al., 2001) whereas HR is generally limited to S and G2 when a sister chromatid is available as a repair template (Rothkamm et al., 2003). Emerging data provide strong evidence that an additional repair pathway(s) may also contribute to resolution of double strand breaks, especially in the absence of NHEJ, although the composition of this pathway as well as its role are not well understood (Audebert et al., 2004; Corneo et al., 2007; Udayakumar et al., 2003; Yan et al., 2007). Although NHEJ has long been labeled ‘‘error prone,’’ it should be emphasized that NHEJ functions efficiently to preserve DNA ends. Furthermore, direct ligation of two DNA ends is a preferable repair choice (over ‘‘error free’’ HR) in G0/G1 when HR using the homologous chromosome or nonallelic homologous sequences could result in deletions, duplications, or loss of heterozygosity. Since most of the genome (in higher eukaryotes) is noncoding, error prone rejoining of DSBs by NHEJ generally has minimal deleterious consequences. Rejoining of DSBs in

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coding regions obviously has the potential to introduce functionally important coding changes, a mechanism exploited by the vertebrate adaptive immune system when it commandeered the NHEJ system (evolutionarily) to resolve breaks during immune receptor assembly. In fact, this exploitation of the error prone nature of NHEJ by the immune system to generate literally billions of unique receptors is (arguably) one of the most clever of all biologic mechanisms. Many excellent recent reviews have provided comprehensive comparisons of the factors and functions of these two pathways, as well as NHEJ’s role in VDJ recombination, and will not be exhaustively reviewed here ( Jung et al., 2006; Lees-Miller and Meek, 2003; Lieber, 2008; Weterings and Chen, 2008). Briefly, NHEJ involves the direct ligation of DNA ends without a requirement for homology. Seven molecules have been shown to be required for NHEJ: Ku70, Ku86, DNA-PKcs, XRCC4, DNA ligase IV, Artemis, and XLF. XRCC4 is a 37-kDa protein that interacts with, and catalytically stimulates, the activity of DNA ligase IV (Grawunder et al., 1997; Li et al., 1995; Modesti et al., 1999). The XRCC4/DNA ligase IV complex carries out the final end-joining step in NHEJ. Artemis is the only nuclease known to function in NHEJ. The function of a newly discovered factor, XLF (Ahnesorg et al., 2006; Buck et al., 2006) that interacts with the XRCC4/ligase IV complex is only now being discerned but in its absence, NHEJ is severely impaired. Additionally, X family polymerases l and m (Nick McElhinny and Ramsden, 2003; Ma et al., 2004; Mahajan et al., 2002) as well as polynucleotide kinase provide activities not entirely essential for NHEJ (Chappell et al., 2002). Three of the essential factors comprise the DNA-dependent protein kinase (DNA-PK) complex, a serine/threonine protein kinase that must be physically associated with DNA to be active (Lees-Miller and Meek, 2003; Meek et al., 2004). Although DNA-PK has been implicated in a variety of other processes—from activation of innate immunity (Chu et al., 2000) to regulation of gene expression (Mo and Dynan, 2002)—its primary role in cellular metabolism is to initiate NHEJ. As discussed above, the primary role of NHEJ is to resolve DNA double strand breaks. Emerging data from our laboratories and others implicate DNA-PK as a central regulator of DNA end access. The focus of this review will be how DNA-PK mechanistically regulates DNA end access (primarily via autophosphorylation) to promote end joining with minimal loss of sequence information. Additionally, it is becoming apparent that DNA-PK may affect other repair pathways, potentially by limiting access of DNA ends to other repair factors. This may have particularly important sequelae in species that express very high levels of DNA-PK and may thus partially explain why DNA-PK may play varying roles in different species.

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During publication, a hypomorphic DNA-PKcs mutation that affects Artemis activation (but not kinase assembly or enzymatic activity) has been described in a human radiosensitive SCID patient (D. van Gent, personal communication).

2. COMPOSITION DNA-PK is composed of the DNA end binding heterodimer, Ku, and the large catalytic subunit, DNA-PKcs. Ku, initially discovered as an autoantigen, consists of two subunits of 70 and 86 kDa (Mimori et al., 1986). Its DNA end binding activity prompted early speculation that Ku might function in DNA repair (Mimori et al., 1986) although this was not formally proven until studies emerged implicating Ku as the defective factor in cells hypersensitive to DNA damaging agents (Liang et al., 1996). Structural studies of the Ku heterodimer reveal that it exists as a ring structure that initially binds DNA and then translocates to more internal regions (Rivera-Calzada et al., 2007; Walker et al., 2001). Jackson and colleagues reported the isolation of the 12 kb DNA-PKcs cDNA in 1995 (Hartley et al., 1995); structural features of DNA-PKcs are depicted in Fig. 2.1. Initial sequence analysis revealed only two recognizable motifs: a leucine rich region (LRR), and the phosphatidylinositol 3- (PI3) kinase domain. Since that time, a caspase site (DEVD) (Song et al., 1996), a Ku interaction domain (Ku) (Jin et al., 1997), and FAT and FATC domains (regions of homology shared between DNA-PKcs and other related kinases, ATM, ATR, FRAP/Tor, TRRAP) have been identified. DNA-PKcs has been shown to possess weak DNA binding activity (without Ku), but only in low (nonphysiologic) salt conditions (Hammarsten

DNA − PKcs

ABCDE cluster 2609 − 2647

JK cluster 1 ∗∗ ∗∗ N cluster

L site LRR

∗

∗∗∗

PQR cluster 2023−2056

Ku

∗∗∗ Capase cleavage FAT

T site 3950 ∗ 4129 PI3K Splice FATC variation

FIGURE 2.1 Schematic of DNA-PKcs showing structural features of the protein. ‘‘*’’ denotes autophosphorylation sites. Only sites that we have evidence as being functionally relevant are shown

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and Chu, 1998). The N terminus and LRR have been implicated in this function (Gupta and Meek, 2005; Meek et al., 2007). Splice variation occurs in the PI3K domain (Convery et al., 2005). In physiologic conditions, Ku bound to DNA (but not free Ku or free DNA) interacts tightly with the large catalytic subunit (DNA-PKcs). Targeting of DNA-PKcs to Ku-bound DNA requires the C terminus of Ku86; this targeting strategy has been conserved in the way related PIKK kinases (ATR and ATM) are targeted to DNA by their own DNA binding co-factors (ATRIP and Nbs1) (Falck et al., 2005). Collectively, Ku and DNA-PKcs assembled onto DNA ends is referred to as the DNA-dependent protein kinase, DNA-PK. A DNA-dependent protein kinase activity was discovered in 1985 (Walker et al., 1985); its identity as Ku and a larger catalytic subunit was demonstrated in 1990 (Lees-Miller et al., 1990). Although the enzyme has been studied intensely for over two decades, DNA-PK’s precise role in NHEJ is still the focus of investigation.

3. DNA BINDING AND KINASE ACTIVATION Ku binds DNA ends in a sequence-independent manner. Ku completely encircles bound DNA; its binding site encompasses approximately two turns of the helix, but only the central 3–4 base pairs are completely surrounded by Ku. When Ku is bound (in the absence of DNA-PKcs), the extreme DNA terminus is bound in an accessible channel (Walker et al., 2001). Ku has strong avidity for DNA with a variety of end structures [including blunt, over-hanged, hair-pinned, and damaged]. Ku can also recognize gaps, nicks, and other discontinuities in double stranded DNA raising the question of whether DNA-PK may affect repair of damage other than DSBs (Rathmell and Chu, 1994). Similarly, DNAPKcs assembles onto Ku bound DNA regardless of end structure (Smider et al., 1998). Elegant studies from Dynan and colleagues utilized photoreactive DNA cross-linking strategies to probe DNA–protein contacts of DNA-PK bound to DNA ends (Yoo and Dynan, 1999; Yoo et al., 1999). As noted above, in the absence of DNA-PKcs, Ku interacts with the extreme termini of DNA ends. In the presence of DNA-PKcs, Ku translocates inward (by about one helical turn) and DNA-PKcs has direct contacts with 10 bp at the terminus of a DNA end. Although DNAPKcs has innate affinity (itself) for DNA ends (as demonstrated using low salt conditions) (Hammarsten and Chu, 1998), Ku is required for targeting DNA-PKcs to damaged DNA in physiologic conditions and in living cells (Drouet et al., 2005). Although DNA end binding by DNA-PK is indifferent to distinct DNA end structures, activation of the kinase is considerably affected by

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end structure. Several studies have concluded that over-hanged ends (either 50 or 30 ) are more robust DNA-PK activators than are DNA ends with blunt termini (Hammarsten et al., 2000; Jovanovic and Dynan, 2006; Smider et al., 1998). Controversy exists as to whether hair-pinned DNA ends (important intermediates in VDJ recombination) that are avidly bound by DNA-PK (Smider et al., 1998; Soubeyrand et al., 2001) also activate the kinase. Recent work by Turchi and colleagues showing that DNA with chemical cross-links near the termini (but not at internal sites), though bound by DNA-PK, do not activate the kinase support the view that free DNA ends are required to activate the kinase (Pawelczak et al., 2005; Turchi et al., 2000). Further, these recent studies suggest that activation must be preceded by some degree of DNA end melting. In fact, Chu and colleagues proposed this very idea from their earlier structural studies (DeFazio et al., 2002). These authors further suggested that kinase activation (by non-annealed DNA ends) occurred in trans. Although there is support for kinase activation in trans, this point is not entirely clear, and others have proposed that a single isolated DNA-PK complex can activate autonomously ( Jovanovic and Dynan, 2006; Pawelczak et al., 2005). Still, the concept of linking synapsis to kinase autophosphorylation [whether kinase activation is in cis or trans] has been expanded by recent work (Reddy et al., 2004) and may represent an important mechanism by which DNA-PK protects DNA ends to preserve genomic integrity.

4. STRUCTURAL STUDIES OF DNA-PK As noted above, the Ku heterodimer exists as a ring structure that completely encircles the DNA, binding the extreme terminus in an accessible channel (Spagnolo et al., 2006; Walker et al., 2001). Association of DNA-PKcs with Ku bound DNA results in translocation of Ku to a more interior location on the DNA (Yoo and Dynan, 1999). Studies from Llorca and colleagues have provided a low-resolution electron microscopy (EM) structure of a DNA-PK synapse (Spagnolo et al., 2006); previous work from these investigators defined three major domains in DNAPKcs (Boskovic et al., 2003; Llorca and Pearl, 2004; Rivera-Calzada et al., 2005). The N-terminal repeat regions comprise the palm domain which itself includes a distal claw and a proximal claw. An ‘‘arm’’ domain connects the N-terminal palm to the head domain composed of the FAT, PI3K, and FATC domains. Assembly of DNA-PKcs onto Ku bound DNA induces a conformational change such that the two globular domains come into contact creating channel(s) where the DNA end resides. Further, Spagnolo et al. demonstrated additional conformation changes in synapsed DNA-PK compared to a single DNA-PK complex (Spagnolo

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et al., 2006). For instance, the FATC domain protrudes substantially, interacting with Ku only in the synapsed structure. The interface between the two complexes is between the claw and arm domains of the two DNA-PKcs subunits. Although Ku does not mediate synaptic interactions, its interaction with the FATC domain may contribute to synapsis by configuring DNA-PKcs into an appropriate conformation for synapsis.

5. TARGETS OF DNA-PK’S ENZYMATIC ACTIVITY DNA-PK’s enzymatic activity is clearly requisite to its role in NHEJ (Kienker et al., 2000; Kurimasa et al., 1999), and significant efforts have been made to define functionally relevant targets of DNA-PK that could explain why. DNA-PK (and the related PIKK family members, ATR and ATM) preferentially targets serines and threonines followed by a glutamine (S-T/Q sites) though other target sites (S-T/hydrophobic residues) have also been reported (Lees-Miller and Meek, 2003). There is a very long list of proteins that are excellent in vitro and in vivo DNA-PK target sites; what is lacking is a clear understanding of the functional relevance of many of these phosphorylation events. Modifying the function of a downstream target is a reasonable explanation for the requirement for DNA-PK’s enzymatic activity in NHEJ; further, most of the known NHEJ factors (XRCC4, Ku70, Ku86, Artemis, DNA-PKcs, and XLF) are excellent in vitro and in vivo DNA-PK targets (Chan et al., 1999; Goodarzi et al., 2006; Leber et al., 1998; Ma et al., 2002, 2005a). However, a long series of mutational studies conclude that phosphorylation of XRCC4, Ku70, Ku86, and Artemis are not functionally important, at least for NHEJ (Douglas et al., 2005; Goodarzi et al., 2006; Lee et al., 2004; Yu et al. 2008). [We consider a site functionally relevant if altering the ability to phosphorylate or dephosphorylate the site affects the capacity of the enzyme to facilitate DNA damage repair in living cells.] To date, DNA-PKcs itself is the only NHEJ factor that can be shown to be a functionally relevant target of its own enzymatic activity (Chan et al., 2002; Cui et al., 2005; Ding et al., 2003; Douglas et al., 2007; Meek et al., 2007; Soubeyrand et al., 2003).

6. DNA-PK’S AUTOPHOSPHORYLATION IS FUNCTIONALLY COMPLEX Early work in the Lees-Miller laboratory demonstrated that autophosphorylation of DNA-PK results in kinase inactivation and dissociation of the kinase’s catalytic subunit (DNA-PKcs) from DNA end bound Ku (Chan and Lees-Miller, 1996; Douglas et al., 2001). Kinase dissociation is

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certainly the most well-studied and well-accepted consequence of DNAPK’s autophosphorylation. However, more recent work has shown that autophosphorylation of DNA-PKcs occurs on many sites (probably more than 30 of the 4,129 residues; K. M., V. D., S. P. L. M., Y. Y., unpublished data), and different phosphorylations have distinct functional consequences. Two major autophosphorylation clusters (termed ABCDE and PQR) (Block et al., 2004; Cui et al., 2005; Ding et al., 2003; Meek et al., 2007; Reddy et al., 2004) spanning residues 2,609–2,647 and 2,023–2,056 have been the most thoroughly studied sites to date and are clearly functionally relevant (Fig. 2.1). Both the ABCDE and PQR clusters function as units in that phosphorylation of any one or two of the five or six sites is functionally sufficient. Several additional DNA-PK autophosphorylation sites have been reported but have either not been studied [S3821, S4026, T4102] (Ma et al., 2005), are not functionally important [for NHEJ, S3205] (Ding et al., 2003), or are potentially functionally redundant. We have described an additional functionally important site (‘‘T,’’ T3950) in the activation loop of DNA-PKcs (Douglas et al., 2007). Relative positions of phosphorylation sites or potential phosphorylation sites that we have evidence as being functionally important (K.M., V.D., unpublished data) but are not completely characterized are denoted as J, K, L, and N (Fig. 2.1). Importantly, none of these sites or combination of sites appears to mediate autophosphorylation-induced dissociation; thus, additional functionally relevant sites remain to be identified (although phosphorylation within the ABCDE cluster may partially contribute to this process, see below).

7. AUTOPHOSPHORYLATION WITHIN TWO CLUSTERS RECIPROCALLY REGULATES DNA END ACCESS The ABCDE and PQR clusters contain six and five conserved sites, respectively. These sites have been studied by generating phosphoablating (alanine) or phospho-mimicking (aspartic acid) mutants at each site. Published data regarding the function of different DNA-PKcs phosphorylation sites is summarized in Table 2.1. Ablation of only 1 or 2 sites in either cluster has little or no functional effect. However, complete ablation of ABCDE phosphorylation (by alanine substitution of all six sites) imparts a severe radiosensitive phenotype, even more radiosensitive than complete lack of DNA-PKcs. In contrast, ablation of PQR phosphorylation imparts only a modestly radiosensitive phenotype. This suggests that blocking ABCDE phosphorylation strongly inhibits progression of NHEJ whereas blocking PQR phosphorylation only modestly impairs progress through NHEJ. Both complete ABCDE and PQR alanine cluster mutants are fully functional kinases, both interact with other

TABLE 2.1

Summary of functional studies of DNA-PKcs phosphorylation site mutants

Wild type Vector A, B, C, D, or E!alac AB, AC, or AC!alac ABCDE!ala ABCDE!asp PQ or R!ala1e PQR!ala PQR!asp ABCDEþPQR!ala T!ala T!asp K!Rf a b c d e f

IR resistance

VDJ rate

Kinase activity

ATP-induced kinase dissociation

þþþþa þ þþþþ þþþ þþ þþþþ þþþ þþþþ þþ þþþ þ þ

þþþþ þþþþ þþþ þ þþ þþþþ þþþ þþþþ þþ þþþ

þþþþ þþþþ þþþþ þþþþ þþþþ þþþþ þþþþ þþþþ þþþþ þþþþ

þþþþ nab þþþþ ntd þþþ nt nt nt nt þþþþ nt na na

HR

þþ þþþþ nt nt þ nt þþþ nt þþþþ þþ nt nt

Nucleotide loss from rejoined breaks

þþþ þþþþþþþþ þþþ þþþ þ þþþ þþþ þþþþþþ þ þþþþþþ þþþ þþþþþþ þþþþþþ

For each functional assay, wild-type, mutant, and vector functions are arbitrarily assigned a range () to (þþþþþþþþ). For some functions, certain mutants are below vector and are thus designated with the lowest value (). Mutants that lack kinase activity cannot be studied for autophosphorylation-induced kinase dissociation and are designated na (not applicable). A, B, C, D, or E!ala mutants were each generated separately and behaved just like wild-type DNA-PKcs. nt (not tested). Combined PQ!ala (three sites) and R!ala (two sites) mutants were studied and behaved just like wild-type DNA-PKcs. K!R is a catalytically active mutant.

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NHEJ factors similar to wild-type DNA-PKcs, both are targeted to sites of DNA damage in living cells, both mutants still autophosphorylate (albeit at reduced levels), and both undergo autophosphorylation-induced kinase inactivation and dissociation (Block et al., 2004; Cui et al., 2005; Ding et al., 2003; Meek et al., 2007; Reddy et al., 2004). The specific defect imparted by blocking either ABCDE or PQR phosphorylation is dysregulated end processing. However, whereas autophosphorylation of sites within the ABCDE cluster promotes end processing, autophosphorylation of sites within the PQR cluster inhibits end processing. This was revealed by sequence analyses of coding joints or rejoined I-Sce1 breaks isolated from cells expressing mutant DNA-PKcs. Joints mediated by the ABCDE!ala mutant have minimal nucleotide loss. Further, biochemical studies show that although ABCDE phosphorylation is not critical for DNA-PK’s interaction with downstream NHEJ factors, it is absolutely requisite for end accessibility to downstream factors (Block et al., 2004; Goodarzi et al., 2006; Povirk et al., 2007; Reddy et al., 2004). These data are consistent with a large body of previous data that correlate kinase activity with end access (Calsou et al., 1999; Uematsu et al., 2007; Weterings et al., 2003). Thus, it was not surprising that autophosphorylation at ABCDE induces end access. In contrast to blocking ABCDE phosphorylation, blocking PQR phosphorylation results in joints with excessive nucleotide deletion. The finding that phosphorylation within the PQR locus strongly inhibits end trimming (while still allowing DNA end access to XRCC4/ligase IV) was not expected. It is fairly remarkable that both blocking and mimicking PQR phosphorylation have such strong (and opposing) effects on end processing without significantly changing the ability to function in NHEJ (as measured by radioresistance and support of VDJ recombination). We suggest that PQR phosphorylation provides DNA-PK with a mechanism to limit end processing (preserving the integrity of the genome) once ends have been aligned. There are several findings that support the model that phosphorylation events within one cluster oppose the effects of phosphorylation within the other cluster. First, phospho-mimicking mutants of either cluster reverse the effects of alanine substitution; notably, joints mediated by phospho-mimicking mutations at PQR display almost no end trimming. Additionally, if autophosphorylation of the two clusters has opposing effects, one might predict that blocking phosphorylation at both clusters should relieve the severe phenotype observed in the ABCDE! ala mutant. Analyses of a combination mutant in living cells confirmed this prediction, supporting a model whereby the ABCDE and PQR sites function reciprocally to regulate DNA end access (Cui et al., 2005). Finally, additional support for the model of co-regulation by the two clusters comes from in vitro studies of the purified enzymes (Meek et al., 2007). Although the ABCDE!ala mutant undergoes autophosphorylation-

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induced kinase dissociation, the kinetics of dissociation are slightly slower than with wild-type DNA-PKcs. However, the combined mutant (ABCDEþPQR!ala) that blocks ‘‘end protecting’’ PQR phosphorylation dissociates exactly analogous to wild type. These data imply that the phosphorylation status of the two clusters can either stabilize or de-stabilize DNA-PK’s interaction with DNA ends, but additional phosphorylation events are required to mediate complete dissociation. An attractive model would be that the phosphorylation status of the two clusters alters DNA-PK’s interaction with DNA ends whereas other phosphorylation events affect stability of the complex by disrupting DNA-PK’s interaction with Ku. It is important to consider that repairing a double strand break (with its two free DNA ends) requires the coordinated function of two assembled DNA-PK complexes. It has been reported that DNA-PK synapsis is required for full kinase activation (DeFazio et al., 2002); however, more recent reports suggest significant activation without synapsis ( Jovanovic and Dynan, 2006). If ABCDE phosphorylation occurred only in trans, this would provide a mechanism to limit end access to modifying enzymes only when appropriately paired ends are synapsed. Experiments using a combination of phosphorylation site mutants and kinase inactive mutants demonstrated that both ABCDE and PQR phosphorylation occurs in trans both in vitro and in living cells. Thus, linking ABCDE phosphorylation to synapsis (whether kinase activation is in cis or trans) provides a mechanism to protect DNA ends until the broken DNA ends are juxtaposed within the DNA-PK synapsis. Finally, these studies indicate that both ABCDE and PQR phosphorylation are primarily autophosphorylation events, suggesting that DNA-PK itself regulates end processing, although it has also been suggested that ATM or ATR can phosphorylate the ABCDE sites (Chen et al., 2007; Yajima et al., 2006).

8. FURTHER DNA-PK AUTOPHOSPHORYLATION IS REQUIRED DURING NHEJ An additional relevant in vivo autophosphorylation site resides within the activation loop (or ‘‘T’’ loop) of the kinase (threonine 3950, termed T) (Douglas et al., 2007). Mimicking phosphorylation at the T site inactivates the kinase, but does not reduce affinity of DNA-PKcs for DNA bound Ku. A DNA-PKcs mutant with the ABCDE, PQR, and T sites substituted to alanine (13 altogether) still substantially autophosphorylates in vitro (50% of wild-type levels) and still undergoes autophosphorylationinduced dissociation (Meek et al., 2007). Thus, another somewhat surprising finding is that autophosphorylation-induced end access and autophosphorylation-induced kinase dissociation are separate events

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mediated by distinct sites. Preliminary work from our laboratories has revealed numerous additional sites within DNA-PKcs that can be autophosphorylated in vitro. To date, eight of these have been studied (by mutational analyses). Five of the eight may be important targets during NHEJ because these mutants do not entirely reverse the radiosensitive phenotype of DNA-PKcs deficient cells. However, no clear function for these sites (with regards to NHEJ) has been determined. Two of the sites may alter the balance between NHEJ and HR (discussed below). Finally, we have also used a mutational approach to try to bypass the absolute necessity for DNA-PK’s kinase activity during NHEJ using numerous phospho-mimicking mutants in combination with kinase inactivating mutants (either on the same molecule or in trans). These studies suggest that ABCDE phosphorylation is required on both sides of the synapse and that additional phosphorylation events, besides PQR and T are required for efficient end joining. Recent work from Povirk and colleagues using a biochemical approach support this conclusion (Povirk et al., 2007). In sum, DNA-PK’s autophosphorylation is both important and functionally complex. Clearly, distinct phosphorylation events have distinct functional consequences. Our current working model is that via a series of autophosphorylation events, DNA-PK undergoes a series of conformational changes that facilitate each step of NHEJ.

9. MODEL OF DNA-PK ACTIVATION A current model of DNA-PK’s activation is as follows (Fig. 2.2). Ku initially binds the two ends of a double strand break, each of which then recruits a DNA-PKcs molecule. In vitro, if DNA-PKcs is not recruited to an end, it is possible for multiple Ku heterodimers to be loaded onto the end (Calsou et al., 1999). It is not known whether this occurs in living cells. Structural studies revealed the presence of distinct channels within the palm domain of DNA-PKcs that likely accommodate DNA. These data fit nicely with an earlier structural EM report that proposed a trans model for DNA-PK activation. After binding, unphosphorylated DNA-PKcs protects DNA ends. Although it is not known whether activation occurs by an interaction of DNA ends with DNA-PKcs in cis or trans, it is clear that some unwinding of the DNA termini is required for DNA-PK to ‘‘sense’’ the DNA end and become activated. Autophosphorylation (in trans) results in a series of conformational changes. ABCDE phosphorylation induces a more open conformation, leaving DNA ends accessible. ABCDE phosphorylation on just one side of the synapse allows end processing (of both DNA strands), but ABCDE must be phosphorylated on both sides of the synapse to promote ligation. PQR phosphorylation induces a conformational change that blocks access of DNA ends to nucleolytic activities,

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but allows ligation, and probably polymerase activities. Although not proven, a reasonable model would be that once a region of microhomology between opposing ends is found, PQR phosphorylation might function to hold the microhomology in place preventing further nucleotide loss, but allowing flap trimming, fill in, and ligation. Phosphorylation of T and additional, undefined sites mediate kinase inactivation and dissociation, respectively. DNA-PKcs can be dephosphorylated (at least partially) by protein phosphatase 5 (Wechsler et al., 2004), probably after dissociation Model of DNA-PK activation Ku

DNA-PKcs FATc

DNA-PKcs/artemis

PI3K FAT

1. Unlike Artemis, in the absence of DNA damage, Ku does not interact with DNA-PKcs. Because of the relative abundance of these two factors, it is likely that some DNA-PKcs is not bound to Artemis.

Head Arm Palm

2. DNA damage is recognized by Ku. The DNA-P Kcs Artemis complex is recruited, but Artemis dissociates from kinase inactive DNA-PK. DNA binding induces a conformational change in DNA-PKcs that results in closer association of the head and palm globular domains. An unsynapsed, DNA-PK bound end may be accessed by the HR pathway.

3. The termini of DNA ends must dissociate to facilitate activation of DNA-PK’s kinase activity. It is not clear whether DNA end melting occurs before or after synapsis or whether DNA-dependent activation is in cis or trans. DNA ends are protected from modification until the kinase is activated and ABCDE autophosphorylation occurs.

4. Synapsis results in a conformational change in DNA-PKcs, including bridging between Ku and the FATc domain. Synapsed DNA ends are sequestered and protected from modification, and HR until kinase activation and autophosphorylation.

FIGURE 2.2 (Continued)

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∗ ABCDE phosphorylation ∗ PQR phosphorylation ∗ T and other phosphorylations 5. ABCDE autophosphorylation in trans promotes access of DNA ends to Artemis and other DNA end modifying factors. With this phosphorylation status, DNA-PK’s interaction with the very termini are somewhat destabilized, but the complex is still stabley assembled at the break. The DNA break would also be accessible to HR at this point, if NHEJ fails.

6. End alignment is “sensed” by DNA-PK resulting in PQR phosphorylation. PQR phosphorylation protects the DNA ends from further nucleotide loss; end processing (based on this alignment) including ligation, fill in synthesis, and flap processing still proceeds. In this configuration, DNA-PK’s interaction is stabilized both by its interaction at the ends and also elsewhere in the complex. PQR phosphorylation strongly inhibits HR.

7. Additional autophosphorylation occurs at T as well as undefined sites to facilitate final steps in NHEJ. This is depicted after end ligation, although it is possible that phosphorylation events are required to facilitate fill in synthesis, flap removal, or ligation. Phosphorylation at sites (not yet identified) that induce dissociation “trump” the effects of ABCDE or PQR phosphorylation on DNA binding.

8. After dissociation, DNA-PKcs is de-phosphorlated by and can be recycled. It is likely that Ku must be removed from the resolved break by proteolysis.

FIGURE 2.2 Model of DNA-PK activation. (See Plate 1 in Color Plate Section.)

from Ku bound DNA. After resolution of the DSB, Ku (because of its ring structure) will likely be trapped on the DNA. It is currently unclear how Ku is removed; however, it has been shown that Ku86 is a target of a cellular protease (Gullo et al., 2008). It is possible that this (undefined) protease degrades Ku to facilitate its removal from repaired DNA.

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The ABCDE and PQR clusters are strongly conserved in vertebrates suggesting a strong benefit for their function. We posit that DNA-PK (composed of three abundant polypeptides) rapidly targets DNA breaks and limits end access until ‘‘downstream’’ repair factors that are expressed at more limiting amounts are targeted to the site of DNA damage. Linking synapsis to trans ABCDE autophosphorylation ensures that end processing, and more importantly ligation, can only occur when an appropriate DNA partner has been bound and aligned. This would be important if DNA-PK binds a single DNA end. Joining of single DNA ends from collapsed replication forks or from mispaired double strand breaks could obviously result in chromosomal aberrations. Speculation about how the cell deals with a single DNA-PK bound end is discussed below.

10. END PROCESSING TO PROMOTE END CONSERVATION Recent studies reveal several interesting aspects of NHEJ’s DNA end processing activities that promote DNA sequence conservation. Artemis is the only nuclease unequivocally involved in NHEJ; it likely contributes to only a subset of DNA breaks including endonucleolytic opening of hair-pinned coding ends during VDJ recombination (Goodarzi et al., 2006; Ma et al., 2002, 2004, 2005b; Niewolik et al., 2006; Riballo et al., 2004; Yannone et al., 2008). Physical association of DNA-PKcs as well as its enzymatic activity are required for Artemis’s endonucleolytic activity (Ma et al., 2002), although the requirement for kinase activity likely reflects a requirement to phosphorylate the ABCDE cluster (Goodarzi et al., 2006; Yannone et al., 2008). In the absence of DNA damage, Artemis is complexed with DNA-PKcs. Because of the relative abundance of DNA-PKcs and Artemis, it is likely that the pool of DNA-PKcs includes both Artemis/DNA-PKcs complexes and DNA-PKcs alone. Work from Calsou, Salles, and colleagues has shown that as DNA-PKcs is targeted to Ku bound DNA, Artemis is released from DNA-PKcs and is only rebound again when the kinase is activated (Drouet et al., 2006). Thus, Artemis is excluded from kinase inactive DNA-PK. Artemis has both endo- and exonuclease activities (Ma et al., 2002). DNA-PKcs strongly suppresses the exonuclease activity of Artemis but allows limited endonucleolytic trimming, likely at regions of single strand to double strand transition (Ma et al., 2002; Yannone et al., 2008). Thus, in combination, Artemis and DNA-PKcs promote relatively limited trimming of 50 overhangs, resulting in 30 end protrusions that can also be cleaved (endonucleolytically) by Artemis. While there is a multitude of evidence that ABCDE phosphorylation is required for Artemis to access internal regions of the DNA termini, there is some disagreement about whether Artemis can access

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the extreme terminus of a bound hairpin in the absence of ABCDE phosphorylation (Ding et al., 2003; Goodarzi et al., 2006). Several studies using primarily in vitro approaches have provided significant new insight into how DNA ends are made ligatable during NHEJ. First, although it has long been known that XRCC4/ligase IV can seal single strand nicks, recent data show that the XRCC4/ligase IV complex facilitates single strand ligation of mismatched DNA termini (Gu et al., 2007a; Ma et al., 2004). Resolving one strand of a double strand break certainly limits sequence loss on the second strand. This would in effect stop excessive end nucleotide loss completely, allowing Artemis to cleave (as flaps) mismatched overhangs, and allow the X family polymerases to fill in resulting gaps. Three X family polymerases l, m, and terminal deoxynucleotidyl transferase (Tdt, a template-independent polymerase that is expressed only in developing lymphocytes) function in NHEJ. All are targeted to the site of damage through interactions with DNA-PK (Ma et al., 2004; Mickelsen et al., 1999; Nick McElhinny et al., 2005). Pol l and pol m likely provide somewhat overlapping function in resolving gaps. However, recent perceptive work from Ramsden and colleagues shows that pol m has the unique ability to direct template-dependent synthesis across a DNA break with no terminal microhomology (Davis et al., 2008; Nick McElhinny et al., 2005). This would undoubtedly facilitate single strand ligation (by XRCC4/ligase IV) of partially aligned termini and explains why pol m deficiency affects only light chain assembly (that occurs when Tdt is not expressed). These authors suggest that (in the absence of pol m) non-templated ‘‘N’’ additions by Tdt facilitate terminal annealing during heavy chain rearrangement. Although the function(s) of XLF are still being investigated, two groups have reported that the newly discovered XLF factor stimulates ligation of mismatched termini by XRCC4/ ligase IV (Gu et al., 2007b; Tsai et al., 2007). Recent elegant structural studies provide an attractive model in support of this conclusion (Andres et al., 2007) Polynucleotide kinase phosphatase that is targeted to DSBs by its interaction with XRCC4 participates in end processing of a subset of DNA breaks (Chappell et al., 2002). An attractive model put forth by Lieber and colleagues is that end processing—either trimming, gap synthesis, or single strand ligation do not necessarily occur in a set order (Gu and Lieber, 2008; Lieber et al., 2008). Thus, end processing during NHEJ is flexible allowing for numerous slightly different ways to repair the same break. An appealing extension of this model (that must still be tested) is that PQR phosphorylation is induced when the two ends are aligned (perhaps at a region of micro-homology). This implies that the synapsed DNA-PK complex can ‘‘sense’’ alignment. PQR phosphorylation functions to ‘‘lock’’ the paired strands in place so that further processing is dictated by that alignment. In this model (after PQR phosphorylation), no additional sequence is lost from the DNA ends, but the ends are still accessible so that fill in

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DNA synthesis, flap processing, and ligation of the two DNA strands can proceed, albeit not in a given order. In sum, numerous investigators have used a variety of approaches to reveal that the DNA-PK synapse provides an accessible platform on which the two DNA ends rest. The current challenge is to decipher how DNA-PK regulates end access and promotes end joining at this synapse.

11. DOES DNA-PK REGULATE DSBR REPAIR PATHWAY CHOICE? How a cell chooses whether to repair a DNA double strand break by NHEJ or by HR is a fundamental and largely unanswered question. There is abundant evidence for competition between HR and NHEJ. This includes reports showing: (1) competition of the two pathways for repair of the same lesions (Frank-Vaillant and Marcand, 2002; Fukushima et al., 2001; Kim et al., 2005; Takata et al., 1998), (2) sensitivity to MMC-induced damage [that is dependent on HR for repair] in paired cell strains based on DNA-PKcs expression (Convery et al., 2005; Pluth et al., 2008), (3) increased HR of substrates in cells that lack DNA-PK (Allen et al., 2002, 2003; Pierce et al., 2001), (4) co-localization of NHEJ and HR factors at the same DNA lesion (Schwartz et al., 2005), and (5) partial rescue of the severe phenotypes associated with deficiency of XRCC4 or ligase IV, by disruption of the DNA-PK complex (in this case, deletion of DNA-PK apparently makes DSBs more accessible to HR, abrogating the severe phenotype) (Adachi et al., 2001; Karanjawala et al., 2002). Because of DNA-PK’s cellular abundance, it seems that there must be more to repair pathway choice than simple competition. All three components of the DNA-PK complex are exceptionally abundant proteins; this is especially true in human cells. In contrast, expression of downstream NHEJ factors and most HR factors is more limited. Further, expression levels of the three component DNA-PK polypeptides are not reduced during S and G2 when HR is more active (Lee et al., 1997). It seems logical that in most cases DNA-PK will be the first repair factor to bind a DSB. Simply put, if the two pathways are in simple competition, DNA-PK would always win. Thus, it seems rationale that a mechanism exists that facilitates HR, even if DNA-PK binds a break first. Data from our laboratories demonstrate that DNA-PK’s phosphorylation status strongly affects a cell’s ability to utilize the HR pathway by its ability to regulate DNA end access. Just as blocking ABCDE phosphorylation inhibits end processing during NHEJ, so does it inhibit end access to the HR pathway. In fact, inhibition of HR likely explains the severe radiosensitivity imparted to cells when ABCDE phosphorylation is blocked. Similarly, the PQR!ala mutant strongly promotes HR (as compared to

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wild-type DNA-PK) just like it promotes end processing during NHEJ. The reciprocal nature of these effects strongly suggests that DNA-PK is an active regulator of DNA end access to the HR pathway. Further, data showing that blocking phosphorylation at PQR in the ABCDE mutant (i.e., the combined mutant) substantially enhances HR and abrogates the severe radiosensitivity observed by blocking ABCDE alone, suggests that phosphorylation of PQR is critical to DNA-PK’s ability to inhibit HR. This fits well with the proposed model of PQR phosphorylation functioning to ‘‘lock’’ two ends in aligned position and thus favoring NHEJ. Since ABCDE and PQR are phosphorylated in trans, DNA-PK bound to a single DNA end (e.g., at a collapsed replication fork) should not be phosphorylated at either ABCDE or PQR. Although the DNA-PK mutant that cannot phosphorylate either cluster cannot function normally to promote NHEJ, it strongly promotes HR (Cui et al., 2005). Thus, it is possible that DNA-PK that cannot find a synaptic partner and thus does not phosphorylate ABCDE or PQR can actually facilitate HR, certainly a beneficial outcome. Recently, we have identified another potential DNA-PKcs phosphorylation site cluster that seems to also serve as a switch between NHEJ and HR (JK, Fig. 2.1). Unlike the first two clusters, phosphorylation at these sites does not appear to affect end processing (K.M., V.D., unpublished data). Although analyses of these sites is still ongoing, our data suggest that the phosphorylation status of DNA-PKcs affects repair pathway choice by regulating end access and perhaps also by regulating factor recruitment.

12. WHY IS DNA-PK SO ABUNDANT? A long-standing question in this field is why primate cells express such high levels of DNA-PK. While DNA-PK is fairly abundant in all mammalian cells, primate cells express 50 times more DNA-PK activity than rodent cells (Finnie et al., 1995). The other NHEJ factors are not so highly expressed in human cells. The high levels of DNA-PK in human cells are somewhat paradoxical in that this does not impart any increased ability to repair DNA damage (Allen et al., 2003). If DNA-PK does not afford human cells with particular resistance to DNA damage, why do human cells universally express such high levels of this huge complex? To try to more fully understand the consequences of expressing different levels of DNA-PK at a cellular level, we are studying the effect of expressing varying amounts of DNA-PKcs in rodent cells (K.M., unpublished data). Although rodent cells expressing human levels of DNA-PKcs and DNA-PK activity (Ku is not limiting) are easily derived, this does not alter radioresistance. However, overexpression of DNA-PKcs in rodent

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cells has a large impact on the cell’s ability to utilize HR to repair an integrated HR substrate (K.M., unpublished data). We suggest that in cells that express abundant DNA-PK, some mechanism must actively promote use of HR (or inhibit NHEJ). [It should be noted that this might differ in human cell lines where it has been shown that haploinsufficiency of DNA-PKcs results in moderate radiosensitivity (Eric Hendrickson, personal communication).] Another long-standing question in this field is why are there no children with defects in DNA-PK even though patients with other factors in this pathway (XLF, Artemis, and DNA ligase IV) have been described. The lack of DNA-PK mutations in humans is even more curious since all three of the spontaneous genetic forms of VDJ associated SCID in animals are DNA-PKcs mutations (SCID mice, dogs, and horses) (Blunt et al., 1996; Ding et al., 2002; Shin et al., 1997). Hundreds (if not thousands) of human SCID mutations have been characterized; none are the result of DNAPKcs or Ku mutations—not even hypomorphic mutations. This has led to the speculation that DNA-PKcs or Ku mutations in humans are lethal. Why should this be? Perhaps DNA-PK plays a secondary role in human cells. It has been suggested that primates may have a unique requirement for DNA repair (and specifically NHEJ) in the brain. Consistent with the idea that neurons have a unique requirement for DNA repair, mice with the most severe NHEJ defects (lack of XRCC4 or ligase IV) die in late embryogenesis because of neuronal apoptosis (Frank et al., 1998; Gao et al., 1998). However, conditional deletion of XRCC4 (only in fetal brain) does not support a unique role for NHEJ in the developing nervous system, but perhaps instead in neural progenitor cells or in the placenta (Yan et al., 2006). Placental defects have been shown to explain a similar phenotype of neuronal apoptosis and embryonic lethality in mice deficient in Rb (MacPherson et al., 2003). At this point, it is unclear whether a specific requirement for DNA-PK in the nervous system can explain the (likely) absolute requirement for DNA-PK for human viability. There is evidence that DNA-PK may be important in all human cells. Support for a particular stringent requirement for DNA-PK in human cells comes from the work of Hendrickson and colleagues (Fattah et al., 2008; Ghosh et al., 2007; Li et al., 2002). They have established that Ku is essential in human somatic cells; even Ku haploinsufficiency results in marked radiosensitivity, genomic instability, and telomeric shortening. We have recently shown that SCID puppies display significant intrauterine growth retardation and are 30% smaller at birth than their normal littermates (K.M., unpublished data). This is surprising since no birth weight differences have been documented in SCID mice. Although canine cells express much less DNA-PK than human cells, dog cells express significantly more than mouse cells (Meek et al., 2001). Perhaps expressing more DNA-PK is a reflection of a more stringent requirement for DNA-PK.

