TTHHÈÈSSEE

En vue de l'obtention du

DDOOCCTTOORRAATT DDEE LL’’UUNNIIVVEERRSSIITTÉÉ DDEE TTOOUULLOOUUSSEE

Délivré par l'Université Toulouse III - Paul Sabatier Discipline ou spécialité : Immunologie

JURY

Pr. Joost van Meerwijk Président Pr. Peter van Endert Rapporteur Pr. Burkhard Becher Rapporteur

Pr. Roland Liblau Directeur de Thèse

Ecole doctorale : Biologie-Santé-Biotechnologies

Unité de recherche : U563 INSERM Directeur(s) de Thèse : Pr. Roland Liblau

Rapporteurs : Pr. Peter van Endert Pr. Burkhard Becher

Présentée et soutenue par Amit SAXENA Le 9 April 2010

Titre : Study of T cell pathogenicity in central nervous

system autoimmunity

Abstract

Multiple sclerosis (MS) is a complex demyelinating disease associated with chronic inflammation of the central nervous system (CNS), axonal loss, and brain atrophy. The CD4+ and CD8+ T cells that are present in the demyelinating lesions are considered to mediate demyelination and axonal damage. An autoimmune process likely contributes to CNS tissue damage in MS, although direct evidence for this is still lacking. A major focus of my work is to understand the mechanisms by which autoreactive T cells contribute to CNS tissue damage.

Although CD8+ T cells mediate effector functions through production of cytokines or by direct cytotoxicity, the mechanisms by which they can cause CNS tissue damage are elusive. In order to assess whether CNS-infiltrating CD8+ T cells could directly induce oligodendrocyte death and demyelination, we developed an original mouse model combining selective expression of influenza hemagglutinin (HA) as a neo-self-Ag in oligodendrocytes with transgenic mice expressing a HA-specific TcR on CD8+ T cells. We demonstrate directly the potential of CD8+ T cells to induce oligodendrocyte death in-vivo, as a likely consequence of direct antigen-recognition.

Untill recently, CD4+ T helper cells have been held responsible for MS immunopathogenesis, partly because certain MHC class II alleles clearly predispose for developing MS. We investigated the pathogenic traits of the CD4+ T cell response targeting the immunodominant epitope of myelin oligodendrocyte protein (MOG). To this end we obtained 2D2 TcR-transgenic C57BL/6 mice, which harbor a large population of MOG-specific CD4+ T cells and spontaneously develop optic neuritis and at a lower prevalence, EAE. Strikingly when we crossed these 2D2 mice with MOG-deficient mice (MOG-/-), we discovered that the 2D2 TcR-transgenic mice developed spontaneous EAE regardless of the presence or absence of the target self-antigen MOG. Therefore we hypothesized, that the 2D2 TcR specific for MOG35-55 recognizes a second CNS antigen. Furthermore, we were able to reveal, that MOG35-55 peptide shares sequence homology with a stretch of neurofilament-medium (NF-M), a cytoskeletal protein expressed in neurons and axons. In addition, this epitope derived from NF-M shares 2D2 TcR contact residues with MOG35-55 when presented in the context of I-Ab. Surprisingly, NF-M15-35 peptide showed heteroclitic response when presented to 2D2 T cells. To test the in-vivo relevance of this observation, we transfer in vitro differentiated Th1 cells from 2D2-Rag2-/- mice in C57BL/6, MOG-/-, and MOG-/- x NF-M-/- mice. We observed no clinical sign of disease in MOG-/- x NF-M-/-

mice, while MOG-/- mice developed slowly progressive disease with delayed onset, as compared to C57BL/6 mice, which developed early classical EAE, suggesting that recognition of MOG and NF-M by the 2D2 CD4+ T cells contribute to disease phenotype and severity.

We refer to this novel observation of self-mimicry, as ‘Cumulative autoimmunity’ where an autoimmune response target two self-autoantigens in the same tissue, resulting in enhanced CNS tissue damage.

1

Acknowledgements

It is an honor for me to thank to Prof. Joost van Meerwijk for his kind

acceptance to be the president on my thesis defense. I would like to thank to Prof.

Peter van Endert and Prof. Burkhard Becher for their kind acceptance to be the

rapporteur for my defense.

I am sincerely grateful to my thesis Director, Prof. Roland Liblau, whose

encouragement, supervision and support from initial to the final level enabled me to

develop an understanding of the subject.

I owe my deepest gratitude to Dr. Lennart Mars, who was generously helpful

and offered invaluable intellectual assistance, support and guidance.

I would like to thank to Dr. Daniel Dunia and Dr. Abdel Saoudi, who spent

their time and shared their knowledge for helping me to complete my thesis. I would

like to extend my thanks to all members of the lab and unit, who have helped me right

from the beginning in accomplishing my pleasant and fruitful stay in the lab. I

appreciate the help of Dr. Cassan during initial days.

I would like to thanks Dr. Kerschen, for his help and support during his stay in

the lab. I would like to thank to Dr. Lazarczyk, Dr. Singh, Dr. Sharma, Dr. Mishra

and Dr. Zein for their friendly support. I offer my regards to all of those who directly

or indirectly become the part of my thesis.

I would like to express my gratitude to everyone from the University Paul

Sabatier, animal house facility, FACS facility, administrative department, and ADR. I

would also like to convey my thanks to French ministry, INSERM, ARSEP and FRM

for providing the financial support and laboratory facilities.

Finally I would like to express my gratitude to my beloved family for their

understanding and endless love, throughout the duration of my studies.

I wish to dedicate my thesis to my beloved family!

2

Table of content

Abstract …………………………………………….......................... 1

Acknowledgements …………………………………………….......................... 2

Table of content …………………………………………….......................... 3

List of figures …………………………………………….......................... 6

Abbreviations …………………………………………….......................... 7

1. Introduction: ………………………………………………………….... 9

1.1. Generation of T-cell responses: ……………………………................... 11

1.1.1. Structure of the major components involved in antigen recognition

1.1.1.1. The major histocompatibility complex ……. ………….………….11

1.1.1.2. The T cell receptor …………………………………….………….15

1.1.1.3. The CD3 complex ……………………………………..………….18

1.1.1.4. The CD4 & CD8 co-receptors ………………………..,………….20

1.1.2. Co-stimulation for naïve T cells and adhesion molecules

1.1.2.1. CD28 ………………………………………………..……………..22

1.1.2.2. Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4) ………..23

1.1.2.3. CD80/CD86 ………………………………………..…………….. 25

1.1.2.4. Inducible co-stimulatory molecule (ICOS) and its ligand (ICOSL).26

1.1.2.5. Programmed death-1 (PD-1) molecule …………….………..…….27

1.1.2.6. Adhesion molecules ……………………….……………………...29

1.1.3. T cell activation

1.1.3.1. Immunological synapse ……………………..…………………….32

1.1.3.2. TcR sensitivity …………………………………………………….34

1.1.4. Heterogeneity of T cell phenotype: Cytokines & T cell differentiation…...35

3

1.2. The T cell development & Immune tolerance: ………………….……………40

1.2.1. The thymic development of the T cell repertoire

1.2.1.1. T cells ontogeny ………………………………………………….. 40

1.2.1.2. Positive selection of thymocytes …………………………………..43

1.2.1.3. CD4 & CD8 lineage commitment ……………………………….. 44

1.2.2. Central tolerance:

1.2.2.1. Role of autoimmune regulator (AIRE) ……………………………48

1.2.2.2. Negative selection of thymocytes ………………………………...51

1.2.3. Peripheral tolerance

1.2.3.1. T cell ignorance …………………………………………………..52

1.2.3.2. T cell anergy …………………………………………………….. 53

1.2.3.3. Apoptosis or Deletion …………………………………………… 55

1.2.4. Regulatory T cells

1.2.4.1. Naturally occurring FoxP3+CD4+CD25+ regulatory T (Treg) cells .58

1.2.4.2. T regulatory type1 (TR1) cells ……………………………………..67

1.3. CNS inflammation and autoimmunity: ………………………………………68

1.3.1. Triggering auto-aggressive immune responses

1.3.1.1. Exposure of tissue-specific antigen ……………………………… 68

1.3.1.2. Cryptic epitope hypothesis ……………………………………….. 71

1.3.1.3. Molecular mimicry ……………………………………………….. 72

1.3.1.4. Dual TcR expression ………………………………………………75

1.3.2. Multiple Sclerosis

1.3.2.1. Generalities ……………………………………………………..…78

1.3.2.2. Animal model for MS: EAE ………………………………………82

1.3.2.3. Pathophysiology ……………………………………………….… 84

1.3.2.4. Effector mechanisms ……………………………………………...91

1.4. General aim of the Thesis …………………………………………..… 99

4

2. Results ……………………………………………………..…………………...100 2.1. Oligodendrocyte-specific CD8 T cells in CNS autoimmunity

2.1.1 Summary ………………………………………………………………101

2.1.2 Publication …………………………………………………………….102

2.2. Neural self-mimicry: ‘Cumulative Autoimmunity’

2.2.1 Summary ………………………………………………………………107

2.2.2 Publication …………………………………………………………….108

3. Discussion & Perspective ……………………………….……………..137

4. Bibliography ……………………………………………………….………150

5. Appendix ………………………………………….……………………….... 206

5

List of Figures

Figure 1 Architecture of MHC-like molecules ….. 13

Figure 2 Structural comparison of αβ and γδ TCRs ….. 17

Figure 3 The CD3 complex and T cell receptor signalling ….. 19

Figure 4 Dynamic processes of antigen recognition and T-cell activation ….. 33

by formation of microclusters and immunological synapse

Figure 5 The cytokine milieu determines CD4+ T cell differentiation ….. 37

and conversion

Figure 6 Different stages of thymocyte development ….. 42

Figure 7 The affinity model of thymocyte selection ….. 43

Figure 8 Environmental cues and nuclear factors that influence CD4/CD8 ….. 45

lineage choice

Figure 9 Aspects of the thymic medulla that are involved in central tolerance ….. 49

Figure 10 Cell surface markers for naturally arising CD4+ Treg cells ….. 60

Figure 11 Basic mechanisms of suppression used by Treg cells ….. 64

Figure 12 The evolution of the molecular mimicry concept and activation of ….. 73

autoreactive T cells via cross-reactivity with foreign antigens

Figure 13 Different scenarios for dual TcR T cells ….. 76

Figure 14 Geographical distribution of MS and migration ….. 79

Figure 15 Genes associated with risk of development of MS ….. 81

Figure 16 Immunopathogenesis of MS ….. 89

6

APC Antigen presenting cell

APL Altered peptide ligand

AIRE Autoimmune regulator

Bim Bcl-2-like protein

BBB Blood brain barrier

BAFF B-cell activating factor

C Constant

CD Cluster of differentiation

CDR Complementarity determining

region

CFA Complete freund’s adjuvant

CFSE 5,6-carboxy-succinimidyl-

flurosceine-ester

CL Clone

CSF cerebrospinal fluid

CTL Cytotoxic T lymphocyte

CTLA-4 Cytotoxic-T-lymphocyte-

associated Antigen-4

D Diversity

DAG Diacylglycerol

DC Dendritic cell

DN Double negative

DNA Deoxyribonucleic acid

DP Double positive

EAE Experimental autoimmune

encephalomyelitis

ERK Extracellular-signal regulated

kinase

Fab Fragment antigen binding

FACS Fluorescent activated cell sorting

FCS Fetal calf serum

FoxP Forkhead box protein

GATA Trans-acting T-cell-specific

transcription factor

GAD Glutamic acid decarboxylase

G-CSF Granulocyte colony stimulating

factor

GTP Guanosine 5’-triphosphate

HA Hemagglutinin antigen

H&E Haemotoxylin and Eosin

HEL Hen egg lysozyme

HLA Human leukocyte antigen

GAD Glutamic acid decarboxylase

GM-CSF Granulocyte/Macrophage-colony

stimulating factor

ICAM Intercellular adhesion molecule

ICOS Inducible costimulator

IDDM Insulin dependent diabetes

mellitus

IDO Indoleamine 2,3-dioxygenase

IFN Interferon

Ig Immunoglobulin

IL Interleukin

IS Immunological synapse

ITAM Immunoreceptor tyrosine based

activation motifs

ITIM Immunoreceptor tyrosine based

inhibition motifs

ITSM Immunoreceptor tyrosine-based

switch motif

J Joining

JNK c-Jun-N-terminal kinase

kD Kilodalton

LAT Linker of activated T cells

LFA Lymphocyte function-associated

antigen

LN Lymph node

LPAM Lymphocyte peyer’s patch

adhesion molecule-1

MAPK Mitogen-activated Protein-

Kinase

MAdCAM Mucosal addressin cell adhesion

molecule

MAG Myelin associated glycoprotein

Abbreviations

7

MBP Myelin basic protein

M-CSF Macrophage-colony stimulating

factor

MIP Macrophage inflammatory

protein

MHC Major histocompatibility

complex

MMP matrix metalloproteinases

MOG Myelin oligodendrocyte protein

MS Multiple sclerosis

NO Nitric oxide

NOD Non obese diabetic

NFAT Nuclear factor of activated T

cells

NK Natural killer

NOD Non-obese diabetic

ODC Oligodendrocyte

p Peptide

PBMC Peripheral blood mononuclear

cells

PBS Phosphate Buffer Saline

PCR Polymerase chain reaction

PKC Protein Kinase C

PLC Phosphoinositide-specific

phospholipase C

PLP Proteolipid protein

PMA Phorbol-12-myristate-13-acetate

PTK Protein tyrosine kinase

RAG Recombinase activating gene

RANTES Regulated upon activation,

Normal T expressed, and

secreted

RNA Ribonucleic acid

ROR Retinoid-related orphan receptor

RPMI Roswell oak memorial institute

medium

RT Room temperature

Runx Runt-related transcription factor

SH Src homology

SLE Systemic lupus erythematosus

SLP SH2 domain containing

leukocyte protein

SP Single positive

TSA Tissue specific antigen

TcR T cell receptor

TGF Transforming growth factor

Th T helper

ThPOK Zinc finger and BTB domain

containing 7B

TN Triple negative

TNF Tumor necrosis factor

Treg Regulatory T cell

V Variable

VCAM Vascular cell adhesion molecule

VLA Very late antigen

ZAP Zeta associated protein

Abbreviations

8

Introduction

Introduction

9

Introduction

1. Introduction:

The immune system has two major components, an innate arm and an adaptive arm.

The innate immune system is a universal and ancient form of host defense against infection.

Innate immune system recognition relies on a limited number of germline-encoded

receptors. These receptors evolved to recognize conserved metabolites produced by

pathogens, but not by the host. Recognition of these molecular structures allows the

immune system to distinguish infectious nonself from noninfectious self. Innate immunity

covers many areas of host defense against pathogenic microbes, including the recognition

of pathogen-associated molecular patterns (PAMPs) (Janeway, 1989). In contrast, the

adaptive immune system involves great variability and rearrangement of receptor gene

segments to generate receptors, which yield myriad of antibodies or T cell receptors (TcRs)

of exquisite specificity for each of potential antigens, additionally the adaptive immune

system is characterized by immunological memory. However, the adaptive immune

response is also responsible for allergy, autoimmunity, and the rejection of allograft.

Because the innate immune responses do not depend on gene rearrangements, they

occur much more rapidly than adaptive immune responses and do so in more stereotyped

way, without tailoring a specific receptor to neutralize the danger signal in the form of

antigen. Instead, in innate immunity, the danger signal (a motif from infectious microbes)

can trigger toll-like receptors (TLRs), C-type lectin receptors (CLRs) to a membrane-based

sensor of danger (Hemmi et al., 2000). In addition, the innate immune system has a

cytosolic system to sense danger, the Nod-like receptors (NLRs), an intracellular system.

The inflammosome is where signals from the TLR system and the NLR system are

integrated. The major constituent of the inflammosome is the cytokine interleukin-1

(Church et al., 2008; Lamkanfi and Dixit, 2009; Martinon et al., 2009). The innate immune

system is made of many cell types, such as macrophages, dendritic cells (DCs), mast cells,

neutrophils, eosinophils and NK cells. These cells can become activated during an

inflammatory response, which is a consistent sign of infection with a pathogenic microbe.

Such cells rapidly differentiate into short-lived effector cells whose main role is to get rid of

the infection. However, in certain cases, the innate immune system is unable to deal with

the infection, and activation of an adaptive immune response becomes necessary. In these

cases, the innate immune system can instruct the adaptive immune system about the nature

10

Introduction

of the pathogenic challenge. It does so through cytokines and chemokines and the

expression of costimulatory molecules, such as CD80 and CD86, on the surface of

specialized antigen-presenting cells (APCs), with DCs as the most important ones that

alarm from infection in virtually all tissues (Banchereau and Steinman, 1998; Fearon and

Locksley, 1996; Janeway, 1989).

The central nervous system (CNS) provides special anatomic barriers to the

movement of cells from the vascular compartment to the parenchyma. A group of

autoimmune diseases of the brain and spinal cord that represents useful model for constitute

a situation which involve antigen-specific T cells and an orchestrated antibody response to

CNS components of the brain or spinal cord. My work is mainly focus on the autoimmune

diseases where the brain and spinal cord itself are attacked.

1.1 Generation of T-cell responses:

1.1.1. Structure of the major components involved in antigen recognition

1.1.1.1 The major histocompatibility complex

The major histocompatibility locus has evolved to be polygenic and highly

polymorphic, and is located on the short arm of chromosome 6 in humans, and on

chromosome 17 in mice (Artzt, 1986; Klein et al., 1982; Ziegler, 1997). In humans the

major histocompatibility complex (MHC) I locus contains 3 classical class I genes, named

human leukocyte antigen-A (HLA-A), HLA-B, HLA-C and non-classical HLA-E and

HLA-G, while the MHC II locus comprises HLA-DR, HLA-DQ and HLA-DP. In mice, the

MHC I locus contains 3 main genes, H-2L, H-2D and H-2K, while the MHC II region

comprises 2 main genes H-2IE and H-2IA, generally abbreviated to IE and IA (Bodmer et

al., 1997; Trowsdale and Campbell, 1992).

Both classes of MHC molecules are heterodimers with similar architectures and are

composed of three domains, one α-helix/β-sheet superdomain that forms the peptide-

binding site and two immunoglobulin (Ig)-like domains (Fig.1) (Bjorkman et al., 1987b;

Brown et al., 1993; Fremont et al., 1996; Madden et al., 1993; Matsumura et al., 1992; Stern

and Wiley, 1994). The overall architecture is the same in both MHC classes, where a seven-

11

Introduction

stranded β-sheet represents the floor of the peptide-binding groove, and the sides are

formed by two long α-helices, that straddle the β-sheet.

In MHC class I molecules, the peptide-binding site is constructed from the heavy

chain only, and an additional 12-kDa light chain subunit, β2-microglobulin (β2m), associates

with the α3 domain of the heavy chain (Fig.1). β2m is a nonglycosylated protein of about

100 amino acids encoded on chromosome 15 in humans and chromosome 2 in mice. Only

one allele is known in humans, while there are seven in mice. There appears to be little

functional significance of the polymorphisms in β2m, although they may subtly affect MHC

class I structure or peptide binding because different β2m alleles can alter cytotoxic T

lymphocyte (CTL) recognition of some antigens (Perarnau et al., 1990). β2m has no

transmembrane domain and is not covalently associated with the extracellular region of the

heavy chain. Because of this, the conformation of the heavy chain is highly dependent on

the presence or absence of β2m chain. The membrane-proximal α3 region of the heavy chain

is an Ig-like domain that contains a binding site for the CD8 receptor on CTL. The α1 and

α2 domains, which are distal to the membrane and interact with the TcR on CTL, fold

together to form a groove that binds and displays peptides. A β-pleated sheet forms the base

of the cleft, and the walls are made of two α helices. The allelic polymorphisms in heavy

chain primarily occur in those residues in and around this cleft, and in this manner, they

alter the peptide-binding specificity of the class I molecules (Bjorkman et al., 1987a;

Parham et al., 1995). The crystal structure of the MHC class I proteins revealed how a

single type of molecule could bind and present so many different peptides. Many of the

molecular interactions are with the peptide's main chain atoms and amino and carboxy

termini, which are features common to all peptides. In addition a limited number of peptide

side-chains extend into pockets along the groove thus imparting some specificity to peptide

binding, although many different peptides can be accommodated (Wilson and Fremont,

1993), and determine general motifs of the peptides capable of binding to particular MHC

class I molecules (Falk et al., 1991). The groove is generally long enough to accommodate

8 or 9 residues in an extended conformation (Madden et al., 1991) with the termini and the

so-called anchor residues buried in specificity pockets that differ from allele to allele

(Fremont et al., 1992; Madden et al., 1993). This binding mode leaves the upward-pointing

peptide side chains available for direct interaction with the TcR. Longer peptides can either

12

Introduction

bind by extension at the C terminus (Stern et al., 1994) or due to the fixing of their termini,

bulge out of the binding groove, providing additional surface area for TcR recognition

(Speir et al., 2001; Tynan et al., 2005).

(a) MHC I (b) MHC II (c) CD1

Figure 1: Architecture of MHC-like molecules

(a) Class I molecules consist of a heavy chain (blue) and a light β2m chain (orange). The peptide-binding site is formed exclusively by elements of the heavy chain (b) Class II molecules; the peptide-binding site is assembled of both subunits. (Rudolph et. al. 2006) (c) Nonclassical MHC molecule mouse CD1d crystal structure loaded with a synthetic variant of alpha-galactosylceramide (alpha-GalCer) (Barral et. al. 2007)

In contrast, the MHC class II molecule are assembled from two heavy chains (αβ) in

which the peptide-binding groove is open at either end, and the peptide termini are not fixed

so that bound peptides are usually significantly longer than in MHC class I (Fig.1). The

MHC class II allows presentation of peptides of 13-18 residues. The peptide backbone in

MHC class II is confined mainly to a poly-proline type II conformation (Stern et al., 1994)

and resides slightly deeper in the binding groove. Thus, the bound peptide is more

accessible for TcR inspection in MHC class I due to its ability to bulge out of the groove,

even for 9-mer peptides, however, in MHC class II the termini particularly the N-terminal

extension, can play a major role in the TcR interaction.

T cells are sensitive to small numbers of antigenic pMHC ligands that are distributed

among an excess of endogenous pMHC complexes on the surface of APCs. Recognition of

antigens in the peptide-binding groove of surface-expressed MHC class I and class II

13

Introduction

molecules by specific TcRs is central to T-cell activation. Two distinct pathways are used

by MHC class I and class II molecules for the presentation of peptide antigens to CD8+ and

CD4+ T cells, respectively (Ackerman and Cresswell, 2004; Kloetzel and Ossendorp, 2004;

Rock et al., 2004; Watts, 2004). The MHC class I antigen presentation pathway is active in

almost all cell types, providing a mechanism for displaying at the cell surface a sample of

peptides derived from proteins that are being synthesized in the cell and are acquired during

initial assembly in the endoplasmic reticulum (Cresswell et al., 2005; Kloetzel and

Ossendorp, 2004; Rock et al., 2004). The MHC class II pathway is constitutively active

only in professional APCs, including DCs, B cells, macrophages and thymic epithelial cells.

The peptide presented by MHC class II molecules are derived from proteins that gain access

to endosomal compartments, providing a way for CD4+ T cells to respond to exogenous

antigens internalized by APCs. The α-chain and β-chain of MHC II molecules are

synthesized in the endoplasmic reticulum (ER) and associate with invariant chain (CD74)

for proper folding, trafficking and protection of the antigen-binding groove (Bryant and

Ploegh, 2004; Cresswell, 1996; Jensen et al., 1999; Watts, 2004).

A third lineage of antigen presenting molecules, distinct from MHC class I and

MHC class II has been identified as CD1, which involves CD1a, CD1b, CD1c, CD1d and

CD1e, and present both foreign and self-lipid antigens to T cells. The CD1 proteins share

sequence homology and overall domain structure with MHC class I molecules, being

constitute of a heavy chain with three extracellular domains that are non-covalently

associated with β2m (Zeng et al., 1997) (Fig.1). On the basis of sequence analysis, the CD1

isoforms can be classified into three groups: group 1 constitutes of CD1a, CD1b and CD1c;

group 2 constitutes CD1d; and group 3 constitutes CD1e. In humans all five CD1 isoforms

(CD1a–CD1e) have been identified, whereas muroid rodents express only CD1d that

include CD1d1 and CD1d2 (Porcelli, 1995). Group 1 CD1 molecules mainly present lipid

antigens to clonally diverse T cells that mediate adaptive immunity to the vast range of

microbial lipid antigens (Beckman et al., 1994; Sieling et al., 1995). By contrast, CD1d

molecules present lipid antigens to natural killer T (NKT) cells (Bendelac et al., 1995).

14

Introduction

1.1.1.2 The T cell receptor

TcRs are cell surface heterodimers consisting of either disulfide-linked α and β or

γ and δ -chains (Brenner et al., 1986; Chien et al., 1987; Hedrick et al., 1984; Koning et al.,

1987; Saito et al., 1984; Winoto and Baltimore, 1989; Yanagi et al., 1984). Sequence

analyses correctly predicted that TcRs would share a domain organization and binding

mode similar to those of antibody Fab fragments (Davis and Bjorkman, 1988).

The αβ TcR comprises 2 membrane proximal constant (C) regions and 2 membrane

distal variable (V) Ig-like domain regions (Garboczi et al., 1996; Garcia et al., 1996a). The

pairing of the membrane distal α and β chain variable regions resembles the Ig variable

domain, such that the αβ combining site resembles the Ig-combining site (Fig.2). The loops

connecting the β sheets located within the pMHC contact area consists of regions with

highly variable amino acid sequences, called complementarity-determining regions (CDRs).

The αβ TcR binds pMHC with relatively low affinity (1–100µM) through CDRs presented

in their variable domains.

Interaction between TcR and pMHC based on the three-dimensional structure of the

pMHC moiety and X-ray crystallographic structure (Davis and Bjorkman, 1988; Machius et

al., 2001) show that CDR1 and CDR2 of both the α and β chains of the TcR interact

preferentially with the helices of the MHC molecule and that contacts to the bound peptide

are made essentially by CDR3 alone (Garboczi et al., 1996; Garcia et al., 1996a; Teng et al.,

1998; Wilson and Garcia, 1997).

The first crystal structures of TcRs with MHC class I molecules led to proposals that

the TcR orientation is approximately diagonal with a mean around 35° (Rudolph and

Wilson, 2002). By contrast, in the first MHC class II complexes, the orientation was

described as being closer to 90° (Hennecke et al., 2000; Reinherz et al., 1999) suggesting a

different binding mode between the MHC classes (Wang and Reinherz, 2002).

Insight into the structural changes that supplement TcR-pMHC engagement must

include crystal structures of the same TcR in its free and bound forms or of the same TcR

bound to different pMHCs. Until recently, only two well-studied systems, the 2C and A6

TcRs, fullfilled these requirements. The 2C system allowed comparison of the free 2C TcR

(Garcia et al., 1996a) with an agonist (Garcia et al., 1998) and a superagonist peptide

(Degano et al., 2000) in complex with the same H-2Kb MHC. This comparison disclosed a

15

Introduction

functional hotspot between the CDR3 loops in the 2C TcR that finely discriminated

between side chains and conformations of centrally located peptide residues through

increased complementarity and additional hydrogen bonds. In the A6 system, altered

peptide ligands (APLs) induced only subtle conformational changes in the TcR. In both the

2C and A6 systems, conformational changes are restricted mainly to the CDR3 loop

regions, and the largest conformational differences were observed when comparing free

versus bound TcRs (Rudolph and Wilson, 2002).

Many studies revealed substantial degeneracy in TcR recognition (Hemmer et al.,

1998; Hunt et al., 1992; Loftus et al., 1999; Pinilla et al., 1999). Multiple structures

comparing bound and unbound TcRs have shown, that the CDR 3 loops are flexible (Garcia

et al., 1998; Reiser et al., 2003). Thus, the conformational flexibility of these regions may

enable polyspecificity through induced-fit recognition of multiple peptides within the

binding cleft of the same MHC molecule.

16

Introduction

Figure 2: Structural comparison of αβ and γδ TCRs.

The top panel is a view down into the binding site, highlighted by the CDR loops. The bottom panel is rotated 90° around the horizontal axis. The light and heavy chains are shaded in light and dark gray, respectively. (Wilson et al. 2002)

Compared with αβ TcRs, much less is known about γδ TcRs (Fig.2). The biological

function of the γδ TcRs is also ill defined. γδ T cells appear to respond to bacterial and

parasitic infections (Morita et al., 1995) and primarily recognize phosphate-containing

antigens (phosphoantigens) from mycobacteria by an unknown mechanism (Belmant et al.,

1999; Morita et al., 1995).

17

Introduction

1.1.1.3 The CD3 complex

The TcR is a complex consisting of the variable αβ chains noncovalently associated

with the nonpolymorphic CD3 proteins. The CD3 proteins exist as a series of dimers

including γε, δε, and ζζ associated with a single αβ heterodimer. These subunits lack

enzymatic activity, but transduce signals via their immunoreceptor tyrosine-based activation

motifs (ITAMs) (Reth, 1989). Tyrosine phosphorylation of the ITAMs serves as docking

sites for interactions with other proteins. The earliest step in intracellular signaling

following TcR ligation is the activation of src-family kinases p56lck and p59Fyn protein

tyrosine kinases (PTKs), leading to phosphorylation of the CD3 ITAMs, followed by

recruitment of Syk kinase family member ZAP-70 (ζ-associated phosphoprotein of 70 kDa)

(Fig.3).

The phosphorylated CD3ζ is a recruitment site of the ZAP-70 PTK (Chan et al.,

1992). The engagement of the TcR leads to Src family PTK activity resulting in ITAM

phosphorylation and recruitment of ZAP-70. This converted the TcR: CD3 with no intrinsic

enzymatic function into an active PTK associated molecular complex able to phosphorylate

a spectrum of substrates leading to a myriad of downstream signals that, when integrated

appropriately along with signals from other co-receptors, lead to T cell activation

(Iwashima et al., 1994).

ZAP-70 targets are the transmembrane adapter protein linker for the activation of T

cells (LAT) and the cytosolic adapter protein Src homology2 (SH2) domain–containing

leukocyte phosphoprotein of 76 kDa (SLP-76) (Bubeck Wardenburg et al., 1996; Zhang et

al., 1998). These two adapters form the backbone of the complex that organizes effector

molecules in a way that allows the activation of multiple signaling pathways. The loss of

either LAT or SLP-76 results in a nearly complete loss of TcR signal transduction

reminiscent of Syk/ZAP-70 or Lck/Fyn double-deficient T cells (Koretzky et al., 2006;

Sommers et al., 2004; Zhang et al., 1999). LAT contains nine tyrosines that are

phosphorylated upon TcR engagement, which bind the C-terminal SH2 domain of PLCγ1,

the p85 subunit of phosphoinositide 3-kinase (PI3K), and the adapters growth factor

receptor-bound protein 2 (GRB2) and GRB2-related adapter downstream of Shc (Gads)

(Sommers et al., 2004). SLP-76 is then recruited to phosphorylated LAT via their mutual

binding partner Gads (Liu et al., 1999). This proximal signaling complex results in the

18

Introduction

activation of PLCγ1-dependent pathways including Ca2+ and diacylglycerol (DAG) induced

responses, cytoskeletal rearrangements, and integrin activation pathways.

Following TcR ligation, PLCγ1 is found in the proximal signaling complex bound to

SLP-76, Vav1, and LAT, where it is phosphorylated and activated by Itk. Activated PLCγ1

then hydrolyzes the membrane lipid producing the second messengers IP3 (inositol 1,4,5-

triphosphate) and DAG. These two messengers are essential for T cell function (Fig.3).

Figure 3: The CD3 complex and T cell receptor signalling

Ligation of the TcR/CD3 results in activation of Src and Syk family PTKs associated with the intracellular CD3 domains that then activate PLCγ1 and Ras-dependent pathways (a) TcR signalling in mid 1990s (b) How the TcR couples to downstream pathways (c) The molecular basis for Ca2+ influx (d) The positive feedback loop responsible for Ras activation. (Smith-Garvin et.al. 2009)

19

Introduction

Ca2+ ions are universal second messengers in eukaryotic cells. The IP3 generated by

TcR-stimulated PLCγ1 activity stimulates Ca2+ permeable ion channel receptors (IP3R) on

the ER membrane, leading to the release of ER Ca2+ stores into the cytoplasm. Depletion of

ER Ca2+ triggers a sustained influx of extracellular Ca2+ through the activation of plasma

membrane Ca2+ release-activated Ca2+ (CRAC) channels in a process known as store-

operated Ca2+ entry (SOCE) (Oh-hora and Rao, 2008).

TcR-induced production of DAG results in the activation of two major pathways

involving Ras and PKCθ. Ras is a guanine nucleotide–binding protein and is required for

the activation of the serine-threonine kinase Raf-1, which initiates a mitogen-associated

protein kinase (MAPK) phosphorylation and activation cascade (Genot and Cantrell, 2000).

1.1.1.4 The CD4 and CD8 co-receptors

The two major subsets of T lymphocytes in the peripheral immune system, the

helper and cytotoxic T cells, are defined by their expression of either the CD4 or the CD8

glycoprotein, respectively. Expression of these molecules, which serve as coreceptors by

interacting with membrane-proximal domains of the MHC molecules (Garcia et al., 1996b;

Genot and Cantrell, 2000), thus permitting simultaneous interaction of the TcR and either

CD4 or CD8 with MHC.

The CD4 is an integral membrane glycoprotein of 55-60 kDa. The primary structure

in humans was deduced from T cell cDNA libraries (Maddon et al., 1987), and indicated a

protein of 458 amino acids. The first 25 amino acids include a cleavage signal, which after

cleavage results in a mature protein of 433 amino acids. Aligning the cDNA sequence with

X-ray crystallography indicated, that the 4 domains of CD4 are similar to the Ig superfamily

(Brady and Barclay, 1996; Maddon et al., 1987). Domain 1 and 3 were classified as variable

(V) domains due to their similarities with the Ig variable region, domain 2 and 4 were

classified as constant (C) domains (Williams and Barclay, 1988). The two membrane distal

domains (D1 and D2) are, like the two membrane proximal domains (D3 and D4), rigidly

packed together, whereas the connection between domain 2 and 3 is flexible. Interestingly,

crystallography revealed CD4 to dimerise via cross-binding of D4 (Wu et al., 1997).

CD8 is a transmembrane glycoprotein composed of a CD8 αα homodimer or a CD8

αβ heterodimer. Each CD8 monomer, CD8α or CD8β, consists of an Ig-V-like domain, an

20

Introduction

extended O-glycosylated stalk region, a single-pass transmembrane domain, and a short

cytoplasmic region. The Ig domains of CD8 bind MHC class I at a site distant from the

TcR-binding site (Chang et al., 2005). CD8αα and CD8αβ bind most but not all MHC class

I and class I-like molecules and usually with a similar affinity/avidity (Kern et al., 1999). A

notable exception is the tenfold higher affinity of CD8αα to the mouse MHC I-like protein,

thymus-leukemia, than CD8αβ (Leishman et al., 2001).

The co-receptor model proposed CD4 and CD8 to function as co-receptors able to

migrate to and interact with the TcR, as well as transduce an independent signal necessary

for complete TcR activation (Janeway et al., 1989). In accordance with this model, D3 of

CD4 has been identified to interact with the TcR/CD3 complex (Mittler et al., 1989; Vignali

et al., 1996; Vignali and Vignali, 1999), whereas D1 binds monomorphic regions on the

MHC class II molecule (Doyle and Strominger, 1987; Fleury et al., 1996). Similarly, the

CD8 co-receptor interacts with MHC class I (Gao et al., 1997).

The T-cell immune response is initiated upon engagement of the TcR and coreceptor

CD4 or CD8, by cognate pMHC complexes presented by APCs. TcR/coreceptor

engagement induces the activation of biochemical signaling pathways that, in combination

with signals from costimulatory molecules and cytokine receptors, direct the outcome of the

response. The intracellular region of CD4 and CD8 binds to Lck (Rudd et al., 1988;

Veillette et al., 1988) and Fyn (zur Hausen et al., 1997). Activation of the these two kinases

is central to the initiation of TcR signaling pathways, and their regulation is tightly

controlled by conformational changes arising from binding of ligands to the SH3 and/or

SH2 domains of the kinases (da Silva et al., 1997; Holdorf et al., 1999; Xu et al., 1999), and

by the phosphorylation and dephosphorylation of two critical tyrosine residues (Palacios

and Weiss, 2004).

A second intracellular signaling component, the LAT, was also found to co-

immunoprecipitate with CD4 and CD8 (Bosselut et al., 1999; Kim et al., 2003).

21

Introduction

1.1.2 Co-stimulation governing the activation of T cells

T-cell activation is mediated by antigen-specific signals from the TcRζ/CD3 and

CD4-CD8-p56lck complexes in combination with additional co-signals provided by

coreceptors such as CD28, inducible costimulator (ICOS), cytotoxic T-lymphocyte antigen-

4 (CTLA-4), programmed death (PD-1), and others. In general terms, CD28 and ICOS

provide positive signals that promote and sustain T-cell responses, while CTLA-4 and PD-1

limit responses. The balance between stimulatory and inhibitory co-signals determines the

ultimate nature of T-cell responses where response to foreign pathogen is achieved without

excessive inflammation or autoimmunity.

1.1.2.1 CD28

CD28 is the most characterized of the Ig family of coreceptors possessing an

intracellular domain with several residues, which are critical for its effective signalling. In

particular, the YMNM motif beginning at tyrosine 170 is critical for the recruitment of

SH2-domain containing proteins, especially PI3K, GRB2 and Gads. CD28 is a 44 kDa

homodimer expressed on both, naïve and activated T cells and binds the ligands B7-1

(CD80) and B7-2 (CD86), using a signature MYPPPY binding motif (Balzano et al., 1992).

Its gene is located on human chromosome 2q33 (Naluai et al., 2000) and mouse

chromosome 1 (Howard et al., 1991).

T-cell activation can occur with a potent TcR signal alone (i.e. high avidity TcR-

pMHC interaction and/or high ligand density), however CD28 coligation is required in most

responses to peptide antigens (Bluestone, 1995; Linsley, 1995; Noel et al., 1996). Without

CD28 coligation, TcR-engagement often induces a non-responsive, anergic state or cell

death (Linsley and Ledbetter, 1993). T cells from CD28-deficient mice show reduced

proliferation in response to peptide antigens (Shahinian et al., 1993), although repeated

antigen stimulation or long-term viral infection can bypass the requirement of CD28

(Kundig et al., 1996). CD28 co-signals can stabilize the mRNA of cytokines and amplify

the activation of nuclear factor of activated T cells (NFAT) and nuclear factor kappa B (NF-

κB) (Kalli et al., 1998; Karin and Ben-Neriah, 2000; Rincon and Flavell, 1994).

22

Introduction

CD28 engagement enhances the production of various cytokines, including IL-1, IL-

2, IL-4, IL-5, TNF, and IFN-γ (Linsley and Ledbetter, 1993; McArthur and Raulet, 1993;

Thompson et al., 1989). In addition it has been shown, that CD28 engagement plays a vital

role in the early development and differentiation of both, Th1 and Th2 T cell subsets (King

et al., 1995; Seder et al., 1994; Van der Pouw-Kraan et al., 1992). In the absence of CD28

signaling, naive T cells are biased towards a Th1 phenotype and no IL-4 is observed when

CD28/B7 interactions are blocked with hCTLA4-Ig (Seder et al., 1994). Recently, it has

been shown that CD28 costimulation downregulate Th17 development in-vitro

(Bouguermouh et al., 2009). Furthermore, CD28 is also important in the homeostasis and

function of regulatory T cells (Tregs), the importance of CD28 signaling in Treg

proliferation is suggested by the effectiveness of anti-CD3 and anti-CD28 treatment to

expand Tregs in-vitro. Accordingly, both mouse and human Tregs can be expanded using

CD3 and CD28 antibodies in the presence of high concentrations of IL-2 (Earle et al., 2005;

Tang et al., 2004). It is also established that mice lacking CD28 or its ligands have

decreased numbers of CD4+CD25+ regulatory T cells (Lohr et al., 2003; Salomon et al.,

2000). In addition CD80/86-blockade experiments have suggested that continual expression

of CD28 ligand in the periphery is necessary to maintain the peripheral Treg compartment

(Salomon et al., 2000).

1.1.2.2 Cytotoxic T lymphocyte-associated antigen-4 (CTLA-4)

CTLA-4, also known as CD152, is an activation-induced type 1 transmembrane

glycoprotein of the Ig superfamily (Brunet et al., 1987). The CTLA-4 gene is located on

chromosome 2 in humans and on chromosome 1 in mice (Dariavach et al., 1988; Ling et al.,

1999). The CTLA-4 gene product contains 223 amino acids, with a 35 amino acid signal

peptide (Brunet et al., 1987; Dariavach et al., 1988), and is found as a covalent homodimer

of 41–43 kDa (Lindsten et al., 1993; Linsley et al., 1995).

The extracellular architecture of CTLA-4 is characterized by a single Ig V-like

domain containing the CD80/CD86 binding site (Brunet et al., 1987; Dariavach et al., 1988;

Linsley et al., 1995; Metzler et al., 1997; Ostrov et al., 2000). Homodimerization of CTLA-

4 is mediated by cysteine-dependent binding at position 122 in the stalk region and by N-

23

Introduction

glycosylation at positions 78 and 110 (Darlington et al., 2005; Linsley et al., 1995). The

simplicity of its structure disguises its critical function as a major negative regulator of T

cell–mediated immune responses (Alegre et al., 2001; Chambers et al., 2001; Rudd et al.,

2002; Salomon and Bluestone, 2001).

The structural similarities and sequence homology between CTLA-4 and CD28

extend to share natural ligands: the B7 molecules CD80 and CD86 (Freeman et al., 1993a;

Freeman et al., 1993b; Linsley et al., 1990). However, a key feature that may explain

CTLA-4's mechanism of action is that CTLA-4 binds CD80 and CD86 with greater affinity

and avidity compared to CD28 (Linsley et al., 1991; Linsley et al., 1994). In general,

CTLA-4 is thought to raise the threshold for TCR signaling, thereby preventing responses

to self-antigens (Chambers et al., 2001; Greenwald et al., 2005). Furthermore, cell extrinsic

and intrinsic mechanisms influence the CTLA-4 function. Cell extrinsic mechanisms

include the secretion of soluble CTLA-4 (sCTLA-4), the production of indoleamine 2,3-

dioxygenase (IDO) and the involvement of Tregs (Rudd, 2008). Cell intrinsic mechanisms

include CTLA-4 ectodomain competition with CD28 for the binding to CD80/86 (Masteller

et al., 2000), the presence of associated phosphatases (Chuang et al., 2000; Lee et al., 1998;

Marengere et al., 1996), blockade of lipid-raft expression (Chikuma et al., 2003; Martin et

al., 2001) and microcluster formation (Schneider et al., 2008). It is likely that most of these

factors contribute to the overall function of CTLA-4 in different contexts.

When engaged to B7, CTLA-4 plays a key role as a negative regulator of T cell

activation (Krummel and Allison, 1995; Walunas et al., 1994), leading to downregulation of

T cell responses and to the preservation of T cell homeostasis and peripheral tolerance

(Tivol and Gorski, 2002). The mechanism of T cell inactivation by CTLA-4 involves

antagonism of CD28-dependent costimulation and direct negative signaling through its

cytoplasmic tail (Carreno et al., 2000). CTLA-4 can exert its inhibitory effects in both,

CD80/CD86 dependent and independent fashions (Grohmann et al., 2002; Ise et al., 2010;

Vijayakrishnan et al., 2004).

CTLA-4 has an intrinsic plasticity for signaling. It refers to the ability of CTLA-4 to

actively deliver, by itself, a negative signal or a positive signal to a T cell, depending on the

ligand it engages and its association/dissociation to protein phosphatase-2A (PP2A). When

engaged to B7, it dissociates from PP2A and acts as an inactivator of T cells (Baroja et al.,

24

Introduction

2002). In contrast, when CTLA-4 is engaged to recombinant ligands 24 and 26 Abs, its

association to PP2A is stabilized and it acts as an activator of T cells (Madrenas et al.,

2004).

1.1.2.3 B7-1 (CD80) and B7-2 (CD86)

Co-signalling molecules are that, whose functions are entirely dependent on TcR

signals (Schwartz, 2003), and they control the TcR signals itself. In the absence of

sufficient TcR signalling, co-signalling molecules lose their function (Chen, 1998).

The two B7 family members, CD80 and CD86, bind to the same two receptors,

CD28 and CTLA-4 (Chikuma and Bluestone, 2003; Coyle and Gutierrez-Ramos, 2001;

Sharpe and Freeman, 2002). These receptors have distinct kinetics of expression and

affinities for CD80 and CD86 (van der Merwe and Davis, 2003). Two additional B7

homologs, B7-H3 (Chapoval et al., 2001) and B7-H4 (Sica et al., 2003) [B7x (Zang et al.,

2003), B7S1 (Prasad et al., 2003)], and another CD28 homolog, BTLA (B and T

lymphocyte attenuator) (Watanabe et al., 2003), have been identified.

