The Immunoregulatory Role of FGL2 as a Novel Effector Molecule of

Treg Cells

In vivo and in vitro studies

By

Itay Shalev

A thesis submitted in conformity with the requirements

for the degree of Doctor of Philosophy

Graduate Department of Immunology

University of Toronto

© Copyright by Itay Shalev 2009 The Immunoregulatory Role of FGL2 as a Novel Effector Molecule of

Treg Cells

Itay Shalev

Doctor of Philosophy, 2009

Department of Immunology, University of Toronto

Abstract

Background and Aim: CD4+CD25+ regulatory T (Treg) cells are important in the maintenance of self-tolerance, regulation of T cell homeostasis, prevention of allograft rejection and in the modulation of immune responses to pathogens. Several groups have recently reported an increased expression of fgl2 in Treg cells by microarray analysis.

FGL2, a member of the fibrinogen-like family, was previously shown to act as an immunomodulator by inhibiting DC maturation and T cell proliferation. Based on these findings, the immunoregulatory role of FGL2 as a novel effector molecule of Treg cells was investigated. Results: In agreement with previous studies, high levels of fgl2

transcripts were detected in Treg cells by real-time PCR. In fgl2-/- mice, an increased

number and percentage of Treg cells were found with a greater expression of Foxp3

compared with fgl2+/+ Treg cells; however, the suppressive activity of fgl2-/- Treg cells

was significantly impaired. Antibody to FGL2 completely inhibited the activity of fgl2+/+

Treg cells in vitro. Consistent with FGL2 contribution to Treg cell activity, targeted

deletion of the gene led to an increased immune reactivity of DC, T cells and B cells, and

the manifestation of glomerular autoimmunity in aged fgl2-/- mice. The importance of

FGL2 as an effector of Treg cells was also demonstrated in MHV-3-induced fulminant

hepatitis. Uninfected susceptible BALB/cJ mice had increased numbers of Treg cells and

ii expression of FGL2 compared to uninfected resistant A/J mice. Following MHV-3

infection, plasma levels of FGL2 in BALB/cJ mice were significantly increased,

correlating with an increased percentage of Treg cells. Treatment with anti-FGL2

antibody completely inhibited Treg cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury and mortality. Adoptive transfer of fgl2+/+ Treg cells

into resistant fgl2-/- mice increased their mortality following MHV-3 infection. Finally,

FGL2 treatment led to prolonged skin graft survival in a murine allotransplant model.

The suppressive activity of FGL2 was mediated through binding to the inhibitory

FcγRIIB receptor expressed on APCs, resulting in inhibition of DC maturation and

induction of B cell apoptosis. Conclusion: These studies indicate that FGL2 plays an

important immunoregulatory role as an effector cytokine of Treg cells; targeting FGL2

may provide a novel therapeutic approach for the treatment of patients with viral hepatitis,

autoimmunity and transplant rejection.

iii Acknowledgments

I wish to sincerely thank my supervisor Dr. Gary Levy for giving me this great

opportunity to work as a PhD student in his lab. His support and guidance helped me to

overcome the many challenges and barriers I faced as a student and researcher. While

providing me with the freedom to work independently, Dr. Levy has inspired me to

achieve my scientific goals and become a better scientist. I would like to acknowledge

my supervisory committee members, Dr. Michael Ratcliffe and Dr. Li Zhang, and the

many collaborators: Dr. David Clark, Dr. M James Phillips, Dr. Myron Cybulsky, Dr.

Jennifer Gommerman, Dr. Mark Cattral, Dr. Oyedele Adeyi, Dr. David Grant, Dr. Ian

McGilvray and Dr. Reginald Gorczynski, for their great contribution to my thesis work.

I would also like to thank all the past and present members of the Levy laboratory

and management that assisted me in one way or another, with special thanks to Andre

Siegel, Charmaine Beal, Anna Kushnir, Camila Balgobin, Dr. Wei He, Dr. Ming Feng

Liu, Dr. Hao Liu, Dr. Nazia Selzner, Justin Manuel, Cheryl Koscik, Yi Zhu, Jianhua

Zhang, Xue-Zhong Ma, Asif Maknojia, Kit Man Wong and Agata Bartczak.

I wish to acknowledge the Training Program in Regenerative Medicine, Canadian

Institutes for Health Research and the Heart and Stroke Foundation of Canada for their

financial support of this thesis and to the Department of Immunology at the University of

Toronto, who made this thesis possible.

To my parents, Rina and Baruch Shalev, and the rest of my family including my

sister and brother-in-law, Ofra and Eytan Saban-Shalev, my brother and sister-in-law, Zvi

Shalev and Victoria Blond and my wife Myong Suk Choi, I would like to thank deeply

for all their love and encouragement.

iv Table of Contents

Abstract ii

Acknowledgements iv

Table of Contents v

List of Tables x

List of Figures xi

List of Abbreviation xiv

List of Publications xvi

Chapter 1: Introduction

1.1. Regulation of the immune system 1

1.1.1. Mechanisms of immunoregulation 3

1.1.1.1. Central tolerance mechanisms 3

1.1.1.1.1. Negative selection 3

1.1.1.1.2. Receptor editing 5

1.1.1.2. Peripheral tolerance mechanisms 6

1.1.1.2.1. Cell-intrinsic mechanisms 6

1.1.1.2.1.1. Ignorance 6

1.1.1.2.1.2. Anergy 7

1.1.1.2.1.3. Phenotypic skewing 11

1.1.1.2.1.4. Activation-induced cell death (AICD) 12

1.1.1.2.2. Cell-extrinsic mechanisms 16

1.1.1.2.2.1. Tolerogenic dendritic cells 16

1.1.1.2.2.2. Regulatory T cells 19

v 1.1.1.2.2.2.1. Regulatory T cells type 1 (Tr1) 21

1.1.1.2.2.2.2. T helper 3 (Th3) 23

1.1.1.2.2.2.3. CD8+ regulatory T cells 25

1.1.1.2.2.2.4. Double negative T cells 29

1.1.1.2.2.2.5. γδ regulatory T cells 31

1.1.1.2.2.2.6. Natural killer T cells 33

1.1.1.2.2.2.7. CD4+CD25+ regulatory T (Treg) cells 34

1.1.1.2.2.2.7.1. Mode of Treg cell action 40

1.2. Fibrinogen-like protein 2 (fgl2/FGL2) 45

1.2.1. Identification of fgl2 45

1.2.2. Fgl2 gene and the encoded protein 46

1.2.3. FGL2 as a regulator of immune responses 49

1.2.4. FGL2 as a marker of regulatory T cells 50

1.3. Hypothesis 51

1.4. Objectives 52

1.5. Goal 53

Chapter 2-4: Results

2 Chapter 2: Targeted Deletion of fgl2 Leads to Impaired Treg Activity and Development of Glomerulonephritis 54

2.1. Summary 54

2.2. Introduction 55

2.3. Materials and Methods 57

2.4. Results 64

2.4.1. Gross morphology, histology and mass of fgl2-/- mice 64

vi 2.4.2. Constitutive FGL2 expression 64

2.4.3. Increased reactivity of B and T cells from fgl2-/- mice 64

2.4.4. Increased numbers and reactivity of DC from fgl2-/- mice 67

2.4.5. Normal spleen architecture and formation of germinal centers (GC) was observed in fgl2 -/- mice 70

2.4.6. Development of autoimmune glomerulonephritis in fgl2-/- mice 70

2.4.7. Fgl2+/+ recipients reconstituted with fgl2-/- BM exhibit the phenotype of fgl2-/- mice 74

2.4.8. Increased expression of fgl2 mRNA in CD4+CD25+ T cells 76

2.4.9. Increased percentage and absolute numbers of Treg cells in fgl2-/- mice compared to fgl2+/+ mice 77

2.4.10. Increased expression of Foxp3 in Treg cells of fgl2-/- mice compared to fgl2+/+ mice 78

2.4.11. Treg cells isolated from fgl2-/- mice have impaired suppressive activity 79

2.4.12. Monoclonal antibody to FGL2 completely blocks the suppressive activity of wild-type Treg cells 81

2.5. Discussion 82

2.6. Contributions 87

3 Chapter 3: The Novel Treg Effector Molecule FGL2 contributes to the Outcome of Murine Fulminant Viral Hepatitis 88

3.1. Summary 88

3.2. Introduction 89

3.3. Materials and Methods 91

3.4. Results 96

3.4.1. Increased percentage and numbers of Treg cells in uninfected BALB/cJ mice compared to A/J mice 96

vii 3.4.2. Increased levels of FGL2 by Treg cells in the plasma of uninfected BALB/cJ mice compared to A/J mice 100

3.4.3. Increased percentage of Treg cells in BALB/cJ mice compared to A/J mice following MHV-3 infection 102

3.4.4. Increased Treg cell infiltration in livers of BALB/cJ mice following viral infection 104

3.4.5. Levels of FGL2 in the plasma of BALB/cJ and A/J mice following viral infection 106

3.4.6. FGL2 is an effector of Treg cell function 107

3.4.7. Anti-FGL2 antibody treatment prolongs the survival of BALB/cJ mice post-MHV-3 infection 108

3.4.8. Adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increases mortality to MHV-3 infection 111

3.5. Discussion 113

3.6. Contributions 118

4 Chapter 4: FGL2 Immunosuppressive Activity in an Allotransplant Model and its Mechanism of suppression 119

4.1. Summary 119

4.2. Materials and Methods 120

4.3. Results 123

4.3.1. FGL2 prolongs the survival of fully mismatched skin allografts 123

4.3.2. FGL2 treatment improves the histology of skin allografts 125

4.3.3. Lymphocytes from FGL2-treated recipients show inhibition of proliferation in response to donor alloantigens compared with untreated recipients 126

4.3.4. FGL2 binding to the inhibitory FcγRIIB receptor leads to inhibition in the maturation of BM-DC 128

4.3.5. FGL2 induces apoptosis in B cells through binding to the inhibitory FcγRIIB receptor 130

viii

4.3.6. FGL2 treatment fails to prolong skin allograft survival in FcγRIIB-/- recipient mice 132

4.3.7. A proposed model of FGL2 immunoregulatory activities 133

4.4. Contributions 136

Chapter 5: Discussion and Conclusions 137

References 152

ix List of Tables

Table 1-1. Treg-associated molecules 44

Table 4-1. Survival of fully mismatched skin grafts with different treatments in FcγRIIβ-/- and FcγRIIβ+/+ recipient mice 135

x List of Figures

Figure 1-1. Central and peripheral tolerance mechanisms for the establishment and maintenance of self-tolerance and immune homeostasis 2

Figure 1-2. Natural and adaptive regulatory T cells 20

Figure 1-3. Reduction or increase in Treg cell number/activity is implicated in various pathologies 39

Figure 1-4. Schematic view of the two forms of FGL2 with their respective function as was previously reported 46

Figure 1-5. Secondary structure prediction of FGL2 protein by SWISS-MODEL 48

Figure 2-1. Increased reactivity of B and T cells from fgl2-/- mice 66

Figure 2-2. Increased numbers and reactivity of DC from fgl2-/- mice 69

Figure 2-3. Kidney defect observed in 7- to 12-mo-old fgl2-/- mice 72

Figure 2-4. The evolution of autoimmune glomerulonephritis 73

Figure 2-5. Fgl2+/+ recipients reconstituted with fgl2-/- BM exhibit the phenotype of fgl2-/- mice 75

Figure 2-6. Increased expression of fgl2 mRNA in CD4+CD25+ T cells 76

Figure 2-7. Increased percentage and absolute numbers of Treg cells in fgl2-/- mice compared to fgl2+/+ mice 77

Figure 2-8. Increased expression of Foxp3 in Treg cells of fgl2-/- mice compared to fgl2+/+ mice 78

Figure 2-9. Treg cells isolated from fgl2-/- mice have impaired suppressive activity 80

Figure 2-10. Monoclonal antibody to FGL2 completely blocks the suppressive activity of wild-type Treg cells 81

Figure 3-1(A-B). Increased percentage of Treg cells in uninfected BALB/cJ mice compared to A/J mice 97

Figure 3-1(C). Increased numbers of Treg cells in uninfected BALB/cJ mice compared to A/J mice 99

Figure 3-2. Increased levels of FGL2 by Treg cells in the plasma of uninfected BALB/cJ

xi mice compared to A/J mice 101

Figure 3-3(A-B). Increased percentage of Treg cells in BALB/cJ mice compared to A/J mice following MHV-3 infection 102

Figure 3-3(C-F). Increased Treg cell infiltration in livers of BALB/cJ mice following viral infection 105

Figure 3-4. Levels of FGL2 in the plasma of BALB/cJ and A/J mice following viral infection 106

Figure 3-5. FGL2 is an effector of Treg cell function 107

Figure 3-6. Effect of monoclonal anti-FGL2 antibody treatment on the course of MHV-3 infection 109

Figure 3-7. The effect of monoclonal anti-FGL2 antibody on liver histology following MHV-3 infection 110

Figure 3-8. Adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increases mortality to MHV-3 infection 112

Figure 4-1. FGL2 prolongs the survival of fully mismatched skin allografts 124

Figure 4-2. FGL2 treatment improves the histology of skin allografts 125

Figure 4-3. Lymphocytes from FGL2-treated recipients show inhibition of proliferation in response to donor alloantigens compared with untreated recipients 127

Figure 4-4. FGL2 binding to the inhibitory FcγRIIB receptor leads to inhibition in the maturation of BM-DC 129

Figure 4-5. FGL2 induces apoptosis in B cells through binding to the inhibitory FcγRIIB receptor 131

Figure 4-6. FGL2 treatment fails to prolong skin allograft survival in FcγRIIB-/- recipient mice 132

Figure 4-7. A proposed model of FGL2 immunoregulatory activities 134

Figure 5-1. Levels of FGL2, produced by Treg cells, contribute to the outcome of the immune response 145

Figure 5-2. Increased plasma levels of FGL2 and staining of Foxp3+FGL2+ Treg cells in explanted liver of patients with chronic HCV infection 146

xii Figure 5-3. Increased expression of fgl2 by both primary and cloned DN Treg cells 150

xiii List of Abbreviation

Ab Antibody ANOVA Analysis of variance APC Antigen presenting cells ATCC American Type Culture Collection BcR; TcR B or T cell receptor BM Bone marrow BM-DC Bone marrow-derived dendritic cells cDNA; DNA Complementary Deoxyribonucleic Acid; Deoxyribonucleic Acid CHO Chinese Hamster Ovary CsA Cyclosporine A CTL Cytotoxic T lymphocytes d Days DC Dendritic cells EC Endothelial cells ELISA Enzyme-linked immunosorbent assay FACS Fluorescence Activated Cell Sorting FBS Fetal bovine serum Fc Constant region fragment FcR Fc receptor FH Fulminant virus hepatitis FITC Fluorescein isothiocyanate FGL2 Fibrinogen-Like Protein 2 FRED Fibrinogen related domain FS Forward scatter g grams (includes pico [p], nano[n], micro [μ], milli [m], kilo [k]) GAPDH Glyceraldehyde 3-phosphate dehydrogenase GC Germinal centre GM-CSF Granulocyte macrophage colony stimulating factor h Hours HBV Hepatitis B virus HCV Hepatitis C virus H&E Hematoxylin and Eosin HRP Horse radish peroxidase IFN Interferon IHC Immunohistochemistry Ig Immunoglobulin IL Interleukin IM Intramuscular Inc. Incorporated IP Intraperitoneal IV Intravenous Kb Kilobase kDa Kilodalton L Liters (includes micro [μ], milli [m], kilo [k])

xiv LPS Lipopolysaccharide M Molar (includes pico [p], nano[n], micro [μ], milli [m]) MACS Magnetic Activated Cell Sorting MFI Mean fluorescence intensity MHC Major histocompatibility complex MHV-3 Murine Hepatitis Virus Strain 3 min Minutes MLR Mixed Lymphocyte Reaction mRNA; RNA Message Ribonucleic acid; Ribonucleic acid NF-kB Nuclear factor-kB PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline PCR; RT-PCR Polymerase Chain Reaction; reverse transcriptase PCR PE Phycoerythrin POD Post-Operative Day RBC Red blood bell rpm Revolutions per minute RT Room temperature SA Strepavidin SD Standard deviation SDS Sodium Dodecyl Sulfate sec Seconds SEM Standard error of the mean SS Side Scatter S-S Di-sulfide bond TGF-β Transforming growth factor beta Th Helper T cell TLR Toll-like receptor TMB 3, 3, 5, 5-Tetramethylbenzidine Treg CD4+CD25+Foxp3+ Regulatory T cells U Units

xv List of Publications

Thesis-related:

Itay Shalev, Kit Man Wong, Katharina Foerster, Yi Zhu, Cecilia Chan, Asif Maknojia, Jianhua Zhang, Xue-Zhong Ma, Xiao Chun Yang, Julia Fang Gao, Hao Liu, Nazia Selzner, David A Clark, Oyedele Adeyi, M. J. Phillips, Reginald R. Gorczynski, David Grant, Ian McGilvray and Gary Levy. The novel CD4+CD25+ regulatory T cell effector molecule fibrinogen-like protein 2 contributes to the outcome of murine fulminant viral hepatitis. Hepatology 2009;49(2):387-97. (Chapter 3 in this thesis)

Hao R. Liu*, Itay Shalev*, Justin Manuel, Wei He, Elisa Leung, Jennifer Crookshank, Ming F. Liu, Jun Diao, Mark Cattral, David A. Clark, , David E. Isenman, Reginald M. Gorczynski, David R. Grant, Li Zhang, Melville J. Phillips, Myron I. Cybulsky and Gary Levy. The FGL2-FcgammaRIIB pathway: a novel mechanism leading to immunosuppression. Eur J Immunol 2008;38(11):3114-26. (Chapter 4 in this thesis)

* These authors contributed equally to this work

Itay Shalev, Hao Liu, Cheryl Koscik, Agata Bartczak, Mojib Javadi, Kit Man Wong, Asif Maknojia, We He, Ming Feng Liu, Jun Diao, Erin Winter, Justin Manuel, Doug McCarthy, Mark Cattral, Jennifer Gommerman, David A Clark, M. J. Phillips, Reginald R. Gorczynski, Li Zhang, Greg Downey, David Grant, Myron I. Cybulsky and Gary Levy. Targeted deletion of fgl2 leads to impaired regulatory T cell activity and development of autoimmune glomerulonephritis. J Immunol 2008;180(1):249-60. (Chapter 2 and 4 in this thesis)

Itay Shalev and Gary Levy. The immunoregulatory role of FGL2 as a novel effector molecule of Treg cells. Invited review (manuscript in preparation).

Mendicino, M., M. Liu, A. Ghanekar, W. He, C. Koscik, I. Shalev, M. Javadi, J. Turnbull, W. Chen, L. Fung, S. Sakamoto, P. Marsden, T. K. Waddell, M. J. Phillips, R. Gorczynski, G. A. Levy, and D. Grant. Targeted deletion of Fgl-2/fibroleukin in the donor modulates immunologic response and acute vascular rejection in cardiac xenografts. Circulation 2005;112:248.

Yi Zhu, Katharina Foerster, Nazia Selzner, Itay Shalev, Oyedele Adeyi, Khattar Ramzi, Gary A. Levy. Development of an ELISA bioassay to measure FGL2 levels in mouse and human plasma. Journal of Immunological Methods (manuscript in preparation).

Thesis-non-related:

Liu, M., M. Mendicino, Q. Ning, A. Ghanekar, W. He, I. McGilvray, I. Shalev, D. Pivato, D. A. Clark, M. J. Phillips, and G. A. Levy. Cytokine-induced hepatic apoptosis is dependent on FGL2/fibroleukin: the role of Sp1/Sp3 and STAT1/PU.1 composite cis elements. J Immunol 2006;176:7028.

xvi De, A. N., E. Baig, X. Ma, J. Zhang, W. He, A. Rowe, M. Habal, M. Liu, I. Shalev, G. P. Downey, R. Gorczynski, J. Butany, J. Leibowitz, S. R. Weiss, I. D. McGilvray, M. J. Phillips, E. N. Fish, and G. A. Levy. Murine hepatitis virus strain 1 produces a clinically relevant model of severe acute respiratory syndrome in A/J mice. J Virol 2006;80:10382.

DeAlbuquerque, N., E. Baig, M. Xuezhong, I. Shalev, M. J. Phillips, M. Habal, J. Leibowitz, I. McGilvray, J. Butany, E. Fish, and G. Levy. Murine hepatitis virus strain 1 as a model for severe acute respiratory distress syndrome (SARS). Adv Exp Med Biol 2006;581:373.

xvii Chapter 1: Introduction

1.1. Regulation of the immune system

The immune system has evolved to protect the host from a wide spectrum of

invading pathogens, while maintaining tolerance to self and controlling excessive immune responses. T and B cells of the adaptive immune system are the main mediators of both protective and harmful immune responses, with the ability to recognize and attack numerous pathogenic microorganisms. Recognition of foreign antigens is achieved through antigen-specific receptors, T cell receptor (TCR) and B cell receptor (BCR) expressed by T and B cells, respectively. The antigen specificity of TCR and BCR is a result of random recombination of the many V, D and J genes that encode the antigen binding sites of these receptors (1). This process can produce more than 109 distinct

receptors that are able to provide an efficient surveillance and elimination of many

disease-causing agents, but potentially may also contain receptors that can react against

self-proteins, leading to the development of autoimmune diseases (2).

In addition to autoimmune attack by self-reactive T and B cell clones, an

uncontrolled immune response of lymphocytes directed against pathogenic agents or

tumor cells can cause collateral damage to normal tissues located in close proximity to

the site of the immune attack. The inflammatory cytokines, which are released during this

response, play a major role in bystander injury (2). To prevent and inhibit these immune-

mediated diseases, a complex network of central and peripheral tolerance mechanisms

exist (Figure 1-1) (3). Failure or breakdown of these tolerance mechanisms, due to

genetic and environmental factors, results in autoimmunity and fatal injuries to the host.

All these regulatory mechanisms will be reviewed here in detail.

1

Figure 1-1. Central and peripheral tolerance mechanisms for the establishment and maintenance of self-tolerance and immune homeostasis. Central tolerance mechanisms that occur in the central lymphoid organs (bone-marrow for B cells and thymus for T cells) include negative selection and receptor editing of lymphocytes bearing self-reactive receptors. Failure of receptor editing leads to deletion of autoreactive lymphocytes by negative selection. Peripheral tolerance mechanisms exist both to control self-reactive lymphocytes that escape central tolerance and to maintain immune homeostasis. Peripheral tolerance can be achieved directly by pathways intrinsic to antigen activation of pathogenic lymphocytes or indirectly via the activity of additional cells (extrinsic pathways). Extrinsic pathways may also affect intrinsic pathways through the secretion of various anti-inflammatory cytokines.

2 1.1.1. Mechanisms of immunoregulation

1.1.1.1. Central tolerance mechanisms

1.1.1.1.1. Negative selection

Central tolerance mechanisms include the negative selection of immature

lymphocytes that are reactive to self-antigens in the primary lymphoid organs (the thymus for T cells and the bone marrow for B cells) (4). Hematopoietic precursors migrate from the bone-marrow to the thymus, where they develop into mature T cells

through several stages. At first, CD4-CD8- double negative (DN) thymocytes undergo a

proliferative expansion and differentiation, resulting in the main population of

CD4+CD8+ double positive (DP) thymocytes that express an unselected repertoire of

TCR. The fate of DP thymocytes is then determined based on their TCR interaction with

self-peptide bound to MHC molecules expressed by epithelial cells and other APCs in the

thymus (5). The majority of DP thymocytes die by neglect due to the failure of their TCR

to engage peptide-MHC ligands. DP thymocytes bearing TCR with low-affinity to MHC-

peptide complexes survive (positive selection), whereas DP thymocytes with high-

affinity receptors to self-antigens undergo apoptosis (negative selection). The selective

process of DP thymocytes ultimately gives rise to CD4+ or CD8+ single-positive mature T

cells, which are self-MHC restricted and self-tolerant (5).