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Certainly; there is clear evidence from emerging data from Hendrickson and colleagues that DNA-PK is particularly important for telomere maintenance in human cells. Still, differing requirements for DNA-PK in different species may have several explanations. Perhaps the fact that DNA-PK deficiency can have different consequences in different species correlates with expression levels and also reflects a particular requirement to more carefully regulate HR. In sum, it seems likely that DNA-PK deficiency is not compatible with human life. Understanding how this large complex functions to orchestrate NHEJ at a double strand break and potentially regulate access of DNA lesions to other repair pathways should provide fundamental knowledge of how DNA-PK promotes genomic stability.

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homologous end-joining pathways of DNA double-strand break repair have overlapping roles in the maintenance of chromosomal integrity in vertebrate cells. EMBO J. 17, 5497–5508. Tsai, C. J., Kim, S. A., and Chu, G. (2007). Cernunnos/XLF promotes the ligation of mismatched and noncohesive DNA ends. Proc. Natl. Acad. Sci. USA 104, 7851–7856. Turchi, J. J., Henkels, K. M., and Zhou, Y. (2000). Cisplatin-DNA adducts inhibit translocation of the Ku subunits of DNA-PK. Nucleic Acids Res. 28, 4634–4641. Udayakumar, D., Bladen, C. L., Hudson, F. Z., and Dynan, W. S. (2003). Distinct pathways of nonhomologous end joining that are differentially regulated by DNA-dependent protein kinase-mediated phosphorylation. J. Biol. Chem. 278, 41631–41635. Uematsu, N., Weterings, E., Yano, K., Morotomi-Yano, K., Jakob, B., Taucher-Scholz, G., Mari, P. O., van Gent, D. C., Chen, B. P., and Chen, D. J. (2007). Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks. J. Cell Biol. 177, 219–229. Walker, A. I., Hunt, T., Jackson, R. J., and Anderson, C. W. (1985). Double-stranded DNA induces the phosphorylation of several proteins including the 90 000 mol. wt. heat-shock protein in animal cell extracts. EMBO J. 4, 139–145. Walker, J. R., Corpina, R. A., and Goldberg, J. (2001). Structure of the Ku heterodimer bound to DNA and its implications for double-strand break repair. Nature 412, 607–614. Wechsler, T., Chen, B. P., Harper, R., Morotomi-Yano, K., Huang, B. C., Meek, K., Cleaver, J. E., Chen, D. J., and Wabl, M. (2004). DNA-PKcs function regulated specifically by protein phosphatase 5. Proc. Natl. Acad. Sci. USA 101, 1247–1252. Weterings, E., and Chen, D. J. (2008). The endless tale of non-homologous end-joining. Cell Res. 18, 114–124. Weterings, E., Verkaik, N. S., Bruggenwirth, H. T., Hoeijmakers, J. H., and van Gent, D. C. (2003). The role of DNA dependent protein kinase in synapsis of DNA ends. Nucleic Acids Res. 31, 7238–7246. Yajima, H., Lee, K. J., and Chen, B. P. (2006). ATR-dependent phosphorylation of DNAdependent protein kinase catalytic subunit in response to UV-induced replication stress. Mol. Cell Biol. 26, 7520–7528. Yan, C. T., Kaushal, D., Murphy, M., Zhang, Y., Datta, A., Chen, C., Monroe, B., Mostoslavsky, G., Coakley, K., Gao, Y., Mills, K. D., Fazeli, A. P., et al. (2006). XRCC4 suppresses medulloblastomas with recurrent translocations in p53-deficient mice. Proc. Natl. Acad. Sci. USA 103, 7378–7383. Yan, C. T., Boboila, C., Souza, E. K., Franco, S., Hickernell, T. R., Murphy, M., Gumaste, S., Geyer, M., Zarrin, A. A., Manis, J. P., Rajewsky, K., and Alt, F.W (2007). IgH class switching and translocations use a robust non-classical end-joining pathway. Nature 449, 478–482. Yannone, S. M., Khan, I. S., Zhou, R. Z., Zhou, T., Valerie, K., and Povirk, L. F. (2008). Coordinate 50 and 30 endonucleolytic trimming of terminally blocked blunt DNA double-strand break ends by Artemis nuclease and DNA-dependent protein kinase. Nucleic Acids Res. 36, 3354–3365. Yoo, S., and Dynan, W. S. (1999a). Geometry of a complex formed by double strand break repair proteins at a single DNA end: Recruitment of DNA-PKcs induces inward translocation of Ku protein. Nucleic Acids Res. 27, 4679–4686. Yoo, S., Kimzey, A., and Dynan, W. S. (1999b). Photocross-linking of an oriented DNA repair complex. Ku bound at a single DNA end. J. Biol. Chem. 274, 20034–20039. Yu, Y., Wang, W., Ding, Q., Ye, R., Chen, D., Merkle, D., Schriemer, D., Meek, K., and LeesMiller, S. P. (2003). DNA-PK phosphorylation sites in XRCC4 are not required for survival after radiation or for V(D)J recombination. DNA Repair (Amst) 2, 1239–1252. Yu, Y., Mahaney, B. L., Yano, K.-I., Ye, R., Fang, S., Douglas, P., Chen, D. J., and LeesMiller, S. P. (2008). In Press.

CHAPTER

3 Thymic Microenvironments for T-Cell Repertoire Formation Takeshi Nitta,* Shigeo Murata,† Tomoo Ueno,* Keiji Tanaka,‡ and Yousuke Takahama*

Contents

1. Introduction 1.1. Overview of T-cell development in thymus 1.2. Stromal components of thymic microenvironments 2. Trafficking of Developing Thymocytes 2.1. Entry of T-precursor cells into thymus 2.2. Outward migration of immature thymocytes 2.3. Relocation of positively selected thymocytes to medulla 2.4. Export of T cells from thymus 3. Cortical Microenvironment 3.1. Formation of thymic cortex 3.2. Positive selection and thymoproteasome 4. Medullary Microenvironment 4.1. Formation of thymic medulla and thymic crosstalk 4.2. NF-kB signaling pathways for medulla formation 4.3. Regulation of mTEC development by TNF superfamily ligands 4.4. Establishment of self-tolerance in medulla: Role of mTEC

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* Division of Experimental Immunology, Institute for Genome Research, University of Tokushima,

Tokushima 770-8503, Japan Laboratory of Protein Metabolism, Graduate School of Pharmaceutical Sciences, University of Tokyo, Tokyo 113-0033, Japan { Laboratory of Frontier Science, Tokyo Metropolitan Institute of Medical Science, Tokyo 113-8613, Japan {

Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00603-2

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2008 Elsevier Inc. All rights reserved.

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4.5. Establishment of self-tolerance in medulla: Role of DC 4.6. Production of regulatory T cells 5. Concluding Remarks Acknowledgments References

Abstract

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Functionally competent immune system includes a functionally competent T-cell repertoire that is reactive to foreign antigens but is tolerant to self-antigens. The repertoire of T cells is primarily formed in the thymus through positive and negative selection of developing thymocytes. Immature thymocytes that undergo V(D) J recombination of T-cell antigen receptor (TCR) genes and that express the virgin repertoire of TCRs are generated in thymic cortex. The recent discovery of thymoproteasomes, a molecular complex specifically expressed in cortical thymic epithelial cells (cTEC), has revealed a unique role of cTEC in cuing the further development of immature thymocytes in thymic cortex, possibly by displaying unique self-peptides that induce positive selection. Cortical thymocytes that receive TCR-mediated positive selection signals are destined to survive for further differentiation and are induced to express CCR7, a chemokine receptor. Being attracted to CCR7 ligands expressed by medullary thymic epithelial cells (mTEC), CCR7-expressing positively selected thymocytes relocate to thymic medulla. The medullary microenvironment displays another set of unique self-peptides for trimming positively selected T-cell repertoire to establish self-tolerance, via promiscuous expression of tissue-specific antigens by mTEC and efficient antigen presentation by dendritic cells. Recent results demonstrate that tumor necrosis factor (TNF) superfamily ligands, including receptor activating NF-kB ligand (RANKL), CD40L, and lymphotoxin, are produced by positively selected thymocytes and pivotally regulate mTEC development and thymic medulla formation.

1. INTRODUCTION 1.1. Overview of T-cell development in thymus The thymus is an organ that supports the development and repertoire formation of T cells (Bevan, 1977; Miller, 1961; Zinkernagel et al., 1978). Thymic parenchyma consists of leukocytic cells called thymocytes, the majority of which belongs to T-lymphoid lineage, and various stromal cells including thymic epithelial cells (TEC) (Le Douarin and Jotereau,1975; Sainte-Marie and Leblond, 1964). Thymic stromal cells provide multiple signals to support manifold processes of thymocyte

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development that are essential for the supply of circulating T cells (Kingston et al., 1985; Williams et al., 1986). In response to these signals, developing thymocytes undergo proliferation, differentiation, and relocation to generate mature T cells that carry a diverse yet self-tolerant repertoire of T-cell antigen receptors (TCRs) (Marrack and Kappler, 1988; Scollay et al., 1988; von Boehmer, 1988). These steps of T-cell development take place in anatomically discrete regions of the thymus where a variety of specialized stromal cells are localized (Bhan et al., 1980; Jenkinson et al., 1980; von Boehmer et al., 2003, 2004). T cells arise from hematopoietic stem cell derived T-lymphoid progenitor cells that migrate to the thymus (Le Douarin and Jotereau,1975). T-cell differentiation is most commonly characterized by the combination of temporally coordinated expressions of cell surface proteins, including CD4, CD8, CD25, and CD44 (Ceredig et al., 1985; Pearse et al., 1989; Penit, 1988; Petrie et al., 1990; Scollay et al., 1984, 1988). Most immature hematopoietic cells that have just entered the thymus are defined as early T-lineage progenitors (Benz et al., 2008; Bhandoola et al., 2003). These cells lack the expression of CD4 and CD8 and therefore belong to CD4/CD8 double-negative (DN) thymocytes (Scollay et al., 1984, 1988). The survival and development of DN thymocytes are supported by delta-like ligands (Anderson et al., 2001; Besseyrias et al., 2007; Manilay et al., 2005; Mohtashami and Zu´n˜iga-Pflu¨cker, 2006; Sambandam et al., 2005; Schmitt and Zu´n˜iga-Pflu¨cker, 2002) and cytokines such as interleukin-7 (Moore et al., 1996; Murray et al., 1989; Peschon et al., 1994; Suda et al., 1990; von Freeden-Jeffry et al., 1995), both of which are produced by cortical TEC (cTEC) (Faas et al., 1993; Schmitt et al., 2004). At DN stage, thymoþ cytes undergo developmental programs through DN1 (CD44 CD25), þ þ þ DN2 (CD44 CD25 ), and DN3 (CD44CD25 ), during which V(D)J rearrangement at TCRg, d, and b loci occurs (Garman et al., 1986; Habu et al., 1987). Two lineages of T cells expressing either TCRab or TCRgd diverge at DN stage (Raulet et al., 1991; von Boehmer, 1988). The assembly of successfully rearranged TCRb and invariant pre-TCRa together with CD3 chains leads to commitment to the TCRab lineage and further differentiation beyond DN3 stage, including proliferation, TCRa-VJ rearrangement, CD25 downregulation, and expression of CD4 and CD8 coreceptors (Saint-Ruf et al., 1994; Shinkai et al., 1993; Yamasaki et al., 2006). These developmental processes of DN thymocytes are associated with the dynamic relocation of the cells in thymic parenchyma. T-lymphoid progenitor cells in adult mouse thymus are mostly localized in the corticomedullary junction, the area between deep cortex and medulla (Lind et al., 2001; Petrie, 2003). Thymocytes migrate toward the capsular region of the thymus during the differentiation to DN2 and DN3 thymocytes. Most DN3 thymocytes are localized in the subcapsular region of the cortex (Benz et al., 2004; Lind et al., 2001) where they further develop to

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pre-DP stage, or DN4 thymocytes, which show low expression of CD4, CD8, CD25, and CD44 and are immediate precursor cells to become CD4/ CD8 double-positive (DP) thymocytes (Petrie et al., 1990; Takahama et al., 1992, Wilson et al., 1989). DP thymocytes expressing TCRab on cell surface are localized in the cortex. DP thymocytes move actively within the cortical microenvironment (Li et al., 2007; Witt et al., 2005), probably seeking TCR interaction with major histocompatibility complex (MHC)-encoded molecules that are associated with self-peptides. Cortical DP thymocytes that interact via their TCRs with the self-peptide-MHC complex are selected for survival or death depending on the avidity of the interaction (Allen, 1994; von Boehmer, 1994). DP thymocytes that receive TCR signals with ligand interactions of weak avidity and nonextensive aggregation are induced to survive and differentiate into mature thymocytes, the process referred to as positive selection (Ashton-Rickardt et al., 1993; Takahama et al., 1994a). By contrast, DP thymocytes that receive TCR signals with ligand interactions of strong avidity and extensive aggregation are destined to die (Ashton-Rickardt and Tonegawa, 1994; Sebzda et al., 1994), a process referred to as negative selection or clonal deletion. During positive selection, the differential kinetics of TCR–ligand interactions determines þ þ cell lineage to become either CD4 CD8 or CD4CD8 single-positive (SP) thymocytes (Singer, 2002; Singer and Bosselut, 2004). Positively selected thymocytes relocate to thymic medulla where they further interact with self-peptides displayed in the medullary microenvironment (Takahama, 2006; Ueno et al., 2004). Medullary TEC (mTEC) express a diverse set of genes representing peripheral tissues (Klein and Kyewski, 2000; Kyewski and Klein, 2006), contributing to the establishment of self-tolerance in thymic medulla. A nuclear factor called autoimmune regulator (AIRE) participates in this promiscuous gene expression in mTEC (Anderson et al., 2005). Consequently, a diverse yet self-tolerant TCR repertoire is formed in the thymus, and mature T cells with such a TCR repertoire are released to the circulation. Thus, T-cell repertoire formation consists of stepwise fate determinations of thymocyte development in different thymic microenvironments (Fig. 3.1). The dynamic relocation of developing thymocytes within thymic microenvironments is crucial for T-cell repertoire selection and will be discussed in Section 2.

1.2. Stromal components of thymic microenvironments The above-mentioned control of T-cell development in the thymus is supported in multiple thymic microenvironments that are formed by different sets of thymic stromal cells. Thymic stromal cells are composed of TEC and other stromal cells including mesenchymal cells, endothelial cells, and

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DN3

b

c

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FIGURE 3.1 Lymphostromal interactions in T-cell development and repertoire formation in the thymus. Thymus seeding with lymphoid progenitor cells is regulated by stroma-mediated attraction and adhesion (signal a). Thymocyte development up to DN3 stage directs cortical thymic epithelial cells (cTEC) development and cortical microenvironment formation (signal b), and is associated with outbound migration within thymus parenchyma toward the subcapsular region. cTEC support positive and negative selection of double-positive (DP) thymocytes (signal c). Positively selected thymocytes differentiate to single-positive (SP) thymocytes and relocate to the medulla through chemotactic attraction by mTEC (signal d). SP thymocytes promote the increase in mTEC cellularity, thereby nurturing medulla formation (signal e). Medullary thymic epithelial cells (mTEC), along with DC, establish negative selection of self-reactive thymocytes (signal f) and produce Treg cells. Mature SP thymocytes are exported from the thymus in response to circulating sphingosine-1-phosphate (S1P).

hematopoietic cells such as dendritic cells (DC) and macrophages (Boyd et al., 1993). TEC consist of at least two major populations, cTEC and mTEC, which play crucial roles in the development and repertoire formation of T cells (will be discussed in Sections 3 and 4). cTEC and mTEC are derived from common progenitor cells that are generated from the endoderm of the third pharyngeal pouch (Blackburn and Manley, 2004; Bleul et al., 2006; Gordon et al., 2004; Manley and Blackburn, 2003; Rossi et al., 2006). The development of TEC is dependent on many transcription factors including Tbx1, Hoxa3, Pax1, and Foxn1 (Lindsay et al., 2001; Manley and Capecchi, 1998; Nehls et al., 1994; Su and Manley, 2000). Thymic mesenchymal cells are also crucial for supporting thymus generation and T-cell development (Anderson et al., 1993; Jenkinson et al., 2003, 2007a; Mu¨ller et al., 2008). The roles of DC in the thymus, particularly in the deletion of self-reactive thymocytes, will also be discussed in Section 4. It is also fascinating to note that thymic stromal cells that support T-cell development are

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supported, in turn, by developing thymocytes. The bidirectional signal exchanges between thymocytes and thymic stromal cells are appreciated as ‘‘thymic crosstalk’’ (Holla¨nder et al., 1995; Ritter and Boyd, 1993; Shores et al., 1991; van Ewijk et al., 1994), and will also be discussed in Sections 3 and 4 (also summarized in Fig. 3.1).

2. TRAFFICKING OF DEVELOPING THYMOCYTES 2.1. Entry of T-precursor cells into thymus Thymic development of T cells is initiated by seeding the thymus with T-lymphoid progenitor cells. Entry of T-lymphoid progenitor cells into the thymus is mediated by at least two different pathways: the vasculature-independent pathway that occurs before vascularization of the thymus during the early stage of embryonic development, and the vasculature-dependent pathway that occurs after vascularization in the late stage of embryonic development and postnatal period (Li et al., 2007; Liu et al., 2006; Rossi et al., 2005). Prevascular colonization of fetal thymus is regulated by chemotactic attraction of T-lymphoid progenitor cells into thymus primordium via interactions between chemokines and chemokine receptors (Li et al., 2007; Liu et al., 2006). Significant roles of two chemokine receptors, CCR7 and CCR9, in fetal thymus colonization during embryonic thymus development have been noted (Liu et al., 2005; Wurbel et al., 2001). Mice deficient for both CCR7 and CCR9 show severely defective fetal thymus colonization before thymus vascularization and selective loss of the first wave of T-cell development generating epidermal gd T cells (Liu et al., 2006). The expression of CCL21, a CCR7 ligand, and CCL25, a CCR9 ligand, is differentially regulated in the third pharyngeal pouch (Liu et al., 2006). During the prevascular period of thymus development, CCL21 is expressed by Gcm2-dependent parathyroid primordium but not by Foxn1-dependent thymic primordium, indicating the role of the parathyroid in supporting fetal T-cell development ( Jenkinson et al., 2007b; Liu et al., 2006). By contrast, CCL25 is expressed predominantly by Foxn1dependent thymic primordium ( Jenkinson et al., 2007b; Liu et al., 2006), in agreement with the severe defect of embryonic thymus seeding in Foxn1deficient nu/nu mice (Cordier and Haumont, 1980; Itoi et al., 2001; Nehls et al., 1996). Another chemokine, CXCL12, is also detectable in the thymicparathyroid epithelium and the perithymic mesenchyme of prevascular embryonic thymus (Bleul and Boehm, 2000; Jenkinson et al., 2007b). However, the number of lymphoid cells accumulating in prevascular fetal thymus is not diminished in mice deficient for CXCL12 or its receptor CXCR4, suggesting that CXCL12/CXCR4 is not required for the

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migration of hematopoietic progenitors to thymic epithelial primordium (Ara et al., 2003). After vascularization of the thymus, T-lymphoid progenitor cells in the thymus are mostly found in the corticomedullary junction where large blood vessels are enriched in the thymus (Lind et al., 2001) as well as in perivascular space surrounding those blood vessels (Mori et al., 2007), suggesting that T-lymphoid progenitor cells enter the thymus through the vasculature and perivascular space. Thymus-seeding T-lymphoid progenitor cells express CCR9 even in postnatal period (Benz and Bleul, 2005; Schwarz et al., 2007) and competitive bone marrow transfer experiments demonstrated that CCR9-deficient T-lymphoid progenitor cells are inefficient in thymus reconstitution (Schwarz et al., 2007; Uehara et al., 2002), suggesting the role of CCR9 in seeding postnatal vascularized thymus with progenitor cells. However, adult mice deficient for CCR9 or doubly deficient for CCR7 and CCR9 show no apparent reduction in the number of thymocytes and T cells in postnatal period (Liu et al., 2006; Uehara et al., 2002). By contrast, postnatal thymus seeding seems to involve adhesive cell–cell interaction between P-selectin, which is expressed by thymic endothelium, and P-selectin glycoprotein ligand-1 (PSGL-1), which is expressed by circulating lymphoid progenitor cells (Rossi et al., 2005; Scimone et al., 2006). Interestingly, the number of resident thymic lymphoid progenitors controls thymic expression of P-selectin (Rossi et al., 2005), suggesting that P-selectin acts as a thymic niche occupancy sensor that gate-keeps thymus seeding of lymphoid progenitor cells (Donskoy et al., 2003; Goldschneider, 2006).

2.2. Outward migration of immature thymocytes Upon entry into the thymus, T-lymphoid progenitor cells begin to differentiate, proceeding to the DN stages of T cell development. Along this developmental process, DN thymocytes migrate outward from the corticomedullary junction to the subcapsular region of the cortex (Lind et al., 2001; Petrie, 2003). Chemokine receptors, especially CXCR4, CCR9, and CCR7, have been noted for their roles in this outward migration of DN thymocytes. DN thymocytes that are deficient for CXCR4 fail to migrate efficiently outward from the corticomedullary junction to the cortex (Plotkin et al., 2003). DN2 thymocytes in CCR7-deficient mice are partially arrested at the corticomedullary junction (Fo¨rster et al., 2008; Misslitz et al., 2004). In CCR9-deficient mice, DN2 and DN3 thymocytes are poorly accumulated in the subcapsular region of the cortex (Benz et al., 2004). Inefficient developmental progression of DN thymocytes is reported in CXCR4-deficient mice and CCR7-deficient mice (Ara et al., 2003; Misslitz et al., 2004), suggesting that the optimal differentiation of DN thymocytes requires chemokine-dependent outward migration in the cortex from the

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corticomedullary junction to the subcapsular region. It is also shown that adhesion molecules, such as integrins a4b1 and a4b7 expressed by DN thymocytes and vascular cell adhesion molecule-1 (VCAM-1) expressed by cTEC, are critical for fixed adhesion between these cells and the optimal movement of DN thymocytes to the subcapsular region (Prockop et al., 2002). The subcapsular region of the cortex expresses transforming growth factor b, which regulates cell-cycle progression of DN thymocytes and their differentiation to DP thymocytes, suggesting that the outward migration of DN thymocytes to the subcapsular region of the cortex may regulate the optimal production of DP thymocytes (Takahama et al., 1994b).

2.3. Relocation of positively selected thymocytes to medulla Newly generated immature DP thymocytes move randomly in thymic cortex, as shown by in vitro real-time imaging of intact thymus lobes (Bousso et al., 2002; Witt et al., 2005). This ‘‘random walk motility’’ is also observed by noninvasive intravital imaging of thymocytes in fish thymus (Li et al., 2007). By contrast, immature thymocytes at early ontogeny before CD4 and CD8 expression appear dormant in the thymus (Li et al., 2007). Thus, the differentiation into DP thymocytes possibly coincides with the acquisition of cellular motility, which represents the activity of cells that dynamically seek TCR interaction with MHC-peptide complex in the cortical microenvironment. DP thymocytes that actively move within the cortex interact with cTEC and pause the motility upon TCR interaction with MHC-peptide ligands (Bhakta et al., 2005). Positively selected thymocytes are induced to survive and to relocate from the cortex to the medulla. Positive selection coincides with the appearance of a thymocyte population that displays rapid and directed migration toward the medulla (Witt et al., 2005). Upon positive selection, DP thymocytes elevate CCR7 expression on the cell surface (Campbell et al., 1999; Ngo et al., 1998; Ueno et al., 2004). CCR7 ligands, CCL19 and CCL21, in the thymus are predominantly expressed by mTEC and mostly localized in the medulla (Ueno et al., 2004). Consequently, positively selected thymocytes are attracted to the medulla through CCR7-mediated chemotaxis. Positively selected mature thymocytes in mice deficient for CCR7 or CCR7 ligands are defective in accumulation in the medulla and are localized in the cortex (Kurobe et al., 2006; Ueno et al., 2004), whereas forced expression of CCR7 in immature thymocytes induces the relocation of premature DP thymocytes to the medulla (Kwan and Killeen, 2004). Mice deficient for CCR7 signals exhibit autoimmunity to peripheral tissues (Davalos-Misslitz et al., 2007b; Ho¨pken et al., 2007; Kurobe et al., 2006). Thymocytes generated without CCR7 ligands are potent in inducing autoimmune exocrinopathy in mice, and thus are defective in

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establishing central tolerance (Kurobe et al., 2006). Therefore, the migration of positively selected thymocytes to the medulla is essential for the establishment of central tolerance (see Section 4.3), and the proper relocation of developing thymocytes within multiple thymic microenvironments is necessary for repertoire formation of T cells (Takahama, 2006). CCR7 in DP thymocytes is reported to be upregulated more prominently in cells that express class I MHC-restricted TCR than in those that express class II MHC-restricted TCR (Yin et al., 2007), and is said to be involved in TCR signaling and negative selection of thymocytes via a yet unknown mechanism (Davalos-Misslitz et al., 2007a).

2.4. Export of T cells from thymus Mature thymocytes that have completed T-cell development and repertoire selection in the thymus are exported from the thymus and delivered via circulation to supply peripheral lymphoid organs. During neonatal period, T-cell emigration from the thymus to the circulation is diminished by the lack of CCR7 (Ueno et al., 2002), possibly because the initial waves of mature thymocytes are defective in CCR7-mediated intrathymic migration to the medulla that is enriched with large vessels. It is also possible that the molecular mechanism of T-cell emigration in newborn period is developmentally regulated and different between neonatal and adult periods, for example, because thymic medulla is not fully formed during newborn period. The emigration of T cells from adult thymus is regulated by sphingosine-1-phosphate (S1P) and S1P receptor 1 (S1P1). S1P is produced by vascular endothelium (Venkataraman et al., 2008), stored in platelets and erythrocytes (Pappu et al., 2007; Yatomi et al., 2001), and present at a higher concentration in blood than in tissues including the thymus (Schwab et al., 2005). In addition to its role as an intracellular signal mediator, S1P serves as an extracellular ligand that activates specific G protein-coupled receptors, regulating chemotaxis and cell survival (Melendez, 2008). S1P1 is an S1P receptor that is highly expressed by mature lymphocytes, and its expression in T cells is elevated during the last stage of thymocyte development (Allende et al., 2004; Kurobe et al., 2006; Matloubian et al., 2004). Mice deficient for S1P1 show severe retention of T cells in both thymus and secondary lymphoid organs, indicating an essential role of S1P in T-cell egress from lymphoid organs including the thymus (Matloubian et al., 2004). It is speculated that mature thymocytes are exported from the thymus through perivascular space, the space surrounded by mesenchymal cells between thymic parenchyma and blood vessels, which is enriched in the corticomedullary junction as well as in thymic medulla (Kato, 1997; Kato and Schoefl, 1989; Mori et al., 2007; Ushiki, 1986). Indeed, the blockade of

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thymocyte egress by FTY720, a pharmacological compound that perturbs S1P- and S1P1-mediated signals, causes the accumulation of mature thymocytes in perivascular space (Kurobe et al., 2006), in agreement with the hypothesis that perivascular space is the path for mature thymocytes to migrate out of the thymus. Other mechanisms that regulate thymic egress of T cells include the expression of integrin a5b1 by mature thymocytes (Cotta-de-Almeida et al., 2004), the downregulation of CD69 expressed by semimature thymocytes (Feng et al., 2002), and the repulsion from CXCL12, a chemokine abundantly expressed in the thymus (Poznansky et al., 2002; Vianello et al., 2005). The roles of transcription factor KLF2 (Carlson et al., 2006) and phosphatidylinositol 3-kinase (Barbee and Alberola-Ila, 2005) in controlling thymocyte export are also noted.

3. CORTICAL MICROENVIRONMENT 3.1. Formation of thymic cortex Stromal cells in thymic cortex are mainly cTEC, many of which are oriented perpendicular to the capsule. During embryogenesis, immature þ þ epithelial cells that express both keratin 5 and keratin 8 (K5 K8 ) as well as þ K5K8 epithelial cells that resemble cTEC are generated before the colonization of lymphoid progenitor cells in thymus primordium (Hamazaki et al., 2007; Klug et al., 1998, 2002). Embryonic generation þ and patterned distribution of TEC subpopulations including K5K8 cTEC-like epithelial cells are not impaired in RAG2/gc double-deficient mice, in which the development of T, B, and NK cells is severely arrested, suggesting that the generation of cTEC does not require thymocytederived signals (Klug et al., 2002). However, later in ontogeny, in RAG2/gc double-deficient mice and in mouse strain tgE26 carrying a transgene encoding human CD3e, in which thymocyte development is arrested at DN1 stage, the meshwork-like architecture of the cortical epithelium in the thymus is disturbed (Holla¨nder et al., 1995; Klug et al., 1998; van Ewijk et al., 2000). TEC in þ þ these mice are mostly arrested at immature K5 K8 stage (Klug et al., 1998) and instead, large cysts are formed in their thymus (Holla¨nder et al., 1995; van Ewijk et al., 2000). These results demonstrate that thymocytemediated signals are required for the maintenance of cTEC and the formation of thymic cortical architecture (Klug et al., 2002). The defective cTEC development in tge26 human CD3e-transgenic mice can be rescued by the transplantation of normal hematopoietic cells into mutant embryos but not into mutant adult mice, suggesting that the thymocyte-induced maintenance of cTEC and cortical

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architecture may occur in a developmentally restricted manner (Holla¨nder et al., 1995). Unlike in RAG2/gc double-deficient mice or in tge26 mice, cTEC development and cortical architecture appear normal in mice deficient for RAG1 or RAG2, in which thymocyte development is arrested at DN3 stage (Klug et al., 1998; van Ewijk et al., 2000), indicating that thymocyte development beyond DN1 stage up to DN3 stage is sufficient for the maintenance of cTEC development and cortical architecture. Thymic cortical architecture is known to be highly susceptible to environmental stress such as corticosteroids or X-ray irradiation, which is likely secondary to the high susceptibility and apoptotic loss of immature DP thymocytes to these stimuli in the cortex (Ashwell et al., 2000; Mathieson and Fowlkes, 1984). Nevertheless, the molecular mechanisms that regulate the development and maintenance of cTEC are largely unknown. Keratin-5-driven disruption of Stat3 in TEC causes severe postnatal thymic hypoplasia including alterations of the cortical epithelial architecture, suggesting that Stat3 in TEC is essential for postnatal maintenance of thymic cortical architecture (Sano et al., 2001). A transgenic mouse strain called Tg66, originally designed to express NK1.1 under the human CD2 promoter, is reported to have a small and disorganized thymus with preferential loss of cTEC and to carry a defect intrinsic to cTEC (Assarsson et al., 2007). These mouse strains may serve as useful models to understand the molecular mechanisms of cTEC development.

3.2. Positive selection and thymoproteasome Numerous studies support the notion that low-affinity self-peptides presented in thymic cortex are responsible for positive selection of developing thymocytes. However, the nature of positively selecting peptides and positively selecting antigen-presenting cells remains elusive (Starr et al., 2003). A single MHC-peptide complex expressed by cTEC can produce a diverse repertoire of T cells (Ignatowicz et al., 1996), suggesting that any peptide that causes low-avidity TCR engagement might be capable of triggering positive selection of thymocytes. It was also suggested that, rather than initial positive selection, subsequent negative selection establishes repertoire formation of T cells (Huseby et al., 2005). Furthermore, it was shown that the experimental capability of inducing positive selection is not limited to cTEC but can be detected in fibroblasts and DC (Hugo et al., 1993; Pawlowski et al., 1993; Yasutomo et al., 2000). It was also shown that developing thymocytes and thymus-reentered T cells can induce positive selection of thymocytes (Choi et al., 2005; Horai et al., 2007; Kirberg et al., 2008; Li et al., 2005). Thus, whether cTEC, or any other cells in thymic cortex, carry any specialized capability to induce positive selection seemed unlikely until recently.

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However, the recent discovery of a novel subunit of the 20S proteasome, b5t, has revealed a unique capability of cTEC to support positive selection of thymocytes (Murata et al., 2007). Proteasomes are multicatalytic protease complexes that are responsible for regulated proteolysis in eukaryotic cells and for the generation of antigenic peptides presented by MHC class I molecules (Kloetzel, 2001; Rock and Goldberg, 1999). The 20S proteasome is responsible for the proteolytic activity of the proteasome and is composed of 28 subunits (two a-rings with a1 to a7 subunits and two b-rings with b1 to b7 subunits). Among these subunits, b1, b2, and b5 are responsible for caspase-like, trypsin-like, and chymotrypsin-like catalytic activities, respectively (Baumeister et al., 1998; Coux et al., 1996). Interferon-g induces the production of a new set of catalytic subunits, b1i, b2i, and b5i, to replace their constitutive counterparts, b1, b2, and b5, thereby forming immunoproteasomes, a proteasome complex that possesses an increased chymotrypsin-like activity and participates in efficient antigen presentation and immune responses (Kloetzel, 2001; Tanaka and Kasahara, 1998). By contrast, the newly identified catalytic subunit b5t is incorporated into the 20S proteasome instead of b5 or b5i, together with b1i and b2i (Murata et al., 2007). Since this novel proteasome containing b5t is specifically expressed in the thymus and exclusively in cTEC, it is termed ‘‘thymoproteasome.’’ In comparison to b5-containing standard proteasomes and b5i-containing immunoproteasomes, b5t-containing thymoproteasomes exhibit reduced chymotrypsin-like activity but normal caspase-like activity and normal trypsin-like activity (Murata et al., 2007). Proteasomes are responsible for the production of MHC class I-binding peptides and are the sole enzymes that determine the C termini of the peptides (Cascio et al., 2001; Rock et al., 2004). Hydrophobic C-terminal anchor residues of the peptides are essential for high-affinity peptide binding into the clefts of MHC class I complexes (Young et al., 1995). Chymotrypsin-like activity carried by b5 and b5i is important for the production of high-affinity MHC class I ligands (Fehling et al., 1994). Thus, it is possible that cTEC generate a unique set of MHC class I-associated peptides that are different from those present in any other cells. þ The generation of CD4CD8 (CD8SP) thymocytes that express high levels of TCRs is severely reduced in b5t-deficient mice. The selective reduction of CD8SP T cells is also observed in the spleen of these mice. By contrast, DP and CD4SP thymocytes as well as peripheral CD4 T cells are unaffected. The absence of b5t does not affect cortical or medullary architecture or overall thymus size, indicating that b5t is essential for neither the development of cTEC nor the generation of normal thymic architecture. These results demonstrate that b5t is required for the development of CD8SP T cells in the thymus and suggest the possibility that b5t is associated with positive selection of CD8SP T cells (Murata et al., 2007).

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It is possible that thymoproteasomes in cTEC may be somehow involved in providing costimulatory signals that are specifically required for the generation of CD8SP T cells rather than CD4SP T cells. However, since the generation of CD8SP T cells is specifically affected in b5t-deficient mice, and since the surface expression of MHC class I molecules on cTEC of b5t-deficient mice are comparable to that of normal mice, b5t-containing thymoproteasomes in cTEC may be involved in producing class I MHCloaded peptides that provide TCR signals required for positive selection of class I MHC-restricted CD8SP T cells (Murata et al., 2007). b5t specifically limits chymotryptic activity that cleaves peptide bonds after hydrophobic amino acid residues in cTEC, and thereby thymoproteasomes predominantly produce low-affinity class I MHC ligands specifically in cTEC. These low-affinity class I MHC ligands may limit the duration and/or avidity of the interaction with TCRs and contribute to inducing positive selection of the majority of CD8SP T cells (Murata et al., 2007; Takahama et al., 2008). The results from b5t-deficient mice reveal that cTEC possess unique protein degradation activity that might lead to the production of a unique set of class I MHC-associated peptides necessary for the generation of CD8 T cells. This unique protein degradation activity of cTEC might not be limited to class I MHC-associated peptides but might also occur in class II MHC-associated peptides, since cathepsin L, a lysosomal protease that is highly expressed by cTEC and not by mTEC (Honey and Rudensky, 2003), is required for the optimal generation of CD4SP T cells (Honey et al., 2002; Nakagawa et al., 1998). A unique lysosomal degradation activity might be functional in cTEC, which is mediated by cathepsin L in a manner analogous to thymoproteasomes. Thus, the unique character of cTEC protein degradation and self-peptide presentation may be pivotal for the positive selection of thymocytes in both CD4SP and CD8SP lineages (Murata et al., 2008; Takahama et al., 2008; Fig. 3.2). These findings on cTEC would not only highlight the significant roles of thymic cortex in T-cell development but also further our understanding of the molecular mechanisms of T-cell repertoire selection.

4. MEDULLARY MICROENVIRONMENT 4.1. Formation of thymic medulla and thymic crosstalk The microenvironment of thymic medulla is mainly composed of mTEC and hematopoietic cells including mature thymocytes and DC. Like cTEC, mTEC are derived from endodermal precursor cells that are generated at the third pharyngeal pouch (Bleul et al., 2006; Rodewald et al., 2001; Rossi et al., 2006).

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FIGURE 3.2 A possible scheme for stepwise T-cell repertoire selection by uniquely displayed self-peptides by cortical thymic epithelial cells (cTEC) and medullary thymic epithelial cells (mTEC). In cTEC, thymoproteasomes likely produce a unique set of class I major histocompatibility complex (MHC)-associated peptides and cathepsin L may participate in producing a unique set of class II MHC-associated peptides. These selfpeptides unique to cTEC may be required for optimal positive selection of cortical thymocytes. In the medulla, mTEC promiscuously express a diverse set of genes including various tissue-specific genes. Peptides encoded by these genes may be displayed with MHC molecules directly by mTEC and/or indirectly by dendritic cells (DC) in thymic medulla for negative selection of self-reactive T cells, establishing T-cell repertoire formation in the thymus. In the periphery, T cells interact with self-peptides for survival and initiate immune responses upon recognition of foreign antigens.

The formation of thymic medulla is associated with the development of mature thymocytes in the thymus. An early study showed that medulla formation is defective in scid mice, in which thymocyte development is arrested at DN3 stage and is restored by the reconstitution with wild-type hematopoietic cells (Shores et al., 1991). Studies of mice deficient for positive selection, including TCRa-deficient mice, ZAP70-deficient mice, and MHC class I and class II double-deficient mice, confirmed that the formation of thymic medulla is dependent on the generation of positively selected mature thymocytes (Gray et al., 2006; Nasreen et al., 2003; Negishi et al., 1995; Philpott et al., 1992; Surh et al., 1992; van Ewijk et al., 1994). In mice deficient for CCR7 or CCR7 ligands, medulla formation is mildly defective (Kurobe et al., 2006; Ueno et al., 2004), suggesting that optimum development of thymic medulla requires the relocation of positively selected thymocytes. Thus, signals produced by positively selected thymocytes crucially regulate mTEC development and medulla formation. This lymphostromal interaction is referred to as thymic crosstalk (van Ewijk et al., 1994). A recent report indicates that positive selection promotes the increase in the number of mTEC and thereby nurtures the formation of thymic medulla (Hikosaka et al., 2008).