CD80 and CD86 have a restricted expression pattern, being expressed mainly by

professional APCs and haematopoietic cells (Chen, 2004). CD80 and CD86 on APCs have

well-recognized roles as T cell costimulatory molecules, but the functional significance of

their expression on T cells is not well understood. CD86 is constitutively expressed on

some resting T cells, whereas CD80 is not present on resting T cells (Taylor et al., 2004).

CD80 and CD86 may influence immune responses through reverse signaling into B7-

expressing APCs (Fallarino et al., 2003; Grohmann et al., 2002; Munn et al., 2004). CD86

on B cells can signal bidirectionally, it stimulates CD28 on T cells and transduces positive

signals into B cells that increase IgG1 and IgE production (Podojil et al., 2004; Podojil and

Sanders, 2003). Basal or constitutive expression of B7 appears to be key for maintaining

regulatory T cell homeostasis and preventing immune responses against self-antigens.

In brief, CD28 first functions by costimulating T cells and preventing the induction

of either anergy or apoptosis (Linsley and Ledbetter, 1993). Second, a CD28 homologue,

CTLA-4, counterbalances CD28 by downregulating the immune response either by

inducing apoptosis or competing with CD28 for its ligands, as both form homodimers and

25

Introduction

bind the ligands CD80 and CD86, using a signature MYPPPY binding motif. CD80 binds

CTLA-4 with much lower affinity than CD28 (van der Merwe et al., 1997) . Third, two

distinct molecules, CD80 and CD86, function as costimulatory ligands for CD28 and

CTLA-4 (Linsley and Ledbetter, 1993). While CD86 dominates in primary responses, the

roles of CD80 and CD86 during an ongoing response depends on the relative expression of

CD28/CTLA-4, the nature and concentration of cytokines in the surrounding milieu, and the

characteristics of the APCs that are encountered. Finally, manipulation of the CD28/B7

pathway can alter the balance of the immune response by influencing the nature of the

cytokines produced in response to antigen exposure.

1.1.2.4 Inducible co-stimulatory molecule (ICOS) and its ligand (ICOSL)

ICOS is a glycosylated disulfide-linked homodimer (Rudd and Schneider, 2003) like

CD28 and CTLA-4. The ICOS cytoplasmic tail contains a YMFM motif that binds the p85

subunit of PI3K, similar to the YMNM motif of CD28 (Coyle et al., 2000), and stimulates

greater PI3K activity than CD28 costimulation (Okamoto et al., 2003; Parry et al., 2003).

However, in contrast to CD28, the ICOS YMFM motif does not allow the binding of

GRB2, which is critical for IL-2 production (Harada et al., 2001; Okamoto et al., 2003).

ICOS signals lead to effector cytokine production and can be inhibited by CTLA-4

engagement (Riley et al., 2001).

ICOS is upregulated on CD4+ and CD8+ T cells following activation and is

expressed on effector and memory T cells (Coyle et al., 2000; Yoshinaga et al., 1999). It is

also upregulated on activated NK cells, and promotes NK cell function (Ogasawara et al.,

2002). ICOS expression is stimulated on T cells by TcR and CD28 signals (Beier et al.,

2000; Coyle et al., 2000; Mages et al., 2000; McAdam et al., 2000). ICOS engagement was

shown initially to selectively produce high levels of IL-10 and IL-4, but in-vivo studies in

several different model systems have demonstrated that ICOS can stimulate production of

both, Th1 and Th2 cytokines during initial priming and during effector T cell responses

(Hutloff et al., 1999; McAdam et al., 2000).

ICOSL has been detected on the surface of T cells, B cells, macrophages, DCs and

other cell types including endothelial cells and some epithelial cells, and its mRNA is

expressed constitutively in lymphoid and nonlymphoid tissues including kidney, liver, lung,

26

Introduction

and testis (Richter et al., 2001; Swallow et al., 1999; Wang et al., 2000; Yoshinaga et al.,

2000). Costimulatory signals through ICOS provide critical T cell help to B cells (Hutloff et

al., 1999; McAdam et al., 2001; Tafuri et al., 2001).

ICOS and CD28 have unique and overlapping functions that synergize to regulate

CD4+ T cell differentiation. Like CD28, ICOS augments T cell differentiation and cytokine

production and provides signals for Ig production (Sharpe and Freeman, 2002; Sperling and

Bluestone, 2001). It also regulates the outcome of autoimmunity in the murine model of

multiple sclerosis (MS), experimental autoimmune encephalomyelitis (EAE) but plays

distinct roles at different times during the pathogenesis of EAE (Chapoval et al., 2001;

Rottman et al., 2001; Sporici et al., 2001). Blocking of ICOS during induction of EAE

exacerbates disease, whereas ICOS blocking during the effector phase of EAE ameliorates

disease (Dong and Nurieva, 2003; Rottman et al., 2001).

Both CD28 and ICOS signaling upregulate Th1 as well as Th2 cytokines; however,

ICOS does not upregulate IL-2 production. Thus, ICOS stimulates T cell effector function

but not T cell expansion. The role of ICOS in stimulating IL-10 production may contribute

to its role in regulating Treg, T cell tolerance, and autoimmunity. The function of ICOS

during in-vivo immune responses appears to depend on timing/stage of immune response as

well as microenvironment, because ICOSL can be expressed on endothelial and epithelial

cells as well as professional APCs.

1.1.2.5 Programmed death-1 (PD-1) molecule

PD-1 is a monomeric member (Freeman et al., 2000; Latchman et al., 2001; Zhang

et al., 2004) of the Ig superfamily (Ishida et al., 1992; Shinohara et al., 1994; Vibhakar et

al., 1997) related to CD28 and CTLA-4, but it lacks the membrane proximal cysteine that

allows these molecules to homodimerize. The PD-1 cytoplasmic domain has two tyrosines,

one that constitutes an immunoreceptor tyrosine-based inhibition motif (ITIM), and the

other an immunoreceptor tyrosine-based switch motif (ITSM) (Shlapatska et al., 2001). PD-

1 is expressed during thymic development primarily on CD4-CD8- cells and double

negative γδ thymocytes (Nishimura et al., 2000) and is induced on peripheral CD4+ and

27

Introduction

CD8+ T cells, B cells, and monocytes upon activation (Agata et al., 1996). NK-T cells also

express low levels of PD-1 (Nishimura et al., 2000).

PD-L1 is a 290 amino acid type I transmembrane protein encoded by the Cd274

gene on mouse chromosome 19 and human chromosome 9. The PD-1 ligands exhibit

distinct patterns of expression; PD-L1 is expressed more broadly than PD-L2 (Dong et al.,

1999; Freeman et al., 2000; Latchman et al., 2001; Tseng et al., 2001). PD-L1 is expressed

on resting cells and upregulated on activated B, T, myeloid, and DCs (Liang et al., 2003). In

contrast to CD80 and CD86, PD-L1 is expressed in nonhematopoietic cells including

microvascular endothelial cells and in nonlymphoid organs including heart, lung, pancreas,

muscle, and placenta (Ishida et al., 2002; Liang et al., 2003; Petroff et al., 2003; Rodig et

al., 2003; Wiendl et al., 2003). The expression of PD-L1 within nonlymphoid tissues

suggests, that PD-L1 may regulate self-reactive T or B cells in peripheral tissues and/or

may regulate inflammatory responses in the target organs.

PD-L2 is a type I transmembrane protein encoded by the Pdcd1lg2 gene adjacent to

Cd274 and separated by only 23 kb of intervening genomic DNA in mouse and 42 kb in

human. There are three PD-L2 splice variants identified from activated human PBMCs (He

et al., 2004; Latchman et al., 2001). PD-L2 is rarely expressed on resting cells and hardly

induced on B cells and T cells. PD-L2 can be induced on macrophages by IL-4 and IFNγ

and on DCs by anti-CD40 antibody, GM-CSF, IL-4, IFNγ and IL-12. It has been reported

that IL-4 induces PD-L2 more strongly than IFNγ, while IFNγ induce PD-L1 more strongly

than IL-4 on macrophages, suggesting that Th1 and Th2 responses mobilize PD-L1 and PD-

L2 differentially (Loke and Allison, 2003).

The lupus-like phenotype of PD-1-/- C57BL/6 mice first suggested a role for PD-1 in

the regulation T cell tolerance and autoimmunity, as these mice exhibit hyperactivation of

the immune system such as splenomegaly and in-vitro augmented proliferation of B cells

(Nishimura et al., 1999; Nishimura et al., 2001). Administration of anti-PD-1 or anti-PD-L1

mAb to one to ten-weeks old prediabetic NOD female mice led to the rapid onset of

diabetes, and it was associated with increased IFN-γ producing glutamate acid

decarboxylase (GAD) reactive splenocytes (Ansari et al., 2003). The expression of PD-L1

on islet cells suggests that it may critically control the responses of self-reactive T cells in

the target organ (Ansari et al., 2003; Liang et al., 2003). PD-1 and its ligands have a

28

Introduction

negative regulatory role in EAE (Latchman et al., 2004; Salama et al., 2003). PD-1, PD-L1,

and PD-L2 are expressed on brain infiltrating inflammatory cells in mice with EAE (Liang

et al., 2003; Salama et al., 2003). PD-L1 is also expressed on vascular endothelial cells,

astrocytes, and microglia. A role for PD-L1 in controlling the responses of self-reactive T

cells is further indicated by the severe clinical EAE that develops following immunization

of PD-L1-/- mice with the myelin oligodendrocyte glycoprotein 35-55 peptide (MOG35-55) or

following the adoptive transfer of MOG35-55 specific T cells into PD-L1-/- recipients

(Latchman et al., 2004).

The PD-1: PD-L1/PD-L2 pathway has critical roles in regulating T cell activation

and tolerance. CTLA-4 and PD-1 have important nonredundant inhibitory functions.

CTLA-4 appears to have a more central role in the lymphoid organs, whereas PD-1 has an

important role in regulating inflammatory responses in peripheral tissues.

1.1.2.6 Adhesion molecules

The continuous recirculation of naïve T cells and their subsequent migration to

tissues following activation is crucial for maintaining protective immunity against invading

pathogens. The preferential targeting of effector and memory T cells to tissues is instructed

during priming and mediated by cell surface expressed adhesion receptors such as integrins.

Integrins are αβ heterodimeric cell surface adhesion molecules that mediate cell-

extracellular matrix (ECM) and cell-cell interactions (Pribila et al., 2004), which are

essential for T cell recirculation, migration into inflammatory sites and for a specific and

effective immune response against foreign pathogens (Hynes, 2002; Shimizu et al., 1999).

Integrin-dependent interactions of lymphocytes and APCs to endothelium regulate the

efficiency and specificity of trafficking into secondary lymphoid organs and peripheral

tissue.

Naïve T cells express a homogeneous array of cell surface molecules that promotes

recirculation through the secondary lymphoid organs of the body, including the spleen,

peripheral lymph nodes (pLN), mesenteric lymph nodes (mLN) and Peyer’s patches (PP) of

the small intestine. Naïve T cells express low levels of the αLβ2 (LFA-1), α4β1 (VLA-4)

and α4β7 (LPAM) integrins, which bind ICAM-1, VCAM-1, and MAdCAM-1,

29

Introduction

respectively (Springer, 1995). The interaction of αLβ2 with ICAM-1 is important for T cell

entry into pLN and for T cell interactions with APCs. The α4β1 ligand VCAM-1 is

expressed at low levels throughout the vasculature, but becomes upregulated on a wide

array of tissue during inflammation (Henninger et al., 1997). MAdCAM-1, the major ligand

for α4β7 is preferentially expressed at steady state in the mLN and Peyer’s patches, where

it promotes entry of naïve T cells into these sites through a high affinity interaction (Berlin

et al., 1993; Erle et al., 1994; von Andrian and Mackay, 2000). Both VCAM-1 and ICAM-1

are also expressed on the vasculature of the inflamed brain. In particular, VCAM-1 is

known to promotes T cell entry through its interaction with α4β1 expressed on activated T

cells (Engelhardt and Ransohoff, 2005).

In addition to mediating adhesion to cell surface and extracellular matrix ligands,

integrins generate a diverse array of intracellular signals. In T cells, LFA-1 and several β1

integrins can provide costimulatory signals for TcR-induced T cell activation (Shiow et al.,

2006; Sprent et al., 1971). Differences in the regulation of IL-2 production by integrins and

CD28 following TcR stimulation demonstrate the unique costimulatory properties of these

molecules. In CD4 T cells, LFA-1-mediated costimulation enhanced IL-2 transcription,

which is not mediated by stabilization of IL-2 mRNA (Abraham and Miller, 2001), in

contrast, CD28 costimulation alters IL-2 levels by stabilizing IL-2 mRNA (Arnold et al.,

2004). Furthermore, LFA-1 and CD28 induce the activation of PI3-K in CD8 T cells

however; PI3-K activity is required only for LFA-1-meditated costimulation (Ni et al.,

2001).

In addition, osteopontin, also known as secreted phosphoprotein1 (SPP1), is a

member of SIBLING (small integrin-binding ligand, N-linked glycoproteins) family of

proteins (Fisher et al., 2001). It is highly expressed in MS lesions (Chabas et al., 2001).

Studies in MS tissue and in models of relapsing-remitting (RR) and progressive EAE

showed that osteopontin might have a role in the progression from RR disease to the more

chronic form (Chabas et al., 2001). Osteopontin expression is induced on inflamed

endothelium in the ECM of the perivascular cuff, and it serves as a ligand for two classes of

adhesion molecules, CD44 and various integrins including VLA-4 (Ashkar et al., 2000).

Furthermore, chemokine receptors served as adhesion molecules especially during

30

Introduction

leukocyte migration, by acting on firm adhesion, locomotion, diapedesis, and chemotaxis

(Cartier et al., 2005).

31

Introduction

1.1.3 T cells activation

1.1.3.1 Immunological synapse

Activation of T cells by APCs requires the formation of a specific and long-lasting

interface between the two cells, known as the immunological synapse (IS). TcR interaction

with pMHC complexes in the IS between the T cell and APC is required for T cell

activation (Norcross, 1984; Paul and Seder, 1994; Valitutti et al., 1995). The required

duration of signaling is on the order of hours, while the activating and inhibitory molecular

interactions in the IS have half-lives on the order of seconds (Iezzi et al., 1998; Matsui et

al., 1994). The mature IS has been defined by arrangement of supramolecular activation

clusters (SMACs) that form within a few minutes of T cell–APC contact (Grakoui et al.,

1999; Monks et al., 1998; Wulfing and Davis, 1998). Physiological T cell activation can be

divided into a series of temporal stages: T cell polarization, initial adhesion, IS formation,

and IS maturation (Grakoui et al., 1999) (Fig.4).

Circulating T cells are rounded and nonpolarized, with a uniform radial distribution

of membrane domains, receptors, and microvilli on the cell surface (Kucik et al., 1996;

Sanchez-Madrid and del Pozo, 1999). These cells are relatively nonmotile and integrin

adhesion molecules are maintained in a low-activity state (Kucik et al., 1996). In order to

extravasate into lymph nodes and inflamed tissues, these T cells must encounter chemokine

gradients before they encounter APC, leading to the rapid polarization of the T lymphocyte.

The close contact areas of IS between polarized T lymphocytes and APCs are established

by adhesion molecules. These close contact areas are achieved indirectly through

cytoskeletal protrusions anchored to larger adhesion mechanisms like LFA-1 and ICAM-1

(Grakoui et al., 1999) or more directly by smaller adhesion receptor pairs like CD2 and

CD58 that would work beside the TcR. CD2 and its ligand CD48 are associated with rafts

in the same fashion like TcR (Yashiro-Ohtani et al., 2000).

32

Introduction

Figure 4: Dynamic processes of antigen recognition and T-cell activation by formation

of microclusters and immunological synapse (Saito et.al. 2006)

Upon T-cell attachment, LFA-1/ICAM-1 complexes cluster in the center and the

MHC complexes are located at the periphery of the synapse. Later, TcR/MHC complexes

become clustered in the center, whereas the LFA-1/ICAM-1 complexes are segregated to

the periphery to form a ring around the TcR/MHC clusters (Grakoui et al., 1999), which is

facilitated by the CD2-CD48 interaction that provides the correct membrane alignment

(Dustin et al., 1996; Dustin et al., 1997; Wild et al., 1999). The formation of the central

cluster gives rise to the immunological synapse that will remain stable for over an hour.

Furthermore it has been shown by intravital two-photon laser scanning microscopy

that T cells undergo rapid, random amoeboid locomotion in secondary lymphoid tissues

(Miller et al., 2003; Miller et al., 2002b). This has been most intensively observed in mouse

lymph nodes, which are subjected to either explanted or in-vivo imaging by two-photon

laser scanning microscopy, which led to the model of stochastic repertoire scanning, in

which T cells, in the T-cell zones move rapidly with average speeds of 10–15 µm/min and

33

Introduction

peak speeds of over 30 µm/min. In another study, it has been shown by two-photon

imaging, that T-cell priming by DCs occurs in three successive stages: transient serial

encounters during the first activation phase are followed by a second phase of stable

contacts culminating in cytokine production, which makes a transition into a third phase of

high motility and rapid proliferation (Mempel et al., 2004).

1.1.3.2 TcR sensitivity

The sensitivity of the TcR for the pMHC complex plays a crucial role in T cell

activation. The TcR discriminates between peptides by as little as a single amino acid, as

was shown when a single amino acid mutation turned an agonistic peptide into an

antagonistic peptide (Allen et al., 1987). The spectrum of peptides presented by the MHC is

generally classified as full agonist, partial agonist APL, non-stimulatory, and antagonist

peptides. Full agonist peptides induce full activation of the peripheral T cells or negative

selection of thymocytes, partial agonist peptides induce incomplete peripheral T cell

activation and positive selection of thymocytes (Kersh and Allen, 1996), and antagonistic

peptides prevent activation of peripheral T cells though they do induce positive selection of

thymocytes (Hogquist et al., 1994).

It has been shown that T cells are sensitive to minute amounts of stimulatory

(agonist) pMHC complexes in a sea of endogenous ligands (Irvine et al., 2002; Krogsgaard

et al., 2005; Purbhoo et al., 2004). T helper cells can stop rolling and transiently increase

cytosolic calcium levels in response to even one agonist-pMHC complex. Ten agonist

complexes appear to be sufficient for sustained calcium release and the formation of the

immunological synapse (Grakoui et al., 1999; Huppa and Davis, 2003; Monks et al., 1998).

Cytotoxic (CD8+) T cells also seem to be very sensitive (Brower et al., 1994; Purbhoo et

al., 2004; Sykulev et al., 1996) and as few as three agonist-pMHC complexes have been

demonstrated to be sufficient for the killing of target cells (Purbhoo et al., 2004). Signalling

induced by agonist ligands can be severely impaired if antagonist-pMHC molecules are

present. Antagonists can be obtained by mutating TcR contact residues of agonistic peptides

(Madrenas et al., 1995; Sloan-Lancaster et al., 1994; Stefanova et al., 2003).

34

Introduction

Because of thymic selection, endogenous ligands bind TcRs much more weakly than

pathogen-derived pMHC molecules. The extraordinary sensitivity of T cells to agonists, and

the weak binding of TcRs to endogenous ligands, has led to the suggestion that these

agonist ligands may be implicated in T cell activation (Irvine et al., 2002; Janeway, 2001;

Krogsgaard et al., 2005; Li et al., 2004; Stefanova et al., 2003; Wulfing et al., 2002). For

CD4+ T cells, a basic model is emerging in which agonist and endogenous ligands act

cooperatively, to trigger TcRs, thereby amplifying signalling when agonists are limiting

(Irvine et al., 2002; Krogsgaard et al., 2005; Li et al., 2004). Spatial localization of a kinase,

Lck, in a signalling complex nucleated by agonist binding to TcRs has been considered to

play a key role in enabling certain endogenous ligands, that associate with such complexes,

to trigger TcRs (Krogsgaard et al., 2005). Recent advancement has also provided evidence

for cooperative interactions between agonist and endogenous ligands in triggering CD8+ T

cells (Anikeeva et al., 2006; Yachi et al., 2005).

1.1.4 Heterogeneity of T cell phenotype: Cytokines & T cell differentiation

There is extensive plasticity in the T-cell response to antigen. Helper CD4+ T cells,

cytotoxic CD8+ T cells, the progression from naïve to effector and memory T cells, and

differentiation into Th1, Tc1, Th2 and Tc2 subsets have long been recognized. However,

the recent identification of multiple distinct subsets of CD4+ cells, like Th17, Th9, and

Th22 and even Treg lineage, highlights a surprising degree of developmental convergence

and late-stage plasticity with particular relevance for autoimmune diseases. More recently it

has become apparent, that T-cell populations display additional diversity in terms of

phenotype, anatomical distribution and effector function. The underlying mechanisms

regulating these processes are poorly understood, although co-stimulatory molecules,

cytokines, chemokines, regulatory cells and the local environment almost certainly play a

role.

Upon interaction with cognate antigen presented by APCs such as DCs, CD4+ T

cells can differentiate into a variety of effector subsets, including classical Th1 cells and

Th2 cells, the more recently defined Th9, Th17, Th22, follicular helper T (Tfh) cells, or

induced regulatory T (iTreg) cells. The differentiation decision is governed predominantly

35

Introduction

by the cytokines in the microenvironment and, to some extent, by the strength of the

interaction of the TCR with pMHC complexes (Boyton and Altmann, 2002).

Th1 cells are characterized by their production of IFN-γ and are involved in cellular

immunity against intracellular microorganisms. IL-12 produced by innate immune cells as

well as IFN-γ produced by both NK cells and T cells polarize naïve T cells toward the Th1

cell differentiation program through action of the signal transducer and activator of

transcription 4 (Stat4), Stat1, and T box transcription factor T-bet (Thieu et al., 2008). Th2

cells produce IL-4, IL-5, and IL-13 and are required for humoral immunity to control

helminths and other extracellular pathogens. Th2 cell differentiation requires the action of

GATA3 downstream of Stat6, and IL-4R (Ansel et al., 2006) (Fig. 5).

Th17 cells produce IL-17A, IL-17F, and IL-22 and play important roles in clearance

of extracellular bacteria and fungi, especially at mucosal surfaces. Th17 cell differentiation

requires retinoid-related orphan receptor (ROR)γt, a transcription factor that is induced by

TGF-β in combination with the proinflammatory cytokines IL-6, IL-21, and IL-23, all of

which activate Stat3 phosphorylation (Chen et al., 2007). Furthermore, T cells producing

IL-22 but not IL-17 are designated as Th22 cells (Duhen et al., 2009). Th17 cells also

produce IL-22, but IL-17 is not obligatorily co-expressed with IL-22, and the fact that some

cells may make only IL-22 does not necessarily qualify them as Th22 cells (Duhen et al.,

2009) (Fig. 5).

TcR stimulation of naïve CD4 T cells in the presence of TGF-β and IL-4 can induce

cells to production IL-9 (Dardalhon et al., 2008; Veldhoen et al., 2008). IL-9 was first

recognized as a Th2 cytokine (Temann et al., 1998). It has been proposed that such IL-9-

producing cells be designated as Th9 cells. TGF-β reprograms the differentiation of Th2

cells towards Th9 cells. T cells producing IL-9 do not produce IL-4, but are nevertheless

associated with Th2-type responses such as helminth-specific immune responses or allergy

(Fig. 5).

36

Introduction

Figure 5: The cytokine milieu determines CD4+ T cell differentiation and conversion

Upon encountering foreign antigens presented by APCs, naive CD4+ T cells can differentiate into Th1, Th2, Th17, iTreg, and Tfh cells. These differentiation programs are controlled by cytokines produced by innate immune cells, such as IL-12 and IFN-γ, which are important for Th1 cell differentiation, and IL-4, which is crucial for Th2 cell differentiation. TGF-β together with IL-6 induces Th17 cell differentiation, whereas iTreg differentiation is induced by TGF-β, retinoic acid (RA), and IL-2. Tfh cell differentiation requires IL-21. (Zhou et.al. 2009)

Given the finding that circulating resting T cells in humans show constitutive TGF-β

signaling (Classen et al., 2007), one can ask if TGF-β is a rate limiting factor for human

Th17 responses. Differences in the requirements for exogenous TGF-β between mice and

humans may apply also to iTreg. Indeed, while TGF-β in the absence of IL-6 readily

induces iTreg differentiation in mice (Bettelli et al., 2006b; Chen et al., 2003), there is no

evidence that TGF-β may do the same in humans (Tran et al., 2007). The iTreg are

functionally similar to thymus derived natural Treg as they are anergic, suppressive, and

capable of inhibiting disease in-vivo. Differentiation of iTreg is under the control of the

transcription factor FoxP3 in both mice and humans (Chen et al., 2003).

Follicular T cells are a subset of helper T cells that regulate the maturation of B cell

responses. These follicular helper T (Tfh) cells are characterized by the expression of

37

Introduction

CXCR5, that mediates their homing and long-term residence in the B-cell follicles

(Breitfeld et al., 2000; Fazilleau et al., 2009a; Schaerli et al., 2000) In addition, Tfh cells

produce IL-21 and express ICOS and PD-1 (King et al., 2008) molecules. Differentiation of

these Tfh cells requires the cytokine IL-21 (Nurieva et al., 2008; Vogelzang et al., 2008),

and may be dependent on the transcription factor Bcl-6 (Fazilleau et al., 2009b).

T helper cells are heterogeneous and somewhat plastic. Many Tregs, just like naïve

CD4 T cells, can be differentiated into various types of T helper cells. Th17 cells are more

plastic than Th1/Th2 cells, suggesting that Th17 cells may represent cells that are not

terminally differentiated. Alternatively, Th17 cells may continue to express important

molecule(s) in determining cell plasticity, which presumably have been repressed in fully

differentiated Th1/Th2 cells. At early stages of T helper cell differentiation or within certain

subsets that are partially differentiated, CD4 T cells can be reprogrammed into different

lineages on receiving appropriate stimulus.

The notion that Th17 cells are derived from the Th1 cell lineage was invalidated by

the findings that these cells express RORγt and can develop in the absence of T-bet or Stat4

(Ivanov et al., 2006; Weaver et al., 2007). However, under homeostatic or inflammatory

conditions, IL-17+IFN-γ+ double-producer cells are easily detected, suggesting that there

may be some intricate relationship between the Th1 and Th17 cell differentiation programs.

Indeed, several recent findings highlight the plasticity of Th17 cells. In-vitro generated

Th17 cells lose IL-17A and IL-17F expression without constant exposure to IL-6 and TGF-

β (Lexberg et al., 2008). Cells polarized in Th17-polarizing conditions for as long as 3

weeks still failed to maintain IL-17A and IL-17F expression and can convert to Th1 or Th2

cells in the presence of IL-12 or IL-4, respectively (Lee et al., 2009; Lexberg et al., 2008).

However, Th17 cells secrete IL-21, which may function together with TGF-β for

maintenance of IL-17 expression in an autocrine manner. In contrast to in-vitro-

differentiated Th17 cells, in-vivo memory CD4+CD62Llo Th17 cells appear more resistant

to conversion (Lexberg et al., 2008). Thus, these findings suggest that there may be

epigenetic differences between in-vitro and in-vivo generated Th17 cells.

TGF-β is profoundly important for establishment of immunological tolerance, at

least in part, because it is required for the differentiation of iTreg cells and for maintenance

of natural Treg (nTreg) cells after they emigrate from the thymus (Chen et al., 2003; Li et

38

Introduction

al., 2006; Marie et al., 2005). Therefore it was surprising to discover that TGF-β is also

essential for the differentiation of proinflammatory Th17 cells (Bettelli et al., 2006b;

Mangan et al., 2006; Veldhoen et al., 2006). Mice deficient in TGF-β lack both FoxP3+

Treg and Th17 cells and have overwhelming autoimmunity largely caused by uncontrolled

Th1 cell activity (Li et al., 2006; Veldhoen et al., 2006). These findings suggest an intimate

link between the Treg and Th17 cell programs of differentiation, and indeed there is

evidence for such a relationship from both in-vitro and in-vivo studies (Beriou et al., 2009;

Bettelli et al., 2006b; Koenen et al., 2008; Yang et al., 2008; Zhou et al., 2008). Therefore,

Th17 cells are reciprocally related to FoxP3+ Tregs given that TGF-β induces FoxP3 in

naive T cells, whereas IL-6 suppresses the TGF-β-driven induction of FoxP3, and TGF-β

plus IL-6 together induce RORγt and the Th17 transcriptional program. At the molecular

level, the balance between Th17 cells and FoxP3+ Tregs is mediated by the antagonistic

interaction of the transcription factors FoxP3 and RORγt.

The proximal signals required for commitment of type 1 and type 2 subsets in CD8+

T cell compartment are similar to their CD4 counterparts (Sad et al., 1995), recently it has

been shown that the IL-17 producing subset of CD8+ T cells follows the same

differentiation program as Th17 cells (Ciric et al., 2009; Hamada et al., 2009; Huber et al.,

2009). However, the importance and function of this subset of CD8 T cells need to be

studied in more depth to better understand its significance. Taken together, the recent

findings suggest that the local microenvironment, through provision of specific cytokines

directs multiple possible alternate fates that T cells can adopt.

39

Introduction

1.2 T cell development and immune tolerance:

1.2.1 The development of the T cell repertoire

The 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.

1.2.1.1 T cells ontogeny

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-cell 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 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 (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, 2004; von Boehmer et al., 2003).

The extensive diversity of the TcR αβ and γδ heterodimers is required to recognize

the endless variety of pathogen-derived peptides and is achieved by a process of random

gene arrangements. The genes encoding each chain comprise various segments that form

the foundation of the TcR diversity. Analogous to the Ig genes, the loci of the α and γ

chains contain variable (V) joining (J) and constant (C) gene segments, whereas the β

chains and δ chain loci include additional diversity (D) segments. Recombination of these

segments is performed by V(D)J-recombinase, an enzymatic complex comprising RAG-1

and RAG-2 (recombinase activating genes) proteins and DNA double strand repair proteins

(Li et al., 1995; Schatz et al., 1992).

40

Introduction

The recent discovery of thymoproteasomes, a molecular complex specifically

expressed in cortical TEC (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 (Murata et al., 2007). Cortical thymocytes that

receive TcR-mediated positive selection signals are destined to survive for further

differentiation and are induced to express CCR7. Being attracted to CCR7 ligands

expressed by medullary TEC (mTEC), they relocate to thymic medulla (Liu et al., 2006a;

Liu et al., 2005). The medullary microenvironment displays another set of unique self-

peptides for trimming the positively selected T-cell repertoire to establish self-tolerance, via

promiscuous expression of tissue-specific antigens (TSAs) by mTEC and efficient antigen

presentation by dendritic cells.

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., 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;

Scollay et al., 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 Zuniga-Pflucker, 2006; Sambandam et al., 2005; Schmitt and Zuniga-

Pflucker, 2002) and cytokines such as IL-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 cTEC (Faas et al., 1993; Schmitt et al., 2004). At the DN stage, thymocytes

undergo developmental programs through DN1 (CD44+CD25-), DN2 (CD44+CD25+), and

DN3 (CD44-CD25+), during which V(D)J rearrangement at TcR γ, δ, and β loci occurs

(Garman et al., 1986; Habu et al., 1987). In order to pass the β-selection point, the β-chain

of the rearranged TcR at the DN3 stage must retain structural properties allowing it to be

presented on the surface of the thymocyte with pre-TcRα. Two lineages of T cells

41

Introduction

expressing either TcR αβ or TcR γδ diverge at DN stage (Raulet et al., 1991; von Boehmer,

1988) (Fig.6).

Figure 6: Different stages of thymocyte development (Sebzda et al. 1999)

The assembly of successfully rearranged TcR β and invariant pre-TcRα together

with CD3 chains leads to commitment to the TcR αβ lineage and further differentiation

beyond DN3 stage, including proliferation, TcRα-VJ rearrangement, CD25 downregulation,

and expression of CD4 and CD8 coreceptors (Saint-Ruf et al., 1994; Shinkai et al., 1993;

Yamasaki et al., 2006). Thymocytes migrate toward the capsular region of the thymus

during the differentiation of DN2 and DN3. Most DN3 thymocytes are localized in the

subcapsular region of the cortex (Benz et al., 2004; Li et al., 2007) where they further

develop to pre-DP stage, or DN4 thymocytes, which show low expression of CD4, CD8,

CD25, and CD44 and are immediate precursor cells of CD4/CD8 double-positive (DP)

thymocytes (Saint-Ruf et al., 1994; Shinkai et al., 1993; Wilson et al., 1989) (Fig. 6).

42

Introduction

1.2.1.2 Positive selection of thymocytes

DP thymocytes express TcR αβ at low levels on the cell surface and move actively

within the cortical microenvironment (Li et al., 2005a; Witt et al., 2005) apparently to

scrutinize for TcR interactions with pMHC molecules. These DP thymocytes are selected

for survival or demise depending on the avidity of the interaction of TcRs with the self-

pMHC complex (Allen, 1994; von Boehmer, 1994). When expressing a TCR that cannot

interact with self-peptide–MHC complexes, they die as a consequence of neglection

referred as death by neglect (Williams and Brady, 2001). DP thymocytes that receive TcR

signals with ligand interactions of weak avidity and confined aggregation are induced to

survive and differentiate into mature thymocytes, the process referred as positive selection

(Ashton-Rickardt et al., 1993; Takahama et al., 1994) (Fig.7).

Figure 7: The affinity model of thymocyte selection (Klein et.al. 2009)

During positive selection, the differential kinetics of TcR–ligand interactions

determines cell lineage to become either CD4+CD8- or CD4-CD8+ single-positive (SP)

thymocytes (Singer, 2002; Singer and Bosselut, 2004). Positively selected thymocytes

43

Introduction

relocate to thymic medulla where they further interact with self-peptides displayed in the

medullary microenvironment (Takahama, 2006; Ueno et al., 2004). Initial αβ TcR signals

in DP thymocytes upregulate CD5 and CD69 surface expression and terminate RAG

expression, which precludes further TCRα gene rearrangements and fixes αβ TCR

specificity. Importantly, since positive selection of DP thymocytes involves differentiation

into either CD4 or CD8 SP T cells, the CD4/CD8 lineage decision is an intrinsic feature of

the positive selection process (Janeway, 1988).

1.2.1.3 CD4 & CD8 lineage differentiation

Synchronized with positive selection, thymocytes undergo alternate commitment to

either the cytotoxic or the helper T cell lineages, generating MHC-class I-restricted SP

CD8+ and MHC class II-restricted SP CD4+ thymocytes. The mechanism by which this

correlation between MHC restriction and functional phenotype is achieved remains elusive,

however recent advancement starting to delineate the process.

Initially it was suggested, that lineage commitment might either be directed by

qualitatively distinct signals initiated upon TcR and coreceptor engagement by class I- or II

MHC ligands (instructive model) (Robey et al., 1991), or occurred randomly and was

followed by a selection step that eliminates thymocytes that express the inappropriate

coreceptor (stochastic-selective model) (Chan et al., 1993; Davis et al., 1993). However,

these models gave inconsistent outcomes, suggesting that they needed to be refined

(Borgulya et al., 1991; Matechak et al., 1996; Robey et al., 1994). A quantitative-instructive

model proposes, that stronger and weaker TcR signals, respectively, promote CD4 and CD8

commitment (Matechak et al., 1996). This is based on the fact that the cytoplasmic tail of

CD4 binds Lck with substantially higher affinity than that of CD8α (Ravichandran and

Burakoff, 1994).

It has been suggested, that lineage commitment is not completed until the transition

from DP to SP, with an intermediate CD4+8lo stage, because many of these cells are not

yet irreversibly committed to a particular lineage (Bosselut et al., 2003; Brugnera et al.,

2000). Indeed, the distinct kinetic-signaling model postulates that lineage commitment is

determined at the CD4+8lo stage, on the basis of the relative ability of coreceptors to

contribute to TcR signaling (Singer and Bosselut, 2004) (Fig. 8).

44

Introduction

Figure 8: Environmental cues and nuclear factors that influence CD4/CD8 lineage

choice (Singer et. al. 2008)

However, the intracellular control of lineage commitment remained elusive before

the discovery of helper T cell-deficient (HD) mice, which carry a spontaneous mutation that

causes MHC class II-restricted thymocytes to mature into SP CD8 rather than CD4 T cells

(Dave et al., 1998; Keefe et al., 1999). The HD mutation has been mapped to the locus

encoding the Zinc-finger transcription factor ThPOK (He et al., 2005; Kappes and He,

2006). The expression pattern of ThPOK during thymic development is tightly regulated in

a manner that indicates a specific role in CD4 commitment. Thus, ThPOK RNA is first

detected at the CD4+8lo stage, and ThPOK mRNA expression is substantially higher in

MHC class II than MHC class I-restricted cells at this stage, consistent with preferential

induction by class II ligands (He et al., 2005). Constitutive expression of ThPOK during

thymic development causes redirection of class I-restricted thymocytes to the CD4 lineage.

45

Introduction

Therefore, ThPOK expression is both necessary and sufficient for CD4 commitment (Fig.

8).

Two properties of ThPOK need to be underlined, first although ThPOK is expressed

in a wide variety of cells, its expression in the thymus is highly lineage specific (He et al.,

2005; Sun et al., 2005), CD4 SP thymocytes express ThPOK, whereas DP and CD8 SP

thymocytes do not. In addition, during MHC-II-induced selection, ThPOK is up regulated

progressively as thymocytes down-regulate CD8. Second, it has been shown, that ThPOK

affects lineage choice but not positive selection, i.e. ThPOK-deficient, MHC-II-restricted

thymocytes becomes CD8 instead of CD4 T cells (Keefe et al., 1999).

Several additional transcription factors also play selective roles in CD4 or CD8

development and might therefore be involved in lineage commitment, notably GATA-3 and

Runx3. Constitutive expression of GATA-3 specifically blocks CD8 development, whereas

its conditional deletion blocks CD4 development (Hernandez-Hoyos et al., 2003; Nawijn et

al., 2001; Pai et al., 2003). However, in neither case affected thymocytes redirected to the

opposite lineage. Hence, GATA-3 is necessary, but not sufficient for CD4 development.

Runx factors clearly play an important role in regulating the CD8 development program,

including the CD8 lineage-specific suppression of CD4 (Sato et al., 2005; Sawada et al.,

1994; Siu et al., 1994; Taniuchi et al., 2002). However, a direct role of Runx factors in

initiating lineage commitment appears unlikely on the basis of results from constitutive

Runx3 transgenic mice. Thus, although development of SP CD4 thymocytes is impaired in

Runx3 transgenic mice, this is corrected by constitutive CD4 expression (Grueter et al.,

2005). CD4 deficiency is known to cause redirection of MHC class II-restricted thymocytes

to the CD8 lineage, presumably by weakening or interrupting TcR signaling (Matechak et

al., 1996; Tyznik et al., 2004). The CD8 population in CD4-deficient mice is heavily

contaminated with MHC class II-restricted T cells; hence the primary effect of Runx factors

on lineage commitment appears to lie on controlling the CD4 expression.

Recent identification of the negative feedback loop between ThPOK and Runx acts

as the keystone of lineage choice in our understanding of CD4 & CD8 differentiation,

preventing the co-expression of both factors in mature thymocytes (Egawa and Littman,

2008; Setoguchi et al., 2008). First, Runx activity is not sufficient for ThPOK repression;

Runx binds the ThPOK silencer not only in DP thymocytes and in CD8 cells but also in

46

Introduction

CD4 cells that express ThPOK (Setoguchi et al., 2008), and there is evidence that additional

segments of the silencer are required for its function (He et al., 2008). Therefore Runx

activity is not sufficient to prevent CD4 differentiation, because enforcing Runx1 or Runx3

expression in thymocytes does not impair CD4 lineage differentiation (Grueter et al., 2005;

Hayashi et al., 2001; Kohu et al., 2005).

In addition to above mentioned lineage specific transcription factors, recent reports

suggested that a previously unknown T cell–specific protein, called 'Themis' (thymus-

expressed molecule involved in selection), is critical for the positive selection of developing

T cells. Themis-/- mice showed a late block in thymocyte selection characterized by fewer

CD4+CD8int, CD4SP and CD8SP thymocytes. Although Themis-/- thymocytes showed no

defect in proximal TcR signaling, but CD4 lineage commitment and maturation was

compromised in a way consistent with signal attenuation, which suggests, that Themis is

involved at a distal point in the TcR signaling cascade. These findings indicate, that Themis

functions to integrate or sustain TcR signaling in CD4+CD8int thymocytes and demonstrate

that this ability is critical for appropriate lineage commitment as well as the development of

CD4SP and CD8SP thymocytes (Fu et al., 2009; Johnson et al., 2009; Lesourne et al.,

2009).

47

Introduction

1.2.2 Central tolerance

Central tolerance is regulated at several levels. First, only those self-antigens that are

presented by thymic APCs can influence central tolerance. So, molecules that regulate the

presentation of self-antigens have a crucial influence on tolerance. Second, when

progenitors encounter such self-antigens, they can undergo apoptosis or acquire regulatory

properties.

1.2.2.1 Role of autoimmune regulator (AIRE)

Most self-reactive T cells are deleted in the thymus, resulting in central tolerance.

This is further supported by regulatory mechanisms outside of primary lymphoid organs,

which are collectively known as peripheral tolerance. The main mechanism of central

tolerance, the negative selection of self-reactive thymocytes, occurs mainly in the medullary

compartment of the thymus (Anderson et al., 2007b; Takahama, 2006). The mTECs express

a large number of genes, including TSAs that are normally present only in specialized

peripheral organs and are apparently not required for the direct function of mTECs

(Derbinski et al., 2005; Kyewski and Klein, 2006). During negative selection, these

encoded TSAs are presented by mTECs or DCs to thymocytes as self-antigens

(Aschenbrenner et al., 2007; Gallegos and Bevan, 2004), leading to the induction of

tolerance by clonal deletion, functional inactivation or clonal deviation of self-reactive T

cells (Hogquist et al., 2005; Palmer, 2003; Yamagata et al., 2004).

The autoimmune regulator (AIRE) protein has been found to be important for the

maintenance of self-tolerance (Nagamine et al., 1997). Mutations in the human AIRE gene

cause autoimmune polyendocrinopathy candidiasis ectodermal dystrophy (APECED), a

syndrome that is characterized by the presence of autoimmunity including autoantibodies

that are specific for multiple self-antigens. This leads to lymphocytic infiltration of

endocrine glands (such as parathyroid, adrenals, endocrine pancreas, ovaries/testes) and

autoimmune disorders localized in these organs (Perheentupa, 2006; Peterson and Peltonen,

2005). Mice with mutations in the AIRE gene have pathological autoimmune features that

are similar to those of patients with APECED, including multiorgan lymphocytic infiltration

and autoantibody production. In recent years, many studies have shown, that AIRE is a

48

Introduction

crucial factor for the promiscuous expression of TSAs in the thymus, and that mutations in

this gene lead to the escape of self-reactive T cells from the thymus, which results in

autoimmunity (Jiang et al., 2005; Kuroda et al., 2005; Niki et al., 2006; Ramsey et al.,

2002). It has been shown, that T cells, in particular Th1 cells, are crucial mediators of the

autoimmune tissue damage in AIRE-deficient mice, whereas B cells have more limited

functions but are still required for early and severe autoimmunity (Devoss et al., 2008;

Gavanescu et al., 2008). Recently, candidiasis in APECED patients was attributed to the

presence of neutralizing autoantibodies to IL-17A, IL-17-F and IL-22 (Puel et al., 2010)

(Fig.9).

Figure 9: Aspects of the thymic medulla that are involved in central tolerance (Kristin

et.al. 2005)

Importantly, mTECs from AIRE-deficient mice have a marked decrease in the

expression of multiple transcripts encoding peripheral TSAs (Anderson et al., 2002;

Derbinski et al., 2005), including the insulin 2 and salivary protein 1 genes, which seem to

49

Introduction

be under the direct control of AIRE (Kont et al., 2008). However, not all transcripts

encoding peripherally expressed antigens are down regulated in the thymus of AIRE-

deficient mice, which indicates that other factors are necessary for the efficient expression

of these genes.

Experimental evidence for the function of AIRE in negative selection was obtained

using mice expressing transgenic TcRs that are specific for neo-self antigens. AIRE-

dependent negative selection of specific TcR-transgenic T cells was observed in transgenic

mice expressing hen egg lysozyme (Kont et al., 2008; Liston et al., 2004a) or ovalbumin

(Anderson et al., 2005) as neo-self antigens under the control of the rat insulin promoter,

which drives the expression of these antigens in mTECs as well as pancreatic β-cells. These

data provided compelling evidence that AIRE is essential for efficient thymic T-cell

deletion of at least some clones reactive to TSAs. Importantly, AIRE-deficient, double-

transgenic mice expressing hen egg lysozyme or ovalbumin antigens under the control of

the rat insulin promoter together with the respective transgenic TcR developed aggressive

diabetes at birth. This suggests that T cells that escape negative selection are capable of

inducing autoimmunity. So, the consequence of AIRE deficiency is thought to be disturb

negative selection of T cells that are specific for self antigens, which could be caused by

defective expression of self antigens by TECs (Villasenor et al., 2005). In support of this

idea, AIRE-deficient mice spontaneously develop autoimmune uveitis and autoimmune

gastritis as a result of self-reactivity against interphotoreceptor retinoid-binding protein

(DeVoss et al., 2006) and mucin 6 antigens (Gavanescu et al., 2007), respectively, the

expression of which is regulated by AIRE in mTECs. In humans, differences in the

expression level of AIRE, together with genetic variances in the insulin and acetylcholine

receptor genes, influence the expression levels of these two established self-antigens of

type-1 diabetes (T1D) and autoimmune myasthenia gravis, respectively (Giraud et al., 2007;

Sabater et al., 2005).