As a consequence of negative selection, immature T cells become tolerant to all

self-antigens presented in the thymus. These self-antigens include those that are

ubiquitously expressed throughout the body and those with a limited expression in only a

few tissues known as tissue-specific antigens (TSAs) (6). Thymic expression of TSA is

primarily expressed by medullary thymic epithelial cells (mTECs) and to some degree in

3 cortical thymic epithelial cells (cTECs). The expression of TSAs by mTECs in the

thymus is critical in the induction of T cell tolerance to these tissue-restricted antigens.

Specific TSAs not expressed in the thymus were targets for autoimmunity mediated by

autoreactive T cells that escaped negative selection (6). The autoimmune regulator

protein (AIRE) has an important role in the expression of TSAs in thymus (7). AIRE-

deficient mice display decreased expression of many tissue specific-genes in mTEC, leading to the development of autoimmune diseases in multiple organs. Similarly to

AIRE-deficient mice, human patients with mutation in the AIRE gene suffer from multiorgan autoimmune syndrome called polyglandular syndrome type 1 (7). Recent studies suggest that AIRE might regulate thymic epithelial differentiation, and therefore indirectly control expression of TSAs in different thymic stromal cell types (7).

The molecular events that occur in thymocytes during negative selection are still

not completely understood, however many important signaling molecules already have

been identified and studied. Engagement of the TCR with a high-affinity peptide-MHC

complex triggers a negative signal pathway mediated by the activation of the serine-

threonine kinase MINK (misshapen-Nck-interactine kinase-related kinase). MINK affects

downstream activation of kinases, including JNK, p38 and ERK. These activated kinases

induce the expression of apoptotic effector molecules, such as Bim, Nur77 and Nor-1,

which are required for apoptosis of thymocytes in response to negative selection signals

(7). Casitas B cell lymphoma (Cbl) proteins negatively regulate TCR signaling, as

inactivation of the Cbl family members, c-Cbl and Cbl-b, results in enhanced negative

selection (8).

4 The mechanism by which TCR interaction with MHC-peptide complexes differentiates between negative and positive selection signals is unclear. Studies using ligands with different affinities for TCR demonstrate that a small increase in binding affinity for TCR results in significant intracellular changes in the localization and activation of signaling molecules, Ras and mitogen-activated protein kinase (MAPK) signaling mediators, and the induction of negative selection (9). Consistent with this, extracellular signal-related kinase 1/2 (ERK1/2) required for positive selection is localized in the cytoplasm of positively selected cells, whereas in negatively selected cells it is found at the cell membrane (7). Furthermore, changes in the conformation of

TCR are observed in thymocytes undergoing negative selection compared to those that are positively selected (10). These conformational changes may lead to the activation of

MINK and other MAP kinase pathways (7). Thus, small changes in the TCR-ligand binding affinities reflected in conformational changes may lead to distinct pathways, allowing for the discrimination of positive and negative selection in the thymus.

1.1.1.1.2. Receptor editing

Mechanisms of central tolerance also include antigen receptor editing during the development of immature lymphocytes in central lymphoid organs. Receptor editing has been described as an alternative and complementary mechanism for negative selection in the maintenance of self-tolerance (11). While in negative selection, self-reactive lymphocytes are deleted in response to self antigens, in receptor editing, these lymphocytes undergo genetic correction of their receptors (11). During this process, autoreactive lymphocytes that bind to self maintain or increase RAG protein expression, which promotes a secondary VDJ recombination. As a result, autoreactive receptors

5 (TCR for T cells or BCR for B cells) are replaced with new receptors that are tested for

self-reactivity (12,13). Receptors that bind with low affinity to MHC-peptide ligands

downregulate RAG protein expression, which signals developing lymphocytes to

progress in their development (12).

1.1.1.2. Peripheral tolerance mechanisms

Both negative selection and receptor editing are critical in the control of the

development of autoreactive lymphocytes; however, these regulatory mechanisms are not

complete. As a result, a fraction of autoreactive lymphocytes escape deletion or receptor

editing and exit to the periphery. Several peripheral tolerance mechanisms coevolved to

control these autoreactive cells and maintain immune homeostasis. These peripheral

mechanisms can be classified into cell-intrinsic mechanisms, which include ignorance,

anergy, phenotypic skewing and apoptosis by activation-induced cell death (AICD), and

cell-extrinsic mechanisms consisting of regulation by APCs and regulatory T cells (14).

1.1.1.2.1. Cell-intrinsic mechanisms

1.1.1.2.1.1. Ignorance

During the activation of T cells, the engagement of TCR with MHC-peptide complexes expressed on APCs is a primary event. The activation of T cells depends on the avidity and duration of the TCR binding with its cognate MHC-peptide. Prolonged

and avid binding of TCR with its cognate antigen favors the activation of T cells. On the other hand, a weak and short encounter of the antigen will not be sufficient to reach the

threshold required for T cell activation (15). As a consequence, self-reactive T cells that

bind self-antigens with low affinity may not be activated. Similarly, self-antigens

presented at low concentration or self-antigens sequestered in inaccessible sites will not

6 trigger activation of autoreactive T cells (3). Therefore, TCR activation threshold serves

as a means of regulating the activation of self-reactive T cells.

1.1.1.2.1.2. Anergy

Encounter of T cells with self-proteins may induce a state of functional

inactivation known as anergy, which is characterized by the inability to respond to

specific antigens by proliferation, cytokine secretion and differentiation. Activation of T

cells requires the engagement of TCR with its cognate MHC-peptide (signal 1) and

costimulation provided by accessory molecules on the membrane of APCs (signal 2). T

cells become anergic when TCR is triggered in the absence of costimulation signals (16).

In the presence of danger signals, such as microbial molecules, APCs present antigens

with the proper costimulatory signals to fully activate antigen-specific T cells. However,

in a non-inflammatory environment lacking pathogen-derived-signals, APCs present the

MHC-antigen without sufficient costimulatory molecules resulting in the induction of T

cell anergy and tolerance (17). The interaction of autoreactive T cells with self-antigens,

which usually occurs in the absence of these danger signals, will therefore favor the

induction of T cell anergy.

Ligation of inhibitory accessory molecules expressed on activated T cells may

also cause anergy. Engagement of these inhibitory receptors with their putative ligands,

the B7 family members on APCs, triggers a negative signal that blocks T cell activation

and may also lead to a hyporesponsiveness state of T cells (18). This negative regulation mediated by the inhibitory receptors has been implicated in the control of both self-

reactive T cells and homeostasis of T cell-mediated immune responses (18). Three types of inhibitory receptors have been identified so far, which include cytotoxic T-lymphocyte

7 associated antigen (CTLA-4), programmed cell death 1 molecule (PD-1) and B- and T-

lymphocyte-attenuator (BTLA/CD272).

The first major coinhibitory receptor of the B7-CD28 family that was identified is

the CTLA-4 molecule. Binding of costimulatory molecules B7-1 (CD80) and B7-2

(CD86) on APCs with costimulatory CD28 on T cells provides the second positive signal

required for the activation of T cells; whereas, CD80/CD86 interaction with the

coinhibitory CTLA-4 on T cells downregulates T cell activation and may also cause

anergy (18). The importance of CTLA-4 as a negative regulator of T cell activation has been highlighted by the phenotype of CTLA-4-deficient mice that exhibit a lethal autoimmune lymphoproliferative disease. In addition, an allelic version of a non coding

3’ region of CTLA-4 contributes to a variety of autoimmune disorders in humans.

Consistent with this, blockade of the receptor leads to increased immune responses (18).

PD-1 is another important coinhibitory receptor that is highly expressed on

activated and anergic T cells (19,20). Engagement of PD-1 with its ligands B7H1 and

B7DC on APCs leads to negative regulation of T cell activation. This inhibition is

mediated through the phosphorylation of immunoreceptor tyrosine-based inhibition motif

(ITIM) in the cytoplasmic region of PD-1, and the subsequent recruitment of the src

homology 2-domain-containing tyrosine phosphatase 2 (SHP-2) that downmodulates

activation signals (21,22). Similar to CTLA-4-deficient mice, loss of PD-1 results in a

breakdown of peripheral tolerance and multiple autoimmune disorders (23).

Polymorphisms of PD-1 are associated with susceptibility to systemic lupus

erythematosus and type I diabetes (24,25). In agreement with these data, blockade of the

PD-1 pathway can augment anti-viral and anti-tumor immunity (26,27). Finally, the fact

8 that the ligand of PD-1 is expressed in nonlymphoid tissues, such as heart and lung,

suggests that the negative regulation of immune responses at sites of inflammation could

be mediated directly by peripheral tissues. This may allow further control of excessive

immune responses and maintenance of self-tolerance (28).

The B and T lymphocyte attenuator (BTLA) was recently identified as the third lymphocyte inhibitory receptor. BTLA is not expressed by naive T cells, but it is induced during activation and remains expressed on Th1 but not Th2 cells (29). Coligation of

BTLA results in downregulation of T-cell proliferation and IL-2 production through an

ITIM-dependent recruitment of the tyrosine phosphatases SHP-1 and SHP-2. T cells from

BTLA-deficient mice exhibit enhanced proliferation in response to antigenic stimulation, and B cells from these mice have increased reactivity to stimulation with anti-IgM antibody. BTLA-deficient mice display increased specific antibody responses and susceptibility to experimental autoimmune encephalomyelitis (29). Consistent with the phenotype of BTLA-deficient mice, treatment of mice with the novel B7 family member

B7-H4, which was suggested to interact with BTLA, leads to inhibition of T-cell responses to antigens (30).

Transcriptional mechanisms regulate the signal transduction events that lead to

anergy induction rather than T cell activation (31). During the full activation of T cells,

calcium/nuclear factor of activated T cells (NFAT) signaling and Ras/MAPK signaling

pathway are fully triggered. As a consequence of these signaling pathways, NFAT

translocates to the nucleus where it cooperates with activator protein 1 (AP1) complex to

induce the expression of T-cell activation-associated genes (32). However, in anergic T

cells, TCR ligation without costimulation leads to excessive activation of calcium/NFAT

9 signaling in the absence of complete activation of Ras/MAPK pathway. This results in

upregulated expression of negative regulatory mediators that participate in the

establishment of T cell anergy (33). These negative regulators include kinases,

phosphatases, proteases and transcriptional repressors that work in concert to inhibit

TCR-CD28 signaling and impose anergy. Among the calcium-induced anergy-associated

factors is diacylglycerol kinase-α (DGK-α), which accounts for the diminished activation

of Ras/MAPK signaling and downstream AP1-dependent transcription (34,35). DGK-α

mediates its activity through the phosphorylation of diacylglycerol (DAG) that reduces

Ras guanyl releasing protein1 (RasGRP1)-dependent activation of Ras. Calcium-induced

anergy-associated factors also include E3 ubiquitin ligases (Cbl-b, Itch and GRAIL) that

inhibit TCR-proximal signaling by promoting the degradation of critical signaling

proteins (36). Phospholipase C-γ1 (PLC- γ1), which is an upstream regulator of Ras

activation, has been previously suggested as a potential target for E3 ligases. Consistent

with E3 ligases role in anergy, mutation or deletion of the E3 ligases Itch and Cbl-b in

mice leads to the development of autoimmune diseases, and over-expression of the E3

ligase GRAIL abrogates T cell proliferation and IL-2 production (36). Unbalanced

activation of calcium/NFAT signaling leads also to upregulated expression of the transcription factors early growth response gene (Egr) 2 and Egr3 (37). It has been proposed that these transcription factors act together with NAFT or independently to

induce the expression of several anergy-associated genes, such as Cbl-b. In support of

this, it has been shown that over-expression of Egr2 and Egr3 upregulates Cbl-b

expression and leads to a decrease in IL-2 production in T cells. Conversely, Egr3-

deficient T cells fail to induce Cbl-b and are resistant to anergy induction (37).

10 Furthermore, two Egr-binding sites have been identified in the promoter of DGK-α,

suggesting that Egr transcription factors are also important for the upregulation of DGK-

α in anergic T cells (36).

1.1.1.2.1.3. Phenotypic skewing

Differentiation of activated CD4+ T cells into distinctive T cell subsets is another

peripheral mechanism that contributes to the control of pathogenic T cell-mediated

diseases. The two major populations of mature T cells are the CD4+ helper T cells and the

CD8+ cytotoxic T cells. Naive CD4+ helper T cells upon activation with self and nonself- antigens can further differentiate into the functionally distinct T helper 1 (Th1) and T helper 2 (Th2) subgroups, which express distinctive sets of cytokines (38-40). Th1 cells express IL-2, IFN-γ and TNF-α and induce cell-mediated immune responses, whereas

Th2 cells produce IL-4, IL-5 and IL-10 and promote humoral immunity. IL-4 and IL-10 secreted by Th2 are known to inhibit Th1 cells, while Th1 cells downregulate Th2 cell function and differentiation through the production of IFN-γ (2,41,42). A balance between Th1 and Th2-type immune responses is important for the protection of the host against various pathogens and the maintenance of peripheral tolerance (43,44).

Accumulated experimental and human data demonstrate that polarization of specific immune responses into a predominant Th1 or Th2 profile results in the development of a wide range of autoimmune and allergy diseases (44). Predominance of Th1-type immune responses accounts for the development of organ-specific autoimmune diseases, including rheumatoid arthritis, multiple sclerosis (MS), liver injury, type 1 diabetes and graft-versus-host disease (GVHD). On the other hand, polarized Th2-type immune

11 responses are associated with systemic lupus erythematosus (SLE) and allergic diseases

(44).

In addition to Th1/Th2 subsets, naive CD4+ T cells may differentiate into other T

cell subpopulations when they are activated by self/tolerogenic antigens. These subsets

include, among others, IL-10-producing type 1 Treg (Tr1) and TGF-β-producing T helper

type 3 Treg (Th3) cells, which play an important role in the regulation of immune

responses and inhibition of autoimmune diseases (45). All the different cytokine-

secreting T cells discussed above are activated specifically by antigen, but mediate their

regulatory activity nonspecifically through the secretion of cytokines. Together these T

cell subsets control the magnitude and class of immune response, leading to the

maintenance of immune homeostasis and self-tolerance in the periphery (45). Tr1, Th3

and other regulatory T cells that are induced in the periphery will be discussed in more

detail as part of the extrinsic mechanisms of peripheral tolerance.

1.1.1.2.1.4. Activation-induced cell death (AICD)

High affinity binding of TCR with MHC-peptide complexes or repetitive stimulation of TCR in the absence of appropriate costimulation may lead to deletion of pathogenic/autoreactive T cells in the periphery in a process known as activation-induced cell death (AICD) (46). This process involves the induction of apoptosis mediated by the

interaction of the Fas receptor (CD95) with its ligand (FasL), which is upregulated on T

cells undergoing AICD. Fas-mediated AICD can be trigged either by the same T cell that

expresses FasL in a cell-autonomous manner (suicide) or by neighboring T cells

(fratricide) (47). FasL can also be induced in nonlymphoid cells, such as intestinal

epithelial cells (IECs), in response to TNF produced by activated lymphocytes. It has

12 been proposed that upregulation of FasL on IECs induces the apoptosis of Fas-bearing

infiltrating lymphocytes, and therefore contributes to peripheral tolerance (48). The

increased expression of FasL by IECs decreases to resting levels following the resolution

of inflammation. The importance of Fas and FasL in self-tolerance and immune

homeostasis is underscored by the phenotype of mice with a mutation in either of these

genes that develop lymphadenopathy and suffer from autoimmune disease (49).

Furthermore, defects in the Fas signaling pathway account for autoimmune

lymphoproliferative syndrome (ALPS) in humans (50,51).

The Fas receptor is a member of the death receptor subfamily, which is part of the tumor necrosis factor (TNF) receptor superfamily. The Fas receptor, as other members of the death receptor subfamily, contains an intracellular death domain (DD) that is essential for the transduction of the apoptotic signaling pathway. This apoptotic pathway is executed through the activation of a caspase cascade (52,53). Oligomerization of Fas upon ligation with FasL leads to the recruitment of the Fas-adaptor protein, Fas- associated DD (FADD), through a DD-DD interaction. Pro-casapase-8 and pro-caspase-

10 are then recruited to FADD through binding of the two death effector domains (DEDs).

The recruitment of FADD and pro-casapase-8/10 to the oligomerized Fas receptor forms the Fas death-inducing signaling complex (Fas DISC), which leads to the autoproteolytic activation of caspase-8/10 (54). These activated caspases can trigger two different types of apoptotic signaling pathways. When Fas DISC and activated caspase-8/10 are present at high levels, caspase-8/10 can directly activate downstream effector caspase-3/6/7 (54).

However, low levels of Fas DISC and subsequently low levels of activated caspase-8/10 require amplification of the apoptotic signal by mitochondria. In this case, low levels of

13 activated caspase-8/10 can cleave the BCL-2-family protein BID to generate truncated

BID (tBID). The pro-apoptotic tBID releases cytochrome c from the mitochondria, which

results in the formation of the apoptosome, caspase-9 activation and subsequent

activation of caspase-3/6/7 (54).

Activated effector caspase-3/6/9 cleave several important cellular components

called the ‘cell-death substrates’. These cell-death substrates include the inhibitor of caspase-activated DNase (ICAD), nuclear lamins and cytoskeleton proteins actin and gelsolin (55). Cleavage of nuclear lamins results in chromatin condensation and nuclear shrinkage, and degradation of ICAD by the effector caspases allows for the activation of

CAD to fragment DNA. Effector caspases also degrade the cytoskeleton proteins actin

and a number of actin-regulatory proteins, such as gelsolin, which results in disruption of

the cytoskeleton, intracellular transport, cell division and signal transduction. Ultimately,

the degradation of all the cell-death substrates by the effector caspases leads to cell

fragmentation, blebbing, apoptotic body formation and cell destruction. The apoptotic

cells are removed by phagocytes that recognize specific apoptotic markers expressed on

the dying cells, such as phosphatidyl serine (55,56).

Regulation of Fas-mediated AICD occurs at different levels of the apoptotic

signaling pathway (54). First, costimulation of CD28 can lead to inhibition of FasL

expression and resistance to apoptosis. Second, expression of the anti-apoptotic BCL-2

family members, such as BCL-2 and BCL-XL, regulate the induction of AICD at the

mitochondria. Finally, various forms of the cellular caspase-8 (FLICE)-inhibitory protein

(c-FLIP), which interact with FADD at the Fas DISC, act as inhibitors of caspase

activation (54). Deceased expression of c-FLIP is associated with sensitization of

14 activated lymphocytes to Fas-induced AICD, whereas increased expression of c-FLIP can

contribute to the development of various autoimmune diseases. In this regard, disease

progression in patients with MS correlates with high levels of c-FLIP, and IFN-β therapy

downregulates the expression of c-FLIP in T cells from patients with MS (57).

Furthermore, over-expression of c-FLIP in activated lymphocytes using a retrovirus-

based gene transfer system leads to a blockade of Fas-induced apoptosis and

autoimmunity (58). These results suggest that c-FLIP plays a major role in the life and

death of T cells, and that activation-dependent downregulation of c-FLIP, which

sensitizes mature lymphocytes to Fas-mediated AICD, is required for the maintenance of

immune homeostasis and self-tolerance.

In addition to Fas/FasL, other mechanisms also contribute to the induction of

AICD, as peripheral deletion in Fas/FasL-deficient mice is reduced but not absent. Indeed, other cell-death receptor and independent cell-death receptor pathways have been implicated in AICD. Ligation of the cell-death receptor TNFR1 with its ligand TNF can induce AICD in some cases. TNF-mediated apoptosis appears to be involved in the late phase of AICD (59-61). Another cell-death receptor that has a role in the induction of

AICD is the TNF-related apoptosis-inducing ligand receptor (TRAILR). Activated mature T cells can produce TRAIL, which can mediate their apoptosis upon binding to

TRAILR (62-64). Mice lacking TRAIL have increased susceptibility to collagen-induced arthritis and streptozotocin-induced diabetes and develop excessive autoimmune responses (65). Independent cell-death receptor mechanisms include the serine protease granzyme B. In response to TCR stimulation, granzyme B is upregulated in Th2 cells causing AICD. Inhibition or deletion of granzyme B abrogates AICD in Th2 cells and

15 increases the production of Th2 cytokines. Furthermore, the loss of granzyme B leads to

enhanced susceptibility to allergen-induced asthma in granzyme B-deficient mice (66).

1.1.1.2.2. Cell-extrinsic mechanisms

1.1.1.2.2.1. Tolerogenic dendritic cells

DC are specialized professional antigen-presenting cells that function as the

principal initiators and modulators of the adaptive immune response. DC act at the

interface of innate and acquired immunity by recognizing pathogens and presenting

pathogen-derived molecules to T cells to initiate the immune response (67-69).

Recognition of various pathogens by DC is achieved through the expression of germline- encoded receptors, which are known as pattern-recognition receptors (PRRs). PRRs can

recognize characteristic molecular structures shared by large groups of pathogens, the so-

called pathogen-associated molecular patterns (PAMPs). Toll-like receptors (TLRs) that

were recently identified as a major family of PRRs recognize and bind to a wide range of

PAMPs (70,71). These PAMPs include components of bacteria, viruses and parasites,

such as lipopolysaccharides, peptidoglycans, lipoarabinomannan, CpG motifs or viral

nucleic acids. The recognition of pathogens through these PRRs induces full activation of

immature DC, accompanied by upregulation of MHC-peptide complex and costimulatory

molecules CD80/CD86, as well as the production of pro-inflammatory cytokines (IL-6,

IL-12, IL-1β, TNF-α, IL-1α). The fully matured DC then migrate to the lymph nodes and

spleen, where they trigger the full activation of antigen-specific T cells. Consequently,

activated T cells are recruited to the site of infection and control the infection (70-72).

In addition to the key role that DC play in the initiation of immune responses, DC

are also important in the induction and maintenance of peripheral T-cell tolerance (73).

16 Peripheral tolerance can be achieved through the activity of partially matured DC that present self-antigens to autoreactive T cells in a tolerogenic manner. During steady-state

(normal) conditions, in the absence of any danger signals such as pathogenic infection, tissue-resident immature DC continuously capture self-antigens from peripheral tissues through the uptake of apoptotic cells and soluble material released in the DC microenvironment (74). Uptake of these self-proteins leads to only partial activation of immature DC, characterized by low or lack of expression of costimulatory molecules and proinflammatory cytokines. These ‘semi-mature’ or partially matured DC migrate via lymphatic or blood vessels from the periphery to the spleen and lymph nodes, where they interact with autoreactive T cells. Ligation of self-reactive TCR in the absence of appropriate costimulatory signals provided by the semi-mature DC, leads to inactivation

(anergy) or deletion of the autoreactive T cells and possibly generation of regulatory T cells (74).

Alternatively, peripheral tolerance can be mediated by distinct DC subpopulations with inherent tolerogenic function. In recent years, several tolerogenic DC subsets have been discovered in both animals and humans. A discrete tolerogenic DC subset lacking the expression of CD4 and OX41 has been described in rats (75). This DC subset constitutively endocytoses and transports apoptotic intestinal epithelial cells to T cell areas of mesenteric lymph nodes, where peripheral tolerance can be induced (75).

Furthermore, the recently identified plasmacytoid DC (pDC) subset found in human peripheral blood can induce antigen-specific anergy in CD4+ T effector cells (76), and appears to play an important role in suppression of Th1 cell-mediated pathological conditions, such as allograft rejection and graft versus host disease (GVHD) (77-79).