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4.2. NF-kB signaling pathways for medulla formation A series of studies have indicated that signaling pathways that lead to the activation of nuclear factor-kB (NF-kB) are required for the development of thymic medulla. Two distinct signaling pathways, classical and nonclassical, are presumed to activate NF-kB (Bonizzi and Karin, 2004; Hoffmann and Baltimore, 2006). Mice deficient for NF-kB-inducing kinase (NIK), IkB-kinase a (IKKa), RelB, or NF-kB2 (p52) exhibit defective thymic medulla formation (Burkly et al., 1995; Heino et al., 2000; Kajiura et al., 2004; Kinoshita et al., 2006; Naspetti et al., 1997; Weih et al., 1995; Zhang et al., 2006; Zhu et al., 2006; Zuklys et al., 2000), indicating the role of nonclassical NF-kB pathway in thymic medulla formation. The defect of medulla formation in NF-kB2-deficient mice is milder than that in mice deficient for other molecules in this pathway. Mice deficient for an IkB family member Bcl-3, which binds to NF-kB2 and modulates transcriptional activity in the nucleus (Bours et al., 1993; Fujita et al., 1993; Hayden and Ghosh, 2004; Nolan et al., 1993), do not cause defective formation of thymic medulla (Zhang et al., 2007). However, the loss of both NF-kB2 and Bcl-3 leads to severe defect in thymic medulla formation, suggesting that NF-kB2 and Bcl-3 share a role in the nonclassical NF-kB pathway for thymic medulla formation (Zhang et al., 2007). The classical NF-kB pathway is also important for thymic medulla formation as the deficiency in mice of tumor necrosis factor (TNF)-receptor-associated factor 6 (TRAF6), a signal transducer activating the classical NF-kB pathway (Inoue et al., 2007), causes severe defect in thymic medulla formation (Akiyama et al., 2005). The TRAF6-mediated classical NF-kB pathway induces RelB expression and therefore relays signals to activate the nonclassical NF-kB pathway in mTEC development (Akiyama et al., 2005). These NF-kB signaling pathways appear to contribute to mTEC development intrinsically within developing mTEC rather than to the transcellular actions of neighboring cells, including developing thymocytes.

4.3. Regulation of mTEC development by TNF superfamily ligands As mentioned above, both intercellular signals derived from developing thymocytes and intracellular NF-kB signals in thymic stromal cells are important for mTEC development. Until recently, however, it was unclear whether these two signaling events are interconnected with each other. Lymphotoxin-b receptor (LTbR), a TNF receptor superfamily (TNFRSF) member that activates the NIK-mediated nonclassical NF-kB signaling pathway (Matsushima et al., 2001; Yin et al., 2001), is the first cell surface receptor that is implicated in linking NF-kB signaling pathways in

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mTEC to cell-to-cell communication. Mice deficient for LTbR show reduced size of thymic medulla and a small number of mature mTEC, and these phenotypes are due to defect intrinsic to mTEC (Boehm et al., 2003; Venanzi et al., 2007). Although one report indicates that LTbR signals could induce AIRE expression in mTEC (Chin et al., 2003), later studies demonstrate that LTbR signals do not regulate AIRE expression in mTEC (Boehm et al., 2003; Seach et al., 2008; Venanzi et al., 2007). However, LTbR signals regulate the expression in mTEC of CCR7-ligand chemokines, which promote cortex-to-medulla migration of developing thymocytes (Seach et al., 2008; Zhu et al., 2007). LTbR-binding ligands are detectable on cell surface of SP thymocytes (Boehm et al., 2003). Indeed, the mRNA levels of LTa and LTb, which are ligands for LTbR, are more strongly detected in SP thymocytes than in DP thymocytes (Hikosaka et al., 2008). However, gross medulla formation is not impaired in mice deficient for LTa, LTb, or LIGHT, another LTbR ligand (Boehm et al., 2003; Rossi et al., 2007; Venanzi et al., 2007), suggesting that LTbR promotes mTEC development through yet unknown ligands or via a ligand-independent manner. Nonetheless, a recent report shows that the expression of AIRE-independent tissue-specific antigens in an AIRE-negative mTEC subpopulation is defective in mice deficient for LTa or LTb, suggesting that LTa and LTb regulate AIRE-independent promiscuous expression of tissue-specific antigens in mTEC (Seach et al., 2008). It is also reported that the overexpression of LTa and LTb induces thymic involution (Heikenwalder et al., 2008). Recent studies have revealed the pivotal roles of receptor activating NF-kB (RANK) and RANK ligand (RANKL), the former being a TNFRSF member and the latter, a TNF superfamily (TNFSF) member, in mTEC development and medulla formation. RANK is more strongly expressed in mTEC than in cTEC and is required for the development of mTEC expressing AIRE (Anderson et al., 2007; Rossi et al., 2007). RANKL þ is expressed by CD4 CD3 lymphoid tissue inducer (LTi) cells and is involved in the differentiation of mTEC during embryogenesis (Rossi et al., 2007; White et al., 2008). RANKL is also expressed by positively selected thymocytes in postnatal mice (Hikosaka et al., 2008). The number of mTEC is reduced in mice deficient for RANKL, and the enforced expression of RANKL in mice deficient for positive selection restores mTEC cellularity and medulla formation, suggesting that RANKL mediates thymic crosstalk signals for the optimal formation of thymic medulla (Hikosaka et al., 2008). Osteoprotegerin (OPG), a naturally occurring soluble RANKL inhibitor that binds to RANKL and inhibits RANKL binding to its signaling receptor RANK, is expressed by mTEC and regulates mTEC development and medulla formation, since mice deficient for OPG exhibit hypercellularity of mTEC and enlarged thymic medulla (Hikosaka et al., 2008).

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Positively selected thymocytes also produce CD40L, another TNFSF member (Fuleihan et al., 1995; Hikosaka et al., 2008). Unlike RANKL, CD40L is not expressed in thymic LTi cells (Hikosaka et al., 2008). The enforced expression of CD40L in vivo produces an enlarged medulla in the thymus, suggesting that CD40L signals can promote thymic medulla formation (Clegg et al., 1997; Dunn et al., 1997). Analysis using fetal thymus organ culture shows that CD40L as well as RANKL facilitates mTEC development through classical and nonclassical NF-kB pathways (Akiyama et al., 2008). Mice deficient for both CD40 and RANKL exhibit severe defect in mTEC development and thymic medulla formation, while the single deficiency of CD40 causes only a mild defect in thymic medulla (Akiyama et al., 2008; Hikosaka et al., 2008). Thus, it is likely that RANKL and CD40 cooperate with each other to promote mTEC development. It is also possible that RANKL and CD40 may sequentially regulate mTEC development; RANKL produced by LTi cells plays a role in mTEC development during embryogenesis, whereas RANKL and CD40L produced by positively selected mature thymocytes essentially promote postnatal increase of mTEC cellularity (Fig. 3.3).

LTi

SP

RANKL

RANKL, CD40L OPG RANK, CD40

RANK AIRE

pTEC CCR7L

mTEC Medulla

FIGURE 3.3 A model of medullary thymic epithelial cells (mTEC) development and thymic medulla formation. Embryonic differentiation of TEC progenitor cells (pTEC) into functionally mature mTEC that express AIRE and CCR7 ligand (CCR7L) chemokines is regulated by receptor activating NF-kB ligand (RANKL)-expressing lymphoid tissue inducer (LTi) cells. In postnatal thymus, positive selection results in the generation of single-positive (SP) thymocytes that express RANKL and CD40L, which promote the proliferation of mTEC and thereby nurture the formation of thymic medulla. The role of osteoprotegerin (OPG), a naturally occurring soluble RANKL inhibitor, is expressed by mTEC and regulates mTEC development and medulla formation.

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4.4. Establishment of self-tolerance in medulla: Role of mTEC Thymic medulla is a specialized microenvironment where developing thymocytes establish tolerance to systemic self-antigens, including peripheral tissue-specific antigens. Indeed, T-cell development within the thymus that is defective in thymic medulla formation leads to the failure in establishing self-tolerance, resulting in autoimmune disorders (Akiyama et al., 2005, 2008; Boehm et al., 2003; DeKoning et al., 1997; Kajiura et al., 2004; Kinoshita et al., 2006; Rossi et al., 2007; Weih et al., 1995; Zhang et al., 2006, 2007; Zhu et al., 2006). In thymic medulla, mTEC express a variety of ‘‘tissue-specific’’ genes, for example, the geneencoding insulin whose expression is restricted to b-islet cells in the pancreas (Heath et al., 1998; Klein et al., 1998; Sospedra et al., 1998; Werdelin et al., 1998). This ‘‘ectopic’’ expression of tissue-specific genes by mTEC is called ‘‘promiscuous gene expression’’ and is responsible for the establishment of self-tolerance through the presentation of tissuespecific antigens to developing thymocytes (Derbinski et al., 2001; Klein et al., 2000; Klein and Kyewski, 2000; Fig. 3.2). AIRE expression is associated with promiscuous gene expression of mTEC. AIRE is a nuclear protein that is predominantly expressed by mTEC (Anderson et al., 2002; Heino et al., 1999, 2000; Zuklys et al., 2000). AIRE deficiency leads to autoimmune polyendocrinopathy syndrome type 1 (APS1), or autoimmune polyendocrinopathy-candidiasisectodermal dystrophy (APECED), in human (Aaltonen et al., 1997; Nagamine et al., 1997), and essentially equivalent organ-specific autoimmune diseases in mouse (Anderson et al., 2002; Kuroda et al., 2005; Ramsey et al., 2002). AIRE-deficient mTEC fail to express a fraction of tissue-specific genes, indicating that AIRE contributes to the establishment of self-tolerance of developing thymocytes through the regulation of promiscuous gene expression (Anderson et al., 2002; Derbinski et al., 2005; DeVoss et al., 2006; Gavanescu et al., 2007; Giraud et al., 2007; Liston et al., 2004). Negative selection of thymocytes that are reactive to tissue-specific antigens is impaired in AIRE-deficient mice (Anderson et al., 2005; Liston et al., 2003, 2004). Thus, AIRE contributes to promiscuous gene expression of mTEC, thereby controlling negative selection of self-reactive thymocytes that are positively selected but not negatively selected in thymic cortex. In addition, AIRE is capable of binding to specific DNA motifs of a variety of genes including several tissue-specific antigens, suggesting that AIRE may regulate the promiscuous transcription of various genes in mTEC nuclei (Ruan et al., 2007). It should be noted, however, that there is an AIRE-independent set of promiscuously expressed genes (Anderson et al., 2005; Derbinski et al., 2005; Kuroda et al., 2005), some of which are even expressed by an mTEC subpopulation distinct from AIRE-positive mTEC (Derbinski et al., 2005;

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Seach et al., 2008). Thus, the promiscuous gene expression of mTEC must be operated by a mechanism that is functional even without AIRE. Indeed, promiscuously expressed genes in mTEC tend to colocalize in clusters in the genome, suggesting that the promiscuous gene expression of mTEC may involve yet unidentified epigenetic regulators of chromosomal remodeling (Derbinski and Kyewski, 2005; Derbinski et al., 2005; Johnnidis et al., 2005). It is also possible that AIRE is a regulator of mTEC development in addition to its being a regulator of promiscuous gene expression (Anderson et al., 2005; Gillard and Farr, 2005). Indeed, AIRE deficiency causes alterations in medullary architecture, suggesting that AIRE regulates mTEC development (Gillard et al., 2007). However, AIRE expression causes proliferative arrest of mTEC (Gray et al., 2007), arguing that AIRE cannot regulate further development of mTEC and AIRE expression should be limited in terminally differentiated mTEC (Gotter and Kyewski, 2004).

4.5. Establishment of self-tolerance in medulla: Role of DC In addition to mTEC, thymic DC critically contribute to the establishment of self-tolerance in thymic medulla (Fairchild and Austyn, 1990; Moore et al., 1994). DC in the thymus are predominantly localized in the medulla, although some DC are sparsely distributed in the cortex (Kurobe et al., 2006). Thymic DC are responsible for the negative selection of Mtv-encoded superantigen-reactive thymocytes (Ferrero et al., 1997; Moore et al., 1994). In the thymus, bone marrow-derived antigen-presenting cells, possibly thymic DC, can also induce the deletion of thymocytes that are reactive to a peripheral tissue-specific antigen that is promiscuously expressed by mTEC (Gallegos and Bevan, 2004). Thus, like peripheral DC, thymic DC participate in the cross-presentation of antigens originally expressed by other cells. Thymic DC acquire tissue-specific antigens from either peripheral tissues or mTEC and present these antigens to developing thymocytes, resulting in negative selection of self-reactive thymocytes. Thymic DC are at least in part intrathymically derived from hematopoietic precursor cells, since lymphoid progenitor cells that seed the thymus are capable of giving rise to DC (Ardavin et al., 1993; Lu et al., 2005; Masuda et al., 2005; Wu and Shortman, 2005; Wu et al., 1996). Apoptosis of mature mTEC may contribute to cross-presentation by thymic DC of mTEC-derived antigens, including promiscuously expressed tissue-specific antigens (Gray et al., 2007). It is also noted that the recruitment of circulating DC into thymic medulla may contribute to intrathymic presentation of peripheral antigens to induce deletion of self-reactive thymocytes (Bonasio et al., 2006). Thus, thymic DC of intrathymic and

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extrathymic origin may contribute to the collection and display of selfantigens in thymic medulla to semimature thymocytes, thereby further trimming thymocyte repertoire by the deletion of self-reactive T cells.

4.6. Production of regulatory T cells In addition to the deletion of self-reactive thymocytes, thymic medulla participates in the establishment of self-tolerance by producing regulatory T (Treg) cells. Thymic production of Treg cells appears essential for the protection from autoimmunity ( Itoh et al., 1999; Sakaguchi et al., 1985), although Treg cells can also be induced in peripheral T cells (Hori et al., 2003). The transcription factor forkhead box P3 (Foxp3) essentially regulates the development and function of Treg cells (Fontenot and Rudensky, 2005; Sakaguchi, 2005). A majority of Foxp3-expressing Treg cells within the thymus are found in the medulla (Aschenbrenner et al., 2007; Fontenot et al., 2005). Intrathymic development of Foxp3-expressing Treg cells requires costimulatory signals, which are available in thymic medulla (Salomon et al., 2000; Spence and Green, 2008; Tai et al., 2005) and NF-kB signaling pathways via NIK, TRAF6, NF-kB2, and Bcl-3, which regulate thymic medulla formation (Akiyama et al., 2005; Kajiura et al., 2004; Zhang et al., 2007). Hassall’s corpuscles, the corpuscular bodies of epithelial cells exclusively detectable in thymic medulla, are reported to play a role in the generation of Treg cells (Watanabe et al., 2005). Hassall’s corpuscles produce thymic stromal lymphopoietin (TSLP), a cytokine that acts on thymic DC to induce the generation of Treg cells (Watanabe et al., 2005). Chemokine signals may direct medullary thymocytes to Hassall’s corpuscles (Annunziato et al., 2000; Chantry et al., 1999). TCR specificity is implicated for its role in thymic development of Treg cells (Coutinho et al., 2005; Hsieh and Rudensky, 2005). Thymic generation of Treg cells requires positive selection with TCR engagement of higher avidity than positive selection for conventional T cells ( Jordan et al., 2001; Kawahata et al., 2002; Picca et al., 2006).

5. CONCLUDING REMARKS The fact that the thymus contains at least two distinct microenvironments, the cortex and the medulla, has been known for over 100 years (Goodall, 1905; Hassall, 1849; Lewis, 1904; Symington, 1898). Studies conducted over a span of 40 years since the discovery of the immunological function of the thymus have remarkably advanced our understanding of lymphocyte biology in relation to T-cell development and selection in the thymus. However, the developmental and molecular biology of thymic stromal

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cells has remained quite vague until recently. Recent achievements have enabled us to study the biology of thymic microenvironments using several molecules that represent key functions of thymic stromal cells. Those molecules include Foxn1, delta-like ligands, AIRE, and b5t. Several molecules that mediate the development of thymic stromal cells, such as RANKL and CD40L, have also been identified. These outcomes should provide a solid foundation for further studies of thymic microenvironments to better understand the molecular mechanism of the development and repertoire formation of T cells and the therapeutic reconstitution of functional thymus for various clinical situations (Gray et al., 2005; van den Brink et al., 2004). Unveiling the many secrets of thymic microenvironments has just begun.

ACKNOWLEDGMENTS We thank present and past members of Takahama laboratory at University of Tokushima, especially Drs. Cunlan Liu, Norimasa Iwanami, Jie Li, Shuhei Tomita, Hirotsugu Kurobe, Hiroshi Nakase, Yoshiko Akamatsu, Michael Sheard, and Mariam Nasreen for fruitful contributions to the studies discussed in this review and Drs. Taishin Akiyama, Graham Anderson, Georg Hollander, Richard Boyd, Nancy Manley, Howard Petrie, and Alfred Singer for critical and helpful discussion. Technical and administrative contributions by Mika Kubo, Fumi Saito-Miyawaki, and Izumi Ohigashi are acknowledged. Support by MEXT in the form of Grant-in-Aids for Scientific Research (Program B and Priority Areas ‘‘Immunological Self’’) is also acknowledged. T. N. is a JSPS Research Fellow.

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CHAPTER

4 Pathogenesis of Myocarditis and Dilated Cardiomyopathy Daniela Cihakova* and Noel R. Rose*,†

Contents

1. Human Myocarditis 1.1. Clinical picture of human myocarditis 1.2. Etiology of Myocarditis 1.3. Genetics of autoimmune myocarditis 1.4. Selected types of myocarditis 2. The Evidence for an Autoimmune Process in Myocarditis 2.1. Autoantibodies 2.2. Immunosuppressive therapy 3. Mouse Models of Myocarditis 3.1. CVB3-induced myocarditis 3.2. Experimental autoimmune myocarditis 4. Role of Proinflammatory Cytokines in Myocarditis 5. Role of T Helper Cells in Myocarditis 5.1. Role of Th1 cells in myocarditis 5.2. Growing evidence of divergent functions of IL-13 and IL-4 5.3. Is myocarditis a Th1- or Th17-driven disease? 6. The Divergent Role of Macrophages in Myocarditis 7. Conclusions/Directions for Future Research Acknowledgments References

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* Department of Pathology, Johns Hopkins University School of Medicine, Baltimore, Maryland, USA {

W. Harry Feinstone Department of Molecular Microbiology and Immunology, The Johns Hopkins University Bloomberg School of Public Health, Baltimore, Maryland, USA

Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00604-4

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2008 Elsevier Inc. All rights reserved.

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Myocarditis is a disease with a variable clinical presentation, ranging from asymptomatic to a fatal outcome. Among the recognized causes of myocarditis are mutations in multiple genes; infection by bacterial, rickettsial, mycotic, protozoan, and viral agents; and exposure to drugs, toxins, and alcohol. Some subtypes of myocarditis, such as giant cell myocarditis or eosinophilic necrotizing myocarditis, are suspected to be caused by an autoimmune inflammation. Several lines of evidence support the involvement of autoimmunity in myocarditis. These include the production of antibodies against relevant self-antigens, the fact that myocarditis symptoms can be relieved by immunosuppressive therapy in some patients, and a co-occurrence of myocarditis with other autoimmune diseases. Most of the evidence that myocarditis is an autoimmune disease comes from animal models. In this chapter, we discuss coxsackievirus B3-induced myocarditis and myosin-induced myocarditis as models of both viral and autoimmune inflammation in the heart. The latest advances in the study of autoimmunity have been concentrated on T helper cells, particularly the newly discovered subset, Th17 cells. Experimental autoimmune myocarditis (EAM), a mouse model of myocarditis induced by cardiac myosin, is partly an IL-17-driven disease. However, we have shown recently in IL-13 knockout mice that the disease can be driven through other pathways, and that the Th1 helper cells also lead to severe heart inflammation. Most importantly, IL-17A knockout mice are not fully protected against EAM and still develop mild myocarditis. The most abundant cells in heart infiltrate in human giant cell myocarditis or EAM are monocyte/macrophages, and there is now evidence that macrophages play a decisive role in the course of EAM.

1. HUMAN MYOCARDITIS 1.1. Clinical picture of human myocarditis The clinical manifestation of myocarditis is highly variable, ranging from no symptoms to heart failure (Rose and Baughman, 1998). Common presenting signs include nonspecific flu-like symptoms, arrhythmias, palpitations, dizziness, syncopes, and left ventricular failure. Electrocardiographic changes are relatively nonspecific (ST elevation, heart block, and low voltage QRS complexes). A definitive diagnosis can be made using a biopsy of the myocardium. According to the Dallas criteria, the diagnosis of myocarditis is based on the presence of mononuclear infiltration and myocyte damage. Yet the use of endomyocardial biopsy is limited by its reported lack of sensitivity

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and a risk of complications. In a common statement, the American Heart Association, the American College of Cardiology, and the European Society of Cardiology summarized clinical scenarios when endomyocardial biopsy should be performed, and emphasized the usefulness of biopsy in establishing the diagnosis of myocarditis in certain clinical settings (Cooper et al., 2007). The lack of sensitivity of the endomyocardial biopsy is most apparent in lymphocytic myocarditis; however, in giant cell myocarditis (GCM) or eosinophilic necrotizing myocarditis the sensitivity is as high as 80–85%. Patients of the latter types are also more likely to have a poor outcome, or require a heart transplant, and they are likely to benefit from immunosuppressive therapy. Detection of certain types of major histocompatibility complex (MHC) expression in a biopsy sample is highly supportive of a diagnosis of immune-mediated myocarditis (Cooper et al., 2007). Echocardiography, scintigraphy, and contrast-enhanced MRI are other useful tools to help diagnose myocarditis. Myocardial scintigraphy with antimyosin monoclonal antibodies has high sensitivity, but because it shows any myocardial necrosis, its specificity is lower (Bergler-Klein et al., 1993). Serum creatinine kinase and troponin T and I levels can help in assessing the extent of myocyte damage (Magnani and Dec, 2006).

1.2. Etiology of Myocarditis In most cases, the etiology of myocarditis and the consequent dilated cardiomyopathy (DCM) is unknown. Some 30–50% of DCM cases are estimated to be of genetic origin with examples of autosomally dominant but also autosomally recessive, X-linked, and mitochondrial inheritance. Mutations in over 20 genes have been reported as causes of DCM—lamin A/C, b-myosin heavy chain, dystrophin, desmin, and tafazzin are examples of some of them (Karkkainen and Peuhkurinen, 2007; Kamisago et al., 2000); however, in the case of other genes, the mutations were often found only in a single family or a small number of families (Karkkainen and Peuhkurinen, 2007). The list of bacterial, rickettsial, mycotic, protozoan, and viral agents capable of causing myocarditis is constantly growing (Magnani and Dec, 2006). Some of the more common viral agents are hepatitis C virus, influenza virus, herpes simplex virus, Epstein-Barr virus, parvovirus B19, and cytomegalovirus. Adenovirus and enterovirus genomes were frequently found in patients with myocarditis (Eckart et al., 2004; Pauschinger et al., 1999). Other common organisms that cause myocarditis are Corynebacterium diphtheriae or Trypanosoma cruzi. One of the more recently identified causes of myocarditis is vaccinia virus. It made headlines a few years ago, when 67 military personnel developed myocarditis within 15 days after smallpox vaccination (Eckart et al., 2004).

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1.3. Genetics of autoimmune myocarditis 1.3.1. MHC II association with myocarditis and DCM Most of the data on the relationship between The human leukocyte antigen system (HLA) and susceptibility to the inflammatory heart disease are obtained from patients with DCM. Several studies reported significant correlation of DCM with MHC class II antigens, mainly HLA-DR4 (Carlquist et al., 1991, 1994; Limas and Limas, 1989; Rodrı´guez-Pe´rez et al., 2007). In addition to HLA-DR4, other associations have been reported, but seem relevant only for certain populations and are probably affected by ethnic origin, sex, age, and geographic parameters (Liu et al., 2006; Lozano et al., 1997; Rodrı´guez-Pe´rez et al., 2007). The MHC II contributes to susceptibility to myocarditis in mice studies in both coxsackie-induced myocarditis and experimental autoimmune myocarditis (EAM). The mice strains on a A/J background (s, a, and f haplotypes) are able to develop severe myocarditis (A/J, H-2a; A.CA, H-2f; and A.SW, H-2s), while strains with a B10 background such as C57BL/10J, B10.A, B10.S, and B10.PL (H-2 b, a, s, u, respectively) are relatively resistant to myocarditis (Neu et al., 1987; Wolfgram et al., 1986).

1.3.2. Myocarditis and non-MHC gene association About 30% of DCM cases in humans, as discussed above, are due to mutations in various non-MHC genes, but the involvement of these mutations in susceptibility to myocarditis is unknown. A case report of a patient with recurrent myocarditis suggested a possible association between myocarditis susceptibility and an alternative splicing of CD45 gene (Tchilian et al., 2006). It is likely that myocarditis, similar to other autoimmune diseases, has a polygenic basis. Although the MHC haplotype is an important genetic factor for EAM susceptibility, the non-MHC genes are also prominent (Neu et al., 1987). We have discovered that two non-MHC loci on murine chromosomes 1 and 6, referred as Eam1 and Eam2, respectively, influence autoimmune myocarditis (Guler et al., 2005). These loci overlap with loci implicated in other autoimmune diseases, such as lupus and diabetes, suggesting that multiple autoimmune diseases might be controlled by similar genetic mechanisms (Li et al., 2008). Additionally, a Canadian group recently reported three loci on chromosome 1 and 4 that control susceptibility to CVB3-induced myocarditis (Aly et al., 2007).

1.4. Selected types of myocarditis 1.4.1. Lymphocytic myocarditis Based on the character of the inflammatory infiltrate in the heart, the most common form of myocarditis is lymphocytic myocarditis. Patients diagnosed with lymphocytic myocarditis are characterized by a sudden onset

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of left ventricular failure typically shortly after a respiratory tract infection, often with a biopsy identifying myocyte necrosis and lymphocytic infiltration. In Europe and North America, the most common viruses associated with myocarditis are the coxsackieviruses of group B (CVB3) and adenoviruses, which share the same cellular receptor (Baboonian and McKenna, 2003). Parvovirus B19 and human herpesvirus 6 have been also frequently reported (Ku¨hl et al., 2005). We can draw a parallel between lymphocitic myocarditis and acute viral myocarditis in CVB3-induced myocarditis in mice. Persistent viral genome can be found in about 70% of the DCM cases, suggesting that the remainder may be due to host response induced by a preceding viral infection (Ku¨hl et al., 2005). How the persistence of viral genomes contributes to the development of DCM is unclear; however, this result suggests that DCM is indeed preceded by subclinical viral myocarditis.

1.4.2. Giant cell myocarditis One of the most rapidly progressing forms of myocarditis is GCM, characterized by the presence of multinucleated giant cells in the heart infiltrate. The prognosis is serious. Immunosuppressive therapy is able to improve survival, but without transplantation only 11% of GCM patients survive 4 years, compared with 44% of patients with lymphocytic myocarditis surviving until this benchmark (Cooper et al., 2007). An autoimmune response is the probable cause of GCM.

1.4.3. Eosinophilic myocarditis The most severe form of eosinophilic myocarditis is necrotizing eosinophilic myocarditis (NEM), which is characterized by acute onset, rapid progression, predominantly diffuse eosinophilic infiltrate, and extensive necrosis. Intensive immunosuppressive treatment is indicated, but prognosis remains dire. The acute onset of NEM, even in previously healthy patients, its poor outcome, and the absence of extracardiac involvement contrast with eosinophilic myocarditis found in hypersensitivity syndrome. Hypersensitivity myocarditis could be accompanied by rash, fever, and peripheral eosinophilia and is often associated with hypersensitivity to a medication. Eosinophilic myocarditis can be also found as a part of hypereosinophilic syndrome, parasite infection, malignancy, and endocardial fibrosis (Cooper et al., 2007).

1.4.4. Dilated cardiomyopathy There is likely a casual relationship between myocarditis and some cases of subsequent DCM, because 9–16% of new onset DCM patients have evidence of prior myocarditis (Felker et al., 2000; Herskowitz et al., 1993). DCM is characterized by chronic left and right ventricular dilatation with normal or reduced left ventricular wall thickness and impaired

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contraction (Daubeney et al., 2006). Patients may present with asymptomatic cardiomegaly or severe congestive heart failure. DCM is the major cause of heart failure in individuals below the age of 40 and a major indication for cardiac transplantation. The 5-year survival rate of patients with DCM is less than 50% (Magnani and Dec, 2007). A large number of etiologic mechanisms have been implicated in DCM, including various infections, metabolic disorders, nutritional deficiency, neuromuscular diseases, toxins, and drugs. In a number of cases, a progression from myocarditis can be traced, and often there is a history suggesting a preceding viral infection.

2. THE EVIDENCE FOR AN AUTOIMMUNE PROCESS IN MYOCARDITIS It is difficult to prove that an autoimmune process causes myocarditis in humans, instead of autoimmunity developing after injury to the heart. There is no report of transient myocarditis in the fetus of a mother with myocarditis, which would clearly show that autoantibodies are involved directly in the pathogenesis of the disease in a way similar to Graves’ disease (Brown, 1996). The heterogeneity of myocarditis is another factor in the difficulty of establishing autoimmunity as one of the etiologic factors causing myocarditis. We have only indirect evidence to suggest that some types of myocarditis are caused by autoimmunity. One of them is the association of myocarditis with other autoimmune diseases, such as lupus or celiac disease. The incidence of myocarditis in lupus is reported to be 3–15%; additionally, patients with lupus often have some kind of pericardial involvement, ranging from asymptomatic pericardial effusion to acute pericarditis (Apte et al., 2008; Tincani et al., 2006). The prognosis of myocarditis associated with lupus is relatively good if immunosuppressive treatment is started in time (Tincani et al., 2006).

2.1. Autoantibodies Immunofluorescence tests using normal human heart tissue as substrate revealed that 59% of patients with myocarditis had cardiac-specific antibodies (Neumann et al., 1990). Konstadoulakis et al. (1993) and Caforio et al. (1992) showed that antibodies to cardiac myosin were present in 66% and 86%, respectively, of patients with DCM. However, many patients with other forms of heart disease also have antibodies to myosin. A further step toward characterizing the antibodies has been taken by distinguishing the subclass of antibodies to myosin in DCM patients. The antimyosin-specific antibodies typical of DCM are mainly in the IgG3 subclass (Warraich et al., 1999). Lauer et al. (2000) found that clinical

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symptoms and left ventricular ejection fraction improved significantly more in myosin antibody-negative group compared with antibodypositive myocarditis patients. In another study, Warraich et al. (2000) found that patients with cardiac myosin antibodies rejected transplanted hearts earlier then antibody-negative patients. It is still unclear, however, whether antibodies to myosin are directly involved in cardiac dysfunction, although immunoabsorption with an anti-IgG column showed clinical benefit in patients with DCM (Felix et al., 2001). In addition to antimyosin antibodies, about 50% DCM patients have antibodies to cardiac b1-adrenoreceptor (Limas et al., 1990). Rats immunized against the second extracellular b1 receptor loop developed severe left ventricular dilatation and dysfunction, suggesting that b1-adrenoreceptor antibodies are likely to play a causal role in DCM. Moreover, serum from these animals can transfer the disease to healthy rats (Jahns et al., 2004). In EAM model myocarditis, a transfer by antimyosin-specific sera was observed only in DBA/2 mice but not in BALB/c or A/J mice (Kuan et al., 1999; Cihakova, unpublished observations). The susceptibility of the DBA/2 strain to antibody-induced myocarditis is due to the extracellular expression of myosin in their heart, supporting the notion that antimyosin antibodies are not able to induce disease in the undamaged heart (Kuan et al., 1999). However, when the injury has already occurred, the antimyosin antibodies can contribute to the damage of cardiac myocytes. Passive transfer of antimyosin antibodies after myocarditis is induced with cardiac myosin led to a greater disease severity compared to mice treated with isotype control (Cihakova, unpublished observation).

2.2. Immunosuppressive therapy The benefits of immunosuppressive therapy constitute additional indirect evidence of the autoimmune origin of some types of myocarditis. Some patients with GCM, eosinophilic myocarditis, granulomatous myocarditis, and lymphocytic myocarditis associated with another autoimmune disease seem to benefit from immunosuppression, which is consistent with the hypothesis of autoimmune origin of these types of myocarditis (Cooper et al., 1997, Ku¨hl et al., 2005). However, placebo-controlled trials did not show increased survival in myocarditis patients treated with immunosuppressive agents (Mason et al., 1995, Parrillo et al., 1989). To fully explore the benefits of immunosuppressive therapy for myocarditis, it may be necessary to distinguish viral and autoimmune myocarditis. Frustaci et al. (2003) found that myocarditis patients who did not respond to therapy with prednisone and azathioprine had viral genome in their hearts, in contrast to patients who benefited from the treatment and usually had heart-reactive autoantibodies. However, the study was not randomized and lacked control groups. It therefore still remains an

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unresolved question whether the patients whose myocarditis or DCM is most likely caused by autoimmune process should be treated differently from patients with myocarditis and DCM of other etiologies.

3. MOUSE MODELS OF MYOCARDITIS 3.1. CVB3-induced myocarditis The most persuasive evidence that some forms of myocarditis are driven by autoimmune processes comes from animal models. There are many animal models of myocarditis. Currently, there are two models of CVB3induced myocarditis. The first one induces acute viral myocarditis with a significant damage to cardiomyocytes, and sudden death of majority of animals within a week of infection (Fuse et al., 2005; Huber et al., 1998). The second is based on using the heart-passaged CVB3 virus (Fairweather and Rose, 2007). In this model, a limited number of all strains of mice appear to develop an acute viral myocarditis following an infection with a cardiotropic strain of CVB3. The peak of inflammation of this early viral myocarditis is 9 days after the infection; the mice usually do not die from the viral myocarditis in this model. The heart infiltration is focal with no necrosis or fibrosis, and consists mostly of macrophages, neutrophils, CD4þ, and CD8þ T cells, some B cells, NK cells, eosinophils, and mast cells (Fairweather et al., 2005b). Most strains of mice recover completely, and have no evidence of the previous inflammatory process within 3 weeks after the infection. However, susceptible strains such as BALB/c, A/J, and SJL/J mice progress to a chronic myocarditis with generalized mononuclear infiltration accompanied by the production of antibodies to cardiac myosin (Afanasyeva and Rose, 2004; Rose et al., 1986). The strains that are susceptible to the second phase of CVB3-induced myocarditis do not all share the same H-2 background. The resistance to chronic myocarditis in B10 or C57BL/6 mice can be overcome by a treatment with LPS, IL-1b, or TNF-a (Fairweather et al., 2005a; Lane et al., 1993).

3.2. Experimental autoimmune myocarditis Immunization with cardiac myosin or with a myocarditogenic peptide derived from the a-cardiac myosin heavy chain emulsified in complete Freund’s adjuvant (CFA) induces a similar monocytic myocarditis in mice strains that are susceptible to the late phase of viral myocarditis, but not in the resistant strains (Neu et al., 1987; Donermeyer et al., 1995; Pummerer et al., 1996). Both late phase CVB3-induced myocarditis and myosininduced EAM have similar characteristics; the disease is accompanied

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Fibrosis pericarditis heart failure

Antibodies Infiltration Induction: activation 0

7

Progression: autoimmunity 14

21

Late: Cardiomyopathy 28

35

42

Time (days postimmunization)

FIGURE 4.1 Phases of experimental autoimmune myocarditis in mice. Mice are immunized with myosin in complete Freund’s adjuvant on days 0 and 7. Around day 21, there is a peak in the infiltration in the heart (solid line) and in antimyosin antibodies levels in the sera of immunized mice (dotted line). After day 21, heart infiltration declines and is replaced with fibrosis. After day 35, signs of dilated cardiomyopathy and heart failure could be detected by echocardiography.

by the production of cardiac myosin specific autoantibodies as well as cardiomyosin-specific T cells. We believe that the reason for the similarities in EAM and late phase of CVB3-induced myocarditis is that the viral infection acts as an adjuvant (Fairweather et al., 2005a,b). To produce EAM, CFA is injected with the antigen (myosin or myocarditogenic peptide) twice in 8 days. During the induction phase of EAM, in the first 10 days after the first immunization, there is no inflammation in the heart, and autoantibodies against cardiac myosin cannot be detected in serum (Cihakova et al., 2004). During the progression phase of EAM, between days 10 to day 21 postimmunization, inflammation increases in the heart, as do cardiac myosin-reactive antibodies in sera. During the late phase of EAM, following day 21 postimmunization, inflammation gradually declines and is replaced by fibrosis (Fairweather et al., 2001) (Fig. 4.1). The time course of CD45þ hematopoietic cells infiltrating the hearts of BALB/c mice with EAM correlates well with the known time course of EAM as determined by classical histopathologic methods (Afanasyeva et al., 2004). Since myocarditis involves myocyte injury, cardiac troponin I (cTnI) levels are elevated as they are in myocarditis patients (Smith et al., 1997). In recent research, a new myocarditis model, induced by immunization with troponin I in CFA, emerged. A/J mice immunized with murine cTnI developed severe myocarditis, with cardiomegaly, fibrosis, reduced fractional shortening, and increased mortality (Go¨ser et al., 2006). Several mice models of spontaneous myocarditis were also published. For example HLA-DQ8 transgenic IAb knockout (KO) NOD mice develop

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spontaneous myocarditis as well as NOD mice carrying the human DQ8 molecule (Hayward et al., 2006; Taneja et al., 2007).

4. ROLE OF PROINFLAMMATORY CYTOKINES IN MYOCARDITIS TNF-a is able to drive the pathogenesis in many rheumatoid diseases, and anti-TNF-a drugs become the success story in treatment of many human autoimmune diseases (Alonso-Ruiz et al., 2008). Interestingly anti-TNF-a drugs also seem to reduce the risk of cardiac disease in rheumatoid arthritis patients (Avouac and Allanore, 2008). Blocking IL-1b or TNF-a ameliorates myocarditis in the mouse model if the blocking is done during the onset of the disease (Fairweather et al., 2004). Additionally, the severity of CVB3-induced myocarditis as well as myosin-induced myocarditis correlates with the levels of IL-1b and IL-18 in the heart (Cihakova et al., 2008; Fairweather et al., 2003).

5. ROLE OF T HELPER CELLS IN MYOCARDITIS 5.1. Role of Th1 cells in myocarditis Until recently, organ-specific autoimmune diseases have been thought to be driven through the Th1 pathway (Charlton and Lafferty, 1995; Ichikawa et al., 2000). However, recent studies show a duality in the role of Th1 cytokines in the pathogenesis of autoimmune disease. The studies show that IFN-g deficiency in KO mice or depletion of IFN-g with antibodies lead to remarkably severe myocarditis with pericarditis and severe fibrosis that lead to DCM and heart failure (Afanasyeva et al., 2001, 2005; Eriksson et al., 2001). Similarly, evidence for a disease-limiting function of IFN-g has been also observed in models of experimental autoimmune encephalomyelitis (EAE), thyroiditis, uveitis, and arthritis (Barin et al., 2003; Chu et al., 2000; Jones et al., 1997; Manoury-Schwartz et al., 1997; Willenborg et al., 1996). In these model disease systems, IFN-g-deficiency by targeted KO mutation or by blockade is associated with disease exacerbation, and IFN-g administration by transgenic overexpression or administration of recombinant cytokine is associated with disease amelioration. However, both IL-12Rb1-deficient mice and STAT4-deficient mice were resistant to myocarditis induction, implying that IL-12 is required for myocarditis development while IFN-g protects against disease (Afanasyeva et al., 2001; Eriksson et al., 2001). The receptor IL-12Rb1 is shared by IL-12 and IL-23; therefore, the data from IL-12Rb1-deficient mice required further clarification. IL-12p35 KO

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mice, which lack only IL-12, developed EAM while IL-12p40 KO mice, which lack both IL-12 and IL-23, are resistant to myocarditis induction. These data suggest that IL-23, rather than IL-12, mediates the autoimmune pathology in diseases such as EAE, experimental arthritis, and EAM (Cua et al., 2003; Murphy et al., 2003, Sonderegger et al., 2006).

5.2. Growing evidence of divergent functions of IL-13 and IL-4 In contrast to a mild disease in IL-4 KO BALB/c mice, IL-13 KO BALB/c mice develop severe CVB3-induced autoimmune myocarditis as well as myocarditogenic peptide-induced EAM (Cihakova et al., 2008). The myocarditis in IL-13 KO mice is so severe that the animals develop marked DCM with impaired cardiac function indicative of heart failure. Interestingly, both arms of the adaptive immune response are upregulated during myocarditis in the absence of IL-13. Production of all subclasses of antimyosin antibodies is increased and T cells are more activated and more abundant during EAM in IL-13 KO mice; additionally, CD4þCD25þFoxp3þregulatory T cell numbers are decreased in the spleens of IL-13 KO mice. However, neither T nor B cells express the IL13Ra1 receptor; that is, they are not directly responsive to IL-13. Therefore, cells of innate immune response are probably responsible for the increased myocarditis in the absence of IL-13. Significant changes in the innate immune cell population of IL-13 KO mice were found in the macrophage subtypes in the heart infiltrate (see below) (Cihakova et al., 2008).