50

Introduction

1.2.2.2 Negative selection of thymocytes

During development, autoreactive thymocytes are eliminated through apoptosis in a

process termed negative selection, which is important to prevent autoimmunity.

Experimental evidence demonstrates, that self-reactive T cells are indeed purged from the

repertoire in the thymus (Kappler et al., 1987). Further studies have demonstrated that

negative selection occurs primarily in the thymic medulla, via presentation of self-antigen-

MHC complexes on DCs, forcing any thymocytes with strong reactivity to undergo

apoptosis (Ashton-Rickardt and Tonegawa, 1994; Sebzda et al., 1994).

The removal of strongly self-reactive T cells has long been considered the sole

purpose of negative selection, however negative selection may play a fundamental role in

moulding the T cell repertoire (Huseby et al., 2005). It has been shown that by limiting

negative selection but leaving positive selection intact, a repertoire of T cells develops that

is highly cross-reactive for many different peptide-MHC complexes. After TcR

rearrangement, most T cells display a very high affinity for MHC, validating the high basal

affinity hypothesis (Huseby et al., 2005).

In the context of negative selection, Cbl proteins may influence signaling pathways

involving Bim, a pro-apoptotic member of the Bcl-2 family, Nur77 family of orphan steroid

nuclear receptors as well as the regulators of the MAPK cascades. Role of Bim and Nur77

have been implicated in apoptosis of autoreactive T cells during negative selection (Bouillet

et al., 2002; Calnan et al., 1995). In NOD mouse strain, Bim and Nur77 among genes that

are upregulated in stimulated wild type but not NOD CD4+CD8+ thymocytes, which is

consistent with their role in negative selection (Liston et al., 2004b; Zucchelli et al., 2005).

In general, thymic selection appears to have two roles: the first one is to bias the

repertoire towards TcRs that are specific for a single complex, and the second to eliminate

undesirable clones with the potential to drive autoimmunity. The rigours of negative

selection are encountered by positively selected thymocytes upon further migration from

the thymic cortex into the thymic medulla (Surh and Sprent, 1994). Negative selection is

most prominent at the cortico-medullary junction removing two thirds of positively selected

thymocytes (Murphy et al., 1990; van Meerwijk et al., 1997).

51

Introduction

Overall the rigorous procedure of neglect and negative selection allow only 3-5% of

double positive thymocytes to survive the transition to end stage single positive cells

committed to either CD4 or CD8 lineage (Huesmann et al., 1991) .

1.2.3 Peripheral tolerance:

Evidence suggests that thymic negative selection most effectively deletes those T

cell precursors that express TcRs with high avidity for self-pMHC complexes expressed on

medullary DCs and mTECs. This implies that peripheral immune tolerance mechanisms are

most important for controlling mature T cells that carry a TcR of relatively low avidity for

self pMHC and that escape to the periphery. The mechanisms responsible for peripheral

tolerance is divided into T-cell intrinsic mechanisms, which act directly on the responding T

cell and T cell extrinsic mechanisms, which evoke additional subsets of cells, including

DCs and Tregs.

1.2.3.1 T cell ignorance

The simplest mechanism of peripheral ‘tolerance’ involves T-cell ignorance of self-

antigens, as the simplest mechanism to avoid an unwanted self-antigen-specific immune

response is to impede the penetration of an immune response in well-defined areas,

designated as immune privileged sites. Several such sites have been identified, including the

brain, the anterior chamber of the eye, and the testis. These sites all seem to be privileged in

their capacity to readily accept allografts / xenografts (Barker and Billingham, 1977).

Ignorance and sequestration are not the direct mechanisms of peripheral tolerance as

neither of the two actively induces unresponsiveness. In the case of ignorance, T cells

bearing a self-reactive TcR may persist without being anergic because they proliferate

normally when challenged ex-vivo with their specific antigen. Nevertheless, these cells do

not mediate a destructive immune response in-vivo except when challenged by the same

antigen presented by different APCs (Ohashi et al., 1991), which suggests that the site and

nature of antigen presentation plays a critical role in how T cells respond to their antigen. In

addition, to mount an immune response a threshold level of antigen is required, which plays

a vital role in T cells ignorance (Kurts et al., 1998).

52

Introduction

Immune privileged sites were originally thought to insulate tissue antigens from

patrolling lymphocytes, however, T cells have been identified to migrate into the brain

(Brabb et al., 2000; Yednock et al., 1992). T cells are not actively excluded, but immune

privilege is maintained by other passive mechanisms, as the brain has very few local APCs

and also lacks lymphatic drainage, which facilitates APC and lymphocyte migration (Barker

and Billingham, 1977; Hart and Fabre, 1981; Wong et al., 1984). In addition to passive

mechanisms, active mechanisms have been identified to maintain the immune privilege by

eliminating or silencing infiltrating lymphocytes. In the anterior chamber of the eye and the

testis, the active mechanism is conferred by expression of Fas-ligand (Fas-L), which

mediates programmed cell death to recruited activated lymphocytes (Griffith et al., 1995).

This suggests that limiting accessibility of the antigen to the immune system by anatomical

sequestration (Alferink et al., 1998; Zinkernagel, 1996) is important for T cell ignorance.

However, ignorance is a reversible state, therefore there is always the risk of

autoimmunity if the self-Ag leaks out of the tissue (Voehringer et al., 2000).

1.2.3.2 T cell anergy

T-cell encounters with self-antigen under certain circumstances and might lead to

functional inactivation, a cell-intrinsic state of unresponsiveness, subsequently termed

anergy, this was shown to be long-lived, associated with defects in cell-cycle progression

and some effector functions, and reversible with strong stimuli.

Clonal T-cell anergy was initially characterized in Th1-cell clones by stimulation

through the TcR without co-stimulation through CD28 (Jenkins et al., 1990; Jenkins et al.,

1987; Quill and Schwartz, 1987). Anergized T-cell clones produced negligible amounts of

IL-2, which was initially thought to be crucial for clonal expansion following T-cell

activation. However, IL-2 receptor knockout mice showed, that IL-2 is mainly implicated in

promoting Tregs to control T cell expansion of activated T cells (Malek, 2003).

Interestingly, clonal T-cell anergy was found not to be a terminal fate, as the addition of

exogenous IL-2 during restimulation could reverse the phenotype (Beverly et al., 1992).

The reduction in IL-2 production by restimulated anergic T cells was found to reside

at the level of IL-2 transcription. In anergic T cells, DNA binding of the activator protein 1

(AP1) heterodimer, comprised of the transcription factors FOS and JUN, was reduced in

53

Introduction

reporter assays, and subsequent studies of signalling pathways in anergic T cells found

defects in the activation of the immediate upstream molecules of the mitogen-activated

protein kinase (MAPK) families (DeSilva et al., 1996; Fields et al., 1996; Kang et al., 1992;

Li et al., 1996). Importantly, anergic T cells had no defects in the activation and nuclear

translocation of nuclear factor of activated T cells (NFAT), which is activated following

calcium flux, principally downstream of TcR cross-linking (Lyakh et al., 1997).

Furthermore, E3 ubiquitin ligases have been described as important mediators of T cell

tolerance (Fathman and Lineberry, 2007; Lin and Mak, 2007). The transmembrane E3

ligase, gene related to anergy in lymphocytes (GRAIL) has been reported to be highly up-

regulated during anergy induction compared with resting/activated T cell clones

(Anandasabapathy et al., 2003), and was subsequently shown to be involved in anergy

induction (Seroogy et al., 2004).

Most studies on the induction of anergy in-vivo utilize T cells bearing a transgenic

TcR. Administration of soluble cognate peptide antigen for the transgenic TcR can induce

an unresponsive state, in cells bearing these transgenic TcR (Kearney et al., 1994). The

signaling pathways responsible for this anergic state are believed to be similar to those

engaged in-vitro. Mice deficient for many of the molecules identified as important for

imposing anergy in-vitro, such as Cbl-b (Bachmaier et al., 2000), Egr-3 (Safford et al.,

2005) as well as mice expressing a dominant negative GRAIL molecule (Seroogy et al.,

2004), are resistant to the induction of peptide-induced anergy in-vivo (Heissmeyer et al.,

2004).

Another model of in-vivo anergy induction, however, involves the transfer of TcR

transgenic T cells into a T cell-depleted host that ubiquitously expresses cognate antigen for

the transferred cells. These transferred T cells also enter into an unresponsive state, called

‘adaptive tolerance’ (Schwartz, 2003). ‘Adaptively tolerant’ T cells are functionally anergic

but differ from clonally anergized cells in their requirements for antigen persistence to

maintain unresponsiveness and by the effect that IL-2 has on unresponsiveness (Rocha et

al., 1993). Later on it has been observed, that the signaling defects between these two

unresponsive states were indeed different, as cells made anergic in-vitro had impaired

activation of the Ras signaling pathway (Chiodetti et al., 2006). In adaptively tolerant cells,

54

Introduction

however, the signal defect was more proximal to the TcR, as these cells displayed impaired

activation of Zap70 kinase activity.

Additionally, a role of PD-L1 molecule has been implicated in maintaining T cell

anergy. Blocking antibodies to PD-L1 or genetic ablation of PD-L1 was sufficient to

reverse established anergy and promote autoimmunity in two different models of

autoimmune diabetes (Fife et al., 2006; Tsushima et al., 2007). Moreover, it was mentioned

that PD-L1 plays a role in the lymphoid organs in the establishment of T cell anergy as well

as in the periphery in the maintenance of the unresponsive state (Tsushima et al., 2007).

This suggests a role of PD-L1 in the establishment of the anergic state in models of both

CD4+ and CD8+ T cell anergy.

Recently it has been shown, that PD-L1 can bind to CD80. In-vitro ligation of PD-

L1 expressed on T cells lacking CD28 and CTLA-4 by a CD80 fusion protein was able to

deliver an inhibitory signal to T cells and inhibit proliferation (Butte et al., 2007).

Interestingly, ligation of CD80 on T cells lacking PD-1 by PD-L1 fusion protein was also

able to inhibit proliferation. Thus, it appeared that the PD-L1:CD80 interaction signaled bi-

directionally, and that expression of either CD80 or PD-L1 on T cells was sufficient to

deliver an inhibitory signal when the B7-1:PD-L1 interaction was engaged (Butte et al.,

2007).

1.2.3.3 Apoptosis/Deletion

Deletion of potential auto-reactive lymphocytes is a vital mechanism in maintaining

self-tolerance. Exterminating self-specific cells from the peripheral lymphocyte repertoire is

an integral part of the negative selection process applied during thymic development.

Both functional unresponsiveness and deletion are effects of incomplete activation

of T cells in the absence of appropriate signals. However, deletion of autoreactive T cells is

the most permanent and irrevocable form of peripheral tolerance. The factors, which

distinguish between these two cell fate decisions, are not completely understood. Antigenic

persistence has been found to be an important variable in determination of either deletion or

survival. To examine the role of antigen persistence, recent adoptive transfer studies of

clone 4 TcR (CL4) transgenic T cells which recognizes the influenza hemagglutinin (HA)

peptide, into Ins-HA mice, constitutively expressing the HA antigen in the pancreatic islet β

cells, showed that after 4 days of initial priming, cells transferred alone proliferated much

55

Introduction

less than cells that were transferred along with an additional activating signal, but still

persisted as a stable pool of surviving T cells (Redmond et al., 2003). These experiments

suggested that the initial priming by a self-antigenic stimulus was not sufficient to result in

complete deletion of the T cell pool. Transferring the transgenic T cells from the original

recipient into a new Ins-HA host resulted in complete deletion of the CL4 T cells, whereas

transfer into an wild type secondary host allowed recovery of a stable population of CL4 T

cells (Redmond et al., 2003). These data proved, that sustained antigenic stimulation in

tolerogenic environments could lead to tolerance by deletion. Furthermore, to assess the

role of the initial tolerogenic antigenic stimulus in either deletion or anergy, they found that

multiple exposures of high doses of antigen resulted in a stable population of antigen-

specific T cells, similar to the effect of a single exposure of antigen, implying that complete

deletion did not take place. Instead these cells appeared to have become refractory to further

stimulation. However, multiple exposures to low doses led to complete deletion of the CL4

donor T cells (Redmond et al., 2005). Later it has been observed, that when T cells are

exposed to a low level of antigenic stimulus under tolerogenic conditions, they are unable to

maintain expression of the anti-apoptotic genes through TcR signaling, and therefore are

steered into a path of death by deletion (Redmond and Sherman, 2005).

Furthermore, the most effective way to prevent autoimmune destruction by a given

T-cell clone is to delete that specificity from the repertoire by activation induced cell death

(AICD). As self-antigens cannot be easily cleared, repetitive TcR engagement might be a

feature of T-cell encounters with self-proteins, which perhaps serves as a trigger for AICD.

A key mechanism underlying AICD in CD4+ T cells is the ligation of the Fas death

receptor by its ligand (Sobel et al., 1993; Suda et al., 1993; Watanabe-Fukunaga et al.,

1992). The physiological importance of Fas signalling was underscored by the finding, that

defects in the Fas pathway in humans are associated with the autoimmune

lymphoproliferative syndrome (ALPS) (Fisher et al., 1995). Recent advancements in the

signalling for AICD include a role for Pten (phosphatase and tensin homolog) (Suzuki et al.,

2001) and potential negative regulation by TGF-β, which has recently been suggested to

inhibit T-cell apoptosis by acting in an intracellular manner.

The apoptosis of mature T cells comprises at least two major forms, antigen driven

apoptosis and apoptosis due to lymphokine withdrawal (Lenardo et al., 1999). Apoptosis

56

Introduction

due to lymphokine withdrawal is a consequence of T cell deprivation of growth factors, a

scenario relevant at the conclusion of an immune response when there is no further antigen

stimulation resulting in the abrogation of cytokine release (Lenardo et al., 1999; Lenardo,

1991). In contrast, the apoptosis of antigen activated mature T cells is mediated by either

Fas-L or TNF (Dhein et al., 1995; Zheng et al., 1995). The selective cytotoxicity towards

mature T cells is predominantly achieved as a result of two interactions. Firstly, contrary to

resting T cells, only IL-2 activated mature T cell express Fas-L and TNF (Zheng et al.,

1998), and second only those T cells whose TcR is actively engaged are susceptible to

autocrine or paracrine Fas mediated apoptosis (Hornung et al., 1997; Lenardo et al., 1999;

Wong et al., 1997).

Consistent with a role for IL-2 in peripheral tolerance, mice deficient in IL-2

production or signalling show autoimmunity (Sadlack et al., 1995; Suzuki et al., 1995;

Willerford et al., 1995). However, it is unclear, if defective AICD is the only abnormality in

these mice, as the absence of IL-2 might lead to abnormalities in thymic selection (Malek et

al., 2000) or production of Tregs. Later studies have shown, that thymus-specific transgenic

expression of IL-2Rβ in IL-2Rβ-/- mice prevented lethal autoimmunity while the peripheral

T cells remained unresponsive to IL-2 which strongly favors a mechanism other than

peripheral AICD for tolerance (Malek et al., 2000; Malek et al., 2002). Importantly, more

studies with mixtures of bone marrow or T cells from either IL-2Rβ-/- or IL-2-/- cells with

wild-type cells showed, that the autoimmunity in these deficient mice was not due to

intrinsic defects but rather lack of a regulatory population (Suzuki et al., 1999; Wolf et al.,

2001). This was further highlighted in experiments, where IL-2Rβ-/- T cells in mice

reconstituted with a mixture of IL-2Rβ-/- T cells and IL-2Rβ+/- bone marrow T cells did not

develop into an abnormally activated phenotype, and already activated IL-2Rβ-/- T cells

were effectively eliminated by IL-2Rβ+/- T cells when both cells were co-transferred to T

cell-deficient host mice (Suzuki et al., 1999).

Importantly, peripheral deletion is rarely complete, and in many models the

remaining cells are anergic, which indicates that removal of a T-cell clone from the

peripheral repertoire is perhaps an overrepresentation of events. Instead, deletion might

simply serve to decrease the precursor frequency to a level at which anergy can be

effectively induced.

57

Introduction

1.2.4. Regulatory T cells

1.2.4.1 FoxP3+CD4+CD25+ naturally occurring regulatory T (Treg) cells

Tregs play an indispensable role in maintaining immunological unresponsiveness to

self-antigens and in suppressing excessive immune responses deleterious to the host. There

are two types of Tregs; natural Tregs (nTreg), which develop in the thymus, and induced

Tregs (iTreg), which are derived from naive CD4+ T cells in the periphery. Tregs utilize a

variety of mechanisms to suppress the immune response. While Tregs are critical for the

peripheral maintenance of potential autoreactive T cells, they can also be detrimental by

preventing effective anti-tumor responses (Kretschmer et al., 2006) and sterilizing

immunity against pathogens (Belkaid, 2007; Rouse et al., 2006).

nTreg cells are generated in the thymus through MHC class II-dependent TcR

interactions resulting in high-avidity selection (Apostolou et al., 2002; Bensinger et al.,

2001; Fontenot et al., 2005; Jordan et al., 2001; Larkin et al., 2008; Modigliani et al., 1996;

Sakaguchi, 2001), however additional selection may take place (van Santen et al., 2004).

They are characterized by the expression of CD25 and the forkhead-family transcription

factor FoxP3, and they have the capacity to suppress in-vitro the activation of other T cells

in a contact-dependent manner (Bluestone and Abbas, 2003; Fontenot and Rudensky, 2005;

Sakaguchi, 2000; Shevach, 2000).

The importance of this T-cell subset, and the role of FoxP3 in its development and

function, is highlighted by experiments of nature in which mutations in FoxP3 result in fatal

autoimmune lymphoproliferative disease. The spontaneous scurfy (sf) mutation in mice is a

loss of function in the FoxP3 gene, which results in a complete loss of Treg cells and death

of the mice at 3-4 weeks of age (Brunkow et al., 2001; Godfrey et al., 1991). Similarly,

humans with IPEX (immunodysregulation, polyendocrinopathy and enteropathy, X-linked

syndrome) also have mutations in the FoxP3 gene and have a range of symptoms that are

consistent with a lack of functional Treg cells (Bennett et al., 2001; Gambineri et al., 2003).

Although nTreg cells develop in a highly controlled thymic microenvironment

(Josefowicz and Rudensky, 2009), FoxP3+ iTreg cells differentiate under more varied

conditions; as an example iTreg cells appear in the mesenteric LNs during induction of oral

tolerance (Coombes et al., 2007; Mucida et al., 2005) and they may continuously

differentiate in the lamina propria of the gut in response to microbiota and food antigens

58

Introduction

(Sun et al., 2007). They are also attracted to mucosa in chronically inflamed tissues

(Curotto de Lafaille et al., 2008), in tumors (Liu et al., 2007), and in transplanted tissues

(Cobbold et al., 2004). Recent finding reveal that FoxP3+ iTreg cell development requires

TcR stimulation and the cytokines TGF-β and IL-2, for both in-vitro and in-vivo

generation. Addition of TGF-β to TcR-stimulated naive CD4+ T cells induced transcription

of FoxP3, acquisition of anergic and suppressive activity in-vitro, and the ability to suppress

inflammation in an experimental asthma model (Chen et al., 2003). TGF-β induced FoxP3

expression in cultures of plate-bound anti-CD3 and CD28-stimulated naive T cells (Chen et

al., 2003) thus, APCs were not required for in-vitro conversion. The mechanism by which

TGF-β induces transcription of FoxP3 involves cooperation of the transcription factors

Stat3 and NFAT at a FoxP3 gene enhancer element (Fantini et al., 2004; Josefowicz and

Rudensky, 2009). Consistent with the in-vitro findings, in-vivo neutralization of TGF-β

impaired oral tolerance (Faria and Weiner, 2005; Miller et al., 1992) and inhibited the

differentiation of antigen-specific FoxP3+ iTreg cells (Mucida et al., 2005).

Phenotype

Naturally occurring Tregs are a heterogeneous population of cells that express

FoxP3 and high levels of CD25, which is considered to be a useful marker for

distinguishing the CD4+ Tregs cells (Asano et al., 1996; Sakaguchi et al., 1995). However,

all T cells express CD25 upon activation, therefore CD25 is hardly a good marker for

unambiguous identification of nTregs. In addition, similar to CD25 expression, CTLA-4

and glucocorticoid-induced tumour necrosis factor receptor family-related protein (GITR)

are highly expressed on nTreg cells, however CTLA-4 and GITR are also expressed by

activated T cells. Nevertheless, several molecules have recently been identified that can

help to distinguish Tregs from other activated T cells (Fig. 10).

59

Introduction

Figure 10 : Cell surface markers for naturally arising CD4+ Treg cells. The most specific and reliable marker for naturally occurring CD4+ Treg cells is the transcription factor FoxP3, which is expressed by the majority of CD25+CD4+ T cells and a fraction of CD25−CD4 T cells. CD45RBlow T cells, GITRhigh T cells, and CTLA-4+ T cells in the CD4+ T cell population include FoxP3-expressing T cells in normal naive mice. (Sakaguchi et.al. 2004)

Latency associated peptide (LAP) forms a latent complex with TGF-β1, one of the

most important cytokines involved in Treg cell development and function (Nakamura et al.,

2004). LAP is useful for distinguishing between Tregs and activated FoxP3- or FoxP3+

non-Tregs (Tran et al., 2009). The expression of LAP characterizes a Treg subset with high

levels of FoxP3, CTLA-4, and GITR (Chen et al., 2008). LAP can be used as a marker for

TGF-β expressing Tregs, which exhibit greater suppression of EAE in-vivo than

CD4+CD25+LAP- cells. In the LAP+ subpopulation, suppression is TGF-β dependent and

is mediated by both cell-to-cell contact and soluble factors. In contrast, the less potent

suppressive function of LAP- cells is mainly cell contact dependent.

60

Introduction

In rats, the folate receptor 4 (FR4) is expressed at high levels by Tregs and is

essential for their maintenance (Yamaguchi et al., 2007). This is in contrast to other naive or

activated T cells. Tregs can thus be distinguished from other activated CD4+ T-cell

subpopulations using a combination of FR4 and CD25 expression.

CD39 (ENTPD1 [ectonucleoside triphosphate diphosphohydrolase-1]; EC 3.6.1.5),

an ectoenzyme that degrades ATP to AMP, represents another surface marker of Tregs

(Borsellino et al., 2007; Deaglio et al., 2007). In mice, it is present on virtually all

CD4+CD25+FoxP3+ Tregs. In humans, CD39 expression is limited to a subset of FoxP3+

regulatory effector/memory-like T cells. These cells have been demonstrated to be present

in dramatically reduced numbers during flares of relapsing/remitting MS (Fletcher et al.,

2009). In addition Tregs also express CD73, an ecto-5'-nucleotidase that converts AMP into

adenosine (Zimmermann, 1992), these suppressor cells are therefore able to catalyze

ATP/ADP eventually into immunosuppressive nucleoside adenosine (Borsellino et al.,

2007; Deaglio et al., 2007; Kobie et al., 2006). Thus, dual expression of CD39 and CD73,

serves as a phenotypic marker for Tregs, and also contributes to their functional suppressive

activity by catalyzing the generation of pericellular adenosine.

Recently, CD127 (IL-7R) expression for discriminating between CD25+ regulatory

and CD25+ activated T cells has been described, because many non-regulatory T-cell

subsets are IL-2-independent and IL-7-dependent (Seddiki et al., 2006). However, Tregs

which require IL-2 for survival, might not require IL-7 or the IL-7R. Eventually, CD127

downregulation on Treg cells was confirmed by using microarray gene-expression-profiling

studies of CD4+CD25hi and CD4+CD25- T cells, and by using quantitative polymerase

chain reaction (qPCR) to confirm the reduction in CD127 mRNA expression (Liu et al.,

2006b). In addition, functional suppressive activity of the CD127low/- population and its

absence in the CD127+ population has been demonstrated (Seddiki et al., 2006). Therefore

CD127low/- also serves a good phenotypic marker for Tregs.

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Introduction

Mechanism of suppression:

The various potential suppression mechanisms used by Treg cells can be grouped

into four basic 'modes of action': suppression by inhibitory cytokines, suppression by

cytolysis, suppression by metabolic disruption and suppression by modulation of DC

maturation or function.

Suppression by inhibitory cytokines

TGF-β is a cytokine with immunoregulatory activity that is present in a latent form

that must be activated in order to be biologically active. The role TGF-β plays in Treg

activity is controversial, as there are conflicting reports in the literature depending on the

experimental model that was used. Recent work has demonstrated the importance of

TGF-β not in the suppressive activity of Tregs, but in their de-novo generation (Shevach et

al., 2008). Thymus-derived Tregs, in the presence of DCs, induced additional Treg

formation from naive precursors in a process termed infectious tolerance. It was proposed

that LAP expressed on the surface of Tregs in combination with TGF-β interact with naive

T cells and transform them into Tregs. Interestingly, DCs lacking the TGF-β-activating

integrin αVβ8 were unable to induce Tregs (Travis et al., 2007). Thus, TGF-β appears

important for Treg expansion and conversion. Additionally, TGF-β has been reported to

play a role in the suppressive function of Tregs. Neuropilin-1 (Nrp1), a suggested marker of

Tregs, has been reported to act as a TGF-β receptor in mice that can bind and activate latent

TGF-β, leading to promotion of suppressive function (Glinka and Prud'homme, 2008).

TGF-β may not necessarily function as a soluble mediator but may bind to surface

molecules, such as LAP and Nrp1, and function as a membrane-bound mediator in a cell-to-

cell contact-dependent manner (Fig. 11).

In addition to TGF-β, IL-10 production by Treg cells has been shown to be essential

for the prevention of colitis in mouse models of IBD (Asseman et al., 1999; Menager-

Marcq et al., 2006). Moreover, it appears that the tumour microenvironment promotes the

generation of FoxP3+ Treg cells that mediate IL-10 dependent, cell-contact independent

suppression (Bergmann et al., 2007). Similarly, IL-10 produced by Treg cells also appears

to be important for IL-10-mediated tolerance in a model of hepatitis induced by

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Introduction

concanavalin A (Erhardt et al., 2007). In addition, Treg-cell-derived IL-10 is also

implicated in the induction of B-cell-enhanced recovery from EAE (Mann et al., 2007).

Interestingly, the Treg cell-specific deletion of IL-10 did not result in the development of

spontaneous systemic autoimmunity, but did result in enhanced pathology in the colon of

older mice and in the lungs of mice with induced airway hypersensitivity, suggesting that

the function of Treg cell-derived IL-10 may be restricted to the control of inflammatory

responses that are induced by pathogens or environmental insults (Rubtsov et al., 2008).

Recently, a new inhibitory cytokine, IL-35, a member of the IL-12 heterodimeric

cytokine family formed by the pairing of Epstein–Barr virus-induced gene 3 (Ebi3) and p35

(IL-12a), has been described to be preferentially expressed by Treg cells and required for

maximal suppressive activity (Collison et al., 2007). Both Ebi3 and IL-12a are

preferentially expressed by mouse FoxP3+ Treg cells (Collison et al., 2007; Gavin et al.,

2007), but not resting or activated effector T cells, and are significantly upregulated in Treg

cells that are actively suppressing (Collison et al., 2007). As predicted for a heterodimeric

cytokine, both Ebi3-/- and IL-2a-/- Treg cells had significantly reduced regulatory activity

in-vitro and failed to control homeostatic proliferation of effector T cells and resolve IBD

in-vivo. This precise phenotype suggested, that IL-35 is required for the maximal

suppressive activity of Treg cells. Importantly, IL-35 was sufficient for Treg-cell activity,

as ectopic expression of IL-35 conferred regulatory activity to naive T cells and

recombinant IL-35 suppressed T-cell proliferation in-vitro (Collison et al., 2007). All these

findings confirm that three inhibitory cytokines, IL-10, IL-35 and TGF-β, are key mediators

of Treg-cell function (Fig. 11).

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Introduction

Figure 11: Basic mechanisms of suppression used by Treg cells (Vignali et.al. 2008)

Suppression by cytolysis

CD8+ T cells and natural killer (NK) cells are known to kill target cells through the

secretion of granzyme molecules (Lieberman, 2003). However, many human CD4+ T cells

also exhibit cytotoxic activity. Consistent with this, activated human Treg cells have been

shown to express granzyme A. Furthermore, Treg cell-mediated target-cell killing was

mediated by granzyme A and perforin through the adhesion of CD18 (McHugh et al.,

2002).

By contrast, as mouse CD4+ T cells are not cytolytic, it was surprising that early

gene expression arrays showed that the expression of granzyme B was upregulated in

mouse Treg cells (Herman et al., 2004; McHugh et al., 2002). It has been shown, that

granzyme B-deficient mouse Treg cells had reduced suppressive activity in-vitro, and that

this granzyme B-dependent suppression appeared to be a perforin-independent one, as

shown by the result of Treg cell-induced apoptosis of effector T cells (Gondek et al., 2005).

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Introduction

The notion that Treg cells might possess cytolytic activity was supported by studies

showing that Treg cells can kill B cells in a granzyme B-dependent and partially perforin-

dependent manner that results in the suppression of B-cell function (Zhao et al., 2006).

More recently, Treg cells were shown to suppress the ability of NK cells and CTLs to clear

tumors by killing these cells in a granzyme B-dependent and perforin-dependent manner

(Cao et al., 2007). Furthermore, Treg cells induce perforin-dependent DC death in tumor-

draining lymph nodes, which limits the onset of CD8+ response in-vivo (Boissonnas et al.,

2010) (Fig.11).

Recently it has been shown, that activated Treg cells induce apoptosis of effector T

cells through a TRAIL–DR5 (tumour-necrosis-factor-related apoptosis-inducing ligand–

death receptor 5) pathway (Ren et al., 2007). Furthermore, galectin-1, which can induce T-

cell apoptosis, has been shown to be upregulated by mouse and human Treg cells and

galectin-1-deficient Treg cells have reduced regulatory activity in-vitro (Garin et al., 2007).

In addition to these findings, more work is required to define the cytolytic mechanisms that

Treg cells use to mediate suppression.

Suppression by metabolic disruption

Several fascinating suppressive mechanisms have been described that could

collectively be referred to a 'metabolic disruption' of the effector T-cell target. There are

some speculative thoughts whether high expression levels of CD25 privilege Treg cells to

consume local IL-2 and therefore starve actively dividing effector T cells by depleting the

IL-2 they need to survive (de la Rosa et al., 2004; Thornton and Shevach, 1998). In

contrast, recent reports from human Treg cells suggested, that IL-2 depletion alone is not

enough for Treg cells to suppress effector T cells (Oberle et al., 2007), therefore more

definitive evidence is still needed.

Recently two new Treg-cell mechanisms have been proposed, that induce the

intracellular or extracellular release of adenosine nucleosides. Concordant expression of the

ectoenzymes CD39 and CD73 on Tregs was shown to generate pericellular adenosine,

which suppressed effector T-cell function through activation of the adenosine receptor 2A

(A2AR) (Borsellino et al., 2007; Deaglio et al., 2007; Kobie et al., 2006). Interestingly,

binding of adenosine to A2AR appears to not only inhibit effector T-cell functions, but also

65

Introduction

to enhance the generation of induced Treg cells by inhibiting IL-6 expression while

promoting TGF-β secretion (Zarek et al., 2008). Both actions favor Treg generation and

expansion over Th17. Recently it has been shown, that nucleus-localized Foxo3 functions in

DCs to inhibit the production of inflammatory cytokines like IL-6, which induced T cell

survival in Foxo3-/- mice resulted from an inability of Foxo3-/- DCs to shut down cytokine

production in response to CTLA-4 signals (Dejean et al., 2009). Treg cells were also shown

to suppress effector T-cell function directly by transferring the potent inhibitory second

messenger cyclic AMP (cAMP) into effector T cells through membrane gap junctions

(Bopp et al., 2007) (Fig.11).

Suppression by targeting dendritic cells

In addition to the direct effect of Treg cells on T-cell function, Treg cells might also

modulate the maturation and/or function of DCs, which are required for the activation of

effector T cells (Bluestone and Tang, 2005). Interestingly, studies using intravital

microscopy have revealed direct interactions between Treg cells and DCs in-vivo. These

interactions were proposed to function in attenuating the effector T-cell activation by DCs

(Tadokoro et al., 2006; Tang et al., 2006) in a process involving the CTLA4, which is

constitutively expressed by Treg cells (Read et al., 2000; Takahashi et al., 1998). More

specifically, the use of CTLA4-specific blocking antibodies or CTLA4-deficient Treg cells

showed, that in the absence of functional CTLA4, Treg-cell-mediated suppression of

effector T cells via DCs was reduced (Oderup et al., 2006; Serra et al., 2003). Importantly,

it was also shown, that Treg cells could condition DCs to express IDO, an enzyme inducing

the production of pro-apoptotic metabolites from the catabolism of tryptophan, resulting in

the suppression of effector T cells. This mechanism is dependent on interactions between

CTLA4 and CD80 and/or CD86 (Fallarino et al., 2003; Mellor and Munn, 2004).

In addition to inducing DCs to produce immunosuppressive molecules, several

studies have suggested, that Treg cells may also downmodulate the capacity of DCs to

activate effector T cells (Houot et al., 2006; Kryczek et al., 2006; Lewkowich et al., 2005;

Serra et al., 2003). Recent reports have also suggested that lymphocyte-activation gene 3

(LAG3) may block DC maturation. LAG3, a CD4 homologue that binds MHC class II

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Introduction

molecules with very high affinity, has a negative regulatory T-cell intrinsic function and is

required for maximal Treg-cell suppression (Huang et al., 2004; Workman et al., 2004).

Finally, Treg cells can also influence immune responses by modulating the

recruitment and function of other cell types. For instance, Treg cell-derived IL-9 has been

shown to recruit and activate mast cells, which are essential regulatory intermediaries in the

establishment of peripheral allograft tolerance (Lu et al., 2006) (Fig.11).

1.2.4.2 Tr1 cells or IL-10-producing cells

Initially, IL-10 was recognized as a Th2 cytokine (Mosmann et al., 1986). Later on,

it was reported, that some cells with regulatory functions express IL-10 and were designated

as Tr1 cells (Groux et al., 1997). However, some conventional Tregs also produce IL-10,

and IL-10 production by these cells is important for controlling inflammation in the gut

(Rubtsov et al., 2008). Nevertheless, it appears that Tr1 cells are different from FoxP3+ IL-

10-producing cells (Roncarolo et al., 2006).

Recently it has been documented, that IL-10 production is also found in the subsets

of Th17 and Th1 cells (Anderson et al., 2007a; Jankovic et al., 2007; McGeachy et al.,

2007), and that IL-10 production by these cells negatively regulates their function. Indeed,

strong TcR stimulation, which leads to sustained Erk activation, results in IL-10 production

in Th17 and Th1 cells (Saraiva et al., 2009), suggesting that IL-10 serves as a critical

negative feedback regulator to control the magnitude of many types of immune responses

and to prevent tissue damage. Therefore, Tr1 cells may very well not be a separate lineage,

but include both IL-10-producing Tregs, as well as those subsets of Th1, Th2 and Th17

cells that are capable of expressing IL-10.

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Introduction

1.3 CNS inflammation and Autoimmunity

1.3.1. Triggering auto-aggressive immune response

Intrathymic deletion of T cells with high avidity for self-antigens restricts the

repertoire of peripheral autoreactive T cells, but this process is incomplete (Ashton-Rickardt

et al., 1994). Lymphocytes with lower avidity for self-antigens or with high avidity for

determinants that are not expressed in the thymus can be found in the periphery (Klein et

al., 2000). The presence of peripheral T cells that react with self-antigens in healthy

individuals (Arnold et al., 1993; Burns et al., 1983) indicates the existence of physiological

regulatory mechanisms that prevents pathological autoimmunity. Autoimmune disease

occurs, when the adaptive immune response is released to mount a response to self-

antigens. The induction of an autoimmune response requires autoantigens to rupture self-

tolerance. Recently it has been shown in different animal models of autoimmunity, how

tolerance can be broken.

1.3.1.1 Exposure of tissue specific antigen

The fact that TSAs are expressed in the thymus makes implausible the hypothesis

that autoimmunity results from a failure to impose central tolerance on the developing T

cell except if gene polymorphisms affect the central tolerance mechanisms or provided if

not all the TSAs are expressed in the thymus. But how genetic polymorphisms in the

regulatory regions of TSA genes might modulate the intrathymic expression levels of the

respective gene remains unclear. One possibility is that these polymorphisms might affect

the access to the TSAs or the DNA binding of transcription co-factors such as AIRE. This

in turn shifts the threshold of self-tolerance to an extent that translates into significant

alterations of disease susceptibility on the appropriate genetic background. However, the

exquisite sensitivity of the central tolerance process to moderate quantitative variations in

TSA expression in this context is quite surprising. Thus, subtle differences in the range of 2

- 4 fold in intrathymic expression of auto-antigens as, obtained in mice with defined copy

numbers of pancreatic or CNS-specific antigens can modulate the susceptibility to

autoimmunity (Chentoufi and Polychronakos, 2002; Miyamoto et al., 2003; Thebault-

Baumont et al., 2003). The threshold range for self-antigens in humans is not well defined.

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Introduction

Carriers of a single allele of an AIRE mutation do not develop autoimmune polyglandular

syndrome type-1 (Vogel et al., 2002). Considering a direct gene-dosage effect between

AIRE and target gene expression levels, as suggested by findings in the AIRE knockout

mouse model (Liston et al., 2004a), minor variations apparently do not predispose to

disease in a polygenic setting in humans. Nevertheless, in type-1 diabetes mellitus, the

IDDM2 (insulin-dependent diabetes mellitus 2) locus shows the highest correlation with

disease susceptibility of all identified disease-associated non-MHC loci. This trait reflects a

polymorphism in variable number of tandem repeats (VNTRs) upstream of the promotor

region of the insulin gene. These VNTR alleles correlate with the level of insulin

transcription in the thymus (Pugliese et al., 1997; Sabater et al., 2005; Vafiadis et al., 1997).

This suggests that there is only limited buffering of the tolerance threshold in humans on a

susceptible background.

Self-antigens expressed in the thymus delete high affinity self-reactive T cells,

whereas low affinity T cells can seed to the periphery (Daniels et al., 2006). This has been

illustrated in studies of the T cell repertoire specific for myelin basic protein (MBP). MBP-

deficient, C3H (H-2k) mice immunized with whole MBP exhibit a strong T cell response to

MBP 79–87, whereas wild type mice exhibit a response that is at least 3 logs lower in

avidity, suggesting that endogenous MBP inactivates the high avidity T cell repertoire

(Huseby et al., 2001b; Targoni and Lehmann, 1998). However, this effect of MBP

expression is not necessarily restricted to the thymus.

Self-reactive cells can escape thymic deletion because the isoform of the antigen

expressed in the thymus differs from that expressed in the target organ. This was

demonstrated in studies of the T cell repertoire specific for myelin proteolipid protein

(PLP), another target antigen in EAE. The predominant form of PLP expressed in the

thymus is a splice variant, DM20, which lacks residues 116–150 of the full-length protein

(Anderson et al., 2000; Klein et al., 2000). Thus, only T cells that react with epitopes within

DM20 would be expected to undergo central tolerance. Indeed, this is to be the case in

studies of the T cell repertoire to PLP in wild type versus PLP-deficient C57BL/6 mice. The

authors described, that expression of DM20 by thymic epithelium was sufficient to confer

T-cell tolerance to PLP in EAE-resistant C57BL/6 mice, whereas PLP-deficient mice

maintained a brisk T cell response upon PLP immunization. They analyzed the PLP-

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Introduction

specific response in thymectomized PLP-deficient mice. They transplanted them with a

wild-type thymus, irradiated them to ablate their immune system and reconstituted them

with PLP-deficient bone marrow cells. They showed in these mice that intrathymic

expression of PLP, presumably by radio-resistant epithelial cells, was sufficient to confer

tolerance on the T-cell repertoire in C57BL/6 mice. In contrast, the major T-cell epitope in

SJL/J mice is only encoded by the CNS-specific exon of PLP, but not by thymic DM20.

Thus, lack of tolerance to this epitope offers an explanation for the exquisite susceptibility

of SJL/J mice to EAE induced by PLP (Klein et al., 2000).

Alternatively, thymically expressed self-antigens may not mediate central tolerance

due to inefficient antigen presentation by MHC alleles present in the individual. This

mechanism has been proposed to explain the escape from tolerance of MBP 1–11 reactive T

cells in H-2k- expressing mice. MBP 1-11, which is expressed in the thymus (Mathisen et

al., 1993), has been shown to bind weakly to I-Au (Kumar et al., 1995) and to form unstable

peptide–MHC complexes (Harrington et al., 1998), resulting in the escape of self-reactive T

cells from the thymus.

Studies based on the transgenic expression of a neo-self antigen have allowed to

clearly delineate the impact of thymic expression of the neo-self antigen at the development

of tolerance. As an example, transgenic mice were raised by crossing mice expressing a

tissue specific transgene encoding hen egg lysozyme (HEL), with mice expressing a

transgenic TcR specific for HEL. The double transgenic mice expressed HEL specifically

on thyroid epithelium, pancreatic islets or systemically (including the thymus). The mice

expressing HEL on thyroid epithelium and pancreatic islets did not express HEL in the

thymus, allowing HEL specific T cells to escape thymic deletion resulting in subclinical

lymphocytic thyroiditis or insulitis (Akkaraju et al., 1997). In contrast, systemic HEL

expression allowed thymic deletion of HEL specific thymocytes, establishing tolerance to

HEL (Akkaraju et al., 1997).

Collectively these examples demonstrate, that the expression of self-antigen in the

thymus shapes the self-reactive repertoire, but in some cases deletion may not be complete

or may not occur at all due to the limited affinity of the MHC molecule for the self-antigen

or due to expression of a splice variant or isoform of the self-antigen.

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Introduction

1.3.1.2 Cryptic epitope hypothesis

This theory is based on epitope sequestration within protein, in that immunogenic

self-epitopes can arise from peptides not usually presented by APCs. Although typical

protein antigens possess numerous immunogenic peptide sequences, only a few of these, the

dominant epitopes, contribute to the development of the T-cell response against the whole

protein. Epitopes that are immunogenic in peptide form, but do not participate in the

immune response against the whole protein, are termed cryptic epitopes (Sercarz et al.,

1993).

Cryptic epitopes have proven important in understanding self–non-self

discrimination and autoimmunity (Lanzavecchia, 1995; Warnock and Goodacre, 1997).

Several studies using transgene-derived neo-self antigens have shown, that immune

tolerance extends only to dominant epitopes, and that T cells specific for cryptic epitopes

may be immune competent (Cabaniols et al., 1994; Cibotti et al., 1992; Moudgil and

Sercarz, 1993; Shih et al., 1997). Some autoimmune diseases are characterized by

determinant spreading, in which the early stages of the disease are characterized by T cells

reactive against one or a few dominant epitopes, while T-cell populations reactive against

cryptic epitopes appear in the later stages of disease (Tuohy et al., 1998).

This potential of cryptic epitopes to unfold an autoimmune disease was indeed

demonstrated in MBP-induced EAE. Significantly, epitope spreading during the chronic

stages of EAE resulted in the activation of T cells responding to various MBP peptides

(Lehmann et al., 1992). Furthermore, the concept of epitope spreading has been

documented in the Theiler murine encephalomyelitis virus-induce demyelinating disease

(TMEV-IDD) mouse model. TMEV, a natural mouse pathogen, is a picornavirus, which

induces a chronic CD4+ T cell-mediated demyelinating disease with a clinical course and

histopathology similar to that of chronic progressive MS (McMahon et al., 2005; Miller et

al., 1990). Demyelination in TMEV-infected mice is initiated by a mononuclear

inflammatory response mediated by CD4+ T cells targeting virus, which chronically

persists in the CNS (Pope et al., 1996). In addition, kinetic and functional studies show, that

T-cell responses to the immunodominant myelin PLP epitope (PLP139-151) did not arise

because of cross-reactivity between TMEV and self-epitopes (Miller et al., 1997), but

because of de-novo priming of self-reactive T cells to sequestered autoantigens released

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Introduction

secondary to virus-specific T cell-mediated demyelination (Lehmann et al., 1992).

Therefore epitope spreading is an important alternate mechanism to explain the etiology of

virus-induced organ-specific autoimmune diseases.