17 A better understanding of the mechanisms employed by DC to induce/maintain

tolerance has led to the development of various in vitro and in vivo strategies to generate tolerogenic DC for therapeutic applications (80,81). Exposure of in vitro-propagated DC

from bone-marrow or peripheral blood to specific culture conditions, such as high levels

of IL-10/TGF-β1, or pharmacological treatments, including cyclosporine and rapamycin,

can promote generation of immature DC with tolerogenic proprieties. Moreover, genetic

transfection systems using recombinant viral vectors or naked DNA to over-express

inhibitory molecules (i.e. CTLA-4-Ig and TGF-β1) in cultured DC can also result in the

induction of tolerogenic DC. Tolerogenic DC that are generated in vitro can be then

injected intravenously into patients to treat autoimmunity and prevent graft rejection

(80,81). In vivo approaches to promote self-tolerance mediated by DC include injection

of apoptotic cells or self-antigens tagged to DC-specific monoclonal antibodies (mAb),

such as DEC-205. The in vivo treatments lead to processing and presentation of self-

antigens derived from apoptotic cells or antigens coupled to DEC-205 mAb on DC in the

context of MHCI and MHCII molecules. Presentation of these antigens without the full

maturation and activation of DC under these steady-state conditions results in the

induction of antigen-specific T-cell tolerance (80,81). Experimental models utilizing the

in vitro and in vivo approaches to induce DC-mediated tolerance have so far shown

promising results. However, further studies are required to evaluate the potential

therapeutic effects of tolerogenic DC in humans.

18 1.1.1.2.2.2. Regulatory T cells

In addition to tolerogenic DC, a major role in the maintenance of peripheral

tolerance has been attributed to the activity of regulatory or suppressor T cells. Early

studies by Gershon et al. nearly 40 years ago described for the first time the existence of

suppressor T cells that can downregulate immune responses of antigen-specific T cells

(82). At the same time, thymectomy studies suggested the production of thymic-derived

regulatory T cells that maintain immunological tolerance. In 1969, Nishizuka et al.

showed that neonatal thymectomy of mice 3 days after birth induced an ovarian

autoimmune disease (83). Furthermore, Penhale et al. reported in 1973 that adult

thymectomy of normal rats followed by sublethal X-irradiation results in the

development of autoimmune thyroiditis associated with the production of tissue-specific

autoantibodies (84). Reconstitution of these thymectomized animals with normal T cells

inhibited the development of autoimmunity in both models (85).

Intensive investigation of regulatory T cells following these early reports

eventually declined in the late 80s and early 90s. Loss of interest in research of regulatory

T cells was mainly due to the lack of reliable markers for identification of these cells that

led to difficulties in isolation and investigation of regulatory T cell activities (86).

However, in recent years with the development of advanced molecular and cellular tools, new cell markers were identified and the function of various cytokines was characterized, leading to reappraisal of regulatory T cells as mediators of self-tolerance (86). As a result of a large number of studies in the field of regulatory T cells performed over the past decade, there is now solid evidence to indicate a crucial role for regulatory T cells in peripheral tolerance. These recent studies demonstrate the existence of various regulatory

19 T cell subsets, which express distinct cytokines and receptors, that function by diverse pathways at different stages of the immune response (87). Together these regulatory T cells maintain self-tolerance and control excessive immune reactions to foreign antigens in the periphery.

Regulatory T cells can be divided into two major subsets based on their ontogeny: the adaptive regulatory T cells, which are induced in the periphery in response to antigen stimulation under tolerogenic conditions, and the naturally occurring regulatory T cells, which are constantly produced by the thymus. The major regulatory T cell subsets that were described over the past decade will be reviewed here (Figure 1-2) with a focus on the most extensively studied Treg cell subset, the CD4+CD25+Foxp3+ regulatory T cells, which was also investigated in our study.

20 Figure 1-2. Natural and adaptive regulatory T cells. Several different types of regulatory T cells exist in the periphery. These cells can be classified into two major subgroups, natural regulatory T cells produced by the thymus and adaptive regulatory T cells that are induced in the periphery upon antigenic stimulation of naive T cells under tolerogenic conditions (such as TGF-β, IL-10 and immature DC). Thymus-derived CD4+CD25+Foxp3+ Treg cells, DN and some subsets of CD8 suppressor cells can also develop in the periphery. Abbreviations: nTreg- naturally occurring CD4+CD25+Foxp3+ Treg cells; iTreg- induced CD4+CD25+Foxp3+ Treg cells; NKT- natural killer T cells; DN- double negative Treg cells; Th3- T helper type 3; Tr1- type 1 regulatory T cells.

1.1.1.2.2.2.1. Regulatory T cells type 1 (Tr1)

Tr1 cells were first described in severe combined immunodeficiency (SCID)

patients, who were successfully transplanted with allogeneic hematopoietic stem cells

(HSCs). CD4+ host-reactive T cell clones that were isolated from these SCID patients

produced high levels of IL-10 and low amounts of IL-2 after antigen stimulation in vitro.

Immunologic reconstitution and induction of tolerance following HLA-mismatched

HSCs transplantation were associated with the presence of IL-10-producing CD4+ T cells

(88). Subsequent studies by Groux et al. demonstrated that ex vivo activation of human

and murine CD4+ T cells in the presence of high levels of IL-10 induced the generation of

IL-10-producing CD4+ T cells with low proliferative responses. These cells produced a unique set of cytokines, characterized by significant high levels of IL-10, TGF-β and IL-5 but low quantities of IL-2 and IFN-γ, and no IL-4 (89). Due to their ability to suppress T- cell immune responses in vitro and in vivo, IL-10-producing CD4+ T cells were termed

regulatory T cells type 1 (Tr1).

Tr1 arise in the periphery following activation of naive T cells with antigen in the

presence of IL-10, and act as important regulators of adaptive immune responses through

the suppression of naive and memory T cell-responses and the inhibition of APC

21 stimulatory activities (90). Although Tr1 are induced in response to antigenic-specific

stimulation, they exert their suppressive function in an antigen non-specific manner

through the production of IL-10 and TGF-β. The release of the anti-inflammatory

cytokines IL-10 and TGF-β is likely the reason for the bystander suppression of Tr1 cells

(90).

To date, the regulatory activity of Tr1 cells has been implicated in various

immunopathologies both in mice and humans (90). The importance of Tr1 cells in the

maintenance of self-tolerance was demonstrated by isolation of Tr1 cells bearing self-

MHC reactive TCR from the peripheral blood of healthy individuals. These cells were

shown to suppress proliferative responses of autoreactive T cells in an antigen-specific

manner via production of IL-10 and TGF-β (91). Furthermore, the number of Tr1 cells isolated from peripheral blood and synovial tissues of patients with rheumatoid arthritis was significantly lower compared to control patients (92). Tr1 cells are also important for the inhibition and prevention of allograft rejection as was demonstrated by several studies.

The presence of Tr1 cells correlated with the absence of graft-versus-host-disease

(GVHD) and long-term graft tolerance in SCID patients that were transplanted with allogeneic HSCs (93). In addition, a high frequency of Tr1 cells was associated with the absence of acute GVHD following bone-marrow transplantation (BMT), while a low proportion of Tr1 cells correlated with development of severe GVHD (94). Moreover, allograft acceptance of kidney and liver was associated with the presence of Tr1 cells, which suppressed naive T cell responses through the production of IL-10 and TGF-β (95).

Clinical trials are currently being performed to evaluate the potential therapeutic

effects of Tr1 cells in the prevention and treatment of GVHD in BMT (96). The clinical

22 protocol used in these trials involves the transfer of ex vivo-generated Tr1 cells to patients with hematological cancers that are treated with HSC transplantation. In the first step, peripheral blood mononuclear cells (PBMCs) are collected from both donor and recipient, and CD34+ donor stem cells isolated from the donor are infused into the host.

Following reconstitution of neutrophils, donor PBMCs are cultured for 10 days in the presence of irradiated host PMBCs and IL-10 to generate IL-10-anergized donor T cells.

This cell preparation contains donor T cells that are anergic to the host and that are enriched for host-specific Tr1 cell precursors, as well as effector T cells that can react against pathogens and tumor cells (96). Treatment of the host with the IL-10-anergized donor T cells could potentially promote immune reconstitution without GVHD and also provide protective immunity against infection and recurrence of cancer (96). A success in these clinical trials could pave the way for Tr1-based immunotherapy in other immune- mediated diseases, such as autoimmunity and allergy.

1.1.1.2.2.2.2. T helper 3 (Th3)

Early studies of oral tolerance led to the identification of a unique CD4+ T cell subset, which was later referred to as the T helper type 3 (Th3) cells. The Th3 cell subpopulation was induced in the gut-associated lymphoid tissues of SJL mice by oral administration of myelin basic protein (MBP). These cells were able to suppress the proliferation and cytokine production of MBP-specific Th1 cells, and inhibit the development of experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis (MS) (97). Although these Th3 cells had the same specificity as encephalitogenic Th1 cells, they differed in their cytokine profile. Th3 cells that were isolated from the mesenteric lymph nodes produced high levels of TGF-β and various

23 amounts of IL-4 and IL-10. Production of TGF-β by Th3 cells accounted for the

inhibition of EAE, as antibodies to the anti-inflammatory cytokine blocked their

suppression (97). In subsequent studies, Th3 cells were also identified in MS patients who were orally treated with MBP and proteolipid protein (PLP). Similar to the mouse model, these treatments induced a significant increase in the frequency of TGF-β- secreting Th3 cells that were found to be specific to MBP and PLP (98).

The gut microenvironment that contains high levels of TGF-β and Th2 cytokines, as well as unique subsets of DC promotes the development of Th3 cells upon encounter with oral antigens (99). Generation of Th3 cells is thought to be important in the induction and maintenance of tolerance to non-pathogenic resident bacteria and potentially immunogenic food antigens. Th3 cells are activated in response to specific antigens but suppress in an antigen-nonspecific manner through the release of TGF-β, and therefore they can mediate bystander suppression. Th3-type cells downregulate both

Th1 and Th2 cells and provide help for B cells in the production of IgA antibodies (99).

The regulatory activity of Th3 cells has been implicated in the control of various

experimental autoimmune disease models other than EAE, including spontaneous

autoimmune diabetes, experimental autoimmune myasthenia gravis and autoimmune glomerulonephritis (45). The importance of Th3 cells was also reported in donor transfusion-induced allograft tolerance and in suppression of lung eosinophilic inflammation (99). Recent studies have also demonstrated a role for Th3 cells in some cases of human autoimmunity and allergy, as reduction in the number of the TGF-β- producing T cells or production of TGF-β was found in the duodenal mucosa of children

24 with food allergy and patients with active chronic idiopathic thrombocytopenic purpura,

respectively (100,101).

1.1.1.2.2.2.3. CD8+ regulatory T cells

In recent years several types of CD8+ T cells with regulatory function have been

identified. These include antigen nonspecific cells that are naturally produced by the

thymus and antigen specific cells that are generated in response to foreign or self-

antigens during the course of the peripheral immune response. In addition, other antigen

specific and nonspecific subsets of CD8+ regulatory T cells have also been induced in

vitro (102). It is important to note that most of the CD8+ regulatory T cells that have been

described so far were characterized by only one or two laboratories using a limited

number of experimental models. Therefore, further studies are required to evaluate the relationship between these subsets and determine whether they represent distinct regulatory cells or the same cells under different activation conditions. Here, I will discuss only the major CD8+ regulatory T cell subsets that have been described in the

literature.

Recent studies defined CD8+CD25+ human thymocytes that share similar

phonotypic and functional properties with naturally occurring CD4+CD25+ T cells produced by the thymus (103). CD8+CD25+ thymocytes express increased mRNA levels

of Foxp3, glucocorticoid-induced tumor necrosis factor receptor (GITR), CCR8, TNFR2

and CTLA-4. Upon activation these cells do not produce cytokines but express surface

TGF-β1 and CTLA-4. Purified CD8+CD25+ thymocytes were found to be anergic in vitro,

and were capable of suppressing the proliferation of CD4+ effector T cells in a contact- dependent manner. Antibodies to TGF-β1 and CTLA-4 abrogated the function of the

25 regulatory cells (103). A similar population of CD8+CD25+ T cells was also detected in

the periphery and thymus of mice lacking MHCII. This cell population expressed Foxp3,

CTLA-4 and IL-10, and was also shown to regulate the response of both CD4+ and CD8+

effector T cells to anti-CD3 stimulation (104). In additional studies, autoreactive human

peripheral blood CD8+CD25+Foxp3+ T cells were expanded and cloned following the

stimulation with autologous LPS-activated DC. They proliferated and produced anti-

inflammatory cytokines, including TGF-β1 and IL-4, upon stimulation with DC in an

autoreactive HLA-restricted manner. A CD8+CD25+Foxp3+ clone inhibited proliferation

and IFN-γ production of CD4+ T cells in vitro through CTLA-4 in a cell-to-cell contact

(105).

A distinct population of human CD8+ regulatory T cells can be induced following

repeated stimulation of peripheral blood mononuclear cells in vitro with allogeneic,

xenogeneic or self-APCs pulsed with antigen (106,107). This CD8+ T cell population

lacks the expression of CD28 but expresses Foxp3. CD8+CD28- T cells are antigen specific, MHC class I-restricted and suppress antigen specific CD4+ T helper cell

responses. They exert their regulatory activity by interacting directly with DC, monocytes

and endothelial cells (ECs). This interaction induces the upregulation of the inhibitory

receptors, immunoglobulin-like transcript 3 (ILT3) and ILT4 on APCs, leading to

inhibition of NF-κB activation and a subsequent reduction in the expression of

costimulatory molecules CD80 and CD86. The suppressed APCs become tolerogenic and

are therefore incapable of inducing and sustaining the full activation of T helper cells

(108). Previous studies have suggested a role for CD8+CD28- T cells in the inhibition of

26 allograft rejection in animals and humans (106,109). The regulatory activity of

CD8+CD28- T cells was also implicated in suppression of EAE (110).

A novel subset of natural regulatory CD8+ T cells has been described in normal

healthy animals. These CD8+ T cells express low levels of surface CD45RC and

following stimulation in vitro produce predominately T helper type 2 cytokines, including

IL-4, IL-10 and IL-13 (111). They also express the transcription factor Foxp3 and CTLA-

4 on their cell membrane. CD8+CD45RClow T cells inhibit the proliferation and

differentiation of CD4 cells into Th1 cells in response to allogeneic APCs via a cell-to- cell contact. These regulatory cells protected against the development of GVHD induced

by CD4+ T effector cells in rats (111). A recent report also showed that allograft

acceptance in major MHC-mismatched cardiac allograft model in rats is mediated by the

regulatory activity of CD8+CD45RClow T cells (112). It was proposed that

CD8+CD45RClow T cells act through the secretion of IFN-γ that in turn induces production of indoleamine 2,3-dioxygenase (IDO) by graft ECs. The immunoregulatory enzyme IDO degrades the essential amino acid tryptophan required for the growth of T cells, and thus its expression suppresses alloreactive T cell responses and promotes allograft tolerance (112).

CD8+CD122+ T cells are defined as naturally occurring IL-10-producing

regulatory T cells (113-115). They directly suppress the proliferation and IFN-γ

production of both CD4+ and CD8+ T cells in vitro. CD8+CD122+ T cells also have

important regulatory function in vivo as indicated by their ability to suppress EAE and

prevent the development of abnormal T cells in CD122-deficient mice (114,116). This

subset exerts their function mainly through the production of IL-10. Deletion of the IL-10

27 gene or antibody against IL-10 abrogated the suppressive activity of CD8+CD122+ T cells

in vitro. However, IL-10-/- CD8+CD122+ T cells were found to have some regulatory

activity in vivo, suggesting that additional unknown factors may contribute to the function of these cells (113).

A second class of IL-10-producing CD8+ regulatory T cells was reported to be

induced in vitro (117). Gilliet et al. showed that stimulation of naive CD8+ T cells with allogeneic CD40 ligand-activated plasmacytoid DC (DC2) resulted in the generation of the regulatory T cells. These cells produced high levels of IL-10 and low IFN-γ, and their generation was dependent on the presence of IL-10 in the cell culture. DC2-primed Treg cells were able to suppress allospecific proliferation of naive CD8+ T cells in response to

monocytes and DC. Similar to CD8+CD122+ T cells, the suppressive activity of DC2-

primed CD8+ T cells was mediated by the production of IL-10 (117).

A distinct regulatory T cell subset expressing TCRαβ and CD8αα has been

recently defined (118,119). High frequencies of these cells can be detected in the

intestinal intraepithelial lymphocyte (IEL) population of the gut (40%), but only small

numbers can be found in the spleen and lymph nodes (<1%). Previous studies showed

that TCRαβ+CD8αα+ Treg cells are derived from the thymus by exposure to self-agonists, and that they are self-reactive. Abundant transcript levels of TGF-β3, lymphocyte activation gene-3 (LAG-3) and fibrinogen-like protein 2 (FGL2) were measured in

TCRαβ+CD8αα+ Treg cells of the IEL population (120). Due to the increased expression

of genes that are involved in immune regulation and the self-reactivity of IEL CD8αα+

Treg cells, it was proposed that these lymphocytes might play a role in regulation of

mucosal immunity (120). In support of this concept, Poussier et al. demonstrated the

28 ability of TCRαβ+CD8αα+ Treg cells to prevent colitis induced by CD4+CD45RBhigh T

cells in SCID mice. This inhibitory activity was dependent on the production of IL-10, as

TCRαβ+CD8αα+ Treg cells from IL-10-deficient mice could not efficiently prevent the

disease (121). In additional experiments, it was shown that TCRαβ+CD8αα+ Treg cells

can inhibit the development of EAE (118). Further studies are required to evaluate the

exact role of these cells and elucidate their mechanism of action.

1.1.1.2.2.2.4. Double negative T cells

The recently identified CD4-CD8-CD3+ regulatory T cell (DN Treg) subset

constitutes 1-3% of peripheral T cells in rodents (122). DN Treg cells express a unique

set of cell surface markers, including TCRαβ, CD25, LFA-1, CD69, CD45, CD30,

CD62L and CTLA-4. Activated DN Treg cells also exhibit a distinct cytokine profile,

characterized by the production of increased amount of IFN-γ, TNF-α and low levels of

TGF-β. However, production of IL-2, IL-4, IL-13 or IL-10 can not be detected in these

cells (123).

DN Treg cells can suppress both CD4+ and CD8+ T cell-mediated immune

responses in vitro and in vivo. In recent years, the regulatory activity of this subset has

been implicated in a number of experimental transplant and autoimmune disease models.

Previous studies showed that stimulation of DN Treg cells with donor antigens activates

the cells to inhibit anti-donor T cell responses and enhance allograft and xenograft

survival in an antigen-specific manner (124,125). In additional experiments, it was also

demonstrated that treatment with activated antigen-specific DN Treg cells can prevent the

development of GVHD and autoimmune type 1 diabetes induced by pathogenic CD8+ T

cells in a mouse model (126,127).

29 The mechanism by which DN Treg cells exert their regulatory activity has been

extensively studied. DN Treg cells act directly on effector T cells in an antigen-specific

fashion through a process that requires cell-to-cell contact. Prior to encounter with the

target cells, DN Treg cells acquire via their antigen-specific TCR allo-MHC peptides

from antigen presenting cells. Presentation of the acquired alloantigens on DN Treg cells

enables them to specifically trap and kill CD4+ and CD8+ T cells that express the same

TCR specificity as the DN Treg cells. Killing of effector T cells is mediated

predominately through Fas/Fas-ligand interactions, although other additional pathways

may contribute to the regulatory activity of this population (128).

The origin and development of DN Treg cells have not been well defined;

however, recent reports have shed light on the underlying mechanisms that are involved

in the generation of DN Treg cells. Ford et al. reported that peripheral DN Treg cells can

be found in the spleen and lymph nodes of thymectomized mice that had been irradiated

and reconstituted with T cell-depleted BM cells, indicating that DN Treg cells can

develop outside of the thymus (129). Indeed, Zhang et al. have identified a new differentiation pathway for the conversion of CD4+ T cells to DN Treg cells (130). The

study showed that peripheral CD4+ T cells can downregulate CD4 expression and

become DN Treg cells via auto/alloantigen-triggered or homeostatic proliferation.

Differentiation of DN Treg cells from mature CD8+ T cell population was not observed

(130). These findings reveal a novel intrinsic homeostatic pathway that provides a deeper

understanding of the peripheral regulatory mechanisms of the immune system.

DN Treg cells have also been identified and characterized in humans (131). These

cells comprised 1-2% of total CD3+ T cells in the blood and lymph nodes of healthy

30 individuals. Similar to DN Treg cells in mice, activated human DN Treg cells produce

high levels of IFN-γ, but no IL-2 and very low levels of IL-10 and IL-4. Human DN Treg cells were shown to exhibit similar functional properties as murine DN Treg cells. DN

Treg cells were able to acquire MHC-peptide complexes from APCs and use them to induce apoptosis and suppress proliferation of antigen-specific cytotoxic T lymphocytes

(CTL) (131). It was recently reported that human DN Treg cells may also play a role in preventing GVHD in patients after allogeneic HSC transplantation (132). Taken together, the results obtained from human and animal studies may potentially lead to the development of novel cell-based therapies using DN Treg cells to prevent allograft rejection and treat autoimmune diseases.

1.1.1.2.2.2.5. γδ regulatory T cells

Less than 5% of total T cells in the peripheral lymphoid tissues express TCR

gamma/delta, which is composed of γ and δ chains instead of the conventional αβ

heterodimers. TCRγδ+ T cells are enriched in the intraepithelial lymphocyte (IEL)

compartments of skin, intestine and genitourinary tract, where they represent up to 50%

of total T cells (133). γδ T cells may be considered part of the adaptive immune response since they rearrange TCR genes and develop into memory cells. On the other hand, γδ T

cells may also be considered a component of the innate immune system, because they

display a restricted TCR repertoire that can respond to PAMPs and self-antigens. In

contrast to αβ T cells, γδ T cells are not MHC-restricted, their development can be thymic dependent or independent and they are capable of recognizing soluble protein and non- protein antigens of endogenous origin (133).

31 γδ T cell function has been implicated in downregulation of immune responses

associated with pathogenic infection, allergens and autoimmune diseases (134). Mice that

lack γδ T cells exhibit exaggerated or accelerated immunopathology when infected with

intestinal Eimeria, Listeria monocytogenes, Mycobacterium tuberculosis and Klebsiella.

In addition, γδ T-cell-deficient mice on a specific background can develop a spontaneous

αβ T cell-dependent cutaneous inflammation (dermatitis), as well as augmented responses to contact allergens and irritants (134). The regulatory activity of γδ T cells has also been reported in GVHD and several experimental models of autoimmune disease, such as type

1 diabetes and lupus erythematosus (133). It is important to note that in certain infections and immunopathologies, γδ T cells may also display a pro-inflammatory phenotype.

Therefore, it has been proposed that γδ T cells could exert different functions depending on their tissue distribution, antigen receptor structure and local microenvironment (133).

A number of possible mechanisms whereby γδ T cells mediate their regulatory

activities have been suggested (135). Production of chemokines and anti-inflammatory

cytokines by γδ T cells (such as TGF-β, IL-10 and thymosin-β4) can regulate the local

homing and maturation of other lymphoid cells. Another possibility is that γδ T cells via

IFN-γ release induce upregulation of Fas ligand on epithelia that then targets pathogenic systemic cells infiltrating the tissue. γδ T cells may also directly kill effector lymphoid cells through Fas-Fas ligand interaction. Finally, it was suggested that secretion of epithelial growth factors (keratinocyte growth factor) by γδ T cells may contribute to

epithelial tissue repair, which in turn provides a physical and chemical barrier to systemic

infiltration of activated immune cells (135).