5.3. Is myocarditis a Th1- or Th17-driven disease? As was discussed above, IL-23 rather than IL-12 is essential for EAM development. IL-23 promotes survival of a new Th subset (called Th17) that produces proinflammatory cytokines IL-17 (Park et al., 2005), and drives pathology of several autoimmune diseases, including EAE, collagen-induced arthritis, and T cell-mediated colitis (Aggarwal et al., 2003; Chen et al., 2006; Langrish et al., 2005; Yen et al., 2006). Studies using a VLP-based vaccination approach showed that neutralization of IL-17 decreased EAM severity (e.g., Sonderegger et al., 2006). We have recently demonstrated that blocking IL-17 with monoclonal antibodies significantly decreased EAM, again confirming that the IL-17 is pathogenic during EAM (Cihakova et al., unpublished data). These experiments suggest that Th17 is also a driving factor of disease pathogenesis in EAM. However, in severe myocarditis in IL-13 KO mice, the IL-17 levels were significantly reduced in their hearts in the peak of EAM compared to BALB/c controls (Cihakova et al., 2008). The disease in the absence of IL-13 is probably driven through the Th1 pathway, since IFN-g was

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Wild type

IL-17 KO

FIGURE 4.2 Genetic absence of IL-17A in IL-17 knockout mice does not protect mice from development of experimental autoimmune myocarditis (EAM). (Left) Heart on day 21 of EAM in WT mouse. (Right) heart on day 21 of EAM in IL-17 knockout mouse. (See Plate 5 in Color Plate Section.)

increased in hearts of IL-13 KO mice. The increase of IFN-g is also responsible for higher levels of ‘‘classically activated’’ macrophages (cMj), which further intensify the disease severity by producing proinflammatory cytokines (Cihakova et al., 2008). Therefore, our data suggest that Th1 cells can induce myocarditis with severity similar to Th17 cells. Recently, we have obtained additional evidence supporting our view that myocarditis can be driven by either Th17 or Th1 pathway. IL-17A KO mice are still able to develop myocarditis that is only slightly reduced in severity compared to WT mice (Baldeviano, Cihakova, and Rose, unpublished data) (Fig. 4.2). Some of the IL-17A KO mice have infiltration in over 30% of the heart indicating that, even without IL-17A, the disease can progress to a severe stage. This finding shows that IL-17A is not necessary for autoimmune disease development. Similar results have been published in an experimental autoimmune uveitis (EAU) model. Luger et al. (2008) showed that EAU can develop in the absence of IL-17 in IL-17A KO mice with a severity similar to WT controls. Therefore, the lack of IL-23 appears to protect mice from myocarditis better than the absence of IL-17. Mice transgenic for IL-23 have elevated levels of IL-1b and TNF-a; both cytokines are essential for myocarditis development as discussed above

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FIGURE 4.3 Experimental autoimmune myocarditis (EAM) is cytokine regulated (schematic). The schematic shows a healthy murine heart (top) and a severe myocarditis on day 21 of EAM (bottom). Th17 as well as Th1 cells can drive the pathogenesis of EAM. IL-13 is protective in myocarditis partially due to the IL-13 induction of alternatively activated macrophages. (See Plate 6 in Color Plate Section.)

(Wiekowski et al., 2001). Additionally, mice deficient in receptor IL-12Rb1 (which is common for both IL-12 and IL-23) had reduced levels of IL-1b that correlated with reduced myocarditis (Fairweather et al., 2003). It is possible that IL-23 promotes inflammatory responses by stimulating production of other proinflammatory cytokines. Thus, IL-23 might be the preferred target for therapeutic intervention in human autoimmune diseases rather than IL-17A. There is also a great need to reexamine role of IFN-g in autoimmune diseases. Our findings from the IFN-g KO mice suggested that IFN-g can be a protective cytokine; however, the protective effect takes place mainly by downregulating IL-17. In certain circumstances, as discussed above, Th1 cells are able to induce a disease comparable in severity to Th17-driven myocarditis (Fig. 4.3).

6. THE DIVERGENT ROLE OF MACROPHAGES IN MYOCARDITIS Macrophages constitute a population that is very diverse in terms of function and phenotype. Traditionally, Th1-derived IFN-g was thought to be a prime activator of T cell-driven macrophage responses, upregulating MHC Class II, iNOS, IL-12, the B7 co-stimulators, and ICAM1 (Adams and Hamilton, 1984). In contrast to IFN-g-elicted cMj, Th2 cytokines IL-4 and IL-13 induce the expression of arginase-1, YM1 (Chi3l3), type A

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scavenger receptor (CD204), and the macrophage mannose receptor (CD206). These ‘‘alternatively activated’’ macrophages (aMj) contrast with the IFN-g-elicted cMj not only in phenotype but also in function. Indeed, IL-13 deficiency leads to decreased numbers of alternatively activated CD206þ and CD204þ macrophages, and increased numbers of cMj. The increased number of cMj in the hearts of IL-13 KO mice with EAM is associated with increased caspase-1 activation. Caspase-1 is an enzyme necessary for the production of an active form of IL-1b and IL-18, which both are also greatly increased in the hearts of IL-13 KO mice (Cihakova et al., 2008). The disease-limiting role of CD11bþ monocytes in EAM was also shown by Valaperti et al. (2008). Similarly, in SIV-infected rhesus monkeys (a model of human HIV-induced myocarditis), Yearley et al. (2007) found that CD163 positive non-cMj are protective.

7. CONCLUSIONS/DIRECTIONS FOR FUTURE RESEARCH As mentioned above, we have assembled evidence that EAM is driven in part by the Th17 pathway in the mouse; however, in the absence of IL-17A an equally severe disease can be driven through the Th1 pathway. It is not yet known whether IL-17 plays a similarly dominant role in the pathogenesis of human myocarditis. Given a lack of immunomodulatory treatments for myocarditis, blocking of the IL-23/IL-17 pathway could be very attractive option. Thus, in myocarditis different pathways can result in comparable disease (Cihakova et al., 2008). Further studies examining the pathogenesis of IL-23/IL-17 in both animal models and humans are needed. Carefully distinguishing the presence or absence of replicating virus in the heart from the presence of an autoimmune response is necessary. We presume that blocking the IL-17/IL-23 pathway might benefit only myocarditis of autoimmune origin. We also need to revisit the role of the Th1 pathway and the role of IFN-g in myocarditis. There is currently no agreement about the role of Th1 cells, since it was shown that Th1 cell lines can induce EAU comparable to the Th17-induced disease (Cox et al., 2008); however, in models of autoimmune colitis and EAE, Th1-induced diseases were milder to Th17-induced diseases (Elson et al., 2007; Langrish et al., 2005). Another new direction in myocarditis research is emphasis on the innate immune response. Monocytes/macrophages are the most abundant cell type in the heart during myocarditis, both in mice and in humans. It is often viewed as a homogenous population with either pathogenic or disease-regulating roles; however, it is possible that the macrophage populations in heart infiltrates are functionally, phenotypically, and kinetically highly heterogenous. Understanding the different functions of the various populations and their interactions with pathogenic

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Th17 cells may reveal previously unappreciated therapeutic targets for new intervention strategies.

ACKNOWLEDGMENTS Authors are pleased to acknowledge funding from NIH grants 5R01HL077611 and 5R01HL067290 from the National Heart, Lung, and Blood Institute, and the Myocarditis Foundation. Jobert Barin and G Christian Baldevianno, graduate students in our laboratory, are currently working on the role of macrophages in EAM and the role of IL-17 in EAM, respectively.

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Rose, R., and Baughman, K. L. (1998). Immune-mediated cardiovascular disease. In ‘‘The Autoimmune Diseases’’ 3rd ednpp. 623–636. Rose, N. R., Wolfgram, L. J., Herskowitz, A., and Beisel, K. W. (1986). Postinfectious autoimmunity: Two distinct phases of coxsackievirus B3-induced myocarditis. Ann. N. Y. Acad. Sci. 475, 146–156. Smith, S. C., Ladenson, J. H., Mason, J. W., and Jaffe, A. S. (1997). Elevations of cardiac troponin I associated with myocarditis. Experimental and clinical correlates. Circulation 95, 163–168. Sonderegger, I., Ro¨hn, T. A., Kurrer, M. O., Iezzi, G., Zou, Y., Kastelein, R. A., Bachmann, M. F., and Kopf, M. (2006). Neutralization of IL-17 by active vaccination inhibits IL-23-dependent autoimmune myocarditis. Eur. J. Immunol. 36, 2849–2856. Taneja, V., Behrens, M., Cooper, L. T., Yamada, S., Kita, H., Redfield, M. M., Terzic, A., and David, C. (2007). Spontaneous myocarditis mimicking human disease occurs in the presence of an appropriate MHC and non-MHC background in transgenic mice. J. Mol. Cell. Cardiol. 42, 1054–1064. Tchilian, E. Z., Gil, J., Navarro, M. L., Fernandez-Cruz, E., Chapel, H., Misbah, S., Ferry, B., Renz, H., Schwinzer, R., and Beverley, P. C. (2006). Unusual case presentations associated with the CD45 C77G polymorphism. Clin. Exp. Immunol. 146, 448–454. Tincani, A., Rebaioli, C. B., Taglietti, M., and Shoenfeld, Y. (2006). Heart involvement in systemic lupus erythematosus, anti-phospholipid syndrome and neonatal lupus. Rheumatology (Oxford) 45(Suppl. 4), iv8–iv13. Yearley, J. H., Pearson, C., Shannon, R. P., and Mansfield, K. G. (2007). Phenotypic variation in myocardial macrophage populations suggests a role for macrophage activation in SIVassociated cardiac disease. AIDS Res. Hum. Retroviruses 23, 515–524. Yen, D., Cheung, J., Scheerens, H., Poulet, F., McClanahan, T., McKenzie, B., Kleinschek, M. A., Owyang, A., Mattson, J., Blumenschein, W., Murphy, E., Sathe, M., et al. (2006). IL-23 is essential for T cell-mediated colitis and promotes inflammation via IL-17 and IL-6. J. Clin. Invest. 116, 1310–1316. Valaperti, A., Marty, R. R., Kania, G., Germano, D., Mauermann, N., Dirnhofer, S., Leimenstoll, B., Blyszczuk, P., Dong, C., Mueller, C., Hunziker, L., and Eriksson, U. (2008). CD11bþ monocytes abrogate Th17 CD4þ T cell-mediated experimental autoimmune myocarditis. J. Immunol. 180, 2686–2695. Warraich, R. S., Dunn, M. J., and Yacoub, M. H. (1999). Subclass specificity of autoantibodies against myosin in patients with idiopathic dilated cardiomyopathy: Pro-inflammatory antibodies in DCM patients. Biochem. Biophys. Res. Comm. 259, 255–261. Warraich, R. S., Pomerance, A., Stanley, A., Banner, N. R., Dunn, M. J., and Yacoub, M. H. (2000). Cardiac myosin autoantibodies and acute rejection after heart transplantation in patients with dilated cardiomyopathy. Transplantation 69, 1609–1617. Wiekowski, M. T., Leach, M. W., Evans, E. W., Sullivan, L., Chen, S. C., Vassileva, G., Bazan, J. F., Gorman, D. M., Kastelein, R. A., Narula, S., and Lira, S. A. (2001). Ubiquitous transgenic expression of the IL-23 subunit p19 induces multiorgan inflammation, runting, infertility, and premature death. J. Immunol. 166, 7563–7570. Willenborg, D. O., Fordham, S., Bernard, C. C., Cowden, W. B., and Ramshaw, I. A. (1996). IFN-gamma plays a critical down-regulatory role in the induction and effector phase of myelin oligodendrocyte glycoprotein-induced autoimmune encephalomyelitis. J. Immunol. 157, 3223–3227. Wolfgram, L. J., Beisel, K. W., Herskowitz, A., and Rose, N. R. (1986). Variations in the susceptibility to coxsackievirus B3-induced myocarditis among different strains of mice. J. Immunol. 136, 1846–1852.

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5 Emergence of the Th17 Pathway and Its Role in Host Defense Darrell B. O’Quinn,1 Matthew T. Palmer,1 Yun Kyung Lee, and Casey T. Weaver

Contents

Abstract

1

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1. Introduction 2. Emergence of the Th17 Pathway 3. Functions of the Th17-Derived Cytokines 3.1. IL-17 3.2. IL-22 3.3. IL-26 4. IL-23/IL-17-Mediated Innate Immunity 5. Th17 Cells and Innate Immunity 6. Th17 Cells and Acquired Immunity 7. IL-23/IL-17-Mediated Responses in Specific Microbial Infections 7.1. Bacteria 7.2. Fungi 7.3. Protozoa 7.4. Viruses 7.5. Other 8. Closing Remarks References

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Recently, a paradigm shift has emerged in T-cell-mediated adaptive immunity. On the heels of the discovery of T cells with immunosuppressive function, so-called regulatory T cells (Tregs), the diversity of effector cells has expanded to include a third helper T cell,

Department of Pathology, University of Alabama at Birmingham, Birmingham, Alabama, USA These authors contributed equally to this work

Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00605-6

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termed Th17. The appreciation that Th17 cells are products of a distinct effector pathway depended critically on observations made during investigations of mouse models of autoimmunity, advanced by discovery of the cytokines IL-17 and IL-23. These studies understandably led investigators to highlight the role played by Th17 cells in autoimmunity. Yet while the dysfunctional behavior of this phenotype as a contributor to inflammatory disease remains a central issue, this pathway evolved to meet a need for host protection against potential pathogens. It has become apparent that the Th17 pathway promotes host defense against certain extracellular bacteria and fungi, but more recent studies also implicate a role in protection against some protozoa and viruses. Here we review the experimental history that ultimately uncovered the existence and nature of Th17 cells, and then turn the reader’s attention to what is currently known about Th17 cells as a bulwark against pathogens.

1. INTRODUCTION The host’s response to different categories of infectious challenge is coordinated in large part by specialized subsets of CD4 T cells, each displaying a relatively unique skill set. This division of labor was initially suggested by the ability of T cells to mediate two types of effects, delayed-type hypersensitivity (DTH) and B cell help, that could be uncoupled (Battisto and Miller, 1962; Janicki and Aron, 1968; Liew and Parish, 1974; Parish, 1971). In polyclonal populations, it was also observed that two types of Th cells could be discriminated based on their respective capacities to provide B cell help (Imperiale et al., 1982; Janeway, 1975; Swierkosz et al., 1979; Tada et al., 1978; Waldmann, 1977). However, formal demonstration of these two phenotypes, termed ‘‘Th1’’ and ‘‘Th2’’ (Tada et al., 1978), could not be achieved using uncloned cells. Mosmann, Coffman, and their coworkers subsequently created a large panel of cloned CD4 T cells and demonstrated that these cells could be grouped according to two sets of shared properties. Th1 cells were defined by their production of interleukin (IL)-2, gamma-interferon (IFNg), and granulocyte-macrophage colony-stimulating factor (GMCSF), whereas Th2 cells were defined by their production of IL-4 (Mosmann et al., 1986b). IL-4 (originally known as B-cell stimulatory factor-1) was found to promote the growth of mast cells and B cells, and strongly enhanced the production of IgE antibody by B cells, whereas IFNg blocked this latter effect (Coffman and Carty, 1986; Coffman et al., 1986; Mosmann et al., 1986a). Th1 cells were subsequently shown to be agents of DTH (Cher and Mosmann, 1987), at least as defined by foot pad swelling. Yokota et al. (1987) discovered that Th2 cells also produce IL-5,

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a cytokine that induces B cells to produce IgA and promotes the formation of eosinophil colonies. Th1 and Th2 cells (Stevens et al., 1988), and their signature cytokines (Snapper and Paul, 1987), were also shown to induce class switching to IgG2a and IgG1, respectively. Collectively, this established the Th1/Th2 hypothesis (Mosmann and Coffman, 1989), a model that would not be fundamentally altered for nearly two decades (for more extensive reviews, see Coffman, 2006; Liew, 2002). But the two helper T-cell phenotypes are not merely distinct, they are polarized. They arise from a common pool of uncommitted naı¨ve T-cell precursors (Hsieh et al., 1992; Le Gros et al., 1990; Seder et al., 1993; Swain et al., 1990) and achieve their distinct maturation states through mechanisms of self-promotion and reciprocal antagonism. Th1 cells produce IFNg, which promotes Th1 differentiation itself and inhibits the development of Th2 cells (Gajewski and Fitch, 1988). Likewise, Th2 cells produce IL-4 which functions, in part, as an autocrine/paracrine cytokine to enforce the development of Th2 cells (Hsieh et al., 1992; Kopf et al., 1993; Le Gros et al., 1990; Seder et al., 1992; Swain et al., 1990) while compromising the development of Th1 cells (Seder et al., 1992). Th2 cells produce IL-10, which blocks the ability of Th1 cells, but not Th2 cells, to be activated by macrophages (Fiorentino et al., 1989, 1991). IL-12 promotes (Hsieh et al., 1993; Manetti et al., 1993; Seder et al., 1993) and inhibits (Manetti et al., 1993) the development of Th1 and Th2 cells, respectively. In vivo, IL-12-deficient mice are compromised in Th1 cytokine responses (Magram et al., 1996) and default to a Th2 response (Mattner et al., 1996). During development, Th1 and Th2 cells become resistant to the influence of IL-4 (Huang and Paul, 1998) and IL-12 (Szabo et al., 1995), respectively. Not surprisingly, then, these phenotypes become increasingly stabilized over time (Murphy et al., 1996; Sornasse et al., 1996). The intracellular signaling pathways that mediate these selfreinforcing and counter-regulatory properties of the two lineages have since been investigated, and are the subject of other reviews (Murphy and Reiner, 2002; Szabo et al., 2003). With regards to Th1 cells, IFNg activates STAT1 (Afkarian et al., 2002) which induces T-bet (Lighvani et al., 2001; Afkarian et al., 2002) which in turn helps drive Th1 development (Szabo et al., 2000) by upregulating the expression of IL-12Rb2 (Afkarian et al., 2002; Mullen et al., 2001). IL-12 activation of STAT4 prolongs Th1 survival and IFN-g expression (Mullen et al., 2001). With regard to Th2 cells, IL-4 activates STAT6 (Kaplan et al., 1996; Shimoda et al., 1996; Takeda et al., 1996) which induces GATA-3 (Ouyang et al., 2000). GATA-3, in turn, upregulates the expression of IL-4 and promotes Th2 development (Zheng and Flavell, 1997). Thus, in both cases, the signature cytokines of Th1 and Th2 cells provide a positive feedback loop to reinforce commitment to their own lineages.

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2. EMERGENCE OF THE Th17 PATHWAY From its inception, the Th1/Th2 hypothesis indicated that Th1 cells might be instigating much of the cell-mediated damage seen in animal models of autoimmunity, and evidence in favor of this view steadily accrued. In experimental autoimmune encephalomyelitis [EAE; formerly known as experimental allergic encephalomyelitis (Steinman, 2007)], depletion of CD4 cells prevents or ameliorates disease (Brostoff and Mason, 1984; Waldor et al., 1985). Mononuclear cells taken from the central nervous system (CNS) and lymphoid organs following induction of EAE secrete IFNg (Mustafa et al., 1991; Olsson, 1992), and T-cell clones that are encephalitogenic exhibit a Th1 cytokine expression profile (Ando et al., 1989; Baron et al., 1993). For its part, IL-12 was reported to participate in Th1-mediated autoimmune diseases (Adorini, 1999). For example, blockade of IL-12 signaling through the use of neutralizing antibodies (Constantinescu et al., 1998; Leonard et al., 1995; Segal et al., 1998) or gene disruption (Segal et al., 1998) protected against EAE. Other experimental forms of autoimmunity were likewise attributed largely to Th1 activity (Brand et al., 2003; Haskins and Wegmann, 1996; Wirtz and Neurath, 2000). Despite the evidence for involvement of Th1 cells in autoimmunity, the role played by its effector cytokines, especially IFNg, was often found to be inconsistent with this association (Rosloniec et al., 2002). The number of published reports on the influence of Th1-associated cytokines [i.e., IFNg, tumor necrosis factor (TNF), and lymphotoxin (LT)] in a diverse set of mouse models is quite large and few of them will be cited here—some studies support a strong role for Th1 cells, many don’t. We focus, instead, on data pertaining to the two models that would later prove decisive in establishing IL-17-producing Th cells as candidate protagonists in at least some forms of autoimmunity. With regard to EAE, LT promotes disease activity (Powell et al., 1990), whereas the literature on TNF is contradictory (Liu et al., 1998; Powell et al., 1990). And IFNg was shown either to have negligible effects, or to be protective (Billiau et al., 1988; Chu et al., 2000; Ferber et al., 1996; Krakowski and Owens, 1996; Powell et al., 1990; Tran et al., 2000; Voorthuis et al., 1990; Willenborg et al., 1996, 1999). With respect to collagen-induced arthritis (CIA), two early reports (Cooper et al., 1988; Mauritz et al., 1988) indicated that IFNg exacerbates the disease, whereas a third suggested a biphasic effect (Boissier et al., 1995). By contrast, a larger number of studies reported no effect, or even a protective effect, of IFNg (Guedez et al., 2001; Kageyama et al., 1998; Manoury-Schwartz et al., 1997; Matthys et al., 1998, 1999; Nakajima et al., 1990; Ortmann and Shevach, 2001; Vermeire et al., 1997; Williams et al., 1993). The respective roles of IL-12 and IFNg in the progression of disease activity in EAE and CIA were, then, seemingly at odds with each other, and the dissonance that resulted would not be relieved until IL-23 was discovered (Oppmann et al., 2000). The studies purporting to show the

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dependence of EAE and CIA activity on IL-12 (a heterodimer designated p70 to distinguish it from its subunits, p40 and p35) had used mice deficient in the IL-12p40 subunit (Segal et al., 1998) and neutralizing antibody raised against IL-12p70 (Constantinescu et al., 1998; Leonard et al., 1995; Matthys et al., 1998; Segal et al., 1998). The antibody was either polyclonal (Leonard et al., 1995; Segal et al., 1998) or monoclonal (i.e., c17.8) (Constantinescu et al., 1998; Matthys et al., 1998). Importantly, the humoral response to IL-12p70 appears to preferentially target the IL-12p40 subunit (Chizzonite et al., 1991) and c17.8 specifically has been shown to do so (Oppmann et al., 2000). This selective targeting of IL-12p40 was unintentional, and ostensibly inconsequential. Nonetheless, there was some cause for concern: mice that are deficient in IL-12p40 are more susceptible to Cryptococcus neoformans (Decken et al., 1998) and Listeria monocytogenes (Brombacher et al., 1999) than are IL-12p35-deficient mice. While this suggested a host protective capacity of IL-12p40 beyond its role in forming IL-12, it would take a computational screen to discover IL-23p19, a protein that heterodimerizes with IL-12p40 to form a cytokine that was termed IL-23 (Oppmann et al., 2000). The gene knockout or neutralization of IL-12p40 therefore compromises signaling not only through IL-12 but through IL-23 as well. This raised the possibility that disruption of IL-12p40 protected against EAE and CIA not by the resulting disruption of IL-12 signaling, and by association Th1 activity, but by undermining some other activity that is mediated by IL-23. Two groups subsequently showed that while IL-12p40 deficiency does protect against EAE, IL-12p35 deficiency is not protective (Gran et al., 2002) and may even exacerbate the disease (Becher et al., 2002). Rostami’s group also showed that deficiency in the IL12-specific subunit of IL-12R (i.e., IL-12Rb2) aggravated EAE (Zhang et al., 2003). These results demonstrated that IL-12 signaling is not the basis for the protective effects of IL-12p40 deficiency in the EAE model. To directly test the role played by IL-23 signaling, Cua and colleagues examined mice deficient in either IL-12p35 or IL-23p19 and showed that IL-23p19 deficiency spared mice from EAE (Cua et al., 2003) and CIA (Murphy et al., 2003), whereas IL-12p35 deficiency was either not protective or even exacerbated disease. This was consistent with earlier work showing that the transgenic overexpression of IL-23p19 leads to widespread inflammation (Wiekowski et al., 2001). These results revealed that the previously inferred relationship linking IL-12 signaling to autoimmunity reflected an incomplete understanding of the function of IL-12p40, and established the premise that IL-23, not IL-12, promotes EAE and CIA. In the absence of supporting roles for IL-12 and IFNg, immunologists were now prepared to embrace a surrogate for Th1 cells, and another player was already waiting in the wings. IL-17, originally called CTLA-8 and now more specifically referred to as IL-17A, is a member of a cytokine family that includes five other members (i.e., IL-17B-F). Prior to the work of two groups in 2003 (Aggarwal et al., 2003; Murphy et al., 2003), it was

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known to be expressed in T cells (Kennedy et al., 1996; Rouvier et al., 1993), including CD4 cells (Aarvak et al., 1999; Fossiez et al., 1996; Infante-Duarte et al., 2000; Yao et al., 1995b), to induce expression of the proinflammatory cytokine IL-6 in other cells (Fossiez et al., 1996; Kennedy et al., 1996; Yao et al., 1995b), and to be relatively abundant in samples taken from patients with rheumatoid arthritis (RA; which is modeled by CIA) (Chabaud et al., 1999; Kotake et al., 1999) and multiple sclerosis (MS; which is modeled by EAE) (Matusevicius et al., 1999). A causal role for IL-171 in CIA was formally established by 2001 (Lubberts et al., 2001) and later confirmed (Lubberts et al., 2004; Nakae et al., 2003). CD4þIL-17þ cells are phenotypically distinct from Th1 and Th2 cells (Aarvak et al., 1999; Infante-Duarte et al., 2000) and co-express GM-CSF and TNF (Infante-Duarte et al., 2000), the latter being a proinflammatory cytokine also produced by Th1 cells. These IL-17producing cells were, then, plausible substitutes for classical Th1 cells as mediators of autoimmunity. A possible link between IL-23 and IL-17-positive T cells was then discovered. In corroboration of the work by Infante-Duarte et al. (2000), Aggarwal et al. (2003) showed that various microbial products induce the secretion of IL-17 into the supernatants of mononuclear splenocyte cultures. It was found that this required the presence of both antigenpresenting cells (APCs) and T cells, suggesting that TLR ligands may induce APCs to secrete a factor that promotes IL-17 secretion from T cells. A pronounced induction of IL-23p19 and IL-12p40 mRNA was measured in DCs exposed to these conditions, and it was found that IL-23 could substitute for the bacterial products in inducing the genes that encode IL-17A and IL-17F and the secretion of IL-17A. It was further shown that LPS-induced production of IL-17A could be decreased by neutralizing IL-12p40 in the supernatant, and could be increased by the use of cells from mice deficient in the IL-12-specific chain of the IL-12 receptor (IL-12Rb2). This indicated not only that IL-23 could substitute for LPS, but that LPS operated through the induction of IL-23 by APCs. Kastelein and co-workers had shown that memory, but not naı¨ve, T cells proliferate in response to IL-23 (Oppmann et al., 2000). Aggarwal et al. therefore sorted CD4 T cells based on their surface expression of CD44 and CD62L and showed that IL-23 strongly induces mRNA for IL-17A and IL-17F, and secretion of IL-17 protein into the supernatants of cultures enriched with memory cells, but not in those enriched with naı¨ve cells. It was therefore concluded that IL-23 acts on memory cells to induce their expression of IL-17. Murphy et al. (2003) showed not only that IL-23p19 deficiency protected against CIA, whereas IL-12p35 deficiency aggravated disease, they 1

IL-17A and IL-17F can form homodimers (e.g., IL-17A/A or IL-17F/F) or heterodimers (e.g. IL-17A/F). In instances in the literature where a distinction is not made, the term ‘IL-17’ is used generically to denote activities that may reflect IL-17A/A or IL-17F/F activity.

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also showed that the gene expression of various inflammatory factors, including IL-17A, was reduced in immunized animals that were deficient in IL-23p19 and IL-12p40, but not in animals deficient in IL-12p35. These findings paralleled the presence of IL-17A-expressing CD4 cells found in draining lymph nodes, being reduced by deficiency in either IL-23p19 or IL-12p40, but enhanced by IL-12p35 deficiency (Murphy et al., 2003). Therefore, IL-23 not only aggravates disease, it also increases both the expression of IL-17 in affected tissue and the presence of CD4þIL17þ cells in draining lymph nodes. Collectively, these results demonstrated the ability of IL-23 to promote the appearance of IL-17-producing T cells in vitro, and established a correlation between IL-23-induced autoimmunity and the induction of Th17 cells in secondary lymphoid organs with their subsequent trafficking to sites of disease. Although these findings replaced the relevance of the IL-12/IFNg axis in EAE and CIA with an IL-23/IL-17 axis, a formal test of the pathogenicity of the IL-17-positive T cells had not yet been done. Cua and coworkers therefore cultured cells, obtained from the draining lymph nodes of mice following antigen immunization, in the presence of IL-12 or IL-23 to obtain populations enriched for cells positive for IFNg or IL-17, respectively (Langrish et al., 2005). CD4þ cells were then purified from the cultures and transferred into susceptible mice. Exposure to IL-23 in vitro rendered the resulting IL-17-producing cells highly encephalitogenic upon transfer, whereas the IL-12-induced Th1 cells were far less pathogenic. While it was possible that pathogenicity was harbored in the many IL-17-negative cells that were present in the transferred populations, the demonstrated propensity of IL-17 to promote inflammation strongly implicated a causal role for the IL-17-positive fraction. But the exclusivity of the Th1/Th2 hypothesis could not be effectively challenged until the origin(s) of Th17 cells was uncovered. Some evidence pointed to a branching off of IL-17-producing cells from a Th1 precursor, whereas other evidence indicated that IL-17-producing cells might constitute a distinct lineage. On the one hand, the receptor for IL-23 is expressed on memory cells but not on naı¨ve cells (Parham et al., 2002), and IL-23 was reported to induce proliferation of memory cells but not of naı¨ve cells (Oppmann et al., 2000) with induction not only of IL-17 (Aggarwal et al., 2003) but of IFNg as well (Oppmann et al., 2000). The association of IL-17 with the Th1 lineage was further emphasized by its co-expression with IFNg in a subset of ‘Th1’ cells, without corresponding expression by Th2 cells (Aarvak et al., 1999). The ability of IL-23 to promote IL-17 producers in these studies only from memory cells and to simultaneously enhance IFNg production suggested that IL-17-producing T cells were closely related to Th1 cells and could not arise independently of them from naı¨ve T cells. Furthermore, resistance to EAE was conferred by deficiency of T-bet and STAT4. Since T-bet and STAT4 induced

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Th1 cells that did not themselves drive EAE, this suggested that IL-23 might divert a Th1 precursor away from the Th1 phenotype and toward the Th17 phenotype, and that this occurs downstream of T-bet and STAT4 signaling (Bettelli and Kuchroo, 2005). On the other hand, Langrish et al. (2005) reported that IL-23 could induce Th17 cells from naı¨ve cells and Dong and colleagues reported that the presence of inducible costimulator (ICOS) during culture of naı¨ve CD4 cells over several days could induce the production of IL-17, without enhancing IFNg production (Nurieva et al., 2003). And in contrast to the implications of the T-bet/STAT4 data, STAT1 signaling, which promotes Th1 development, opposed the induction of EAE (Bettelli et al., 2004; Nishibori et al., 2004), suggesting that a Th1 precursor may not be required in order to generate EAE. The developmental pathways leading to committed Th1 and Th2 phenotypes from naı¨ve precursors were well defined by this point in time, thereby enabling a rigorous test of the dependence of Th17 development on the key components of those pathways, with a corresponding resolution to the question of this cell’s developmental origins. In concurrent studies, our group (Harrington et al., 2005) and Dong and co-workers (Park et al., 2005) reported results establishing that IL-17-producing effectors did not arise from a common Th1 precursor. On the basis of the known reciprocal antagonism exhibited between Th1 and Th2 cells, it was reasoned that the signature cytokines of these phenotypes might likewise antagonize the development of Th17 cells. Both groups indeed found that IFNg and IL-4 are inhibitory and that neutralization of these cytokines enhanced the yield of Th17 cells in culture. Under these conditions, and in the presence of IL-23, Th17 cells developed from naı¨ve T cells. The cultures included some IFNg-positive cells, but expression of IL-17 and IFNg was for the most part mutually exclusive. Critically, the use of T cells deficient in STAT1 (Harrington et al., 2005), STAT4 (Harrington et al., 2005; Park et al., 2005), STAT6 (Harrington et al., 2005; Park et al., 2005), or T-bet (Harrington et al., 2005) rendered those cells prone to Th17 development, not resistant to it. The cytokines and signaling pathways that advance the development of Th1 and Th2 cells are, then, comprehensively antithetical to the development of Th17 cells. Collectively, this demonstrated that the Th17 lineage is distinct from the other two, and thereby expanded the Th1/Th2 hypothesis to include a new member—a possibility that the founders of the hypothesis foresaw (Mosmann and Coffman, 1989). Soon thereafter, the early developmental signals that lead to programming of Th17 effectors were discovered, and critical role for coordinate TGF-b and IL-6 signaling in early Th17 differentiation was defined (Bettelli et al., 2006; Mangan et al., 2006; Veldhoen et al., 2006). This was accompanied by the finding that IL-23 was dispensable for induction of

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Th17 developmental commitment (Mangan et al., 2006; Veldhoen et al., 2006); its inducible receptor component, IL-23R, being upregulated downstream of these signals. The subsequent discoveries of a transcription factor associated with Th17 development, RORgt, by Littman, Cua, and co-workers (Ivanov et al., 2006), and the independent finding by three groups that IL-21 acts as an essential autocrine factor promoting Th17 development downstream of IL-6 signaling and upstream of IL-23R expression (Korn et al., 2007; Nurieva et al., 2007; Zhou et al., 2007) further advanced our understanding of the Th17 developmental pathway (Fig. 5.1) and have been the subject of more in-depth reviews to which the reader is referred (Bettelli et al., 2008; McGeachy and Cua, 2008; Weaver et al., 2007). Thus, although originally defined on the basis of the eponymous cytokine, IL-17, Th17 cells are now more broadly defined by their expression of the transcription factors RORgt, RORa, STAT3, and IRF4; and secretion of the cytokines IL-17(A), IL-17F, IL-21, and IL-22; and in humans and other species, also IL-26. The emergence of IL-17-producing CD4þ T cells as a distinct subset of T helper cells has had profound effects upon our understanding of IL-23 TGF-β IL-12Rβ1 IL-23R IL-6 IL-21 STAT3

SMADs STAT3 RORγ t

IL-23R IL-1R

Th17 IL-23R IL-1RI

IL-17A/F

IL-17(A) IL-17F IL-22

Tn IL-12Rβ1 T-bet STAT1

IFNγ

IL-12R

IFNγ

IL-12Rβ2

IL-18R

IL-18Rα STAT4

IL-12Rβ1

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IL-12Rβ2

IL-12

IFNγ

Neutrophilic inflammation/ microbicidal bacteria fungi

Monocytic inflammation/ intracellular killing bacteria protozoans viruses

IL-4 STAT6

IL-4

Th2 GATA-3

GATA-3

Mucosal clearance helminthes

IL-4 IL-5 IL-13

FIGURE 5.1 Developmental divergence of effector CD4 T-cell lineages. This schematic emphasizes the key pathways in differentiation of the Th17, Th1, and Th2 effector lineages and the major pathogen classes for which they coordinate innate and adaptive immunity. See text for details.

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immunity. As in studies of autoimmunity, the Th1/Th2 paradigm failed to explain certain paradoxes of host protection, especially with respect to Th1 cells, which the discovery of Th17 cells is beginning to rectify. Since its discovery, inflammatory responses involving the production of IL-17 have had a strong association with neutrophilic inflammation and barrier defense (i.e., skin and mucosae), as has been reviewed elsewhere (Kolls and Linden, 2004). Early observations of dendritic cell production of IL-23 in response to bacteria and the discovery that IL-23 promoted Th17 responses led to the view that this pathway of T helper cell development was an evolutionary adaptation specifically directed at extracellular bacteria. Indeed, clear evidence has been presented suggesting that the Th17 arm of the inflammatory response is pivotal in the control of certain pathogenic bacteria. This is especially supported by the finding that IL-17 and IL-22, the latter a more recent addition to the Th17 cytokine repertoire (Zheng et al., 2007), both independently and cooperatively upregulate epithelial secretion of proteins that have direct bactericidal activity, such as b-defensins (Liang et al., 2006). The Th17 link to protection against specific classes of pathogens is further supported by more recent human disease associations. Milner et al. (2008) have observed that individuals with autosomal dominant hyper-IgE syndrome (HIES), which is characterized by susceptibility to particular extracellular bacterial and fungal infections, lack the ability to generate Th17 cells. However, an organism represents a niche that other organisms can potentially utilize for their survival. Thus, whether beneficial or harmful, evolution has contrived many strategies that allow organisms to utilize one another for their survival. Of course, these tactics extend beyond those for bacteria alone, and while the different subsets of effector T cells have clearly developed with specialized functions for handling particular types of invaders, pathogens have little respect for our attempts to neatly model immune responses and tend to defy simple categorizations into Th1-, Th2- or Th17-orchestrated immunity. This is particularly true for attempts to categorize host protection into Th1- or Th17-predominant responses, due to findings that T-cell-derived IFNg is invariably found in association with IL-17-producing T helper cells in inflammatory reactions in vivo. Indeed, T cells co-expressing IL-17 and IFNg occur not infrequently. Whether these are primarily Th1 cells expressing IL-17 or Th17 cells expressing IFNg is a question that remains to be answered. In the remaining sections of this review, we present an overview of current knowledge regarding the role of IL-23/IL-17-mediated innate and adaptive immune responses in repelling microbial assaults, with special emphasis on the Th17 lineage. We discuss many examples of IL-23/ IL-17/Th17-mediated host defense against bacterial pathogens. But we also discuss emerging reports that indicate a broader spectrum of pathogenic organisms that may recruit this pathway. As we hope to illustrate,

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while IL-23/IL-17-mediated immune responses are especially suited to dealing with extracellular bacterial infections, emerging evidence reveals that Th17-induced immunity is also increasingly associated with certain fungal, protozoal, and viral infections.

3. FUNCTIONS OF THE Th17-DERIVED CYTOKINES 3.1. IL-17 Originally identified as CTLA-8, IL-17 (IL-17A) cDNA was first cloned from activated murine lymphoid cells and used to identify the gene in rats and humans (Rouvier et al., 1993). After identifying the murine receptor (Yao et al., 1995a), the same group later isolated a cDNA encoding human IL-17A from a CD4þ library, and found that high levels of hIL-17A were expressed in peripheral blood CD4þ T cells upon stimulation. They furthermore demonstrated that hIL-17A fusion protein and supernatants from cells transfected with hIL-17A induced IL-6 and IL-8 production and enhanced surface expression of ICAM-1 in fibroblasts (Yao et al., 1995b). It was soon found that hIL-17A stimulates epithelial, endothelial, and fibroblastic cells to secrete GM-CSF and prostaglandin E2, in addition to IL-6 and IL-8, and that co-culture of hIL-17A-treated fibroblasts with CD34þ hematopoietic progenitors induced their development into mature neutrophils (Fossiez et al., 1996). Since then, IL-17 has been repeatedly shown to induce expression of various inflammatory cytokines and chemokines (Table 5.1) and thus contribute to the maturation and recruitment of leukocytes, especially neutrophils. Most recently, Liang et al. (2007) have shown that IL-17A and IL-17F form homo- and heterodimers, induce CXCL1 expression in vitro and CXCL5 in vivo, and that direct administration of IL-17A/F or IL-17F/A into the airways of mice significantly increased neutrophils and chemokine expression. IL-17F/F homodimers had limited pro-inflammatory effects in this model, the basis for which remains unclear.