1.3.1.3 Molecular mimicry

The concept of molecular mimicry is based on a structural similarity between a

pathogen and self-antigens. The similarity could be expressed as shared amino acid

sequences or similar conformational structure between the antigens (Fig.12). In molecular

mimicry, the exogenous antigen needs not to be present or to infect the target tissue of the

autoimmune process. Molecular mimicry is characterized by an immune response to an

environmental agent that cross-reacts with a host antigen, resulting in disease (Albert and

Inman, 1999; Oldstone, 1998). This hypothesis has been implicated in the pathogenesis of

diabetes, lupus and MS (Albert and Inman, 1999; Fujinami and Oldstone, 1985; Gran et al.,

1999; Oldstone, 1998). In addition, implication of molecular mimicry has been described in

some forms of Guillain-Barré syndrome (GBS), an example of damage to the peripheral

nervous system (PNS) mediated by an immune reaction after infection with bacteria

Camyllabacter jejuni due to share expression of the ganglioside GM1 (Ang et al., 2004;

Yuki and Odaka, 2005).

Providing support to this hypothesis is the observation that a given TcR can cross-

react with different pMHC complexes and therefore a single T cell can respond to different

peptides (Gregersen et al., 2006; Lang et al., 2002; Marrack et al., 2008; Wucherpfennig

and Strominger, 1995). This implies, that responses to microbial antigens could result in the

activation of T cells, which are cross-reactive with self-antigens. Similarly, monoclonal

antibodies have also been found to recognize both microbial and self-antigens (Fujinami et

al., 1983).

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Introduction

Figure 12: The evolution of the molecular mimicry concept and activation of

autoreactive T cells via cross-reactivity with foreign antigens (Sospedra et.al. 2005)

The potential for molecular mimicry has been documented in many animal models

of autoimmune disease, such as herpes simplex virus (HSV)-associated stromal keratitis,

(Zhao et al., 1998) and models of type I diabetes (Christen et al., 2004). This mechanism of

molecular mimicry is also involved in the TMEV-IDD model of MS, a severe rapid-onset

demyelinating disease of the CNS induced by intracerebral or peripheral infection with

TMEV that has been engineered to express the immunodominant self epitope from myelin

PLP 139–151 (Olson et al., 2001). Several bacterial and viral peptides that mimic PLP 139–

151 or MBP 1-11 have been identified and have been used in models that directly address

the possibility that autoimmune diseases could be induced by molecular mimicry (Carrizosa

et al., 1998; Gautam et al., 1998) (Fig. 12).

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Introduction

Possibly relevant to human disease, bacterial peptides mimic the MBP epitope 85–

99 and induce demyelinating disease in mice that transgenically express a human MBP 85-

99 specific TcR and the appropriate HLA class II molecule that can present the peptide

(Greene et al., 2008).

This mechanism of molecular mimicry has been indirectly implicated in MS.

Indeed, high viral loads that occur during symptomatic primary infection of EBV and result

in infectious mononucleosis, are associated with an increased risk of developing MS

(Nielsen et al., 2009; Thacker et al., 2006), and could prime these polyspecific T-cell

responses. Accordingly, patients with MS have predominant clonal expansions of T cells

that are specific for EBV nuclear antigen 1 (EBNA1), the EBV antigen that is most

commonly targeted by CD4+ T cells in healthy virus carriers, and EBNA1-specific T cells

recognize myelin antigens more frequently than autoantigens that are not associated with

MS (Lunemann et al., 2008).

Although all these findings demonstrate, that molecular mimicry is a plausible

hypothesis that can explain the link between infection and autoimmunity, evidence for this

phenomenon in human autoimmune diseases is still need to be established, except for some

forms of GBS.

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Introduction

1.3.1.4 Dual TcR expression

During T cell ontogeny successful rearrangement of the TcR β chain VDJ regions

results in the allelic exclusion of the alternative TcR β locus (Fenton et al., 1988;

Krimpenfort et al., 1989; Uematsu et al., 1988). Conversely, TcR α chain rearrangement

occurs simultaneously on both chromosomes and successful rearrangement does not

abrogate expression of the alternative allele, though α chain rearrangement is abrogated due

to down-regulation of RAG expression after positive selection of the expressed αβ TcR

(Borgulya et al., 1992; Petrie et al., 1993). This allows thymocytes to develop into T cell

clones expressing one TcR β chain complexed with either of two TcR α chains, effectively

estabilishing dual TcR expression. Some of these clones can express two functional MHC-

restricted TcRs (Hardardottir et al., 1995; Morris and Allen, 2009). If the initially expressed

αβ TcR is not selected, T cells can be rescued from apoptosis by coexpression of a second

TcR that can mediate positive selection and appear in the periphery as dual TcR expressing

T cells (Borgulya et al., 1992; Brandle et al., 1992; Padovan et al., 1995; Petrie et al., 1993).

These dual TcR expressing T cells could be activated by engagement of either TcR; this

dual-specific T cell activation could potentially provide T cells with a mechanism to evade

tolerance (Padovan et al., 1993; Simpson et al., 1995). In-vivo observations indicated that

thymocytes expressing low levels of an auto-reactive TcR could escape negative selection

provided they express a second non-self specific TcR (Zal et al., 1996) (Fig.13).

Interestingly, an alternative approach has indicated a function for the expression of

dual TcRs in autoimmune induction in-vivo. Mice expressing a transgenic HA-specific TcR

in combination with systemic expression of transgenic HA fail to develop autoimmunity

due to central tolerance and anergy. However, this state of tolerance can be by-passed if

high levels of an irrelevant TcR are expressed on the HA-specific T cells, permitting

insulitis and mild diabetes (Sarukhan et al., 1998).

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Introduction

Figure 13: Different scenarios for dual TcR T cells. (a) Tourist visa. Thymocytes express one TcR (1), which is incapable of interacting with peptide-self-MHC ligands presented on thymic epithelium. Thus, due to ongoing TcR α rearrangement, a second TcR (2) is expressed, composed of the original TcR β chain and a new TcRα. This second TcR mediates positive selection, and, in the periphery, it can recognize foreign peptides presented by the selecting MHC. The first TcR (1) does not contribute to the useful TcR repertoire, however, it may infrequently detect foreign antigen. (b) Illegal emigrant. During thymocyte selection, the first TcR (1) exhibits high affinity for an MHC-peptide ligand expressed in the thymus. Consequently, this TcR is down regulated (shown as an orange TcR-1 α-chain), and RAG expression re-induced. Subsequent TcRα rearrangement produces TcR (2), which rescues the thymocyte from deletion. In the periphery, low expression of the first TcR will result in autoimmunity if it is not excised from the TcRα locus. (c) Dual citizenship. During thymocyte selection, both TcRs (1 and 2) may contribute synergistically to positive selection. Neither TcR is capable of efficiently selecting the thymocyte by itself however; both are self-restricted and are equally capable of detecting nonself peptides presented by the selecting MHC molecule(s) in the periphery. Thus, the repertoire of useful TcRs is expanded (He et.al. 2002)

Most discussion has focused on the dangers of dual TcR expression because one of

the TcRs on dual TcR cells does not go through thymic selection. This may allow

autoreactive TcRs to sneak into the mature T cell repertoire under the cover of a original

TcR that drives positive selection of a given thymocyte. However, it has been shown that

the dual TcR cells can extend the TcR repertoire for foreign antigens by rescuing functional

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Introduction

TcRs that cannot be selected on single TcR cells and can, therefore, benefit the immune

system (He et al., 2002).

Recently it has been shown in a dual-TcR transgenic murine model, that tolerization

of the self-reactive TcR does not disrupt signaling or function mediated via the second

receptor, suggesting that tolerance can be maintained proximally at the level of the self-

reactive TcR (Teague et al., 2008).

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Introduction

1.3.2. Multiple Sclerosis

1.3.2.1 Generalities

MS is a chronic demyelinating inflammatory disease that affects the CNS and

usually begins in young adulthood (McFarlin and McFarland, 1982a, b). The disease affects

0.05–0.15% of Caucasians and women more frequently than men. MS leads to substantial

disability through deficits of sensation and of motor, autonomic, and neurocognitive

function (Noseworthy et al., 2000). In 80–90% of cases, MS starts with a relapsing–

remitting course (RR-MS). Over time, the number of relapses decreases, but most patients

develop progressive neurological deficits that occur independently of relapses (the so-called

secondary progressive phase). In 10–20% of patients, MS begins with a primary progressive

course (PP-MS) without acute relapses. In general, the progression rate in RR-MS is

comparable to that of PP-MS as soon as the patients enter the secondary progressive phase

(Kremenchutzky et al., 1999). Imaging studies have revealed differences between RR-MS

and PP-MS. In patients who suffer from RR-MS, acute CNS lesions with spontaneous

resolution are frequently detected, even in the absence of clinical attacks (Miller et al.,

1998). These lesions are usually located in the white matter, and are often characterized by

a disturbance of the BBB and local oedema that are compatible with an inflammatory

process. By contrast, when progressing to the secondary phase and in patients with PP-MS,

such inflammatory activity is much less conspicuous (Miller et al., 1998). Global brain

atrophy, however, is more dominant in the progressive stage and seems to correlate with

disability (Fox et al., 2000; Losseff et al., 1996). These findings indicate, that early in the

disease, ongoing inflammatory activity is present in most patients and is responsible for the

relapsing–remitting course, whereas a distinct process might be operative in the progressive

phase of the disease, when inflammatory activity diminishes despite faster evolution of

disability.

The prevalence of MS varies significantly depending on the genetic background of

the population considered (Compston and Coles, 2008). MS is highly prevalent in

Caucasians, but only rarely observed in Asians or Africans. Even in areas with high MS

prevalence, these ethnic groups are at much lower risk than Caucasians. Moreover, the risk

of developing the disease is significantly higher for family members of patients with MS

(Ebers et al., 1995). By contrast, the prevalence in MS-patient’s spouses and adopted family

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Introduction

members does not differ from that of the general population (Fig.14). These findings argue

for a strong genetic predisposition to MS, and have prompted a large number of linkage and

association studies to identify disease loci and alleles. The results of genomic screens in MS

indicate that a number of different genes (estimated to date 30-100), each having a

relatively small contribution, in different potential combination are involved in the

susceptibility to MS (Fugger et al., 2009). The large variations in susceptibility loci between

the studies also indicate heterogeneity in disease alleles, even in Caucasian populations. The

role of genetic factors is even more complex, as they seem to be involved not only in

disease manifestation, but also in the clinical course of MS (Chapman et al., 2001; Fazekas

et al., 2001) .

Figure 14 Geographical distributions of MS and migrations (Compston et.al. 2008) The five continents are depicted to show medium prevalence of MS (orange), areas of exceptionally high frequency (red), and those with low rates (grey-blue). Some regions are fairly uncharted and these colors are only intended to provide an impression of the geographical trends. Major routes of migration from the high-risk zone of northern Europe, especially including small but informative studies, are shown as dotted arrows. Studies involving migrants from low-risk to high-risk zones are shown as solid arrows.

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Introduction

However, genome-wide association studies and candidate gene association studies

revealed, that MS is associated with many common genetic variants including mostly

immunologically relevant genes (Fig. 15).

HLA controls immune responses and its association with MS indicates the primary

involvement of the immune system in this disease. MS was one of the first conditions

proven to be HLA associated involving primarily HLA class II loci. HLA-class II alleles of

the HLA-DR2 haplotype are more strongly associated to MS (Jersild et al., 1973; Madsen et

al., 1999). Three candidate risk genes of this haplotype, HLA-DRB1*1501 (HLA-DR2b),

HLA-DRB5*0101 (HLA-DR2a) and HLA-DQB1*0602 (HLA-DQ6), are tightly associated

with MS susceptibility. Recently, HLA-DRB1*1501 allele has been implicated as the main

susceptibility allele in MS as conditioning on either HLA-DRB1 or the most significant

HLA class II haplotype block found no additional block or SNP association independent of

the HLA class II genomic region (Lincoln et al., 2005; Oksenberg et al., 2004). In addition

it has been shown, that carriers of HLA-DRB1*1501 had a median of 4 spinal cord lesions

compared with 2 lesions for noncarriers (P < .001)(Sombekke et al., 2009).

Furthermore, more genetic studies have identified additional susceptibility loci

within the HLA class I region. The HLA-A and HLA-C are candidate susceptibility loci,

and the disease-associated alleles at these loci are HLA-A*0301, which predisposes to

disease (Fogdell-Hahn et al., 2000; Harbo et al., 2004) and HLA-A*0201 and HLA-C*05

which have a protective effect (Brynedal et al., 2007; Yeo et al., 2007) (Fig.15).

Although the MHC region certainly plays a dominant role in the development of

MS, non-MHC genetic variants contribute to disease susceptibility. At least 16 non-MHC

genes have been identified so far to be associated with MS including the immunological

genes (Fig.15), with the possible exception of the C-type lectin (CLEC16A) and the killer

cell lectin-like receptor subfamily B, member 1 (KLRB1), all genes encode protein which

are known to be important for T cell responses, cytokine receptors for IL-7 and IL-2 (Hafler

et al., 2007; Lundmark et al., 2007), signal transducers tyrosine kinase-2 (TYK2) for

cytokines, such as interferons, IL-10, IL-12 (Ban et al., 2009) and cell adhesion molecules

(like CD58) (Reich et al., 2005) and CD226 (Hafler et al., 2009), . The neurological genes

include amiloride-sensitive cation channel 1, neuronal (ACCN1; also known as ASIC2)

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Introduction

(Bernardinelli et al., 2007) and kinesin family member 1B (KIF1B) (Aulchenko et al.,

2008) (Fig. 15). This latter gene has yet to be confirmed (De Jager et al., 2009).

Figure 15: Genes associated with risk of development of MS (Fugger et.al. 2009)

Epidemiological studies have provided evidence for an additional, environmental

component in the disease process. Migration from high to low-risk areas before adolescence

reduces the risk of developing MS (Gale and Martyn, 1995). In areas with homogeneous

ethnic populations, MS prevalence varies, being higher in areas with moderate climate

(Lauer, 1994). One proposed causative factor is the decrease in sunlight exposure

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Introduction

depending on the latitude. UV radiation may exert its effects either by influencing

immunoregulatory cells or by the biosynthesis of vitamin D (Hayes, 2000). EAE data and

the association of a vitamin D receptor polymorphism support the latter notion with MS in

Japan (Simon et al., 2010). The underlying factors are not completely understood, moreover

environmental factors can influence disease course, as MS relapses are often associated

with common viral infections (Sibley et al., 1985).

Overall, clinical studies indicate at least two distinct phases to characterize MS, one

that is dominated by acute relapses and one by steady progression. Both genetic and

environmental factors seem to contribute synergistically to the manifestation and

progression of the disease.

1.3.2.2 Animal Model: Experimental autoimmune encephalomyelitis

EAE represents a useful animal model for MS (Rivers et al., 1933; Whitehouse et

al., 1969), as it mimics some of its key clinical and histological features and also served to

develop several new therapeutic strategies, now implemented in patients (Copaxone,

Natalizumab, FTY720).

The minimal requirement for inducing CNS inflammation in the various EAE

models is activation of myelin-reactive T cells in the peripheral immune system (Goverman

et al., 1993; Zamvil and Steinman, 1990). EAE can be induced by several ways in

susceptible experimental animals, as follows:

Actively induced EAE

Immunization of susceptible inbred strains of rats or mice with CNS protein extract

(purified myelin proteins or myelin derived peptides), in CFA causes paralytic disease in

most strains, often starting with limp tail and hind legs and paralysis progressing towards

the upper limbs. The rapidly unfolding neurological defects are accompanied by a dramatic

loss of weight.

Not all animal strains and species respond in the same way to encephalitogenic

immunization. For example, MOG is a strong encephalitogen, especially in C57BL/6 mice,

but also DA rats and marmosets (Mendel et al., 1995), but not in PL/J mice or Lewis rats.

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Introduction

Passive transfer of EAE

EAE can be transferred to naïve hosts by myelin-specific T-cell clones isolated from

actively primed syngenic donors. The encephalitogenic T cells are classically CD4+ T

lymphocytes producing IFN-γ and TNFα, recognizing autoantigenic peptide epitopes in the

context of MHC class II determinants (Liblau et al., 1995). Recent evidence, however,

suggests that these populations include a subset of CD4+ T cells that preferentially secrete

IL17 rather than IFN-γ, the Th17 cells (Bettelli and Kuchroo, 2005; Haak et al., 2009). It

currently appears that both Th1 and Th17 have pathogenic potentials, and that they

complement each other as the ratio between Th1:Th17 dictate T cell infiltration and

inflammation in the brain parenchyma (Jager et al., 2009; Stromnes et al., 2008).

Although the most common models of transferred EAE involve CD4+ T cells, some

reports describe the role of MBP-specific CD8 T cells in EAE (Huseby et al., 2001a).

Alternatively, MOG-reactive CD8 T cells were isolated from wild-type C57BL/6 mice

immunized with MOG. The T cells were primed by conventional active immunization, but

upon culturing with antigen the CD8 T cells overgrew their CD4 counterparts. Enriched

CD8 cell populations transferred EAE, which was characterized by a particularly high

proportion of CD8 T cells in the CNS infiltrates (Sun et al., 2001).

Spontaneous EAE

Untill recently there were no naturally occurring animal models of spontaneous EAE

comparable to the NOD mouse model of autoimmune diabetes. However, a number of

recent transgenic mouse strains have been generated that spontaneously develop a variety of

EAE phenotypes. The first transgenic mice harbouring the genes for MBP-specific TcRs

showed spontaneous EAE at variable rates, depending on the microbial status of their

breeding facility (Goverman et al., 1993). Spontaneous EAE development with severe

outcome and complete penetrance was also demonstrated in PLP139-151 TcR transgenic mice

(Waldner et al., 2000).

Furthermore, 2D2 TcR transgenic mice that possess CD4 T cells specific for the

MOG35–55 peptide on the C57Bl/6 background develop spontaneous optic neuritis, the first

signs of disease in a large proportion of MS patients. In addition, some transgenic mice

(4%) also develop bonafide EAE (Bettelli et al., 2003).

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Introduction

Recently, two groups described double transgenic mice that developed a

spontaneous EAE syndrome strikingly resembling human neuro-myelitis optica (Devic’s

disease), an autoimmune disease related to MS (Bettelli et al., 2006a; Krishnamoorthy et al.,

2006). The animals possess T cells with receptors specific for a MOG epitope, at the same

time, 30% of their B cells bind a conformational MOG epitope and secrete autoantibodies

of the same specificity. The mice develop optico-spinal EAE as a result of cooperation

between T and B cells recognizing the same encephalitogenic autoantigen. This is

important, since evidence is emerging, that in a substantial proportion of patients with MS,

autoimmune T and B cells contribute to the pathogenesis, as B cells are efficient APCs for

processing intact myelin antigen and subsequent activation and pro-inflammatory

differentiation of T cells (Weber and Hemmer, 2009).

1.3.2.3 Pathophyisology of MS

CNS demyelination in MS is mediated by an inflammatory response characterized

by the recruitment of CD4 and CD8 T cells as well as macrophages in the white matter. The

antigenic targets of this immune response is currently unknown but may include myelin

self-antigens resulting in myelin destruction (Sospedra and Martin, 2005). While

considering the pathogenesis of MS, the disease process mainly comprises of an initial

phase of inflammation, which leads to demyelination, and this results in axon loss. The loss

of axons is considered to be the ultimate cause of the permanent clinical disability that

occurs during the disease.

Glial cells and myelin composition

Glial cells are non-neuronal cells that maintain homeostasis, form myelin, and

provide support and protection to the neurons and axons. In the human brain, there is

roughly one glial cell for every neuron with a ratio of about two neurons for every three

glial cell in the cerebral gray matter (Azevedo et al., 2009). The three types of CNS glial

cells are astrocytes, microglia, and oligodendrocytes.

Astrocytes are star-shaped glial cells that provide physical support to neurons and

clean up debris within the brain. They also provide neurons with some of the chemicals

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Introduction

needed for proper functioning and help control the chemical composition of the fluid

surrounding neurons. Finally, astrocytes play a role in providing nourishment to neurons.

Microglia are the smallest of the glial cells, which act as phagocytes to clean up

CNS debris. Microglia protect the brain from invading microorganisms and are thought to

be similar in nature to macrophages.

Oligodendrocytes are the myelin producing cells; arise during development from

oligodendrocyte precursor cells, which can be identified by their expression of a number of

antigens, such as ganglioside GD3, the NG2 chondroitin sulfate proteoglycan (Dawson et

al., 2000), and platelet-derived growth factor-α receptor subunit (Scolding et al., 1998). The

myelin sheath provides insulation to the axon that allows electrical signals to propagate

more efficiently. Myelin is mainly composed of 80% lipid and 20% protein.

The most abundant CNS myelin protein (about 50%) is PLP. It is highly

hydrophobic and evolutionarily conserved across species. PLP is a four transmembrane

domain myelin protein, which binds strongly to other copies of itself on the extracellular

side of the membrane. In a myelin sheath, as the layers of myelin wraps come together, PLP

will bind itself and tightly hold the cellular membranes together.

In mice, there are two main transcripts, one encoding the full-length 276 amino acid

isoform, and one encoding DM-20, an isoform that lacks 35 amino acids and is mainly

expressed in brain and spinal cord prior to myelination but also in lymphoid organs, where

full-length PLP is barely found as previously discussed in (section 1.3.1.1) (Bruno et al.,

2002; Seamons et al., 2003).

MBP is the second most abundant myelin protein (30%–40%) after PLP. It contains

170 amino acids and is relatively easy to isolate owing to its physicochemical

characteristics, and was the first to be used extensively in EAE. The highly basic MBP is

positioned at the intracellular face of myelin membranes, and via interactions with acidic

lipid moieties it is involved in maintaining the structure of compact myelin. MBP is found

in significant quantities in both central and peripheral myelin, and MBP transcripts have

also been detected in the thymus and in peripheral lymphoid organs (Bruno et al., 2002).

EAE can be induced with MBP in several mouse and rat strains, guinea pigs, and nonhuman

primates (Wekerle et al., 1994).

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Introduction

MOG, a 218 amino acid transmembrane glycoprotein of the Ig superfamily, is much

less abundant (0.01%–0.05%). It is located at the outermost lamellae of compact myelin and

on the outer surface of the oligodendrocyte membrane, therefore it is directly accessible to

antibodies and believed to be relevant as a target for both cellular and humoral immune

responses in MS. MOG is expressed late in myelination and is only found in the

brain/spinal cord and the optic nerve, not in peripheral nerves. Furthermore, MOG is either

absent or weekly expressed in lymphoid organs (Bruno et al., 2002; Seamons et al., 2003).

MOG-induced EAE is best examined in C57/BL6 mice, in which the MOG(35–55) peptide

induces a chronic, nonrelapsing EAE (Mendel et al., 1995). The recent 2D2 MOG TcR

transgenic mouse model showed spontaneous EAE with inflammation, demyelination, and

axonal damage in brain and spinal cord in a small fraction of animals, while 35% developed

spontaneous optic neuritis (Bettelli et al., 2003). The relatively higher expression of MOG

in the optic nerve has been proposed as one explanation for the involvement of the optic

nerve.

Myelin associated glycoprotein (MAG) is a 626 amino acid large myelin

glycoprotein located at the inner surface of the myelin sheath opposing the axon surface. It

accounts for less than 1% of total myelin protein in the CNS and is even less abundant in

the peripheral nervous system (PNS). Anti-MAG IgM response in polyneuropathies

revealed the pathogenic significance of this protein (Steck et al., 1983).

2’, 3’-Cyclic nucleotide 3’phosphodieserase (CNPase) exists in two splice variants

(CNPase I and II, 46 kDa and 48 kDa) and makes up 3%–4% of total myelin protein.

Demyelination

The pathology of MS is defined as an inflammatory process, associated with focal

plaques of primary demyelination in the white matter of the brain and spinal cord.

Inflammatory infiltrating cells mainly consist of T cells, activated macrophages and

microglia. In active lesions this inflammatory process is accompanied by a profound

disturbance of the BBB (Hochmeister et al., 2006; Kirk et al., 2003), the local expression of

proinflammatory cytokines and chemokines as well as of their cognate receptors (Cannella

and Raine, 1995; Huang et al., 2000). Complete demyelination is associated by a variable

degree of acute axonal injury and axonal loss (Ferguson et al., 1997; Trapp et al., 1998).

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Introduction

Furthermore, demyelination-associated axon degeneration was increased when

remyelination was inhibited (Irvine and Blakemore, 2008).

Although axon degeneration is accepted as a major cause of persistent disability in

MS (De Stefano et al., 1998; Griffiths et al., 1998), little is known about the mechanisms of

how inflammatory demyelination leads to neuronal damage. Evidence based from animal

models suggested, that myelin-specific T cells are activated outside the CNS, followed by

upregulation of adhesion molecules and chemokine receptors, allowing them to adhere, role

along and finally transmigrate through the endothelium (Charo and Ransohoff, 2006;

Engelhardt and Ransohoff, 2005). Once in the perivascular space, autoreactive T cells get

reactivated by local APCs, such as DCs (Greter et al., 2005), and thereby gain access to

CNS parenchyma. Proinflammatory cytokines released by activated T cells and activated

microglia lead to further inflammatory cell recruitment, finally resulting in an aggressive

immune attack against the myelin sheath. (Fig.16)

Antibodies to various myelin proteins and lipids of the myelin sheath are secreted by

B cells that have migrated to the brain or from serum that has extravasated across the BBB

(Steinman, 2001). Activated T cells are targeted to CNS proteins and their distribution

throughout the lesion is reflected in the expression pattern of MHC molecules. MHC class

II is sufficiently displayed only on professional APCs (e.g. DCs, B cells, macrophages),

whereas MHC class I can be expressed by all cells in the inflammatory milieu of the CNS

(Dandekar et al., 2001; Neumann et al., 1995). Accordingly, CD4+ T cells are

predominantly found in perivascular cuffs and the meninges, whereas CD8+ T cells also

seem to invade the parenchyma of the inflamed lesion. On contact with their cognate

antigen, the T cells arrest and locally mediates their effector functions (Kawakami et al.,

2005). CD4+ T cells recruit macrophages, which release proinflammatory cytokines and

toxic molecules, such as nitric oxide (NO), IL-1, IL-6, TNF-α, TNF-β and matrix

metalloproteinases (MMPs) which is involved in the killing of oligodendroglial cells. CD8+

T cells also directly attack MHC class I-expressing cells such as oligodendrocytes (Saxena

et al., 2008) and neurons (Fig.16).

The combined effect of antibodies and inflammatory milieu damages myelin and

induces the macrophages to phagocytose large fragments of the myelin sheath. In addition,

macrophages and T cells produce osteopontin, which further induces Th1 cytokines IFN-γ

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Introduction

and IL-12, while downregulating Th2 cytokines like IL-10. Th1 cytokines may induce

exacerbations of MS (Bielekova et al., 2000; Chabas et al., 2001; Kappos et al., 2000;

Steinman, 2001). Overall, this attack of immune cells and inflammatory mediators

including cytokines, osteopontin, and NO produce areas of demyelination, impairing

electrical conduction along the axon and producing the pathophysiologic defect.

Histological analysis of MS tissue has demonstrated deposition of antibodies and

complement on the myelin sheath of a substantial fraction of lesions, but also indicated

significant heterogeneity in the composition of demyelinating lesions. These lesions have

been classified in four categories based on inflammatory features and on myelin

oligodendrocyte pathology (Lassmann et al., 2001). The first two patterns (P1 and P2) are

characterized by demarcated regions of infiltrating T cells (P1) and activated macrophages

(P2), centered on small veins, however, areas of demyelination were observed in both

patterns (P1 & P2). The presence (P2) or absence (P1) of antibody and complement

deposition distinguishes these two types of lesions, suggesting a different level of B cell

involvement. Infiltrating T cells and macrophages were also found in other two patterns (P3

& P4) of lesions, but these two patterns are characterized by an oligodendrocyte defect with

a preferential loss of MAG (P3) or nearly complete loss of oligodendrocytes and complete

lack of remyelination (P4). The IgG found in P2 lesions may directly contribute to disease

progression, as patients with P2 lesions respond to therapeutic plasma exchange (Keegan et

al., 2005).

However, in contrast to the role of T cells in demyelination, recently it has been

shown, that myelin phagocytosis is not due to CD4 T-cell-mediated macrophage activation.

It is an innate scavenging response of macrophages to the presence of degenerate and dead

myelin, with T and B cell activity largely restricted to recently demyelinated tissue. In

detail, the sequence of changes that occurs in tissue bordering actively expanding MS

lesions starts with a loss of oligodendrocytes in the presence of activated microglia with few

other inflammatory cells. Macrophages then appear and scavenge the degenerate and dead

myelin, again with relatively few other inflammatory cells present. Following the removal

of all myelin from axons, and while the tissue remains packed with lipid-filled macrophages

and steeped in IgG, activated T cells, B cells, and IgG-positive plasma cells accumulate in

large numbers, together with increasing numbers of differentiated oligodendrocytes.

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Introduction

Although adaptive immune activity is easily seen in recently demyelinated tissue, there are

early cases of MS in which large MS lesions expand rapidly in the absence of IgG-positive

plasma cells within the lesions and with relatively few lymphocytes present in perivascular

spaces or in the parenchyma (Henderson et al., 2009).

Figure 16: Immunopathogenesis of MS (Hemmer et.al. 2005)

The acute neurologic dysfunction that occurs during a relapse is speculated to be due

to a depolarization-induced conduction block at demyelinated regions of axons (Kornek and

Lassmann, 2003). One way to reduce long-term disability in patients with MS would be to

promote remyelination of axons (Stangel and Hartung, 2002). Some remyelination does

occur in MS lesions, but the myelin is usually thinner than before, with shorter internodes

(Stangel and Hartung, 2002). Moreover, several features are predicted to inhibit

remyelination in MS: the loss of oligodendrocytes and oligodendrocyte precursors as a

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Introduction

consequence of attack by immune cells; inhibitory signals produced by inflammation; the

obstruction of oligodendrocytes by astrocytic scarring; and the reduced receptiveness of

injured axons to remyelination (Hohlfeld, 2002).

Axonal loss

Loss of axons is a key anatomic determinant of disability in MS, but its mechanisms

still remain elusive. Axonal injury is believed to occur as a consequence of demyelination

and was recently shown to be a feature even of the early disease stages (Kuhlmann et al.,

2002). Initially, the MS lesion was considered to be an area of demyelination with

preservation of axons. However, it has been observed, that destruction of axons does occur,

with axon loss being reported in normal-appearing white matter (Neumann, 2003). Axon

damage and transection occur in both acute and chronic inflammatory plaques and have

been observed in inactive and chronic lesions from patients with clinical disease durations

ranging from 2 weeks to 27 years (Trapp et al., 1998). Axon transection is irreversible and

is most abundant in areas of inflammation. Later axon damage may result from changes in

the microenvironment in the CNS or from sublethal injury during acute inflammatory

events, resulting in decreased long-term survival. Therefore, the following mechanisms

have been proposed for demyelination-induced axon loss: a direct effect through the loss of

trophic influences from oligodendrocytes; a consequence of sustained demyelination-

induced conduction block; and indirectly through the increased vulnerability of the exposed

axon (Halfpenny et al., 2002). The progressive disability that occurs in MS is believed to

result from cumulative axon damage and degeneration, along with neuron loss due to

Wallerian degeneration. Dropout of damaged axons over time may result in premature

stress and damage to surviving axons within the same pathways, thereby producing further

loss and functional impairment. Brain white matter contains, by volume, 46% axons, 24%

myelin, 17% glia, and 13% blood vessel, blood, and tissue fluid (Miller et al., 2002a).

Given the large amount of myelin in brain tissue, demyelination is of course also a factor to

consider in atrophy. Therefore, brain atrophy can be explained by axonal loss, that may be

caused either by tissue loss within the lesion or by Wallerian degeneration which may

especially be expected in spinal cord atrophy (Lovas et al., 2000). Atrophy is most evident

in progressive forms of MS and may be a predictor for the conversion from RR-MS to SP-

MS, once a threshold of axon transection is exceeded.

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Introduction

1.3.2.3 Effector mechanisms of multiple sclerosis

The immune responses in MS are mainly targeting the cellular components of the

CNS that are normally inaccessible to the immune system because of their location behind

the BBB. Entry of immune cells into the CNS depends on their state of activation and their

ability to respond to adhesion molecules and chemokine signals that induce them to cross

the BBB. The inflammatory infiltrates in active MS lesions consist of a mostly perivascular

accumulation of CD4 and CD8 T cells, monocytes, and B cells.

Penetration of the BBB by activated lymphocytes take place in a synchronized way.

First, the endothelial cells in the CNS, which are nonfenestrated and connected through

tight junctions, are induced to express vascular cellular adhesion molecule (VCAM) and

MHC class II molecules by IFN-γ and TNF-α, which are released in the inflammatory

response (Allavena et al., 2010; Cannella and Raine, 1995). In inflammatory conditions of

the CNS, the expression of adhesion molecules and chemokines is induced on BBB

endothelium and the choroid plexus epithelium, providing additional traffic signals for

circulating leukocytes. In inflamed murine vessels, CD8+ T lymphocytes from MS patients

preferentially roll via P-selectin glycoprotein ligand-1 (PSGL-1), whereas CD4+ T

lymphocytes roll via α4-integrin (Battistini et al., 2003). During EAE, both α4-integrin and

PSGL-1-mediated leukocyte rolling occur in brain vessels (Kerfoot and Kubes, 2002).

After chemokine activation, cell surface integrins on leukocytes undergo

conformational changes and engage in interactions of higher affinity with endothelial cell

adhesion molecules. Integrin activation, mediated by signalling through G-protein-coupled

receptors, is essential for leukocyte arrest under flow conditions. Chemokines and their

receptors deliver such signals (Piccio et al., 2002; Vajkoczy et al., 2001). Chemokines

produced by BBB endothelium during EAE include the lymphoid ‘homeostatic’

chemokines CCL19 and CCL21 (Alt et al., 2002; Columba-Cabezas et al., 2003).

Numerous other chemokines are produced in the inflamed brain, both in EAE and in MS,

and encephalitogenic lymphocytes express a substantial number of chemokine receptors.

It has been described, that the adhesion molecules ICAM-1 and VCAM-1 were

upregulated on CNS microvascular endothelial cells during EAE (Baron et al., 1993;

Steffen et al., 1994). Perivascular inflammatory cells surrounding ICAM-1 and VCAM-1-

positive murine venules stain positive for LFA-1 (αLβ2-integrin) and α4β1-integrin, but

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Introduction

not for L-selectin or α4β7-integrin. Furthermore, adhesion of encephalitogenic T

lymphocytes to brain endothelial cells in vitro is mediated by α4–VCAM-1 and LFA-1–

ICAM-1 interactions (Laschinger and Engelhardt, 2000). Overall, these observations clearly

implicate α4β1–VCAM-1 and probably also LFA-1–ICAM-1 in CNS inflammation during

EAE.

However, recently it has been shown, that activated leukocyte cell adhesion

molecule (ALCAM also known as CD166) is upregulated substantially during

inflammatory stimuli, along with ICAM-1. More notably, the upregulation of VCAM-1

after inflammatory stimulus is marginal relative to that of ALCAM. During the

transmigration of lymphocytes across BBB endothelial cells, the ALCAM ligand CD6

localizes together with ALCAM and forms a transmigratory cup. Furthermore, post-mortem

sections from patients with MS and mice with EAE have higher expression of ALCAM,

and antibody blockade of ALCAM significantly lowers the accumulation of CD4+ T cells

and decreases the severity and delays the onset of disease in EAE models (Cayrol et al.,

2008). All these novel findings suggest, that ALCAM-CD6 interactions seem to replace

VCAM-1–VLA-4 interactions in mediating the transmigration of CD4+ T cells across the

BBB.

During diapedesis across the inflamed BBB, leukocytes migrate by a transcellular

pathway through endothelial cells, leaving tight junctions intact (Engelhardt and Wolburg,

2004). Recently, CD99, a unique highly O-glycosylated protein expressed on leukocytes,

was localized in endothelial cell-to-cell-contacts (Schenkel et al., 2002). CD99 antibodies

inhibit the migration of encephalitogenic T lymphocytes across brain endothelial

monolayers in vitro, suggesting the involvement of CD99 in T-lymphocyte recruitment

across the BBB in vivo (Bixel et al., 2004).

Once the activated lymphocytes have extravasated, they must still pass through a barrier of extracellular matrix comprised of type IV collagen before entering into the CNS. α-1-integrin may play a role in binding to collagen type IV (Steinman et al., 2002). Following contact with collagen, immune cells secrete enzymes such as MMPs, which allow the activated lymphocytes to gain access to the white matter surrounding axons of the CNS. The MMPs are a family of structurally and functionally related enzymes involved in the degradation of the extracellular matrix as well as the proteolysis of myelin components in MS (Hartung, 1995; Rosenberg, 2002).

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Introduction

CD4 + T cell function in MS

The role of CD4+ T cells in MS is supported indirectly by the fact that certain HLA

class II alleles represent the strongest genetic risk factor for MS (Oksenberg and Hauser,

2005), presumably via their role as antigen-presenting molecules to pathogenic CD4+ T

cells. Initially, MS was mainly considered as a CD4+ Th1-mediated autoimmune disease

(Hafler, 2004; Martin et al., 1992), which is further evident by the cellular composition of

brain and cerebrospinal fluid (CSF)-infiltrating cells and findings from the EAE model

(Zamvil and Steinman, 1990). Recently it has been shown that Th17 cells also play a

pivotal role in the pathogenesis of EAE (Becher et al., 2003; Jager et al., 2009; Langrish et

al., 2005; Reboldi et al., 2009), further emphasizing the importance of CD4+ T cells. In MS,

the role of Th17 cells is more difficult to explore, but there are some evidence implicating

IL-17 producing cells in the pathogenesis. Indeed, IL-17 and IL-6 are among the most

highly expressed genes in MS lesions (Lock et al., 2002), and elevated levels of IL-17 has

been reported in the serum and CSF of MS patients (Matusevicius et al., 1999).

Furthermore, it has been shown both in-vitro and in-vivo, that human Th17 cells might be

well competent to breach the BBB and infiltrate the CNS parenchyma (Kebir et al., 2007;

Tzartos et al., 2008). Altogether these findings suggest, that Th17 cells may play an

important role in MS pathogenesis.

Recent studies have suggested that Foxp3+ Tregs are capable of migrating into the

CNS under inflammatory conditions and may actively modulate the function of effector

cells within the target organ itself. During EAE, peripherally expanded Tregs are found to

accumulate within the CNS (Korn et al., 2007), and Foxp3+ Tregs were found enriched in

the CSF of MS patients (Feger et al., 2007a; Venken et al., 2006). Although there is no

difference in the number of Tregs between MS and control patients, Tregs play a likely role

in MS pathogenesis, since patients with MS demonstrates reduced Treg function (Haas et

al., 2005).

Recently a novel population of nTreg cells has been identified in human peripheral

blood. These cells can be either CD4+ or CD8+, they lack FoxP3 expression, and they are

distinguished from other nTreg cells by the expression of human leukocyte antigen G

(HLA-G) (Feger et al., 2007b; Wiendl et al., 2005). They exhibit potent suppressive

properties that are partially mediated by HLA-G and Ig-like transcript 2.

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Introduction

HLA-G-expressing Treg cells accumulate at sites of inflammation in acute relapses

of MS. Indeed, those patients uniformly show higher frequencies of HLA-G-expressing

cells in the CSF than in peripheral blood (Feger et al., 2007b; Wiendl et al., 2005).

CD8 + T cell function in MS

Although evidence for the importance of CD4+ T cells exists in MS, the involvement

of CD8+ T cells cannot be neglected as CD8+ T cells are easily demonstrated in

inflammatory lesions (Traugott et al., 1983). CD8+ T cells are present at the lesion edge as

well as perivascular regions. In addition, a higher frequency of CD8+ T cells recognizing

myelin proteins in patients with MS than in healthy controls has been reported (Crawford et

al., 2004), although these data are controversial (Berthelot et al., 2008). TcR analysis of

brain tissue from patients with MS has shown oligoclonal expansion of CD8+ T cells in

lesions based on TcR analysis (Babbe et al., 2000; Junker et al., 2007), and more strikingly

in the CSF (Jacobsen et al., 2002). This suggests antigen-driven activation of CD8+ T cells

during the course of MS.

Furthermore, CD8+ T cells have been shown to mediate EAE (Ji and Goverman,

2007), and transgenic mice over-expressing the costimulatory molecule CD86 on microglia

develop disease mediated mainly by CD8+ T cells (Brisebois et al., 2006). Conversely, mice

lacking CD8+ T cells develop a less severe encephalomyelitis (Koh et al., 1992).

Interestingly, oligodendrocyte do express detectable levels of MHC class I molecules in MS

lesions (Hoftberger et al., 2004), and this may be functionally relevant as myelin-specific

CD8+ T cells induce lysis of HLA-matched oligodendrocyte in-vitro (Jurewicz et al., 1998).

Altogether these findings further strengthened the observations for an active role of CD8+ T

cells in CNS autoimmunity.

CD8+ T cells found in MS lesions might also have a regulatory function on disease

progression. These cells demonstrate both immune suppressive and pathogenic

characteristics (Babbe et al., 2000; Correale and Villa, 2008; Johnson et al., 2007). CD8+ T

cells can mediate suppression on CD4+ T cells proliferation through the secretion of

perforin (Johnson et al., 2007). Although it appears plausible that CD8+ T cells are

important players in MS pathogenesis, the precise subset of CD8+ T cells that infiltrates the

CNS in MS remains to be defined. However, a recent report suggested a significant increase

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Introduction

in the number of IL-17+ T cells in the active areas of the lesions (Tzartos et al., 2008).

Importantly, these included CD8+ and CD4+ T cells, which expressed IL-17 at similar

frequencies (Tzartos et al., 2008). It might have been overlooked in EAE, where the antigen

and/or adjuvant used in the immunization procedure introduces a bias toward CD4+ T cell

priming, and possibly toward pathogenic Th17 responses (Bettelli et al., 2008; Gold et al.,

2006). Interestingly, CD8+ T cells from mice deficient in the transcription factors T-bet and

eomesodermin differentiate excessively into IL-17-secreting cells. On viral infection, these

mice experience development of a overwhelming CD8+ IL-17+ T-cell mediated

autoinflammatory syndrome (Intlekofer et al., 2008). However, the importance of CD8+ IL-

17+ T cells in MS pathogenesis remains to be established.

It can be speculated that the MS lesion is initiated by CD4+ T cells but the

amplification and damage could be mediated by CD8+ T cells, which is supported by data

showing that CD8+ T cells can directly attach to and damage axons (Medana et al., 2001).

B-cell function in MS

The elimination or reprogramming of self-reactive B cells through B-cell receptor

(BCR) editing takes place in bone marrow during the B-cell development and in spleen and

lymph nodes during the B-cell maturation (Hardy and Hayakawa, 2001). During the

establishment of tolerance, cytokines such as IL-7 and B-cell activating factor (BAFF) play

a significant role (Goodnow et al., 2005).

B cells and antibodies also play a role in MS pathogenesis. There is a very consistent

presence of oligoclonal IgG bands in CSF but not in the serum of patients with MS,

indicating an intrathecal Ig production (Cross et al., 2001; Obermeier et al., 2008). These

high concentrations are caused by a local IgG response that mainly involves the IgG1 and

IgG3 isotypes (Archelos et al., 2000; Losy et al., 1990; Walsh and Tourtellotte, 1986).

Indeed, the occurrence of an oligoclonal intrathecal antibody response is still an important

marker for chronic CNS inflammation in MS and bears a diagnostic value. Interestingly, the

antibody response seems to be stable over long periods and to involve a limited number of

clonotypes (Walsh and Tourtellotte, 1986). It has been further complemented by

investigations of antibody-secreting B cells in the CNS and CSF of patients with MS, which

suggest that there may be an intense antigenic stimulation, possibly by a relatively restricted

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Introduction

number of antigens leading to compartmentalized clonal expansion (Baranzini et al., 1999;

Colombo et al., 2000; Qin et al., 1998). Furthermore, recently a novel method, coupling the

Ig proteomes with the B cell transcriptome in the CSF of MS patients showed that the

oligoclonal Ig bands correspond to the clonally expanded B cells, have been described as

diagnostic hallmarks of MS (Obermeier et al., 2008). However the antigenic target(s) of

these oligoclonal IgG are still unknown.

Furthermore, recent evidence implicates a role of BAFF in the pathogenesis of

autoimmune diseases. Elevated levels of soluble BAFF have been observed in sera and

target organs of mouse models of SLE (Gross et al., 2000), in collagen-induced arthritis

(Zhang et al., 2005), or in chemically induced autoimmunity (Zheng et al., 2005). BAFF

levels were increased in the brains of patients with MS (Krumbholz et al., 2005), and

correlated with disease activity and titers of pathogenic autoantibodies (Mackay et al., 2007;

Piazza et al., 2010).

However, in addition to maturing into antibody producing plasma cells, B cells have

the robust capacity to present antigen and interact with T cells (Dalakas, 2008). Moreover,

B cells are abundant producers of both proinflammatory (TNF, IFN-γ) and regulatory (IL-

10) cytokines. In addition, B cells have the capacity to form ectopic lymphoid structures or

germinal centers, which have been found in patients with MS (Dalakas, 2008; Magliozzi et

al., 2007; Serafini et al., 2004; Takemura et al., 2001).