32 1.1.1.2.2.2.6. Natural killer T cells

Natural killer T (NKT) cells are defined as lymphocytes that coexpress NK-cell-

surface receptors (NK1.1/CD161) with a semi-invariant T-cell receptor. This TCR

consists of an invariant α chain preferentially paired with restricted variable β chains

(136). In mice, most NKT cells have an invariant Vα14-Jα18 rearrangement paired with

Vβ8.2, Vβ2 or Vβ7 chains, while the homologous population of human NKT cells

expresses an invariant Vα24-Jα18 rearrangement paired with Vβ11. In contrast to other T

lymphocytes which recognize peptides in the context of classical MHC class I or II

molecules, NKT cells interact with glycolipids presented by the non-classical MHC class

I-like molecule CD1d (137). Upon activation, NKT cells produce large amount of Th1

(including IFN-γ and TNF-α) and Th2 (IL-4, IL-10 and IL-13) cytokines potentially

enabling them to act as powerful regulators of the immune system. Since NKT cells can

simultaneously express both Th1 and Th2 cytokines, they can either up- or downregulate

immune responses (138).

In addition to the function of NKT cells in tumor rejection and resistance to

pathogenic infection, these cells have also been implicated in promotion of tolerance induction in various autoimmune disorders and in allograft acceptance (139). Data obtained from animal models of MS and type1 diabetes provided the best evidence for the role of NKT cells in inhibition of autoimmune diseases. The protective effect of NKT cells in these well-characterized models was associated with either a Th2 polarization or a suppression of autoreactive T cells. Experimental mouse models also showed that NKT cells were required for allograft tolerance and inhibition of GVHD, involving NKT cell-

33 dependent generation of downstream allospecific Treg cells and IL-4 production,

respectively (139).

The relevance of NKT cells in clinical autoimmunity has been reported by a

number of studies. In these studies, it was shown that reduced numbers of Vα24 NKT

cells were associated with the development of type 1 diabetes, MS, systemic lupus

erythematosus, systemic sclerosis and rheumatoid arthritis (140). Similar to the murine

studies, it appears that human NKT cells can mediate their suppressive activity by

shifting the balance of the immune response from a Th1 pathogenic response to a

protective Th2 immunity. It is worth noting that in certain experimental settings and

models the effect of NKT cells was not beneficial or was even harmful to the host.

Therefore, further studies are required to evaluate the factors that determine the net

outcome of NKT cell function in different conditions of disease. A better understanding of the mechanisms whereby NKT cells exert their immunoregulatory activities will help

for the development of novel potential therapies for the treatment of a wide range of

diseases.

1.1.1.2.2.2.7. CD4+CD25+Foxp3+ regulatory T cells

Naturally occurring CD4+CD25+Foxp3+ regulatory T (Treg) cells were discovered

over a decade ago by studies of Sakaguchi et al. (141). In the pursuit of Treg cell

identification markers, Sakaguchi found a small (5-10% of peripheral CD4+ T cells and

5% of CD4+CD8- thymocytes) but distinct CD4+ T cell subset, which expresses high

levels of the IL-2 receptor α-chain (CD25). Athymic nude mice that were infused with

syngeneic splenic T cells depleted of Treg cells produced a broad spectrum of

autoimmune diseases, including thyroiditis, gastritis, insulitis, sialoadenitis, adrenalitis,

34 oophoritis, glomerulonephritis and polyarthritis. Co-transfer of a small number of Treg cells with CD25- splenic T cells prevented these autoimmune developments in a dose- dependent manner (141). Infusion of Treg cells was also shown to inhibit excessive immune responses of effector T cells to allo and xeno antigens in nude mice. Moreover, the appearance of Treg cells in the periphery of normal mice 3 days after birth correlated with the development of autoimmunity in mice that were thymectomized around the same day. Reconstitution of the thymectomized mice with Treg cells inhibited the autoimmune disease. Taken together, these early studies demonstrated for the first time the existence of thymic-derived Treg cells that are essential for the maintenance of self-tolerance and negative control of immune responses to non-self antigens.

Shortly after the discovery of CD25 as an identification marker for Treg cells, in vitro assays were established to study the function of Treg cells (142). Using these in vitro assays it was shown that Treg cells can suppress the proliferation of stimulated

CD4+ and CD8+ T cells in the presence of APCs. Treg cells were found to be anergic and failed to produce IL-2 upon stimulation. However, TCR-stimulated Treg cells were capable of proliferating in the presence of high levels of IL-2. The in vitro assays also showed that Treg cells act through a direct contact with their target cells without the need of soluble factors. The simple and highly reproducible in vitro assay together with CD25 discovery eventually led to the identification of human CD4+CD25+ Treg cells with phenotypic properties very similar to those found in rodents (143).

Expression of CD25 molecules and the presence of the growth factor IL-2 are critical for the generation and maintenance of Treg cells. Accordingly, deletion of CD25 or IL-2 in mice and genetic deficiency of CD25 in humans result in reduced numbers of

35 Treg cells and development of severe autoimmune disorders. Furthermore, treatment of

naive normal mice with neutralization antibody to circulating IL-2 causes a decrease in

Treg cell numbers and autoimmunity (144). The main source of IL-2 responsible for Treg

cell survival and activation in vivo is activated effector T cells, and it is was therefore

proposed that IL-2 production in sites of inflammation serves as a negative feedback that

regulates and controls adaptive immune responses (144).

Rudensky, Sakaguchi and colleagues have identified the specific expression of

Foxp3 (forkhead box P3), a member of the forkhead/winged-helix family of transcription factors, in CD4+CD25+ Treg cells (145,146). This transcription factor has been described

as a master regulator for Treg cell development and function. Disruption of Foxp3 leads

to impaired generation and activity of Treg cells and results in a fatal lymphoproliferative

disorder in mice. Similarly, humans with mutations in the gene encoding Foxp3 suffer

from a genetic disease known as IPEX (immune dysregulation, polyendocrinopathy,

enteropathy, X-linked syndrome), which is characterized by multiorgan autoimmune

disease, allergy and inflammatory bowel disease (IBD) (147). The importance of Foxp3

as a key control molecule in Treg cells was further supported by recent studies, which

demonstrated the ability of Foxp3 transduction to induce a Treg cell phenotype in naive T

cells (148). Foxp3-transduced cells exhibited regulatory activity, hyporesponsivness upon

stimulation, low production of IL-2 and upregulation of Treg-associated molecules (such

as CD25, CTLA-4 and GITR) (148). Several reports have recently suggested that Foxp3

controls Treg lineage development through direct or indirect activation and repression of

hundreds of genes. These target genes include those that encode nuclear factors

controlling gene expression and chromatin remodeling, plasma membrane proteins, as

36 well as cell signaling proteins. Foxp3 appears to regulate gene expression by forming a

transcription complex with other key transcription factors, such as NFAT, NFκB, AP-1

and AML/Runx1 (149).

In addition to natural Foxp3+ Treg cells that are constantly produced by the

thymus, naive T cells in the periphery may also acquire Foxp3 expression and convert

into Treg cells with functional and phonotypic similarity to their natural counterpart.

Induced Treg (iTreg) cells may arise following in vitro antigenic stimulation of naive T

cells in the presence of TGF-β. Both IL-2 and retinoic acid (a vitamin A metabolite)

promote TGF-β-dependent generation of iTreg, while IL-6 inhibits this process and

induces differentiation of naive CD4+ T cells into Th17 inflammatory cells (150).

Chronic antigenic stimulation could also induce Foxp3+ Treg cells in the periphery

through a process that involves antigen presentation in the absence of a ‘danger signal’,

such as may occur with food antigens in the gut (102). Further studies are still required to

assess the long-term stability of iTreg in the periphery, and to evaluate their contribution

to the peripheral pool of Foxp3+ Treg cells and their physiological significance in the induction of tolerance (150).

The identification of Foxp3 as a specific marker for Treg cells has revealed two

major differences between human and mouse Treg cells. In humans, naive T cells can

readily express low and transient levels of Foxp3 upon TCR stimulation, whereas similar

expression in mouse Treg cells has not been observed. The induced expression of Foxp3

in human naive T cells, however, does not confer suppressive function, and may serve as

a T-cell-intrinsic mechanism that can regulate the development of pathogenic T cells

(151). Furthermore, expression levels of CD25 and Foxp3 correlate well in murine Treg

37 cells (90% of CD25+ T cells are Foxp3 positive) allowing for their efficient isolation

based on the expression of high levels of CD25. In contrast, human Foxp3+ Treg cells can

either express high or intermediate levels of CD25 which make their isolation more

difficult, given the fact that CD25 at various levels can also be detected on activated T

cells (102). Collectively, these data suggest that the analysis and isolation of human Treg cells based on Foxp3 and CD25 levels should be performed with more caution.

With the discovery of Foxp3 and CD25 allowing for direct molecular and

functional investigation of Treg cells, there is now compelling evidence in mice and humans that these cells are critical for the negative control of various pathological and physiological immune responses (152). In recent years many laboratories worldwide have demonstrated the importance of Treg cells in the prevention of a broad spectrum of autoimmune diseases (in experimental models for type 1 diabetes, MS, rheumatoid arthritis, inflammatory bowel disease and systemic lupus erythematosus), as well as the inhibition of GVHD and allograft rejection. Treg cells have also been implicated in the negative control of excessive immune responses associated with pathogenic infection and cancer, which in some cases could be harmful to the host leading to chronic infection and cancer progression (152). Manipulation of Treg cells in the clinical settings provides a novel therapeutic approach for the treatment and prevention of a variety of human diseases. Increasing Treg cell number or enhancing their suppressive activity may lead to the inhibition of autoimmunity and the induction of tolerance to non-self antigens, including allografts, allergens and commensal bacteria. On the other hand, a reduction in the number or function of Treg cells could augment host immunity against bacterial infection and tumor cells, resulting in viral and tumor eradication (Figure 1-3) (150). To

38 date, there are two clinical trials which examine the ability of Treg cells to prevent

GVHD following HSC transplantation (96). In these studies, either freshly isolated Treg

cells or ex-vivo expanded Treg cells (with CD3- and CD28-specifc antibodies and high

doses of IL-2) from the donor are infused into the transplant recipient to inhibit and

prevent the development of the T cell-mediated pathology (96).

Figure 1-3. Reduction or increase in Treg cell numbers/activity is implicated in various pathologies. Reduction in Treg cell numbers or activity results in the development of autoimmunity, allergy and graft rejection. Increase in Treg cell numbers or function may cause susceptibility to chronic infection and tumor development.

39 1.1.1.2.2.2.7.1. Mode of Treg cell action

Treg cells possess TCRs that can recognize both self and non-self antigens, and appear to be in an activated state, as evident by the expression of high levels of activation markers and adhesion molecules (153). Unlike the hyporesponsiveness they exhibit in vitro, a large number of Treg cells continuously proliferate in vivo probably due to their interaction with self-antigens and commensal bacteria in the periphery. Antigenic stimulation is required to activate the suppressive function of Treg cells, however, once activated they downregulate immune responses in an antigen-nonspecific manner (153).

The regulatory cells can suppress the proliferation and activation of many different types of immune cells, including T cells, DC, B cells, NK cells and NKT cells both in vitro and in vivo (150). In the past few years there has been a significant progress in defining the effector molecules that Treg cells use to mediate their regulatory activity (Table 1-1).

Based on their molecular characterization and function, effector molecules of Treg cells can be classified into four groups: immunosuppressive cytokines, molecules involved in metabolic disruption, cytolytic molecules and membrane-associated molecules that downmodulate the activation of effector cells.

Secretion of inhibitory cytokines has been proposed as a major pathway by which

Treg cells exert their function. Among these inhibitory molecules, TGF-β and IL-10 have been reported as key mediators of Treg cell suppression (154). Interestingly, TGF-β and

IL-10 are both dispensable for the function of Treg cells in vitro, as their deletion or neutralization using a specific antibody does not alter Treg cell suppression (154).

However, the inhibitory role of TGF-β and IL-10 in vivo has been demonstrated in various experimental models, including IBD, leishmania skin infection, type 1 diabetes,

40 transplantation and tumor models. Furthermore, loss of TGF-β in mice leads to the

development of a lethal autoimmune disease associated with a reduction in Treg cell

numbers, while mice that lack IL-10 display severe intestinal inflammation in response to

normal flora (154,155). The inhibitory cytokine IL-35, a member of the IL-12 family, has

recently been described as a potent suppressive molecule of Treg cells. IL-35 is

preferentially produced by Treg cells and its transcript levels are markedly upregulated in

Treg cells that are actively suppressing. The important contribution of IL-35 to Treg cell

function has been demonstrated both in vitro and in vivo in an animal model of IBD (156).

Several Treg effector molecules are involved in the metabolic disruption of the

effector cell-target. These molecules include the inhibitory second messenger cyclic

AMP (cAMP), which can be transferred by Treg cells into target cells through membrane

gap junctions. Upon entrance to the target cell, cAMP inhibits IL-2 production and

proliferation (157). Recent studies also described the unique activity of the ectoenzymes

CD39 (ectonucleoside triphosphate diphosphohydrolase 1) and CD73 (ecto-5’-

nucleotidase) expressed predominately on the surface of Treg cells. The ectoenzymes catalyze the generation of pericellular adenosine that delivers a negative signal to effector

cells by interaction with the adenosine receptor 2A (A2A) (158). In addition to the role of

CD25 in the maintenance and generation of Treg cells, it has been suggested that the high

level expression of the receptor is used by Treg cells to absorb the local IL-2 required for

non-Treg cell survival. The deprivation of the essential growth factor IL-2 leads to

apoptosis of naive and effector T cells (159).

In addition to regulation by suppression, Treg cells can also mediate their activity by killing effector cells. This process involves the release of cytolytic molecules, such as

41 granzyme A/B, perforin or through the TRAIL-DR5 (tumor-necrosis-factor-related apoptosis-inducing ligand-death receptor 5) pathway (160). Accordingly, granzymes are highly expressed in Treg cells and their deficiency results in the reduction of Treg cell function. Moreover, target cells that over-express a specific inhibitor of granzyme are

resistant to death by apoptosis (160). Similarly, increased expression of the cytolytic

galectin-1 has been observed in both human and mouse Treg cells, and Treg cells isolated

from galectin-1-deficient mice show impaired activity in vitro (160).

The expression of a number of inhibitory membrane-associated molecules that

can downmodulate effector cell function has also been implicated in Treg-mediated suppression. The most characterized surface molecule expressed by Treg cells is the negative regulator CTLA-4, which plays a critical role in the function of Treg cells (154).

Treg-derived CTLA-4 mediates its activities through interaction with the costimulatory molecules CD80 and CD86 on DC, leading to downregulation of the maturation markers or induction of the potent suppressive enzyme IDO in the antigen presenting cells, and subsequent suppression of T cell activation and function (154). CTLA-4 may also directly suppress effector T cells by binding to the accessory molecules, which are expressed on the cells upon activation. Furthermore, it has also been suggested that signaling via CTLA-4 transduces a costimulatory signal to Treg cells which is required for their activation. CTLA-4-deficient mice develop a severe autoimmune disease, however, Treg cells isolated from these mice display normal regulatory function in vitro

(154). Recent studies have identified another inhibitory membrane-associated molecule on Treg cells known as lymphocyte-activation gene 3 (LAG-3) (161). LAG-3 is a CD4- related adhesion protein that binds to MHC class II molecules expressed on DC and other

42 antigen presenting cells. Binding of LAG-3 to MHCII molecules induces a negative

signal that inhibits DC maturation and function. Antibodies against LAG-3 block Treg

cell suppression in vitro and in vivo, and Treg cells from LAG-3-deficient mice exhibit reduced function in vitro. However, loss of LAG-3 in mice does not result in the development of autoimmunity (161).

As over-suppression meditated by Treg cells could hinder the ability to elicit an

effective immune response to microbes and tumor cells, control of the magnitude of Treg

cell activity is important for the benefit of the host (154). Indeed, during an inflammatory

response associated with pathogenic infection or cancer, a number of inflammatory cytokines (such as IL-6 and TNF-α) produced by mature DC have a negative effect on the activation and expansion of Treg cells. Also, the binding of microbial particles to TLR

(TLR8 and TLR2) expressed by Treg cells triggers an inhibitory signal in the cells. Lastly, in response to inflammatory cytokines, strong TCR activation and GITR stimulation, effector T cells may become resistant to Treg-mediated suppression (154). The inhibition

of Treg function together with the resistance of effector T cells to suppression permits the development of an efficient immunity during the inflammatory response.

The data presented here suggest that the relative contribution of Treg-associated

molecules to the suppressive activity of the cells may depend on various factors,

including the genetic background of the host, the stimulant antigen, the experimental system and the site and type of the immune response. Dissecting the contribution of Treg molecules in these different settings would be a key issue in the future of Treg cell research, which may help explain the contradictory results obtained from the in vitro and in vivo studies. Ultimately, a better understating of how Treg cells work and defining the

43 molecules they use could potentially lead to the development of novel therapeutic

approaches for the treatment of a wide rang of immune-mediated diseases.

Table 1-1. Table shows some of the main Treg-associated molecules that have been described in the literature. Deletion of most of the effector genes leads to the development (to some degree) of autoimmune diseases. Treg cells from the respective gene-deficient mice, however, in some cases exhibit normal suppressive activity in vitro. Similarly, antibodies to the respective proteins block Treg cell activity in vivo, but in some occasions fail to do so in vitro. Further work is required to fully understand the controversy between the in vivo and in vitro results.

44 1.2. Fibrinogen-like protein 2 (fgl2/FGL2)

1.2.1. Identification of fgl2

Koyama et al. identified from a cytotoxic T cell library a T cell development gene

and named it pT49 (162). Subsequently, our group identified from macrophages an

identical gene and named it fgl2 (163). The encoded protein showed high homology to

the β and γ chains of fibrinogen and was therefore referred to as a fibrinogen-like protein

(musfiblp). Subsequent studies by Ruegg and Marazzi et al. identified the human

homologue of the musfiblp by screening a human small intestine cDNA library (164,165).

They referred to the human protein as fibroleukin/FGL2. FGL2 was shown to be

spontaneously secreted by both CD4+ and CD8+ T cell in vitro with preferential

expression in CD45R0+ memory T cells. In vivo, FGL2 production was detected in the

extracellular matrix of the lamina propria mucosa of the small intestine that is rich in T

lymphocytes. The function of FGL2 secreted by T cells was not evaluated in these studies,

but it was suggested that the protein might play a role in regulation of physiological activities at mucosal sites (164,165).

In reticuloendothelial (RE) cells, FGL2 is expressed as a membrane-associated

protein, which acts as a prothrombinase enzyme with the ability to generate thrombin

directly from prothrombin (166). The procoagulant activity of membrane-associated

FGL2 has been implicated in the pathogenesis of various human and experimental

models, including fulminant and chronic hepatitis, allo- and xenograft rejection and fetal

loss syndrome (167). Taken together, it appears that RE cells express FGL2 as a

membrane-associated molecule that is involved in the coagulation process, while T cells

45 produce a secreted form of the protein, which may possess a regulatory function (Figure

1-4).

Figure 1-4. Schematic view of the two forms of FGL2 with their respective function as was previously reported. Macrophages and endothelial cells express a membrane- associated FGL2, which acts as a prothrombinase, while T cells produce a secreted from of the protein that is proposed to play a role in regulation of immune responses.

1.2.2. Fgl2 gene and the encoded protein

The fgl2 gene has been localized to chromosome 7 and 5 in humans and mice, respectively. The human and murine fgl2 genes are composed of two exons that are separated by one intron. The fgl2 promoter contains cis element consensus sequences for the binding of various transcription factors, including Ets, AP1, Sp1, TCF1, Ikaros and

CEBP. Recruitment of these transcription factors to the promoter may contribute to the differential transcription and expression patterns of fgl2 across cell and tissue types, as was observed by previous studies (168). For instance, Ikaros and TCF1 could account for

46 the expression in lymphocytes, while members of the Ets family of transcription factors

may be important for fgl2 expression in vascular endothelium. In addition, a specific

member of the CEBP family of acute-phase proteins (CEBP/a) may contribute to the

constitutive expression of the gene in one cell type, whereas the recruitment of another

member of this family (CEBF/b) may be responsible for the induction of fgl2 expression

in another cell type (168). The identification of similar cis acting-elements in mouse and

human promoters suggests that fgl2 expression is regulated in a similar manner in both

organisms.

The fgl2 gene encodes a protein of 432 amino acids in mice and 439 amino acids

in humans. The deduced protein sequence contains a predicted signal peptide, five N-

linked glycosylation sites and conserved cysteine residues, suggesting that FGL2 may be

expressed as an oligomeric protein. Indeed, under non-reducing conditions the molecular

mass of the protein is 250-300 kDa and in reducing condition it is 64-70 kDa, indicating

that FGL2 in its natural state forms a tetrameric complex (164). Based on sequence and

structural analysis, it is predicted that the encoded protein is composed of two major regions, the N-terminal domain and the carboxyl-terminus (Figure 1-5). The N-terminal domain is proposed to have a linear conformation due to the presence of α-helical region and several conserved cysteine residues, which can promote coiled-coil formation. By a combination of site-directed mutagenesis and production of truncated proteins, it was shown that the serine 89 residue of the N-terminal domain accounts for the procoagulant activity of FGL2 (169). The 229-amino-acid-long carboxyl-terminus consists of a highly conserved globular domain, known as the fibrinogen-related domain (FRED) that is characteristic of the fibrinogen-related protein superfamily. This domain has been

47 reported to be responsible for the regulatory activity of several members of the

fibrinogen-related superfamily, and recently was also shown to account for FGL2-

mediated immunoregulation (170). The overall identity between the mouse and human

FGL2 is 78%, but within the FRED domain the two proteins share 90% homology (166).

The high identity shared by the mouse and human FGL2 indicates the conserved and

important function of the protein.

Figure 1-5. Secondary structure prediction of FGL2 protein by SWISS-MODEL. The protein consists of two major domains, the linear N-terminal (domain 1) and the globular C-terminal (domain 2). Domain 1 contains the serine 89 residue, which is responsible for the procoagulant activity of the protein, whereas the highly conserved domain 2 is proposed to account for the regulatory function of FGL2.

48 1.2.3. FGL2 as a regulator of immune responses

Although the prothrombinase activity of the membrane-associated FGL2

expressed by RE cells has been well-established by many studies, the exact role of T cell-

secreted FGL2 has remained undefined. Recent investigations by our group and other

laboratories have suggested that FGL2 might be important in the regulation of adaptive

immune responses. Furthermore, other members of the fibrinogen-related superfamily

that contain the FRED domain were previously shown to have an immunoregulatory

function in addition to their role in coagulation (170). These members include the soluble

tenascin that can inhibit T cell activation in response to alloantigens and Con A, and also

fibrinogen that has the ability to trigger a series of intracellular signaling events and

cellular responses upon interaction with integrins. Other members of this family also

include angiopoietins and ficolins, which have also been shown to regulate immune

responses (170).

Clinical studies have also suggested a role for FGL2 in immunoregulation. Kohno et al. showed that expression of fgl2 is downregulated in patients with both acute and chronic adult T cell leukemia/lymphoma (171). Patients with acute and chronic hepatitis

B infection expressed high levels of FGL2 in their livers (167). In addition, genomic analysis revealed a polymorphism in the fgl2 gene in patients that are susceptible to severe acute respiratory syndrome (SARS) and severe periodontitis (172,173). Lastly, a recent study has demonstrated abundant levels of FGL2 protein in livers of patients with

hepatocellular carcinoma (174). Collectively, these clinical data propose that FGL2 might be involved in regulation of immunity; however, direct evidence for this is still lacking.