3.2. IL-22 IL-22, originally called IL-10-related T-cell inducible factor (IL-TIF), was cloned from human cDNA by Dumoutier et al. (2000a). IL-22 is a member of the extended IL-10 family, which in addition to IL-10 and IL-22, includes IL-19, IL-20, IL-24, and IL-26 (Fickenscher et al., 2002). Although itself a product of hematopoietic cells, expression of the receptor for IL-22, which is composed of the IL-22Ra chain and the IL-10Rb—the latter shared with the IL-10 receptor, is not expressed by hematopoietic cells, but is preferentially expressed by epithelial cells, particularly in skin, pulmonary and intestinal tissues, as well as pancreas, liver, and kidney

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TABLE 5.1 Chemokines and other inflammatory proteins induced by IL-17

Proteins CXCL1, CXCL5, and CXCL8 G-CSF and GM-CSF IL-6 Prostaglandin E2 b-Defensin 2 S100A7, S100A8, and S100A9 MMP3 and MMP9 VEGF Responding cells Fibroblasts Epithelial cells Keratinocytes Mesothelium Endothelial cells Synoviocytes Osteoblasts

(Boniface et al., 2005; Gurney, 2004) (Figure. 5.2.) While also being shown to provide protection in hepatitis (Pan et al., 2004; Radaeva et al., 2004; Zenewicz et al., 2007) and to have a pro-inflammatory role in psoriasis (Boniface et al., 2007; Sa et al., 2007; Zheng et al., 2007), a protective role for IL-22 in host defense against pathogens was first suggested by the observation that it induced an acute phase response similar to that of IL-6 (i.e., the induction of acute phase reactants) (Boniface et al., 2005; Gurney, 2004). IL-22 induces expression of b-defensins, S100 protein family members, chemokines, matrix metalloproteinases (MMPs), among others, and stimulates keratinocyte migration and hyperplasia, characteristics that indicate involvement in innate immunity, inflammatory processes, and wound healing (Boniface et al., 2005; Sa et al., 2007; Wolk and Sabat, 2006; Wolk et al., 2004, 2006). Only recently has IL-22 been found to be closely associated with the Th17 subset. Two groups have demonstrated IL-22 production from Th17 cells. Liang et al. (2006) observed high level expression of IL-22 in TGFb/ IL-6 polarized Th17 cells compared to Th1 or Th2 cells. Maintenance of IL22 production from these cells was dependent on IL-23. In addition to observing IL-23-induced secretion of IL-22 from gd T cells, CD8þ T cells, monocytes, and murine and human naı¨ve T cells, Zheng et al. (2007) also found that IL-22 was preferentially produced by Th17 cells polarized from a naı¨ve population in the presence of IL-23 under Th1/Th2 neutralizing conditions. Stimulation with IL-6 similarly induced IL-22

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Cornea Skin

Nasopharynx

IL-23 Lung

DC

Th17

Liver

IL-17/IL-22

Stomach Pancreas

Intestine

Genital tract

FIGURE 5.2 The IL-23/IL-17/IL-22 pathway affects host defense at many tissue sites, primarily at sites of interface between the host and environment, such as mucosal surfaces.

expression. Interestingly, this group found that TGF-b inhibited IL-6induced IL-22 secretion from Th17 cells. Whether or not IL-23 could override this effect (e.g., simultaneous stimulation with TGF-b, IL-6, and IL-23) was not examined.

3.3. IL-26 Similar to IL-17, AK155/IL-26 was first identified from herpesvirus saimiri-transformed T cells (Knappe et al., 2000). Another member of the IL-10 family, the coding sequence for IL-26 is located in close proximity to that for both IL-22 and IFNg in the genome of most species studied to date, with the notable exception of rodents (Dumoutier et al., 2000b; Igawa et al., 2006; Knappe et al., 2000). The synteny of IL26 (and IL22) with IFNG suggests possible regulatory overlap for these three genes, although there is currently little data on this. Despite limited published data regarding its function, like its close relative IL-22, IL-26 appears to target epithelial cells through a receptor composed of the IL-20Ra and IL-10Rb chains (Hor et al., 2004). And because of its production by T cells, it is likely that this cytokine plays a role in normal and pathological immunity

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(Fickenscher and Pirzer, 2004). The elimination of a functional Il26 gene in rodents by a remote insertional event suggests a potentially compensatory role for IL-22 or another of the IL-10 family members (e.g., IL-24) in these species, and cautions against too broad an extrapolation of rodent IL-22 effects to humans or other species.

4. IL-23/IL-17-MEDIATED INNATE IMMUNITY As previously mentioned, recruitment of neutrophils has been established as a primary function of IL-17 (Kolls and Linden, 2004; McKenzie et al., 2006). Smith et al. (2007) have recently observed that treatment with IL-23, a potent inducer of IL-17, restores circulating neutrophil counts in mice deficient for both IL-12p40 and b2 integrin. However, IL-17A and IL-17F homo- and heterodimers appear to have differential activity in mediating neutrophil recruitment and chemokine expression (Liang et al., 2007). Accordingly, an anti-IL-17A-specific mAb prevented airway neutrophilia induced by intranasal administration of ovalbumin following adoptive transfer of Th17-polarized DO11.10 cells, while administration of an IL-17F-specific mAb did not. Furthermore, direct administration of IL-17A/A homodimer and IL-17A/F heterodimer into the airways increased neutrophils and chemokine expression, while IL-17F/F did not. Many cell types that have a role in host defense are capable of secreting IL-17. Expression of IL-17 by ab TCRþ or gd TCRþ CD4CD8 cells was reported soon after this cytokine’s discovery (Kennedy et al., 1996; Stark et al., 2005; Umemura et al., 2004). IL-17-producing splenocytes are expanded in various strains of adhesion molecule-deficient mice, yet even in wild-type mice, the majority of IL-17þ splenocytes are gd T cells, and a smaller but significant portion are Vb8þ NKT-like cells (Ley et al., 2006). CD8þ T-cell depletion reduces IL-17 levels in bronchoalveolar lavage (BAL) fluids from mice infected with Klebsiella pneumoniae, and IL-17 is secreted by CD8þ T cells cultured in supernatants from dendritic cells stimulated with K. pneumoniae (Happel et al., 2003). Upregulation of IL-17 mRNA was observed in BAL neutrophils from wild-type (WT) and T- and B-cell-deficient SCID mice following airway administration of LPS (Ferretti et al., 2003). And although not in the context of host defense, IL-17 expression by microglia, astrocytes, and oligodendrocytes in active EAE lesions has been reported (Kawanokuchi et al., 2007; Tzartos et al., 2008). Recently, Michel et al. (2007) have demonstrated IL-17 expression by liver NK1.1neg invariant NKT cells following stimulation with a-GalCer or either of the microbial antigens Borellia burgdorferi-derived glycolipid IIc (BdGL-IIc) and Sphingomonas wittichii-derived galacturonic acid-containing glycosphingolipid antigen (GalA-GSL).

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With regard to host defense, the most studied among non-ab T cells that produce IL-17 is the gd T-cell subset. IL-23 alone is a potent inducer of IL-17 secretion from these cells. Although gd T cells are the major source of IL-17 in naı¨ve mice as well as Mycobacterium tuberculosis-infected mice (Lockhart et al., 2006), its secretion by gd T cells or other cell types is apparently unnecessary in this setting as the absence of either IL-23 or IL-17 receptor signaling did not affect bacterial load during primary infection (Aujla et al., 2007; Khader et al., 2005). To the contrary, Shibata et al. (2007) demonstrated a critical role for IL-17 derived from Vg1þ gd T cells for protection against intraperitoneal injection of an avirulent strain of Escherichia coli, and in some settings these cells can also induce host injury, as recently shown in studies conducted by Romani et al. (2008). In a mutant mouse model of chronic granulomatous disease (infection with Aspergillus fumagatus), overwhelming Vg1þ gd T-cell reactivity associated with high levels of IL-17 and defective regulatory T-cell activity lead to acute inflammatory lung injury that was partially ameliorated by IL-17 neutralization or gd T-cell depletion.

5. Th17 CELLS AND INNATE IMMUNITY As discussed above, many hematopoietic cell types have been reported to be IL-17 competent. However, in light of their antigenic specificity, provision of B-cell help and production of epithelial-activating cytokines, particularly IL-17 and IL-22, Th17 cells appear to be uniquely situated at the interface of innate and acquired immunity. IL-17 can be rapidly induced from T cells in response to infection. Intratracheal inoculation of K. pneumoniae in mice induces a >100-fold increase in expression of IL-17A and IL-17F mRNA in the lung within 24 h of inoculation (Happel et al., 2005). Although host defense is also dependent on IL-12 and IL-23, the direct contribution of IL-17 in this setting was evidenced by >70% mortality within 7 days in the absence of IL-17 receptor signaling (Happel et al., 2005; Ye et al., 2001a). Furthermore, administration of recombinant IL-17A 12 h after inoculation significantly reduced bacterial load 36 h postinoculation in IL-12p40-deficient animals (Happel et al., 2005). The origin of IL-17 in these studies was both CD4þ and CD8þ cells. A 90% reduction in BAL IL-17 concentration resulted from Ab depletion of both subsets, while only a 45% decrease was observed when only one subset was depleted (Happel et al., 2003). Injection of Bacteriodes fragilis into the peritoneal cavity induced a similar early induction of IL-17 (4 h, measured in the peritoneal fluid). Examination of the resulting abdominal abscesses revealed a co-localization of CD4þ and IL-17þ cells in the fibrotic wall surrounding these lesions, which were significantly reduced by the administration of an IL-17 neutralizing Ab at the time of B. fragilis

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injection (Chung et al., 2003). IL-17 is also rapidly induced (24 h) from pulmonary CD4þ cells following intranasal inoculation with 107 CFU Mycoplasma pneumoniae (Wu et al., 2007). These observations suggest that Th17 cells, or a related CD4þ subtype, can be rapidly activated within hours of pathogen entry. The early expression of IL-17 during infection is logical given that it induces both neutrophil recruitment, as discussed above, and expression of antimicrobial proteins (Table 5.1). IL-23 is believed to initiate this response, but whether or not activation occurs in an antigen-independent manner is unknown and deserves further investigation. The direct activation of effector T cells by microbial ligand engagement of expressed PAMP receptors is possible (reviewed in (Kabelitz, 2007). Induction of proliferation and cytokine secretion from CD8þ cytotoxic lymphocytes without antigen stimulation has been demonstrated to occur by this mechanism through TLR2 recognition of lipidated outer surface protein OspA of Borrelia burgdorferi (Sobek et al., 2004). Although dependent on the presence of TCR stimulating aCD3, the engagement of TLR2, which is constitutively expressed in the CD45ROþ memory population, induces the same effect in CD4 T cells (Komai-Koma et al., 2004; Liu et al., 2006). Observations suggesting a similar MyD88-dependent role for TLR9 were also recently reported (Gelman et al., 2006; LaRosa et al., 2007). It is likely that the source of IL-17 in these instances is not entirely from prototypical Th17 cells, but rather cells of other CD4þ lineages such as NKT cells (Ley et al., 2006; Michel et al., 2007) or CD4þCD8þ cells (Huang et al., 2007). However, observations made by Sutton et al. and our own laboratory allow for the possibility of rapid IL-17 secretion from mature Th17 cells through an antigen-independent pathway. In vitro, IL-1 and IL-18 synergize with IL-23 to induce IL-17 expression from Th17-polarized effectors independently of TCR stimulation (Sutton et al., 2006; Lee et al., unpublished data). In humans, IL-21 synergizes with IL-7 and IL-15 to drive the antigen-independent proliferation of central memory and effector memory T cells ex vivo (Onoda et al., 2007), and IL-15 has been demonstrated to be a potent inducer of IL-17 (Ferretti et al., 2003; Hoeve et al., 2006; Ziolkowska et al., 2000). Therefore, it is not unlikely that the initiation of an acute inflammatory response might stir resident effectormemory Th17 cells into action via a TCR-independent mechanism even in anticipation of an antigen-specific response. This could be particularly beneficial in the intestines, where Th17 cells are normally enriched in the lamina propria (Ivanov et al., 2006; Mangan et al., 2006). The absence of this population in germ-free mice (Niess et al., 2008) suggests a role for these cells in the maintenance of innate barrier defenses to microbial invasion. Nevertheless, as noted earlier, there are many paths leading to the induction of IL-17 from various cell types, and it is too early to

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speculate on the contribution or relevance of its acute secretion from Th17 cells at environmental interfaces at this time.

6. Th17 CELLS AND ACQUIRED IMMUNITY Th17 cells also fulfill the traditional roles of T helper cells, such as in directing B-cell antibody class switching and activation of cytotoxic T cells. Bordetella bronchiseptica infection induces a strong antigen-specific Th17-cell response in the lung (Siciliano, Skinner and Yuk, 2006). Resistance to Bordetella pertussis challenge was observed following immunization of mice with a whole cell vaccine (Fennelly et al., 2008; Higgins et al., 2006), and vaccination with Mycobacterium tuberculosis ESAT-6 antigen elicited a protective antigen-specific Th1 and Th17 response (Khader et al., 2007). The importance of Th17 cells in conferring immunity to fungal infection is suggested by induction of Candida albicans-specific Th17 cells in mice and their presence in the peripheral circulation in humans (Acosta-Rodriguez et al., 2007; LeibundGut-Landmann et al., 2007). Antigen-specific production of IL-17 was observed from splenocytes following immunization with pneumococcal proteins, which induced antibody-independent protection against Streptococcus pneumoniae colonization (Basset et al., 2007). And even immunization with rotavirus VP6/LT (R192G) yielded antigen-specific lamina propria CD44high Th17 cells and protective immunity (Smiley et al., 2007; VanCott et al., 2006). In many of the reports discussed above, antigen-specific IFNg expression by CD4 T cells that co-expressed IL-17, and even expression of IL-17 by CD8þ cells, was also observed. As yet, there are no conclusive reports in which an absolute requirement for prototypical Th17 cells in the development of a protective acquired immune response has been demonstrated through rigorous in vivo experimentation. The functions of Th1 and Th17 cells in the acquisition of protective adaptive immunity appear to be somewhat overlapping, as suggested by the finding that either an IL-23/IL-17 or an IL-12/IFNg response is sufficient to provide protection against M. tuberculosis (Khader et al., 2005). In many cases, immune responses to infections previously believed to be Th1-mediated have now been shown to include both IFNg- and IL-17-producing effectors, suggesting the possibility of cooperation between the two subsets (Khader and Cooper, 2008). In addition, the frequent observation of CD4þ cells double positive for expression of both IL-17 and IFNg has been taken as evidence for a close relationship between the Th1 and Th17 lineages. Alternatively, our own recent studies have shown that Th17 cells can give rise to IFNg producers, contingent on the cytokine environment following initial Th17 commitment (Lee et al., submitted for publication). This suggests that Th17 cells might subserve dual functions in host

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defense, producing strongly pro-inflammatory IL-17-producers that coordinate early neutrophilic responses, yet also giving rise to IFNg-producers that can mediate transition to a more chronic, monocytic pattern of inflammation as needed. Further infectious studies using mice specifically deficient in factors critical for Th17 development (e.g., RORgt and RORa) should permit more definitive studies of the specific requirement for Th17 cells in different types of infection, permitting examination of the unique protective functions that these cells mediate.

7. IL-23/IL-17-MEDIATED RESPONSES IN SPECIFIC MICROBIAL INFECTIONS It is reasonable that T helper cell responses to infection have been historically segregated in the context of the Th1/Th2, and more recently, Th1/ Th2/Th17 paradigms. Within this framework, host defense against intracellular pathogens has largely been attributed to Th1 responses, defense against mucosal parasites to Th2 responses, and defense against extracellular bacteria and fungi attributed to Th17 responses (Fig. 5.1). While this concept holds in general, there are notable exceptions, complicated by the apparent overlap in certain functions of the Th17 and Th1 subsets. Emerging data suggest the IL-23/IL-17 axis has evolved to defend against a wider variety of pathogenic organisms than initially appreciated, which will be considered here on the basis of pathogen classes (Table 5.2).

7.1. Bacteria The first evidence for increased expression of IL-17 associated with infection was reported by Luzza et al. (2000) in studies of Helicobacter pylori-induced gastritis in humans. Treatment of gastric lamina propria mononuclear cell cultures with an anti-IL-17 mAb resulted in significantly decreased IL-8 secretion, and supernatants from an IL-17 stimulated gastric epithelial cell line, MKN 28, promoted in vitro polymorphonuclear leukocyte migration that was inhibited by neutralizing IL-8 but not IL-17. A seminal discovery contributing to the eventual characterization of the Th17 lineage was made by Infante-Duarte et al. (2000) in experiments examining induction of T helper cell differentiation in response to Borrelia burgdorferi and Mycobacterium bovis bacillus Calmette-Gue´rin (BCG). Murine TCR transgenic T cells primed with antigen in the presence of lysates from B. burgdorferi or M. bovis BCG strain Danish yielded IFNgproducing cells, but also yielded CD4þ cells that which produced IL-17, TNFa, and frequently also GM-CSF, but not IFNg or IL-4. The relevance of this finding to human disease was established with the identification of IL-17þ TNFaþ T helper cells in the synovial fluid of patients with Lyme

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Organisms that have been linked to IL-23/ IL-17 pathway

Organism

Helicobacter pylori Borellia burgdorferi Mycobacterium bovis BCG Klebsiella pneumoniae Pseudomonas aeruginosa Bordetella pertussis Bordetella bronchiseptica Mycobacterium tuberculosis

IL-23/IL-17 pathway beneficial or deleterious?

Reference

?

Luzza et al., 2000

Infection? Deleterious—Lyme arthritis Deleterious

Burchill et al., 2003; Infante-Duarte et al., 2000 Cruz et al., 2006

Beneficial Deleterious

Aujla et al., 2008; Happel et al., 2005 Dubin and Kolls, 2007

Beneficial Beneficial?

Higgins et al., 2006 Siciliano et al., 2006

?

Khader and Cooper, 2008; Khader et al., 2007 Yu et al., 2007

Porphyromonas gingivalis Salmonella enterica Typhimurium Citrobacter rodentium

Beneficial

Escherichia coli Streptococcus pneumoniae Staphylococcus aureus Corynebacterium xerosis

Beneficial ?

Godinez et al., 2008; Raffatellu et al., 2007 Aujla et al., 2008; Mangan et al., 2006 Shibata et al., 2007 van Beelen et al., 2007

?

van Beelen et al., 2007

?

Nisseria menigitidis

?

Bacteroides fragilis Enterococcus faecalis

Deleterious

van Beelen et al., 2007 van Beelen et al., 2007 Chung et al., 2003

Helicobacter muridarum

? Beneficial

Deleterious (dual association with E. coli in gnotobiotic IL-10-deficient mice) Deleterious (CD45RBhi)

Kim et al., 2007

Jiang et al., 2002 (continued)

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TABLE 5.2

(continued)

Organism

Helicobacter hepaticus Helicobacter bilis Candida albicans

Aspergillus fumagatus Cryptococcus neoformans Pneumocystis carinii Arthroderma benhamiae Cryptosporidium parvum Toxoplasma gondii

IL-23/IL-17 pathway beneficial or deleterious?

Deleterious (CD45RBhi) Deleterious (CD45RBhi) Beneficial—IV challenge Deleterious— mucosal challenge

Shomer et al., 1997

Deleterious—mucosal challenge ? ?

Rudner et al., 2007

?

Shiraki et al., 2006

Beneficial

Ehigiator, McNair and Mead, 2007 Kelly et al., 2005

Leishmania amazonensis Vaccinia virus

Beneficial

Herpes simplex (HSK) HIV Human rhinovirus

Beneficial

Theiler’s murine encephalomyelitis virus Respiratory syncytial virus

Cahill et al., 1997

LeibundGutLandmann et al., 2007 Bozza et al., 2008; Zelante et al., 2007 Bozza et al., 2008; Zelante et al., 2007 Kleinschek et al., 2006

Beneficial—clearance Deleterious— inflammation Beneficial— immunization ?

Eimeria acervulina

Reference

? Beneficial Deleterious

?

Ding et al., 2004 Xin, Li and Soong, 2007 Kohyama et al., 2007 Kim et al., 2008 Maek et al., 2007 Wiehler and Proud, 2007 Al-Salleeh and Petro, 2007; Petro, 2005 Hashimoto et al., 2005 (continued)

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TABLE 5.2

135

(continued)

Organism

Human T-cell leukemia virus type 1 Schistosoma mansoni Syphacia obvelata Trichuris muris

IL-23/IL-17 pathway beneficial or deleterious?

Reference

Dodon et al., 2004

Deleterious—HAM/ TSP Deleterious

Rutitzky et al., 2008

? Deleterious

Bugarski et al., 2006 Owyang et al., 2006

arthritis, suggesting an association between CD4þ T-cell-derived IL-17 and B. burgdorferi-induced immunopathology. It had previously been shown that B. burgdorferi-specific CD4þ T cells induce arthritis in rodent models (Lim et al., 1995a, b) independently of IL-4 or IFNg (Brown and Reiner, 1999). These observations, coupled with the association of IL-17 with synovial damage (Chabaud et al., 2000) and osteoclastogenesis (Kotake et al., 1999), lead Burchill et al. (2003) to consider its role in experimental Lyme arthritis. They observed that treatment with anti-IL-17 or anti-IL-17R mAb inhibited lesion development in IFNg-deficient mice following vaccination and challenge with B. burgdorferi. The role of IL-23/IL-17-mediated immune responses in contributing to host defense against pathogenic bacteria in the respiratory tract has received much attention, especially with regard to secondary infections due to HIV/AIDS. The increase in cases of HIV-associated K. pneumoniaeinduced bacterial pneumonia led Ye et al. (2001b) to investigate the contribution of IL-17 in mediating resistance to this organism. Intranasal inoculation of IL-17 receptor-deficient mice led to 100% mortality within 48 h compared to only 40% mortality in controls. Significantly lower levels of G-CSF and MIP-2 in these mutants were associated with a significant delay in neutrophil recruitment and greater bacterial dissemination. Happel et al. (2003) later observed IL-23-induced T-cell expression of IL-17, and that IL-23 and IL-17 were induced in a TRL4-dependent manner in response to K. pneumoniae infection. These findings were followed by the observation that mice deficient in IL-17R, IL-23p19, IL-12p35, or IL-12p40 suffered accelerated and increased mortality compared to WT mice infected with K. pneumoniae (Happel et al., 2005). Administration of IL-17 significantly reduced bacterial load in IL-23p19deficient mice, but a protective role for IFNg was revealed by a less profound reduction in treated IL-12p40-deficient animals. Recently, Aujla et al. (2008) have demonstrated that human primary bronchial epithelial cells express the receptor for the Th17-associated

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cytokine IL-22. Treatment of these cells with IL-17, IL-22, or both resulted in the upregulation of several host defense genes and increased production of G-CSF and IL-6. Rapidly induced IL-22 transcription and protein production were observed in the lungs of K. pneumoniae-infected mice. IL-22 antibody blockade in infected mice resulted in 100% mortality within 24 h of infection, which was significantly sooner than that observed in IL-17A-deficient mice and WT controls. Dissemination of the infection was also significantly increased in WT and IL-17A-deficient animals treated with anti-IL-22 antibody. Mice deficient in IL-23 also exhibit increased susceptibility and dissemination of the infection. Treatment with either IL-17 or IL-22 or both significantly reduced the number of bacteria recovered in the lung and spleen, suggesting that IL-22 expression requires IL-23. Interestingly, IL-17 and/or IL-22 induced the upregulation of several host defense genes in primary mouse tracheal epithelial cells (MTEC). Among these was the gene encoding lipocalin2 (Lcn2), an iron sequestering antimicrobial protein. IL-22-induced killing was significantly inhibited in infected MTEC cells derived from Lcn2/ mice. Finally, T cells isolated from the hylar lymph nodes of P. aeruginosainfected cystic fibrosis patients produced significantly greater amounts of IL-17A, IL-17F, and IL-22. IL-23 is rapidly induced following inoculation with Mycoplasma pneumoniae in mice (Wu et al., 2007). Lung CD4 T-cell-derived IL-17A and IL-17F are also increased. Interestingly, while lung IL-17C mRNA was unaffected, IL-17A and IL-17F, along with neutrophil recruitment and activity, were reduced by blocking IL-12p19 in vivo. The observation that Bordetella pertussis induced IL-23 in infected monocyte-derived dendritic cells in vitro (Spensieri et al., 2006) was followed by two reports revealing a role for Th17 cells in host defense against Bordetella organisms. Siciliano et al. (2006) showed that B. bronchiseptica-infected macrophages specifically induced the secretion of IL-17, but not IFNg, from CD4þ splenocytes in vitro, and that cells isolated from the lungs of infected mice 7 days postinoculation secreted large amounts of IL-17 when restimulated with antigen, but very little IFNg. Immunization with a whole cell B. pertussis vaccine induced a protective Th1 and Th17 response that was dependent on TLR4. Immunity was significantly reduced by prior administration of anti-IL-17 mAb. IL-17 was also found to activate macrophage killing of B. pertussis (Higgins et al., 2006). Perhaps not surprisingly, therefore, it has recently been observed that the type III secretion system of B. pertussis subverts this response (Fennelly et al., 2008). However, anti-pathogen IL-23/IL-17 immune responses are not always beneficial. Following their observation of elevated levels of IL-23 and IL-17 in the sputum of cystic fibrosis patients infected with Pseudomonas aeruginosa (McAllister et al., 2005), Dubin and Kolls found that inflammation was reduced while bacterial dissemination was unaffected in IL-12p19-deficient mice compared to WT controls. This suggests that the

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IL-23/IL-17 axis does not contribute to host defense in this setting, but rather likely promotes the airway destruction and bronchiectasis observed in CF patients (Dubin and Kolls, 2007). A similar pathological role for IL-17-producing T cells in mice infected with M. bovis BCM has also been implicated (Cruz et al., 2006). Mycoplasma tuberculosis infection was shown to elicit the early induction of IL-23 in infected lungs. Secretion of IL-17 in this setting was observed primarily from CD4CD8 gd T cells (Lockhart et al., 2006). Scriba et al. (2008) have recently found distinct subpopulations of antigen-specific IL-17þ or IL-22þ CD4þ memory T cells in the peripheral blood of M. tuberculosis-exposed individuals. Interestingly, antigen-specific production of IL-22, unlike IL-17, was not suppressed by treatment with Th1 cytokines or IFNg. In addition, these populations were reduced in the peripheral blood of individuals with active tuberculosis, while IL-22 was elevated and IL-17 was not detected in BAL fluids. Whether these cells contribute to Mycobacterium-initiated tissue destruction has not been determined. Prior vaccination appears to dramatically alter the immune response and outcome of subsequent M. tuberculosis challenge. Khader et al. reported that under these circumstances, IL-23 was essential for the development of an accelerated IFNg response by lung CD4þ T cells, inhibition of bacterial growth, and directing the development of lung Th17 cells (Khader and Cooper, 2008; Khader et al., 2007). Antibody neutralization of IL-17 decreased chemokine expression and the recruitment of Th1 cells. The authors proposed a model of Th17/Th1 cooperativity in which vaccination establishes a population of lung Th17 cells, which upon rechallenge induce chemokine-mediated recruitment of Th1 cells that inhibit bacterial growth. IL-23/IL-17-mediated immune responses likewise contribute to host defense in the alimentary tract (Yu et al., 2007). Yu et al. demonstrated a protective role for IL-17 in Porphyromonas gingivalis-induced bone loss in mice. Neutrophil recruitment is a key regulator of host defense here, as evidenced by the observation that humans with defects in neutrophil adhesion or trafficking are extremely susceptible to periodontal disease (Baker, 2000; Genco, 1996). Despite evidence for osteoclastogenic role for IL-17 in arthritis (Yago et al., 2007), in this model IL-17RA-deficient mice exhibited enhanced periodontal bone destruction associated with reduced chemokine expression and neutrophil migration to bone. In the stomach, Helicobacter pylori infection is associated with increased expression of IL-23, IL-17, and IL-21 (Caruso et al., 2008; Caruso et al., 2007). IL-21 enhances the secretion of MMP-2, MMP-9, and induces gelatinase synthesis by gastric epithelial cells in a dose-dependent fashion (Caruso et al., 2007). Eradication of infection decreases IL-17 and IL-21 expression (Caruso et al., 2007; Luzza et al., 2000). Scott Algood et al. showed the recruitment of H. pylori antigen-specific CD4þ T cells during

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the adaptive phase of the immune response in the mouse model. The appearance of these cells correlated with elevated IL-17 mRNA transcripts at 22 days, which transitioned to elevated IFNg mRNA transcripts at 34 days (Scott Algood et al., 2007). In the intestine, Salmonella enterica serotype Typhimurium rapidly induces IL-17 and IL-22 expression in both the bovine ligated ileal loop and streptomycin-pretreated mouse models (Godinez et al., 2008; Raffatellu et al., 2007). In our own laboratory, we have shown that the murine pathogen and causative agent of infectious colonic hyperplasia, Citrobacter rodentium, causes rapid mortality in 100% of infected IL-12p19deficient mice (Mangan et al., 2006). In WT mice, the inflammatory response is characterized by accumulation in the colonic lamina propria of IL-17þ cells, and to a lesser extent, IFNgþ cells. In this model, CD4þ T cells, in addition to TCRb and CD40L, are required for survival and development of a protective humoral response (Bry and Brenner, 2004; Bry et al., 2006). Although there is an absolute requirement for IL-23, mice with defects in the IL-12/IFNg pathway or IL-17RC-deficient mice do not succumb to infection, suggesting that initial protection can be mediated in the absence of either Th1 or Th17 responses (O’Quinn and Weaver, unpublished data). Interestingly, the absence of IL-23 results in accelerated bacterial growth during the initial phase of the infection prior to the appearance of infiltrating T cells, suggesting a defect in innate immunity possibly involving altered induction of antimicrobial protein secretion (O’Quinn et al., unpublished data). Indeed, Zheng et al. (2008) have recently demonstrated a requirement for IL-23 in inducing IL-22, which they show directly increases expression of antimicrobial genes such as RegIIIb and RegIIIg by the involved intestinal epithelium. Mice deficient in IL-22 quickly succumbed to C. rodentium infection associated with an absence of Reg protein expression. Protection was partially restored by reconstitution of infected IL-22-deficient mice with recombinant murine or human RegIIIg (100% mortality in IL-22-deficient vs. 50% mortality in IL-22-deficient plus rmRegIIIg). Within the peritoneal cavity, the IL-23/IL-17 pathway also appears to have a role in host defense against introduced gut flora, but via an alternative mechanism (Fig. 5.2). In a mouse model of intraperitoneal infection with Escherichia coli, Shibata et al. (2007) found a rapid, but transient, induction of IL-17. The subsequent influx of neutrophils could be blocked by administration of IL-17 neutralizing Ab, but resulted in impaired clearance of the bacteria. The dominant IL-17-producing cell population in this setting was demonstrated to be Vd1þ gd T cells, from which secretion of IL-17 depends both on TLR4 and IL-23. Other bacteria have been found to induce IL-17 responses in vitro. van Beelen et al. (2007) found that human monocyte-derived dendritic cells pulsed with several of the bacteria previously mentioned could elicit the

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secretion of IL-17 from unfractionated peripheral blood CD4þ T cells. In addition to S. pneumoniae, E. coli, and K. pneumoniae, they found that Staphylococcus aureus, Corynebacterium xerosis, and Neisseria menigitidis were equally effective in inducing this response. These data suggest that in the human system both Gram-positive and Gram-negative bacteria may induce, and be targets of, systemic Th17 responses. However, as was observed with B. burgdorferi and some respiratory infections, gut-associated bacteria can elicit damaging immune processes mediated by the IL-23/IL-17 pathway. Outer membrane protein from P. gingivalis stimulated IL-17 expression from peripheral blood mononuclear cells, and these cells occurred at a higher frequency in periodontitis patients (Oda et al., 2003). Bacteroides fragilis injected into the peritoneal cavity of mice induces the formation of abscesses. Chung et al. demonstrated that the development of these lesions depended on the presence of ab TCRþ IL-17-producing CD4þ T cells that localize to the abscess wall. Furthermore, the administration of IL-17 neutralizing Ab blocked formation of abscesses (Chung et al., 2003). Severe colitis lesions, with significantly elevated levels of IL-12p40 in the colon and antigen-specific IL-17producing CD4þ T cells in mesenteric lymp nodes (MLN), developed in gnotobiotic IL-10-deficient mice colonized with only E. faecalis and E. coli (Kim et al., 2007). Elson et al. (2007) observed that a cecal bacteria-reactive Th17-polarized cell line induced more severe colitis upon adoptive transfer into SCID recipients than did comparable Th1-polarized cells. Furthermore, the administration of a blocking anti-IL-23 mAb prevented lesion formation and ameliorated active disease. Additionally, in the CD45RBhigh T-cell transfer colitis model, in which IL-23 and IL-17 are active (Liu et al., 2007), disease has been monoassociated with colonization of Helicobacter muridarum ( Jiang et al., 2002). H. hepaticus or H. bilis, when combined with defined flora, also induce colitis in this model (Cahill et al., 1997; Shomer et al., 1997).

7.2. Fungi The involvement of IL-17 in mediating host defense against fungal infections was first suggested by the observation that IL-17RA-deficient mice exhibited accelerated dose-dependent mortality following intravenous injection of Candida albicans (Huang et al., 2004). Recently, many new insights have been gained regarding the role of the IL-23/IL-17 immune responses in mediating host defense against this organism and other fungi. Interestingly, important differences in the immune response have been observed depending on the route of challenge (systemic vs. mucosal), fungal morphology (yeast or hyphae), and APC phenotype. Dectin-1, a C-type lectin that is a pattern recognition receptor for fungal b-glucans, has been implicated in the induction of IL-23 that promotes anti-fungal

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Th17 responses. LeibundGut-Landmann et al. (2007) showed that curdlan, a b-glucan derived from yeast cell walls, induced IL-6, TNF-a, and IL-23 secreting bone marrow-derived dendritic cells (BMDC) dependent on dectin-1–Syk–CARD9 signaling. Curdlan-treated BMDCs directed the development of Th17 cells in vitro, and curdlan acted as a Th17 adjuvant in vivo. Furthermore, systemic infection of mice with C. albicans induced a CARD9-dependent Th17 immune response. Acosta-Rodriguez et al. (2007) followed with the observation of C. albicans-specific RORgtþ Th17 cells in the CCR6þCCR4þ subset of human peripheral blood memory T cells. As previously mentioned, route of entry appears to be an important factor in directing the development of the resulting immune response. In WT, IL-12p19-, IL-12p35-, and IL-12p40-deficient mice challenged via the mucosa with either C. albicans (intragastric) or Aspergillus fumigatus (intranasal), Zelante et al. (2007) observed that only IL-12p35-deficient animals exhibited reduced survival and significantly increased pathogen load at 3 and 7 days compared with WT controls. This was associated with significantly elevated levels of IL-23 and IL-17 in stomach homogenates, increased numbers of IL-17þ and IL-4þ CD4þ T cells in MLN, and histopathology revealing severe inflammation in the stomach mucosa. Although a similar pattern of cytokine and receptor expression to that induced by Candida in the MLN was observed in the thoracic lymph nodes of A. fumigatus-infected animals, infection did not affect survival time or fungal load in IL-12p35-deficient mice compared to WT controls. Antibody blockade with anti-IL-23 decreased the fungal load in both Candida and Aspergillus infected mice, whereas anti-IL-17 only reduced fungal load in Aspergillus infected animals. Anti-IL-23 treatment resulted in higher fungal load in IFNg-deficient mice while decreasing fungal load in WT, IL-4-, and IL-12p35-deficient animals, suggesting that IL-23 provides IL-12p35-dependent resistance in the absence of IFNg. In a series of in vitro experiments, Zelante et al. (2007) found that the yeast form of Candida, as opposed to hyphae, induced greater expression of IL-23 from conventional dendritic cells via MyD88-dependent signaling of TLR2 and TLR4. The first part of this observation is supported by evidence from experiments with Aspergillis showing adoptive transfer of antigen-pulsed plasmacytoid DCs confer protection, while conventional DCs exacerbate infection (Romani et al., 2006). Differences in inflammatory response based on the morphologic form of A. fumagatus spores, and thus b-glucan expression, have been shown previously (Hohl et al., 2005; Steele et al., 2005). With regard to the signaling pathways mentioned above, De Luca et al. (2007) have recently presented supporting evidence showing that control of fungal growth and balanced reactivity to Candida mucosal challenge requires signaling through MyD88 and TRIF to allow for appropriate Treg function. In this setting, TRIF-deficient CD11cþ MLN DCs stimulated

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with either yeast or hyphae secreted IL-6 and much greater levels of IL-23 compared to WT controls. These cells induced Th17 differentiation from both TRIF-deficient and WT naı¨ve CD4þ cells, whereas WT DCs induced IL-10 expression from both WT and TRIF-deficient naı¨ve CD4þ T cells. These results indicate that DC TRIF signaling is required for activation of Tregs, an observation supported in vivo by the increased Th17 and reduced Treg populations in WT mice adoptively transferred with Candida-pulsed TRIF-deficient DCs (De Luca et al., 2007). Zelante et al. (2007) had also revealed that IFNg enhanced neutrophil fungal killing, whereas killing of both Candida and Aspergillus was inhibited by IL-23 and IL-17 via antagonism of indoleamine 2,3-deoxygenase (IDO) induction. TRIF-deficient DCs were also found to be deficient in IDO upon stimulation with fungus. Although immunogenic, these DCs were capable of inducing Treg development from naı¨ve T cells in the presence of tryptophan catabolites (kynurenins) (De Luca et al., 2007). Taken together, these results help to explain the previously paradoxical observation of fungal persistence in the face of a chronic inflammatory response by revealing that a Th17 response favors this outcome. Studies from Bozza et al. (2008) have reinforced this concept by demonstrating that DC-expressed Toll IL-1R8 (Tir8), a suppressor of TLR/IL-1R signaling, is also involved in the skewing of the inflammatory response away from the Th17 pathway. They found enhanced susceptibility and increased inflammation in C. albicans and A. fumigatus infected Tir8-deficient mice associated with activation of Th17 cells. Conversely, infected IL1R-deficient mice exhibited less inflammatory pathology and suppressed Th17 activation. Other studies suggest that the IL-23/IL-17 pathway is also induced in mediating host defense against systemic Cryptococcus neoformans (Kleinschek et al., 2006), Pneumocystis carinii mucosal challenge (Rudner et al., 2007), and the dermatophyte Arthroderma benhamiae (Shiraki et al., 2006).

7.3. Protozoa Relatively little information exists regarding the role of the IL-23/IL-17 pathway in regulating host defense against protozoa (Fig. 5.3). The limited available data is restricted to the related apicomplexan coccidia Cryptosporium parvum and Toxoplasma gondii, intestinal parasites that cause potentially life threatening disease in immunodeficient patients and human fetuses, respectively, and the vector borne parasite Trypanosoma cruzi, the causative agent of Chaga’s disease. Administration of recombinant IL-12 or IL-18 significantly reduced fecal oocyte shedding in C. parvum-infected IL-12p40-deficient mice (Ehigiator et al., 2007). However, administration of recombinant IL-23, similar to IFNg, also reduced parasite shedding, indicating that, while

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sub-dominant to IL-12, IL-23 can contribute to host defense in this model as well. Recent studies with T. gondii suggest IL-23/IL-17 mediated inflammation is both beneficial and injurious. Kelly et al. (2005) found that almost 80% of IL-17R-deficient mice challenged perorally with T. gondii cysts, succumbed to infection by day 22 postinfection, associated with an increase in parasite burden in the tissues and reduced PMN influx. Histological examination of the affected tissues revealed that T. gondii tachyzoites were evident in the lamina propria of IL-17R-deficient animals, while WT mice had no evidence of parasites. Interestingly, IL17R-deficient mice had slightly less severe fatty change in hepatocytes and much less mucosal damage in the small intestine compared to WT controls. Other recent data indicate a requirement for suppression of Th17 responses to protect the host against protozoal infections. In the absence of IL-27 signaling, mice injected intraperitoneally with T. gondii cysts developed severe CD4þ T-cell-dependent neuroinflammation (Stumhofer et al., 2006). These lesions were characterized by significantly elevated transcripts of IL-17 mRNA, and antigen restimulation of brain mononuclear cells revealed the presence of high frequencies of both Th17 cells and IL-17-producing CD8þ T cells. Treatment with IL-27 suppressed the secretion of IL-17 from these cells in vitro, and also blocked the development of Th17 cells from naı¨ve precursors via a STAT1-dependent pathway. Additionally, in the absence of the G-protein-coupled bradykinin B2 receptor (B2R), mice infected intraperitoneally with T. cruzi display higher parasitemia and mortality rates compared to wild type (Monteiro et al., 2007). These changes were associated with the development of antigen-specific Th17 cells in the heart and spleen. The adoptive transfer of immature WT CD11cþ DCs conferred resistance to B2R-deficient recipients, induced Th1 development, and suppressed the development of Th17 cells. In other studies involving protozoa, in ovo immunization of chicken embryos with an Eimeria acervulina subunit vaccine in combination purified plasmid DNA encoding the chicken IL-17 gene revealed that IL-17 acted as an adjuvant by inducing higher serum antibody responses and reduced oocyst shedding in challenged 14-day-old chicks (Ding et al., 2004). Also, Leishmania amazonensis-infected DCs have been shown to preferentially activate CD4þ T cells to a IFNglow IL-10high IL-17high phenotype in vitro and in vivo (Xin et al., 2007). Collectively, these results indicate that IL-23/IL-17 signaling plays a contributing role in regulating host defense against protozoal infection, particularly with regard to induction of neutrophil recruitment and subsequent parasite killing. However, in the absence of regulatory mechanisms to suppress the Th17 response, severe inflammatory sequelae arise that cause tissue injury and increased mortality. No data have yet been reported regarding Plasmodium

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falciparum and Babesia, but it is possible that similar processes are also engaged by these significant protozoan parasites as well.