There is increasing evidence that B cells and their products are involved in MS

pathogenesis and that treatment targeting these cells may provide long-lasting benefits. A

recent report provides evidence, that follicles rich in CD20+ B cells are found in the brains

of patients with secondary-progressive MS (Magliozzi et al., 2007). The memory B cells,

late plasmablasts and long-lived plasma cells migrate not only to the bone marrow, spleen

and lymphoid tissues, but also to the brain, where they transform into antibody-secreting

cells after encountering their antigen (Knopf et al., 1998). Interactions between the

homeostatic chemokines CXCL13, CXCL10 and CXCL12 secreted from the endothelial

cell wall and their respective receptors on B cells are fundamental for B-cell homeostasis

not only within the lymphoid follicles but also within the brain (Alter et al., 2003; Dalakas,

2008). These molecules are upregulated in the brains of patients with MS, allowing the

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Introduction

recruitment and transmigration of antibody-producing B cells into the brain (Meinl et al.,

2006; Ritchie et al., 2004).

Recent advancement of disease modifying therapies including IFN-β, glatiramer

acetate, and natalizumab, on B-cell function are now coming to be better appreciated as a

therapeutic target, as no T-cell-specific therapy has shown much clinical efficacy to date in

either phase II or phase III trials. Treatment with anti-CD20 (rituximab) activates

complement factors to attack the lipid raft environment on CD20+ B cells and induces

apoptosis which significantly reduced inflammatory brain lesions and clinical relapses (Bar-

Or et al., 2008; Hauser et al., 2008). In addition to targeting B cells, another approach is to

target BAFF, which is one of the cytokines responsible for B-cell maturation.

Mast cell function in MS

Mast cells are derived from CD34+ hematopoietic progenitor cells and initiate their

differentiation in the bone marrow under the influence of c-kit ligand and IL-3 (Rodewald

et al., 1996). The mast cells release a number of mediators immediately upon activation,

such as TNF-α, IL-4, histamine, heparin, serotonin, kinins and proteases. A second class of

synthesized mediators include the IL-12, IL-13, IL-15 and IL-16, chemokines, and growth

and angiogenesis factors, such as vasoactive intestinal peptide (VIP), vascular endothelial

growth factor (VEGF) and platelet-derived growth factor (PDGF), as well as prostaglandins

and leukotrienes. Together, these molecules have profound effects on inflammatory cell

activation and recruitment (Galli et al., 2005).

TNF-α and VIP produced by mast cells can also drive Th17 maturation independent

of IL-6 (Kimura et al., 2007). Mast cells can play a role in Th17 differentiation via its

secretion of IL-6, TGF-β, TNF-α and VIP (Kanbe et al., 1999; Kimura et al., 2007; Mekori

and Metcalfe, 2000). Th2 cells produce IL-4, IL-5, and IL-13, which are associated with

allergic reactions and maturation of mast cells and have recently suggested to be implicated

in MS (Pedotti et al., 2003a; Pedotti et al., 2003b).

Mast cells are found perivascularly surrounding the endothelial cells and the

pericytes that together form the BBB, at the ventricles (Esposito et al., 2002) and close to

MS plaques (Ibrahim et al., 1996). They are critical for the regulation of BBB permeability,

(Ibrahim et al., 1996) disruption of which precedes clinical or pathologic signs of MS

97

Introduction

(Minagar and Alexander, 2003; Stone et al., 1995). In particular, acute stress can disrupt the

BBB through secretion of corticotropin-releasing hormone (CRH), which activates human

umbilical cord blood–derived cultured mast cells (Theoharides and Konstantinidou, 2007).

In fact, human mast cells express CRH-R1, activation of which leads to selective secretion

of VEGF (Cao et al., 2006). Release of MBP can stimulate mast cells to secrete VEGF,

which could increase BBB permeability (Johnson et al., 1988; Theoharides et al., 1993).

The mast cell proteolytic enzyme, tryptase, is elevated in the CSF of MS patients

(Rozniecki et al., 1995) and can cause widespread inflammation by stimulating protease-

activated receptors (PARs) (Malamud et al., 2003; Molino et al., 1997). Tryptase can also

damage myelin directly (Dietsch and Hinrichs, 1991; Johnson et al., 1988). Furthermore,

tryptase can activate peripheral blood mononuclear cells isolated from MS patients to

release TNF-α, IL-1β, and IL-6, which are possibly involved in the pathogenesis of MS

(Martino et al., 2000). Moreover, the genes found to be most upregulated in plaques from

deceased patients with MS include those for tryptase, for the IgE receptor (FcεRI) and for

the histamine-1 receptor, all associated with mast cells (Bomprezzi et al., 2003; Lock et al.,

2002).

Therefore, mast cells could participate in the pathogenesis of MS through multiple

actions like promoting adhesion molecule expression, BBB disruption, demyelination,

fibrosis/plaque formation, immune cell chemoattraction, PAR stimulation, T cell

stimulation and vasodilation. Chemokines produced by mast cells enhance T cell

recruitment to the site of inflammation, in particular CCL2, CCL3, CCL4, CCL5, and IL-16

(Salamon et al., 2005) and leukotriene B4 regulates T cell migration (Ott et al., 2003).

Histamine released from mast cells can promote Th1 and Th2 cell activation (Jutel et al.,

2001). Mast cell-derived IL-4 worsens EAE severity by enhancing the encephalitogenic

potential of Th1 cells (Gregory et al., 2006).

Overall, mast cells and their ability to release numerous potent inflammatory

mediators upon activation, can directly or indirectly activate T cells coming in contact with

them at the BBB.

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Introduction

1.4 General aim of the Thesis

The association of MS with MHC genes, the nature of the inflammatory white

matter infiltrates, similarities with animal models, and the observation that MS can be

treated with immunomodulatory and immunosuppressive therapies support the hypothesis

that autoimmunity plays a major role in the disease pathogenesis. Evidence supports the

idea, that activated CD4+ T cells are the major mediators of the disease. However,

increasing evidence suggest that CD8 T cell contribution to the disease process might have

been underestimated.

Therefore the main aim of my thesis is to investigate the mechanisms by which

autoreactive CD4 and CD8 T cells can contribute to the oligodendrocyte loss and

demyelination, a major pathological feature of MS. During these studies we made the

surprising observation of spontaneous development of autoimmune EAE, even in the

absence of the target antigen MOG. Therefore we investigated this paradoxical observation.

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Results

Results

100

Results

Oligodendrocyte-specific CD8 T cells in CNS autoimmunity

Summary Genetic studies in humans revealed an independent role for HLA class-I genes in

MS susceptibility. In addition, neuropathologic studies demonstrated that CD8+ T cells are

the most numerous inflammatory infiltrate in MS lesions at all stages of lesion

development. CD8+ T cells are also capable of damaging neurons and axons in vitro. To

assess the pathogenic impact of CD8+ T cells on oligodendrocytes, we generated mice,

referred as double knock-in (DKI) mice, in which the well-studied model antigen HA is

selectively expressed on oligodendrocytes.

Introduction of HA-specific CD8+ T cells by TcR transgenesis did not result in any

sign of central or peripheral tolerance, or any CNS pathology in the DKI animals. The

exclusive expression of HA in oligodendrocytes, and the resulting lack of T-cell silencing,

is very reminiscent of some natural myelin self-antigens such as MOG. Transfer of pre-

activated HA-specific CD8+ T cells from CL4 TCR transgenic mice in DKI recipients led to

inflammatory lesions in the optic nerve, spinal cord, and brain. These lesions associating

CD8+ T-cell infiltration with focal loss of oligodendrocytes, demyelination, and microglia

activation, were very reminiscent of active MS lesions. In order to trace these HA-specific

CD8+ T cells in vivo, we labeled them with green florescent protein (GFP). Staining for

GFP and Granzyme B revealed, that HA-specific Tc1 cells containing Granzyme B

granules were detected within CNS lesions of DKI mice, some of the Granzyme B+ T cells

were found in close apposition to carbonic anhydrase II or cyclic nucleotide 3’

phosphodiesterase+ oligodendrocytes. We also detected GFP+ Tc1 cells with polarized

Granzyme B+ granules facing an oligodendrocyte, suggesting directed degranulation.

These findings can provide important new insights with regard to CNS tissue

damage mediated by CD8+ T cells specific for a sequestered antigen and in extension for

the understanding of the role of CD8+ T cells in human MS.

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Cutting EdgeCutting Edge

Cutting Edge: Multiple Sclerosis-Like Lesions Induced byEffector CD8 T Cells Recognizing a Sequestered Antigenon Oligodendrocytes1

Amit Saxena,2*† Jan Bauer,2‡ Tanja Scheikl,*† Jacques Zappulla,*† Marc Audebert,*†

Sabine Desbois,*† Ari Waisman,§ Hans Lassmann,‡ Roland S. Liblau,3*†

and Lennart T. Mars*†

CD8 T cells are emerging as important players in mul-tiple sclerosis (MS) pathogenesis, although their directcontribution to tissue damage is still debated. To assesswhether autoreactive CD8 T cells can contribute to thepronounced loss of oligodendrocytes observed in MSplaques, we generated mice in which the model Ag in-fluenza hemagglutinin is selectively expressed in oligo-dendrocytes. Transfer of preactivated hemagglutinin-specific CD8 T cells led to inflammatory lesions in theoptic nerve, spinal cord, and brain. These lesions, associ-ating CD8 T cell infiltration with focal loss of oligoden-drocytes, demyelination, and microglia activation, werevery reminiscent of active MS lesions. Thus, our studydemonstrates the potential of CD8 T cells to induce oligo-dendrocyte lysis in vivo as a likely consequence of directAg-recognition. These results provide new insights with re-gard to CNS tissue damage mediated by CD8 T cells andfor understanding the role of CD8 T cells in MS. TheJournal of Immunology, 2008, 181: 1617–1621.

T he immune effector mechanisms contributing to tissuedamage in multiple sclerosis (MS)4 are only partiallyelucidated. The heterogeneity of MS likely reflects a

varying contribution of humoral factors, T cell subsets, and ac-tivated macrophages/microglia. Indirect evidence suggests thatthe role of CD8 T cells has been largely underestimated to date.Indeed, CD8 T cells accumulate within active MS lesions wherethey often outnumber CD4 T cells (1–3). These CNS-infiltrat-ing CD8, but not CD4, T cells exhibit oligoclonal expansion, alikely consequence of their local Ag-driven activation (3).Moreover, myelin-reactive cytotoxic CD8 T cells have beenidentified in MS patients, sometimes more frequently than in

controls (4, 5). Finally, active MS plaques exhibit MHC class-Iexpression on CNS oligodendrocytes and neurons/axons (6),which become potential targets of CD8 T cells. Altogether,these data suggest that CD8 T cells may be mediators ratherthan regulators of CNS inflammation and damage in MS.

Although the loss of oligodendrocytes, the CNS myelin-pro-ducing cells, is a key feature of MS lesions, the precise contri-bution of CD8 T cells to oligodendrocyte death and to CNSdemyelination is unknown. It has been shown in vitro that my-elin-specific CD8 T cells can kill isolated HLA-matched oligo-dendrocytes (4), but data in vivo are less compelling. Previousstudies have shown that CD8 T cells are necessary for the de-velopment of a full-blown pathology in animal models of neu-roinflammation, but their Ag specificity is still unknown (2, 7,8). Similarly, a role for CD8 T cells in demyelination has beenclearly illustrated in viral models of CNS inflammation, but themechanisms involved have remained contentious (9, 10). My-elin-specific CD8 T cells can adoptively transfer autoimmuneencephalomyelitis but the lesions were reminiscent of ischemicinjury with the demyelination associated with more global tis-sue damage, and few CD8 T cells actually infiltrated the CNSparenchyma (11, 12). Therefore, the direct effect of CD8 Tcells on oligodendrocytes and myelin in vivo is not clear.

In this study, to test whether CNS-infiltrating CD8 T cells candirectly induce oligodendrocyte death and demyelination, we de-veloped a mouse model combining selective expression of influenzahemagglutinin (HA) as a neo-self-Ag in oligodendrocytes withtransgenic mice expressing a HA-specific TCR on CD8 T cells.

Materials and MethodsMice, generation, and characterization of the Rosa-Stop-HA knock-inmice

The CL4-TCR mouse expresses a TCR specific for the influenza virusHA512–520 peptide on most CD8 T cells (13). The MOGi-Cre knock-in mouse

*Institut National de la Sante et de la Recherche Medicale, Unite 563, Toulouse, France;†Universite Toulouse III, Paul-Sabatier, Toulouse, France; ‡Center for Brain Research,Medical University of Vienna, Vienna, Austria; and §First Medical Department, JohannesGutenberg University, Mainz, Germany

Received for publication April 25, 2008. Accepted for publication June 9, 2008.

The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked advertisement in accordance with 18 U.S.C.Section 1734 solely to indicate this fact.1 This work was supported by European Union Grant LSHM-CT-2005-018637, Institut Na-tional de la Sante et de la Recherche Medicale, and the French Multiple Sclerosis SocietyARSEP.

2 A.S. and J.B. contributed equally to this study.3 Address correspondence and reprint requests to Dr. Roland Liblau, Institut National dela Sante et de la Recherche Medicale-Unite 563, Hopital Purpan, Boîte Postale 3028,31024 Toulouse Cedex 3, France. E-mail address: rolandliblau@hotmail.com4 Abbreviations used in this paper: MS, multiple sclerosis; CAII, carbonic anhydrase II;CNPase, cyclic nucleotide 3�-phosphodiesterase; DKI, double knock-in; GrB, granzymeB; HA, hemagglutinin; MOG, myelin oligodendrocyte glycoprotein; MS, multiplesclerosis.

Copyright © 2008 by The American Association of Immunologists, Inc. 0022-1767/08/$2.00

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102

(where MOG is myelin oligodendrocyte glycoprotein) (14) was backcrossed�10 times on the BALB/c background.

A conditional expression cassette, encompassing the open reading frame oftransmembrane HA placed 3� of a Stop sequence (neoR and a trimer of SV40polyadenylation sites) flanked by LoxP sites (Fig. 1A), was inserted into thePacI-AscI sites of the pROSA26PA vector (15). This gene-targeting vector wasthen electroporated into 129SV embryonic stem cells. Two embryonic stem cellclones exhibiting homologous recombination were injected into C57BL/6 blas-tocysts to generate chimeras that transmitted the Rosa-Stop-HA allele. Theseknock-in mice, referred to as Rosa26tm(HA)1Libl, were crossed seven times ontothe BALB/c background.

Cre-mediated recombination of the Rosa26-Stop-HA locus was assessed byPCR on genomic DNA from different organs. To analyze HA expression, RNAwas extracted using TRIzol, contaminating DNA was removed by DNaseI di-gestion (Promega), and a quantitative RT-PCR or a nested RT-PCR approachwas followed.

In vitro differentiation, GFP transduction, adoptive transfer ofHA-specific Tc1 cells, and in vivo cytotoxicity

HA-specific Tc1 cells were generated as described and routinely contained�98% pure CD8�CD3�V�8.2� T cells (13). GFP transduction of the HA-specific Tc1 cells was performed using the pLGFPSN retroviral vector and theGP�E-86 packaging cell line. At day 6, living CD8 T cells were collected byFicoll density separation and naive CD8 T cells or Tc1 cells were injected i.v.into immunocompetent recipient mice. Mice were assessed daily. For in vivocytotoxicity, BALB/c mice were injected or not injected with 2 � 106 HA-

specific Tc1 cells. The next day they all received 20 � 106 HA-pulsed and 20 �106 control peptide-pulsed splenocytes stained with a high (CFSEhigh) or low(CFSElow) concentration of CFSE, respectively. After 24 h, spleens from therecipient mice were analyzed by flow cytometry.

Histopathology

At the specified time points, mice were perfused with 4% paraformaldehyde inPBS. Tissues were removed and embedded in paraffin. Five micrometer-thicksections were stained with H&E and Luxol fast blue/periodic acid Schiff myelinstain. Immunohistological staining and confocal laser microscope analyses wereperformed as described previously (13, 14).

Results and DiscussionNaive HA-specific CD8 T cells are “indifferent” in mice that expressHA as an oligodendroglial neo-self Ag

For this study, we designed a mouse model system ensuring spe-cific expression of HA in oligodendrocytes by using a double

FIGURE 1. Generation and characterization of DKI mice. A, Schematicrepresentation of the knock-in mice. Top row, The targeted Rosa26 locus in theRosa26tm(HA)1Libl mice contains the LoxP-flanked Stop cassette and the HAsequence. Filled triangles indicate the LoxP sites. Middle row, The Cre sequenceinserted in the mog gene in MOGi-Cre mice. Bottom row, The Cre-mediatedrecombination of the Rosa26 locus in DKI mice allows transcription of HA. P1,P2, P3, and P4 represent the position of the primers used. B, Cre-mediated re-combination was assessed by PCR on genomic DNA from different organs usingP1 and P2 primers. One representative experiment from a total of three is shown. C,Transcription of HA in different tissues was assessed by nested RT-PCR usingP1/P2 then P3/P4 primers. Actin was used to evaluate cDNA quality. No HA sig-nal was obtained when the reverse-transcription step was omitted. Similar resultswere obtained in three different mice per group. Tissue abbreviations: B, brain;Sc, spinal cord; T, thymus; Sp, spleen; K, kidney; Li, liver. D, Double immu-nostaining detection of lacZ expression (blue) in oligodendrocytes (CNPase;red) of a R26R reporter mouse crossed with a MOGi-Cre mouse. White mattertracts in the corpus callosum (left), spinal cord (middle), and optic nerve (right)(original magnification: �260) are shown.

FIGURE 2. CD8 T cell indifference in DKI mice and characterization ofeffector CD8 T cells. A, Gated CD8� CD4� lymph node cells from CL4-TCR(dotted line) or DKI � CL4-TCR (solid line) mice were stained with Kd:HA512–520 pentamer (top) or anti-CD62L mAb (bottom). Similar data were ob-tained for seven mice per group. B, Proliferation of purified CD8� T cells fromCL4-TCR (dotted line) or DKI � CL4-TCR mice (black line) stimulated withHA512–520 peptide. These results are representative of five independent exper-iments. C, Following a 6-day culture, the HA-specific CD8 T cells from CL4-TCR mice differentiated into IFN-�-producing, granzyme B� T cells (left).Following transduction with a retroviral vector encoding GFP, most of the HA-specific Tc1 cells were brightly fluorescent at day 6, just before their adoptivetransfer (right). D, The HA-specific Tc1 cells exhibit Ag-specific cytotoxicity invivo. The histograms are gated on CFSE-labeled cells; the ratios of CFSEhigh toCFSElow peaks indicated that the Ag-specific lysis in this experiment was61.5 � 2.8% (n � 3 mice/group).

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knock-in (DKI) approach. First, we generated mice, referred toas Rosa26tm(HA)1Libl, in which the HA coding sequence was in-troduced in the ubiquitously active Rosa26 locus but where HAtranscription was prevented by an upstream LoxP-flanked Stopcassette (Fig. 1A). The Rosa26tm(HA)1Libl mice were thencrossed with the MOGi-Cre mice (14), which express Cre spe-cifically in oligodendrocytes (Fig. 1A). The resulting DKI miceexcise the Stop cassette due to MOG-controlled Cre expression,leading to restricted HA expression to oligodendrocytes. TheDKI mice exhibited no spontaneous phenotype.

PCR analyses of the genomic DNA of DKI andRosa26tm(HA)1Libl mice confirmed that Cre-mediated recombi-nation occurred only in the CNS of DKI mice (Fig. 1B). As aresult, HA transcripts were detected by quantitative RT-PCRonly in the brain, spinal cord, and optic nerve of DKI mice(data not shown), although HA protein expression in the CNSwas below detection levels using immunohistochemistry.Moreover, using a sensitive nested RT-PCR approach, HARNA was undetectable in extra-neurological tissues (Fig. 1C).Further indirect evidence of oligodendrocyte specificity of thetransgene expression was provided upon crossing MOGi-Cremice with the R26R reporter mice, which are similar in designto the Rosa26tm(HA)1Libl mice apart from the substitution of HAby lacZ. In these mice, LacZ expression was only detected inoligodendrocytes (Fig. 1D).

Interestingly, we did not observe any neurological signs uponcrossing the DKI mice with CL4-TCR mice (25 CL4-TCR �DKI mice followed for up to 9 mo). Moreover, we did not de-tect any TCR down-regulation or deletion of HA-specific CD8T cells, as assessed by Kd:HA512–520 pentamer staining (Fig.2A). Rather, peripheral CD8 T cells appeared “indifferent” toHA, as they exhibited mostly a naive phenotype(CD25�CD69�CD62LhighCD44intermediate and retained fullproliferative capacity in response to the HA512–520 peptide (Fig.2, A and B). These data are consistent with a lack of expressionand probably also of cross-presentation of HA in the lymphoid

organs of DKI mice. Therefore, the MOG-driven HA proteinbehaves as a sequestered oligodendrocyte Ag in DKI mice.

Transfer of effector HA-specific CD8 T cells into HA-expressing miceresults in CNS inflammation and demyelination

We then decided to test whether effector CD8 T cells can me-diate oligodendrocyte cell death and demyelination in vivo. Ef-fector T cells were first generated by in vitro activation of Kd:HA512–520 pentamer� CD8 T cells obtained from CL4-TCRmice using HA peptide, IL-2, and IL-12. The resulting Tc1cells produce large amounts of granzyme B (GrB) and IFN-�(Fig. 2C, left). These cells exhibit potent cytotoxicity to HA-loaded target cells in vivo (Fig. 2D).

Next, we transferred these HA-specific Tc1 cells into DKIand control mice. Following i.v. injection of 3 � 107 HA-spe-cific Tc1 cells, but not naive HA-specific CD8 T cells, �40%of the DKI mice developed an overt monophasic disease peak-ing at day 8–10 and waning by 4 wk posttransfer. The clinicalmanifestations included weight loss and, in the more severecases, tremors, reduced mobility, and difficulty to right whenoverturned without overt paralysis (Table I). In the littermatecontrols, only one animal exhibited weight loss and none devel-oped any neurological signs (p � 0.0004). This Ag-specific,CD8-mediated disease also provides strong evidence for func-tional HA protein expression in vivo in DKI mice.

Upon histological analysis, all DKI mice injected with Tc1cells demonstrated clear CNS pathology from day 5 onwards(Table I). Parenchymal infiltrates were never observed in con-trol littermates injected in parallel with HA-specific Tc1 cells.The pathology of DKI mice was dominant in the optic nerve(Figs. 3 and 4) and spinal cord (Fig. 4) but also affected fre-quently the cerebellum, fornix, and periventricular areas of thebrain. Inflammatory lesions were never found in the peripheralnervous system or in non-nervous tissues. Quantification re-vealed that T cell infiltration started on day 5 in the spinal cord,reached its peak on day 9, and declined by day 28 (Fig. 5A). On

Table I. Summary of the clinical and histological signs of Tc1-injected mice

Day of Sacrifice Genotype (No. of Mice) Weight Loss at Day 9a

Spinal Cord Optic Nerve

Inflammation Demyelination Inflammation Demyelination

Day 2.5 DKI (n � 3) NAb 0/3 0/3 0/3 0/3Control micec (n � 3) NA 0/3 0/3 0/3 0/3

Day 5 DKI (n � 4) NA 4/4 0/4 3/3 1/3Control mice (n � 3) NA 0/3 0/3 0/3 0/3

Day 9 DKI (n � 13) 5/13 9/11 0/11 9/9 5/9Control mice (n � 18) 0/18 0/16 0/16 0/16 0/16

Day 18 DKI (n � 5) 3/5 5/5 1/5 4/4 4/4Control mice (n � 5) 0/5 0/2 0/2 0/2 0/2

Day 28 DKI (n � 5) 2/5 5/5 3/5 4/4 4/4Control mice (n � 9) 1/9 0/9 0/9 0/9 0/9

Day 56 DKI (n � 3) 1/3 0/3d 0/3 0/3d 0/3e

Control mice (n � 5) 0/5 0/5 0/5 0/5 0/5

a Body weight �95% of the weight before Tc1 injection.b NA, Not applicable.c Rosa26tm(HA)1Libl, MOGi-Cre, and nontransgenic littermates.d Absence of perivascular inflammatory infiltrates but some diffuse tissue infiltration by CD3� cells.e Focal areas of reduced myelin density with thin myelinated sheaths, suggesting remyelination.

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day 56, there was only mild diffuse infiltration of the CNS byCD3� T cells. In the optic nerves we found focal areas withreduced myelin density and thin myelin sheaths, suggestingremyelination.

We also transferred GFP-transduced Tc1 cells (Fig. 2C,right), allowing us to trace the CNS-invading, HA-specificCD8 T cells. Staining for GFP and GrB revealed that HA-spe-cific Tc1 cells containing GrB� granules were detected withinCNS lesions of DKI mice (Fig. 4c). Some of the GrB� T cellswere found in close apposition to carbonic anhydrase II (CAII)or cyclic nucleotide 3�-phosphodiesterase� (CNPase) oligo-dendrocytes (Fig. 4, c and d). We also detected Tc1 cells withpolarized GrB� granules facing an oligodendrocyte, suggestingdirected degranulation (Fig. 4d). In the inflamed lesions, MHCclass I (�2-microglobulin) expression was detected on oligoden-drocytes (Fig. 5B). The CD8 T cell infiltration was associatedwith focal activation of microglia (Fig. 3, h, l, p, and t). Besides

the presence of CD8 T cells and activated microglial cells, le-sions consisted of CD4 T cells (1–13% of T cells depending onthe time point) and small numbers of B220� B cells (mostly inthe perivascular space).

Oligodendrocyte and myelin pathology was studied on sec-tions stained for Luxol fast blue, CNPase, and CAII (Figs. 3 and4). Oligodendrocyte apoptosis, detected by nuclear condensa-tion (Fig. 3g and Fig. 4, e–h), was present at early time points inoptic nerves and spinal cords of DKI recipients. In the opticnerve of DKI mice, the number of apoptotic oligodendrocyteswas 6.9 � 3.5 per section on day 5 and 15.3 � 4.8 on day 9,whereas they were virtually absent in control mice. On day 5,some vacuolization and early demyelination was found in theoptic nerve (Fig. 3e). From day 9 on, most of the animals ex-hibited extensive demyelination in the optic nerve (Fig. 3, i, m,and q) and more limited demyelination in the brain and spinal

FIGURE 3. Optic nerve pathology in DKI mice following injection of HA-specific Tc1 cells. a–d (day 2.5), Staining shows the absence of myelin pathol-ogy (Luxol fast blue/periodic acid Schiff and CNPase) and of T cell infil-tration (CD3). e– h (day 5), Large numbers of CD3� cells (f) and activatedmicroglia (h) have infiltrated the parenchyma. The myelin stain shows somevacuolization (e) but no demyelination (e and g). Oligodendrocytes with con-densed nuclei (inset in g) are found, indicating apoptosis. i–l (day 9), Myelinstains (i and k) reveal vacuolization and some demyelination. The number of Tcells (j) has declined (see Fig. 5A, right), but the MAC-3 staining (l) showsphagocytic microglia. m–p (day 18), Extensive demyelination is seen in the left(L) optic nerve (m). Demyelination is ongoing in the right (R) optic nerve, withmyelin degradation products in PAS� macrophages. T cells (n) and largephagocytic cells (p) are still present. The CNPase staining (o) shows some re-maining oligodendrocytes in the left optic nerve. q–t (day 28), Both opticnerves show extensive demyelination (q) and ongoing inflammation (r and t).CNPase staining shows some reactivity in the left optic nerve (s), which mayindicate remyelination. All figures have a �100 original magnification with theexception of q (�26), s (�70), and the inset in g (�1000).

FIGURE 4. Detection of CD8-mediated oligodendrocyte killing in the spi-nal cords and optic nerves of DKI animals on day 9. a and b, GFP (green) andCNPase (red) double staining (�450). a, Some GFP� HA-specific CD8 T cellslie in close apposition to oligodendrocytes (arrowheads) in the optic nerve. b,An HA-specific CD8 T cell in contact (arrowhead) with an oligodendrocyte. cand d, Triple staining for GrB (green), GFP (blue), and CAII (red) (�1400). c,A GrB� HA-specific CD8 T cell is next to a row of oligodendrocytes. d, An-other example of a HA-specific T cell in close apposition to an oligodendrocyte,with its GrB-containing granules polarized toward the surface of the oligoden-drocyte. e–h, Staining for oligodendrocytes (green), with TO-PRO-3 nuclearcounterstain. e, One of the CAII� oligodendrocytes shows nuclear condensa-tion (arrowhead), indicative of apoptosis (�850). f, Apoptosis (arrowhead) of aCAII� oligodendrocyte in the optic nerve (�2000). g, Nuclear condensation(arrowhead) in a CNPase� oligodendrocyte. The inset shows this condensednucleus without CNPase staining (�1500). h, An oligodendrocyte with nuclearcondensation and fragmentation into apoptotic bodies (�1800).

105

cord. In addition, staining for nonphosphorylated neurofila-ment revealed severe axonal damage in active demyelinating le-sions by day 19 (Fig. 5, C and D). Importantly, Ab-mediateddepletion of CD4 T cells in vivo had no impact on the severityof CNS inflammation and demyelination (data not shown), ex-cluding their pathogenic contribution in this model. Collec-tively, our data show that demyelinating lesions, sharing manyof the attributes of an active MS plaque, can arise when CD8 Tcells target oligodendrocytes.

The experimental system described here allows the clear as-sessment of the individual role of cytotoxic CD8 T cells by un-coupling them from other adaptive immune mechanisms. Ge-netic GFP labeling of the transferred CD8 T cells permitted theunequivocal tracing of the HA-specific CTLs in situ. Strikingly,we show that numerous HA-specific CD8 T cells enter theCNS and optic nerve parenchyma and colocalize with oligo-dendrocytes, with occasional figures of tight apposition be-tween the two cell types. These data strongly suggest that directcell contact-mediated cytotoxicity plays a central role in oligo-dendrocyte death and demyelination. It is, however, possiblethat soluble inflammatory mediators synergize to induce tissuelesions.

In conclusion, we generated a mouse model to study the con-tribution of T cell subsets on CNS tissue damage. An analogoustransgenic model has been recently used to investigate CD4 Tcell reactivity to OVA expressed specifically in oligodendrocytes(16). We focused on oligodendrocyte-specific CD8 T cells be-cause little in vivo information is currently available regardingthe pathogenicity of this subset and because both CD8 T cellinfiltration and loss of oligodendrocytes are essential features ofMS lesions. The novel finding provided by this study is thateffector CD8 T cells exhibit a potent deleterious effect on oli-godendrocytes, resulting in an inflammatory demyelinating pa-thology resembling active MS lesions. The exclusive expressionof HA in oligodendrocytes is very reminiscent of some myelin

self-Ags such as MOG, which are not (or barely) expressed inthe lymphoid tissue and therefore fail to tolerize the T cell rep-ertoire (17). This carries an obvious risk of activation of auto-reactive CD8 T cells with subsequent development of autoim-munity. Collectively, these data reinforce the idea that CD8 Tcells represent relevant therapeutic targets in MS (1, 2).

AcknowledgmentsWe thank A. Flugel, E. Dardillac, A. Saoudi, and D. Dunia for input on the project.

DisclosuresThe authors have no financial conflict of interest.

References1. Liblau, R. S., F. S. Wong, L. T. Mars, and P. Santamaria. 2002. Autoreactive CD8 T

cells in organ-specific autoimmunity. Emerging targets for therapeutic intervention.Immunity 17: 1–6.

2. Neumann, H., I. M. Medana, J. Bauer, and H. Lassmann. 2002. Cytotoxic T lym-phocytes in autoimmune and degenerative CNS diseases. Trends Neurosci. 25:313–319.

3. Junker, A., J. Ivanidze, J. Malotka, I. Eiglmeier, H. Lassmann, H. Wekerle, E. Meinl,R. Hohlfeld, and K. Dornmair. 2007. Multiple sclerosis: T-cell receptor expression indistinct brain regions. Brain 130: 2789–2799.

4. Jurewicz, A., W. E. Biddison, and J. P. Antel. 1998. MHC class I-restricted lysis ofhuman oligodendrocytes by myelin basic protein peptide-specific CD8 T lympho-cytes. J. Immunol. 160: 3056–3059.

5. Crawford, M. P., S. X. Yan, S. B. Ortega, R. S. Mehta, R. E. Hewitt, D. A. Price,P. Stastny, D. C. Douek, R. A. Koup, M. K. Racke, and N. J. Karandikar. 2004. Highprevalence of autoreactive, neuroantigen-specific CD8� T cells in multiple sclerosisrevealed by novel flow cytometric assay. Blood 103: 4222–4231.

6. Hoftberger, R., F. E. Aboul, W. Bruck, C. Lucchinetti, M. Rodriguez,M. Schmidbauer, K. Jellinger, and H. Lassmann. 2004. Expression of major histo-compatibility complex class I molecules on the different cell types in multiple sclerosislesions. Brain Pathol. 14: 43–50.

7. Brisebois, M., S. P. Zehntner, J. Estrada, T. Owens, and S. Fournier. 2006. A patho-genic role for CD8� T cells in a spontaneous model of demyelinating disease. J. Im-munol. 177: 2403–2411.

8. Ip, C. W., A. Kroner, M. Bendszus, C. Leder, I. Kobsar, S. Fischer, H. Wiendl,K. A. Nave, and R. Martini. 2006. Immune cells contribute to myelin degenerationand axonopathic changes in mice overexpressing proteolipid protein in oligodendro-cytes. J. Neurosci. 26: 8206–8216.

9. Evans, C. F., M. B. Horwitz, M. V. Hobbs, and M. B. Oldstone. 1996. Viral infectionof transgenic mice expressing a viral protein in oligodendrocytes leads to chronic cen-tral nervous system autoimmune disease. J. Exp. Med. 184: 2371–2384.

10. Haring, J. S., L. L. Pewe, and S. Perlman. 2002. Bystander CD8 T cell-mediateddemyelination after viral infection of the central nervous system. J. Immunol. 169:1550–1555.

11. Huseby, E. S., D. Liggitt, T. Brabb, B. Schnabel, C. Ohlen, and J. Goverman. 2001.A pathogenic role for myelin-specific CD8� T cell in a model for multiple sclerosis.J. Exp. Med. 194: 669–676.

12. Sun, D., Z. Huang, D. Liu, C. Coleclough, H. Wekerle, and C. S. Raine. 2001. My-elin antigen-specific CD8� T cells are encephalitogenic and produce severe disease inC57BL/6 mice. J. Immunol. 166: 7579–7587.

13. Cabarrocas, J., J. Bauer, E. Piaggio, R. Liblau, and H. Lassmann. 2003. Effective andselective immune surveillance of the brain by MHC class I-restricted cytotoxic T lym-phocytes. Eur. J. Immunol. 33: 1174–1182.

14. Hovelmeyer, N., K. Kranidioti, G. Kassiotis, T. Buch, F. Frommer, L. Von Hoch,D. Kramer, L. Minichello, G. Kollias, H. Lassmann, and A. Waisman. 2005. Apo-ptosis of oligodendrocytes via Fas and TNF-R1 is a key event in the induction ofexperimental autoimmune encephalomyelitis. J. Immunol. 175: 5875–5884.

15. Srinivas, S., T. Watanabe, C. S. Lin, C. M. William, Y. Tanabe, T. M. Jessell, andF. Costantini. 2001. CRE reporter strains produced by targeted insertion of EYFP andECFP into the ROSA26 locus. BMC Dev. Biol. 1: 4.

16. Cao, Y., C. Toben, S.-Y. Na, K. Stark, L. Nitschke, A. Peterson, R. Gold, A. Schimpl,and T. Hunig. 2006. Induction of experimental autoimmune encephalomyelitis intransgenic mice expressing ovalbumin in oligodendrocytes. Eur. J. Immunol. 36:207–215.

17. Delarasse, C., P. Daubas, L. T. Mars, C. Vizler, T. Litzenburger, A. Iglesias, J. Bauer,B. Della Gaspera, A. Schubart, L. Decker, et al. 2003. Myelin/oligodendrocyte gly-coprotein-deficient (MOG-deficient) mice reveal lack of immune tolerance to MOGin wild-type mice. J. Clin. Invest. 112: 544–553.

FIGURE 5. T cell infiltration and axonal damage in the CNS of DKI mice.A, Quantification of CD3� T cells in the spinal cord and optic nerve of DKImice at different time points after Tc1 cell injection (Table I). B, �2-Micro-globulin expression by oligodendrocytes in the spinal cord on day 9 (�650). Cand D, Axonal injury in demyelinating lesions of the optic nerve, detected withSMI311 staining nonphosphorylated neurofilaments (�250). By day 9 (C)only some axonal spheroids are present (arrows), whereas at day 18 (D) massiveaxonal destruction is observed.

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Results

Neural self-mimicry: Cumulative autoimmunity

Summary

To assess the impact of the limited MOG expression in lymphoid tissues on the

ontogeny of MOG-reactive T cells we obtained the 2D2 TcR transgenic mice, in which the

CD4+ T cell population expresses an I-Ab-restricted-TcR specific for the immunodominant

MOG35-55 peptide and crossed them with MOG-/- mice. We unexpectedly discovered that

2D2 TcR mice develop spontaneous EAE irrespective of the presence or absence of the

target self-antigen MOG.

Therefore we hypothesized, that 2D2 TcR specific for MOG35-55 recognizes a second

autoantigenic target in the CNS. In order to identify the cross-reactive antigen in MOG-/-

mice, our collaborators in Germany, fractionated the CNS tissue from these MOG-/- mice

and this material was presented to 2D2-transgenic T cells. They identified a response of

2D2 T cells to one of the fraction, which was later found by conventional biochemical

method and subsequent mass spectrometry, to contain an axonal protein, NF-M, a cross-

reactive agonist for the 2D2 TcR.

An epitope derived from NF-M (NF-M15-35), when presented in the context of I-Ab,

has similar TcR contact residues compared to MOG35-55. Consequently, antigen-

presentation of this epitope permitted the full activation of CD4+ T cells from 2D2 TcR

transgenic mice. This cross-reactivity extends to other MOG-specific TcR from C57BL/6

mice.

To test the in-vivo relevance of these observations, we transferred in-vitro differentiated Th1 cells from 2D2-Rag2-/- mice, in C57BL/6, MOG-/- and MOG-/- x NF-M-/- mice. We observed no clinical sign of disease in MOG-/- x NF-M-/- mice, while MOG-/- mice develop EAE with delayed onset, as compared to C57BL/6 mice, which developed early classical EAE. We referred to this novel observation as ‘cumulative autoimmunity’ to designate autoreactive T cells clones that target more than one cognate self-epitope and consequently exhibit additive pathogenicity.

107

Myelin-specific T cells also recognize neuronalautoantigen in a transgenic mouse model ofmultiple sclerosisGurumoorthy Krishnamoorthy1, Amit Saxena2, Lennart T Mars2, Helena S Domingues1,3, Reinhard Mentele4,5,Avraham Ben-Nun6, Hans Lassmann7, Klaus Dornmair1,4,9, Florian C Kurschus1,8,9, Roland S Liblau2,9 &Hartmut Wekerle1

We describe here the paradoxical development of spontaneous experimental autoimmune encephalomyelitis (EAE) in transgenic

mice expressing a myelin oligodendrocyte glycoprotein (MOG)-specific T cell antigen receptor (TCR) in the absence of MOG. We

report that in Mog-deficient mice (Mog–/–), the autoimmune response by transgenic T cells is redirected to a neuronal cytoskeletal

self antigen, neurofilament-M (NF-M). Although components of radically different protein classes, the cross-reacting major

histocompatibility complex I-Ab–restricted epitope sequences of MOG35–55 and NF-M18–30 share essential TCR contact positions.

This pattern of cross-reaction is not specific to the transgenic TCR but is also commonly seen in MOG35–55–I-Ab–reactive T cells.

We propose that in the C57BL/6 mouse, MOG and NF-M response components add up to overcome the general resistance of this

strain to experimental induction of autoimmunity. Similar cumulative responses against more than one autoantigen may have a role

in spontaneously developing human autoimmune diseases.

Organ-specific autoimmune disease is a key group of inflammatorydisorders that includes rheumatoid arthritis, type 1 diabetes mellitus,thyroiditis and multiple sclerosis. The prevailing thinking is that thepathogenic changes are typically initiated and driven by T cells, whichexpress receptors for autoantigens restricted to, or enriched within, theparticular target tissues.

Unfortunately, it has been impossible so far to identify, withcertainty, which autoantigens are the targets in individual humans.One reason for this limitation is the complexity of the humanautoimmune response. Indeed, there is evidence that in one personmore than one self antigen may be the target of the autoimmuneattack and that, in addition, the profile of target autoantigens mayfluctuate over time1. Furthermore, the peripheral immune repertoireof healthy humans contains a large number of T cells specific formany, if not all, autoantigens potentially related to autoimmunediseases2. There is no practical assay to distinguish T cells with highpathogenic potential from nonpathogenic counterparts and, more-over, to identify in humans the T cells participating in the patho-genesis from those that are uninvolved.

We report here a new mechanism of autoimmunity, ‘cumulativeautoimmunity’, that may provide a solution to this dilemma.Cumulative autoimmunity designates an autoimmune response that

targets more than one particular cognate autoantigenic target at thesame time, and the accumulation of these responses results in a tissueattack of enhanced vigor.

We observed a cumulative autoimmune response in transgenic micewith a TCR selected for reactivity to MOG peptide35–55, who developspontaneous EAE in the presence of MOG and, unexpectedly, also inits absence. We found that in Mog-deficient mice, the transgenic T cellsrecognize a peptide fragment of the medium-sized neurofilament NF-M.

RESULTS

Spontaneous EAE in the absence of MOG

In an experiment designed to detail the role of the autoantigen inspontaneous autoimmunity, we bred transgenes encoding the MOG-specific TCR 2D2 (ref. 3) and immunoglobulin heavy chain specificfor MOG (IgHMOG) (ref. 4) either separately or together into Mog–/–

mice5 (Fig. 1). To our surprise, spontaneous EAE developed in Mog-deficient 2D2 transgenic mice (2D2 � Mog–/–) with incidence andkinetics indistinguishable from those of wild-type (WT) counterparts.Between 15% and 20% of 2D2 � Mog–/– mice developed spontaneousEAE (Fig. 1a and Supplementary Table 1 online). Mog–/– mice andthe Mog-deficient IgHMOG mice (IgHMOG � Mog–/–), whose B cells,but not T cells, are specific for MOG, remained healthy (Fig. 1a

Received 10 June 2008; accepted 29 April 2009; published online 31 May 2009; doi:10.1038/nm.1975

1Department of Neuroimmunology, Max Planck Institute of Neurobiology, Martinsried, Germany. 2Institut National de la Sante et de la Recherche Medicale, Unite 563,Universite Toulouse III, Paul-Sabatier, Toulouse, France. 3PhD Program in Experimental Biology and Biomedicine, Center for Neuroscience and Cell Biology, Universityof Coimbra, Coimbra, Portugal. 4Institute of Clinical Neuroimmunology, University Hospital Grosshadern, Ludwig-Maximilians University, Munich, Germany.5Department for Protein Analytics, Max Planck Institute of Biochemistry, Martinsried, Germany. 6Department of Immunology, The Weizmann Institute of Science,Rehovot, Israel. 7Center for Brain Research, Medical University of Vienna, Vienna, Austria. 8Present address: I. Medizinische Klinik und Poliklinik, Johannes GutenbergUniversitat, Mainz, Germany. 9These authors contributed equally to this work. Correspondence should be addressed to H.W. (hwekerle@neuro.mpg.de).

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and Supplementary Table 1). Fifty percent of double-transgenic2D2 � IgHMOG mice (also known as OSE/Devic mice6) spontaneouslydevelop opticospinal myelitis3,6, but, in a limited cohort ofMOG-deficient 2D2 � IgHMOG mice, fewer than 15% of the micedeveloped spontaneous EAE, a proportion similar to the one seenwith Mog-deficient 2D2 mice but substantially lower than thatin 2D2 � IgHMOG Mog-sufficient counterparts (Fig. 1a andSupplementary Table 1).

Clinically, spontaneous EAE was indistinguishable between Mog-sufficient and Mog-deficient transgenic mice. In all groups, diseasestarted between 7 and 10 weeks of age, with classical paralytic EAEsigns and, in a minority of cases, with a spastic component (Supple-mentary Table 1 and Supplementary Movies 1–6 online).

The lesions in Mog-sufficient and Mog-deficient groups were indis-tinguishable (data not shown), and they were restricted to the opticnerve and spinal cord6,7. In addition, we have now observed inflam-matory infiltrates in the trigeminal ganglia, spinal ganglia, spinal rootsand peripheral nerves, despite the absence of MOG within thesetissues (Fig. 1c and Supplementary Table 2 online). However, inmice immunized with MOG35–55, the acute EAE lesions were presentonly in the central nervous system (CNS) and not in the peripheralnervous system (PNS; Fig. 1d).