49 The role of FGL2 in regulation of adaptive immune responses was first shown by

Chan et al. (170), who studied the molecular and functional properties of the protein in vitro. A recombinant FGL2, which was generated in a baculovirus expression system, inhibited the proliferation of T cells in response to stimulation with anti-CD3/CD28 antibodies, Con A and alloantigens. The inhibitory effect of FGL2 on T cells was mediated through suppression of DC maturation, characterized by the inhibition of NF-

κB nuclear translocation and downregulation of the maturation markers CD80 and MHC class II molecules. The suppressive effects of FGL2 were abrogated in the presence of specific antibodies directed against the C-terminal domain of the molecule, indicating that the carboxyl FRED region accounts for the regulatory activity of the molecule. The recombinant protein also polarized allogeneic T cell responses towards a Th2 cytokine profile with increased production of IL-10 and IL-4, and decreased secretion of Th1 cytokines, including IFN-γ and IL-2 (170). In another study, it was shown that the absence of FGL2 was associated with accelerated cellular rejection in a xenotransplant model (175).

1.2.4. FGL2 as a marker of regulatory T cells

A number of groups have recently reported that regulatory T cells have increased fgl2 gene transcription detected by microarray gene analysis. Herman et al. were the first to report the abundant levels of fgl2 mRNA in Treg cells isolated from pancreas lesions, possibly involved in the prevention of experimental type 1 diabetes (176). Subsequent studies by Rudensky et al. have also detected high expression of fgl2 in Treg cells isolated from wild-type Foxp3gfp mice and IL-2-/- mice. Upregulation of fgl2 expression

was observed in Treg cells from IL-2-/- mice that were treated with recombinant IL-2 for

50 24 hours (177). Rudensky and colleagues further showed a positive correlation between

Foxp3 and fgl2 expression, however, the expression of fgl2 was not controlled directly by

Foxp3 binding to the promoter of the gene (178).

In addition to CD4+CD25+ Treg cells, other subsets of regulatory T cells have also

been found to express high levels of fgl2. A recent report showed that CD8αα+ regulatory

T cells in the intestine of mice over-express fgl2, which was suggested to play a role in the function of these cells (120). Our laboratory has detected high production of FGL2 protein and mRNA in both primary and clones of DN regulatory T cells. Interestingly, high levels of FGL2 were produced by a functional clone of DN T cells, but were undetectable in a nonfunctional clone. Furthermore, increased expression of fgl2 in

primary DN T cells correlated with their activation in vitro (detailed information in the

Discussion section). Finally, Anegon et al. found increased expression of fgl2 in

CD8+CD45RClow regulatory T cells that mediate allograft tolerance in a rat model

(personal communication). The role of FGL2 in these different types of regulatory T cells

is currently being evaluated by us and the respective groups.

1.3. Hypothesis

Based on these observations we postulate that T cell-expressed FGL2 contributes

to the suppressive activity of CD4+CD25+ Treg cells. Furthermore, we propose that lack of FGL2 (or its blockade) would lead to impaired function of Treg cells, accompanied by increased reactivity of immune cells and development of an autoimmune disease

(Chapter 2). We propose that increased levels of FGL2 produced by Treg cells would impede the ability of the host to mount an effective immune response against viral infection, resulting in increased susceptibly to the virus (Chapter 3). Since FGL2 can

51 inhibit T cell alloantigenic responses in vitro, we predict that treatment with FGL2 in vivo could prevent allograft rejection. We propose that FGL2 mediates its suppressive effects through binding to an inhibitory receptor expressed on antigen presenting cells (Chapter

4).

1.4. Objectives

To study the immunoregulatory role of FGL2 as an effector cytokine of CD4+CD25+

Treg cells we propose to do the following:

1. Characterize the function and phenotype of Treg cells in mice that lack FGL2

expression (fgl2-/- mice) compared to littermate control mice, including

measurement of fgl2 expression in wild-type Treg cells and FGL2 antibody

blocking studies (Chapter 2).

2. Evaluate the contribution of Treg-expressed FGL2 in the pathogenesis of viral

infection utilizing the well-established model of MHV-3-induced fulminant

hepatitis. This study includes characterization of the Treg cell compartment and

FGL2 production levels in susceptible and resistant mouse strains, FGL2 antibody

blocking studies (in vitro and in vivo), as well as adoptive transfer of fgl2+/+ Treg

cells into resistant fgl2-/- mice (Chapter 3).

3. Examine the immunoregulatory properties of FGL2 in vivo by using an

allotransplant model. In this model, skin graft recipient mice will be treated for

several days with recombinant FGL2 to study the suppressive effect of the protein

on graft survival and histology. In this chapter, the mode of FGL2 action will also

be assessed by both in vitro and in vivo studies (Chapter 4).

52 1.5. Goal

Understanding the mechanism by which Treg cells mediate suppression and defining the molecules that are involved in their activity would allow for the development of novel therapeutic strategies to treat and prevent autoimmune disorders as well as graft rejection. This could be achieved by the use of FGL2 or FGL2 analogs as an immunosuppressant therapy. Treatment with anti-FGL2 antibodies to neutralize the suppressive effect of the protein may potentially enhance immune responses against pathogens and tumor cells, resulting in clearance of chronic infections and inhibition of cancer development.

53 Chapter 2: Targeted Deletion of FGL2 Leads to Impaired regulatory T cell Activity

and Development of Autoimmune Glomerulonephritis

2.1. Summary

Mice with targeted deletion of fibrinogen-like protein 2 (fgl2) spontaneously developed autoimmune glomerulonephritis with increasing age, as did wild-type recipients reconstituted with fgl2-/- bone marrow. These data implicate FGL2 as an

important immunoregulatory molecule and led us to identify the underlying mechanisms.

Deficiency of FGL2, produced by CD4+CD25+ regulatory T (Treg) cells, resulted in

increased T cell proliferation to lectins and alloantigens, T helper 1 (Th1) polarization,

and increased numbers of antibody-producing B cells following immunization with T-

independent antigens. Dendritic cells (DC) were more abundant in fgl2-/- mice and had

increased expression of CD80 and MHCII following LPS stimulation. Treg cells were

also more abundant in fgl2-/- mice, but their suppressive activity was significantly

impaired. Antibody to FGL2 completely inhibited Treg cell activity in vitro. Collectively,

these data suggest that FGL2 contributes to Treg cell activity and inhibits the

development of autoimmune disease.

54 2.2. Introduction

FGL2/fibroleukin is a member of the fibrinogen-related protein superfamily of

proteins (179,180). FGL2 was first cloned from human cytotoxic T cells, and its protein

structure deduced from the nucleotide sequence showed a high degree of homology to

fibrinogen β and γ subunits (162). FGL2 has been shown to play an important role in

innate immunity as an immune coagulant expressed by activated reticuloendothelial cells

(macrophages and endothelial cells) (181-183) and has been implicated in the pathogenesis of several inflammatory disorders, including viral hepatitis

(163,167,184,185), allo and xenograft rejection (175,186,187) and cytokine induced fetal loss (188,189). In a previous report, we showed that antibody to FGL2 ameliorated allograft rejection (187). Furthermore, cardiac grafts from fgl2-/- mice were resistant to

thrombosis, suggesting a role for endothelial cell fgl2 expression in the fibrin deposition

associated with allo and xenotransplantation rejection. In contrast, Hancock and Ning

showed that fgl2-/- mice rejected fully mismatched allografts, supporting the contention

that the endothelium but not leukocytes maybe more important for fibrin deposition in

allotransplant rejection (187,190).

In addition, Marazzi et al. reported that both CD4+ and CD8+ T cells spontaneously

secrete FGL2 with preferential expression in CD45R0+ memory T cells (164,165). They

suggested that secreted FGL2, which is devoid of prothrombinase activity, may have

immune regulatory activity (164). Our lab provided the first evidence that FGL2 also

plays a role in acquired immune responses by demonstrating its immunosuppressive

activity on dendritic cells (DC) and T cells (170). In vitro, recombinant FGL2 inhibited T

cell proliferation in response to anti-CD3/CD28 antibodies, Concanavalin A (Con A) and

55 alloantigens. Moreover, FGL2 promoted a Th2 cytokine profile in allogeneic cultures and

inhibited DC maturation by preventing NF-κB nuclear translocation (170).

Recently, Rudensky, Herman and Kronenberg have reported increased expression of

fgl2 in regulatory T cells including CD4+CD25+Foxp3+ regulatory T (Treg) cells and

CD8αα+ T cells, and proposed that fgl2 is a putative regulatory T cell effector gene

(120,148,176-178,191,192). In this study, we showed that Treg cell function was completely inhibited by antibody to FGL2. We further demonstrated that suppressive activity of Treg cells from fgl2-/- mice was impaired, resulting in increased DC, T and B

cell immune reactivity and development of autoimmune renal disease. Collectively, these

data support the notion that FGL2 contributes to Treg cell function and has important

immunoregulatory activity.

56 2.3. Materials and Methods

Mice

Fgl2-/- and fgl2+/+ mice were previously generated and the methodology for their production has previously been described (167). Briefly, fgl2-/- mice were generated by

transfecting 129Sv embryonic stem cells with the knockout construct (also derived from

129Sv). These stem cells were then injected into C57BL/6 blastocysts and the resultant chimeras were backcrossed for 10 generations onto a C57BL/6 background (167). fgl2+/+

mice were used as controls. BALB/c and FcγRIIB-/- mice were obtained from the Jackson

Laboratories (Bar Harbor, ME). Mice were housed in the animal facility at Princess

Margaret Hospital and treated under the guidelines provided by the University Health

Network. Male and female mice used for assays were 6 to 8 weeks old unless specified in

the text.

Tissue mass and quantification

Mice were anesthetized with pentobarbital. Blood was collected by cardiac

puncture, and serum samples were stored at -80oC. Tissues were dissected and fat and

connective tissue were removed. Prior to weighing, tissues were rinsed with PBS to

remove blood and excess fluid was removed.

Tissue fixation and sectioning

Frozen tissue was prepared by embedding in OCT (Fisher)-filled cryomolds

(TissueTek) and snap frozen in liquid nitrogen. For paraffin-embedded preparations, dissected tissues were immersed in 10% formalin and fixed for 24 hours, which were then embedded in paraffin and sectioned in the Pathology Department at the Hospital for

57 Sick Children. Paraffin sections, 5 microns thick, were stained using standard H&E and

periodic acid-Schiff (PAS) staining protocols (167).

Renal function

Serum creatinine was analyzed in the Department of Biochemistry, Univeristy

Health Network using an Ektachem 700 analyzer as previously described (167). Urine was collected from mice and analyzed for pH, presence of protein, blood, glucose and ketones by Labstix purchased from Bayer HealthCare Inc. (Toronto, ON, Canada).

Histopathology and immunohistochemistry

Kidneys were obtained and histological sections were stained with PAS or H&E

(193). Glomerular and vascular deposition of IgG, IgM and IgA was determined by

staining frozen sections with rat anti-IgG, anti-IgA and IgM monoclonal antibody,

followed by alkaline phosphatase labeled goat anti-rat Ig conjugates as previously

described (193). C3 deposits were examined by direct staining with alkaline phosphatase

labeled goat anti-mouse C3 (Cappel Laboratories, West Chester, PA).

Treg cell isolation

For real-time PCR gene studies, CD4+CD25+ Treg cells and CD4+CD25- T cells

were purified with an EasySep® Mouse CD4+ T Cell Enrichment Kit (StemCell

Technologies Inc.) according to manufacturer’s instructions followed by FACS using a

FACSAria cell sorter (Becton Dickinson, CA). Purity of cell populations was ≥ 98%. For

other experiments, CD4+CD25+ Treg cells and CD4+CD25- T cells were purified using a

FACSAria cell sorter with purity of ≥ 90-95%.

58 Splenocyte isolation

Spleens were dissected, flushed with PBS using a syringe and needle and cut up

into small pieces (< 0.5cm), which were passed through a nylon filter to separate cells

from connective tissue. Mononuclear cells were separated from erythrocytes using a

Lympholyte M density separation medium (Cedarlane, Burlington, Canada). To release

DC, spleens were digested with collagenase D (Roche Diagnostics, Laval, Canada) for 45 minutes at 37oC, cell suspensions were then separated using Lympholyte M.

DC Generation from bone marrow (BM) precursors and stimulation with LPS

BM was isolated and cultured as previously described (170). Briefly, BM was

flushed from tibia and femurs, and erythrocytes and T cells were subsequently removed

using Lympholyte M and magnetic-activated cell sorting (MACS) (Miltenyi Biotec,

Auburn, CA), respectively. The remaining BM fractions were cultured on plastic Petri dishes (NUNC) for 2 hours to remove adherent macrophages. BM was cultured for 7 days in RPMI media supplemented with 10% FBS, 800 U/ml GM-CSF and 500 U/ml IL-

4 (Sigma). After 7 days, cultures were stimulated with 1 µg/ml O5:B55 LPS (Sigma).

T cell proliferation assay

Splenocytes were cultured for 3 days in α-MEM supplemented with 10% FBS,

after which 1µCurie 3H-thymidine (Amersham Biosciences) was added to each well. 18

hours later, cells were harvested using the UNIFILTER-96 Filtermate Harvester (Perkin

Elmer) and counted by Packard Microplate Scintillation Counter. One-way Mixed

Lymphocyte Reaction (MLR). Fgl2-/- or fgl2+/+ responder splenocytes (4x105/100 µl) were stimulated with irradiated BALB/c splenic mononuclear cells (4x105/100 µl). Stimulator

BALB/c splenocytes were irradiated with 30Gy using Nordin Gamma-Irradiator to

59 prevent cell division. Con A stimulation. Splenocytes were plated at a concentration of

2x105 cells/well in 200 µl of media and stimulated with 5 µg/ml Con A (Sigma).

Flow cytometry

Antibodies used for flow cytometry. Detection antibodies included: FITC-B220,

PE-B220, FITC-CD11b, FITC-CD11c, PE-CD11c, PE-Cy5-CD11c, FITC-CD3, PE-Cy5-

CD3, FITC-CD4, PE-Cy7-CD25, PE-Foxp3, PE-CD40, FITC-CD8α, PE-CD8α, PE-

CD80, PE-CD86, PE-IgD, FITC-IgM and PE-Cy5- NK1.1. Isotype controls included:

FITC-armenian hamster IgG, FITC-mouse IgG2a, PE-mouse IgG2a, PE-mouse IgG2b,

FITC-rat IgG2a, PE-rat IgG2a, PE-Cy5-rat IgG2a, PE-rat IgG2b and PE-Cy5-rat IgG2b.

All antibodies were obtained from Cedarlane, BD Pharmingen or eBiosciences.

Cell labeling and analysis. Cell suspensions were washed and suspended in PBS

7 containing 3.5% mouse serum (Cedarlane) at a final concentration of 1x10 cells/ml in

order to block Fc receptors. 100 µl of the cell suspension was aliquoted into 5 ml

polypropylene test tubes and antibody was added and incubated for 30 minutes at 4oC.

Antibody binding was assessed using a Coulter Epics-XL-MCL flow cytometer and data were analyzed using CXP/RXP software (Beckman Coulter). Live cells were gated according to forward and side scatter parameters.

Annexin V and propidium iodide (PI) labeling. To 100 µl of cell suspension (containing

1x106 cells), 10 µl of Annexin-V (Southern Biotech) was added and incubated at 4oC for

15 minutes. 380 µl of binding buffer was added along with propidium iodide (Southern

Biotech) and incubated for another 15 minutes prior to analysis by flow cytometry.

60 ELISA

Supernatants were removed from MLR cultures at 24 and 48-hour time points.

Cytokine profiles were obtained using IL-4, IL-2 and IFN-γ ELISA kits (Pierce). ELISA

was performed according to the kit instructions. Plates were coated with anti-cytokine

antibody; supernatant samples were incubated on the plates for 2 hours at 37oC. Cytokine

binding was detected by using a secondary HRP-conjugated anti-cytokine antibody. TMB

substrate was used as a chromogenic substrate. Absorbance was measured at 450nm.

ELISPOT

B cells were purified from spleens of mice using the MACS purification system.

Purified B-cells (1x103 per well) were incubated on Millipore plates coated with anti-IgM

and detected with a biotin-conjugated secondary antibody. Spots were developed using the AEC substrate set (BD Biosciences) and detected with ELISPOT Reader series 3A

Analyzer. Antibody production was induced by injection of LPS, NP-Ficoll or NP-CGG.

Immunization protocol

Mice were intravenously injected with LPS (Sigma) at a dose of 1.5 µg/g of body

weight. NP-Ficoll (Sigma) and NP-CGG (Biosearch Technologies) were given by intraperitoneal (IP) injection at 30 µg and 100 µg doses respectively. Mice were euthanized 5 days after LPS injection and 12 days following treatment with NP-Ficoll or

NP-CGG. Alum precipitation of NP-CGG was performed to prepare the reagent for injection. First, 400 μl of aluminum potassium sulfate (Sigma) per 100 µg NP-CGG were combined. Protein was precipitated by the addition of 1N potassium hydroxide and the precipitate was then washed 3 times with PBS and 100 µg NP-CGG with alum was injected IP (194).

61 Generation of BM chimeras

Reciprocal transplants were performed between fgl2+/+ and fgl2-/- donors and

recipients. Donor BM cells were prepared as previously described for the generation of in

vitro DC cultures. Two days prior to irradiation and 2 weeks after transplantation, mice

were given clavamox in their drinking water to prevent infection. Recipients were

irradiated with 11Gy using a Nordon Gamma-Irradiator and immediately intravenously

injected with 5x106 BM cells. Two months after injection, chimeric mice were euthanized

and cells were harvested and analyzed.

Real-time PCR

Following the purification of splenic CD4+CD25+ and CD4+CD25- T cells by

FACS, RNA was extracted from the cells and the levels of fgl2 mRNA expression were

measured by real-time PCR. fgl2 expression levels were normalized to HPRT, GAPDH

and RPL13, which served as house-keeping genes. 2-ΔΔCT calculations were used to

present levels of fgl2 expression in CD4+CD25+ relative to CD4+CD25- T cells.

Suppression assay

In vitro suppression assays used cultures of 2x104 CD4+CD25- T cells from fgl2+/+

mice as responder cells, together with 8x104 irradiated splenocytes as APCs and titrated numbers of CD4+CD25+ Treg cells from either fgl2-/- or fgl2+/+ mice as suppressor cells.

Cultures were stimulated with Con A (1 µg/ml) for 72 hours and 3H-thymidine was added

for the last 18 hours to measure proliferation of effector T cells. For antibody blockade

studies, titrated concentrations of a monoclonal antibody to FGL2 (#H00010875-M01

monoclonal IgG2a antibody, Abnova, Taiwan) were added to the cell cultures of CD4+

62 effector T cells and CD4+CD25+ Treg cells sorted from wild-type mice, at a 1:4 suppressor:responder cell ratio in the presence of APCs and Con A.

Statistical analysis

Statistical significance was assessed by a Student's t-test or by ANOVA; differences with P ≤ 0.05 were considered significant.

63 2.4. Results

2.4.1. Gross morphology, histology and mass of fgl2-/- mice

The generation of fgl2-/- mice has been previously described (167). Gross

morphology and histology of tissues and organs from young (6-8 weeks) and old (7 months) fgl2-/- and fgl2+/+ mice were compared. In young fgl2-/- mice, all organs appeared

normal as assessed by gross appearance, histological analysis and weight; however, 25%

fewer Peyer's Patches were found in the small intestine. Decreased number of intestinal

Peyer’s Patches and follicles was also found in older fgl2-/- mice. While 6 to 8-week-old

fgl2-/- and fgl2+/+ control mice exhibited similar body weights, at 7 months of age, fgl2-/-

mice were significantly smaller in size and weight (P ≤ 0.05). By 7 months of age, kidney

size and weight were smaller in 25% of fgl2-/- mice as compared to fgl2+/+ mice. Spleens

were enlarged in fgl2-/- mice compared to littermate controls (data not shown).

2.4.2. Constitutive FGL2 expression

The LacZ reporter gene inserted into exon 1 of the fgl2-/- construct was used to

examine constitutive FGL2 expression (167). β-galactosidase activity was detected in

lymphoid organs, including bone marrow (BM), thymus, lymph nodes and spleen. β-

galactosidase staining was also detected in the stomach and intestine, which was

localized primarily to the lamina propria, consistent with reports by others (164).

Presence of FGL2 protein was compared by immunohistochemistry. In particular heart

and intestine stained strongly for presence of FGL2 (data not shown).

2.4.3. Increased reactivity of B and T cells from fgl2-/- mice

Previous studies from our laboratory suggested that FGL2 has immunosuppressive

activity (170). To further explore the effects of FGL2 on the immune system, we

64 examined the proportion and activity of T and B cells from fgl2-/- mice. While we found

similar proportions of T cells (CD4+ and CD8+) and B cells (IgM+ and IgD+) in the

spleens of fgl2-/- and fgl2+/+ mice, T and B cells from fgl2-/- mice were shown to have

increased reactivity and effector T cells were polarized towards a Th1 response. T cells

from fgl2-/- mice had increased proliferation in response to Con A (P ≤ 0.04) and in a one

way mixed lymphocyte reaction (MLR) (P ≤ 0.05) (Figure 2-1A), consistent with our

previous in vitro data, which showed that FGL2 inhibited T cell proliferation (170). Th1

and Th2 cytokine profiles were analyzed by ELISA. IFN-γ, a Th1 cytokine, was found to

be markedly increased, whereas the production of the Th2 cytokine IL-4 was diminished

in supernatants from the MLR cultures of fgl2-/- responder cells (Figure 2-1B).

Type-1 (TI-1), Type-2 (TI-2) T cell-independent and T cell-dependent (TD) B cell

responses were analyzed by ELISPOT. In response to the TI antigens, LPS (P ≤ 0.02) and

NP-Ficoll (P ≤ 0.03), increased numbers of antibody-producing B cells from fgl2-/- mice were seen (Figure 2-1C and 1D). However, the amount of antibody produced per cell as indicated by the size of the spot was similar in B cells from fgl2+/+ and fgl2-/-mice. Fgl2-/-

mice immunized with the T-dependent antigen NP-CGG had similar numbers of

antibody-producing B cells and equivalent antibody production per cell as fgl2+/+ mice.

These data indicate that FGL2 is involved in the regulation of T-independent B cell

responses, which require help from antigen-presenting cells (APCs), but not T-dependent

B cell responses.

65

Figure 2-1. Increased reactivity of B and T cells from fgl2-/- mice. (A) T cell proliferation in response to Con A stimulation and in a MLR as measured by [3H]thymidine (1µCi) incorporation; n = 6. (B) Levels of IFN-γ (left) and IL-4 (right) in supernatants from MLR reactions, taken at 24 h poststimulation as assessed by sandwich ELISA; n = 3. (C and D) Mice were injected with LPS, NP-Ficoll, or NP-CGG. At 5–12 days after injection, spleens were isolated and subjected to ELISPOT analysis. Representative pictures of triplicate ELISPOT wells from fgl2+/+ and fgl2-/- mice stimulated with LPS are shown. The number of spots per well (top right) is representative of the number of B cells producing Ab per well. Average spot size (bottom right) is a measure of the amount of Ab being produced per B cell; n = 3. Graphs in all panels show mean ± SEM.