7.4. Viruses Viral immunity is primarily mediated through the combined innate activity of natural killer cells and induction of cytotoxic T cells (CTLs) during the adaptive phase. As viral immune responses are characterized by secretion of IFNg, which suppresses Th17 development, it might be expected that this pathway would play a minor role, if any. However, recent evidence suggests that the IL-23/IL-17 pathway may be enlisted for some forms of antiviral protection. Evidence from Kohyama et al. (2007) indicates that IL-23 is involved in mediating resistance to vaccinia virus infection. Engineered recombinant vaccinia virus (VV) expressing IL-12 (VV-IL-12) and expressing IL-23 (VV-IL-23) were generated. Substantial mortality was observed in BALB/c mice inoculated with wild-type VV (VV-WT), while all mice inoculated with VV-IL-23 survived. In addition, IL-12p40-deficient mice survived infection with VV-IL-23, whereas 100% mortality resulted from infection with VV-WT in IL-12p40-deficient controls. IFNg-deficient mice did not eliminate VV-IL-12, but did eradicate VV-IL-23 indicating that IFNg is essential for IL-12-mediated resistance, but dispensable for IL-23regulated resistance. Further, treatment with an anti-IL-17 mAb resulted in a significant increase in viral titers in VV-IL-23-infected IFNg-deficient mice, although the effect was limited and IL-17-deficient mice survived infection with VV-IL-23. Herpetic Stromal Keratitis (HSK) is an immunopathogenic disease triggered by infection of the cornea with herpes simplex virus (HSV). IL-17R is constitutively expressed by human corneal fibroblasts, and corneas from patients with HSK exhibit increased expression of IL-17. In cultured human corneal fibroblasts, IL-17 was observed to synergize with TNF-a to induce IL-6 and IL-8 secretion as well several chemokines and MMP-1 (Maertzdorf et al., 2002). IL-23 and IL-17 are elevated in the murine cornea after highdose HSV-1 infection, triggering the induction of IL-6 and MIP-2 both in vitro and in vivo (Kim et al., 2008). Transiently reduced corneal opacity due to fewer infiltrating neutrophils was observed in IL-17R-deficient mice following HSV-1 infection (Molesworth-Kenyon et al., 2007). IL-17-induced secretion of GM-CSF by human corneal fibroblasts and human corneal epithelial cells resulted in significantly delayed neutrophil apoptosis (Duan et al., 2007). Infection in IFNg-deficient mice was associated with elevated IL-17 levels and accelerated corneal opacity, but HSV-1 growth and clearance in IL-17-deficient mice was similar to that of WT controls (Molesworth-Kenyon et al., 2007). Although viral clearance was enhanced, increased disease severity was observed in infected IL-23p19-deficient

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Gut lumen Bacteria

Enterocyte proliferation, increased tight junctions, & increased antimicrobial defenses

Enterocyte M cell

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TNFa MIP-3a IL-1,6,8,18 MCP-3 IP-10 I-TAC RANTES TECK

DC

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B cell

LP Capillary

TNFa MIP-1a IL-8 IL-6

a4b7

LFA-1

FIGURE 5.3 The role of the IL-23/IL-17 pathway at mucosal surfaces. As exemplified in the gut, where Th17 cells are normally enriched, bacteria are recognized and taken up by resident dendritic cells (DCs). Microbe-activated DCs traffic to secondary immune tissues to induce differentiation of Th17 cells, as well as present antigen and release IL23 and IL-1 locally to activate Th17 release of pro-inflammatory cytokines, such as IL-17 and IL-22. Local DCs may also participate in the production and/or regulation of TGF-b and IL-6, which promote Th17 development. Th17 cells may provide B-cell help, through actions of IL-17 and IL-21. Epithelial cells are key participants in the orchestration of this process by responding to IL-17 and IL-22 to secrete factors that complement the actions of IL-17 in recruiting PMNs to the inflammatory site, alter secretion of mucins, enhance epithelial junctions, and secrete microbicidal factors that resist microbial breach of epithelial integrity (see text for details).

mice, an observation associated with increased IL-12 and elevated frequency of Th1 cells and reduced Th17 cell numbers. These data suggest that IL-12/IFNg responses in the absence of IL-23 may be deleterious in this setting (Kim et al., 2008). Respiratory syncytial virus (RSV) is the leading infectious cause of respiratory failure and bronchiolitis in infants and young children. Premature infants exhibit decreased levels of STAT1-regulated type I interferons, and are at increased risk for RSV infection. Respiratory syncytial virus induces increased mucus production and expression of gob-5 and Muc5AC in infected STAT1-deficient mice. IL-17, which regulates

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Muc5AC expression, and IL-23p19 were expressed in the lungs of STAT1 mutants, but not in WT mice (Hashimoto et al., 2005). In studies with primary human bronchial epithelial cells, IL-17A synergistically enhanced human rhinovirus (HRV)-induced epithelial production of the neutrophil chemoattractant, IL-8, as well as human b defensin-2 in vitro. Although viral uptake and replication were unaltered in this system, this result suggests that IL-17A can modify epithelial responses to HRV in a manner that would be expected to recruit relevant inflammatory cells to the airways (Wiehler and Proud, 2007). IL-17 has also been implicated as a contributor to viral-induced pathogenic inflammation. Intracranial infection with Theiler’s murine encephalomyelitis virus (TMEV), used as a model for the study of multiple sclerosis, leads to infection of CNS microglia, macrophages, oligodendrocyte, and astrocytes and induces demyelinating disease associated with anti-myelin CD4þ T cells in strains of susceptible mice. Upregulation of IL-23 has been reported in TMEV-infected macrophages (Al-Salleeh and Petro, 2007; Petro, 2005), although a direct role for IL-23-induced cytokines (e.g., IL-17) has not yet been established in this model. Interestingly, emerging reports indicate a possible role for Th17 cells in protection against HIV infection. Studies have shown that, while not associated uniquely with Th17 cells, IL-22 contributes to antiviral immunity. Activated T cells from repeatedly HIV-1-exposed, uninfected individuals overproduce several proteins involved in the innate immune response. IL-22 from CD4þ cells, along with peroxiredoxin II from CD8þ cells, was significantly elevated in exposed, uninfected individuals, suggesting a correlation between IL-22 responses and host resistance to HIV1 infection. Misse et al. (2007) propose that IL-22 induces the production of acute-phase serum amyloid A (A-SAA), which was demonstrated to be elevated in uninfected individuals. A-SAA inhibited in vitro HIV-1 infection of DCs, which are susceptible to infection with HIV-1 via CCR5. A-SAA downmodulated CCR5 expression, and preincubation of DCs with A-SAA reduced viral load 100-fold as compared with cells preincubated in the absence of A-SAA. Also, a lysine to glutamine substitution in IL-10RB, a shared subunit of receptors for IL-10 and IL-22, has been strongly associated with clearance of hepatitis B virus (HBV) in Gambian families (Frodsham et al., 2006). Infection with immunodeficiency viruses may have profound effects on Th17-mediated immunity. In a recent report, Raffatellu et al. (2008) found that macaques infected with simian immunodeficiency virus (SIV) were depleted of Th17 cells in the intestinal mucosa, resulting in susceptibility to dissemination of intestinal Salmonella typhimurium infection due to impaired mucosal barrier functions. In SIV-negative macaques, the gene expression profile induced by S. typhimurium in ligated ileal loops was characterized by Th17-type responses, including the expression of

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IL-17 and IL-22. Mice deficient in IL-17R expression showed increased dissemination of S. typhimurium, consistent with a critical role for IL-17 in maintenance of mucosal resistance to S. typhimurium. Collectively, these data suggest that HIV-1 infection subverts Th17 immunity in the intestinal mucosa, although any potential protective effects of the Th17 pathway in resistance to HIV-1 have not been directly established. Human T-cell leukemia virus type 1 (HTLV-1) is the causative agent of adult T-cell leukemia and is linked to the inflammatory neurological condition HTLV-1 associated myelopathy/tropical spastic paraparesis (HAM/TSP). Tax protein, a product of HTLV-1, upregulates the expression of human IL-17 in infected CD4þ T cells and Tax-expressing Jurkat T cells, and was linked to activation of the IL-17 promoter via the CREB/ ATF pathway (Dodon et al., 2004). Accordingly, IL-17 mRNA expression in T lymphocytes from a HAM/TSP patient was found to be 17-fold higher than that observed in unstimulated HTLV-1-negative T lymphocytes cultured in the presence of IL-2. Whether this induces a protective response or might deviate immunity toward a non-protective Th17 response remains to be determined.

7.5. Other As noted above, infection can elicit IL-23/IL-17-mediated immune responses with negative consequences. This seems to be especially true with regard to certain parasitic infections. IL-21 is expressed at high levels by Th17 cells, but not Th2 cells (Onoda et al., 2007; Wei et al., 2007). In the absence of IL-21 receptor signaling, reduced granulomatous inflammation and liver fibrosis were observed following infection with the helminth, Schistosoma mansoni. However, these results were attributed to a marked reduction in Th2 cytokine expression and function. Th1 responses were similarly reduced (Pesce et al., 2006). Recently, Rutitzky et al. (2008) have observed decreased immunopathology induced by Schistosoma egg antigens in IL-23p19-deficient mice associated with a decrease in IL-17 in granulomas. Infection of mice with the helminth Syphacia obvelata produced significant alterations in hematopoiesis characterized by increased myelopoiesis and erythropoiesis. Bone marrow myeloid and erythroid progenitors from S. obvelata-infected mice displayed altered sensitivity to IL-17 when compared to uninfected controls. Specifically, early erythroid progenitors from uninfected mice treated with IL-17 were induced to proliferate, whereas late erythroid progenitors were inhibited. Progenitor cells of both types from S. obvelata-infected mice were unresponsive to IL17 (Bugarski et al., 2006). Infection of mice with the helminth Trichuris muris in the absence of IL-25, a Th2 promoting cytokine, resulted in the increased expression of IFNg and IL-17 accompanied by severe intestinal inflammation (Owyang et al., 2006). Taken together, these results indicate

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that suppression of Th17-related cytokines in this setting prevents harmful inflammatory sequelae.

8. CLOSING REMARKS The dominance of the human species is a matter of perspective. Viewed in terms of population and biomass, we are in the minority. There are an estimated five nonillion (5 1030) prokaryotes representing a biomass of around 200 quadrillion grams (200 1015 g). Humans are greatly outnumbered by comparison, with an estimated population of 6 billion (6 109) and biomass of 220 trillion grams (220 1012 g). Indeed, the combined number of prokaryotic organisms living on the skin of one individual is estimated to be 300 million, which is dwarfed by the estimated 70 trillion bacteria in the colon (Whitman et al., 1998). Fortunately, only a very small percentage of these organisms cause disease in man. Only recently have we begun to fully understand how we can coexist in a world dominated by microbes. Evolutionary adaptations that have arisen over millions of years of continuous interactions between prokaryotes and eukaryotes have produced a myriad of strategies by both to enable mutual survival in the constant struggle for symbiotic equilibrium. Along with basic physical barriers such as skin and mucous, antimicrobial proteins and receptors allowing the recognition of elemental microbial components are just some of the innate mechanisms that contribute to host defense and allow us to rebuff ongoing challenges by the microbial world. As highlighted herein, IL-23/IL-17-mediated immune responses have emerged as central players in host defense, both through rapid recruitment of early innate responses that act to enhance barrier function at skin and mucosal surfaces, and through the Th17 effector lineage, which fills a previous void in our understanding of the adaptive immune response that complements the traditional roles played by Th1 and Th2. As evidenced by the variety of cell types demonstrated to secrete IL-17 and IL-22 (and IL-26, as well as other IL-10 family members not yet fully characterized), diverse immune cells can be involved in mediating protection via this pathway. The IL-23-induced rapid secretion of IL-17 and IL-22 from hematopoietic cells, such as iNK T cells and gd T cells in primary infections or effector-memory T cells upon rechallenge, suggest they are part of the first line of defense against invasion. As we have seen, although recruitment of this pathway for host protection is most evident for certain extracellular bacteria and fungi, new studies implicate protective roles for other pathogens, including some protozoa and viruses. Given the relatively recent appreciation of the Th17 pathway and the roles for several of its associated cytokines in protective responses, the

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diversity of organisms that elicit and are controlled by this pathway is likely to broaden. In every conflict there is the risk of friendly fire. A prime example of this type of collateral damage is Lyme arthritis initiated by the IL-23/ IL-17-mediated response to Borellia infection (Burchill et al., 2003). Similarly, IL-23 has also been associated with a demyelinating autoimmune disease resembling multiple sclerosis in genetically susceptible mice infected with Theiler’s murine encephalomyelitis virus (Petro, 2005). Chronic intestinal inflammation resulting from dysregulated Th17 responses to the intestinal microbiota is involved in the pathogenesis of inflammatory bowel diseases (Elson et al., 2007; Yen et al., 2006). And the unregulated IL-23/IL-17-mediated response to an infectious organism itself can also contribute to impaired clearance and chronic inflammation in some cases, as exemplified by mucosal challenge with C. albicans in IL-12p35-deficient mice (Zelante et al., 2007). Clearly, then, host defense via the IL-23/IL-17/IL-22 pathway is an evolutionary adaptation to many forms of pathogens, but route of entry, morphologic form, and the genetic and immune status of the host dramatically affect the outcome of its induction. Continued advances in our understanding of the organisms whose clearance is facilitated by this immune pathway and specific mechanisms by which this can be achieved without untoward effects on the host should provide new opportunities for improved vaccine design while informing novel approaches to the prevention and treatment of chronic inflammatory diseases.

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CHAPTER

6 Peptides Presented In Vivo by HLA-DR in Thyroid Autoimmunity Laia Muixı´, In˜aki Alvarez, and Dolores Jaraquemada

Contents

. List of Abbreviations 1. Introduction 1.1. Endocrine autoimmunity 1.2. Autoimmune thyroid disease as a model of tissue-restricted autoimmune responses 1.3. HLA class II and AITD 1.4. Tolerance to thyroid antigens 1.5. The role of antigenic peptides in the autoimmune responses in situ 2. Natural Peptides: The Constitutive Ligands of MHC Molecules 2.1. Natural ligands of class II MHC molecules: Binding properties and promiscuity 2.2. Some technical considerations: The analysis of MHC-associated peptides from tissue samples 3. Natural Ligands for HLA Class II in Autoimmune Thyroid Tissue 3.1. Are HLA class II natural ligands related to the function of their specific T cells? 3.2. The case of Tg 3.3. TPO and TSHR peptides 4. Concluding Remarks Acknowledgments References

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Immunology Unit, Institut de Biotecnologia i Biomedicina (IBB), and Department of Cell Biology, Physiology and Immunology, Universitat Auto`noma de Barcelona (UAB), Campus de Bellaterra, 08193 Barcelona, Spain Advances in Immunology, Volume 99 ISSN 0065-2776, DOI: 10.1016/S0065-2776(08)00606-8

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2008 Elsevier Inc. All rights reserved.

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The association of the major histocompatibility complex (MHC) genes with autoimmune diseases together with the ectopic expression of class II molecules by epithelial cells of the target tissue gives to these molecules a central role in the pathogenesis of the disease, in its regulation and in the persistence of the immune response in situ. HLA-DR molecules expressed by thyroid follicular cells in thyroid autoimmune diseases are compact molecules stably associated with peptides. The nature of these peptides is of vital importance in the understanding of the disease, since these MHC-II-peptide complexes are going to be recognized by both effector and regulatory T cells in situ. In this chapter, we review the current state of the analysis of naturally processed peptides presented by MHC class II molecules in the context of autoimmunity and we discuss our data of natural HLA-DR ligands eluted from Graves’ disease affected thyroid glands, from where autoantigen-derived peptides have been identified. Key Words: Human, thyroid autoimmunity, peptides, antigen presentation, MHC. ß 2008 Elsevier Inc.

LIST OF ABBREVIATIONS T1D, type 1 diabetes; AITD, autoimmune thyroid diseases; GD, Graves’ disease; HT, Hashimoto’s thyroiditis; DCs, dendritic cells; Tg, thyroglobulin; TPO, thyroid peroxidase; TSHR, thyroid stimulating hormone receptor; TFC, thyroid follicular cells; MHC, major histocompatibility complex; SNP, single nucleotide polymorphism; mTEC, medullary thymic epithelial cells; AIRE, autoimmune regulator; MS, mass spectrometry; B-LCL, B-lymphoblastoid cell line; APC, antigen-presenting cell; EAT, experimental autoimmune thyroiditis

1. INTRODUCTION 1.1. Endocrine autoimmunity Autoimmune endocrine diseases are mostly classical organ-specific T-cell-mediated diseases of multifactorial etiology and a strong genetic component. The autoimmune attack affects endocrine glands and is directed to molecules expressed by cells of the target organs. Together with type 1 diabetes (T1D), the most common autoimmune endocrine disorders are the autoimmune thyroid diseases (AITD), including Graves’

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disease (GD) and Hashimoto’s thyroiditis (HT). The pathogenesis of these diseases involves an effector (humoral or cellular) response dependent on self-antigen-specific T cells, which results in the dysfunction of the target organ (Roep, 2003; Weetman, 2004). Because of the constant presence of the target autoantigens in the affected tissue, the in situ immune response produces local chronic inflammation leading in the long term to tissue destruction and loss of function, causing the clinical symptoms of the disease. The tissues affected by these chronic immune responses against self-antigens are infiltrated by cellular components of the immune system, mainly T cells and macrophages, but also by dendritic cells (DCs), B cells, and plasma cells. In many cases, this infiltrate is anatomically and functionally organized as in secondary lymphoid organs, with the formation of T-cell areas as well as functional B-cell follicles, constituting the so-called tertiary lymphoid tissue (Aloisi and Pujol-Borrell, 2006; Armengol et al., 2001; Drayton et al., 2006; Salomonsson et al., 2003; Serafini et al., 2004). This slowly sustained autoimmune response has allowed sometimes the identification of effectors, although the diverse autoantigens, that is, the tissue-specific molecules that are specifically recognized by these effectors, are often unknown. Although T1D is by far the most important endocrine autoimmune disease for its prevalence in public health, the most relevant studies of its etiology have not been done in human autoimmune pancreas but in the NOD mouse model due to the limited access to tissue samples. The diverse forms of autoimmune thyroidopathies, together with Addison’s disease, are the most common organ-specific human autoimmune disorders. In contrast to TD1, the availability of human autoimmune thyroid tissue samples allows its direct analysis, and makes the AITD very interesting models for studying human autoimmunity.

1.2. Autoimmune thyroid disease as a model of tissue-restricted autoimmune responses Our studies have been directed at the AITD as models of endocrine autoimmunity. Several reasons make thyroid autoimmunity a good model to study human tissue-restricted autoimmune processes: diverse forms of in situ immune responses ranging from organ hyperfunction due to the effect of agonistic stimulating antibodies, to hypofunction due to several causes including blocking antibodies and cytotoxic T cells; the three main autoantigens in AITD, thyroglobulin (Tg), thyroid peroxidase (TPO), and the thyroid stimulating hormone receptor (TSHR), are well defined and biochemically and genetically characterized; both B and T cells specific for these three antigens have been isolated from thyroid infiltrates (Armengol et al., 2001; Dayan et al., 1991; Londei et al., 1985; Pichurin et al., 2004; Weetman et al., 1986); mouse experimental models for

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nearly all forms of AITD have been obtained (Kim-Saijo et al., 2003; Kong, 2007; Kotani et al., 1990; Nagayama, 2007; Nagayama et al., 2002; Shimojo et al., 1996), including models using HLA-DR transgenic mice (Kong et al., 2007); and finally thyroidectomy is relatively common therapy for some AITD such as GD so a large amount of tissue can be available for different studies from the strictly morphological to the clonal analysis of relevant T or B cells and their receptors. Finally, the thyroid is the most commonly affected organ in human autoimmunity. Therefore, even if the treatments for AITD are effective, nonaggressive, and relatively inexpensive, thyroid autoimmunity remains of public interest. Human GD is a disease mediated by agonistic stimulating autoantibodies to the TSHR. The affected tissue is characterized by a heterogeneous cellular infiltration consisting of mostly T cells but also macrophages, DCs, B cells, and plasma cells, and a local immune response to the above-mentioned thyroid autoantigens, while maintaining the organ structure. The thyroid in GD is considered a tertiary lymphoid tissue, with evident formation of T-cell zones and germinal centers (Aloisi and Pujol-Borrell, 2006; Armengol et al., 2001). Tissue resident DCs, macrophages, and B cells infiltrating the target organ express class II HLA molecules (HLA-II) and can process and present antigen to T cells. In a recent report, a constitutive role of islet resident DC to capture beta cell antigens and present them to T cells in the draining lymph node to maintain tolerance has been proposed for T1D (Calderon et al., 2008). A parallel role for thyroid resident DCs, in close contact with endocrine cells, could be proposed for maintaining the tolerance to thyroid antigens and thus should be studied. In addition, the main hormone secreting cells of the thyroid parenchyma, called thyroid follicular cells (TFC), are polarized epithelial cells with a very important endocytic activity at their apical end. From there, they endocytose Tg-rich colloid that is processed to generate thyroid hormones and released by exocytosis to the capillaries in the basal pole (Marino and McCluskey, 2000; Marino et al., 2000). This high capacity of endocytosis of extracellular material, together with their expression of HLA-II induced by the chronic inflammation settled in the tissue, makes TFC capable of presenting self-antigen to T cells (Hanafusa et al., 1983). The importance of nonprofessional antigen presenting cell (APCs) such as TFC in the context of the disease is unknown, but it is clear that the autoimmune response is maintained and modulated through the interaction between immune cells and cells of the target organ, with a strong influence of the local microenvironment. In this regard, it has been established that many TPO-specific T cells and most TPO-specific autoantibodies in human AITD develop in response to TPO presented by intact TFC, rather than by TPO released by damaged TFC and presented by resident DC (McLachlan and Rapoport, 2007). Data for other thyroid autoantigen are not so clear.

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1.3. HLA class II and AITD Although the exact etiology of the autoimmune thyroid disease remains unknown, there is solid evidence that genetic factors are a major influence in the development of AITD, and it is widely accepted that susceptibility genes, including major histocompatibility complex (MHC) and non-MHC genes, in conjunction with environmental factors, such as diet iodine, initiate an autoimmune response in the thyroid ( Jacobson and Tomer, 2007a; Tomer and Davies, 2003). Recently several AITD susceptibility genes have been identified including some polymorphisms of immunoregulatory genes such as CTLA-4 or CD40 ( Jacobson and Tomer, 2007b), and genes with tissue-restricted expression like Tg and the TSHR ( Jacobson and Tomer, 2007b). Although susceptibility to AITD is influenced by all these genes, MHC genes are by far the most important ( Jacobson et al., 2008). HLA-DR genes specifically play a major role in the etiology of GD. Although statistically the association of AITD with HLA class II alleles is less strong than that of other autoimmune diseases including T1D, most studies agree in that GD is associated with HLA-DR3 ( Jacobson et al., 2008; Tomer and Davies, 2003). The frequency of DR3 in GD patients is 40–55% compared to 15–30% in the general population, giving a relative risk for HLA-DR3 (HLA-DRB1*0301) positive individuals of 3 (Farid et al., 1980; Mangklabruks et al., 1991), and it is generally agreed that HLA-DR3 is the primary susceptibility gene for GD (Zamani et al., 2000). This concept has been confirmed by family studies (Heward et al., 1998) and reinforced by the studies on DR3-transgenic mouse models of experimental thyroiditis (Kong et al., 1996). In addition, HLA-DQA1*0501 has also been shown to be associated with GD in Caucasians (Barlow et al., 1996; Marga et al., 2001; Yanagawa et al., 1993). The exact mechanism of this association is not known, and there is a relative large number of patients who are HLA-DR3 negative, suggesting that HLA-DR is important, but one of many factors influencing the pathogenesis of GD. In a recent series of studies, it has been postulated that the presence of the amino acid Arg at position 74 (R74) of the HLA-DRb chain is the critical element for conferring susceptibility to GD, whereas other amino acids at that position (Ala or Gln) are protective (Ban et al., 2004; Simmonds et al., 2005). HLA-DR3 (DRB1*0301) is the only allele of the HLA-DRB1 locus that contains the R74 residue, with the exception of some uncommon alleles such as HLA-DRB1*0116, *0422, *1107, *1476, and *1525. Interestingly, a high number of alleles of the HLA-DRB3 gene, commonly associated with DR3 and other DR alleles, also have Arg at position 74. Specifically, all alleles of DRB3*01 except *0107 are positive for DRb-Arg74, in addition to DRB3*0204, *0219, *0222, and *0303 (sequence data from IMGT database, http://www.ebi.ac.uk/cgi-bin/

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imgt/hla/). Population studies of DRB haplotypes have shown that HLADR3 (i.e., DRB1*03 alleles) is mostly associated with DRB3*0101 (83%) versus only 16% of DRB3*0202 (Tang et al., 2002). Other DRB1 alleles that associate with DRB3*0101 are DRB1*1401 (100%), DRB1*1301 (63%), and DRB1*1201 (50%). In contrast, 97% of the DR11 haplotypes present association with DRB3*0202 and only 2% with DRB3*0101 (Tang et al., 2000). HLA-DR3-negative GD patients can therefore express other DR molecules with Arg at position 74 of the beta chain, suggesting that the presence of R74 may indeed be a key element to confer susceptibility to GD and at least explain in part the relatively weak association of GD with the HLA-DR3 allele. In addition, the existence of a synergistic effect of R74 together with homozygosis for a single nucleotide polymorphism (SNP) in exon 33 of the Tg gene suggests an interaction between these alleles in conferring susceptibility to GD. A possible mechanism can be that the presence of R74 would favor the direct interaction of pathogenic TSHR or Tg peptides with the DR binding groove or their exposition to the TCR, allowing their efficient recognition by specific T cells. Polymorphic changes on the Tg gene could also facilitate the generation of pathogenic epitopes if they induce sequence modifications that generate new processing sites (Hodge et al., 2006).

1.4. Tolerance to thyroid antigens Tolerance to self-antigens is established in the thymus (central tolerance) and induced and maintained in the periphery (peripheral tolerance). The deletion of T cells whose TCRs bind peptides derived from self-proteins with high affinity is efficiently achieved by the involvement in thymic selection of medullary thymic epithelial cells (mTECs) expressing the autoimmune regulator (AIRE) (Anderson et al., 2005; Derbinski et al., 2005). AIRE is a transcriptional mediator that plays an important role in the promiscuous expression of tissue-specific autoantigens in the thymus (Derbinski et al., 2001; Kyewski et al., 2002; Smith et al., 1997; Sospedra et al., 1998). To generate an appropriate level of tolerance to autoantigens, the mTECs of the thymus transcribe a partial internal representation of the genome, including genes codifying tissue-specific self-antigens, covering essentially all organs. This permits the accessibility to developing T cells of self-antigens with physically or temporally restricted expression (Kyewski and Derbinski, 2004). This expression is complex and very variable. Some antigens are expressed by all individuals, whereas others are not homogeneously expressed. Expression varies with time, may be restricted to some regions of the genes, and may generate a different array of epitopes in the thymus if comparing with the cells expressing that same

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genes in the periphery (Bruno et al., 2002; Klein et al., 2000; Pugliese et al., 1997). It has been demonstrated that the genes encoding thyroid-specific autoantigens (Tg, TPO, and TSHR) are expressed in human thymus (Sospedra et al., 1998; Spitzweg, Joba and Heufelder, 1999). More specifically, the Tg gene is expressed by most mammalian thymi, but at least in mouse, thymus Tg transcripts are truncated and maybe not all possible Tg epitopes are accounted for (Li and Carayanniotis, 2005). Besides, compared to Tg, the level of expression of TPO and TSHR transcripts is lower and there are interindividual variations (Sospedra et al., 1998). Therefore, at least theoretically, tolerance to Tg in the thymus should be achieved more efficiently both by the elimination of high reactive T cells and by the generation of peptide-specific regulatory T cells. Indeed higher levels of antigen in thymic medullary epithelium were observed in transgenic mice carrying hen egg lysozyme antigen controlled by the Tg promoter, compared to the H-2Kb promoter (Liston et al., 2004). The trigger of immune responses to Tg, TPO, or TSHR in AITD should then be different or require different thresholds if affected by the level of expression of these three genes in the thymus. Thus, the genetic component of AITD should include the differential expression of the specific autoantigens’ in the thymus, in addition to the effect of MHC and other genes. These variations could presumably be controlled by polymorphisms on the genes’ promoter regions, as for the insulin gene in T1D. So if the role of MHC molecules in AITD is the efficient presentation of self-antigens to effector or regulatory cells in the thyroid, at the same time as presenting peptides in the thymus to establish central tolerance, a modulation of this role in periphery by the degree of thymus expression of each of the autoantigens can be expected.

1.5. The role of antigenic peptides in the autoimmune responses in situ MHC class I and II molecules are expressed at the cell surface of antigenpresenting cells, associated by default with peptides derived from selfmolecules that, in case of infection, will be displaced by peptides from the pathogen’s antigens. Class II MHC (human HLA-II) molecules are transmembrane glycoproteins which mostly bind peptides derived from exogenous or cell membrane proteins to be presented to CD4þ T lymphocytes. The correct expression of stable peptide-HLA-II complexes at the surface depends mainly on two class II-pathway-specific chaperones, the invariant chain and HLA-DM, which permit the stabilization of the complex in the endocytic pathway by favoring the binding of ‘‘high stability’’ peptides (Arndt et al., 1997; Avva and Cresswell, 1994; Kropshofer et al., 1997; Vogt et al., 1997). Expression of HLA-II is normally restricted to

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professional APCs, that is, DCs, macrophages, B cells, and thymic epithelial cells (TECs), but it is induced in other cell types by inflammatory conditions as shown in vivo in epithelial cells from the target organs of autoimmune diseases including AITD (Bottazzo et al., 1983; Guardiola and Maffei, 1993; Hanafusa et al., 1983; Lucas-Martin et al., 1988). This expression of MHC molecules is related to the in situ maintenance of the autoreactive response, although the mechanisms involved are as yet unknown. The nature of the T-cell populations that recognize peptides in the context of MHC expressed by the epithelial cells or by the APC that share the space in the infiltrated tissue is unknown, as are many of the autoantigens recognized by the infiltrating T cells. Our group has demonstrated that the expression of HLA-II in GD TFC is accompanied by the expression of the two main chaperones of the class II pathway, invariant chain and HLA-DM. The level of HLA-DM expression is sufficient to allow correct peptide loading into the HLA-II molecules and hence the expression of stable HLA-II-peptide complexes at the cell surface (Catalfamo et al., 1999; Lucas-Martin et al., 1988). Therefore, we can assume that the HLA-II-expressing epithelial cells in human AITD are capable of presenting self-proteins to infiltrating CD4þ cells in situ. Thus, class IIþ TFC should play a role in the regulation of the sustained autoimmune response in the target tissue. As a consequence, the peptide repertoire displayed at the surface of HLA-II-expressing epithelial cells must be relevant to understand self-reactive T-cell responses in situ. This role should be complementary to that played by other HLA-IIexpressing cells in the infiltrated thyroid glands, namely, resident and infiltrating DCs, endothelial cells, activated T cells, B cells, and macrophages (Catalfamo et al., 1999). To identify the target antigens and the different functions played by the multiple T-cell populations infiltrating the tissue, we and other have studied cloned T cells from AITD thyroid infiltrating lymphocytes, including CD4þ cells, but also CD8þ, gamma delta T cells, NKT cells, and a large number of regulatory cells. The data show that most T cells infiltrating the organ-expressed TCRs capable of recognizing autologous thyroid epithelial cells but in most cases with a very low affinity, impairing the characterization of their function as well as of their specificity (Catalfamo et al., 1996; Dayan et al., 1991; Londei et al., 1985; Roura-Mir et al., 1993, 1997, 2005). The difficulty in characterizing T-cell specificity in the thyroid infiltrates, together with the expression of stable peptideassociated HLA-DR molecules by the TFC, led us to search for the antigenic specificities of the in situ responses by identifying which were the peptides that naturally associated with the class II molecules in the tissue. This approach, called by some ‘‘reverse immunology’’, has been used in other systems, both for MHC class I and class II molecules (Dengjel et al., 2006; Paradela et al., 1998; Viatte et al., 2006).

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2. NATURAL PEPTIDES: THE CONSTITUTIVE LIGANDS OF MHC MOLECULES 2.1. Natural ligands of class II MHC molecules: Binding properties and promiscuity The identification of peptide epitopes that naturally associate to MHC class II molecules is an important objective in immunological research. Studies characterizing HLA-DR-associated peptides have mostly been done using B-lymphoblastoid cell lines (B-LCLs) as the source of DRpeptide complexes. These studies have led to the identification of the allele-specific structural motifs common to peptides that associate to most DR and some DQ alleles (Chicz et al., 1993; Rammensee et al., 1995; Singh and Raghava, 2001), as well as to the generation of large databases of natural ligands for MHC class II molecules (Rammensee et al., 1999; Sathiamurthy et al., 2003; Singh and Raghava, 2001). On the basis of these data, predictive computational tools have been developed as alternative approaches, although there is still a large number of inaccuracies in these predictions (Gowthaman and Agrewala, 2008; Wang et al., 2008). Experiments to identify class II-associated peptides are expensive and time consuming, and the difficulty increases when trying to identify or predict natural DR ligands from nonlymphoid cell types, both for the identification of peptides and for epitope prediction. In these cells, HLA-II expression is more variable, access to samples is normally limited, and there may be variations in the processing mechanisms leading to the generation of class II ligands. This is more so when dealing with highly differentiated hormone-secreting epithelial cells, such as TFC or islet-b cells. Nevertheless, the real diversity of antigens targeted by T cells in any autoimmune tissue can only be inferred from the peptide repertoireassociated in situ to class II molecules. In a previous work, we analyzed the peptide repertoire associated with HLA-DR4 molecules expressed by a transfected insulinoma cell line. We detected a heterogeneous pool of self-peptides derived from cell surface proteins and proteins from internal cell compartments, including the cytosol and the secretory pathway, some of which corresponded to tissue-specific proteins (Muntasell et al., 2002, 2004). These data showed that the repertoire of peptides associated with DR4 in epithelial cells was different from that of DR4-associated peptides from lymphoblastoid cells, while keeping the right structural DR4-binding motif. The data showed about 50% coincidence in the distribution of proteins generating class II ligands, mostly from the cell membrane. The differences revealed that many of the peptides associated with DR4 in epithelial cells derived from proteins of the cytosol and secretory vesicles, whereas the B cells showed a larger number of peptides derived from proteins resident in endocytic vesicles (Muntasell et al., 2002).

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However, this material gave only an indirect assessment on the repertoire of peptide ligands occupying the groove of HLA-II molecules in real-life autoimmune tissue. Nevertheless, a recent study has demonstrated the reliability of this strategy by using a more differentiated mouse insulinoma cell line expressing the diabetogenic I-Ag7 molecules (Suri et al., 2008). They identified naturally processed peptides displayed by I-Ag7, some of which derived from secretory granules and other proteins expressed by normal b cells. But the access to differentiated endocrine cell lines is very limited and cultured cells lack the local environment of a whole tissue sample. Therefore, studies from ex vivo tissue samples are still necessary to evaluate the real repertoire of DR-associated natural peptides from autoimmune tissue that can be recognized by T-cells in situ. Little has been done with human tissue, where sample heterozygosis adds to the other major difficulties. The first published work on HLA-DRpeptide purification from human tissue in 1995, identified 17 peptides from 50 g of the spleen of a patient with rheumatoid arthritis (Gordon et al., 1995). Later, 55 peptides were sequenced from 9 human intestine samples from patients with inflammatory bowel disease (Oshitani et al., 2003). More recently, a large number of HLA class II-associated peptides were identified from primary renal carcinoma samples (Dengjel et al., 2006). Finally, a recently published study described sequences of HLADR3-bound peptides from bronchoalveolar lavage cells of sarcoidosis patients that allowed the identification of antigens targeted by the autoimmune response (Wahlstrom et al., 2007). Even by comparing this limited set of studies, one can realize the influence of the specific characteristics of each tissue in the repertoire of peptides displayed by class II molecules. Whereas peptides from the spleen were derived from proteins of very diverse origin, that is, serum albumin, human erythroid protein, 60S ribosomal proteins L31 and L35, VCAM-1, human immunoglobulin lambda chain, or cathepsin-S, the peptides from intestinal samples were nearly all (51/55) from exogenous proteins, including antigens from Escherichia coli, Saccharomyces cerevisiae, or Caenorhabditis elegans. In contrast, the repertoire of peptides isolated from human tumoral tissue was more similar to that of epithelial-transfected cells (Muntasell et al., 2002), where in addition to human serum proteins and membrane components, many of the source proteins that generated class II ligands were from nuclear or cytosolic origin (Dengjel et al., 2006). Peptides bound to the groove of MHC class II molecules have a binding core of 8–9-amino acid residues as class I peptides, but have protruding ends, extended outside the groove by a variable number of residues. Since the groove is open at both ends, the flanking sequences are exposed and can influence MHC binding and stability or T-cell recognition (Dai et al., 2008; Lovitch et al., 2006; Sant’Angelo et al., 2002). Class II MHC-associated peptides are often selected in ‘‘nested sets’’ sharing a common 9-mer core (residues P1-P9), with varying lengths of flanking

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residues at both ends (Muntasell et al., 2002; Suri et al., 2006). Another property of class II peptides compared to class I is their degree of promiscuity, that is, the capacity of many peptides to be good binders to more than a single allele of HLA-II (Chicz et al., 1993; Hammer et al., 1993). During antigen processing, class II ligands are generated and displayed on the surface of APCs to direct immune responses to foreign antigens and to guide self-tolerance. The production of the MHC class II ligands occurs in endolysosomal compartments and thus involves extracellular proteins that travel to this location from the outside of the cell by endocytosis or proteins derived from internal cell components that reach the endocytic pathway via vesicle fusion or by targeted protein degradation (Castellino et al., 1997; Cresswell, 1994; Germain, 1994; Watts, 1997). Exogenous and cytoplasmic antigens are processed by distinct routes within APCs. Exogenous antigens including membrane self-proteins are internalized, processed, and bound to class II molecules within endolysosomal compartments. Proteins from different intracellular vesicles or granules are often degraded by fusion of these organelles with the same endocytic compartments. In contrast, cytoplasmic or nuclear autoantigens can be partially degraded in the cytosol and the resulting peptides translocated to endolysosomal compartments for further trimming and intersection with class II molecules (Lich et al., 2000). A consideration to take into account when comparing processing by different cell types concerns the specific proteases involved in the degradation pathways. It is well known that APC have very efficient processing mechanisms that may be different from those of cells whose cellular machinery is dedicated to hormone synthesis (Haque et al., 2007; Muntasell et al., 2002; Suri et al., 2008). Highly specialized cells like the TFC have very high endocytic activity at their apical membrane and express several proteases involved in thyroid function, including cathepsin L, a protease expressed by TECs but not by professional APCs (Nakagawa et al., 1998). Multiple pathways are thus involved in the association of putative ligands to the nascent class II molecules and therefore different peptides generated from the same proteins in different cell types are expected (Li et al., 2005). Moreover the binding properties of the different alleles of MHC class II can be variable, selecting peptides with very different allele specific structural characteristics although some very different alleles can end up selecting similar peptides, as described for the peptides binding to the human and mouse main diabetogenic alleles, HLA-DQ8 and H-2 I-Ag7 (Suri et al., 2005).