2D2-transgenic T cells recognize a non-MOG CNS autoantigen

EAE in the Mog–/– mice might be explained by the incomplete deletionof Mog. The Mog knockout strain initially used in this study wascreated by the insertion of a cassette containing the LacZ andneomycin resistance genes behind the Mog promoter, leaving theMOG coding sequence intact5, which could leave some aberrantMOG expression in 2D2 � Mog–/– mice. However, in line with theoriginal description of the Mog–/– mice5, western blot analyses withmonoclonal as well as polyclonal antibodies did not detect anyresidual MOG protein expression in Mog–/– mice (Supplementary

Fig. 1 online). To further exclude faulty MOG expression, we bred2D2 TCR–transgenic mice with another line of Mog-knockout mice inwhich the 5¢ end of MOG exon 2 encoding the immunodominantMOG35–55 epitope was deleted and replaced by the Cre recombinasegene (MogCre/Cre)8 (Supplementary Fig. 2 online). 2D2-transgenicmice crossed onto the MogCre/Cre background also developed sponta-neous EAE at the same rate as 2D2-transgenic mice (Fig. 1b).

Spontaneous EAE in 2D2 � Mog–/– mice could also have beencaused by T cells recruited from the endogenous repertoire or byT cells with dual TCR expression—expressing both 2D2 and endo-genous receptor chains. Indeed, whereas in 2D2-transgenic mice mostCD4+ T cells use the transgenic TCR, there is also a considerablepopulation with endogenous receptors (data not shown). In theabsence of MOG, alternative TCRs might be stimulated to mountan attack against an alternative CNS target autoantigen. However,FACS analysis of 2D2 and 2D2 � Mog–/– thymus and spleen did notreveal any considerable differences in cell number or in activationmarkers such as CD25, CD44 and CD62 ligand (SupplementaryFig. 3 online) or MOG-specific forkhead box P3–positive T regulatorycells (Supplementary Fig. 4 online). In addition, the CNS infiltrateswere predominantly composed of transgenic T cells with only a minorpopulation of endogenous T cells (Supplementary Fig. 5 online).Furthermore, we noted spontaneous EAE in two out of six triple-transgenic Rag2-deficient 2D2 � Mog–/– mice (2D2 � Mog–/– �Rag2–/–), whose T cells express exclusively the transgenic TCR,indicating that transgenic T cells, not endogenous T cells, are theprincipal agents in the observed EAE.

Finally, the transgenic TCR might recognize an endogenous cross-reactive epitope. We tested some of the known encephalitogenicproteins and peptides such as myelin basic protein (MBP), S-100calcium-binding protein, beta chain (S100b) and proteolipid protein(PLP) amino acids 139–151, but none of them activated the2D2-expressing T cells (Fig. 2a). Then we compared crude myelinpreparations from Mog+/+ and Mog–/– CNS isolated by classicalprotocols9,10. Both preparations activated MOG-specific 2D2-expres-sing cells to a comparable degree when presented by syngeneic bonemarrow–derived dendritic cells (BMDCs) (Fig. 2a) but did notstimulate ovalbumin-specific OT-II TCR transgenic T cells, whichwe used as negative controls. Furthermore, myelin-induced prolifera-tion of 2D2-transgenic T cells was blocked by antibodies to CD4 andmajor histocompatibility complex (MHC) class II but not by antibodyto CD1d or control rat antibodies (Fig. 2b).

a b

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Figure 1 Paradoxical development of spontaneous EAE in MOG-specific 2D2

TCR–transgenic mice in two different Mog-deficient strains. (a) Spontaneous

incidence of EAE-like disease observed in transgenic mice carrying MOG-

specific TCR (2D2), B cell receptor (IgHMOG) or both, on Mog-sufficient and

Mog-deficient C57BL/6 backgrounds. Shown is the survival curve analysis of

the mice that were observed for a minimum of 7 weeks after birth. 2D2,

n ¼ 440; 2D2 � Mog–/–, n ¼ 218; 2D2 � IgHMOG, n ¼ 258; 2D2 �IgHMOG � Mog–/–, n ¼ 48; IgHMOG � Mog–/–, n ¼ 63; Mog–/–, n ¼ 279.

(b) Incidence of spontaneous EAE in MOG-specific 2D2–transgenic mice

on a Mog-deficient background (different than in a). 2D2, n ¼ 199;

2D2 � MogCre/Cre, n ¼ 140; MogCre/Cre, n ¼ 23. (c) Nervous system

pathology of 2D2-transgenic mice with spontaneous EAE. Infiltration,

demyelination and axonal damage in trigeminal ganglia were revealed by

H&E, luxol fast blue (LFB) and Bielschowsky silver impregnation (Biel),

respectively. The infiltrates are composed of macrophages (Mac3) as wellas CD3+ T cells. Scale bars, 100 mm. (d) Nervous system pathology of

C57BL/6 mice immunized with MOG35–55. The acute EAE lesions of CNS

and PNS parts of the trigeminal nerve and ganglion were visualized by

staining for macrophages (Mac3) and CD3+ T cells. The bottom images

show the respective isotype control antibody staining. Scale bars, 100 mm.

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NF-M peptide cross-reacts with MOG-specific T cells

We systematically fractionated CNS tissue from Mog–/– mice (Fig. 3).We employed a purification regime composed of homogenization,lipid extraction and purification of the urea-dissolved proteins by ion-exchange and gel chromatography (Supplementary Fig. 6 online).Among the chromatography fractions presented to 2D2-expressingT cells by syngeneic antigen-presenting cells (BMDCs), we elutedseveral antigenic fractions from the anion-exchange column (Fig. 3a).Fractionation of Mog-sufficient CNS tissue led to a similar profile(Fig. 3b). Parallel fractionation of extracts from WT and Mog–/– CNSwith the cation exchange column yielded similar results (Supplemen-tary Fig. 7a,b online).

Gel filtration chromatography of both pooled positive fractions(fractions 16 and 17) from the anion exchange column resulted in onefraction (fraction 9) that was recognized by 2D2-expressing T cells(Fig. 4a). SDS-PAGE analysis revealed two prominent bands withmolecular masses of approximately 68 kDa and 150 kDa (Fig. 4b). Weexcised both bands from the gel, in-gel digested them with trypsinand subjected them to matrix-assisted laser desorption-ionization–time-of-flight mass spectrometry. We identified the 68-kDa protein asthe light chain of mouse neurofilament (NF-L) and the 150-kDaprotein as NF-M.

We tested the exclusive cross-reactivity of the NF-M with MOG-specific 2D2-transgenic T cells by an analysis of CNS proteins fromNF-M–deficient (Nefm–/–) mice. None of the ion-exchange chroma-tography fractions from Nefm–/– mice activated 2D2-transgenic T cells(Fig. 3c and Supplementary Fig. 7c). In addition, we also found thatthe crude myelin preparations from both Mog–/– and WT mice thatcontained abundant amounts of NF-M (Supplementary Fig. 8aonline), and not those from the Mog–/– � Nefm–/– mice, activated2D2-transgenic T cells (Fig. 2a and Supplementary Fig. 9 online).Full-length MOG, which is highly hydrophobic owing to its threemembrane-spanning helices, was found mainly in the urea-insolublefraction (data not shown). We detected only trace amounts ofMOG in the urea-soluble extract but not in any of the 2D2-transgenicT cell–activating fractions from Mog-sufficient mice (Supplementary

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Figure 2 MOG-specific T cells respond to myelin from Mog–/– mice.

(a) Proliferation, as measured by 3H-thymidine incorporation assay, of spleen

cells from 2D2-transgenic or control OT-II mice (2 � 105 cells per well)

together with BMDCs (5 � 104 cells per well) cultured with the indicated

proteins, peptides (20 mg ml–1) or myelin preparations (1 ml per well) from

Mog–/– and WT mice. pC1, rat MBP68–84; pC2, guinea pig MBP45–67; p81,

guinea pig MBP69–83. (b) Proliferation, as measured by 3H-thymidine

incorporation assay, of 2D2-transgenic spleen cells together with BMDCs

incubated with rat MOG (20 mg ml–1) or myelin suspension (1 ml per well)

preparations from Mog–/– and WT mice. The antibodies to MHC II (Anti-MHC

II), CD1d (Anti-CD1d) or CD4 (Anti-CD4) or control rat IgG2a and rat IgG2b

antibodies were added at 10 mg ml–1. Proliferation of T cells from a and b

was measured by labeling with 3H-thymidine during the last 16 h of a 72-h

assay. Shown is the mean ± s.e.m. of triplicate measurements. Statistical

significance was analyzed by analysis of variance. ***P o 0.001.

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cFigure 3 Fractionation of CNS proteins from Mog–/–, WT and Nefm–/– mice.

(a–c) Proliferation, as measured by 3H-thymidine incorporation assay,

of 2D2 spleen cells in response to anion exchange column fractions of

CNS tissue extracts from Mog–/– (a), WT C57BL/6 (b) and Nefm–/– (c) mice.3H-thymidine incorporation was measured during the last 16 h of the 72-h

assay. Shown is the mean ± s.e.m. of triplicate measurements. Shown is a

representative of two individual protein purifications (with different pools of

Mog–/– mice, WT mice or Nefm–/– mice) and stimulation experiments. The

narrow elution profile of the Mog–/– CNS extract is due to the use of Mono Q

column material instead of Source Q material, which was used for WT and

Nefm–/– experiments.

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Fig. 8b). Hence, MOG is lost quantitatively during lipid extractionand urea solubilization, and these data exclude the presence ofadditional cross-reactive antigens, at least in the urea-soluble fractionwe examined.

An in silico search identified a seven–amino acid peptide of NF-Mnearly identical to the core region of the antigenic peptide MOG38–50

(Fig. 4c), which spans from Tyr40 to Val47and contains the amino acids Arg41, Phe44,Arg46 and Val47, which are known to be thecrucial contact amino acids for the 2D2 TCRand other MOG-specific T cell lines11,12.These amino acids are completely preservedat identical positions in NF-M18–30; that is,positions Arg21, Phe24, Arg26 and Val27.Another candidate peptide, NF-M225–237,also showed some homology to MOG butlacked the essential residues of the coreregion (Fig. 4c).

2D2 T cells responded vigorously to thesynthetic peptide NF-M18–30 but not toNF-M225–237 (Fig. 5a). In addition,NF-M18–30 induced strong proliferation of2D2 � Rag2–/– transgenic T cells, indicatingthat the cross-reactivity is intrinsic to thetransgenic 2D2 TCR (Fig. 5b). This cross-reactivity is not limited to the syntheticMOG and NF-M peptides. 2D2 T cells alsorecognized the naturally processed recombi-nant MOG and NF-M proteins produced inE. coli (Fig. 5a,b). Notably, NF-M and MOGpeptides mixed in a 1:1 ratio induced pro-liferation and cytokine secretion of 2D2-expressing T cells isolated either from thespleen or from the CNS of mice withspontaneous EAE without any signs of toler-ance or anergy (Fig. 5a and SupplementaryFig. 10 online).

2D2-transgenic T cell responses to MOGand NF-M peptides were similar but notidentical. In dose-dependent proliferationtests, NF-M18–30 peptide was superior toMOG35–55 and MOG38–50 peptides in indu-cing proliferation (Fig. 5a,b). We confirmed

this finding in cytokine assays, in which NF-M stimulated largerinterferon-g (IFN-g), interleukin-17 (IL-17), IL-2 and IL-10 releasesthan did MOG (Fig. 5c).

To clarify whether the cross-reactivity of 2D2-expressing T cells withNF-M reflects a ‘private’ clonotypic response (a response by a singleT-cell clone) or represents a more general cross-reactivity betweenMOG35–55- and NF-M–specific T cells, we isolated fresh MOG35–55-specific T cells from C57BL/6 mice, expanded them and tested themfor reactivity with NF-M. Indeed, a polyclonal MOG35–55-specific

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Figure 4 Identification of a protein that cross-reacts with MOG-specific

2D2–transgenic T cells. (a) Proliferation, as measured by 3H-thymidine

incorporation assay, of 2D2 spleen cells in response to the T cell–activating

fractions (fractions 16 and 17 from Fig. 3a) from Mog–/– mice. The fractions

were pooled and further separated by gel filtration chromatography.

Proliferation was measured during the last 16 h of the 72-h assay. Shown is

the mean ± s.e.m. of triplicate measurements. (b) Silver staining of a 2D2

T cell-activating fraction from Mog–/– mice. T cell-activating fraction (fraction

9) from several similar gel filtrations were pooled and concentrated by re-

chromatography with a Mono Q column. These fractions were resolved on a

10% Tris-glycine gel and stained by silver staining. F9, gel filtration fraction

before concentration. (c) Amino acid alignment of the immunodominant

epitopes of MOG and NF-M. MOG35–55 and the minimal epitope MOG38–50

were aligned with NF-M. Shown in red and underlined is the amino acid

identity of MOG and NF-M peptides.

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Figure 5 NF-M reacts specifically with 2D2-transgenic T cells. (a) Proliferation, as measured by3H-thymidine incorporation assay, of 2D2 splenocytes cultured with increasing concentrations of the

indicated proteins, peptides or mixtures. Shown is a representative of more than three individual

experiments consisting of more than six mice. (b) Proliferation, as measured by 3H-thymidine

incorporation assay, of splenocytes from 2D2 � Rag2–/– mice cultured with the indicated proteins and

peptides. Means ± s.e.m. of triplicate measurements are shown. (c) Quantification of cytokines released

by MOG-specific 2D2-transgenic T cells in response to cross-reactive NF-M peptide and protein. 2D2splenocytes were cultured for 3 d with the indicated peptides and proteins in a dose-dependent fashion.

The concentrations of the cytokines secreted by the T cells were measured in the supernatants in a

sandwich ELISA composed of specific antibody pairs. The data were combined from three independent

experiments. Each data point represents two to seven mice per group. Means ± s.e.m. are shown.

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T cell line, which used Va and Vb regions distinct from the 2D2 TCR(Supplementary Fig. 11a online), readily responded to NF-M18–30 butnot to NF-M225–237 or ovalbumin (Ova) amino acids 323–339(Fig. 6a). Conversely, T cell lines from NF-M–immunized mice,also with Va and Vb gene segments different from those in the 2D2TCR (Supplementary Fig. 11b), cross-reacted with MOG protein andpeptides (Fig. 6b).

In vivo recognition of NF-M by MOG-specific T cells

To determine whether 2D2-transgenic T cells find their alternativetarget (that is, NF-M) in vivo during EAE, we transferred MOGpeptide–activated, 2D2-transgenic CD4+ T cells to Rag2–/– orRag2–/– � Mog–/– mice. Whereas both recipient groups developedEAE, in Mog-deficient Rag2–/– mice the disease was delayed (Supple-mentary Fig. 12 online).

We next examined whether NF-M–activated 2D2-T cells can alsoinduce EAE. NF-M–activated 2D2-expressing T cells triggered EAE inWT hosts, which by incidence and kinetics was comparable to EAEcaused by MOG-activated 2D2-expressing T cells (Supplementary

Fig. 13a,b online). To assess the impactof soluble MOG peptide tolerization, wetransferred in vitro MOG peptide–activated2D2-transgenic T cells into irradiated WTor Mog–/– mice and injected Ova323–339 orMOG35–55 peptides intravenously. Weobserved that treatment with MOG peptidedelayed the onset of EAE in WT C57BL/6recipients and, more notably, also in Mog–/–

mice (Supplementary Fig. 13a). Similarly,MOG peptide tolerization lowered theencephalitogenic activity of 2D2-expressingT cells activated in vitro with NF-M peptide(Supplementary Fig. 13b).

Furthermore, we tested the encephalito-genic potential of ‘genuine’ NF-M–specificT cell lines, that is, CD4+ T cells isolatedfrom NF-M–primed C57BL/6 mice and pro-pagated by serial NF-M–specific activation.T cells from an NF-M–specific line trans-ferred to C57BL/6 mice induced severe EAE(Fig. 6c). The pathology in these mice wasclosely similar to that seen after transfer of2D2-expressing cells in MOG-deficient mice.Lesions were most pronounced in the spinalcord and featured inflammation by T cellsand macrophages (Fig. 6c), confluent demye-lination and severe axonal loss (data notshown). We transferred NF-M peptide–activated transgenic T cells derived from2D2 � Rag2–/– mice into both Mog–/– andWT mice. These monoclonal 2D2 � Rag2–/–

T cells induced EAE in Mog–/– mice, albeitwith a delayed onset as compared to WTrecipients, confirming the autonomous cap-ability of 2D2-expressing T cells to recognizethe alternative target, NF-M (Fig. 6d).

The lesions developing in Mog–/– recipientmice injected with 2D2 � Rag2–/– T cellswere severe, with large infiltrates in the spinalcord and cerebellum (Fig. 6d) and, remark-ably, also in the trigeminal ganglia and in

peripheral nerves (data now shown). These findings are reminiscent ofthe pathology seen in the spontaneous EAE of 2D2 � Mog–/– mice.

To confirm that the 2D2-transgenic T cell–mediated lesions inthe Mog–/– recipient mice are due to in vivo recognition of NF-M,we transferred activated 2D2 � Rag2–/– T cells into Mog–/– � Nefm–/–

double-knockout mice. None of the double-knockout mice showedany clinical signs of EAE, and there were no lesions in the CNS(Fig. 6d).

DISCUSSION

C57BL/6 mice are resistant to the induction of most T cell–mediatedorgan-specific autoimmune diseases. In these mice, immunizationwith classical autoantigens elicits vigorous T cell responses butcommonly fails to produce a clinical autoimmune disease. In contrast,immunization with MOG35–55 peptide reliably induces clinical EAE,thus overcoming resistance of C57BL/6 mice13. Our observations mayprovide an unexpected clue explaining the unusual autoimmunepotential of MOG35–55. We show that CD4+ T cells selected fromC57BL/6 mice for reactivity to MOG35–55 also respond to an epitope

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C57BL/6

Figure 6 In vitro and in vivo cross-reactivity between NF-M– and MOG-specific T cells. (a,b) Proli-

feration, as measured by 3H-thymidine incorporation assay, of MOG- and NF-M–specific T cell lines.

A MOG35–55-specific T cell line (a) and NF-M–specific T cell line (b) generated from WT mice primed

with its respective antigen were tested with the indicated protein and peptides (20 mg ml–1). The

proliferation was measured during last 16 h of the 72-h assay. Shown is the mean ± s.e.m. of triplicate

measurements. (c) Clinical course of EAE induced by the NF-M–specific T-cell line. Naive WT C57BL/6

mice were lightly irradiated (400 rad) and injected intravenously with 12 � 106 (n ¼ 3 mice) or

6 � 106 (n ¼ 7 mice) activated T cells specific for NF-M15–35 peptide. The T-cell line was derived

from NF-M15–35–immunized WT C57BL/6 mice. Shown is the mean clinical score of mice fromthree experiments. Histopathology analysis of spinal cords (right) shows typical EAE pathology, with

inflammatory infiltrates comprised of polymorphonuclear cells, T cells and macrophages or activated

microglia. Scale bars, 100 mm. (d) EAE induced by 2D2 T cells in WT C57BL/6 and Mog–/– but not in

Mog–/– � Nefm–/– double-knockout mice. We transferred 15 � 106 2D2 Rag2–/– T helper type 1 cells

into lightly irradiated (300 rad) syngenic WT (n ¼ 15), Mog–/– (n ¼ 15) and Mog–/– � Nefm–/– double-

knockout (n ¼ 9) C57BL/6 mice. Left, EAE clinical score of mice from three (WT, Mog–/–) or two

(Mog–/– � Nefm–/–) independent experiments. EAE frequency is significantly lower in double-knockout

recipients as compared to either of the two other groups (X2; P o 1 � 10�6). Kinetics of EAE differs

significantly (Log-rank test; P o 10�4) between WT and Mog–/– mice. Data represent mean ± s.e.m.

Right, representative images comprising inflammatory infiltrates in the spinal cord of Mog–/– and

Mog–/– � Nefm–/– mice revealed by H&E, CD3 and Mac3 staining. Scale bars, 1 mm.

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of the medium-sized neurofilament, NF-M. We propose that thecombined response to the two target structures may overcome theinnate resistance of C57BL/6 mice to autoimmune diseases.

It has previously been reported that the 2D2-transgenic C57BL/6mice that harbor large populations of MOG-specific CD4+ T cells tendto spontaneously develop optic neuritis and EAE3. This trendis markedly increased in the presence of MOG-specific transgenicB lymphocytes6,7. Paradoxically, however, as reported here, 2D2 micedevelop spontaneous EAE also in the absence of MOG, the primaryencephalitogenic target.

This was discovered in mice with disrupted exon 1 of the Moggene5, and it was confirmed in another cohort of Mog-deficient 2D2-transgenic mice, whose Mog gene was deleted by an independentknock-in strategy8, excluding residual, atypical MOG material in theseknockout animals as a possible encephalitogenic target.

We fractionated CNS tissue from Mog-knockout mice and foundmaterial that was presented to and recognized by 2D2-transgenicT cells. We identified the autoantigenic component, by classicalbiochemical methods and subsequent mass spectrometry, as NF-M.The salient target epitope of NF-M was finally determined by anin silico search for sequences related to the MOG35–55 motif recognizedby the 2D2 clone11. Given the marked degeneracy of peptide recogni-tion by T cells14, cross-reaction of NF-M by 2D2 at the peptide levelmay not be very unexpected. However, less trivially, we confirmed NF-M as the stimulatory autoantigen at the protein level using both CNSwhite matter protein extracts and recombinant proteins.

Neurofilaments, including NF-M, are produced by neurons andalso by some glial cells15. They were characterized recently as auto-antigens in actively induced EAE and as possible targets in multiplesclerosis, too. Immunization of Biozzi ABH (antibody high, AB/H,ABH) mice with the light form of neurofilament, NF-L, causes EAE ina moderate proportion of treated animals16. Also, autoantibodies toNF-M have been detected in the cerebrospinal fluid of some indivi-duals with multiple sclerosis17.

The CD4+ T cell repertoire of 2D2-transgenic mice is dominated bythe transgenic, MOG-reactive TCR but is by no means monoclonal.The NF-M–specific response could have been effected either by T cellsfrom the residual endogenous repertoire or by T cells that escapedallelic exclusion and use endogenous TCR chains along with thetransgenic one. We ruled out both possibilities, as 2D2-expressingT cells from Rag2-knockout mice showed a similar heteroclitic(stronger) cross-recognition of NF-M in vitro and in vivo, indistin-guishable from their Rag2-sufficient counterparts. Furthermore, weobserved spontaneous EAE in 2D2 � Rag2–/– � Mog–/– mice, suggest-ing the autonomous role of transgenic T cells in the cross-recognition.

EAE was readily mediated by T cell lines selected for reactivity toeither MOG (2D2) or NF-M and by 2D2-expressing T cells activatedby NF-M. In contrast, we were unable to induce disease by immuniza-tion with NF-M using protocols that allow active disease induction byMOG35–55 (data not shown). This discrepancy between active andpassive EAE induction is, however, not exceptional. It has beenpreviously described for other models, including EAE induced inLewis rats by glial fibrillary acidic protein18 and S100-b19 andMBP-induced EAE in BALB/c mice20.

Autoimmune cross-reactivity between MOG and NF-M has beendiscovered and analyzed in one clonal model, 2D2-transgenic T cells,but has been confirmed in other I-Ab–restricted MOG- andNF-M–specific CD4+ T cells. MOG- or NF-M–primed polyclonalT cell populations isolated from WT C57BL/6 mice show extensivecross-reactivity between NF-M and MOG proteins and their salientepitopes, respectively. Of note, these populations rarely use Va3.2 and

Vb11, the variable chains used by the 2D2 clone, indicating that MOGand NF-M cross-reactivity is not limited to the 2D2 TCR.

Our in vitro results formally establish the cross-reactivity of 2D2 andother MOG35–55 peptide–specific T cells with NF-M, but do theseT cells respond to NF-M in vivo, and might there be additional cross-recognized autoantigens? In vivo NF-M–specific responses are suggestedby several lines of evidence. We found lesions in trigeminal and spinalganglia, tissues which even in WT mice are devoid of MOG autoantigenbut contain NF-M. Furthermore, 2D2 � Rag2–/– T cells transferred intoMog–/– � Nefm–/– double-knockout recipients failed to develop EAE.This latter observation, together with the loss of autoantigenic potentialof myelin from double-knockout white matter, also rules out unknownautoantigens acting in addition to MOG and NF-M.

How would cross-reactive T cells respond to the simultaneouspresence of both MOG and NF-M? Would the response componentsadd up or would there be tolerization? Several observations suggestthat MOG35–55-specific T cells respond both to the MOG epitopes aswell as to the cross-reactive NF-M epitopes at the same time. In vitro,T cells isolated from CNS and spleen respond to both antigens in anadditive fashion. In vivo, transfer of activated 2D2-expressing T cellscaused substantially earlier appearance of EAE in Mog-sufficient micecompared to Mog–/– mice. Also, the clinical picture of Mog-sufficientand Mog-deficient 2D2-transgenic mice is very similar. However, aselective anti-MOG response component seems to prevail in trans-genic mice with transgenic TCRs plus B cell receptors. In the presenceof MOG, in T-B double-transgenic mice the incidence of spontaneousEAE rises to rates of 50% and more. In the absence of MOG,these double-transgenic mice develop EAE at proportions similarto their single-TCR transgenic counterparts. The MOG-dependentelevation of spontaneous EAE frequency, noted in double-transgenic2D2 � IgHMOG mice6, is not seen in the absence of MOG.

To our knowledge, this is the first description of immunologicalself-mimicry, that is, the response of one T cell population to twoindependent target autoantigens in the same tissue, MOG and NF-M.Is such a response an exception to the norm or is it common? Asmentioned, the MOG and NF-M response does not seem to be uniqueto the MOG-reactive 2D2 clone studied here but is also noted inpolyclonal MOG- and NF-M–reactive T cell populations from C57BL/6mice. Furthermore, another case of self mimicry, though betweenstructures from different tissues was reported by another group, whodescribed in the Dark Agouti (DA) rat cross-reaction between MOG-specific T cells and an epitope of the milk protein butyrophilin21.

The dual response of T cells against two target autoantigens expressedwithin the same target tissue could have major implications for organ-specific autoimmune disease. It could have an additive role in deter-minant spreading (development of an immune response distinct fromthe initial disease-causing epitope) in the course of an autoimmuneresponse. Beyond this, we propose that in C57BL/6 mice autoimmuneresponse components directed against MOG and NF-M may accumu-late to overcome the general resistance of these mice to induction ofEAE. T cells with similar cumulative double self-reactivity could act asdominant pathogens in human multiple sclerosis, and genetic factorsfavoring bireactive T cells would enhance susceptibility to the disease.This study should provide a way to identify such T cells in humans andappreciate their role in the pathogenesis of multiple sclerosis.

METHODS

Methods and any associated references are available in the onlineversion of the paper at http://www.nature.com/naturemedicine/.

Note: Supplementary information is available on the Nature Medicine website.

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ACKNOWLEDGMENTSMogCre/Cre, Nefm–/–, 2D2 and Mog–/– mice were generously provided by A. Waisman(Johannes Gutenberg University of Mainz), J.-P. Julien (Laval University),V.K. Kuchroo (Harvard Medical School) and D. Pham-Dinh (INSERM UMR 546).We thank F. Lottspeich for granting us permission to use his mass spectrometer.We thank L. Penner and I. Arnold-Ammer for technical support. This projectwas supported by the Deutsche Forschungsgemeinschaft (Sonderforschungsbereiche(SFB) 571, Projects A1 and B6) and the Max Planck Society. H.S.D. is supportedby a PhD fellowship (Portuguese Fundacao para a Ciencia ea Tecnologia (FCT)program SFRH/BD/15885/2005). Part of the study (conducted by H.W., R.L.and H.L.) was funded by the EU Project Neuropromise (PL 018637), andA.B.-N. was supported by the Israel Science Foundation and the NationalMultiple Sclerosis Society of New York (RG3195B8/2). A.B.-N. is an Alexandervon Humboldt Prize Awardee.

AUTHOR CONTRIBUTIONSG.K. performed most of the experiments. G.K. and H.W. designed the studyand wrote the manuscript with input from co-authors. A.S., L.T.M. and R.S.L.contributed EAE and T cell data. K.D. supervised protein purification andmass spectrometry and performed in silico searches. R.M. performed massspectrometry. H.S.D. assisted in EAE experiments. A.B.-N. performed T cellline transfer EAE experiments. H.L. performed and interpreted histology.F.C.K. designed experiments and performed protein purification.

Published online at http://www.nature.com/naturemedicine/

Reprints and permissions information is available online at http://npg.nature.com/

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7. Bettelli, E., Baeten, D., Jager, A., Sobel, R.A. & Kuchroo, V.K. Myelin oligodendrocyteglycoprotein-specific T and B cells cooperate to induce a Devic-like disease in mice.J. Clin. Invest. 116, 2393–2402 (2006).

8. Hovelmeyer, N. et al. Apoptosis of oligodendrocytes via Fas and TNF-R1 is a key eventin the induction of experimental autoimmune encephalomyelitis. J. Immunol. 175,5875–5884 (2005).

9. Norton, W.T. & Poduslo, S.E. Myelination in rat brain changes in myelin compositionduring maturation. J. Neurochem. 21, 759–773 (1973).

10. Chantry, A., Gregson, N.A. & Glynn, P. A novel metalloproteinase associated with brainmyelin membranes. Isolation and characterization. J. Biol. Chem. 264, 21603–21607(1989).

11. Petersen, T.R. et al. Characterization of MHC- and TCR-binding residues of themyelin oligodendrocyte glycoprotein 38–51 peptide. Eur. J. Immunol. 34, 165–173(2004).

12. Ben-Nun, A. et al. Anatomy of T cell autoimmunity to myelin oligodendrocyteglycoprotein (MOG): prime role of MOG44F in selection and control of MOG-reactiveT cells in H-2b mice. Eur. J. Immunol. 36, 478–493 (2006).

13. Mendel, I., Kerlero de Rosbo, N. & Ben-Nun, A. A myelin oligodendrocyte glycoproteinpeptide induces typical chronic experimental autoimmune encephalomyelitis in H-2b

mice: fine specificity and T cell receptor Vb expression of encephalitogenic T cells. Eur.J. Immunol. 25, 1951–1959 (1995).

14. Wucherpfennig, K.W. & Strominger, J.L. Molecular mimicry in T cell–mediatedautoimmunity: viral peptides activate human T cell clones specific for myelin basicprotein. Cell 80, 695–705 (1995).

15. Kelly, B.M., Gillespie, C.S., Sherman, D.L. & Brophy, P.J. Schwann cells of themyelin-forming phenotype express neurofilament protein NF-M. J. Cell Biol. 118,397–410 (1992).

16. Huizinga, R. et al. Immunization with neurofilament light protein induces spasticparesis and axonal degeneration in Biozzi ABH mice. J. Neuropathol. Exp. Neurol. 66,295–304 (2007).

17. Bartos, A. et al. Elevated intrathecal antibodies against the medium neurofilamentsubunit in multiple sclerosis. J. Neurol. 254, 20–25 (2007).

18. Berger, T. et al. Experimental autoimmune encephalomyelitis: the antigen specificity ofT-lymphocytes determines the topography of lesions in the central and peripheralnervous system. Lab. Invest. 76, 355–364 (1997).

19. Kojima, K. et al. Experimental autoimmune panencephalitis and uveoretinitis in theLewis rat transferred by T lymphocytes specific for the S100b molecule, a calciumbinding protein of astroglia. J. Exp. Med. 180, 817–829 (1994).

20. Abromson-Leeman, S. et al. T cell responses to myelin basic protein in experimentalautoimmune encephalomyelitis-resistant BALB/c mice. J. Neuroimmunol. 45, 89–101(1993).

21. Stefferl, A. et al. Butyrophilin, a milk protein, modulates the encephalitogenicT cell response to myelin oligodendrocyte glycoprotein in experimental autoimmuneencephalomyelitis. J. Immunol. 165, 2859–2865 (2000).

ART ICL ES

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ONLINE METHODSTransgenic mice. We bred MOG-specific TCR transgenic mice (2D2)3 and

B cell knock-in IgHMOG (also known as Th)4 on a C57BL/6 background into

Mog–/– mice5 to obtain 2D2 � Mog–/– and IgHMOG � Mog–/– and 2D2 �IgHMOG � Mog–/– mice. We obtained WT C57BL/6 mice from the animal

facility of the Max Planck Institute of Biochemistry. We bred Rag2–/– mice and

OT-II mice (Jackson Laboratories) in the conventional animal facilities along

with other transgenic mice. We bred a second Mog–/– strain (MogCre/Cre)

harboring the insertion of the gene encoding Cre recombinase in the first

exon of Mog8 with the 2D2-transgenic mice in the specific pathogen–free

animal facility of Institut Federatif de Recherche (IFR30). We also maintained

Nefm–/– mice22 in the specific pathogen–free animal facility of IFR30.

We routinely monitored a cohort of transgenic mice of the above genotypes

at least one or two times a week for clinical EAE signs. The EAE disease scores

were according to the classic scale6. To determine other neurological abnorm-

alities, we lifted the mice by their tail and allowed them to grab the grid of a

cage by their front limbs (see Supplementary Movies 1–6). We noted the

clasping and hyperextension of hind limbs within 10–30 s of holding them by

the tail.

All animal procedures were in accordance with guidelines of the Committee

on Animals of the Max Planck Institute of Neurobiology or the Midi-Pyrenees

Ethic Committee on Animal Experimentation and with the license of the

Regierung von Oberbayern (or from the French Ministry of Agriculture).

Peptides and proteins. Mouse MOG35–55 (MEVGWYRSPFSRVVHLYRNGK),

mouse MOG38–50 (GWYRSPFSRVVHL), mouse NF-M18–30 (TETRSSFSRVSGS),

mouse PLP139–151 (HSLGKWLGHPDKF), Ova323–339 (ISQAVHAAHAEINEAGR),

mouse MOG90–110 (SDEGGYTCFFRDHSYQEEAA), rat MBP pC1 (MBP68–84)

(HYGSLPQKSPRSQDENPV), guinea pig MBP pC2 (MBP45–67) (GSDRAAP

KRGSGKDSHHAARTT) or guinea pig MBP p81 (MBP69–83) (YGSLPQKSQR

SQDEN) were synthesized either by BioTrend or by the core facility of Max

Planck Institute of Biochemistry. We obtained mouse NF-M225–237 (LQDEVA

FLRSNHE) from Metabion. We purified the peptides by HPLC to 495%

purity and analyzed them by mass spectrometry.

We purified recombinant soluble rat MOG protein (MOG1–125)23, mouse

full-length NF-L and mouse head domain fragment of NF-M (NF-M ‘head’;

NF-M1–102) (Supplementary Methods online) from bacterial inclusion bodies.

We purchased S100b and ovalbumin from Sigma. We purified guinea pig MBP

and rat MBP using standard protocols.

Histology. We perfused mice with 4% paraformaldehyde in PBS and stored

them in the same fixative for 24 h. We stained adjacent serial sections of CNS

and PNS with H&E, luxol fast blue or Bielschowsky silver impregnation. We

also stained some sections with CD3-specific (Serotec) and Mac3-specific (BD

Biosciences) antibodies. We stained adjacent sections with the respective

isotype controls.

Adoptive transfer EAE. For 2D2 � Rag2–/– T cell transfer, we purified CD4+

T cells from 2D2 � Rag2–/– mice and stimulated them in vitro with 20 mg ml–1

of NF-M15–35 peptide in the presence of 20 ng ml–1 IL-12 and 1 ng ml–1 IL-2

(both from R&D Systems) and irradiated syngeneic splenocytes. On day 6, we

re-stimulated viable cells with splenocytes and 20 mg ml–1 of NF-M15–35 in

the presence of 20 mg ml–1 IL-12 and 1 mg ml–1 IL-2. On day 9, we

injected Ficoll-purified T helper type 1 cells into lightly irradiated (300 rad)

syngeneic recipients.

Myelin purification. We purified crude myelin from Mog–/–, WT C57BL/6 or

Mog–/– � Nefm–/– CNS tissues according to previously published protocols9,10.

Briefly, we pooled brain and spinal cord and homogenized it in 0.32 M sucrose

in 10 mM Tris HCl, pH 7.4. Then we centrifuged at 15,000g and washed the

pellet twice with 0.32 M sucrose solution. Finally, we suspended the pellet in

0.32 M sucrose and overlaid on to 0.85 M sucrose and centrifuged at 26,000g.

We collected the myelin at the interface, washed it twice and suspended it in

1 ml of sterile PBS.

Proliferation assay. For the analysis of fractions from biochemical separations,

we mixed spleen cells from 2D2 or OT-II mice (2 � 105 cells) with

LPS-activated BMDCs from WT C57BL/6 mice (5 � 104 cells) together with

1 in 50-diluted fractions.

Unless otherwise mentioned, in all other T cell proliferation experiments, we

cultured 2 � 105 spleen cells with 20 mg ml–1 peptides and proteins. We

performed all proliferation assays in triplicate. We measured the T cell

proliferation by the incorporation of 3H-labeled thymidine during the last

6 h of a 48-h culture or the last 16 h of a 72-h culture.

Enzyme-linked immunosorbent assay. We assayed cell culture supernatants

with antibody pairs or kits for IFN-g, IL-2 (both from BD Biosciences), TNF-a(Peprotech), IL-10 (R&D Systems) or IL-17 (eBioscience) according to the

manufacturer’s instructions.

T cell lines. We established antigen-specific T cell lines from C57BL/6 mice

immunized with MOG35–55, NF-M15–35 or NF-M ‘head’ in complete Freund’s

adjuvant supplemented with 5 mg ml–1 Mycobacterium tuberculosis (strain

H37Ra) using established protocols. We collected spleen and draining lymph

nodes 10–12 d after immunization and stimulated them with respective antigen

at 20 mg ml–1. We supplemented T cell cultures with recombinant mouse IL-2

(Peprotech) and supernatant from concanavalin A–stimulated mouse spleen

cells on days 0, 3 and 5. We purified live T cells by Nycoprep gradient (Progen

Biotechnik) and repeated stimulation every 7–10 d.

Statistical analyses. We analyzed spontaneous EAE incidence by Kaplan-Meier

survival curve analysis, and we analyzed adoptive transfer EAE data and

proliferation assays by analysis of variance. We used GraphPad Prism for all

statistical analyses. We considered P values less than 0.05 to be significant.

22. Jacomy, H., Zhu, Q.Z., Couillard-Despres, S., Beaulieu, J.M. & Julien, J.P. Disruptionof type IV intermediate filament network in mice lacking the neurofilament mediumand heavy subunits. J. Neurochem. 73, 972–984 (1999).

23. Amor, S. et al. Identification of epitopes of myelin oligodendrocyte glycoprotein for theinduction of experimental allergic encephalomyelitis in SJL and Biozzi AB/H mice.J. Immunol. 153, 4349–4356 (1994).

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Supplementary Figure 1. Western blot analysis of brain and spinal cord homogenates from Mog–/–

and wild type (Wt) mice. Equal amounts of CNS homogenates were resolved on a 12.5% Tris-

Glycine gel and probed with rabbit antibody to MOG (N-terminal). The membrane was stripped and

re-probed with β-actin-specific antibody. The protein bands were visualized by ECL system. #1, #2-

wild type mice and #3 and #4-Mog–/–

mice. β-actin is shown for loading control for homogenates.

Supplementary information:

Myelin-specific T cells co-recognize neuronal autoantigen in a transgenic mouse

model of multiple sclerosis

Gurumoorthy Krishnamoorthy, Amit Saxena, Lennart T. Mars, Helena S. Domingues, Reinhard Mentele,

Avraham Ben-Nun, Hans Lassmann, Klaus Dornmair, Florian C. Kurschus, Roland Liblau & Hartmut

Wekerle

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Results

116

Supplementary Figure 2. Sequence of the targeted locus in the Mog–/– mice (Hövelmeyer et al.,

2005). The Cre-recombinase was inserted in the Mog locus with concomitant deletion of exon 1 and

part of exon 2. The coding sequence of the MOG 35–55 was highlighted in red. Arrows delimit the

sequence which was deleted after homologous recombination.

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Results

117

a

c

b

d e

44.8

37.7

3.7

13.8

44.8

37.7

3.7

13.8

39.9

41.7

2.9

15.6

39.9

41.7

2.9

15.6

CD4C

D8

Supplementary Figure 3. Genetic ablation of Mog does not alter the development and activation

status of MOG-specific transgenic T cells. (a) Splenic and thymic cell numbers from age matched

2D2 and 2D2 x Mog–/– mice. n= 3–4 per group. (b) Representative FACS plot of thymic CD4 and

CD8 staining. Thymic cells from 2D2 (top panel) and 2D2 x Mog–/– (bottom panel) mice were stained

with fluorochrome labeled CD4 and CD8 antibodies. Numbers indicate the percentage of cells in the

respective quadrant. (c) Thymic T cell subsets. Thymic cells were stained as in b and the frequency

of the CD4+, CD8

+, CD4

+CD8

+(DP; double positive), CD4

-CD8

-(DN; double negative) cells were

determined. n = 3– 4 per group. (d) and (e) Frequency and expression of activation markers in T

and B cells. Spleen cells from 2D2 and 2D2 x Mog–/–

mice were stained with the indicated

antibodies. Frequency of the cells expressing the indicated lineage or activation markers was

plotted. n = 3 mice per group. The figures show mean ± s.e.m. Statistical significance was analyzed

by ANOVA. n.s; not significantNature Medicine: doi:10.1038/nm.1975

Results

118

Supplementary Figure 4. Genetic ablation of MOG does not alter the frequency and absolute

number of 2D2 Foxp3+ regulatory T cells. (a) Representative FACS plot illustrating the frequency of

Foxp3+ cells amongst TCR-Vα3.2+Vβ11+ 2D2 CD4+ peripheral T cells from age-matched 2D2 (left

panel) and 2D2 x Mog–/– (right panel) mice (n = 3 mice per group). (b) Absolute numbers of CD4+

2D2 T cells (left panel) and of regulatory Foxp3+

CD4+

2D2 T cells (right panel) in the spleen of age-

matched 2D2 and 2D2 x Mog–/–

mice (n = 3 per group). n.s; not significant

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Results

119

0.46 96.75

1.60 1.20

0.46 96.75

1.60 1.20

a

b

CD4

CD

8

CD4

CD

8

V�3.2

V�

11

V�3.2

V�

11

Supplementary Figure 5. CNS infiltrating mononuclear cells from sick 2D2 (a) and 2D2 x Mog–/– (b)

mice predominantly contain transgenic T cells. CNS mononuclear cells were isolated by percoll

gradient and stained with fluorochrome labeled antibodies. T cells expressing TCR transgenes

(V�3.2, V�11) were analyzed on the CD4+CD8- gated cells. Numbers indicate the percentage of

cells in the respective quadrant.

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120

Supplementary Figure 6. Fractionation scheme of CNS proteins from Mog–/–, WT and Nefm–/–

mice. Flow chart of the strategy applied to purify the CNS proteins. CNS tissues were homogenized,

de-lipidated, and solubilized in urea. Mog, a transmembrane protein is found in the urea-insoluble

fraction. Urea-soluble material was used for ion-exchange chromatography and tested for T cell

activating potential and finally separated with gel filtration. The positive fractions were separated by

SDS-PAGE and proteins were identified by mass spectrometry.

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a b

c

Supplementary Figure 7. Cation exchanger fractions from wild type and Mog–/– but not Nefm–/–

mice showed similar activation profiles. (a) Mog–/–, (b) WT C57BL/6 and (c) Nefm–/– mice CNS

tissue extracts were separated through a Source S column and fractions were tested in a T cell

proliferation assay (4 �l/well) using 2D2 or OT-II spleen cells as responders. T cell proliferation of

was measured by labeling with3H-thymidine during the last 16 h of 72 h assay. Shown is the mean

± s.e.m of triplicate measurements representative of two individual protein purifications (with

different pools of Mog–/–

mice, WT mice or Nefm–/–

mice) and stimulation experiments. c.p.m, counts

per minute.Nature Medicine: doi:10.1038/nm.1975

Results

122

a

b

Supplementary Figure 8. Localization of NF-M and MOG in CNS extracts and in the ion-exchange

chromatography fractions. (a) CNS homogenate, urea-soluble extracts and crude myelin from Mog–/–

, WT and Nefm–/– mice were separated on a SDS-PAGE and probed with NF-M specific antibody.

The same membrane was stripped and re-probed with antibody to GFAP antibody. Myelin, whole

homogenate and urea-soluble extracts from WT and Mog–/–

mice contained abundant amounts of

NF-M whereas Nefm–/–

mice CNS urea extract was devoid of any detectable NF-M protein. Anti-

GFAP immunoblot shows the presence of similar amounts of GFAP protein in all the urea extracts

and in the crude myelin of the WT and Mog–/–

mice. (b) Whole CNS homogenate, urea soluble

proteins and fractions of the ion-exchange chromatography from WT mice were separated on a 10%

Tris-Glycine gel. Equal volumes of the fractions were loaded on the gel. Antibody to MOG (N-

terminal) was used to probe for MOG. The protein bands were visualized by ECL system.

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Results

123

a

b

Supplementary Figure 9. Mog–/– x Nefm–/– myelin do not contain 2D2 T cell activating material.