66 2.4.4. Increased numbers and reactivity of DC from fgl2-/- mice

In the spleen of fgl2-/- mice, we observed a 30% increase in the proportion and

absolute numbers of DC (CD11c+MHCII+) (P ≤ 0.01) (Figure 2-2A), while the

proportions of macrophages (CD11b+MHCII+) were similar between fgl2-/- and fgl2+/+ mice. A significant increase in the number of DC obtained from in vitro BM cultures was seen in fgl2-/--derived DC cultures (P ≤ 0.01) (Figure 2-2B). We further examined the

proportions of DC subsets in lymphoid tissues based on the expression of B220, CD11b

and CD8α. In fgl2-/- mice, we observed an increase in all plasmacytoid and myeloid DC

subsets rather than an increase in a specific subset (data not shown).

The increase in the number of DC present in the spleen and BM of fgl2-/- mice could

be explained by an abnormal proliferation of DC precursors or an increase in DC lifespan.

Forward and side scatter plots showed that DC derived from fgl2+/+ mice at 24 and 72

hours post-LPS stimulation contained more dead cells than cultures of DC derived from

fgl2-/- mice. Cultures were stained with Annexin V and propidium iodide to assess for

apoptosis. At 24 hours post-LPS stimulation, there was a 3-fold increase in the proportion

of DC staining positive for Annexin V in fgl2+/+ cultures as compared to cultures of DC

from fgl2-/- mice. At 72 hours, nearly all DC from fgl2+/+ mice stained positive for

propidium iodide (Figure 2-2C), whereas in fgl2-/- cultures, a smaller population of DC

stained positive. Thus in the absence of fgl2, apoptosis of LPS-stimulated DC in culture is

both decreased and delayed.

Unstimulated DC generated from BM cultures from both fgl2-/- and fgl2+/+ mice

expressed similarly low levels of CD80, CD86, CD40, MHCII, MHCI and DEC205. DC from both fgl2-/- and fgl2+/+ mice after LPS stimulation showed increased expression of

67 these maturation markers. However, we observed higher levels of CD80 and MHCII

expression in DC from fgl2-/- mice (Figure 2-2D). These data collectively suggest that DC

from fgl2-/- mice are increased in numbers and achieve greater activation following LPS

stimulation.

The migration rate of DC from the bridging junction to peri-arteriolar lymphoid

sheath (PALS) of the spleen following LPS stimulation was assayed by

immunohistochemistry. Murine DC in the spleen reside in the bridging junctions between

the PALS and B cell follicle, and upon antigenic stimulation, migrate into the PALS. At

time 0 (pre-injection), splenic DC from both fgl2+/+ and fgl2-/- mice were localized to the

bridging junctions between the PALS and B cell follicle. By 2-4 hours post-LPS injection,

DC migrated to the PALS in both fgl2+/+ and fgl2-/- mice. However, comparison of DC

staining in the PALS at 30 minutes and 1 hour post-LPS stimulation showed that DC in fgl2-/- mice moved into the PALS at a faster rate (Figure 2-2E), although this was not

statistically significant.

68

Figure 2-2. Increased numbers and reactivity of DC from fgl2-/- mice. (A) The percentage of DC (CD11c+MHCII+) and macrophages (CD11b+MHCII+) in spleen of fgl2+/+ and fgl2-/- mice. Graph shows mean ± SEM; n = 6. (B) Representative flow cytometry plot of BM-derived DC stimulated with 1 µg/ml LPS; n = 6. (C) BM-derived DC from fgl2+/+ and fgl2-/- mice were harvested at 24 h and 72 h after LPS stimulation and stained with PI and Annexin V. Live and dead cells were first differentiated on the basis of forward and side scatter (left). Numbers beside outlined areas indicate a representative percentage of dead cells in the designated gate. Representative flow cytometry values of Annexin V+ DC at 24 h (left) and PI+ DC at 72 h (right) are shown. (D) Expression of surface molecules on DC with and without LPS stimulation by flow cytometry. BM-derived DC were cultured with 800 U/ml GM-CSF and 500 U/ml IL-4 for 7 days. A total of 1 µg/ml LPS was then added and cells were cultured for another 18 h to induce maturation and induction of high levels of costimulatory molecules. Control cultures did not receive LPS and therefore represent the expression profile of immature DC. (E) Kinetics of DC movement in the spleens of fgl2+/+ and fgl2-/- mice. Mice were i.v. injected with 1.5 µg/g of body weight LPS and sacrificed at times 0, 30 min, 1, 2, and 4 h after receiving LPS. Frozen sections were subsequently stained with CD11c (brown) and B220 (blue). Movement from the bridging junction into the PALS is complete between 2 and 4 h. Sections at 1 h and 2 h are similar to sections at 30 min and 4 h, respectively (data not shown).

69 2.4.5. Normal spleen architecture and formation of germinal centers (GC) was

observed in fgl2 -/- mice

In order to assess whether fgl2 is important for splenic organization and germinal

center formation, fgl2-/- and fgl2+/+ mice were injected with saline (control) or NP-CGG

(a T cell-dependent antigen). Spleens from each group were harvested at 12 days post-

immunization and analyzed by immunohistochemistry and flow cytometry. Distribution

of T and B cells was visualized by staining tissue sections with anti-CD3 and anti-B220

antibodies (data not shown). In both fgl2+/+ and fgl2-/- spleens, a clear interface between the T cell-rich PALS and surrounding B cell follicles was observed. The MAdCAM-1- stained marginal sinus, which separates the marginal zone from the outer follicular region of the B cell follicle was evident in both fgl2-/- and fgl2+/+ spleens. Furthermore, extensive peanut agglutinin (PNA) staining, the GC B cell marker, was observed in the

PALS of both fgl2-/- and fgl2+/+ spleens following immunization with NP-CGG. This was

confirmed by flow cytometry analysis of GC B cells (B220+GL7+Fas+).

2.4.6. Development of autoimmune glomerulonephritis in fgl2-/- mice

With age, fgl2-/- mice lost weight and by 6 months, twenty-five percent of fgl2-/- aged mice developed severe glomerulonephritis, with characteristically small and yellow kidneys (Figure 2-3A). Staining of kidneys with the periodic acid-Schiff reaction (PAS) revealed extensive infiltration of mononuclear cells and interstitial fibrosis (Figure 2-3B).

Many renal tubules had collapsed and were surrounded with fibrin, and mesangial expansion was evident in the glomerulus (Figure 2-3C). Hemosiderin staining of macrophages indicated the presence of hemorrhage within the kidney (Figure 2-3D).

Such kidney defects were not observed in fgl2+/+ mice at any age up to 9 months (n=20).

70 To further explore the renal abnormality observed in 6-12-month old fgl2-/- mice, kidneys were harvested from fgl2-/- mice at 1, 3 and 6 months and examined by routine histology and stained for the presence of immunoglobulins (IgA, IgG and IgM).

Histological examination of kidneys at different time points was done on H&E stained sections (Figure 2-4A). Kidney structure was normal at 1 month. In particular the glomeruli were normal, aside from very minimal mesangial thickening. By 3 months there was focal segmental mesangial thickening and mild increase in cellularity of glomeruli (Figure 2-4A). By 6 months the glomerular changes were severe and widespread with generalized increase in cellularity, marked mesangial thickening and marked decrease in vascularity. Additionally, at 6 months there was also a heavy interstitial inflammatory cell infiltration comprised predominantly of lymphocytes. There was also marked tubular loss, interstitial sclerosis and fibrosis (Figure 2-4A).

Immunoperoxidase staining for IgG, IgM, and IgA was strongly positive in glomeruli and in infiltrating cells (Figure 2-4A). Kidneys from wild-type mice were normal at all times.

C3 deposits were detected in kidneys from fgl2-/- mice at 1, 3 and 6 months coincident with deposits of immunoglobulins.

Urine and serum analysis were also performed at different time points and revealed progressive increases in the levels of protein (albumin) and blood in the urine (Figure 2-

4C) and increases in serum creatinine (Figure 2-4B) in fgl2-/- mice with age. In contrast, in wild-type mice the levels of albumin and blood in the urine (unpublished data) and serum creatinine (Figure 2-4B) were normal at all times. The levels of IgG and IgM autoantibodies against dsDNA, ssDNA or chromatin in aged fgl2-/- mice were normal and comparable to aged wild-type mice (data not shown).

71

Figure 2-3. Kidney defect observed in 7- to 12-mo-old fgl2-/- mice. (A) Gross appearance of a fgl2-/- kidney that developed a severe glomerulonephritis compared with the littermate control. (B-D) Histology of the defective kidney from fgl2-/- mice stained with PAS. (B) Intense cellular infiltration and fibrin deposition (10x magnification). (C) Mesangial expansion of the glomerulus (asterisk) and collapsed tubules surrounded by fibrin (arrow) (40x magnification). (D) Brown staining (arrow) is hemosiderin staining of macrophages and indicates the presence of hemorrhage (40x magnification).

72

Figure 2-4. The evolution of autoimmune glomerulonephritis. (A-1) Kidney from wild-type mouse at 6 mo. Normal glomerulus. HE, x 400. A-2–A-10, Kidneys from fgl2-/- mice. (A-2) At 1 mo, glomeruli show slight focal mesangial thickening. Tubules are normal. PAS x 400. (A-3) At 3 mo, glomeruli show slight mesangial thickening and slightly increase in cellularity. Capillary loops widely patent. Tubules are normal. PAS x 400. (A-4) At 6 mo, glomeruli show prominent mesangial thickening and there is increase in cellularity. Capillary loops hard to visualize. Tubules are normal. PAS x 400. (A-5) At 6 mo, another area showing marked glomerular changes with global mesangial thickening, sclerosis, and loss of vascularity. Note also loss of tubules, interstitial inflammatory cell infiltrate, sclerosis, and collapse of parenchyma. PAS x 200. (A-6) At 6 mo, IgG isotype control. Note glomerulus in the center with increased cellularity and surrounding renal parenchyma with inflammatory cell infiltrate and fibrosis. Severe glomerulonephritis. This control immunoperoxidase stain is negative. IP x 400. (A-7) At 6 mo, immunoperoxidase stain for IgG. The glomerular mesangium is positively stained.

73 IP x 400. (A-8) At 6 mo, immunoperoxidase stain for IgM. The glomerular mesangium is positively stained. IP x 400. (A-9) At 6 mo, immunoperoxidase stain for IgA. The glomerular mesangium is positively stained. IP x 400. (A-10) At 6 mo, immunostaining of cellular infiltrate for IgG. Many of the infiltrating cells are positive. The same result was seen with IgM and IgA (data not shown). IP x 400. (B) Levels of creatinine in the serum of fgl2-/- and fgl2+/+ mice. Data represent as mean ± SEM; n = 5, *, P ≤ 0.05. (C) Urinalysis of fgl2-/- mice. Urinalysis of fgl2-/- mice was performed at different time points. The levels of blood, ketones, glucose, protein, and pH are shown. Blood levels are shown as + for mild, ++ for moderate, and +++ for high. Protein levels are presented as mean ± SEM; n = 5.

+/+ -/- 2.4.7. Fgl2 recipients reconstituted with fgl2 BM exhibit the phenotype of fgl2-/-

mice

We generated BM chimeras in order to determine whether the etiology of the

increased immune reactivity of fgl2-/- mice was hematopoietic. Mice were sublethally

irradiated and fgl2+/+ mice were reconstituted with fgl2-/- BM, whereas fgl2-/- mice were

reconstituted with fgl2+/+ BM. Populations of macrophages, T cells, B cells and DC

purified from each of the transplant groups by magnetic cell sorting (MACS) were

genotyped to confirm that reconstitution had occurred (unpublished data). By three

months after BM reconstitution, elevated levels of creatinine were measured in the serum

of fgl2+/+ mice reconstituted with fgl2-/- BM (Figure 2-5A), and one of these mice

developed a severe kidney defect as seen in fgl2-/- aged mice. The kidney was small in

size (Figure 2-5B) and displayed the following histological features: intense cellular

infiltrate, fibrin deposition, expanded mesangial matrix of some glomeruli and collapsed

tubules. Hemosiderin staining was also present indicating hemorrhage (Figure 2-5C).

Mice were followed for up to 9 months and similar kidney abnormalities were seen in all

fgl2+/+ mice reconstituted with fgl2-/- BM.

In addition, fgl2-/- BM-reconstituted mice exhibited increased proliferation of T cells in response to alloantigens and Con A (Figure 2-5D), increased numbers of DC (Figure

74 2-5E) with increased expression of cell surface CD80 and MHCII compared to fgl2+/+

BM-reconstituted mice (Figure 2-5F).

Figure 2-5. Fgl2+/+ recipients reconstituted with fgl2-/- BM exhibit the phenotype of fgl2-/- mice. Fgl2-/- and fgl2+/+ mice were irradiated with 11 Gy of γ-irradiation before injection with BM from fgl2+/+ and fgl2-/- mice, respectively; assays were performed following BM reconstitution. (A) Levels of creatinine in the serum of fgl2-/- and fgl2+/+ BM-reconstituted mice at different time points (indicated by number of months) following BM reconstitution. Data represent mean ± SEM; n = 5, *, P ≤ 0.05. (B) Gross appearance of a defective kidney from fgl2-/- BM-reconstituted mice, which developed a severe glomerulonephritis. The kidney was small in size and displayed histological abnormalities. (C) Evidence of mesangial expansion (arrowhead), collapsed tubules (arrow), cellular infiltrates, fibrin deposition, and hemosiderin staining (asterisks) were shown with PAS staining. (D-F) Two months following BM reconstitution, splenocytes, and BM were harvested from each treatment group for analysis of the development and function of T cells and DC. (D) T cell proliferation in response to Con A or in a MLR. Data represent as mean ± SEM. (E) Flow cytometry of DC (CD11c+MHCII+) in spleen of fgl2-/- and fgl2+/+ BM-reconstituted mice. Numbers indicate a representative percentage of cells in the designated gate. (F) Representative expression of costimulatory molecules on the surface of BM-derived DC by flow cytometry.

75 2.4.8. Increased expression of fgl2 mRNA in CD4+CD25+ T cells

Previous studies have reported increased expression of fgl2 mRNA transcripts in

Treg cells and suggested a role for fgl2 as a putative Treg cell effector gene (148,176-

178,191,192). To examine this possibility, we first assessed the expression of fgl2 in Treg

cells. CD4+CD25+ T cells and CD4+CD25- T cells were purified from spleens of

C57BL/6 mice by fluorescent activated cell sorting (FACS) and fgl2 transcript levels

were measured by real-time PCR. A 6-fold increase of fgl2 mRNA in CD4+CD25+ T cells

compared to CD4+CD25- T cells was observed (Figure 2-6).

Figure 2-6. Increased expression of fgl2 mRNA in CD4+CD25+ T cells. The levels of fgl2 mRNA in purified CD4+CD25+ T cells and CD4+CD25- T cells as measured by real- time PCR. Fgl2 expression levels in both T cell subsets were first normalized to HPRT, GAPDH and RPL13, which served as house-keeping genes. 2-ΔΔCT calculations were used to present levels of fgl2 expression in CD4+CD25+ relative to CD4+CD25- T cells. Graph shows the mean ± SEM from three independent experiments of two to four mice in each group.

76 2.4.9. Increased percentage and absolute numbers of Treg cells in fgl2-/- mice compared to fgl2+/+ mice

To determine if fgl2 was important for the generation and maintenance of Treg cells,

we analyzed the proportion of Treg cells in the lymphoid tissues of fgl2+/+ and fgl2-/- mice.

Following isolation, mononuclear cells isolated from various lymphoid tissues were

stained with monoclonal antibodies against CD4, CD3, Foxp3 and CD25 and analyzed by

flow cytometry. A statistically significant increase in the percentage of Treg cells was

observed in all lymphoid tissues of fgl2-/- mice compared to littermate controls (Figure 2-

7). This increase corresponded to a higher absolute number of Treg cells in all lymphoid

organs (Figure 2-7).

Figure 2-7. Increased percentage and absolute numbers of Treg cells in fgl2-/- mice compared to fgl2+/+ mice. Flow cytometry measurement of CD4+Foxp3+ Treg cell percentage in spleen, lymph nodes, and thymus (top) of fgl2-/- and fgl2+/+ mice; calculated absolute CD4+Foxp3+ Treg cell number in lymphoid organs (bottom) of fgl2-/- and fgl2+/+ mice. Graphs show the mean ± SEM from three independent experiments of three to four mice in each group. Student’s t-test was used to calculate statistical differences between fgl2-/- and fgl2+/+ mice.

77 2.4.10. Increased expression of Foxp3 in Treg cells from fgl2-/- mice compared to

fgl2+/+ mice

To measure the expression levels of Foxp3 in Treg cells from fgl2+/+ and fgl2-/- mice,

the mean fluorescence intensity (MFI) of Foxp3 expression was compared within the

gated CD4+CD25+ T cell population. Treg cells from fgl2-/- mice had increased levels of

Foxp3 expression compared with littermate controls in the thymus, spleen and lymph

nodes (Figure 2-8). Statistical differences were found in thymus and spleen between

fgl2+/+ and fgl2-/- mice.

Figure 2-8. Increased expression of Foxp3 in Treg cells of fgl2-/- mice compared to fgl2+/+ mice. Expression levels of Foxp3 in Treg cells of fgl2+/+ and fgl2-/- mice were measured by flow cytometry as the mean fluorescence intensity (MFI) of Foxp3 expression within the gated CD4+CD25+ T cell population. Graphs show the mean ± SEM from three independent experiments of three to four mice in each group. Student’s t-test was used to calculate statistical differences between fgl2-/- and fgl2+/+ mice.

78 2.4.11. Treg cells isolated from fgl2-/- mice have impaired suppressive activity

We directly assessed the effect of targeted deletion of fgl2 on the ability of Treg cells

to suppress effector CD4+ T cell proliferation. Purified CD4+CD25- T cells from fgl2+/+

mice were cultured for 4 days in the presence of irradiated splenocytes, which served as a

source of APCs, Con A (1 µg/ml), and in the presence of titrated numbers of CD4+CD25+

T cells purified from either fgl2-/- or fgl2+/+ mice. 3H-thymidine incorporation was used to

measure the proliferation of effector CD4+ T cells. Treg cells from fgl2-/- mice were less

efficient in suppressing CD4+ T cell proliferation compared to Treg cells from fgl2+/+

mice at all ratios used (Figure 2-9). Surprisingly, at ratios 1:16 and 1:32 of Treg cells to

effector T cells, we observed stimulation and proliferation of CD4+ T cells in cultures to

which Treg cells from fgl2-/- mice had been added. One possible explanation could be a

contamination of effector T cells from fgl2-/- mice, which were previously shown to have

a hyperproliferative response to Con A and alloantigens (Figure 2-1A).

79

Figure 2-9. Treg cells isolated from fgl2-/- mice have impaired suppressive activity. In vitro suppression assays using cultures of CD4+CD25– T cells from fgl2+/+ mice as responder cells, together with APCs and titrated numbers of CD4+CD25+ Treg cells from either fgl2-/- or fgl2+/+ mice as suppressor cells. [3H]Thymidine incorporation was used to measure the proliferation of effector T cells. The activity of Treg cells was expressed as a percent suppression of CD4+ effector T cell proliferation in the absence of Treg cells. Graph shows the mean ± SEM from three independent experiments of two mice in each group. Student’s t-test was used to calculate statistical differences between fgl2-/- and fgl2+/+ mice.

80 2.4.12. Monoclonal antibody to FGL2 completely blocks the suppressive activity of wild-type Treg cells

In a separate set of experiments, we examined the ability of anti-FGL2 monoclonal

antibody to block Treg cell activity. Titrated concentrations of anti-FGL2 antibody were

added to cultures of effector T cells and Treg cells at a 1:4 suppressor:responder cell ratio in the presence of APCs. Anti-FGL2 antibody completely blocked the suppressive activity of Treg cells in a dose dependent manner (Figure 2-10). In contrast, an isotype control antibody had no inhibitory effect.

Figure 2-10. Monoclonal antibody to FGL2 completely blocks the suppressive activity of wild-type Treg cells. Titrated concentrations of anti-FGL2 antbody were added to the cultures of CD4+CD25– T cells and CD4+CD25+ Treg cells at a 1:4 suppressor:responder cell ratio in the presence of APCs. [3H]Thymidine incorporation was used to measure the proliferation of effector T cells following 4 days of culture. Graph represents the mean ± SEM from three independent experiments of two mice in each group.

81 2.5. Discussion

FGL2 consists of 432 amino acids and contains a C-terminal fibrinogen-related

domain (FRED), which is a highly conserved region found in the β and γ chains of fibrinogen and is characteristic of proteins within the fibrinogen superfamily (179,180).

Members of this functionally diverse superfamily include the extracellular matrix proteins tenascin, angiopoietin, growth factors and ficolin (170). These proteins have been shown to exhibit coagulant and angiogenic activity as well as immunoregulatory

activity (179,180,195). Similar to other members of the fibrinogen superfamily, our

laboratory has recently discovered that FGL2 inhibits murine DC maturation and adaptive T cell immune responses (170), in addition to its role in innate immunity through its ability to activate the coagulation system (163,167,175,184-189).

In this present study, we examined the effects of targeted deletion of fgl2 on the

immune system and its role as a negative regulator in vivo. As we had hypothesized, fgl2

deficiency resulted in increased immune reactivity. We first observed increased numbers

of DC in the spleen and BM-derived cultures from fgl2-/- mice following LPS stimulation.

This was accompanied by an enhanced expression of maturation markers including CD80

and MHCII, as well as an increased life span of DC from fgl2-/- mice. The increased

numbers and activation of DC seen in the fgl2-/- mice were consistent with the inhibitory

effect of FGL2 on DC in vitro, which we have previously reported (170). T cells from

fgl2-/- mice showed greater proliferation in response to Con A and alloantigens. High

levels of IFN-γ, a Th1 cytokine, and low levels of IL-4, a Th2 cytokine, were detected in allogeneic cultures of fgl2-/- responder T cells, demonstrating polarization towards a Th1

immune response. Again, these findings are consistent with in vitro data, showing the

82 ability of FGL2 to inhibit T cell proliferation and promote a Th2 cytokine profile (170).

Also, as one might have predicted based on previous studies, we found that DC from

fgl2-/- mice had increased CD80 expression but no change in the expression of CD86

(170).

Hancock et al. also examined the effects of targeted deletion of fgl2 on immune responses, in particular Th1 activity (190). They reported that Th1 responses and T cell proliferation in response to bacterial and viral infections were comparable in fgl2-/- and

fgl2+/+ mice. In addition, they showed that the proportions of B cells, macrophages, and

CD4+ and CD8+ T cell populations were similar. This is consistent with our findings that equivalent proportions of B cells, T cells and macrophages are found in the spleens of

fgl2-/- and fgl2+/+ mice. Our group and Hancock et al. are in agreement with the fact that

the Th1 response in fgl2-/- mice is not diminished or impaired. Our group has in fact

reported an increase in the Th1 immune response. The transplant data in both studies

(187,190) were also similar with comparable rejection rates in fgl2-/- and wild-type

recipient mice. Again, our group has also extended these findings to report an increased

survival of grafts from fgl2-/- mice (187), an issue which was not examined by Hancock et

al. (190). While some differences seen in the phenotype of our fgl2-/- mice compared with

mice reported by Hancock et al. (190) could be attributed to the different methodologies

used to generate the two sets of mice (167,190), we suggest that differences in

experimental design are equally plausible as an explanation. For example, Hancock et al. used a potent immunogen to examine immune responses and reported a high IFN-γ level

in serum (40 ng/ml)(190); In contrast, we have used a lesser stimulus in vitro which

generated only 4 ng/ml of IFN-γ (Figure 2-1B). Hancock et al. suggested that FGL2

83 might contribute to pathologically significant coagulative responses under certain

conditions (190), just as we have shown in response to MHV-3 but not to endotoxin (167).