2.2. Some technical considerations: The analysis of MHC-associated peptides from tissue samples Studies of the peptide repertoires associated with MHC molecules started in the early 90s. With them, the structural features of class I and class II natural ligands could be defined, and their parental proteins identified.

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The first experiments were done by immunoprecipitation of class I MHC molecules, acid elution of peptides and Edman sequencing of the nonfractionated peptide pool. They demonstrated that some peptide positions were conserved (Falk et al., 1991), suggesting a structural ‘‘binding motif’’ for each MHC allele, defined by the ‘‘anchor residues’’ of the peptides bound to it. As mentioned above, peptides bound to class II MHC molecules differ from the class I ligands in their length and in the particularity of having extending ends outside the binding site, which made sequencing by Edman degradation not as efficient as for class I ligands, since class II anchor residues could not easily be defined using pool sequencing. However, Edman degradation was used for the identification of very abundant class II ligands (Rudensky et al., 1991). But important advances in the study of natural ligands of MHC molecules came from the incorporation of mass spectrometry (MS), facilitating the identification of individual sequences that conformed the peptide repertoire (Huczko et al., 1993; Hunt et al., 1992a; Kubo et al., 1994). MS made possible the identification of many individual class II natural ligands and the description of the structural features of the peptide repertoires associated with many different class II alleles. A characteristic feature of class II peptides was the common finding that many peptides conforming these repertoires were grouped in nested sets, with a common binding core that interacted with the binding site and different length extensions of the same protein sequence at both ends of the core (Bluestone et al., 1982; Chicz et al., 1992, 1993; Hunt et al., 1992b; Lippolis et al., 2002). The classical source of HLA molecules and the corresponding peptides has been B-LCLs expressing known HLA alleles. There is a large collection of HLA homozygous B-LCL available from the International HLA Workshop Repository (http://www.ihwg.org/cellbank/). B-LCLs grow very efficiently in culture and express high levels of cell surface HLA molecules. Initially, the low sensitivity of the Edman sequencing technique and of the mass spectrometers available required the use of high cell numbers (3 1010 cells were normally used) (Verreck et al., 1996). Improvement of MS devices has increased their accuracy and sensitivity. Thus, the new generation of mass spectrometers has made possible to obtain good enough spectra from much lower amounts of peptides. Our laboratory has recently described the peptide repertoire of HLA-DR10 (DRB1*1001) by sequencing 238 natural ligands of this allele from a total of 2 109 cells (Alvarez et al., 2008). Currently larger numbers of sequences can be obtained from even lower amounts of cells (Lippolis et al., 2002; Muixı´ L., unpublished). This enhanced sensitivity allow to obtain larger number of spectra from decreasing sample sizes but gives rise to a new problem, that is, the analysis of such amount of data. Thus, the advances in the

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development of new and more accurate search engines have been an important complement to the improvement of MS devices. These allow the analysis of a very high number of spectra while decreasing the time that the researcher must spend to obtain the sequences. However, it is still necessary the confirmation of individual sequences to avoid false assignations. Since class I molecules are expressed by practically all nucleated cells, class I peptides have been easily isolated also from ex vivo material in different studies (Schirle et al., 2000; Weinschenk et al., 2002). The isolation of natural ligands of class II molecules from these samples is by far more complex because HLA-II is only expressed in normal conditions in professional APCs. However, recently some reports have described successful attempts to purify and sequence natural ligands associated with class II molecules from different ex vivo sources (Dengjel et al., 2006; Wahlstrom et al., 2007). It is then possible after the recent technical advances in MS and in the sequence identification programs, to yield information of the class II-associated peptide repertoires from primary tissues.

3. NATURAL LIGANDS FOR HLA CLASS II IN AUTOIMMUNE THYROID TISSUE 3.1. Are HLA class II natural ligands related to the function of their specific T cells? The identification of the autologous peptides presented by class II MHC molecules in the affected tissue is important for understanding autoimmune diseases: it gives a bone fide account on the antigens that T-cell types present in the infiltrates may be recognizing and it is a step to the understanding of autoreactive T-cell selection. Whatever the mechanism triggering GD, the aberrant expression of HLA class II molecules on TFC may enhance thyroid autoimmunity via direct thyroid autoantigen presentation. But this expression may also be involved in the regulation of the response by presenting autoantigens to one or more populations of regulatory cells. In view of the heterogeneity of the lymphocytic infiltration of the tissue, containing both effectors and regulatory T cells, it is likely that depending on the peptide presented, both functions may be played by the TFC. In a recent report, we have characterized a number of natural peptides bound to HLA-II molecules from thyroid glands of GD patients, using MS techniques (Muixı´ et al., 2008). Because we analyzed whole tissue samples from several glands without applying cell separation techniques (Fig. 6.1), the repertoire of peptides corresponded to all the HLA-DR molecules in the tissue. The data demonstrated the heterogeneity of the class II-bound

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Frozen thyroid samples

Mechanic homogenization

2 h lysis with 0.5% NP40

HLA-DR immunoaffinity purification (mAb L243, B8.11.2) Acid elution H2O 0.1%TFA

Fractionation HPLC-UV

mAU

Ultrafiltration 10 KDa cutoff

Min Molecular mass determination MALDI-TOF

Mass (m/z)

Peptide sequencing with nanoESI, MALDI-TOF/TOF

FIGURE 6.1 Protocol for the analysis of DR-associated peptides from thyroid samples of GD patients.

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peptides, the generation in the tissue of nested sets of peptides as well as the overwhelming dominance of peptides deriving from ubiquitous self-proteins. We also demonstrated that tissue-specific peptides are generated for their presentation by HLA-DR molecules. A more refined analysis will be required to determine the putative APCs presenting each peptide, but the use of whole tissue samples has advantages, since it has provided complementary information on the tissue milieu. In addition, separation of TFC from the digested tissue requires a minimum of 24 h of culture (Catalfamo et al., 1999; Roura-Mir et al., 1997; Sospedra et al., 1995) that induces downregulation of class II expression, protein degradation, and maybe the modification of the peptide repertoire. Moreover, the yield of thyroid cells obtained from these cultures is low making the use of single-cell-type samples not feasible. In this work, we were able to identify more than 150 peptides that allowed the partial analysis of the repertoire of peptides associated with DR in autoimmune thyroids. All peptides complied with the known common features of HLA-DR natural ligands: they were variable in length, formed ‘‘nested’’ sets with a common core and varying flanking sequences, and were derived from proteins present in different compartments, including the extracellular milieu, the plasma membrane, vesicular compartments, and the cytosol. Most peptides came from proteins that would be degraded in the endocytic pathway (Table 6.1). Some were extracellular peptides derived from serum soluble proteins such as complement factors, apolipoprotein, hemoglobin, soluble factors like macrophage migration inhibition factor (MIF), or alpha-1-antitripsin. Membrane proteins were the next most abundant peptide generators, most from class II MHC molecules or from different cadherins. The next set of ‘‘endocytic’’ peptides corresponded to extracellular matrix proteins, where collagen peptides dominated. Cathepsins were the only endocytic vesicle residents from where peptides were identified. Within the extracellular peptides, a separate section corresponded to colloid components. Tg and albumin are the most abundant components of the colloid (Baggio et al., 1996) and of the whole thyroid tissue. Although albumin is also a serum protein, the colloid stores large amounts of it, and it seems likely that it would be mostly captured from there. Incidentally, many of the serum proteins generating peptides, such as hemoglobin and complement components, can also be found in the colloid and hemoglobin has been reported to be increased in hyperplasic thyroid tissue (Ribeiro-Carvalho et al., 2002). Most interestingly, we identified eight different peptides corresponding to the well-defined thyroid autoantigen Tg. Two of them where obtained each from TB448 and TB449, and the remaining six were all from the TB471 sample. We will discuss Tg peptides in the next section. Although we do not know if any of these

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TABLE 6.1 Peptides generated in the endocytic pathway Mr (Da)

Sequence

Size (aa)

1770.0 2014.2 1678.9 1765.8 1636.8 1395.6 1690.9

DIPELVNMGQWKIRA YPKSLHMYANRLLDHR REIFNMARQRSR EGLTFQMKKNAEELK GLTFQMKKNAEELK GLTFQMKKNAEE GNRIAQWQSFQLEGG

15 16 12 15 14 12 15

1633.9 GNRIAQWQSFQLEG

14

1171.6 1683.9 3196.9 3327.4 3344.8 3473.6 2744.4 2174.0 3095.5 1528.2 2755.4 3262.7

11 16 31 32 32 33 32 21 40 15 24 29

VLSPADKTNVK VLSPADKTNVKAAWGK vlspadktnvkaawgkvgahageygaealer vlspadktnvkaawgkvgahageygaealerm vlspadktnvkaawgkvgahageygaealerm vlspadktnvkaawgkvgahageygaealermf ktnvkaawgkvgahageygaealerm AAWGKVGAHAGEYGAEALERM aawgkvgahageygaealermsfpttk VGAHAGEYGAEALER FLSFPTTKTYFPHFDLSHGSAQVK FLSFPTTKTYFPHFDLSHGSAQVKGHGKK

Source protein

Id no.

L.

Sample

Complement C3 (194–208) Apolipoprotein B (1200–1215) Apolipoprotein B (427–439) A-IV Apolipoprotein (239–253) A-IV Apolipoprotein (240–253) A-IV Apolipoprotein (240–251) Alpha-2-macroglobulin (172–186) Alpha-2-macroglobulin (172–185) Hemoglobin a chain (1–11) Hemoglobin a chain (1–16) Hemoglobin a chain (1–31) Hemoglobin a chain (1–32) Hemoglobin a chain (1–32) Hemoglobin a chain (1–33) Hemoglobin a chain (7–32) Hemoglobin a chain (12–32) Hemoglobin a chain (12–40) Hemoglobin a chain (17–31) Hemoglobin a chain (33–56) Hemoglobin a chain (33–61)

P01024 P04114 P04114 P06727 P06727 P06727 P01023

E E E E E E E

TB448 TB448 TB448 TB471 TB471 TB471 TB449

P01023

E

TB449

P69905 P01922 P01922 P01922 P01922 P01922 P01922 P01922 P01922 P01922 P01922 P01922

E E E E E E E E E E E E

TB449 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471

3134.6 1833.7 2341.2 2996.5 1087.5 3427.9 2792.5 1700.0 2301.3 1815.0 1571.6 1311.6 952.4 3274.7 3387.8 1308.6 1448.7 2582.2 2139.1 1910.9 1274.6 1308.2 1421.8 2305.6

FLSFPTTKTYFPHFDLSHGSAQVKGHGK TYFPHFDLSHGSAQVK TYFPHFDLSHGSAQVKGHGKK VADALTNAVAHVDNMPNALSALSDLHAHK LRVDPVNFK AAHLPAEFTPAVHASLDKFLASVSTVLTSKYR AAHLPAEFTPAVHASLDKFLASVSTVL KFLASVSTVLTSKYR LVTLAAHLPAEFTPAVHASLDK DKFLASVSTVLTSKYR FLASVSTVLTSKYR ASVSTVLTSKYR VHLTPEEK VHLTPEEKSAVTALWGKVNVDEVGGEALGRL VHLTPEEKSAVTALWGKVNVDEVGGEALGRLL LVVYPWTQRF VVAGVANALAHKYH VGAHAGEYGAEALERMFLSFPTTK FESFGDLSTPDAVMGNPKVK FESFGDLSTPDAVMGNPK LLVVYPWTQR LVVYPWTQRF LLVVYPWTQRF PmFIVNTNVPRASVPDGFLSE

28 16 29 29 9 32 27 32 22 16 14 12 8 31 32 10 14 24 20 18 10 10 11 21

Hemoglobin a chain (33–60) Hemoglobin a chain (41–56) Hemoglobin a chain (41–61) Hemoglobin a chain (62–90) Hemoglobin a chain (91–99) Hemoglobin a chain (110–141) Hemoglobin a chain (110–136) Hemoglobin a chain (127–141) Hemoglobin a chain (106–127) Hemoglobin a chain (126–141) Hemoglobin a chain (128–141) Hemoglobin a chain (130–141) Hemoglobin b chain (1–8) Hemoglobin b chain (1–31) Hemoglobin b chain (1–32) Hemoglobin b chain (32–41) Hemoglobin b chain (133–146) Hemoglobin b chain (17–40) Hemoglobin b chain (42–61) Hemoglobin b chain (42–59) Hemoglobin d chain (31–40) Hemoglobin d chain (32–41) Hemoglobin d chain (31–41) Macrophage MIF (2–22)

P01922 P01922 P01922 P01922 P01922 P01922 P01922 P01922 P69905 P69905 P69905 P69905 P02023 P02023 P02023 P02042 P02042 P01922 P68871 P68871 P02042 P02042 P02042 P14174

E E E E E E E E E E E E E E E E E E E E E E E E

TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB449 TB449 TB449 TB449 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB471 TB449 TB449 TB449 TB449

181

(continued)

182

TABLE 6.1

(continued)

Mr (Da)

Sequence

Size (aa)

2089.6 1914.4 2384.6 2245.3 1886.9 1829.9 1613.9 1374.6 1501.8 1737.6 1906.0

PmFIVNTNVPRASVPDGFL IVNTNVPRASVPDGFLSE IVNTNVPRASVPDGFLSELTQQ LAQATGKPPQYIAVHVVPDQL GTQGKIVDLVKELDRDT TQGKIVDLVKELDRDT TQGKIVDLVKELDR KAVLTIDEKGTEA KAVLTIDEKGTEAAG VHKAVLTIDEKGTEAAG AHLLILRDTKTYMLAF

19 18 22 21 17 16 14 13 15 17 16

1758.9 AHLLILRDTKTYMLA

15

1631.9 1296.7 1774.1 1629.8 1676.8 1567.0

16 10 15 15 15 15

SWNSGALTSGVHTFPA DRVYIHPFHL KVKKIYLDEKRLLAG DVGVYRAVTPQGRPD DVGEYRAVTELGRPD TNVPPEVTVLTNSPV

Source protein

Id no.

L.

Sample

Macrophage MIF (2–20) Macrophage MIF (5–22) Macrophage MIF (5–26) Macrophage MIF (27–47) Alpha-1 antitripsin (188–204) Alpha-1 antitripsin (189–204) Alpha-1 antitripsin (189–202) Alpha-1 antitripsin (359–371) Alpha-1 antitripsin (359–373) Alpha-1 antitripsin (357–373) Alpha-1 acid glycoprotein (117–132) Alpha-1 acid glycoprotein (117–131) Ig gamma-1 C chain (40–55) Angiotensin (34–43) SPARC-like 1 protein(556–570) DQB1*0602 b chain (75–89) HLA-II b chain DRB1*4 (72–86) HLA class II, DR a chain (108–122)

P14174 P14174 P14174 P14174 P01009 P01009 P01009 P01009 P01009 P01009 P02763

E E E E E E E E E E E

TB449 TB449 TB449 TB449 TB471 TB471 TB471 TB471 TB471 TB471 TB471

P02763

E

TB471

P08157 ANHU Q14515 P03992 P13760 P01903

E E E M M M

TB449 TB449 TB471 TB448 TB448 TB449

1310.8 WRLEEFGRFA

10

1409.8 VWRLEEFGRFA

11

1744.0 WLRNGKPVTTGVSETV

16

1845.1 TWLRNGKPVTTGVSETV

17

2846.4 AVTELGRPDAEYWNSQKDLLEQKR

24

2690.5 AVTELGRPDAEYWNSQKDILEQK

23

1489.0 EDIVADHVASYGVN

14

1937.1 EQFYVDLDKKETVWH 1397.7 RFDSDVGEFRAV

15 12

1257.6 FDSDVGEYRAV

11

1626.9 KEYFAIDNSGRIIT 1684.0 GKEYFAIDNSGRIIT

14 15

1609.8 SVPRYLPRPANPDE

14

HLA classe II, DR a chain (68–76) HLA classe II, DR a chain (67–76) HLA class II DR a chain (146–161) HLA class II, DR a chain (145–161) HLA class II, DRB1–4 b chain (78–101) HLA class II, DRB1–4 b chain (78–100) HLA class II, DQ(2,6,3) a chain (24–37) MHC class II (61–75) HLA class II, DR, DP DQ cadena b (68–79) HLA class II, DRB3–1 cadena b (69–79) Cadherin-5 (197–210) Cadherin-5 (196–210) Epithelial Cadherin (E-Cadherin) (793–806)

P01903

M

TB449

P01903

M

TB449

P01903

M

TB449

P01903

M

TB449

P13760

M

TB471

P13760

M

TB471

P04226

M

TB449

Q95HB9 P01911

M M

TB449 TB449

P79483

M

TB449

P33151 P33151

M M

P12830

M

TB449 TB449, TB471 TB471

183

(continued)

184

TABLE 6.1 Mr (Da)

(continued)

Sequence

Size (aa)

1728.1 QLQPDNSTLTWVKPT 1594.0 AAGLLSTYRAFLSSH

15 15

1522.8 AGLLSTYRAFLSSH

14

1915.1 DPSPSPVLGYKIVYKPVG

18

1661.0 RLPIIDVAPLDVGAPD

16

1863.0 RPKDYEVDATLKSLNN

16

1530.4 GTLIIRDVKESDQG

14

1795.0 YGGELRFTVTQRSQPG

16

1633.0 GGELRFTVTQRSQPG

15

1720.0 IRASYAQQPAESRVSG

16

1895.1 DVPKWISIMTERSVPH 1857.0 IGSSYFPEHGYFRAPE

16 16

Source protein

Id no.

L.

Sample

Phospholipase C (883–897) Collagen a1 chain (XV) (1243–1257) Collagen a1 chain (XV) (1244–1257) Collagen a1 chain (XII) (2137–2154) Collagen a1 chain (I) C-term propeptide (1436–1451) Collagen a2 chain (I) C-term propeptide (1129–1144) Heparan sulfate proteoglycan (462–475) Heparan sulfate proteoglycan (1005–1020) Heparan sulfate proteoglycan (1006–1020) Heparan sulfate proteoglycan (1089–1104) Annexin II (208–223) Multimerin 2 (851–866)

Q9HBX6 M TB449 P39059 ECM TB448, TB449 P39059 ECM TB448, TB449 Q5VYK1 ECM TB471 P02452

ECM TB448

P08123

ECM TB448

P98160

ECM TB449

P98160

ECM TB448

P98160

ECM TB448

P98160

ECM TB448

P07335 Q9H8L6

ECM TB448 ECM TB449

1442.8 SRAGLELGAEETI

14

1660.8 YDHNFVKAINAIQK 1827.0 NIFSFYLSRDPDAQPG 1687.0 LKKYLYEIARRHP 2030.0 1220.5 1670.0 1970.9 1274.4 1880.8 1477.7 1059.4

CPTPCQLQAEQAFLRTVQ SRTSGLLSSWK LSSVVVDPSIRHFDV QVDQFLGVPYAAPPLAER IGSSQDDGLINR AKAVKQFEESQGRTSSK AKAVKQFEESQGR LSLQEGSKT

Q14112

ECM TB449

14 16 13

Nidogen-2 (NID-2) (1167– 1180) Cathepsin C (170–183) Cathepsin D (227–241) Serum albumin (159–171)

P53634 P07339 P02768

V V Co

18 11 15 18 12 17 13 10

Tg (726–743) Tg (1080–1090) Tg (2098–2112) Tg (2224–2241) Tg (2508–2519) Tg (2520–2536) Tg (2520–2532) Tg (2756–2765)

P01266 P01266 P01266 P01266 P01266 P01266 P01266 P01266

Co Co Co Co Co Co Co Co

Tg, thyroglobulin; MIF, migration inhibition factor; MHC, major histocompatibility complex.

TB448 TB448 TB448, TB449 TB448 TB471 TB449 TB471 TB471 TB471 TB471 TB471

185

186

Laia Muixı´ et al.

peptides are associated with class II molecules expressed by the TFC rather than by other class II expressing cells, the common presence of colloid components together with the important endocytic activity at the apical membrane of the TFCs suggested that at least some of these peptides can indeed be associated with DR molecules expressed by TFC. An attractive possibility would be if tissue DC could also capture colloid antigens by the capability of ‘‘crossing’’ the thyroid epithelium with their dendritic extensions, as found for DC in the intestinal epithelium (Chieppa et al., 2006; Vallon-Eberhard et al., 2006). Otherwise the repertoire of ‘‘endocytic peptides’’ was as expected. With the exception of Tg, all other source proteins were nontissue-specific and peptides were similar to others from the same origin described in other works (Dengjel et al., 2006; Muntasell et al., 2002). All represented ubiquitous and abundant tissue components and many coincided with those isolated from HLA-DR molecules in endocrine epithelial cell transfectants (Muntasell et al., 2002). The peptides putatively generated in the cytosolic pathway were somehow different. Cytosolic peptides derived mostly from enzymes, although none of them was exclusive of thyroid tissue. These peptides were quantitatively less abundant and accordingly, fewer nested sets were found. However, some have been previously found in other studies, including various histone and GAPDH peptides (Table 6.2) (Dengjel et al., 2006; Muntasell et al., 2002). Since most of the samples were heterozygous and there was only partial coincidence in the HLA class II typing between them, one of the uncertainties was if the peptides could really be identified as binders to one or other of the DR molecules expressed by each sample. An important issue that arose from these results was therefore the assignment of a sequence to an allele when several are present, as expected from samples of human tissue. The difficulty is based on the designations of the core segment. The main anchor residues vary greatly and are influenced by various other residues in the anchor sites. Prediction algorithms for HLA binding can be used to provide an indication that peptides identified by MS techniques actually bind the HLA of interest, but a confirmation can only come from direct binding studies with purified MHC molecules. Furthermore, a final assignment of any peptide sequence to an HLA allele can never be completely certain unless T-cell data are generated; even binding data may not be enough when discussing about self-peptides, since their binding affinities are often low and peptide binding experiments with class II molecules are not very efficient. However, when we are dealing with a large number of peptides, none of the above is feasible. Therefore, bioinformatic analysis using prediction algorithms is a useful tool if at least some confirming binding data can be provided.

TABLE 6.2 Peptides generated in the cytosolic pathway Mr (Da)

Sequence

Size (aa)

Source protein

Id no.

L.

Sample

2031.2 1511.7 1637.6

AFTPEGERLIGDAAKNQLT AKFEELNMDLFR AKVAVLGASGGIGQPLSL

19 12 18

P11021 P11021 P40926

ER ER mit

TB449 TB471 TB449

1600.9

RGNSIIMLEALERV

14

Q15357

N

TB449

1919.3 1416.9 1175.7 1999.0

RLLLPGELAKHAVSEGTK RLLLPGELAKHAV FLENVIRDAV PAAPAAPAPAEKTPVKKKAR

18 13 10 20

P62807 P62807 P62805 P10412

N N N N

TB449 TB449 TB449 TB471

2044.0 2171.8 1331.7 1733.9 1004.6

S(Acet)ETAPAAPAAAPPAEKAPVKK S(Acet)ETAPAAPAAAPPAEKAPVKKK GAPAAATAPAPTAHK GELEKEEAQPIVTKY IKEESKTAV

21 22 15 15 9

P16403 P16403 Q92522 Q1KMD3 P49454

N N N N N

TB471 TB471 TB471 TB449 TB449

2953.2

sgnfggsrnmggpygggnygpggsggsggyggr

33

BIP (67–85) BIP Protein (325–336) Malate deshidrogenase (25–42) small G nuclear ribonucleoprotein (63–76) Histone H2B K (99–116) Histone H2B. (99–111) Histone H4 (61–70) Histone H1.4 (Histona H1b) (5–24) Histone H1.2 (1–21) Histone H1.2 (1–22) Histone H1 (129–143) hnRNP U-like 2 (611–625) Kynetocor Protein CENP-F (2346–2354) hnRNP A2/B1 (318–350)

P22626

N

TB471 (continued)

187

188

TABLE 6.2 (continued) Mr (Da)

Sequence

Size (aa)

Source protein

Id no.

L.

Sample

1613.8 1449.8

KPGQFIRSVDPDSPA EDFRDGLKLMLL

15 12

O14745 P12814

C C

TB448 TB448

1214.6 1739.8 1763.8 1650.2 1735.8 1362.7 1521.0 2043.2

TVDGPSGKLWR DGRGALQNIIPASTGAAK LISWYDNEFGYSNR ISWYDNEFGYSNR SWYDNEFGYSNRVV VVDLMAHMASKE DGVIKVFNDMKVR TGGASGLGLATAERLVGQGASAV

11 18 14 13 14 12 13 23

P04406 P04406 NP002037 NP002037 P04406 NP002037 P23528 Q99714

C C C C C C C C

TB471 TB471 TB449 TB449 TB449 TB449 TB449 TB449

1687.1

RVMTIAPGLFGTPLLT

16

Q99714

C

TB449

1362.4

mLLIPTSFSPLK

12

Q9UFN0

C

TB449

1730.9

FDPIIEDRHGGYKPS

15

P12277

C

TB449

1643.8

FDPIIEDRHGGYKP

14

P12277

C

TB449

1714.4

AVPSPPPASPRSQYNF

16

NHERF (174–188) a Actinin 1(57–68) or a actinin 4 (76–87) GAPDH (186–196) GAPDH (197–214) GAPDH (310–323) GAPDH (311–323) GAPDH (312–325) GAPDH (324–335) Cofilin-1 (8–20) 3-Hydroxyacyl-CoA dehydrogenase type II (16–38) 3-Hydroxyacyl-CoA dehydrogenase type II (192–207) NipSnap4 protein (236–247) B chain, creatine kinase (89–103) B chain, creatine kinase (B-CK) (89–102) Serin protease (134–149)

O43464

C

TB449

189

2013.4 1926.0

AVPSPPPASPRSQYNFIAD GRMHAPGKGLSQSALPYR

19 18

1956.9

QVTQPTVGMNFKTPRGPV

18

1059.5

IPDWFLNR

1324.5

LITPAVVSERLK

12

1807.0

IYTRNTKGGDAPAAGEDA

18

1470.7

YYKVDENGKISR

12

1688.6

ASGNYATVISHNPETK

16

1457.6

AGNLGGGVVTIERSK

15

1894.0

AYVRLAPDYDALDVANK

17

1504.9

RYSVDIPLDKTVV

13

1699.9

TGKNKWFFQKLRF

13

1709.9

TGAAPIIDVVRSGYYK

16

2167.3

LLTEAPLNPKANREKMTQI

19

8

Serin protease (134–152) Ribosomal protein 40S S13 (1–18) Ribosomal protein 40S S17 (107–134) Ribosomal protein 40S S18 (79–86) Ribosomal protein 40S S25 (67–78) Ribosomal protein S25 (108–125) Ribosomal protein 40S 27 (29–40) Ribosomal protein 60S L8 (128–143) Ribosomal protein 60S L22 (52–66) Ribosomal protein 60S L23a (136–152) Ribosomal protein 60S L27 Ribosomal protein 60S L27 (123–135) Ribosomal protein 60S L27a (94–109) Actin (106–124)

O43464 P62277

C C

TB449 TB471

P08708

C

TB471

P62269

C

TB471

P62851

C

TB471

P62851

C

TB449

P62979

C

TB471

P62917

C

TB471

P35268

C

TB471

P62750

C

TB471

P61353

C

TB449

P61353

C

TB471

P46776

C

TB471

P63261

C

TB449 (continued)

190

TABLE 6.2 (continued) Mr (Da)

Sequence

Size (aa)

Source protein

Id no.

L.

Sample

2094.0

SYELPDGQVITIGNERFR

18

P60709

C

TB471

1161.4

EITALAPSTMK

11

P60709

C

TB471

1543.8

DTNADKQLSFEEF

13

Actin (alpha, beta, gamma) (239–256) Actin (alpha, beta, gamma) (316–326) Calgranulin B (67–79)

P06702

C

TB449

Natural HLA-II Peptides in Autoimmunity

191

For assigning sequences to each allele, having a well-defined binding motif is needed to allow the search for the right anchor residues of the core. Some items as the presence or not of the relevant P1 residue are considered very definitive and are indeed so in our experience (Alvarez et al., 2008; Muntasell et al., 2004). In our samples, assigning sequences to alleles with strong binding motifs was relatively easy, for example, DR3, where in addition to the P1, a Glu or Asp residue is favored in position P4 of the core. Other motifs are less well defined, as for DR7, so the degree of confidence was lower. In some alleles, such as DR8 (DRB1*0801), there is not a defined motif. In addition, DR8 peptides tend to be longer than the usual DR-associated peptides, so no clear assignment could be done. For the final assignment, bioinformatic tools were used and several published analysis systems tested and the results were mostly coincident between them (Muixı´ L., unpublished data). For completeness, we finally chose one of these programs (ProPred, www.imtech.res.in/raghava/propred/) that scan all possible P1 to P9 residues in each peptide sequence for a large number of HLA-DR alleles (n ¼ 51). The binding affinity of the peptides once the allele is assigned is relevant, particularly when studying selfpeptides in an autoimmune tissue. The balance between high and low binders may be important for determining tolerance to a given epitope in the thymus, so maybe we should not expect many high binders to be good candidates as target epitopes. Lower binders, in contrast, may have to compete in the thymus for a given allele and one result may be the absence of tolerance to that epitope. A systematic approach comparing all the peptides is not feasible right now, but comparing binding affinities of some peptides with their capacity of activating or regulating T-cell responses would be very informative. Studies of the T-cell populations recognizing the peptides should be needed for such an analysis.

3.2. The case of Tg The isolation of Tg peptides in three samples of GD thyroids with different DR haplotypes can be relevant to the study of AITD and of autoimmunity in general. Table 6.3 shows the details of each of the Tg peptides identified. Except for two of the peptides (Tg2520–2532 and Tg2520– 2536), no nested sets were found. The alleles to which the peptides were assigned were first calculated by the ProPred program (www.imtech.res. in/raghava/propred/), and only two assignments were possible: Tg726– 743 from TB448 was apparently associated with DR51 (DRB5*0101 of the DR15 haplotype) and showed an intermediate score for DR15 and Tg2098– 2108 from TB449 showed association to DR3 with relatively high scores (Muixı´ et al., 2008). No allele included in the ProPred database appeared to associate to any of the Tg peptides from sample TB471 (DRB1*0301, B1*0801). The absence of theoretical binding do not necessarily exclude

192 TABLE 6.3

Thyroglobulin peptides isolated from human GD thyroid samples

Sample

HLA-DR

Mr (Da)

Sequence

Size (aa)

Protein

TB448

DRB1*1501 DRB1*0407 DRB1*0301 DRB1*1501 DRB1*0301 DRB1*0801

2031.00

CPTPCQLQAEQAFLRTVQ

18

Tg (726–743)

1669.99

LSSVVVDPSIRHFDV

15

Tg (2098–2108)

1477.69 1880.78 1970.94 1274.40 1220.52 1059.40

AKAVKQFEESQGR AKAVKQFEESQGRTSSK QVDQFLGVPYAAPPLAER IGSSQDDGLINR SRTSGLLSSWK LSLQEPGSKT

13 17 18 12 11 10

Tg (2520–2532) Tg (2520–2536) Tg (2224–2241) Tg (2508–2519) Tg (1080–1090) Tg (2756–2765)

TB449 TB471

Tg, thyroglobulin.

Natural HLA-II Peptides in Autoimmunity

193

DRB1*0301 REVEALTM binding assay 1428.7

1400 1200 975.7

1000 800 600 353.3

400

Tg2508−2519

0 M-tuberculosis Ab 85B 14−27

Tg2225−2241

0

75.1

E-cadherin 196−210

0

Tg2098−2112

0

Tg2756−2765

0

Tg1080−1090

16.4

Tg2224−2240

200

Tg2520−2532

% Relative to pass/fail control

1600

FIGURE 6.2 Cell-free binding assay. HLA-DR3 binding experiments were performed by ProimmuneÒ , using their cell-free class II REVEALTM binding assay (Muixı´ et al., 2008). Sequences of the different peptides were: Tg2520–2532 (AKAVKQFEESQGR); Tg2224– 2240 (QVDQFLGVPYAAPPLAE); Tg2225–2241 (VDQFLGVPYAAPPLAER); Tg2508–2519 (IGSSQDDGLINR); Tg1080–1090 (SRTSGLLSSWK); Tg2756–2765 (LSLQEPGSKT); Tg2098– 2112 (LSSVVVDPSIRHFDV) assigned to DR3 by ProPred; E-Cadherin 196-210 (GKEYFAIDNSGRIIT) assigned to DR3 by ProPred; DR3 positive control peptide, Mycobacterium tuberculosis Ab 85B 14–27 (PSPSMGRDIKVQFQ).

the binding of the peptide to that particular allele, it may bind weakly although the probability is low (see Fig. 6.2). Besides, they could either be binding to DR8 (DRB1*0801), or to the DRB3 allele associated with DR3 (DRB3*0101), for which there are not well-defined motifs.

3.2.1. Peptide binding analysis of all Tg peptides yielded some results In a first set of experiments, the 16 peptides from TB448 were synthesized and analyzed for binding to HLA class II negative bare lymphocyte syndrome (BLS) cells transfected with either HLA-DR15 (DRA1*0101/ DRB1*1501) or HLA-DR51 (DRA1*0101/DRB5*0101), of the DR2 haplotype (Norrby-Teglund et al., 2002) by a competition assay (Arndt et al., 2000). In brief, peptides to be tested were incubated with the cells at 37 C for 3 h in the presence of a control biotinilated peptide specific of each HLA-DR allele (MBP83–99 and MBP85–98 for DR15 and DR51, respectively). Cells were then lysed and HLA-DR-biotinilated peptide

194

Laia Muixı´ et al.

complexes were quantified by ELISA by time-resolved fluorescence, using a europium-streptavidin label. The binding capacity of each peptide was measured as the percent inhibition of control peptide binding. The data are summarized in Table 6.4. Most peptides could be assigned to one or the other allele (or both) and a few could only be theoretically assigned to the other DRB1 allele expressed by the donor (DRB1*0407), for which no transfectants were available. The results showed that Tg726– 743 had some moderate binding capacity to both DR15 and DR51. Of the other peptides, some showed considerable biding capacity to DR15, and some were positive for DR51, but with lower levels. Those that were negative or very low for both alleles, showed theoretical binding to DR407 (DRB1*0407). Only one peptide (collagen alpha 1436–1451) showed no binding. Modeling of the Tg726–743 peptide associated with D15 and DR51 separately suggested that the association was more stable with DR51 (Muixı´ et al., 2008). With Tg peptides from samples TB449 and TB471, a cell-free peptide binding assay could be done, only with DR3 and DR15 molecules, since we did not have any of the other alleles available in soluble form. The DR3 association of Tg2098–2108 was confirmed, showing a good but relatively weak binding affinity, as compared to the control peptide and to the binding to a nontissue-specific peptide from the same thyroid sample, that was also assigned to DR3, E-cadherin 196–210. None of the other Tg peptides showed positive binding to DR3 (Fig. 6.2). In summary, the Tg peptides capable of binding to one of the HLA-DR alleles tested, showed moderate binding affinity. Thyroglobulin is a very large autoantigen of 2749 aa that is extensively modified by iodination and other posttranslational events. Iodinated Tg peptides are highly immunogenic and can trigger thyroid autoreactive T cells. Iodine can modify the processing of Tg by APC, resulting in the generation of pathogenic epitopes (Carayanniotis, 2007; Dai et al., 2005; Jiang et al., 2007; Verginis et al., 2002). However, all the Tg peptides that were isolated from our samples were noniodinated epitopes. Although the pathological relevance of the Tg peptides that we have identified cannot be determined without in vivo studies, they were compared to Tg epitopes identified by other means in studies with both human and mouse AITD. Table 6.5 shows a summary of such epitopes. The most interesting peptide by far was shown to be the DR3-associated Tg2098–2112 peptide from TB449. Flynn et al. (2004) had used a computerbased predicted set of 39 synthetic DR3-binding Tg peptides that were screened against T cells from EATþDR3-transgenic mice. Only one out of four peptides capable of stimulating T-cell responses, LSSVVVDPSIRHFDV (identical to Tg2098–2112), could induce the disease both by direct peptide immunization and by adoptive transfer of peptide-activated T cells. Putting the data together, the in vivo generation

TABLE 6.4

Binding of TB448 peptides to DR15 and DR51 alleles

Sequence

Size

Protein

GGELRFTVTQRSQPG

15

DIPELVNMGQWKIRA

15

YGGELRFTVTQRSQPG

16

DVGVYRAVTPQGRPD

15

AAGLLSTYRAFLSSH

15

AGLLSTYRAFLSSH

14

CPTPCQLQAEQAFLRTVQ

18

YPKSLHMYANRLLDHR

16

IRASYAQQPAESRVSG

16

Heparan sulfate proteoglican (1006–1020) Complement C3 (194–208) Heparan sulfate Proteoglican (1005–1020) DQB1*0602 b chain (75–89) Collagen a1 (XV) (1243–1257) Collagen a 1 chain (XV) (1244–1257) Thyroglobulin (726–743) Apolipoprotein B (1200–1215) Heparan sulfate proteoglican (1089–1104)

% Inhib DRB1*1501 (DR2b)

Corea

% Inhib DRB5*0101 (DR2a)

Corea

Theoretical binding to DRB1*0407b

59

LRFTVTQRS

16

LRFTVTQRS

NO

96

LVNMGQWKI

6

35

LRFTVTQRS

26

LRFTVTQRS

NO

30

VGVYRAVTP

19

YRAVTPQGR

NO

97

LSTYRAFLS

20

LLSTYRAFL

NO

91

LSTYRAFLS

43

LLSTYRAFL

NO

40

LQAEQAFLR

32

LQAEQAFLR

NO

93

LHMYANRLL

22

LHMYANRLL

NO

10

IRASYAQQP

23

YAQQPAESR

NO

LVNMGQWKI NO

195

(continued)

196

TABLE 6.4

a b

(continued)

Sequence

Size

Protein

EDFRDGLKLMLL

12

YDHNFVKAINAIQK

14

KPGQFIRSVDPDSPA

15

RPKDYEVDATLKSLNN

16

NIFSFYLSRDPDAQPG

16

LKKYLYEIARRHP

13

RLPIIDVAPLDVGAPD

16

a actinin 1 (57–68) or a actinin 4 (76– 87) Cathepsin C (170–183) NHERF (174– 188) Collagen a2 (I) C-Term propeptide (1129–1144) Cathepsin D (227–241) Serum Albumin (159–171) Collagen a1 (I) C-term peptide (1436–1451)

% Inhib DRB1*1501 (DR2b)

Corea

% Inhib DRB5*0101 (DR2a)

Corea

Theoretical binding to DRB1*0407b

25

FRDGLKLML

31

FRDGLKLML

NO

5

VKAINAIQK

43

VKAINAIQK

FVKAINAIQ

7

IRSVDPDSP

9

IRSVDPDSP

FIRSVDPDS

15

YEVDATLKS

13

YEVDATLKS

YEVDATLKS

2

IFSFYLSRD

4

IFSFYLSRD

FYLSRDPDA

18

LKKYLYEIA

16

YLYEIARRH

YLYEIARRH

11

IIDVAPLDV

12

LPIIDVAPL

NO

Best core sequence according to ProPred (http://www.imtech.res.in/raghava/propred). Best core sequence fitting to the DRB1*0407 motif (http://www.syfpeithi.de). Bold letters in core sequences correspond to anchor residues

TABLE 6.5

Summary of pathogenic thyroglobulin epitopes from mouse models of experimental thyroiditis

Position

Historical name Sequence

Type of T-cell MHC restriction epitope

1–12

T4(5)

NIFEYQVDAQPL

k, s

179–197 181–195 410–424 418–432 1518–1532 1671–1710

hTg179 hTg181 hTg410 hTg418 p1518 F40D

2079–2093 p2079 2340–2359 p2340

NTTDMMIFDLVHSYNRFPD TDMMIFDLVHSYNRF QSQQFSVSENLLKEA ENLLKEAIRAIFPSR GSGKAFCVDGEGRRL KRFEPTGFQNMLSGLY NPIVFSASGANLTDA HLFCLLACD LSSVVVDPSIRHFDV QVAALTWVQTHIRGFGGDPR

2730–2743 – 2550–2561 T4 (2553)

EAT induction

T-cell Peptideresponse specific in vitro IgG Ref.