Spleen cells from (a) 2D2 or (b) control OT-II mice (2 x 105 cells/well) together with irradiated

syngenic BM-DCs (5 x 104 cells/well) were cultured with the indicated myelin preparations (3, 1 or

0.3 � l/well) from Mog–/–, WT and Mog–/– x Nefm–/– mice. Proliferation was measured by 3H-thymidine

incorporation assay. While myelin preparations from WT and Mog–/–

mice stimulated 2D2 and not

control OT-II T cells in a dose dependent manner, myelin from Mog–/–

x Nefm–/–

mice failed to

activate 2D2 T cells. c.p.m, counts per minute.

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Results

124

Bla

nk

MO

G38

-50

NF-M

18-3

0

MO

G38

-50

+NF-M

18-3

0(1

:1)

Ova

323-

339

0

250

500

750

1,000CNS cells-2D2

[3H

]in

co

rpo

ratio

n(c

.p.m

.)

a b

Bla

nk

MO

G35

-55

NF-M

15-3

5

MO

G35

-55

+NF-M

15-3

5(1

:1)

0

500

1,000

1,500

2,000

2,500

IFN-�

IL-17

pg

ml-1

Supplementary Figure 10. CNS-infiltrating T cells from sick 2D2 mice respond to both NF-M and

MOG. (a) The mononuclear cells were isolated from pooled brain and spinal cords of sick 2D2

transgenic mice by percoll gradient. 5 x 104 mononuclear cells were mixed with 2 x 105 irradiated

spleen cells from C57BL/6 mice. Cultures were added with 20 μg ml-1 of MOG 38–50, NF-M 18–30,

Ova 323–339 or 1:1 mixture of MOG and NF-M peptides. T cell proliferation was measured by3H-

thymidine incorporation during the last 16 h of 72 h assay. The figure shows mean ± s.e.m of

triplicate measurements. c.p.m, counts per minute (b) CD4+

T cells were isolated from percoll

purified CNS-infiltrating mononuclear cells of sick 2D2 transgenic mice (n = 3). 2 x 104

CD4+

T cells

were cultured with 12.5 x 104

irradiated T cell-depleted C57BL/6 splenocytes. The cultures were

stimulated with either 10 �g ml-1

of MOG 35–55, 10 μg ml-1

of NF-M 15–35, or a 1:1 mixture of both

peptides. Unstimulated (blank) cultures were used as controls. IFN-� and IL-17A secretion was

measured in culture supernatants after 72 h. The histogram presents the mean � s.e.m of triplicate

measurements.

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Results

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2�V

3�V

4�V5.

1,5.

2

�V

6�V

7�V8.

1,8.

2

�V

8.3

�V

9�V 10

b

�V

11�V

12�V

13�V

14�V

17�V

0

1

2

3

%Liv

ece

lls

2�V

3�V

4�V5.

1,5.

2

�V

6�V

7�V

8.18

.2

�V

8.3

�V

9�V 10

b

�V

11�V

12�V

13�V

14�V

17�V

0

10

20

30

40

50

60

70%

Liv

ece

lls

a

2�V 3.

2�V

8.3

�V11

�V

0.0

0.2

0.4

0.6

0.8

%Liv

ece

lls

b

2�V 3.

2�V

8.3

�V11

�V

0.0

0.5

1.0

1.5

2.0%

Liv

ece

lls

Supplementary Figure 11. TCR repertoire analysis of a MOG 35–55 specific and NF-M specific T

cell lines. (a) MOG 35–55 specific (b) NF-M specific T cell lines were generated from WT C57BL/6

after immunization and repeated rounds of in vitro stimulation with MOG 35–55 or rNF-M. TCR

expression was analyzed using V� & V� screening panel antibodies by FACS. Shown are the % live

cells expressing the indicated TCR chain. The T cell lines were primarily of CD4+

phenotype.Nature Medicine: doi:10.1038/nm.1975

Results

126

**

***

***

***

***

***

Supplementary Figure 12. 2D2 T cells induce EAE in Mog–/– mice. Transgenic 2D2 spleen cells

were polarized in vitro into Th1 and Th17 cells as described in methods. Th1 and Th17 cells were

mixed at 1:4 ratio. 5 x 106 mixed cells were transferred i.v to Rag–/– (n = 4) or Rag–/– x Mog–/– (n = 4)

recipients and clinical EAE was monitored. The experiment was repeated once with similar results.

Statistical significance was analyzed by ANOVA. **p<0.01, *** p<0.001.

Nature Medicine: doi:10.1038/nm.1975

Results

127

0 10 20 30 40

0

1

2

3

4

5B6 irradiated + Ova323-339

B6 irradiated + MOG 38-50

Time (d) after transfer

Me

an

EA

Esc

ore

a

b

Supplementary Figure 13. Tolerance induced by high dose soluble MOG inhibits 2D2 T cell

induced EAE. 2D2 spleen cells were stimulated with MOG 35–55 (a) or NF-M 18–30 (b) and

polarized in vitro into effector Th1 and Th17 cells. 3 - 5 x 106 mixed cells (Th1:Th17; 1:4) were

transferred i.v to irradiated WT C57BL/6 mice (B6) or Mog–/– mice (n = 3 per group) and these mice

injected with 100μg of the indicated peptide i.v on days 1, 3 and 5. Clinical signs of EAE were

monitored in these groups of mice. MOG 35–55 peptide treatment delayed the EAE onset in WT and

Mog–/–

recipients in both MOG and NF-M stimulated 2D2 T cell induced EAE.

Nature Medicine: doi:10.1038/nm.1975

Results

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Strain Clinical signs Incidence Onset(weeks)

2D2 EAE 62/440

Clasping & hyperextension of limbs 7/440

EAE + clasping & hyperextension 13/440

Total signs 82/440 (18.6%) 7.2 ± 2.1

2D2 x Mog–/–

EAE 27/218

Clasping & hyperextension of limbs 6/218

EAE + clasping & hyperextension 7/218

Total signs 40/218 (18.3%) 8.1 ± 1.9

2D2 x IgHMOG

EAE 141/258 (54.7%) 7.8 ± 5.4

2D2 x IgHMOG

x Mog–/–

EAE 4/48

Clasping & hyperextension of limbs -

EAE + clasping & hyperextension 3/48

Total signs 7/48 (14.5%) 10.2 ± 3.8

IgHMOG

x Mog–/–

NA 0/63 (0%) NA

Mog–/–

NA 0/279 (0%) NA

Supplementary Table 1. Neurological abnormalities observed in transgenic mice.

Mice of the indicated genotypes were monitored regularly for the appearance of signs of EAE, hind

limb clasping and spasticity (hyperextension of the hind limbs). Clasping and hyperextension were

noted when the mice were lifted by their tail. Mice that were observed for a minimum of 7 weeks

after birth were included in the analysis. The onset of clinical signs were expressed as postnatal

weeks � SD. NA- Not applicable

Nature Medicine: doi:10.1038/nm.1975

Results

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AnimalNo

GenotypeClinicalscore

Other neurological symptoms SC Inf SC DM Brain Inf Brain DM PNS

7778 2D2 0 - 0.1 PV FO, 0 TG, RO

10106 2D2 0 - 0 0 0 0 0

9916 2D2 1 hind limb clasping/hyperextension M dors 2 M, Trig Trig TG, RO

9911 2D2 1 hind limb clasping/hyperextension M dors 1 M, Trig 0 TG, RO

9399 2D2 1.5 hind limb clasping 0 0 FO, ON ON 3 RO

9912 2D2 1.5 hind limb clasping/hyperextension 0.2 dors 2 ON ON 3 TG, RO

9408 2D2 4 - 0.8 3 ON ON 3 TG, RO

7961 2D2 4 - 2.3 2 M, ON 1 TG, RO

7682 2D2 x Mog–/–

0 - 0 0 0 0 0

9364 2D2 x Mog–/–

0 - 0 0 0 0 0

9337 2D2 x Mog–/–

0 - 0 0 0 0 0

9369 2D2 x Mog–/–

0 hind limb clasping/hyperextension 0 dors 1 0 0 TG, RO

9366 2D2 x Mog–/–

0 hind limb hyperextension 0.1 0 0 0 TG, RO

9567 2D2 x Mog–/–

1 hind limb clasping 0 0 ON 0 TG, RO

7678 2D2 x Mog–/–

2 - 2.1 3 Cer 0 0

9352 2D2 x Mog–/–

2.5 Front limb extension 0.3 2 ON ON 3 TG, RO

7767 2D2 x Mog–/–

4 - 0.3 2 ON ON 3 TG, RO

7763 2D2 x Mog–/–

4 - 5 4 Cer 0 TG, RO

9730 2D2 x Rag–/–

X Mog–/–

3.5 - 0.1 2 Cer Cer 2 RO

10062 2D2 x IgHMOG

x Mog–/–

1 hind limb clasping/hyperextension 0 dors 1 0 0 TG, RO

9693 2D2 x IgHMOG

x Mog–/–

1 hind limb clasping/hyperextension 0 dors 1 0 0 TG, RO

9363 2D2 x IgHMOG

x Mog–/–

3.5 - 0.4 3 Trig, ON ON 4 Trig 3 TG, RO

10053 2D2 x IgHMOG

/ IgHMOG

x Mog–/–

0 hind limb clasping/hyperextension 0 0 0 0 TG, RO

9038 Mog–/–

0 - 0 0 0 0 0

9362 Mog–/–

0 - 0 0 0 0 0

Supplementary Table 2. Histological analysis of CNS and PNS in transgenic mice.

Mice of the indicated genotypes and clinical phenotypes were analyzed for the histological signs of inflammation and demyelination

in the CNS and PNS. SC Inf: Perivascular infiltrates and SC cross section; DM1: single perivascular fibers demyelinated; DM2:

perivascular or subpial DM; DM3: profound confluent DM (1/3 to half of spinal cord, one optic nerve); DM4: extensive demyelination

(more than half of SC cross section, both optic nerves); Dors 1–2: demyelination exclusively in superficial dorsal column (probably

secondary Wallerian degeneration due to pathology in spinal roots); M: meninges; Trig: central protion of trigeminal root; FO: fornix;

ON: optic nerve; Cer: cerebellum; Obl: oblongata; PNS: inflammation and damage in peripheral nervous system; TG: trigeminal

ganglia; RO: spinal rootsNature Medicine: doi:10.1038/nm.1975

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Supplementary methods

Intracellular staining

For intracellular cytokine staining, we stimulated T cells for 4 h with PMA (50

ng ml–1), ionomycin (500 ng ml–1) and Brefeldin A (5 μg ml–1) (Sigma Aldrich). We

first stained extracellularly with PercP-conjugated CD4-specific antibody (RM4-5),

fixed and permeabilized with 2% paraformaldehyde and saponin buffer. Then we

stained intracellularly with FITC or allophycocyanin (APC)-conjugated antibody to

IFN-γ (XMG1.2) and PE-conjugated antibody to IL-17 (TC11-18H10) (all from BD

Pharmingen). We acquired Samples on a FACSCalibur (BD Biosciences) and

analysed with CellQuest software (BD Biosciences).

Purification of CNS proteins by chromatography

We pooled brain and spinal cord from Mog–/–, Nefm–/–, or WT mice (5 mice each),

homogenized in 5 ml water and 45 ml chloroform/methanol (1:2). We constantly

stirred the homogenates overnight at 4 °C. We centrifuged the sample, removed the

aqueous and chloroform phase, and the insoluble material was lyophilized. We

dissolved the de-lipidated, lyophilized CNS material in 6 M urea, 20 mM NaCl, in 20

mM phosphate buffer and centrifuged. We subjected urea-soluble proteins to cation

(“Source S”) or anion (“Source Q”) exchange column chromatography at a flow rate

of 1 ml min–1. We eluted Bound proteins from the columns by linear salt gradients

from 20 mM to 2 M NaCl and 1 ml fractions were collected. Fractions which induced

the 2D2 T cell proliferation were identified by a proliferation assay (see below). We

pooled positive fractions from the Source Q column, dialyzed against 6 M urea, 20

mM NaCl, in 20 mM degassed phosphate buffer pH 7.4 in the presence of 1.2 mM

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dithiothreitol, and loaded on a Mono Q anion exchanger that was equilibrated in the

above buffer, but contained 0.5 mM dithiothreitol. We eluted at 1 ml min–1 by a linear

gradient from 20 mM to 2 M NaCl for 40 ml. We further separated the fractions that

were positive in the T cell activation assay by gel filtration chromatography on a

Superose 12 column at a flow rate of 0.5 ml min–1. We collected 1 ml fractions and

tested for 2D2 TCR activation. We concentrated the positive fraction by re-

chromatography on a Mono Q column. All columns were from GE Healthcare. In

some experiments we omitted the initial non-reducing anion-exchange

chromatography without any changes in the elution and T cell activation profiles.

Antibodies and flow cytometry

We purchased antibodies to IA/IE (2G9), CD4 (GK1.5) and CD1d (1B1), rat

IgG2a isotype control (R35-95) and rat IgG2b isotype control (A95-1) from BD

Biosciences and used at 10 μg ml–1 for blocking experiments. For FACS analysis, we

purchased the following fluorochrome labeled antibodies against mouse antigens

from BD Biosciences: CD4 (RM4-5), CD3e (145-2C11), CD69 (H1.2F3), CD62L

(MEL-14), CD44 (IM7), CD8a (53-6.7), Vα3.2 (RR3-16), Vβ11 (RR3-15), CD19

(1D3), CD25 (PC61), Foxp3 (FJK-16S). Vα and Vβ TCR screening panel antibodies

were from BD Biosciences. We acquired samples on a FACSCalibur (BD

Biosciences) and data were analysed with CellQuest software (BD Biosciences).

T cell polarization and adoptive transfer EAE

We polarized 2D2 T cells in vitro towards Th1 and Th17 phenotype using

previously published protocols with some modifications21, 22. Briefly, we mixed 20 x

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106 spleen cells (per well in 6 well plates, total volume of 3 ml) from 2D2 mice with

20 x 106 irradiated (3000 rad) spleen cells from IgHMOG mice and stimulated with 20

μg ml–1 rMOG plus Th1 and Th17 polarizing cytokines and antibodies: IL-12 (10 ng

ml–1), IL–18 (25 ng ml–1) and antibody toIL-4 (10 μg ml–1, clone 11B11) for Th1

polarization; human TGF-β1 (3ng ml–1), IL-6 (20 ng ml–1), IL-23 (10 ng ml–1),

antibodies to IL-4 (10 μg ml–1) and IFN-γ (10 μg ml–1, clone R4-6A2) for Th17

polarization. Th1 cells were supplemented with IL-2 (10 ng ml–1) and Th17 cells with

IL-23 (10 ng ml–1) at day 3. We purchased all cytokines are from Peprotech except

IL-23 (R&D Systems) and IL-18 (MBL), and purified from hybridoma culture

supernatants by protein G chromatography. We purified CD4+ T cells on day 5 by

Dynal CD4 negative selection kit (Invitrogen), and stimulated again like above on day

6. We used irradiated (3000 rad) spleen cells from IgHMOG mice as APCs for second

stimulation. Three days after second stimulation, we purified live activated blast cells

by Nycoprep gradient (Progen Immuno Diagnostica) and injected 5 x 106 cells mixed

Th1 & Th17 cells (1:4; Th1:Th17) intravenously (i.v) via tail vein into Rag2–/–, Mog–/– x

Rag–/– or irradiated (400 rad) WT and Mog–/– mice. For tolerance induction, we

injected 100μg of Ova 323-339 or MOG 35-55 or MOG 38-50 peptide intravenously

on 1, 3 and 5 days post T cell transfer. We monitored the polarization efficiency on

day 5 and day 9 by intracellular cytokine staining (see flow cytometry).

Western blotting and gel staining

We analyzed column fractions and CNS extracts by SDS PAGE on 10% or 12.5%

gels. Gels were stained by silver for analytical purposes, by Coomassie Blue for

mass spectrometry, or blotted onto nitrocellulose membranes and proteins were

detected either by antibodies to MOG (N-terminal) (Sigma; M0695 or R&D systems;

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BAM2439) or NF-M (Acris antibodies; NF-09). We stripped the membranes and re-

probed with β-actin (Sigma; AC-74) or GFAP-specific (Santa cruz; sc-6170)

antibodies for loading controls.

Mass spectrometry

We destained coomassie blue stained gels and excised appropriate bands. The

disulfide bonds were reduced by dithiothreitol, alkylated with iodoacetamide, and

proteins in-gel digested by trypsin (Roche) as described23. We analyzed peptides by

MALDI-TOF using a Bruker ultraflex II TOF/TOF spectrometer (Bruker Daltronik). We

analyzed peptide masses using the program "Mascot" (Matrix Science) and the non-

redundant NCBI mouse database.

Construction of mouse NF-L and NF-M expression vector and

purification

We amplified full-length mouse NF-L from mouse brain cDNA by PCR using

the following primers (5’-ATT ACA TAT GAG TTC GTT CGG CTA CGA-3’ and 5’-

ATT ACT CGA GAT CTT TCT TCT TAG CCA CCT-3’) and cloned in-frame into

pET21c vector. After sequence verification, we transformed the plasmid into

BL21star competent cells. For protein expression, we grew NF-L transformed

bacteria in LB medium containing 1% glucose overnight. We seeded the overnight

culture (1:10) into fresh LB medium and grown till OD600 reaches 0.6 and cultures

were induced with 1 mM IPTG. After 3 h, we harvested the cells and suspended in

lysis buffer (50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8.0). We lysed

the cells by sonication and centrifuged at 20,000 g for 15 min at 4 °C. We washed

the pellet containing the aggregated NF-L twice and dissolved in binding buffer (8 M

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Urea, 100 mM NaH2PO4, 40 mM imidazole pH 7.4). We cleared the sample by

centrifugation and loaded into the Ni2+ charged chelating sepharose column. We

washed the column with the binding buffer and eluted the column bound NF-L with

elution buffer (8M Urea, 100 mM NaH2PO4, 0.5 M imidazole pH 7.4). Finally we

dialyzed the fractions containing NF-L against 10 mM sodium phosphate, pH 7.4

buffer.

We amplified mouse NF-M 1-102 aa (head domain) from mouse brain cDNA

using primers (5’-TGC ATA TGA GAG GAT CGC ATC ACC ATC ACC ATC ACA

TGA GCT ACA CGC TGG AC-3’ and 5’-TTA AGC TTT ACT GCT CTT TCT CGT

TAG AGC-3’) containing N-terminal 6-his tag coding sequences. We cloned The

PCR product into pRSET-B vector and sequence verified. We transformed a

sequence verified plasmid clone into BL21-AI competent cells (Invitrogen) and frozen

as glycerol stocks. For the purification of the recombinant protein, we inoculated

bacteria in LB medium contaning 0.1% glucose and grew until the OD600 reached

0.6–1.0. We inoculated this pre-culture (1:10) into fresh LB medium and allowed to

grow until 0.4 OD600, then induced with 1 mM IPTG and 0.2 % arabinose. Bacterial

cultures were grown further for 3 h at 37 °C and processed as above. Finally, we

dialyzed the protein against 20 mM sodium acetate pH 3.0 or 5.0. We analyzed the

purity of the recombinant proteins by coomassie stained gel.

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Discussion & Perspectives

Discussion & Perspectives

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Discussion & Perspectives

Discussion & Perspective

Implication of oligodendrocyte-specific CD8 T cells in MS

Emerging genetic data highlight the independent role of HLA class-I genes in

conferring susceptibility to chronic human inflammatory diseases such as T1D (Nejentsev

et al., 2007) and MS (Brynedal et al., 2007). These data provide strong incentive to further

investigate the contribution of MHC class-I-restricted CD8+ T cells in the induction and

development of these diseases.

We addressed these issues in a mouse model of CNS inflammation in which CD8+ T

cells specifically target oligodendrocytes. In addition, as described in the introduction

several evidence points to the role of CD8+ T cells in MS (Friese and Fugger, 2009;

Perchellet et al., 2004). To understand the possible contribution of CD8+ T cells in CNS

tissue damage. Several groups have investigated their implication and role in experimental

inflammatory diseases of the CNS. CD8+ T cells are necessary for the development of a

full-fledged pathology in animal models of neuroinflammation, but their antigen specificity

is still unknown (Brisebois et al., 2006; Ip et al., 2006; Neumann et al., 2002). Myelin-

specific CD8+ T cells can adoptively transfer autoimmune encephalomyelitis, but the

lesions were reminiscent of ischemic injury with the demyelination associated with more

global tissue damage, and few CD8+ T cells actually infiltrated the CNS parenchyma

(Huseby et al., 2001a; Sun et al., 2001). Furthermore it has been shown recently that a

myelin-specific TcR, derived from a CD8+ T cell clone from a MS patient, actuates MS-like

disease in humanized mice expressing the MS-associated MHC class I alleles HLA-A*0301

and the HLA-A*0201 allele, which has a protective effect (Friese et al., 2008). These data,

although based on humanized animals, further strengthen the involvement of CD8+ T cells

in MS pathogenesis. However, the exact targets of infiltrating CD8+ T cells in the CNS and

how they might exert their neurotoxicity remains to be defined.

It has been described before, that CD8+ T cells can cause demyelination by targeting

oligodendrocytes. Using a model system in which lymphocytic choriomeningitis virus

(LCMV) proteins were selectively expressed in oligodendrocytes by transgenesis, it was

possible to test whether LCMV Armstrong infection, which does not infect the CNS

parenchyma, could initiate CNS autoimmune demyelination due to the imposed molecular

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Discussion & Perspectives

mimicry. Indeed, CD8+ T cell-mediated demyelination was clearly illustrated in this viral

model but the effector mechanisms involved have remained unexplored (Evans et al.,

1996).

Recent reports suggested, that CD8+ T cells could control herpes simplex virus type

1 (HSV-1) infection via both lytic and non-cytolytic mechanisms. CD8+ T cell lytic

granules can block the HSV-1 life cycle through a non-lytic mechanism, and granzyme B

can directly cleave ICP4, a viral protein required for efficient transcription of early and late

viral genes (Knickelbein et al., 2008). However, neuronal damage in LCMV acute

meningoencephalitis was not mediated by lytic activity of CD8+ T cells, but rather by their

recruitment and activation of myelomonocytic cells (Kim et al., 2009). Thus, the part

played by perforin and granzyme B in neurodegeneration may vary according to the model

used. Therefore, the direct effect of CD8+ T cells on oligodendrocyte lysis and the

consequent myelin damage in vivo remains elusive.

In order to assess whether CNS-infiltrating CD8+ T cells can directly induce

oligodendrocyte death and demyelination, we developed a mouse model combining

selective expression of influenza HA as a neo-self-antigen in oligodendrocytes (using the

MOG promoter) with transgenic mice expressing a HA-specific TcR on CD8+ T cells

(Bercovici et al., 2000; Morgan et al., 1996). Given the fact, that MOG is the sequestered

CNS antigen (Bruno et al., 2002), we first assessed whether in our mouse model HA is also

expressed as a sequestered antigen. Therefore we crossed our MOG-HA mice with CL4-

TcR transgenic mice and followed them for any development of spontaneous disease.

Interestingly we did not observe any neurological sign. We did not observe evidence for T

cell tolerance such as TcR downregulation or deletion of HA-specific CD8+ T cells, which

is consistent with the lack of expression or possible cross presentation of HA in the

lymphoid organs.

Perhaps the innocuous behavior of HA-specific CD8+ T cells in the CL4-TcR x

MOG-HA mice despite their high avidity for HA (Cabarrocas et al., 2003) could be due to

low levels of HA expression, its strict expression beyond the BBB and/or the weak local

expression of MHC class-I. Indeed, it has been clearly shown, that below a certain antigen

dose, specific CD8+ T cells do not respond to tissue self-antigen (Kurts et al., 1999).

However, other parameters could determine the CD8+ T-cell fate as a seemingly indifferent

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Discussion & Perspectives

phenotype, which has been reported for high avidity MBP-specific CD8+ T cells (Perchellet

et al., 2004) despite detectable MBP expression in lymphoid organs. In addition, CFSE-

labeled naïve CL4-TcR CD8+ T cells failed to proliferate following their adoptive transfer

in MOG-HA mice. This result was notably different from an earlier study, showing that in

GFAP-HA mice expression of HA by lymph node stromal cells induced strong proliferation

of HA-specific CD8+ T cells in vitro and in vivo (Magnusson et al., 2008). This further

accentuates the lack of expression or cross-presentation of HA in the lymphoid organs of

MOG-HA mice. Therefore, HA protein expression driven by MOG promoter behaves as a

sequestered antigen, thus supporting the model to mimic MOG in the involvement of

autoimmune demyelinating disease.

As shown previously, in-vitro activated HA-specific CD8+ T cells can readily cross

the BBB (Cabarrocas et al., 2003). Consequently we transferred HA-specific Tc1 cells in

immunocompetent MOG-HA mice. A plaque-like demyelination pathology was induced. In

addition, staining for amyloid precursor protein (APP) revealed some axonal pathology.

Importantly, we demonstrate, that demyelinating lesions, sharing many of the attributes of

an active MS plaque, can arise when CD8+ T cells target oligodendrocytes.

Our system allows the clear assessment of the individual role of CD8+ T cells by

uncoupling the impact of cytotoxic CD8+ T cells from other adaptive immune mechanisms.

Genetic GFP-labeling of the transferred CD8+ T cells also permitted to unequivocally trace

the HA-specific cytotoxic T cells in situ. Strikingly, we show here that CD8+ T cells enter

the CNS and optic nerve parenchyma and form close anatomical interactions with

oligodendrocytes. The data strongly suggest, that direct cell contact-mediated cytotoxicity

plays a central role in the oligodendrocyte death and demyelination observed in our model.

Indeed, numerous T cells, among which an important proportion is of HA-specific cytotoxic

T cells, were invading the CNS of MOG-HA mice. There was a strong co-localization

between HA-specific CD8+ T cells and oligodendrocytes with occasional figures suggesting

close apposition between the two cell types. It is however possible that soluble

inflammatory mediator contributes in several synergistic ways to tissue lesions. Indeed,

IFN-γ and TNF promote up-regulation of MHC class-I molecules on oligodendrocytes

(Sobottka et al., 2009; Turnley et al., 1991), rendering these cells more susceptible to CD8-

mediated cytotoxicity. In addition, chemokines and MMP create the appropriate milieu to

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Discussion & Perspectives

favor migration across the BBB of additional immune cells. Finally, inflammatory

cytokines favor the activation of resident microglial cells and enhance their phagocytic

properties. In line with this hypothesis, our unpublished data showed, that transfer of IFN-γ-

deficient HA-specific preactivated CD8+ T cells in MOG-HA mice led to a much-reduced

phenotype.

It has been shown in vitro, that myelin-specific cytotoxic CD8+ T cells can kill

isolated HLA-matched oligodendrocytes (Jurewicz et al., 1998) but data in vivo were less

compelling. Similarly, a role for CD8+ T cells in demyelination has been clearly illustrated

in viral models of CNS inflammation but the mechanisms involved have remained elusive

(Evans et al., 1996; Rivera-Quinones et al., 1998), as even activated CD8+ T cells of

irrelevant specificity can mediate bystander demyelination in this setting (Dandekar et al.,

2004).

In the same line of research another independent group describe the similar outcome

of CD8+ T cell mediated oligodendrocyte lysis, while using ovalbumin (OVA) sequestered

as a neo-self antigen in the cytosol of oligodendrocytes (Na et al., 2008). They show, that

endogenously generated CD8 T-cells expressing the MHC class I-restricted, OVA-specific

OT-I receptor are highly encephalitogenic in ODC-OVA mice, inducing demyelinating

lesions reminiscent of MS plaque. This encephalitogenic property mainly depends on the

production of IFN-γ by the OVA-specific CD8+ T-cells, similar to our observation.

The investigation on the role of CD8 T cells in CNS inflammatory diseases is not

confined to animal models. Indeed, in MS patients, a German team suggests that at least a

part of the pervasive T-cell clones belong to the CD8+ compartment. Because of the shared

TcR usage it is assumed that some of these T cells respond to a common antigen (Junker et

al., 2007). Therefore our findings could provide a helpful link to understand MS pathology.

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Discussion & Perspectives

Implication of self-molecular mimicry in MS

Activation of autoreactive T cells is critical for the induction of autoimmune

diseases. In humans, T cell clones specific for myelin antigens are present in the circulation

of both MS patients and healthy individuals. Autoreactive T cell clones demonstrate a

significant degree of functional degeneracy in antigen recognition. Cross-reactivity against

other myelin antigens has been observed in MBP-reactive T cell clones (Mycko et al.,

2004). Furthermore, recent studies characterizing TcR degeneracy have determined, that the

frequency of T cells with degenerate TcR is higher in patients with MS than in healthy

controls, and that a subset of these cells recognize myelin antigen (Zhang et al., 2008).

These findings support the hypothesis, that non-self antigens may activate cross-reactive T

cells during the course of an infection.

A number of viral epitopes that trigger autoreactive T-cell clones, including a

peptide from human cytomegalovirus have been identified (Hemmer et al., 1997). Other

infectious agents, including EBV, Chlamydia pneumoniae, hepatitis B virus, and

Haemophilus influenzae, have also been reported to express cross-reactive epitopes

(Croxford et al., 2005; Fujinami et al., 1983; Lang et al., 2002; Lenz et al., 2001). Such

molecular mimicry by viral or bacterial proteins could initiate autoimmune pathogenesis by

myelin-reactive T cells (Fujinami and Oldstone, 1985; Wucherpfennig and Strominger,

1995). Molecular mimicry during the course of an infection could provoke development of

MS in genetically susceptible individuals. However, there is little direct support providing a

link between infection and MS. The most studied epidemiological candidate to date is EBV,

which has a slightly higher prevalence rate in MS patients than in healthy controls

(Ascherio and Munger, 2007). Recently it has been suggested, that prevalence of EBV

infection, as measured by EBV seropositivity, was linked to HLA haplotype (De Jager et

al., 2008).

A number of T cell clones specific for foreign epitopes lacked significant multi-

specificity, suggesting that the recognition of multiple epitopes is more frequent among

autoreactive T cell clonotypes (Cai and Hafler, 2007). In the same line it has been shown

that single TcR also bind to distinct self-peptides from different autoantigens or non-

overlapping peptides derived from the same autoantigen. For instance CD4+ T cells cloned

from peripheral blood of healthy subjects and expanded in an autologous-mixed

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Discussion & Perspectives

lymphocyte response recognizing multiple autoantigenic epitopes, which include a number

of peptides derived from autoantigens associated with T1D and MS, such as GAD, insulin,

proinsulin, and MBP (Cai and Hafler, 2007). In addition, a relatively high frequency of

GAD65-specific CD4+ T-cell clones recognizes two non-overlapping GAD65 peptides and

this promiscuity correlates with increased avidity/affinity and an enhanced capacity to

mediate insulitis (Li et al., 2008).

Collectively these studies described, that multiple cross-reactivity persists towards

self-antigens among autoreactive T cells in both human and mice, therefore self-mimicry

may play a role in T-cell mediated autoimmunity. Interestingly, it has been demonstrated

that TcRs specific for viral or other foreign antigens recognize non-overlapping epitopes

from the same antigen and these promiscuous T-cell clones may represent a subset of CD4+

and CD8+ T-cells with enhanced effector functions (Brehm et al., 2002; Chen et al., 2001;

Riedl et al., 2006).

MOG differs from other myelin autoantigens because of its immunodominance

(Amor et al., 1994; Genain et al., 1995) and its highly encephalitogenic properties upon

immunization of susceptible animals (Bernard et al., 1997; Iglesias et al., 2001; Mendel et

al., 1995). In addition it has been shown, that MOG is a direct target for humoral response

(Litzenburger et al., 1998), and that the anti-MOG response is a major pathogenic

component of the autoimmune response directed against myelin, which is associated with

lack of immune tolerance to MOG (Delarasse et al., 2003).

The 2D2 TcR-transgenic C57BL/6 mice, which harbor large populations of MOG-

specific CD4+ T cells, tend to spontaneously develop optic neuritis and more rarely bona

fide EAE (Bettelli et al., 2003). Strikingly, when we crossed these 2D2 mice with MOG-/-

mice, we observed, that about 16% of the 2D2 TcR-transgenic mice developed spontaneous

EAE regardless of the presence or absence of MOG. Therefore we hypothesized, that 2D2

TcR specific for MOG35-55 recognizes a second autoantigenic target. This paradoxical

observation was made independently in two different cohorts of 2D2-transgenic mice, one

with disrupted exon1 of the MOG gene (Delarasse et al., 2003), and the other in which the

MOG gene was deleted by an independent knock-in strategy (Hovelmeyer et al., 2005),

excluding residual, atypical MOG material in these knockout animals as a possible

encephalitogenic target.

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Discussion & Perspectives

In silico search for sequence related to the MOG35-55 motif recognized by the 2D2 T

cell clone (Petersen et al., 2004) revealed the stimulatory autoantigenic epitope of NF-M.

Unexpectedly, this NF-M18-30 peptide induced proliferation and cytokine secretion by 2D2

CD4+ T cells at lower concentrations than MOG35-55 peptide. We used 2D2 TcR transgenic

cells from Rag2-/- mice to exclude a role for dual TcR expression and for the endogenous

TcR repertoire. These results further strengthen our observation that 2D2 cross-reactive T

cells recognize their alternative autoantigenic target in the absence of MOG, and that this

cross-recognition is restricted to MOG specific TcR. Given the marked degeneracy of

peptide recognition by T cells, this observation was not very surprising (Wucherpfennig and

Strominger, 1995). Nevertheless we confirmed NF-M as the stimulatory autoantigen at the

protein level using both CNS white matter protein extracts and recombinant proteins.

Polyclonal T cell populations primed with either MOG or NF-M isolated from

C57BL/6 mice show extensive cross-reactivity between NF-M and MOG proteins and their

salient epitopes, respectively. Of note, these populations rarely use Vα3.2 and Vβ11, the

variable chains used by the 2D2 clone, indicating that MOG and NF-M cross-reactivity is

not limited to the 2D2 TcR. Indeed, we now have evidence for such cross-reactivity in other

I-Ab-restricted MOG and NF-M specific CD4+ T cells expressing distinct TcRs. This

implies that the peripheral T cell repertoire of C57BL/6 mice contains both a MOG35-55 and a

NF-M15-35 specific T cell pool and both pools may overlap. The size of this overlap can be

assessed by I-Ab restricted tetramers specific for MOG and NF-M. In addition, the

frequency of cross-reactive clones can be assessed after MOG35-55 and NF-M15-35

immunization. These cells will then be used to study the pathogenicity afforded by single or

cross-reactive TcRs in autoimmune diseases. This can be achieved by using retro-transgenic

mice.

Neurofilaments, including NF-M, are produced by neurons and also by some glial

cells (Kelly et al., 1992). It can be the target of autoantibodies in a number of autoimmune

situations. For example, the acetylcholine receptor-like epitope of the NF-M was identified

by peptide epitope mapping as an autoantigenic determinant of myasthenia gravis (Schultz

et al., 1999). A significant proportion of rheumatoid arthritis patients with neuropathy had

abnormal antibody levels to one or more neurofilaments (Salih et al., 1998). Neurofilaments

were recently characterized as autoantigens in actively induced EAE. Immunization of

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Discussion & Perspectives

Biozzi ABH (antibody high, AB/H, ABH) mice with the light form of neurofilament, NF-L,

causes EAE in a moderate proportion of treated animals (Huizinga et al., 2007).

Furthermore, autoantibodies to NF-M have been detected in the cerebrospinal fluid of some

individuals with MS (Bartos et al., 2007).

Based on our results, showing cross-reactivity between two different neural self-

antigens i.e. MOG and NF-M, one could ask what is the relative pathogenic impact of MOG

and NF-M as target antigens for the 2D2 T cells? Therefore we generated mouse lines

lacking one or both autoantigenic target antigens and showed, that MOG was important for

rapid onset of EAE. In the same line our unpublished data revealed that, when transferring

in-vitro differentiated Th1 cells from 2D2-RAG-/- donor mice into recipients lacking NF-M,

an EAE with the same kinetics as wild type recipients was induced. Disease severity was

however decreased in this situation, suggesting that NF-M may play a role in disease

severity.

In contrast, we were unable to induce disease in wild type C57BL/6 mice by

immunization with NF-M using protocols that consistently allow active disease induction

by MOG35–55. This discrepancy between active and passive EAE induction is, however, not

exceptional. It has been previously described for other models, including EAE induced in

Lewis rats by glial fibrillary acidic protein (GFAP) (Berger et al., 1997) and S100-β

(Kojima et al., 1994) and MBP-induced EAE in BALB/c mice (Abromson-Leeman et al.,

1993). It has been shown that when EAE was induced by the adoptive transfer of CD4+ T

cell lines specific for the MBP, MOG and S100-β antigen, as in the MBP induced EAE, the

spinal cord was more severely affected with only minor inflammation in the forebrain. In

contrast, MOG induced a far higher density of lesions in the periventricular and cerebellar

white matter. S100-β and GFAP mediated severe inflammation particularly in the gray

matter. In addition to these topographic differences, antigen specificity also influenced the

extent of both parenchymal inflammation and macrophage activation in the CNS.

We refer to the observation of self-mimicry, as ‘Cumulative autoimmunity’ where

an autoimmune response targets two self-autoantigens in the same tissue, consequently

exhibit additive pathogenicity resulting in enhanced CNS tissue damage. A similar kind of

additive pathogenicity has been described among GAD65 multispecific CD4+ T cell clones,

which exhibit increased pathogenicity upon adoptive transfer in NOD.scid recipients (Li et

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Discussion & Perspectives

al., 2008). One important mechanistic issue is whether cumulative autoimmunity galvanizes

tissue destruction by increasing the availability of auto-antigen at a given site or by

diversifying the cellular and anatomical targets of the autoimmune response. MOG and NF-

M co-localize in the CNS white matter, with the axons expressing NF-M and

oligodendrocytes expressing MOG. Therefore the cross-presentation of both antigens by

local APCs could exacerbate the intensity of a given demyelinating lesion by increasing the

density of agonistic ligands (both I-Ab:MOG35-55 and I-Ab:NF-M15-35). However, MOG is

absent from the myelin sheath in the PNS making NF-M the only target antigen, which

could indicate that cumulative autoimmunity permits the broadening of anatomical and

cellular targets aggravating disease by multiplying inflammatory lesions. Indeed, while

transferring in-vitro differentiated Th1 cells from 2D2-Rag2-/- mice, we found lesions in the

tissues which are devoid of MOG autoantigen but contain NF-M, such as the trigeminal and

spinal ganglia, spinal roots and peripheral nerves. This observation is reminiscent of our

histological observations in diseased 2D2 TcR transgenic mice, having inflammatory

lesions at sites devoid of MOG.

This cumulative autoimmunity is attributed to TcR cross-reactivity. Although much

is known about TcR recognition of foreign antigens (Rudolph et al., 2006), the structural

and biophysical principles governing TcR recognition of self have only recently begun to be

elucidated. Recent studies of self-recognition by autoreactive TcR suggested, that

unconventional topologies are possible because of the unique CDR3 sequences created

during rearrangement (Hahn et al., 2005; Li et al., 2005b; Maynard et al., 2005; Nicholson

et al., 2005). This topology reduces the interaction surface with peptide and alters the

geometry for CD4 association. Several structures of autoimmune TcR pMHC complexes

have now been reported, such as the complex between human TcR Ob.1A12 and MBP85–99

presented by HLA-DR2b (Hahn et al., 2005) the complex between human TcR 3A6 and

MBP89–101 presented by HLA-DR2a (Li et al., 2005b) and the complex between mouse TcR

172.10 and MBP1–11 presented by I-Au (Maynard et al., 2005). TcR Ob.1A12 and 3A6 were

isolated from MS patients, and humanized mice transgenic for these TcR and for DR2b or

DR2a develop symptoms typical of EAE. TcR 172.10 was derived from a T-cell clone that

causes EAE following transfer into mice. Significantly, all three autoimmune TcRs engage

pMHC class II with unconventional binding topologies compared with TcR specific for

145

Discussion & Perspectives

foreign antigens. However, current structural database for MHC class II-restricted TcR is

limited as compared to MHC class I restricted TcR (Rudolph et al., 2006) therefore it is

difficult to define the full range of docking topologies for MHC-class II-restricted TcR. The

3A6–MBP–DR2a complex provides further evidence of unusual interactions between

autoimmune TcR and self-pMHC. Similar to Ob.1A12, 3A6 TcR binds its self-ligand with

low affinity (KD > 200 µM) (Li et al., 2005b). Therefore, it would be interesting to assess

whether 2D2 TcR binds self-peptides such as MOG and NF-M with different affinity.

Would this, as a consequence, influence disease severity or onset? All these important

issues need to be addressed in detail.

We have demonstrated immunological self-mimicry in the same tissue, where one T

cell population with specific TcR to MOG35–55, recognizes another independent target self-

autoantigen, i.e. NF-M. However, another case of self-mimicry between structures from

different tissues was reported in the Dark Agouti (DA) rat in which MOG-specific T cells

reacted to an epitope of the milk protein butyrophilin (Stefferl et al., 2000). Transmucosal

exposure to butyrophilin peptide can modulate disease activity in MOG-induced EAE. If

transferable to humans, this may suggests, that in the context of an appropriate HLA

haplotype, dietary exposure to butyrophilin may modulate the pathogenic autoimmune

response to MOG in MS.

Furthermore, whether molecular mimicry between neural self-antigens could

contribute to cross-reactive T cell tolerance is not defined. As mentioned earlier, MOG is

barely expressed in the thymus and periphery, consequently, no detectable T and B cell

tolerance can be observed against MOG in C57BL/6 mice (Delarasse et al., 2003).

However, NF-M expression can be detected in human thymic medulla by immuno-

histochemistry (Marx et al., 1996) and in murine non-lymphoid thymic stromal cells

(Screpanti et al., 1992). Moreover due to its presence in the PNS, NF-M is likely to be

present in local draining lymph nodes (Grant and Pant, 2000; Oldstone, 1987; Perrot et al.,

2008). Therefore our future study will assess whether the expression of NF-M can impact

the capacity of the T cell repertoire to respond to MOG35-55 and vice versa.

Finally identification of neural self-mimicry as a cumulative trigger in CNS

autoimmunity defines a novel mechanism that perpetuates EAE. This mechanism permits

the spreading of antigenic and cellular targets due to the plasticity of the TcR. Molecular

146

Discussion & Perspectives

mimicry between auto-antigens is therefore likely to be a universal mechanism propagating

organ-specific autoimmune disease.

Concluding remarks

Gene-modified mouse models have been extremely useful to decipher the

mechanisms involved in immune tolerance and inflammatory disease development. We

generated a mouse model to study the contribution of individual T-cell subsets on CNS

tissue damage. We have focused on oligodendrocyte-specific CD8+ T cells, as little in vivo

information was available regarding the pathogenicity of this subset at the beginning of this

study. Furthermore, both CD8+ T-cell infiltration and loss of oligodendrocytes are essential

features of MS lesions. The novel findings provided by my work are that, whereas naïve

CD8+ T-cells specific for a CNS-restricted self-antigen persist but remain innocuous,

effector CD8+ T cells exhibit a potent deleterious effect on oligodendrocytes, resulting in an

inflammatory demyelinating pathology reminiscent of active MS lesions. The exclusive

expression of HA in oligodendrocytes, and the resulting lack of T-cell silencing, is very

reminiscent of some natural myelin self-antigens such as MOG, which is barely expressed

in the thymus and therefore fails to exert efficient censoring of the T cell repertoire

(Delarasse et al., 2003; Fazilleau et al., 2006). Tolerance to these sequestered antigens,

which relies solely on passive mechanisms (sequestration, low antigen dose), carries an

obvious risk of activation of autoreactive CD8+ T cells, with subsequent development of

autoimmunity. Identification of the stimuli that may reverse this operationally tolerant state

will be a matter of further studies. Adoptive transfer of activated oligodendrocyte-specific

CD8+ T cells in MOG-HA mice has provided unequivocal evidence of their in vivo potential

to induce oligodendrocyte apoptosis and resulting demyelination, a likely consequence of

direct antigen-recognition. Since our data show, that CD8+ T cells induce inflammatory

demyelinating lesions closely resembling active MS lesions, they reinforce the idea that

CD8+ T cells represent attractive therapeutic targets in MS (Friese and Fugger, 2005, 2009;

Liblau et al., 2002; Neumann et al., 2002; Sobottka et al., 2009).

In addition, we discovered the implication of self-mimicry in CNS autoimmunity

due to the paradoxical development of spontaneous EAE in 2D2 x MOG-/- mice despite the

absence of its target antigen, MOG. A cross-reactive epitope was identified as NF-M.

147

Discussion & Perspectives

Importantly, this cross-reactivity is not restricted to the transgenic TCR but is commonly

seen in short term cell lines from MOG35-55 or NF-M15-35 immunized mice. Our collaborative

study reveals the identification of neural self-mimicry as a cumulative trigger, which

defines a novel mechanism in CNS autoimmune disease. However, it is not clear, how the

effector mechanisms targeting MOG and NF-M in vivo cumulate during CNS

autoimmunity. Similarly it is important to understand the impact of neural self-mimicry on

immune tolerance. These are obvious directions for future studies in our laboratory.

Overall, we propose that in C57BL/6 mice autoimmune response components

directed against MOG and NF-M may accumulate to overcome the general resistance of

these mice to induction of EAE. T cells with similar cumulative double self-reactivity could

act as dominant pathogens in human MS, and genetic factors favoring bi-specific T cells

(possibly the HLA class II alleles) would enhance susceptibility to the disease. Our study

should provide a way to identify such T cells in humans and acknowledge their role in the

pathogenesis of MS.