Hancock et al. and we have raised the possibility that FGL2 plays important roles in

responses, which have yet to be fully evaluated, and our data are not in disagreement with

this prediction.

With age, our fgl2-/- mice lost weight compared to littermate controls and developed

glomerulonephritis and renal failure as indicated by histology and by increased levels of

serum creatinine and protein and blood in the urine. The first evidence to suggest that the

renal disease may be autoimmune was the marked infiltration of mononuclear cells in the

kidneys and deposits of IgG, IgM, IgA and complement (C3) in association with renal

disease. Secondly, the disease was re-capitulated in BM chimeras (fgl2-/- BM Æ fgl2+/+

mice). We examined the effect of aging on both numbers and function of Treg cells in

fgl2-/- mice. One-year-old fgl2-/- mice had significantly reduced numbers of Treg cells and

their function was impaired similar to results seen in young fgl2-/- mice (unpublished

data). Alteration in CD4+CD25+ Treg cell numbers or activity has been shown to be

associated with autoimmune diseases (144). We therefore propose that loss of Treg cell function in fgl2-/- mice accounts for the increased immune reactivity and the development

of the glomerulonephritis. In favor of the important role for FGL2 in Treg cell

suppressive activity is the observation that Treg cells treated with antibody to FGL2 have

no suppressive activity.

Increased numbers of Treg cells and Foxp3 expression in fgl2-/- mice were observed.

Increased levels of IL-2 in supernatants from the MLR cultures of fgl2-/- responder cells

(unpublished data) might explain the increased numbers of Treg cells, which bear CD25

84 and thus proliferate in response to IL-2. However, Treg cells from fgl2-/- mice exhibited

decreased suppressive activity compared to Treg cells from fgl2+/+ mice. These results

suggest that FGL2 is an important effector molecule of Treg cells and that the increased numbers of Treg cells and Foxp3 expression might serve as a compensatory mechanism for the diminished Treg cell activity in fgl2-/- mice. Other molecules including TGF-β, IL-

10, CTLA-4, LAG-3, CD39 and galectin-1 have also been reported to account for the regulatory activity of Treg cells. However, antibody to these molecules in some cases had

no effect or only minimally inhibited Treg cell activity in vitro in contrast to antibody to

FGL2 (154,158,161,196,197). The data generated in this report and by others suggest that

the suppressive activity of Treg cells is complex involving many redundant pathways. In

support of this concept, Treg cells from both TGF-β-/- and CTLA-4-/- mice exhibited

normal activity (154) .

In addition to CD4+CD25+ Treg cells, fgl2 might be important to the function and/or

development of other regulatory T cells subsets. We have observed a marked increase in

the expression levels of fgl2 mRNA and FGL2 protein in CD4-CD8- double negative (DN)

T cells and a loss of fgl2 expression in a DN mutant clone that is devoid of suppressor

activity. Recently, CD8αα+ T cells were likewise shown to express increased levels of fgl2 suggesting that fgl2 may have an important role in other Treg cell populations (120).

The fact that CD8αα+ T cells are critical for intestinal tolerance suggests that fgl2 may

also play a role in maintaining tolerance in the intestine.

Clinical evidence also suggests that FGL2 negatively regulates the immune system.

Hepatitis B and hepatitis C patients express increased levels of FGL2 (166,170) and DC

derived from patients with chronic HCV and HBV infections have attenuated responses

85 to maturation stimuli (lower CD86 expression), impaired T cell stimulating capabilities

and decreased IFN-γ production (198,199). Furthermore, these chronic viral HBV and

HCV-infected patients are incapable of mounting protective T cell responses (200).

Kohno et al. have demonstrated a loss of fgl2 transcripts in patients with acute and

chronic adult T cell leukemia, a lymphoproliferative disorder of T-helper cells,

suggesting a potential role for FGL2 in the regulation of T cell proliferation (171).

The mechanism whereby FGL2 exerts its immunoregulatory activity was also

investigated. BM chimera studies demonstrated that hematopoietic cells from fgl2-/- mice

mediate the increased immune reactivity seen in fgl2-/- mice. The increased numbers of antibody-producing B cells in response to TI antigens, shown by ELISPOT and our DC studies, suggest that FGL2 fulfills its immunoregulatory function through its effects on

APCs. In favor of this notion, FGL2 has been demonstrated to bind specifically to the low-affinity FcγRIIB and FcγRIII, which are present on DC and B cells (201,202). The binding of FGL2 to these receptors led to inhibition of DC maturation and induction of B cell apoptosis (Figure 4-4 and 4-5 in chapter 4). It has also been reported that glomerular mesangial cells within the kidney express high levels of inhibitory FcγRIIB receptors

(203), and Ravetch et al. have shown that FcγRIIB-/- mice on a C57BL/6 background

develop glomerulonephritis (204). Thus, we would expect that fgl2-/- mice, which lack the

ligand for FcγRIIB, would develop glomerulonephritis similar to FcγRIIB-/- mice.

Collectively, the results of the present study suggest that fgl2 accounts, at least in

part, for the suppressive activity of Treg cells. Loss of FGL2 results in significant

immune dysregulation and glomerulonephritis.

86 2.6. Contributions

Fgl2-/- mice and fgl2+/+ littermate control mice were generated and bred by Dr.

Liasum Fung in the laboratory of Dr. Gary Levy. Subsequent breeding and genotyping

were assisted by Dr. Wei He and Zhumei Kang. The studies in figures 1, 2 and 5

including immunostaining of kidneys and β-gal staining were performed by Cheryl

Koscik with assistance of Mojib Javadi for some of the experiments. Cheryl Koscik also

performed analysis of splenic architecture, gross morphology, histology and mass of

fgl2-/- and fgl2+/+ littermate control mice. Paraffin-embedding and sectioning, frozen

tissue sectioning and staining for histological analysis were performed by the Department

of Pathology at the Hospital for Sick Children. Expert histological analysis and

pathological guidance were provided by Dr. M. James Phillips of the Department of

Pathology at the Hospital for Sick Children. Doug McCarthy and Dr. Jennifer

Gommerman of the Department of Immunology at the University of Toronto assisted with the splenic architecture analysis and the DC migration study. Cell separation using

Fluorescence-Activated Cell Sorting (FACS) was performed by Joyce Pun. Figures and

text in this chapter have been adapted from Shalev et al. J Immunol 180:249-260

(copyright © 2008 by The American Association of Immunologists, Inc.) with written

permission.

87

Chapter 3: The Novel Treg Effector Molecule FGL2 Contributes to the Outcome of

Murine Fulminant Viral Hepatitis

3.1. Summary

Fulminant viral hepatitis (FH) remains an important clinical problem in which the

underlying pathogenesis is still not well understood. Here, we present insight into the

immunological mechanisms involved in FH caused by murine hepatitis virus strain 3

(MHV-3), indicating a critical role for CD4+CD25+ regulatory T (Treg) cells and production of the novel Treg effector molecule FGL2. Prior to infection with MHV-3, susceptible BALB/cJ mice had increased numbers of Tregs and expression of fgl2 mRNA

and FGL2 protein compared to resistant A/J mice. Following MHV-3 infection, plasma

levels of FGL2 in BALB/cJ mice were significantly increased, correlating with increased

percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg

cell activity and protected susceptible BALB/cJ mice against MHV-3-liver injury and

mortality. Adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increased

their mortality to MHV-3 infection, whereas transfer of peritoneal exudate macrophages

had no adverse effect. Conclusions: This study demonstrates that FGL2 is an important

effector cytokine of Treg cells that contributes to susceptibility to MHV-3-induced FH.

The results further suggest that targeting FGL2 may lead to the development of novel

treatment approaches for acute viral hepatitis infection.

88 3.2. Introduction

Naturally occurring CD4+CD25+ regulatory T (Treg) cells have been

demonstrated to play an important role in maintenance of peripheral self-tolerance (144).

Depletion or functional alteration of Treg cells in mice results in the development of

autoimmune disease (144). In athymic nude mice, transfer of syngeneic splenic cells depleted of Treg cells produces autoimmune disease that is preventable by the co-transfer of small numbers of Treg cells (144). In addition to their role in the control of self- tolerance, Treg cells are also involved in regulation of T cell homeostasis, modulation of

immune responses to cancer, pathogens and alloantigens, as well as the prevention of allograft rejection (144).

Treg cells have been implicated in suppressing T cell immune responses in viral

and bacterial infections (205). Patients with chronic hepatitis B (HBV) and hepatitis C

virus (HCV) infection have increased numbers of Treg cells, which impair immune

responses against HBV and HCV, thus leading to viral persistence and chronic infection

(206,207). It has been shown that circulating and liver-resident Treg cells actively

influence the anti-viral immune response and disease progression in patients with HBV

(208). Moreover, depletion of Treg cells in mice results in enhancement of HBV-specific

+ CD8 T cell responses primed by DNA immunization (209). The role of Treg cells in

acute viral hepatitis, however, has not been reported.

FGL2, a member of the fibrinogen-related superfamily of proteins known to be

secreted by T cells (164,165), has recently been reported by a number of groups to be

highly expressed by Treg cells and has been proposed to have a role in Treg effector

function (176,177,192). In this study, we addressed the contribution of Treg-expressed

89 FGL2 to the pathogenesis of MHV-3-induced murine FH. The results show that the

frequency of Treg cells and Treg cell expression of fgl2 mRNA and FGL2 protein are

higher in lymphoid tissues of uninfected BALB/cJ mice compared to uninfected A/J mice.

Post-MHV-3 infection, Treg cells were increased correlating with elevated levels of

FGL2 in the plasma of susceptible BALB/cJ mice. Treatment with FGL2 antibody

blocked Treg cell activity in vitro and protected against MHV-3-induced liver injury and

mortality in vivo. Adoptive transfer of wild-type Treg cells to resistant fgl2-/- mice resulted in increased mortality post-MHV-3 infection. Collectively, the results support the concept that Treg cells and the effector cytokine FGL2 are logical targets for molecular manipulation in the development of novel treatment approaches for patients with viral hepatitis.

90 3.3. Materials and Methods

Mice

Female BALB/cJ and A/J mice aged 6 to 8 weeks (Jackson Laboratories) were

maintained in micro isolator cages and housed in the animal colony at the Toronto

General Hospital, University of Toronto, and fed standard lab chow diet and water ad

libitum. The Animal Welfare Committee approved all protocols.

Virus

MHV-3 was obtained from the American Type Culture Collection, Manassas,

VA. It was first plaque-purified and then expanded in murine 17CL1 cells to a

concentration of 1x107/ml. Virus-containing supernatants were collected and

subsequently stored at −80oC until use. Mice were infected with 100 PFU by the intraperitoneal (ip) route.

Viral infection and treatment with anti-FGL2 antibody

Mice received an ip injection of 100 PFU MHV-3 and were monitored daily for

symptoms of disease, including ruffled fur, tremors, and lack of activity. Additionally,

BALB/cJ mice were treated with 25, 50 or 100 μg of an IgG2a monoclonal anti-FGL2

antibody (1F4.2) or an isotype control antibody daily for 7 days pre-MHV-3 infection and

7 days post-MHV-3 infection by tail vein injection.

Viral titers

Livers were removed aseptically, cut into small pieces, and homogenized in 10%

ice-cold Dulbecco’s modified essential medium (DMEM) utilizing a Polytron

91 homogenizer (Fisher Scientific, Whitby, Ontario, Canada). Viral titers were determined

as described previously (210).

Isolation of Treg cells

CD4+CD25+ Treg cells and CD4+CD25- T cells were purified from spleens and

lymph nodes with either the EasySep® Mouse CD4+ T Cell Enrichment Kit (StemCell

Technologies Inc.) or with the magnetic-activated cell sorting system (MACS, Miltenyi

Biotech Inc., Auburn, CA.) according to manufacturers instructions followed by FACS using a FACSAria cell sorter (Becton Dickinson, CA). Purity of cell populations was

greater than 95% as assessed by flow cytometry.

Tissue isolation

Mice were sacrificed on days 0, 1, 2, 3 and 8 (only A/J mice) post-MHV-3

infection. Blood was collected via cardiac puncture and serum was stored at −80oC.

Livers were collected for histology and immunohistochemistry (211). To quantify the

effect of anti-FGL2 antibody on liver histology, a digitalized image analysis system (HP-

88; Hewlett Packard Co. Ltd., Mississauga, Ontario) was used (212). The areas of

necrosis were encircled, yielding a percentage representing the proportion of diseased

liver in that particular section. For each animal, three random sections were assayed in

this fashion and the mean ± SEM was calculated. Frozen tissue was embedded in OCT

(Fisher)-filled cryomolds (TissueTek), snap-frozen in liquid nitrogen and stored at −80oC

for immunohistochemistry.

Flow cytometry

Cell suspensions obtained from spleen, lymph nodes (inguinal, axillary,

mesenteric and cervical) and thymus were washed and suspended in PBS containing 5%

92 7 mouse serum (Cedarlane) at a final concentration of 1x10 cells/ml in order to block Fc

receptors. Following staining of the cell membrane with anti-CD4-FITC, cells were fixed

and permeabilized for intracellular staining with anti-Foxp3-PE and anti-rat IgG2a-PE isotype control (eBiosciences) according to the manufacturer's instructions. Cell staining was assessed using a Coulter FC500 flow cytometer and data were analyzed using

CXP/RXP software (Beckman Coulter). Live cells were gated according to forward and

side scatter parameters.

Sandwich ELISA

Plates were coated and incubated overnight with 2 μg/ml monoclonal anti-FGL2

(6H12) (IgG1) as a capture antibody. Plasma samples (50 μl) were added to each well,

and following a 1-hour incubation at 37oC and 3 washes with TTBS, the wells were

incubated with 2 μg/ml polyclonal rabbit anti-FGL2 antibody (187) for 2 h at 37oC. The

plate was washed again and polyclonal anti-FGL2 binding was detected with a secondary

HRP-conjugated anti-rabbit antibody. Tetramethlybenzidine (TMB) was then added and

absorbance was measured at 450nm using an ELISA plate reader.

Real-time PCR

Following the purification of splenic CD4+CD25+ Treg cells from either A/J or

BALB/cJ mice by FACS, total RNA was extracted from the cells and the levels of fgl2

mRNA expression were measured by real-time PCR (RT-PCR). The levels of fgl2 were

normalized to the housekeeping genes HPRT, GAPDH and RPL13. Expression of fgl2

mRNA in CD4+CD25+ Treg cells from BALB/cJ mice relative to CD4+CD25+ Treg cells

from A/J mice was calculated by the 2-ΔΔCT method and corrected for the absolute

numbers of CD4+CD25+ Treg cells in each strain.

93 Immunohistochemical staining for infiltrating Treg cells in liver tissue

Infiltrating cells in liver tissue of A/J and BALB/cJ mice were characterized both pre- and post-MHV-3 infection by indirect immunohistochemistry using purified anti-

Foxp3 antibody or isotype negative control antibody and a secondary HRP-conjuated anti-rat antibody according to the manufacturer's instructions (eBioscience).

Suppression assay

Cultures were set up consisting of 4x104/well CD4+CD25- T cells (responder) and

CD4+CD25+ Treg cells (suppressor) at different suppressor:responder ratios in the presence of 2x105/well irradiated syngeneic antigen-presenting cells (APCs) and anti-

CD3 antibody (0.5 μg/ml). Monoclonal antibody to FGL2 (10 µg/ml) (Abnova, Taiwan) or isotype negative control antibody (10 µg/ml) were added to these cultures, which were then cultured for 96 hours and proliferation determined by incorporation of 3H-thymidine.

Adoptive transfer studies

Fgl2-/- mice (n=15/group) were infused with 120-300x103 purified wild-type Treg cells by intravascular injection (iv) 1h prior to MHV-3 infection. Fgl2-/- mice

(n=10/group) were injected ip with 5x106 wild-type peritoneal-exudate macrophages

(PEMs) or 5x106 wild-type splenocytes. Fgl2-/- mice (n=20/group) which did not receive infusions of Treg cells and fgl2+/+ mice (n=10/group) were used as controls. Survival rate and H&E staining of livers from infected mice were analyzed to assess the effect of Treg- expressed FGL2 on the survival and severity of MHV-3 disease.

Statistical analysis

Results are reported as mean and standard error of mean (SEM) unless otherwise specified. One-way or two-way ANOVA followed by the Bonferroni test for post-hoc

94 analysis were used for group comparison. Rates of animal survival were calculated using the Kaplan-Meier method and compared between groups with the log rank test.

Differences with P ≤ 0.05 were considered significant.

95 3.4. Results

3.4.1. Increased numbers and percentage of Treg cells in uninfected BALB/cJ mice compared to A/J mice

The percentage of Treg cells in the lymphoid organs of uninfected BALB/cJ and

A/J mice was compared based on cells co-expressing CD4 and Foxp3 identified by flow cytometry. Spleens of BALB/cJ mice contained a 1.6-fold increase in the proportion of

Treg cells compared to A/J mice (3.3% compared to 2% respectively). The proportions of

Treg cells (5.9%) found in inguinal, axillary, mesenteric and cervical lymph nodes of uninfected BALB/cJ mice were higher than that of A/J mice (5.1%). Naturally occurring

Treg cells composed 0.71% of the total thymic cell population in BALB/cJ mice, while only 0.29% Treg cells were present in A/J thymus (Figures 3-1A and 3-1B). The increased proportion of Treg cells found in lymphoid tissues of BALB/cJ mice reflected an increase in the absolute numbers of Treg cells in BALB/cJ mice compared to A/J mice

(Figure 3-1C).

96

Figure 3-1A. Increased percentage of Treg cells in uninfected BALB/cJ mice compared to A/J mice. Representative flow cytometry plots displaying Treg cell proportions in the lymphoid tissues of uninfected BALB/cJ (bottom) and A/J mice (top) based on CD4 and Foxp3 co-expression. For thymic cells, the Treg population was analyzed by gating on the CD4+CD8- population. Data were collected from 3 independent experiments of 3-4 mice in each group.

97

Figure 3-1B. Increased percentage of Treg cells in uninfected BALB/cJ mice compared to A/J mice. Graphs show the % mean ± SEM of Treg cell proportions in the lymphoid tissues of uninfected BALB/cJ and A/J mice based on CD4 and Foxp3 co- expression as detected by flow cytometry. For thymic cells, the Treg population was analyzed by gating on the CD4+CD8- population. Data were collected from 3 independent experiments of 3-4 mice in each group. Comparison between groups was performed using a one way ANOVA for statistical analysis.

98

Figure 3-1C. Increased numbers of Treg cells in uninfected BALB/cJ mice compared to A/J mice. Graphs show the mean ± SEM of absolute numbers of Treg cells in the lymphoid tissues of uninfected BALB/cJ and A/J mice as calculated based on the percentage of total Treg cells multiplied by the total number of cells for each organ. Data were collected from 3 independent experiments of 3-4 mice in each group. Comparison between groups was performed using a one-way ANOVA for statistical analysis.

99 3.4.2. Increased levels of FGL2 by Treg cells in the plasma of uninfected BALB/cJ mice compared to A/J mice

The expression of fgl2 mRNA and FGL2 protein by Treg cells and plasma FGL2 levels in BALB/cJ and A/J mice were first compared. Treg cells from BALB/cJ mice had a 1.6-fold higher level of fgl2 mRNA compared to A/J mice after normalization to reference gene expression (Figure 3-2A). By ELISA, a 4-fold increase in levels of FGL2 was detected in cell culture media of Treg cells isolated from BALB/cJ mice compared to

Treg cells isolated from A/J mice (Figure 3-2B).

Consistent with the higher fgl2 mRNA and FGL2 protein expression by BALB/cJ

Treg cells, we detected 124 ± 36 ng/ml of FGL2 in the plasma of BALB/cJ mice compared to 79 ± 17 ng/ml in A/J mice at baseline prior to infection with MHV-3 (Figure

3-2C).

100

Figure 3-2. Increased levels of FGL2 by Treg cells in the plasma of uninfected BALB/cJ mice compared to A/J mice. (A) Calculated levels of fgl2 mRNA expression in CD4+CD25+ Treg cells of BALB/cJ mice relative to CD4+CD25+ Treg cells of A/J mice as measured by RT- PCR; n=3. (B) FGL2 levels in culture supernatants of freshly isolated Treg cells from BALB/cJ or A/J mice that were cultured for 4 days as measured by ELISA. Data show the mean ± SEM of three independent experiments of 5 mice in each group. (C) Graph shows the mean ± SEM of the levels of plasma FGL2 in A/J and BALB/cJ mice as determined by ELISA; n=15. Comparison between groups was performed using a one way ANOVA for statistical analysis.

101 3.4.3. Increased percentage of Treg cells in BALB/cJ mice compared to A/J mice

following MHV-3 infection

BALB/cJ and A/J mice, which were infected with 100 PFU MHV-3 ip, were

sacrificed on days 1, 2, 3 post-infection (p.i.) and on day 8 p.i. all remaining A/J mice

were sacrificed and the proportion of Treg cells in their lymphoid organs was assessed by

flow cytometry. At all time points p.i., the percentage of Treg cells was higher in spleens

of BALB/cJ mice compared to A/J mice (Figure 3-3A). However, the percentage of Treg cells in spleens of both strains decreased following MHV-3 infection. Post-MHV-3

infection a marked increase in the percentage of Treg cells in the thymus of BALB/cJ

mice compared to A/J mice was observed (Figure 3-3B).

102

Figure 3-3A-B. Increased percentage of Treg cells in BALB/cJ mice compared to A/J mice following MHV-3 infection. The percentages of Treg cells in the spleen (A) or thymus (B) of BALB/cJ and A/J mice are shown at different time points following viral infection. Graphs represent the mean ± SD from 2 independent experiments of 3-4 mice in each group. A two way ANOVA with a Bonferroni test for post hoc analysis were used to compare means.

103 3.4.4. Increased Treg cell infiltration in livers of BALB/cJ mice following viral

infection

Prior to infection, Treg cells were undetectable in both mouse strains. On day 1

post-MHV-3 infection, livers of BALB/cJ mice had high numbers of Foxp3+ Treg cells,

whereas Foxp3-staining was negative in livers of MHV-3-infected A/J mice (Figure 3-3C

and 3-3D). However, on day 2 and 3 p.i., small numbers of Treg cells were detected in

livers of A/J mice, whereas at these time points the livers of BALB/cJ mice were largely

necrotic making it difficult to assess Foxp3 staining (data not shown). MHV-3 was not detected in livers from BALB/cJ mice at 1 day p.i., whereas low viral titers were found in

A/J mice (1.3x102 PFU/g). On day 2 and 3 p.i., viral titers increased in BALB/cJ mice

(day 2- 5.3x105 ± 1.32 PFU/g; day 3- 7.2x106 ± 2.63 PFU/g) and in A/J mice (day 2-

2.6x103 PFU/g; day 3- 3x103 ± 1.23 PFU/g).

104

Figure 3-3C-F. Increased Treg cell infiltration in livers of BALB/cJ mice following viral infection. Representative Foxp3+ Treg cell staining in liver of A/J (C) or BALB/cJ mice (D) at day 1 post-MHV-3 infection (magnification x 400). Immunostaining of spleen of uninfected BALB/cJ mice with anti-Foxp3 antibody (E) used as a positive control or with isotype negative control antibody (F).

105 3.4.5. Levels of FGL2 in the plasma of BALB/cJ and A/J mice following viral

infection

By ELISA, levels of FGL2 in BALB/cJ mice rose significantly p.i. correlating

with disease progression. Within 2 days p.i., levels of FGL2 rose from a baseline of 124

±36 ng/ml to 443 ± 166 ng/ml, reaching a maximal level of 1589 ± 75 ng/ml on day 3 p.i.