Tg

Direct Adoptive

Tg

Weak

k>s

ND

Eb DR3 Eb DR3 DR3 K

Dominant

hTg hTg hTg hTg hTg hTg

þ Weak þ ND ND þ

þ ND ND ND

þ þ ND þ þ ND

þ Low ND ND Low þ

DR3 Ek, DR3

Subdominant hTg Subdominant hTg

þ Weak

þ ND

þ þ

Low þ

GLREDLLSLQEPGS STDDYASFSRAL

K Ak

hTg Subdominant h, mTg

þ þ

þ þ

ND þ

2596–2608 p2596

YGHGSLELLADVQ

Ak

Subdominant h, mTg þ

þ

þ

þ

2496–2512 TgP1, p2495

GLINRAKAVKQFEESQG

Ek

Cryptic

m, rTg þ

ND

þ

þ

121–130

p117

VQCWCVDTEGMEVYGT

Ak

mTg

Weaka þa

þa

þa

181–192

p179

NTTDMMIFDLIHNYNR

Ek, Ak

mTg

Weak

þ

–

(Kong et al., 1995; Wan et al., 1997) (Brown et al., 2008) (Flynn et al., 2004) (Brown et al., 2008) (Flynn et al., 2004) (Flynn et al., 2004) (Texier et al., 1992)

(Flynn et al., 2004) (Karras et al., 2003; Karras et al., 2005) (Hoshioka et al., 1993) (Dai et al., 1999; Hutchings et al., 1992; Kong et al., 1995) (El Hassani et al., 2004; Verginis et al., 2002) (Chronopoulou and Carayanniotis, 1992; Dai et al., 1999) (Li and Carayanniotis, 2006) (Li et al., 2007)

197

(continued)

TABLE 6.5

(continued)

198 Position

Historical name Sequence

EAT induction Tg

Direct Adoptive

T-cell Peptideresponse specific in vitro IgG Ref.

181–195 306–316

mTg179 p304

NTTDMMIFDLIHNYNRFPD GHYQTVQCQTEGMCW

E Ak

mTg mTg

þ a

þa

þ þa

þ Low

306–320 409–423 1577–1591 1823–1837 1931–1945

p306 mTg409 p1579 p1826 p1931

YQTVQCQTEGMCWCV HYQRLSESRSLLREA LVQCLTDCANDEA GDMATELFSP KVVLNDKVNNFYTRL

Ak Eb Ak Ak Ak

Subdominant Subdominant Subdominant Subdominant

mTg mTg mTg mTg mTg

þ Weak Weak Weak Weaka

þ weak þ þ þa

þ þ þ þ þa

Low

2102–2116 2496–2504 2499–2507 2532–2543 2542–2552 2694–2705 2696–2707

p2102 TgP1 TgP1 P2529 P2540 – –

SMAQDFCLQQCSRHQ LINRAKAVK RAKAVKQFE EDSDARILAAAVWYYSL VWYYSLEHSTDDYAS CSFWSKYIQTLK FWSKYIQTLKDA

Ak Ek , A s Ak, As Ek , A k Ak As As

Subdominant Subdominant

mTg mTg mTg mTg mTg mTg mTg

þ Weak Weak Weak þ ND

þ ND ND ND ND ND þ

þ þ þ þ þ þ

þ þ þ þ þ þ

FWSKYIQTLKDADGAKDA

As

Subdominant rTg

Weak

þ

þ

þ

2696–2713 TgP2, p2695 a

Type of T-cell MHC restriction epitope b

Only the iodinated form of the peptide. Underlined shared sequences with peptide eluted from GD thyroid samples. ND, not determined.

(Brown et al., 2008) (Li and Carayanniotis, 2006) (Verginis et al., 2002) (Brown et al., 2008) (Verginis et al., 2002) (Verginis et al., 2002) (Li and Carayanniotis, 2006) (Verginis et al., 2002) (Rao et al., 1994) (Rao et al., 1994) (Li et al., 2007) (Li et al., 2007) (Rao et al., 1999) (Rao and Carayanniotis, 1997) (Carayanniotis et al., 1994)

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of a potential pathogenic Tg peptide presented by DR3 in a human thyroid affected by GD was demonstrated. Our data also confirm the suggestion by the authors that the peptide was likely a natural DR3 ligand and consequently that a computer algorithm can predict a peptide identical in size and sequence to a naturally processed peptide. Other natural Tg peptides could be related to previously published pathogenic peptides, although not such direct relationship was found. Tg 2756–2765 from TB471 is a small peptide (LSLQEPGSKT) that could not be assigned to any DR allele of those analyzed. Its sequence is part (underline) of a shared immunogenic T-cell epitope between hTg and hTPO (Tg2730–2743, in the alternative nomenclature) that is related to the development of experimental autoimmune thyroiditis in H-2k haplotype mice (Hoshioka et al., 1993). The sequence of the epitope is GLREDLLSLQEPGS and it is located at the C terminus of the Tg next to a hormonogenic tyrosine residue. Two TB471 adjacent peptides: IGSSQDDGLINR (Tg2508–2519) and AKAVKQFEESRGRTSSK (Tg2520–2536) are contained in a sequence that has been shown to be a cryptic peptide in mouse experimental thyroiditis (TgP1, p2495, or Tg2495–2511, corresponding to the sequence GLINRAKAVKQFEESQG of the mouse Tg). This peptide is only generated when mice are immunized with highly iodinated Tg (Dai et al., 2002). The same region of the Tg sequence was used to delineate the 9-mer (2495–2503) corresponding to the minimal T-cell epitope to induce EAT in Ek mice, capable to be presented by several MHC alleles (Rao et al., 1994). This epitope, despite its small size, still included part of the same two natural peptides. In the same work, another 9-mer (2498–2506) from the same region was also pathogenic for Ak mice. Comparative analysis of the different peptides should be performed to identify a relationship between the natural human peptides and the homologous mice pathogenic epitopes. It remains to be clarified whether DR51-Tg726–743 or any other DR-Tg complex expressed in human GD thyroids are also potentially pathogenic or related to the regulation of the autoimmune response. The binding data of Tg peptides compared to other natural ligands from the same sample are interesting (Muixı´ et al., 2008). When analyzing the binding to HLA-DR3, we found that one of the ubiquitous abundant peptides (E-cadherin 196–210) did bind to HLA-DR3 with high affinity (Fig. 6.2). Binding of the same allele by the putatively pathogenic Tg2098– 2112 peptide was however much lower. The affinity of this peptide, albeit positive, is below 50% of the affinity of the cadherin peptide for the same allele. Being a protein involved in multiple function and present in most tissues we can presume that tolerance to cadherin should be efficient and no reactive T cells are expected to be found in the periphery. If these two peptides are generated in vivo in the thymus, the Tg peptide would have

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difficulty in competing for the binding to DR3 and some cells reactive to this epitope could more readily escape selection. Further analysis of peptide binding affinities and identification of T cells recognizing HLA-DR complexes with ubiquitous or restricted peptides will give answers as to the potential role of the different peptides in the disease.

3.3. TPO and TSHR peptides No peptides from these two antigens were detected in our studies. It is obvious that the abundance of these other two molecules is nothing compared to Tg, and a great improvement of the sensitivity of the technique will be necessary to identify them (Rapoport and McLachlan, 2007). In the meantime, there are data available on TSHR peptides’ affinity for DR3 and their relation to T-cell responses against the antigen. A study on the binding properties of TSHR to HLA-DR3 showed that DR3 molecules bind TSHR T-cell reactive peptide epitopes with intermediate affinity but nonimmunogenic peptides were mostly poor or no binders. However, there was a single peptide with the highest binding affinity that was not capable of eliciting T-cell responses (Sawai and DeGroot, 2000). A larger analysis of binding specificity of TSHR peptides showed that peptides bound to susceptible DR alleles with intermediate or low affinity, whereas the peptides binding to protective allele HLA-DR7 were high binders (Inaba et al., 2006). T-cell epitopes from TPO are well defined (McLachlan and Rapoport, 2007), most of them belong to a region of the TPO molecule with homology with granulocyte myeloperoxidase, but no clear immunodominant peptide has been identified. Processing by TFC appears to be essential for recognition of at least one cryptic TPO epitope (Quaratino et al., 1995), although B-cell processing by means of specific immunoglobulins uptake appear to be important since TPO autoantibodies influence T-cell recognition of some epitopes (Guo et al., 1996; Quaratino et al., 2005).

4. CONCLUDING REMARKS The issue of the utility of peptides as vaccines for cancer or autoimmunity should be reopened in view of new data. MHC-bound peptides are indeed potentially very interesting but their role in T-cell recognition is very variable and they can be used as inhibitory as well as enhancing agents. The line between these two functions is anyway fragile, since there is no evidence of any difference between peptides recognized by effectors or by regulatory cells. Analysis of tolerogenic versus activating peptides could give some answers, but the identification of T cells and their

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function from the thyroid infiltrates, capable of recognizing each peptide, is still necessary.

ACKNOWLEDGMENTS The authors thank Drs. Merce` Martı´ and Carme Roura-Mir for help with the manuscript, and for a lot of previous thyroid work. To all the other people that were involved in the work that generated these data, M. Carrascal, C. Pinilla, E. Borra`s, M. Cata´lfamo, L. Serradell, A. Muntasell, M. Costa, M. P. Armengol, M. Sospedra, A. Lucas-Martı´n, G. Obiols, J. Abian, X. Daura, and R. Pujol-Borrell. And special thanks to the thyroid sample donors. This work was supported in part by Grants SAF2000-0131-C02-01, SAF2003-08843-C0201, and SAF2006-08928 from the Spanish Ministry of Education and by the Eurothymaide Integrated European Project LSHB-CT-2003-503410. L. M. is a FPI fellow of the Spanish Ministry of Education.

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SUBJECT INDEX A Acquired immunity, 131–132 Activation induced cytosine deaminase (AID), 7–8 Activation model, DNA-PK ABCDE phosphorylation, 44 PQR phosphorylation, 44–46 repair factors, 47 Acute-phase serum amyloid A (A-SAA), 145 Aspergillus fumigatus, 140 Autoimmune myocarditis autoantibodies, 100–101 immunosupressive therapy, 101–102 Autoimmune thyroid diseases (AITD) antigenic peptides, 172 HLA class II binding motifs, 191 cytosolic peptides, 186 endocytic peptides, 186 extracellular peptides, 179, 186 HLA-DR genes, 169–170 R74 synergistic effect and SNP, 170 Tg peptides, 191–200 TPO and TSHR peptides, 200 thyroid antigen tolerance autoantigens, 170–171 self-antigens, 170 thymus, 171 tissue-restricted autoimmune response autoantigens, 167 thyroid follicular cells (TFC), 168 Autophosphorylation ABCDE clusters ablation, 40 dysregulated end processing, 42 distinct functional consequences, 40 kinase dissociation, 39 NHEJ, 43–44 PQR clusters ablation, 40 dysregulated end processing, 42

B Bacteria, IL-23/IL-17-mediated responses Bordetella organisms, 136 Borrelia burgdorferi, 132, 135 Helicobacter pylori infection, 137–138 HIV-associated K. pneumoniae, 135 in intestine, 138 Mycoplasma tuberculosis infection, 137 respiratory infections, 139 Bordetella pertussis, 136 C Candida albicans, 139–141 Cis-regulatory elements IgH locus accessibility hypothesis, 10–16 allelic exclusion, 8–10 CH genes, 3 class switch recombination, 7–8 control, 16–23 DNaseI hypersensitive sites, 3–4 murine, 4 rearrangements, 8–10 somatic hypermutation, 7–8 transcripts, 5 V(D)J recombination, 4–7 T-cell receptor (TCR) variable, 2–3 Collagen-induced arthritis (CIA), 118–120 Common lymphoid progenitor (CLP) cells, 8 Cortical microenvironment thymic cortex cortical thymic epithelial cells, 68–69 transgenic mouse strain, Tg66, 69 thymocyte positive selection and thymoproteasome costimulatory signals, 71 interferon-g, 70 MHC ligands, 71 proteolysis, 70 Cortical thymic epithelial cells (cTEC), 68–69, 72

211

212

Subject Index

Coxsackieviruses of group B (CVB3) myocarditis IL-13 and IL-4, 105 lymphocitic myocarditis, 99 mouse model, 102 myosin-induced EAM, 102–103 proinflammatory cytokines, 104 D Dendritic cells (DC), 77–78 Dilated cardiomyopathy (DCM) antibodies, 100–101 characteristics, 99–100 etiology, 97 IFN-g deficiency, 104 IL-13 and IL-4, 105 MHC II association, 98 DNA-dependent protein kinase (DNA-PK) abundance embryonic lethality, 51 genomic stability, 52 homologous recombination (HR), 51 rodent cells, 50 telomere maintenance, 52 activation model ABCDE phosphorylation, 44 PQR phosphorylation, 44–46 repair factors, 47 autophosphorylation ABCDE clusters, 40–43 distinct functional consequences, 40 kinase dissociation, 39 NHEJ, 43–44 PQR clusters, 40–43 composition DNA binding co-factors, 37 initial sequence analysis, 36 DNA binding and kinase activation, 37–38 double strand break repair cellular abundance, 49 homologous recombination (HR) pathway, 49–50 reports, 49 double strand break repair pathway, 49–50 end processing Artemis, 47 NHEJ, 47–48 polynucleotide kinase phosphatase, 48 enzymatic activity, 39

structural studies, 38–39 Double strand break repair pathway, 49–50 E Eosinophilic myocarditis, 99 Experimental autoimmune encephalomyelitis (EAE), 118–119 Experimental autoimmune myocarditis (EAM) antibodies, 101 CVB3-induced myocarditis, 103 cytokine regulation, 107 IL-13 and IL-4, 105 macrophages, 108 non-MHC gene association, 98 Experimental autoimmune uveitis (EAU) model, 106 F Fungi, IL-23/IL-17-mediated responses Candida and Aspergillus, 140–141 toll IL-1R8 (Tir8), 141 G Giant cell myocarditis (GCM), 99 Graves’ disease DR-associated peptide analysis, 177–178 etiology, 169–170 HLA-II expression, 172 Tg peptides, HLA-DR cell-free binding assay, 193 TB448, 193–196 TB449, 194 TB471, 199 H Helicobacter pylori, 137–138 Herpes simplex virus (HSV), 143 Herpetic stromal keratitis (HSK), 143 HLA class II, thyroid autoimmunity binding motifs, 191 cytosolic peptides, 186 endocytic peptides, 186 extracellular peptides, 179, 186 HLA-DR genes, 169–170 R74 synergistic effect and SNP, 170 Tg peptides, 191–200 TPO and TSHR peptides, 200 Human rhinovirus (HRV), 145 Human T-cell leukemia virus type 1 (HTLV-1), 146

213

Subject Index

I IL-17, 125–126 IL-22 IL-23/IL-17/IL-22 pathway, 127 production, 126 IL-26, 127–128 IL-10-related T-cell inducible factor (IL-TIF). See IL-22 Immunoglobulin heavy (IgH) chain locus accessibility hypothesis germline transcripts, 10–12 spatial organization and nuclear positioning, 12–16 allelic exclusion chromatin changes, 9 CLP cells and RAG activity, 8 feedback-mediated mechanism, 9 CH genes, 3 class switch recombination activation induced cytosine deaminase (AID), 7–8 B-cells, 7 repetitive switch regions, 7 control chromatin state, 22–23 DQ52 promoter, 16–17 intronic enhancer (Em), 18–19 potential regulatory elements, 21–22 regulatory region and I promoters, 20–21 VH promoters, 17–18 DNaseI hypersensitive sites, 3–4 murine, 4 rearrangements chromatin changes, 9 CLP cells and RAG activity, 8 somatic hypermutation activation induced cytosine deaminase (AID), 7–8 B-cells, 7 repetitive switch regions, 7 transcripts, 5 V(D)J recombination coding ends (CE), 7 exons, 4 pre-B-cells, 4, 6 Innate immunity, Th17 pathway, 129–131 Intronic enhancer (Em), 18–19 K Klebsiella pneumoniae, 128–129, 135

L Leucine rich region (LRR), 36 Lymphocytic myocarditis, 98–99 Lymphotoxin-b receptor (LTbR), 73 M Macrophages, 107 Medullary microenvironment medullary thymic epithelial cells (mTEC) CD40L, 75 LTbR, 73–74 RANK and RANKL, 74–75 NF-kB signaling pathways, 73 regulatory T cells, 78 self-tolerance, DC, 77–78 self-tolerance, mTEC autoimmune regulator (AIRE), 76–77 promiscuous gene expression, 76 thymic medulla and thymic crosstalk, 71–72 Medullary thymic epithelial cells (mTEC), 73–77 Mycobacterium bovis bacillus CalmetteGue´rin (BCG), 132 Myocarditis autoimmune myocarditis, 98 autoimmune process autoantibodies, 100–101 immunosupressive therapy, 101–102 clinical manifestation, 96–97 etiology, 97–98 macrophages, 107–108 mouse models CVB3-induced myocarditis, 102 experimental autoimmune myocarditis, 102–104 proinflammatory cytokines, 104 T helper cells IL-13 and IL-4, 105 Th1 cells, 104–105 Th1-or Th17-driven disease, 105–107 types dilated cardiomyopathy, 99–100 eosinophilic myocarditis, 99 giant cell myocarditis, 99 lymphocytic myocarditis, 98–99

214

Subject Index

N Nonhomologous end joining (NHEJ), 43–44, 47–48 Nuclear factor (NF)-kB signaling pathways, 73 P Phosphatidylinositol 3-kinase, 36 Potential regulatory elements, 21–22 Proinflammatory cytokines, 104 Protozoa, Th17 pathway mucosal surfaces, 141, 144 T. gondii, 142 R Receptor activating NF-kB (RANK), 74 Respiratory syncytial virus (RSV), 144 S Salmonella typhimurium, 145–146 Simian immunodeficiency virus (SIV), 145 Sphingosine-1-phosphate (S1P), 67 Syphacia obvelata, 146 T T-cell antigen receptors (TCRs), 61 T-cell repertoire formation cortical microenvironment thymic cortex, 68–69 thymocyte positive selection and thymoproteasome, 69–71 lymphostromal interactions, 63 medullary microenvironment medullary thymic epithelial cells (mTEC), 73–75 NF-kB signaling pathways, 73 regulatory T cells, 78 self-tolerance, DC, 77–78 self-tolerance, mTEC, 76–77 thymic medulla and thymic crosstalk, 71–72 stromal components thymic crosstalk, 64 thymic epithelial cells, 63 thymic mesenchymal cells, 63 T-cell antigen receptor, 61 thymic parenchyma, 61 thymocytes, trafficking

export, 67–68 outward migration, 65–66 relocation, 66–67 T-precursor cells, 64–65 TCR. See T-cell antigen receptor TEC. See Thymic epithelial cells Theiler’s murine encephalomyelitis virus (TMEV), 145 Th17 pathway acquired immunity, 131–132 emergence of autoimmune diseases, 118 EAE and CIA, 118–119 effector CD4 T-cell lineages, 123 IL-23 and IL-17, 119–120 IL-12 signaling, 119 naı¨ve cells, 121–122 organisms survillence, 124 IL-17, 125 IL-22, 125–127 IL-26, 127–128 IL-23/IL-17-mediated innate immunity expression of, 128 gd T-cell subset, 129 IL-23/IL-17-mediated responses bacteria, 132, 135–139 fungi, 139–141 organisms in, 133–135 protozoa, 141–143 Syphacia obvelata, 146 viruses, 143–146 and innate immunity IL-17 in, 129 microbial activation, 130 Lyme arthritis, 148 microbes in, 147 Th1 and Th2 cells, 116–117 Thymic epithelial cells (TEC), 63 Thymocytes, trafficking export phosphatidylinositol 3-kinase, 68 sphingosine-1-phosphate (S1P), 67 transcription factor KLF2, 68 outward migration corticomedullary junction, 65 VCAM-1, 66 relocation autoimmune exocrinopathy, 66 medulla, 66–67 random walk motility, 66 T-precursor cells chemokine receptors, 64

215

Subject Index

T-lymphoid progenitor cells, 64–65 vasculature-independent and dependent pathways, 64 Thyroglobulin (Tg) endocytic peptides, 186 extracellular peptides, 179 Graves’ disease, HLA-DR cell-free binding assay, 193 TB448, 193–196 TB449, 194 TB471, 199 thymus, 171 Thyroid autoimmunity antigenic peptides, 172 HLA class II binding motifs, 191 cytosolic peptides, 186 endocytic peptides, 186 extracellular peptides, 179, 186 HLA-DR genes, 169–170 R74 synergistic effect and SNP, 170 Tg peptides, 191–200 TPO and TSHR peptides, 200 thyroid antigen tolerance autoantigens, 170–171 self-antigens, 170 thymus, 171 tissue-restricted autoimmune response

autoantigens, 167 thyroid follicular cells (TFC), 168 Tissue-restricted autoimmune process, thyroid disease autoantigens, 167 thyroid follicular cells (TFC), 168 Toxoplasma gondii, 141–142 T-precursor cells chemokine receptors, 64 T-lymphoid progenitor cells, 64–65 vasculature-independent and dependent pathways, 64 Type 1 diabetes (T1D), 166–167 V Vaccinia virus (VV), 143 Vascular cell adhesion molecule-1 (VCAM-1), 66 Viruses, IL-23/IL-17-mediated responses herpes simplex virus (HSV), 143–144 HIV infection, 145 HTLV-1, 146 human rhinovirus (HRV), 145 respiratory syncytial virus (RSV), 144 Theiler’s murine encephalomyelitis virus, 145 vaccinia virus (VV), 143

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CONTENTS OF RECENT VOLUMES Volume 85 Cumulative Subject Index Volumes 66–82

Volume 86 Adenosine Deaminase Deficiency: Metabolic Basis of Immune Deficiency and Pulmonary Inflammation Michael R. Blackburn and Rodney E. Kellems Mechanism and Control of V(D)J Recombination Versus Class Switch Recombination: Similarities and Differences Darryll D. Dudley, Jayanta Chaudhuri, Craig H. Bassing, and Frederick W. Alt Isoforms of Terminal Deoxynucleotidyltransferase: Developmental Aspects and Function To-Ha Thai and John F. Kearney Innate Autoimmunity Michael C. Carroll and V. Michael Holers Formation of Bradykinin: A Major Contributor to the Innate Inflammatory Response Kusumam Joseph and Allen P. Kaplan Interleukin-2, Interleukin-15, and Their Roles in Human Natural Killer Cells Brian Becknell and Michael A. Caligiuri Regulation of Antigen Presentation and Cross-Presentation in the Dendritic Cell Network: Facts, Hypothesis, and Immunological Implications Nicholas S. Wilson and Jose A. Villadangos Index

Bernard Malissen, Enrique Aguado, and Marie Malissen The Integration of Conventional and Unconventional T Cells that Characterizes Cell-Mediated Responses Daniel J. Pennington, David Vermijlen, Emma L. Wise, Sarah L. Clarke, Robert E. Tigelaar, and Adrian C. Hayday Negative Regulation of Cytokine and TLR Signalings by SOCS and Others Tetsuji Naka, Minoru Fujimoto, Hiroko Tsutsui, and Akihiko Yoshimura Pathogenic T-Cell Clones in Autoimmune Diabetes: More Lessons from the NOD Mouse Kathryn Haskins The Biology of Human Lymphoid Malignancies Revealed by Gene Expression Profiling Louis M. Staudt and Sandeep Dave New Insights into Alternative Mechanisms of Immune Receptor Diversification Gary W. Litman, John P. Cannon, and Jonathan P. Rast The Repair of DNA Damages/ Modifications During the Maturation of the Immune System: Lessons from Human Primary Immunodeficiency Disorders and Animal Models Patrick Revy, Dietke Buck, Franc¸oise le Deist, and Jean-Pierre de Villartay

Volume 87

Antibody Class Switch Recombination: Roles for Switch Sequences and Mismatch Repair Proteins Irene M. Min and Erik Selsing

Role of the LAT Adaptor in T-Cell Development and Th2 Differentiation

Index

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218

Contents of Recent Volumes

Volume 88 CD22: A Multifunctional Receptor That Regulates B Lymphocyte Survival and Signal Transduction Thomas F. Tedder, Jonathan C. Poe, and Karen M. Haas Tetramer Analysis of Human Autoreactive CD4-Positive T Cells Gerald T. Nepom Regulation of Phospholipase C-g2 Networks in B Lymphocytes Masaki Hikida and Tomohiro Kurosaki Role of Human Mast Cells and Basophils in Bronchial Asthma Gianni Marone, Massimo Triggiani, Arturo Genovese, and Amato De Paulis A Novel Recognition System for MHC Class I Molecules Constituted by PIR Toshiyuki Takai Dendritic Cell Biology Francesca Granucci, Maria Foti, and Paola Ricciardi-Castagnoli The Murine Diabetogenic Class II Histocompatibility Molecule I-Ag7: Structural and Functional Properties and Specificity of Peptide Selection Anish Suri and Emil R. Unanue RNAi and RNA-Based Regulation of Immune System Function Dipanjan Chowdhury and Carl D. Novina Index

Lysophospholipids as Mediators of Immunity Debby A. Lin and Joshua A. Boyce Systemic Mastocytosis Jamie Robyn and Dean D. Metcalfe Regulation of Fibrosis by the Immune System Mark L. Lupher, Jr. and W. Michael Gallatin Immunity and Acquired Alterations in Cognition and Emotion: Lessons from SLE Betty Diamond, Czeslawa Kowal, Patricio T. Huerta, Cynthia Aranow, Meggan Mackay, Lorraine A. DeGiorgio, Ji Lee, Antigone Triantafyllopoulou, Joel Cohen-Solal Bruce, and T. Volpe Immunodeficiencies with Autoimmune Consequences Luigi D. Notarangelo, Eleonora Gambineri, and Raffaele Badolato Index

Volume 90 Cancer Immunosurveillance and Immunoediting: The Roles of Immunity in Suppressing Tumor Development and Shaping Tumor Immunogenicity Mark J. Smyth, Gavin P. Dunn, and Robert D. Schreiber

Volume 89

Mechanisms of Immune Evasion by Tumors Charles G. Drake, Elizabeth Jaffee, and Drew M. Pardoll

Posttranscriptional Mechanisms Regulating the Inflammatory Response Georg Stoecklin Paul Anderson

Development of Antibodies and Chimeric Molecules for Cancer Immunotherapy Thomas A. Waldmann and John C. Morris

Negative Signaling in Fc Receptor Complexes Marc Dae¨ron and Renaud Lesourne

Induction of Tumor Immunity Following Allogeneic Stem Cell Transplantation Catherine J. Wu and Jerome Ritz

The Surprising Diversity of Lipid Antigens for CD1-Restricted T Cells D. Branch Moody

Vaccination for Treatment and Prevention of Cancer in Animal Models

Contents of Recent Volumes

Federica Cavallo, Rienk Offringa, Sjoerd H. van der Burg, Guido Forni, and Cornelis J. M. Melief Unraveling the Complex Relationship Between Cancer Immunity and Autoimmunity: Lessons from Melanoma and Vitiligo Hiroshi Uchi, Rodica Stan, Mary Jo Turk, Manuel E. Engelhorn, Gabrielle A. Rizzuto, Stacie M. Goldberg, Jedd D. Wolchok, and Alan N. Houghton Immunity to Melanoma Antigens: From Self-Tolerance to Immunotherapy Craig L. Slingluff, Jr., Kimberly A. Chianese-Bullock, Timothy N. J. Bullock, William W. Grosh, David W. Mullins, Lisa Nichols, Walter Olson, Gina Petroni, Mark Smolkin, and Victor H. Engelhard Checkpoint Blockade in Cancer Immunotherapy Alan J. Korman, Karl S. Peggs, and James P. Allison Combinatorial Cancer Immunotherapy F. Stephen Hodi and Glenn Dranoff Index

Volume 91 A Reappraisal of Humoral Immunity Based on Mechanisms of AntibodyMediated Protection Against Intracellular Pathogens Arturo Casadevall and Liise-anne Pirofski Accessibility Control of V(D)J Recombination Robin Milley Cobb, Kenneth J. Oestreich, Oleg A. Osipovich, and Eugene M. Oltz Targeting Integrin Structure and Function in Disease

219

Donald E. Staunton, Mark L. Lupher, Robert Liddington, and W. Michael Gallatin Endogenous TLR Ligands and Autoimmunity Hermann Wagner Genetic Analysis of Innate Immunity Kasper Hoebe, Zhengfan Jiang, Koichi Tabeta, Xin Du, Philippe Georgel, Karine Crozat, and Bruce Beutler TIM Family of Genes in Immunity and Tolerance Vijay K. Kuchroo, Jennifer Hartt Meyers, Dale T. Umetsu, and Rosemarie H. DeKruyff Inhibition of Inflammatory Responses by Leukocyte Ig-Like Receptors Howard R. Katz Index

Volume 92 Systemic Lupus Erythematosus: Multiple Immunological Phenotypes in a Complex Genetic Disease Anna-Marie Fairhurst, Amy E. Wandstrat, and Edward K. Wakeland Avian Models with Spontaneous Autoimmune Diseases Georg Wick, Leif Andersson, Karel Hala, M. Eric Gershwin,Carlo Selmi, Gisela F. Erf, Susan J. Lamont, and Roswitha Sgonc Functional Dynamics of Naturally Occurring Regulatory T Cells in Health and Autoimmunity Megan K. Levings, Sarah Allan, Eva d’Hennezel, and Ciriaco A. Piccirillo BTLA and HVEM Cross Talk Regulates Inhibition and Costimulation

220

Contents of Recent Volumes

Maya Gavrieli, John Sedy, Christopher A. Nelson, and Kenneth M. Murphy The Human T Cell Response to Melanoma Antigens Pedro Romero, Jean-Charles Cerottini, and Daniel E. Speiser Antigen Presentation and the Ubiquitin-Proteasome System in Host–Pathogen Interactions Joana Loureiro and Hidde L. Ploegh Index

Volume 93 Class Switch Recombination: A Comparison Between Mouse and Human Qiang Pan-Hammarstro¨m, Yaofeng Zhao, and Lennart Hammarstro¨m Anti-IgE Antibodies for the Treatment of IgE-Mediated Allergic Diseases Tse Wen Chang, Pheidias C. Wu, C. Long Hsu, and Alfur F. Hung Immune Semaphorins: Increasing Members and Their Diverse Roles Hitoshi Kikutani, Kazuhiro Suzuki, and Atsushi Kumanogoh Tec Kinases in T Cell and Mast Cell Signaling Martin Felices, Markus Falk, Yoko Kosaka, and Leslie J. Berg Integrin Regulation of Lymphocyte Trafficking: Lessons from Structural and Signaling Studies Tatsuo Kinashi Regulation of Immune Responses and Hematopoiesis by the Rap1 Signal Nagahiro Minato, Kohei Kometani, and Masakazu Hattori Lung Dendritic Cell Migration Hamida Hammad and Bart N. Lambrecht Index

Volume 94 Discovery of Activation-Induced Cytidine Deaminase, the Engraver of Antibody Memory Masamichi Muramatsu, Hitoshi Nagaoka, Reiko Shinkura, Nasim A. Begum, and Tasuku Honjo DNA Deamination in Immunity: AID in the Context of Its APOBEC Relatives Silvestro G. Conticello, Marc-Andre Langlois, Zizhen Yang, and Michael S. Neuberger The Role of Activation-Induced Deaminase in Antibody Diversification and Chromosome Translocations Almudena Ramiro, Bernardo Reina San-Martin, Kevin McBride, Mila Jankovic, Vasco Barreto, Andre´ Nussenzweig, and Michel C. Nussenzweig Targeting of AID-Mediated Sequence Diversification by cis-Acting Determinants Shu Yuan Yang and David G. Schatz AID-Initiated Purposeful Mutations in Immunoglobulin Genes Myron F. Goodman, Matthew D. Scharff, and Floyd E. Romesberg Evolution of the Immunoglobulin Heavy Chain Class Switch Recombination Mechanism Jayanta Chaudhuri, Uttiya Basu, Ali Zarrin, Catherine Yan, Sonia Franco, Thomas Perlot, Bao Vuong, Jing Wang, Ryan T. Phan, Abhishek Datta, John Manis, and Frederick W. Alt Beyond SHM and CSR: AID and Related Cytidine Deaminases in the Host Response to Viral Infection Brad R. Rosenberg and F. Nina Papavasiliou Role of AID in Tumorigenesis Il-mi Okazaki, Ai Kotani, and Tasuku Honjo

Contents of Recent Volumes

Pathophysiology of B-Cell Intrinsic Immunoglobulin Class Switch Recombination Deficiencies Anne Durandy, Nadine Taubenheim, Sophie Peron, and Alain Fischer Index

Inherited Complement Regulatory Protein Deficiency Predisposes to Human Disease in Acute Injury and Chronic Inflammatory States Anna Richards, David Kavanagh, and John P. Atkinson Fc-Receptors as Regulators of Immunity Falk Nimmerjahn and Jeffrey V. Ravetch

Volume 95

Index

Fate Decisions Regulating Bone Marrow and Peripheral B Lymphocyte Development John G. Monroe and Kenneth Dorshkind

Volume 97

Tolerance and Autoimmunity: Lessons at the Bedside of Primary Immunodeficiencies Magda Carneiro-Sampaio and Antonio Coutinho B-Cell Self-Tolerance in Humans Hedda Wardemann and Michel C. Nussenzweig Manipulation of Regulatory T-Cell Number and Function with CD28Specific Monoclonal Antibodies Thomas Hu¨nig Osteoimmunology: A View from the Bone Jean-Pierre David Mast Cell Proteases ˚ brink, Gunnar Pejler, Magnus A Maria Ringvall, and Sara Wernersson Index

Volume 96 New Insights into Adaptive Immunity in Chronic Neuroinflammation Volker Siffrin, Alexander U. Brandt, Josephine Herz, and Frauke Zipp Regulation of Interferon-g During Innate and Adaptive Immune Responses Jamie R. Schoenborn and Christopher B. Wilson The Expansion and Maintenance of Antigen-Selected CD8þ T Cell Clones Douglas T. Fearon

221

T Cell Activation and the Cytoskeleton: You Can’t Have One Without the Other Timothy S. Gomez and Daniel D. Billadeau HLA Class II Transgenic Mice Mimic Human Inflammatory Diseases Ashutosh K. Mangalam, Govindarajan Rajagopalan, Veena Taneja, and Chella S. David Roles of Zinc and Zinc Signaling in Immunity: Zinc as an Intracellular Signaling Molecule Toshio Hirano, Masaaki Murakami, Toshiyuki Fukada, Keigo Nishida, Satoru Yamasaki, and Tomoyuki Suzuki The SLAM and SAP Gene Families Control Innate and Adaptive Immune Responses Silvia Calpe, Ninghai Wang, Xavier Romero, Scott B. Berger, Arpad Lanyi, Pablo Engel, and Cox Terhorst Conformational Plasticity and Navigation of Signaling Proteins in Antigen-Activated B Lymphocytes Niklas Engels, Michael Engelke, and Ju¨rgen Wienands Index

222

Contents of Recent Volumes

Volume 98 Immune Regulation by B Cells and Antibodies: A View Towards the Clinic Kai Hoehlig, Vicky Lampropoulou, Toralf Roch, Patricia Neves, Elisabeth Calderon-Gomez, Stephen M. Anderton, Ulrich Steinhoff, and Simon Fillatreau

Juan Rivera, Nora A. Fierro, Ana Olivera, and Ryo Suzuki B Cells and Autoantibodies in the Pathogenesis of Multiple Sclerosis and Related Inflammatory Demyelinating Diseases Katherine A. McLaughlin and Kai W. Wucherpfennig

Cumulative Environmental Changes, Skewed Antigen Exposure, and the Increase of Allergy Tse Wen Chang and Ariel Y. Pan

Human B Cell Subsets Stephen M. Jackson, Patrick C. Wilson, Judith A. James, and J. Donald Capra

New Insights on Mast Cell Activation via the High Affinity Receptor for IgE

Index

Model of DNA-PK activation Ku

DNA-PKcs FATc

DNA-PKcs/artemis

PI3K FAT

1. Unlike Artemis, in the absence of DNA damage, Ku does not interact with DNA-PKcs. Because of the relative abundance of these two factors, it is likely that some DNA-PKcs is not bound to Artemis.

Head Arm Palm

2. DNA damage is recognized by Ku. The DNA-P Kcs Artemis complex is recruited, but Artemis dissociates from kinase inactive DNA-PK. DNA binding induces a conformational change in DNA-PKcs that results in closer association of the head and palm globular domains. An unsynapsed, DNA-PK bound end may be accessed by the HR pathway.

3. The termini of DNA ends must dissociate to facilitate activation of DNA-PK’s kinase activity. It is not clear whether DNA end melting occurs before or after synapsis or whether DNA-dependent activation is in cis or trans. DNA ends are protected from modification until the kinase is activated and ABCDE autophosphorylation occurs.

4. Synapsis results in a conformational change in DNA-PKcs, including bridging between Ku and the FATc domain. Synapsed DNA ends are sequestered and protected from modification, and HR until kinase activation and autophosphorylation.

PLATE 1

(Continued)

∗ ABCDE phosphorylation ∗ PQR phosphorylation ∗ T and other phosphorylations 5. ABCDE autophosphorylation in trans promotes access of DNA ends to Artemis and other DNA end modifying factors. With this phosphorylation status, DNA-PK’s interaction with the very termini are somewhat destabilized, but the complex is still stabley assembled at the break. The DNA break would also be accessible to HR at this point, if NHEJ fails.

6. End alignment is “sensed” by DNA-PK resulting in PQR phosphorylation. PQR phosphorylation protects the DNA ends from further nucleotide loss; end processing (based on this alignment) including ligation, fill in synthesis, and flap processing still proceeds. In this configuration, DNA-PK’s interaction is stabilized both by its interaction at the ends and also elsewhere in the complex. PQR phosphorylation strongly inhibits HR.

7. Additional autophosphorylation occurs at T as well as undefined sites to facilitate final steps in NHEJ. This is depicted after end ligation, although it is possible that phosphorylation events are required to facilitate fill in synthesis, flap removal, or ligation. Phosphorylation at sites (not yet identified) that induce dissociation “trump” the effects of ABCDE or PQR phosphorylation on DNA binding.

8. After dissociation, DNA-PKcs is de-phosphorlated by and can be recycled. It is likely that Ku must be removed from the resolved break by proteolysis.

Progenitor cell

DN3

b

c

DP

cTEC

a

d ETP

e SP f

mTEC

PLATE 2

PLATE 3

T cell

LTi

SP

RANKL

RANKL, CD40L OPG RANK, CD40

RANK AIRE

pTEC CCR7L

mTEC Medulla

PLATE 4

Wild type

IL-17 KO

PLATE 5

PLATE 6

IL-23 TGF-β IL-12Rβ1 IL-23R IL-6 IL-21 STAT3

SMADs STAT3 RORγ t

IL-23R IL-1R

Th17 IL-23R IL-1RI

IL-17A/F

IL-17(A) IL-17F IL-22

Tn IL-12Rβ1 T-bet STAT1

IFNγ

IL-12R

IFNγ

IL-12Rβ2

IL-18R

IL-18Rα STAT4

IL-12Rβ1

Th1

IL-12Rβ2

IL-12

IFNγ

Neutrophilic inflammation/ microbicidal bacteria fungi

Monocytic inflammation/ intracellular killing bacteria protozoans viruses

IL-4 STAT6

IL-4

Th2 GATA-3

GATA-3

IL-4 IL-5 IL-13

PLATE 7

Mucosal clearance helminthes

Cornea Skin

Nasopharynx

IL-23 Lung

DC

Th17

Liver

IL-17/IL-22

Stomach Pancreas

Intestine

Genital tract

PLATE 8

Gut lumen Bacteria

Enterocyte proliferation, increased tight junctions, & increased antimicrobial defenses

Enterocyte M cell

IL-23 TGFb Th17

IL-1

T ce ll help

IL-17 & IL-22 Lamina propria

TNFa MIP-3a IL-1,6,8,18 MCP-3 IP-10 I-TAC RANTES TECK

DC

B cell

IL-17

LP Capillary

TNFa MIP-1a IL-8 IL-6

a4b7

LFA-1

PLATE 9

Frozen thyroid samples

Mechanic homogenization

2 h lysis with 0.5% NP40

HLA-DR immunoaffinity purification (mAb L243, B8.11.2) Acid elution H2O 0.1%TFA

Fractionation HPLC-UV

mAU

Ultrafiltration 10 KDa cutoff

Min Molecular mass determination MALDI-TOF

Mass (m/z)

Peptide sequencing with nanoESI, MALDI-TOF/TOF

PLATE 10