148

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Appendix

Appendix

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Appendix

Appendix List of Publications:

Main Projects during my PhD Thesis

1. Krishnamoorthy G, Saxena A, Mars LT, Domingues HS, Mentele R, Ben-Nun A,

Lassmann H, Dornmair K, Kurschus FC, Liblau RS, Wekerle H. 2009. Myelin-specific T cells also recognize neuronal autoantigen in a transgenic mouse model of multiple sclerosis. Nat Med 15: 626-32

2. Saxena A, Bauer J, Scheikl T, Zappulla J, Audebert M, Desbois S, Waisman A,

Lassmann H, Liblau RS, Mars LT. 2008. Cutting edge: Multiple sclerosis-like lesions induced by effector CD8 T cells recognizing a sequestered antigen on oligodendrocytes. J Immunol 181: 1617-21

Collaboration with Dr. Nathalie Vergnolle within Toulouse-Purpan Research Center

3. Hyun E, Ramachandran R, Cenac N, Houle S, Rousset P, Saxena A, Liblau RS, Hollenberg MD, Vergnolle N. 2010. Insulin modulates protease-activated receptor 2 signaling: implications for the innate immune response. J Immunol 184: 2702-9

Work performed in India prior to joining the Phd Thesis

4. Ghosh J, Swarup V, Saxena A, Das S, Hazra A, Paira P, Banerjee S, Mondal NB,

Basu A. 2008. Therapeutic effect of a novel anilidoquinoline derivative, 2-(2-methyl -quinoline-4ylamino)-N-(2-chlorophenyl)-acetamide, in Japanese encephalitis: correlation with in vitro neuroprotection. Int J Antimicrob Agents 32: 349-54

5. Swarup V, Ghosh J, Ghosh S, Saxena A, Basu A. 2007. Antiviral and anti-

inflammatory effects of rosmarinic acid in an experimental murine model of Japanese encephalitis. Antimicrob Agents Chemother 51: 3367-70

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The Journal of Immunology

Insulin Modulates Protease-Activated Receptor 2 Signaling:Implications for the Innate Immune Response

Eric Hyun,* Rithwik Ramachandran,* Nicolas Cenac,†,‡ Steeve Houle,* Perrine Rousset,†,‡

Amit Saxena,†,‡ Roland S. Liblau,†,‡ Morley D. Hollenberg,* and Nathalie Vergnolle*,†,‡

Given the anti-inflammatory effects of insulin in human and animal studies done in vivo and given the signaling pathways in com-mon between insulin and the protease-activated receptor 2 (PAR2), a G protein-coupled receptor, we hypothesized that insulinwould have an impact on the inflammatory actions of PAR2. We found that low doses or concentrations of insulin in thesubnanomolar range reduced PAR2-induced inflammation in a murine paw edema model, attenuated PAR2-induced leukocytetrafficking in mouse intestinal venules, and reduced PAR2 calcium signaling in cultured dorsal root ganglion neurons andendothelial cells. This effect of insulin to attenuate PAR2-mediated inflammation was reversed when cells were preincubated withLY294002 (a PI3K inhibitor) and GF 109203X (a pan-protein kinase C inhibitor). The enhanced inflammatory effect of PAR2

observed in vivo in an insulin-deficient murine type 1 diabetes model was attenuated by the local administration of insulin at theinflammatory site. Our data point to an anti-inflammatory action of insulin that targets the acute innate inflammatory responsetriggered by PAR2. The Journal of Immunology, 2010, 184: 2702–2709.

I ncreasing evidence points to an association between diabetesand chronic activation of the innate immune system, withelevated low-grade inflammation. Insulin has been shown to

exert anti-inflammatory properties in a number of settings, in-cluding several animal models of inflammation (summarized inRef. 1). To date, the anti-inflammatory action of insulin has beenattributed to its effects on mononuclear cells (2), possibly via thesuppression of endotoxin-induced proinflammatory transcriptionfactors and their gene targets. In a cardiac ischemia‑reperfusionmodel, the ability of insulin to reduce infarct size has been at-tributed to its antiapoptotic action, mediated via PI3K, Akt (pro-tein kinase B), Bcl-2–associated death promoter, and reduced NOproduction via NO synthase phosphorylation (reviewed in Ref. 1).However, a modulatory role for insulin on inflammatory signalsinduced by mediators signaling through G protein-coupled re-ceptors is still largely unknown. Serine proteases, through theactivation of protease-activated receptors (PARs), are now con-sidered as key mediators of the innate immune response (3).Several studies have shown the involvement of this family of Gprotein-coupled receptors in both acute and chronic inflammatorystates (3, 4). PARs belong to a unique four-member G protein-coupled receptor family (PAR1–4), which are proteolytically ac-

tivated by the unmasking of a cryptic N-terminal sequence thatbecomes a tethered receptor-activating ligand (5). In isolation,synthetic peptides having sequences in common with the pro-teolytically revealed tethered ligand (e.g., SLIGRL-NH2, derivedfrom PAR2) can selectively activate PARs in the absence of en-zymatic cleavage. By cleaving and activating PAR2, trypsin andother serine proteases, such as tryptase or tissue kallikreins, havebeen found to trigger a variety of inflammatory responses, in-cluding edema, leukocyte infiltration, and pain (6–8). These in-flammatory actions of trypsin, due in large part to a neurogenicmechanism (9), are mimicked by the receptor-selective PAR2-activating peptide (PAR2-AP) SLIGRL-NH2. A number of studieshave highlighted, particularly for PAR2, a prominent inflammatoryrole as a mediator of innate immunity, singling out PAR2 as a newmolecular target for anti-inflammatory and analgesic treatments(10, 11). Although some studies have identified factors that areable to control the expression of PAR2, no studies have identifiedso far the endogenous signaling pathways that can modulate itsproinflammatory effects.In the context of an association among diabetes, low insulin

levels, and an inflammatory state and in view of the prominent roleof proteases in initiating inflammatory processes via PAR2, wehypothesized a potential interaction between insulin and PAR2

agonists to affect inflammation. This possible interaction betweeninsulin and PAR2 signaling might relate pathophysiologically toinflammation in the setting of diabetes.To test the hypothesis that PAR2 and insulin signaling pathways

can interact in the triggering of an acute inflammatory response, weused: 1) a PAR2-selective agonist, SLIGRL-NH2, to activate PAR2

without affecting other PARs, like trypsin would do, and 2) ap-propriately low doses or concentrations of insulin (1 nM in vitro;150 pmol, administered locally in vivo) with or without activationof PAR2 by SLIGRL-NH2. The PAR2-selective agonist and insulinwere used to evaluate several inflammation-related targets: 1) anin vivo paw edema inflammation model for which we have pre-viously documented the inflammatory action of trypsin and PAR2

(6), 2) an in vivo leukocyte‑endothelium interaction model, docu-mented with intravital microscopy (7), and 3) a PAR2-stimulatedcalcium signaling model, using the two cell types that have been

*Department of Physiology and Pharmacology, University of Calgary, Calgary, Al-berta, Canada; †Institut National de la Sante et de la Recherche Medicale, U563,Centre de Physiopathologie de Toulouse Purpan, Centre Hospitalier UniversitairePurpan; and ‡Universite Toulouse III Paul Sabatier, Toulouse, France

Received for publication July 7, 2009. Accepted for publication December 25, 2009.

This work was supported by an Institut National de la Sante et de la RechercheMedicale Avenir research grant, Foundation Bettencourt-Schueller (to N.V.), Foun-dation Schlumberger (to N.V.), and operating grants from the Canadian Institutes ofHealth Research (to N.V. and M.D.H.).

Address correspondence and reprint requests to Dr. Nathalie Vergnolle, Institut Na-tional de la Sante et de la Recherche Medicale, U563, Centre de Physiopathologie deToulouse Purpan, BP 3028, Centre Hospitalier Universitaire Purpan, 31024 ToulouseCedex, F-31000 France. E-mail address: nathalie.vergnolle@inserm.fr

Abbreviations used in this paper: DRG, dorsal root ganglion; HMEC, human micro-vascular endothelial cell; MPO, myeloperoxidase; PAR, protease-activated receptor;PAR-AP, PAR-activating peptide; PKC, protein kinase C.

Copyright! 2010 by TheAmericanAssociation of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.0902171

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shown to play an important role in PAR2-induced inflammation,isolated dorsal root ganglion (DRG) neurons (12) and culturedmicrovascular endothelial cells (13). Further, we evaluated the in-flammatory action of SLIGRL-NH2 in mice rendered diabetic viapancreatic b cell elimination.

Materials and MethodsMice

Six- to eight-week-old C57BL/6 mice were obtained from Charles RiverLaboratories (Wilmington, MA) or Janvier (St. Quentin Fallavier, France).RIP-HA mice expressing influenza hemagglutinin (HA) on pancreatic isletb cells under the control of a rat insulin promoter (14) were bred at theINSERM U563 Animal Care Facility. CL4-TCR transgenic mice expresson most CD8 T cells a TCR specific for the influenza virus HA512–520

peptide‑Kd complex (15). RIP-HA and CL4-TCR transgenic mice werebackcrossed at least 10 times onto the BALB/c background. Animals werekept under pathogen-free conditions and given free access to food andwater. All of the experimental protocols were approved by local AnimalCare and Ethic Committees and followed the guidelines of the Canadianand French Councils on Animal Care.

Chemicals

SLIGRL-NH2 (a receptor-selective PAR2-AP) and a control peptideLRGILS-NH2 (the reverse sequence of PAR2-AP that cannot activatePAR2) were obtained from the protein synthesis facility at the University ofCalgary (Calgary, Alberta, Canada). HPLC and mass spectrometry wereused to assess the purity of the peptides. Peptides that were .95% pure(HPLC and mass spectrometry criteria) were dissolved for paw edemastudies in isotonic 0.9% NaCl (saline) or for intravital studies in an80:10:10 (v/v/v) saline solution supplemented with Tween 80 and ethanol(80% saline, 10% ethanol, and 10% Tween 80). This vehicle is necessaryto enable the peptide to gain access to the tissue. Carrageenan and re-combinant yeast-produced human insulin (I2643, 27.5 U/mg) were fromSigma-Aldrich (St. Louis, MO). The PI3K inhibitor LY294002 was fromCayman Chemical (Ann Arbor, MI), and the protein kinase C (PKC) in-hibitor GF 109203X was from Tocris Bioscience (Ellisville, MO).

Paw edema

Paw edema was monitored with an electronic caliper (resolution 0.01;Mitutoyo,Aurora, IL) usingC57BL/6mice, in keepingwith previous studieswith rats (6). Basal paw thickness (at time 0)wasmeasured, and immediatelythereafter 10 ml solution containing various test compounds was adminis-tered via intraplantar injection. The test substances included vehicle (iso-tonic saline), SLIGRL-NH2 (50 mg), LRGILS-NH2 (50 mg), insulin (150pmol), and carrageenan (200 mg). The inflammatory agonists (peptides orcarrageenan) were administered either alone, or in combination with insulin(e.g., SLIGRL-NH2 plus insulin [50 mg and 150 pmol, respectively] andcarrageenan plus insulin [200 mg and 150 pmol, respectively]). Isotonicsaline served as a vehicle control for all of the experiments using insulin. Thechange in paw thickness wasmeasured every hour for 6 h and recorded as thedifferences between the measurement at each time point and the basalmeasurement done immediately prior to the intraplantar injections. After thelast paw edema measurement, the animals were sacrificed by cervical dis-location, and paws were removed for the extraction and measurement ofmyeloperoxidase (MPO) activity as an index of granulocyte infiltration.Similar procedures were followed for in vivo experiments with kinase in-hibitors, but 15 min prior to the intraplantar injections with SLIGRL-NH2 orSLIGRL-NH2 plus insulin, mice received an intraplantar injection of 2 mldistilled water to cause osmotic shock, immediately followed by a 5ml in-traplantar injection of LY294002 (1 mg) or GF 109203X (1 mg).

MPO activity

Tissues collected forMPO assay were processed as outlined previously (16).In brief, dissected paw samples were homogenized in 0.5% hexadecyl-trimethylammonium bromide solution (dissolved in phosphate buffer so-lution). The homogenized tissues were centrifuged at 13,000 3 g for 4min. Supernatants were placed into a 96-well plate, and 1% hydrogenperoxide/O-dianisidine dihydrochloride-containing buffer was added. ODreadings were at a wavelength of 450 nm for 1 min at 30 s intervals.

Intravital microscopy

Mice were anesthetized by a mixture of xylazine (10 mg/kg; MTCPharmaceuticals, Cambridge, Ontario, Canada) and ketamine (200 mg/kg;Rogar/STB, London, Ontario, Canada) by i.p. injection. Mice were given

100 ml rhodamine 6G (Sigma-Aldrich) in NaCl 0.9% (0.3 mg/kg, ad-ministered i.v. in 0.1 ml). The dosage of rhodamine 6G used in this studyhas been shown to have no effect on leukocyte kinetics while effectivelylabeling leukocytes and platelets (17). Microcirculation was observedusing an inverted fluorescent microscope (Nikon, Melville, NY) witha 203 objective lens by epi-illumination at 510‑560 nm, using a 590 nmemission filter. After anesthesia, a midline abdominal incision was made,and a segment from the small intestine (jejunum) was exteriorized care-fully. The exposed intestine was placed on top of a viewing pedestal andsuperfused with bicarbonate-buffered saline (pH 7.4). After 10 min ofequilibration, single venules (20–40 mm in diameter) were selected andvisualized, with images recorded for 5 min. The end of the first 5 min ofrecording was designated as time 0. Immediately thereafter at the jejunalsite of visualization, agonist or control peptides were administered by anintraluminal injection: SLIGRL-NH2 (100 mg), LRGILS-NH2 (100 mg),or SLIGRL-NH2 plus insulin (150 pmol). The polypeptides were ad-ministered in 0.1 ml physiological isotonic saline (pH 7.4), supplementedwith 10% (v/v) absolute ethanol and 10% (v/v) Tween 80. The controlmice were treated with vehicle alone. Additional images of the selectedvenules then were recorded for 5 min intervals at times 20, 35, 50, 65, and80 min. Upon video playback, leukocyte rolling, adherence, and vesseldiameter were measured. Leukocyte flux was defined as the number ofleukocytes per minute moving at a velocity less than that of the eryth-rocytes that passed a reference point in the venule. A leukocyte wasconsidered adherent to the vessel wall if it remained stationary for 30 s orlonger. The change in vessel diameter and leukocyte flux was evaluated asthe differences between the values at each interval and the basal valueobserved at time 0. The parameters measured were plotted over the 80 mintime interval of observation, and the area under the curve was calculated.

Endothelial and primary afferent neuronal DRG cell cultures

Human microvascular endothelial cells (HMEC-1) (Centers for DiseaseControl and Prevention, Atlanta, GA) were grown in a cell culture flaskcontaining growth medium MCDB 131 (Invitrogen, Burlington, Ontario,Canada) supplemented with epidermal growth factor (10 ng/ml), hydro-cortisone (1 mg), 10% FBS, and 1% penicillin/streptomycin.

DRG neurons were isolated from C57BL/6 mice as described previously(12, 18, 19). DRGs were rinsed in HBSS and incubated in this solutioncontaining 27 mg/ml papain (Sigma-Aldrich) for 10 min at 37˚C. Afterbeing washed first with L-15 buffer (Leibovitz’s L-15 medium [In-vitrogen], supplemented with 2 mM glutamine [Sigma-Aldrich] and 10%FBS [Invitrogen]) and then with HBSS, DRGs were incubated in HBSScontaining 1 mg/ml collagenase type I (Sigma-Aldrich) and 4 mg/mldispase II (Sigma-Aldrich). L-15 wash buffer then was added to neutralizeenzymatic activities, and the suspension was centrifuged at 600 3 g for 5min. Neurons in the pellet were suspended in DMEM (Invitrogen) con-taining 2.5% FBS, 2 mM glutamine, 1% penicillin/streptomycin, and 10mM each of cytosine arabinoside, flurodeoxyuridine, and uridine (all fromSigma-Aldrich). Cells were plated on CC2 LabTek II culture slides (Nunc,Domique Dutscher, Brumath, France) for measurements of calcium sig-naling assay Ca2+ (see below). Neuronal cultures were maintained for 24 hor more to enable a regeneration of cell surface receptors, such as thePARs, that may have been affected by papain treatment.

Imaging to measure calcium transients in HMECs and DRGs

Mobilization of intracellular Ca2+ in HMECs was measured using cellmonolayers in accord with methods described previously (20). In brief,HMECs were seeded in glass-bottom petri dishes (MatTek, Ashland, MA).HMECs grown to 90% confluence were incubated for 30 min at 37˚C withserum-free MCDB 131 growth medium containing sulfinpyrazone (0.25mM) and fluo-3-acetoxymethyl ester (2.5 mM) (Molecular Probes, Eugene,OR). Cell monolayers then were washed briefly with dye-free calciumassay buffer (pH 7.4) containing NaCl (150 mM), KCl (3 mM), CaCl2 (1.5mM), glucose (10 mM), HEPES (20 mM), and sulfinpyrazone (25 mM)and then covered with 500ml assay buffer. Test agonists diluted in this assaybuffer were added to the monolayers, and the elevation of the intracellularcalcium concentration was monitored. Increase in fluo-3-acetoxymethylester fluorescence, an index of increased intracellular calcium concentration,was measured using an inverted microscope with a 320 objective. Thefluorescence signal was analyzed by ImageMaster system software (PhotonTechnology International, London, Ontario, Canada) with excitation at 480nm and emission recorded at 530 nm through a dichroic filter cube with theappropriate filter and an intensified charge-coupled device video camera(Photon Technology International). The magnitude of the calcium signalobserved was expressed as a percentage (% A23187) of the signal caused inthe same cell monolayers by the calcium ionophore A23187 (4 mM; Sigma-Aldrich).

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The uptake of calcium indicator dye by DRG neurons was achieved overa 30min incubation at 37˚C inHBSS (pH7.45) (Invitrogen) containing 0.1%BSA (Sigma-Aldrich) and 2.5 mM probenecid (Sigma-Aldrich), supple-mentedwith 10mMfluo-3-acetoxymethyl (Invitrogen), and 20%pluronic F-127 (Sigma-Aldrich). After the incubation period, the plates were washedtwice with the dye-free solution and placed into a 37˚C incubator in the darkfor 30 min. The increase in cellular fluorescence (excitation at 460‑490 nm;emission at 515 nm) was recorded using an inverted microscope (Zeiss,Oberkochen, Germany) and a (310, 0.5 numerical aperture objective). Im-ages were acquired using a charge-coupled device camera (Zeiss) andMetamorph software (Molecular Devices, Sunnyvale, CA). Acquisitionparameters were kept constant within each experiment. A kinetic of 200recordings (one per second) was performed. From 10 to 65 s, neurons wereexposed to agonists, then from 65 to 155 s, neurons were exposed toHBSS‑BSA‑probenecid buffer containing 50mMthapsigargin (Calbiochem,SanDiego, CA), 5mM ionomycin (Calbiochem), and 10mMEGTA (Sigma-Aldrich). Finally, from 155 to 200 s, neurons were exposed toHBSS‑BSA‑probenecid buffer containing 120 mM CaCl2. Regions of in-terest were fitted around the perimeter of one neuron, and intensity variationsfor each region of interest were measured using Metamorph software. In-tracellular calcium concentrations [Ca2+]i in nanomolar were calculatedusing the equation [Ca2+]i = Kd (F 2 Fmin)/(Fmax 2 F), where Kd is thedissociation constant of the Ca2+‑fluo-3-acetyoxymethyl ester complex (390nM) and F represents the fluorescence intensity of the cells expressed as theratio between the highest fluorescencemeasurement between 10 and 65 s andthe baseline. Fmin corresponds to the minimum fluorescence between 65 and155 s, and Fmax represents the maximum fluorescence between 155 and 200s. The data were expressed as the increase in fluorescencewith respect to thebaseline fluorescence intensity measured during the 10 s time period beforethe addition of agonists. Cells were treated with various compounds in-cluding SLIRGRL-NH2 (100 mM) with or without added native or boiledinsulin (1 nM). The impact of insulin on signaling was monitored in theabsence or presence of the PI3K inhibitor LY294002 (10 mM), the PKCinhibitor GF 109203X (10 mM), or both. When assessed for its effects in thecalcium signaling assays, insulin was added to the monolayers for 5 minbefore the addition of an agonist. The effects of the PI3K and PKC inhibitorson the effects of insulin were assessed for monolayers treated for at least 30min with the inhibitors before the addition of insulin or any other testcompounds.

Induction of diabetes in mice

To induce diabetes, influenza HA-specific Tc1 cells were generated. A totalof 107 spleen and lymph node cells from CL4-TCR transgenic mice werestimulated with 1 mg/ml HA512–520 peptide in DMEM supplemented with10% FCS, 1 ng/ml IL-2, and 20 ng/ml IL-12 (R&D Systems, Minneapolis,MN) with irradiated syngeneic spleen cells. At day 6, viable cells werecollected by Ficoll density separation and used in adoptive transfer ex-periments to mediate b cell destruction so as to induce insulin-deficientdiabetes. These cells routinely contained .98% pure CD8+CD3+Vb8.2+

T cells coexpressing IFN-g and granzyme B (21). A total of 5 3 106 ofthese effector Tc1 cells were injected i.v. (retro-orbital vein) into immu-nocompetent recipient mice. Control (nondiabetic) mice were injected i.v.with isotonic PBS (pH 7.4). Glycosuria levels were assessed daily (Glu-kotest strips; Roche Diagnostic Systems, Mannheim, Germany) to monitorthe onset of diabetes, and experiments were performed with diabetic micefor which urine glucose levels were $3 g/dl for more than two consecutivedays. Saline-treated nondiabetic littermate mice served as controls.

Statistical analysis

One-way ANOVA followed by the Student-Newman-Keuls test was used toassess statistical difference among experimental groups. All of the resultsare expressed as mean 6 SEM. Statistical significance was assessed ac-cording to a p value ,0.05.

ResultsPAR2-induced inflammation is modulated by insulin treatment:reduced paw edema

Intraplantar injection of PPAR2-AP (SLIGRL-NH2) induced in-flammation that was characterized by edema and granulocyte in-filtration, as indicated by increases in paw thickness and MPOactivity, respectively (Fig. 1A, 1B). Mice that received a co-injection of insulin along with PAR2-AP also showed a significantincrease in paw thickness as compared with saline-treated mice,but the edema observed in the presence of insulin was significantly

lower at the 2, 3, 5, and 6 h time points, as compared with the micetreated with PAR2-AP alone (Fig. 1A, open circles). The increasein MPO activity induced by PAR2-AP injection (Fig. 1B, gray bar)was abolished when insulin was coadministered along with PAR2-AP (Fig. 1B, black bar). These anti-inflammatory effects were notcaused by insulin that had been treated by boiling before its co-administration with SLIGRL-NH2 (Fig. 1C, 1D for edema andMPO activity, respectively). The anti-inflammatory effect of in-sulin in the paw edema assay seen with PAR2-AP-induced in-flammation was not observed when inflammation was caused bythe intraplantar injection of carrageenan (Fig.1E, 1F).

Insulin treatment reduces PAR2-triggered leukocyte recruitment

Because the inflammatory action of PAR2 in the pawedemamodel isknown to involve the endothelium-dependent recruitment of in-flammatory leukocytes (7, 11), we evaluated the effects of insulin onPAR2-induced leukocyte rolling and adhesion onto the endothelialwall in vivo. Leukocyte trafficking in the microcirculation of in-testinal venules was monitored both before and at different timesafter the intraluminal administration of PAR2-AP alone or in com-bination with insulin. Intraluminal administration of PAR2-AP,SLIGRL-NH2, but not the reverse PAR2-inactive peptide, induceda significant increase in the area under the curve for the fluxof rollingleukocytes and their adherence to the vessel wall (Fig. 2A, 2B).

FIGURE 1. Effects of insulin on paw inflammation. Changes in pawthickness (A, C, E) were measured every hour for 6 h using an electroniccaliper and reported as a difference between the measurement at each timepoint and the basal measurement. MPO activity (B, D, F), a marker ofgranulocyte infiltration, was measured from the paws isolated 6 h after theinjection of indicated solutions. A–F, C57BL6 mice received intraplantarinjections of saline, PAR2-AP (SLIGRL-NH2, 50 mg per paw), insulin (150pmol), PAR2-AP (50 mg per paw) plus insulin (150 pmol) (native orboiled), carrageenan (200 mg), or carrageenan (200 mg) plus insulin (150pmol). #p, 0.05, significantly different from mice injected with saline; pp, 0.05, significantly different from mice injected with PAR2-AP alone.Values are shown as mean 6 SEM (n = 8).

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These prominent effects of PAR2-AP on leukocyte trafficking wereabolishedwhen insulinwas coadministered with PAR2-AP (Fig. 2A,2B). The data demonstrated that low doses of insulin were able toreverse the marked effects of PAR2 activation on increasing leuko-cyte recruitment to the venule.

Insulin reduces endothelial cell Ca2+ signaling in response toPAR2 activation

Because leukocyte trafficking along intestinal venules depends onendothelial function and the expression by both leukocytes and theendotheliumofadhesionmoleculesandbecausePAR2beenshowntoplay a role in the upregulation of ICAMs in endothelial cells (22),wehypothesized that insulinwould be able to affect PAR2-AP signalingin HMECs (23). To test this hypothesis, we evaluated the ability ofinsulin tomodulate PAR2-triggered calcium signaling in these cells.Treatment of HMECs with PAR2-AP elicited an elevation of in-tracellular calcium concentration in the HMECs (Fig. 3A, 3C). Theincrease in HMEC intracellular calcium concentration caused byPAR2-AP observed after PAR2 activation was attenuated signifi-cantly by preincubation of the cells with insulin for either 1 or 5 min(0.1, 1, and 10 nM; Fig. 3A). The inhibitory effects of insulin on thePAR2 agonist-induced calcium signal in endothelial cells appeared

to be receptor-specific because the elevation of HMEC intracellularcalcium concentration caused by activation of PAR1 with the PAR1-activating peptide (PAR1-AP) TFLLR-NH2 was not reduced bypretreatment with insulin (0.1 and 1 nM) but, in contrast, was in-creased slightly at the highest concentration of insulin used (10 nM)(Fig. 3B). Similarly, in vivo, PAR1-AP-induced edema was not re-duced by coinjection with insulin at all of the time points after in-traplantar injection, except at the first hour, where insulin treatmentslightly increased PAR1 agonist-induced edema (Fig. 3D).

Insulin reduces PAR2-stimulated Ca2+ signaling in primarycultures of DRG neurons

Our previous studies have shown that PAR2 agonist-induced pawedema involves a neurogenic mechanism, with activation of thereceptor on sensory neurons that in turn releases the vasoactiveneurokinin substance P and calcitonin gene-related peptide througha calcium-dependent mechanism (9). Given the ability of insulin toreduce the edema response (Fig. 1), we wondered whether insulinmight modulate the action of PAR2 in sensory neurons. To explorethis possibility, we evaluated the effect of insulin on PAR2-mediatedcalcium signaling in isolated sensory DRG cells. As describedpreviously (12, 18, 19), activation of PAR2 in sensory neurons

FIGURE 3. Effects of insulin on calcium flux in en-dothelial cells and onPAR1-AP-induced edema.Calciumflux in endothelial cells induced by PAR2-AP (SLIGRL-NH2, 100mM)with orwithout insulin (at 0.1, 1, or 10 nMwith 1 or 5 min preincubation) (A,C) and in the presenceor absence of the PI3K inhibitor (LY294002, 50 mM) orPKC inhibitor (GF 109203X, 100 mM) (C) or in endo-thelial cells stimulated by PAR1-AP (TFLLR-NH2, 100mM)with orwithout insulin treatment (0.1, 1, and 10 nM,1 min preincubation) (B). pp , 0.05; pppp , 0.005,significantly different from PAR2-AP treatment (A,C) orfrom PAR1-AP treatment (B).Values, representing theincrease in intracellular calcium concentration (E530), asa percentage of the increase caused by 4 mMA23187 (%A23187) are shown as mean6 SEM. Observations weremade for a minimum of 44 cells for each condition. D,Changes in paw thickness were measured every hour for6 h using an electronic caliper and reported as the dif-ferences between the measurements at each time pointand the basal measurement. C57BL6 mice received in-traplantar injections of PAR1-AP (TFLLR-NH2, 50 mgper paw), insulin (150 pmol), or PAR1-AP (50 mg perpaw) plus insulin (150 pmol). pp , 0.05, significantlydifferent frommice injectedwith PAR1-APalone.Valuesare shown as mean6 SEM (n = 8 in each group).

FIGURE 2. Effects of insulin on PAR2 agonist-induced changes in leukocyte flux and leukocyte adherence. Changes in the flux of rolling leukocytes (A)and the number of leukocytes adherent to the vessel wall (B) in mouse intestinal venules were measured by intravital microscopy under basal conditions andimmediately after intraluminal administration of vehicle (80% saline, 10% ethanol, and 10% Tween 80), PAR2-AP (SLIGRL-NH2, 100 mg), reverse peptide(LRGILS-NH2, 100 mg), or PAR2-AP plus insulin (100 mg and 150 pmol, respectively). Graphs represent the area under the curve of values measured overa 5 min time span at 15 min intervals, over a period of 80 min (a total of six readings). #p , 0.05, significantly different from vehicle administration; pp ,0.05, significantly different from PAR2-AP administration. Values are shown as the mean 6 SEM (n = 7–10).

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isolated frommouse DRGs led to rapid mobilization of intracellularcalcium (Fig. 4). Cultured DRGs preincubated with insulin (1 nM)for either 1 (data not shown) or 5min showed a substantial reductionin the elevation of intracellular calcium concentration caused byPAR2-AP (Fig. 4A). The proportion of cultured neurons that re-sponded to PAR2-AP with an increase in calcium signaling alsowasreduced significantly when the cultures were treated with insulin (1nM) (Fig. 4B). In contrast, when the cultured DRG neuronal cellswere preincubatedwith boiled insulin (1 nM), thePAR2-AP–inducedelevation of intracellular calcium concentrationwas not significantlydifferent from that of cells treated with PAR2-AP alone (data notshown).

Intracellular signaling pathways associatedwith insulin-inducedreduction of PAR2 signals

To assess the mechanism(s) by which insulin was able to diminishPAR2 calcium signaling at a cellular level, either HMECs or pri-mary afferent neurons were preincubated with either LY294002(an inhibitor of PI3K) or GF 109203X (a nonselective inhibitor ofPKC) prior to monitoring the effects of insulin on PAR2-inducedcalcium mobilization in those cell types. The PI3K and PKC in-hibitors on their own had no effect on the ability of PAR2-AP toelevate intracellular calcium concentration in either HMECs (Fig.3C) or DRGs (Fig. 4A). However, in the presence of either in-hibitor, the ability of insulin to reverse the PAR2-triggered in-crease in intracellular calcium concentration was blocked in boththe HMECs (Fig. 3C) and the cultured DRGs (Fig. 4A). This re-versal of the inhibitory action of insulin also was reflected in termsof the proportion of responding cells in the DRG cultures (Fig.4B). Thus, in both the HMECs and the cultured primary afferentneurons, the ability of insulin to diminish the cell response toPAR2-AP (elevated intracellular calcium concentration) appearedto depend on the PI3K and PKC signaling pathways.We have investigated in vivo whether PI3K or PKC inhibitors,

when injected into the mouse paw before the injection of PAR2

agonist, or the coinjection of PAR2 agonist and insulin can modifythe inflammatory parameters of edema and granulocyte infiltration(MPO activity). Pretreatment with the PI3K or PKC inhibitorsshowed no significant effect on PAR2-induced paw edema andincreased MPO activity (Fig. 5A, 5B). However, in mice that re-ceived a pretreatment with those inhibitors into the paw, the abilityof insulin to significantly reduce PAR2-induced paw edema, andincreased MPO activity was blocked (Fig. 5C, 5D). This resultsuggests that in vivo as well the ability of insulin to reduce PAR2

agonist-induced inflammation depends on the PI3K and PKCsignaling pathways.

Inflammatory response to PAR2-AP in the paw edema model isincreased in mice with type 1 diabetes

Because our data showed that insulin could diminish PAR2-in-duced inflammation, ostensibly via an effect on the endotheliumand sensory nerve components, we hypothesized that the in-

flammatory effect of PAR2 activation might be affected in thesetting of insulin-deficient diabetes. To test this hypothesis, weevaluated PAR2-triggered paw inflammation in mice rendereddiabetic via the immune destruction of pancreatic b cells. Inkeeping with the data in Fig. 1, nondiabetic RIP-HA transgenicmice that received an intraplantar injection of the PAR2 agonistSLIGRL-NH2 developed an inflammatory response characterizedby an increase in paw diameter (Fig. 6A, open squares) and aninflux of inflammatory cells (increased MPO activity; Fig. 6B,white bars). In support of our working hypothesis, the in-flammatory response of the diabetic RIP-HA mice to the intra-plantar administration of SLIGRL-NH2 was enhanced, in terms ofboth edema (Fig. 6A, solid squares) and increased neutrophil in-filtration (enhanced MPO activity; Fig. 6B, solid histograms).Thus, the insulin-deficient diabetic mice appeared to be moreprone to a PAR2-mediated inflammatory response. Acute admin-istration of insulin together with the PAR2 agonist in diabetic micewas able to inhibit PAR2 agonist-induced edema significantly, tothe same extent as in control mice (Fig. 6C). This result suggestsfurther that the increased inflammatory response associated withPAR2 activation is due to the lack of modulatory effects of insulinin the diabetic mice.

DiscussionThe main finding of our study was that insulin, at low doses, canattenuate PAR2-mediated inflammation. The concentrations ofinsulin used in vitro were in the physiological range (1 nM). It isdifficult to predict the absolute concentrations of insulin reachedin the experiments done in vivo, even though comparatively lowdoses were administered. This action of insulin to mitigate theacute inflammatory effects of PAR2 activation was reflected notonly by diminished edema and neutrophil infiltration(MPO ac-tivity) caused by the intraplantar administration of a PAR2 agonistbut also in terms of markedly attenuated PAR2-induced leukocytetrafficking and a reduction in intracellular calcium signalingtriggered by PAR2 in sensory neuronal cells (DRGs) and endo-thelial cells. Thus, insulin action can be seen to diminish the in-flammatory effects of PAR2 in an intact tissue by targetinga number of key sites: the endothelium (intracellular signaling andleukocyte adherence), the sensory nerve that can release vasoac-tive neuropeptides (calcium signaling), and vascular permeability(edema in vivo). All of these sites have been documented to playroles in PAR2-triggered neurogenic inflammation (9, 24). The timeframe of the PAR2-triggered effects on leukocyte trafficking (seenwithin 15‑20 min) suggests a rapid P-selectin–initiated processfollowed by interactions with constitutively expressed endothelialVLA4-VCAM1/LFA1-ICAM1 (25). This mechanism, reversed byinsulin, would merit further study.Our data provide a new link between the action of insulin and the

innate immune response system, a topic that has attracted con-siderable attention over the past decade (1, 26). To date, in the

FIGURE 4. Effects of insulin on the increase in in-tracellular calcium concentration (nanomolar) (A) andnumber of responding neurons (B) in primary afferentDRG cultures stimulated by PAR2-AP (SLIGRL-NH2,100mM) in the presence or absence of insulin (1 nM,with5minpreincubation), and in thepresenceorabsenceof thePI3K inhibitor (LY294002, 50 mM) or the PKC inhibitor(GF 109203X, 100 mM) (C). ppp , 0.01, significantlydifferent from PAR2-AP–treated neurons. Values, repre-senting the increase in calcium concentration (nano-molar), are shown as mean 6 SEM. Observations weremade for a minimum of 92 cells for each condition.

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setting of diabetes, the role of the innate immune system has beenviewed as diminishing insulin responses, due to inflammatorycytokine action (24). In contrast, our data now reveal that re-ciprocally insulin action can diminish a number of inflammatoryparameters triggered by an acute stimulation of the innate immunesystem itself, as typified by PAR2 activation. Because the PAR2

system appears to play a prominent role in inflammatory diseases,such as arthritis (27) and intestinal inflammatory disease (3, 4, 11,28), one can suggest that PAR2 activation also may enhance in-flammatory responses in type 1 and type 2 diabetes. It is thus ofmuch interest that insulin itself can be a negative regulator of theacute inflammatory action of PAR2. This action of insulin is inkeeping with its anti-inflammatory and cardioprotective effectsobserved in several animal models in vivo (1) and with the clinicaluse of rigorous insulin therapy in humans in an intensive caresetting (29). However, it can be noted that rigorous treatmentof patients with type 2 diabetes with insulin or metformin to op-timize blood glucose levels failed to reduce chronic inflammatory

biomarkers compared with those of control patients [e.g., high-sensitivity C-reactive protein (30)]. Thus, the data that we reportmay apply only to an acute and not a chronic inflammatory pro-cess in diabetic humans. This issue merits further study.The increased paw inflammation caused by SLIGRL-NH2 in

insulin-deficient diabetic mice was in complete accord with ourother data, because in these animals the anti-inflammatory actionof insulin would be diminished due to the reduced insulin levels.Indeed, local treatment of the diabetic mice with low doses ofinsulin, administered at the inflammatory site so as not to affectsystemic insulin levels, attenuated PAR2-induced inflammation.Notwithstanding, the enhanced inflammation triggered by PAR2 inthe diabetic animals also might have been influenced by hyper-glycemia, which also is recognized to have proinflammatory ef-fects (1). Thus, in a type 1 diabetes setting mimicked by themurine model that we used, one can suggest that increased in-flammation resulting from an acute activation of the innate im-mune response would be due to both hyperglycemia and a relative

FIGURE 5. Proinflammatory effects of intraplantarinjection with PAR2-AP (SLIGRL-NH2, 50 mg perpaw) or PAR2-AP plus insulin (150 pmol per paw) inmice that had received a pretreatment into the pawwith vehicle, the PI3K inhibitor (LY294002, 1 mg), orthe PKC inhibitor (GF 109203X, 1 mg). Changes inpaw thickness (A, C) and increases in paw MPO ac-tivity (B, D). Changes in paw thickness (electroniccaliper) were measured every hour over a 6 h timeperiod and are reported as the differences between themeasurements at each time point and the basal mea-surement at time 0. MPO activity, a marker of gran-ulocyte infiltration, was measured from paws taken 6 hafter the injection of the indicated solutions. Signifi-cant differences caused by the PI3K and PKC in-hibitors compared with mice injected with PAR2-AP incombination with insulin alone were observed: pp ,0.05; ppp , 0.01. Values are shown as means 6 SEM(n = 6 for diabetic groups and n = 4 for controls).

FIGURE 6. Proinflammatory effects of intra-plantar injection with PAR2-AP (SLIGRL-NH2, 50mg per paw), PAR2-AP plus insulin (150 pmol perpaw), or control peptide (LIGILS, 50 mg/paw) indiabetic or controlmice. Changes in paw thickness (A,C) and increases in pawMPO activity (B). Changes inpaw thickness (electronic caliper) were measuredevery hour over a 4 h time period and are reported asthe differences between the measurements at eachtime point and the basal measurement at time 0. MPOactivity, a marker of granulocyte infiltration, wasmeasured frompaws taken 6 h after the injection of theindicated solutions. #p, 0.05, significantly differentfrom mice of the corresponding group but injectedwith control peptide (LRGILS-NH2) inA or SLIGRL-NH2alone inC.pp,0.05, significantly different fromcontrol mice injected with PAR2-AP (SLIGRL-NH2)(A, C). Values are shown as means6 SEM (n = 6 fordiabetic groups and n = 4 for controls).

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reduction in the anti-inflammatory action of insulin. This increasein inflammation, due to protease activation of the innate immuneresponse, also may occur in a type 2 diabetes setting, where hy-perglycemia is accompanied by a reduced sensitivity to the actionof insulin. Thus, an interaction between the actions of insulin andthe inflammatory effects of endogenous activation of PAR2 maybe of importance in both type 1 and type 2 diabetes.The mechanism(s) whereby insulin was able to mitigate the in-

flammatory response to the PAR2 agonist appears complex, in-volving both the PI3K and PKC signaling pathways. Reciprocalinteractions between signaling by insulin and G protein-coupledreceptors have been known for over 30 y, best typified by the in-hibitory effect of insulin on norepinephrine, adrenocorticotropichormone, and glucagon-stimulated lipolysis in isolated adipocytes(31). More recently, it has been demonstrated that for certain Gprotein-coupled receptor-mediated increases in intracellular calciumconcentration (vasopressin, bradykinin, angiotensin II, neurotensin,and bombesin) insulin can enhance rather than inhibit signalingvia a mammalian target of rapamycin-dependent pathway (32, 33).However, our data demonstrating an insulin-mediated decreaserather than an increase in PAR2-triggered calcium signaling are inkeeping with the ability of insulin-like growth factor I and insulin toinduce phosphorylation and desensitization of the G protein-cou-pled a1B and a1D adrenergic receptors (34, 35). In those studies, asin ours, inhibitors of PI3K and PKC blocked receptor phosphory-lation (on serines in the a1D adrenoceptor) caused by the growthfactors. It can be noted that the concentrations of insulin used inother studies done in vitro (100 nM or higher) all have been wellabove the near-physiological concentrations that we employed forour cell culture work (∼1 nM). Significantly, our data showed thatinsulin treatment desensitized PAR2-mediated HMEC calcium sig-naling but did not decrease or even slightly increased calciumsignaling via PAR1 (Fig. 3). Thus, in summary, activation of theinsulin receptor may differentially affect signaling not only by thePARs but also by other G protein-coupled receptors. This ability ofinsulin to affect G protein-coupled receptor signaling in a differen-tial manner may explain why inflammation triggered by carra-geenan was not affected by insulin treatment, whereas PAR2-induced inflammation was reduced.To conclude, our data reveal an unexpected inhibitory interaction

between insulin and the acute inflammatory arm of the innateimmune response triggered by PAR2. The data bring into focusa potential pathophysiological role for serine proteases, such asthose of the coagulation pathway. In principle, those enzymes, viaPAR2, can trigger the innate inflammatory response in the settingof type 1 and type 2 diabetes. Thus, in the setting of the insulindeficiency of type 1 diabetes or the insulin signaling resistance intype 2 diabetes, the diminished impact of insulin signaling mayenhance the inflammatory effects of the innate immune response.

AcknowledgmentsWe are grateful to Dr. Alun Edwards for helpful discussions concerning theissue of diabetic neuropathic pain and insulin action.

DisclosuresThe authors have no financial conflicts of interest.

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Author : Amit SAXENA Titre : Etude de la pathogénicité des cellules T dans l'auto-immunité

du système nerveux central Directeur de Thèse : Pr. Roland Liblau Lieu et date de soutenance : 09 avril 2010, Toulouse

Multiple sclerosis (MS) is a complex demyelinating disease associated with chronic inflammation

of the central nervous system (CNS), axonal loss, and brain atrophy. The CD4+ and CD8+ T cells that are present in the demyelinating lesions are considered to mediate demyelination and axonal damage.

I developed an original mouse model combining selective expression of influenza hemagglutinin (HA) as a neo-self-Ag in oligodendrocytes with transgenic mice expressing a HA-specific TcR on CD8+ T cells. I demonstrate directly the potential of CD8+ T cells to induce oligodendrocyte death in-vivo, as a likely consequence of direct antigen-recognition.

I investigated the pathogenic traits of the CD4+ T cell response targeting the immunodominant epitope of myelin oligodendrocyte protein (MOG). To this end we obtained 2D2 TcR-transgenic C57BL/6 mice, which harbor a large population of MOG-specific CD4+ T cells. Strikingly when we crossed these 2D2 mice with MOG-deficient mice (MOG-/-), we discovered that the 2D2 TcR-transgenic mice developed spontaneous EAE regardless of the presence or absence of the target self-antigen MOG. Therefore we hypothesized, that the 2D2 TcR specific for MOG35-55 recognizes a second CNS antigen. Furthermore, we were able to reveal, that MOG35-55 peptide shares sequence homology with a stretch of neurofilament-medium (NF-M), a cytoskeletal protein expressed in neurons and axons. In addition, this epitope derived from NF-M shares 2D2 TcR contact residues with MOG35-55 when presented in the context of I-Ab. Surprisingly, NF-M15-35 peptide showed heteroclitic response when presented to 2D2 T cells. Using gene-deficient mice, we demonstrate that the co-recognition of MOG and NF-M by the 2D2 CD4+ T cells contributes individually to disease phenotype and severity.

We refer to this novel observation of self-mimicry, as ‘Cumulative autoimmunity’ where clonal autoimmune response targets two self-autoantigens in the same tissue, results in enhanced CNS tissue damage.

Key words : CNS autoimmunity, multiple sclerosis, autoreactive T cells, EAE.

Discipline : Immunology

INSERM U563, Centre de Physiopathologie de Toulouse Purpan Equipe auto-immunité et immunorégulation Pavillion Lefebvre, Bât B 4eme étage CHU Purpan BP 3028 Toulouse (FRANCE)