MHV-3-resistant A/J mice showed low basal levels of FGL2, which rose to 138 ± 80

ng/ml by day 3 p.i. but returned to normal levels by day 8 (Figure 3-4).

Figure 3-4. Levels of FGL2 in the plasma of BALB/cJ and A/J mice following viral infection. Levels of FGL2 protein in the plasma of BALB/cJ or A/J mice are shown at different time points following MHV-3 infection. Data represent mean ± SEM of 4-6 mice in each group. A two way ANOVA with a Bonferroni test for post hoc analysis were used to compare means.

106 3.4.6. FGL2 is an effector of Treg cell function

To evaluate whether FGL2 accounts for the immunosuppressive activity of Treg

cells as has been suggested (176,177), a standard in vitro suppression assay was

performed. Monoclonal antibody to FGL2 (10 µg/ml) or isotype negative control (10

µg/ml) were added to co-cultures of CD4+CD25- T cells (responder) and CD4+CD25+

Treg cells (suppressor) at different suppressor:responder ratios in the presence of syngeneic APCs and anti-CD3 antibody (0.5 μg/ml). Addition of monoclonal anti-FGL2 completely inhibited Treg cell activity at all suppressor:responder ratios assessed, whereas the isotype control antibody had no inhibitory effect on Treg cell function

(Figure 3-5).

Figure 3-5. FGL2 is an effector of Treg cell function. Monoclonal antibody to FGL2 (10 µg/ml) or isotype negative control (10 µg/ml) were added to co-cultures of

107 CD4+CD25- T cells (responder) and CD4+CD25+ Treg cells (suppressor) at different suppressor:responder ratios in the presence of syngeneic APCs and anti-CD3 antibody (0.5 μg/ml). Anti-FGL2 antibody significantly blocked the suppressive activity of Treg cells at all suppressor:responder ratios. Data represent mean ± SEM of two independent experiments. A two way ANOVA with a Bonferroni test for post hoc analysis were used to compare means.

3.4.7. Anti-FGL2 antibody treatment prolongs the survival of BALB/cJ mice post-

MHV-3 infection

To further evaluate the contribution of FGL2 to the pathogenesis of MHV-3-

induced liver disease, BALB/cJ mice were infected with MHV-3 and treated with an

IgG2a monoclonal antibody to FGL2 (182). Treatment with the monoclonal antibody

1F4.2 daily for 7 days pre-infection and 7 days p.i. by tail vein injection markedly

reduced hepatic necrosis, inhibited viral replication, and prolonged the survival of MHV-

3-infected BALB/cJ mice in a dose-dependent manner. All animals that received 100 μg

of IF4.2/day survived, whereas untreated mice and animals treated with isotype negative

control antibody died within 2-5 days p.i. (Figure 3-6A). Untreated MHV-3-infected

BALB/cJ mice developed histologic evidence of liver disease by day 3 p.i. (Figures 3-6

and 3-7). In contrast, mice infected with MHV-3 but treated with antibody to FGL2

showed a marked reduction in liver necrosis in a dose-dependent fashion (Figure 3-7).

Morphometric image analysis revealed that the proportion of necrotic liver tissue was

significantly different between anti-FGL2-treated and untreated mice post-MHV-3

infection (Figure 3-6B). Liver histology from all survivors at 10 and 14 days p.i. was

normal. Coincident with reduced liver necrosis and increased survival, viral titers from

livers of infected, IF4.2-treated mice were markedly reduced compared to infected,

untreated mice at all time points (Figure 3-6C).

108

Figure 3-6. Effect of monoclonal anti-FGL2 antibody treatment on the course of MHV-3 infection. (A) BALB/cJ mice were either untreated (0) or pre-treated with 25, 50 or 100 μg of monoclonal anti-FGL2 antibody (1F4.2) for 7 days before infection with 100 PFU of MHV-3. Antibody treatment was continued in treated animals for 7 days p.i. and survival was studied (n=10 mice/group). The graph represents Kaplan-Meier cumulative survival. There was a significant difference in animal survival between the untreated and FGL2-treated groups (log-rank test). (B) Morphometric analysis of liver necrosis following MHV-3 infection. At 24, 48, 72 and 96 hours p.i., a marked difference in the proportion of liver necrosis was seen between untreated and monoclonal antibody IF4.2-treated groups. (C) The effect of treatment with monoclonal antibody on viral replication. High titers of virus were recovered from untreated, MHV-3-infected animals at all time points. In contrast, monoclonal antibody treatment at doses of 25, 50 and 100 μg attenuated the titers of virus recovered from the liver in a dose-dependent manner. Graphs show the mean ± SEM of 5 mice in each group. The star in panel B and C indicates the statistical significance of each treatment group compared with untreated MHV-3-infected control mice.

109

Figure 3-7. The effect of monoclonal anti-FGL2 antibody on liver histology following MHV-3 infection. Liver sections from MHV-3-infected and untreated mice (A) showed massive hepatic necrosis with hemorrhage and fibrin deposition on day 4 p.i. (arrows). In contrast, in mice that received pre- and p.i. treatment (B) with 25 μg of antibody, necrosis was markedly reduced; (C) with 50 μg of antibody, liver was near normal with only portal inflammatory infiltrates (arrows) and minimal necrosis; and (D) with 100 μg of antibody, there was near-normal histology (H&E stain: x 225).

110 3.4.8. Adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increases

mortality to MHV-3 infection

To directly investigate the contribution of Treg-expressed FGL2 to the outcome

of MHV-3 infection, resistant fgl2-/- mice were infused with wild-type Treg cells and

their survival and liver histology examined (Figure 3-8). Fgl2-/- mice not receiving wild-

type Treg cells nearly all survived MHV-3 infection (Figure 3-8A). However, adoptive

transfer of Treg cells from fgl2+/+ mice into fgl2-/- mice resulted in increased mortality to

MHV-3 infection, at rates similar to those seen in fgl2+/+ mice. Similarly, infected-fgl2-/-

mice that were reconstituted with splenocytes from fgl2+/+ mice, which contained Treg

cells, all died within 3-5 days after infection (Figure 3-8A). In contrast, fgl2-/- mice that

were infused with peritoneal-exudate macrophages (PEMs) from fgl2+/+ mice nearly all

survived MHV-3 infection (Figure 3-8A). These results support a role for Treg-expressed

FGL2 in the pathogenesis of MHV-3-induced FH. Livers harvested from infected mice

prior to death demonstrated widespread hepatic necrosis associated with marked fibrin

deposition in both fgl2+/+ and fgl2-/- mice that had received Treg cells, whereas livers

were near normal in fgl2-/- mice (Figure 3-8B). FGL2 was detected in the plasma of fgl2-/-

mice, which received Treg cells from fgl2+/+ mice (22.26 ± 2.5 ng/ml), whereas it was

undetectable in fgl2-/- mice. Levels of alanine transaminase (ALT) increased in the serum

of infected-fgl2-/- mice that were reconstituted with fgl2+/+ Treg cells compared to fgl2-/- mice which were not infused with fgl2+/+ Treg cells (Figure 3-8C) and correlated with

development of liver disease.

111

Figure 3-8. Adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increases mortality to MHV-3 infection. (A) Fgl2+/+ mice (n=10), fgl2-/- mice (n=20), fgl2-/- mice infused with fgl2+/+ Treg cells (n=15), fgl2-/- mice infused with fgl2+/+ PEMs (n=10) and fgl2-/- mice infused with fgl2+/+ splenocytes (n=10) were infected with MHV- 3 (100 PFU) and survival was studied. Fgl2-/- mice infused with fgl2+/+ Treg cells had increased mortality post-MHV-3 infection compared to resistant fgl2-/- mice, which did not receive fgl2+/+ Treg cells (log-rank test). (B) Representative H&E staining of liver harvested 4 days following MHV-3 infection. A liver lobule is shown from fgl2-/- mice infused with fgl2+/+ Treg cells. A terminal hepatic vein (central vein) is present in the center of the micrograph and on the far left is part of a portal tract with a bile duct. There is complete acute necrosis involving the entire lobular parenchyma aside from a narrow rim of surviving hepatocytes in zone 3, area immediately surrounding the hepatic vein (arrows). The diffuse pink staining material which replaces normal hepatocytes throughout the lobule represents the residua of totally necrotic liver parenchymal cells. There is over 90% necrosis of liver parenchyma. The pathology is that of massive acute hepatic necrosis of the liver. The extent of the necrosis is estimated as over 90% necrosis (H&E stain: x 300). (C) Alanine transaminase (ALT)

112 levels in the serum of fgl2-/- mice which had been infused with or without fgl2+/+ Treg cells. Graph shows mean ± SEM of 3 mice in each group.

3.5. Discussion

In the present study, the contribution of Treg-expressed FGL2 to the pathogenesis

of MHV-3-induced fulminant hepatitis (FH) was examined. Uninfected, susceptible

BALB/cJ mice had increased percentage/number of Treg cells in lymphoid organs

accompanied by increased FGL2 expression compared to resistant A/J mice. Post-MHV-

3 infection, BALB/cJ mice had a significant increase in Treg cells and plasma levels of

FGL2 versus low levels of plasma FGL2 and Treg cells detected in A/J mice. Treatment

with antibody to FGL2 increased survival of susceptible mice and adoptive transfer of

Treg cells from fgl2+/+ mice into fgl2-/- mice resulted in increased mortality to MHV-3

infection. Collectively, these results support the hypothesis that Treg-expressed FGL2 contributes to the pathogenesis of MHV-3-induced FH.

It has been reported that Treg cells over-express a subset of Th2 response genes

including IL-10 and TGF-β (213), suppressing Th1 responses and resulting in Th2

polarization (214,215). BALB/cJ mice, which are known to exhibit a Th2-phenotype

(216), have increased numbers of Treg cells compared to A/J mice that predominantly

mount Th1 immune responses (217). Others have shown that BALB/cJ mice display non-

protective Th2 responses and these mice are susceptible to infection with other pathogens,

including the intracellular parasite Leishmania major, while mice which develop a Th1

immune response are protected from infection (218).

Post-MHV-3 infection, Treg cells increased in the thymus of susceptible

BALB/cJ mice, whereas they were reduced in the spleens of both resistant and

113 susceptible mice. We propose that the increase in Treg cells in the thymus of BALB/cJ

mice is an attempt of susceptible mice to suppress the pro-inflammatory cytokine storm

that is known to occur following MHV-3 infection (217,219). It has been proposed that

inflammatory cytokines, including TNF-α and IL-6, can reduce the numbers of Treg cells and inhibit Treg cell function, providing a potential explanation for the findings in the

present study (154,220).

Various molecular and cellular events have been proposed to explain the

mechanism by which Treg cells suppress immune responses. These include cell-to-cell

contact-dependent suppression, cytotoxicity, and immunosuppressive cytokine secretion

(154). Some studies have suggested that anti-inflammatory cytokines, such as IL-10 and

TGF-β, are important mediators of Treg cell activity in vivo (154). However, the

importance of these cytokines remains controversial, since several reports have

demonstrated that antibodies against IL-10 and TGF-β fail to block Treg cell suppressive

function (154). Also, Treg cells from TGF-β-deficient mice have normal suppressive

activity in vitro and can prevent development of autoimmune disease (154).

Recently, it has been reported that Treg cells have increased expression of fgl2

mRNA and it has been suggested that FGL2 might be an important Treg effector

molecule (176,177,192). The fgl2 gene was first cloned by Koyama et al. from cytotoxic

T lymphocytes (CTL) and the encoded protein has been classified as a member of the

fibrinogen–related superfamily based on its homology to the β and γ chains of fibrinogen

(162). Previously we reported that in addition to its role in innate immunity as a

membrane associated prothrombinase, FGL2 may also have an important role in adaptive

immune responses, similar to other members of the fibrinogen-like family of proteins,

114 which include tenascin and angiopoietins (170). Recombinant FGL2 was shown previously to suppress T cell proliferation to alloantigens, anti-CD3/CD28 antibodies and

Con A (170). FGL2 also has been shown to inhibit maturation of bone marrow-derived

DC and polarized an allogeneic immune response towards a Th2 cytokine profile (170).

In fgl2-deficient mice, Th1 cytokine levels and activity of DC, B and T cells were all increased (221).

Levels of FGL2 in the plasma correlated with numbers of Treg cells in both resistant and susceptible strains of mice pre- and post-MHV-3 infection, suggesting that

Treg cells are a major source of FGL2. The development of a Th2 immune response in

BALB/cJ mice following MHV-3 infection (217) fits with the demonstrated effect of

FGL2 to promote production of Th2 cytokines and subsequent inhibition of Th1 immunity (170).

To evaluate the importance of FGL2 for Treg cell function, we assessed the effect of a monoclonal antibody against FGL2 on Treg cell activity in vitro. Antibody to FGL2 completely inhibited Treg cell function, consistent with the hypothesis that FGL2 is an important effector molecule for Treg cell activity. Further evidence for the importance of

FGL2 to Treg cell function is the observation that Treg cells from fgl2-deficient mice have impaired function and that fgl2-deficient mice develop autoimmune glomerulonephritis with increased age (221). Based on these findings, we proceeded to investigate the contribution of FGL2 as a putative effector cytokine of Treg cells to the pathogenesis of MHV-3 infection in vivo. The data presented here demonstrated that antibody directed to the C terminal domain of FGL2, which is known to account for its immunosuppressive activity (170), protected mice from the lethality of MHV-3 infection.

115 Others have reported that treatment with antibody against TGF-β, a known important

Treg effector molecule, also resulted in prolonged survival of susceptible strains to

MHV-3 infection further supporting our hypothesis that Treg cells are important in the

pathogenesis of MHV-3-induced FH (222). In addition, fgl2+/+ Treg cells that were

transferred to fgl2-/- mice but not PEMs increased mortality to MHV-3 infection, further

supporting a role for Treg-expressed FGL2 in the outcome of the infection.

Although the transfer of PEMs, in numbers comparable to frequency of

macrophages in the spleen, did not adversely affect the survival of MHV-3-infected-

fgl2-/- mice, this does not negate the importance of membrane-associated FGL2

prothrombinase production by reticuloendothelial cells (REs) as previously reported

(167). The lack of effect of macrophages might be due to the fact that insufficient cell

numbers of fgl2+/+ PEMs were transferred into fgl2-/- mice to recapitulate the endogenous

production of FGL2 by liver-resident REs. It should be noted that macrophages from A/J

mice can produce FGL2 to IFNγ in vitro, yet do not generate FGL2 to MHV-3 in vivo

(163,184,223). Taken together, the data demonstrate that production of FGL2 is tightly regulated and supports the important role of Treg-expressed FGL2 in the pathogenesis of

MHV-3-induced FH.

The mechanism by which FGL2 mediates its immunosuppressive activity is

currently under intensive investigation. Recent data from our group have demonstrated

that FGL2 binds to the inhibitory FcγRIIB receptor expressed primarily on antigen- presenting cells (APCs) (201,202,221). This FGL2-FcγRIIB interaction was shown to induce B cell apoptosis and inhibit DC maturation (201,202,221). We have shown that

Treg cells isolated from A/J mice have equipotent immunosuppressive activity to Treg

116 cells from BALB/cJ mice, indicating that differences in resistance and susceptibility

between BALB/cJ and A/J mice can not be explained by the lack or reduced suppressive

function of Treg cells in resistant mice. However, in contrast to BALB/cJ mice, FGL2

does not bind to the inhibitory FcγRIIB receptor on DC and B cells from A/J mice due to

an allelic polymorphism of the FcγRIIB receptor, but binds rather to the activating

FcγRIII expressed on APCs (201). We postulate that the reduced production of FGL2 in

vivo and binding of FGL2 only to the FcγRIII may account for the protective Th1

response in A/J mice and resistance of A/J mice to MHV-3 infection.

Other factors which may reflect differences in the genetic background of the host

including the increased production of pro-inflammatory cytokines, such as IFN-γ and

TNF, in BALB/cJ mice compared to A/J mice could also contribute to resistance/susceptibility to MHV-3 infection of these two strains of mice as has been reported. Both IFN-γ and TNF can induce fgl2 linking these observations to the pathogenesis of MHV-3 (217,224,225).

The results of this study clearly demonstrate that FGL2 is an important effector cytokine of Treg cells that contributes to host susceptibility to MHV3-induced FH. As we have shown that patients with FH and chronic HBV and HCV infection have increased levels of FGL2 (166,167), the data presented here in concert with human studies suggest that measurement of FGL2 levels in the plasma may be useful in predicting the outcome of both experimental and human viral hepatitis and may provide a rationale for targeting

FGL2 for the treatment of patients with acute and chronic viral hepatitis.

117 3.6. Contributions

Measurements of FGL2 levels by ELISA were performed by Katharina Foerster,

Yi Zhu, Cecilia Chan and Jenny Yang. Kit Man Wong assisted with the real-time PCR experiments. Infection of mice with MHV-3 was performed by Xue-Zhong Ma. Infusion of cells (IV injection) in the Treg adoptive study was performed by Julia Fang Gao and

Reginald M.Gorczynski. Paraffin-embedding and sectioning, frozen tissue sectioning and

staining (IHC and H&E) were performed by the Department of Pathology at the Hospital

for Sick Children and by the Pathology Department at the Toronto Centre for

Phenogenomics. Expert histological analysis and pathological guidance were provided by

Dr. M. James Phillips of the Department of Pathology at the Hospital for Sick Children

and by Dr. Oyedele Adeyi of the Department of Laboratory Medicine and Pathobiology

at the Toronto General Hospital. Cell separation using Fluorescence-Activated Cell

Sorting (FACS) was performed by Joyce Pun and Michelle Tseng. Material from the article entitled: “The novel Treg effector molecule FGL2 contributes to the outcome of murine fulminant viral hepatitis” Shalev et al. Hepatology 49:387-397 (copyright © 2009 by The American Association for the Study of Liver Diseases, Inc.) was reused in this chapter. This material was reprinted with permission of Wiley-Liss, Inc. a subsidiary of

John Wiley & Sons, Inc.

118

Chapter 4- FGL2 Immunosuppressive Activity in an Allotransplant Model and its

Mechanism of Suppression

4.1. Summary

The suppressive activity of FGL2 was tested in vivo in a murine allotransplant model. Skin grafts from BALB/cJ mice were transplanted onto C57BL/6 mice; the recipients were treated with recombinant FGL2 (IV 20 μg day 0, 3, 6 and 9) and monitored for skin graft survival and other assays. Survival of skin grafts was markedly prolonged with FGL2 treatment from 7.8 ± 1.99 to 15 ± 2.56 days (P < 0.001). Grafts were fully protected from rejection as long as FGL2 was administered, but after discontinuation of treatment grafts were rejected within 9 days. Histological analysis of skin grafts from FGL2-treated recipients on day 6 POD revealed well preserved skin structure with less cellular infiltration and hemorrhage compared with untreated recipients. In addition, lymphocytes from recipients treated with FGL2 in contrast to lymphocytes from untreated recipients showed inhibition of proliferation in response to donor alloantigens. The suppressive activity of FGL2 was mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs, as FGL2 treatment failed to prolong allograft survival in FcγRIIB-deficient recipients. Consistent with these data, recombinant

FGL2 binding to FcγRIIB resulted in inhibition of DC maturation and induction of B cell apoptosis. Collectively, these data demonstrate that treatment with FGL2 leads to prolonged survival of fully mismatched allografts, and that the suppressive activity of

FGL2 is mediated through binding to the inhibitory FcγRIIB receptor expressed on APCs.

119 4.2. Materials and Methods

Mice

C57BL/6J, BALB/cJ and FcγRIIB-/- mice were purchased from Jackson

Laboratory (Bar Harbor, Maine, USA). Female mice at 2 to 4 months of age were used for all experiments. All mice were housed in specific pathogen free conditions and maintained at the Toronto General Hospital animal facility under the specific guidelines provided by the University Health Network.

Skin transplantation

Full thickness abdominal skin from donor BALB/cJ mice was grafted onto the dorsum of recipient C57BL/6J, FcγRIIB-/- or BALB/cJ mice (autotransplant). Skin grafts were sutured in place and covered with a band aid. Recipient mice were either untreated or treated with four IV injections of 20 μg FGL2, fibrinogen or Fc tag on days 0, 3, 6, and

9. Cyclosporine A (CsA) (0.5 mg) was administrated to recipient mice by a subcutaneous injection on days 0, 3, 6, and 9 for protocol CsA-1 or every two days until day 20 for protocol CsA-2. Graft survival was followed daily by visual inspection until the time of rejection, which was defined as a complete loss of viable skin.

Skin graft collection and histology

Skin grafts were harvested on day 6 post-transplantation and were fixed using

10% formalin. Following fixation, grafts were embedded in paraffin and sectioned in the

Pathology Department at the Hospital for Sick Children. Paraffin sections, 5 microns thick, were stained using standard H&E staining protocol (167).

120 Mixed lymphocyte reaction

Lymphocytes were isolated from FGL2-treated or untreated C57BL/6J recipients on day 6 post-transplantation, and were cultured with irradiated (2000 rad) splenocytes obtained from naive BALB/cJ mice. Cells were cultured at 37°C in a humidified (5%

CO2) incubator in a complete α-MEM media supplemented with 10 % fetal bovine serum

(FBS). Wells were pulsed with 1 μCi tritiated thymidine (Amersham Life Sciences,

Amersham, UK) for the last 18 hours of the 4-day culture to measure the proliferation rates of lymphocytes isolated from both groups.

FGL2 suppressive effect on DC

The effect of FGL2 on the LPS-induced maturation of BM-derived DC was examined by adding recombinant FGL2 to DC cultures during LPS-induced maturation

(170). BM cells from C57BL/6J and FcγRIIB-/- mice were prepared and cultured for 7 days in the presence of GM-CSF and IL-4 to derive immature DC. Immature DC were stimulated with LPS (200 ng/ml) to reach final maturation for 2 days in the presence of fibrinogen (negative control) or FGL2 (10 μg/ml). The expression of surface maturation markers, including CD80, CD86 and MHCII molecules, were measured with specific antibodies and isotype controls by flow cytometry analysis.

FGL2 induces apoptosis in B cells

The A20 B cell line was obtained from ATCC (Manassas,VA) and the A20IIA1.6 mutated B cell line was a kind gift from Dr. James Booth (Sunnybrook Research Institute,

Toronto, ON). The cells were stained with anti-FcγRIIB/III antibody (2.4G2, BD

PharMingen) and biotinylated FGL2 (2 μg) as previously described (201). 2x105 A20 and

A20IIA1.6 cells were cultured with RPMI media containing 10% FBS in 48 well tissue

121 culture plates (Corning, MA). Cells treated with 500 nM staurosporine, 500 nM FGL2 or

500 nM fibrinogen (negative control) for 12 hours, were stained with either propium iodide (PI) or FAM-VAD-FMK (poly caspases activation marker) and analyzed by flow cytometry for cell apoptosis.

Statistical analysis

A one-way ANOVA was used to compare statistical differences between groups in the mixed lymphocyte reaction study. Rates of animal survival were calculated using the Kaplan-Meier method and compared between groups with the log rank test.

Differences with P ≤ 0.05 were considered significant.

122 4.3. Results:

4.3.1. FGL2 prolongs the survival of fully mismatched skin allografts

To study the immunosuppressive role of FGL2 in vivo, we utilized a murine model of allotransplantation. A series of full thickness, fully mismatched allo-skin transplants were performed in the presence or absence of treatment with FGL2. Survival of skin grafts from BALB/cJ (H-2d) to C57BL/6J (H-2b) mice that received four IV injections of 20 μg FGL2 on days 0, 3, 6 and 9 was markedly prolonged from 7.8 ± 1.99 days to 15.0 ± 2.56 days (P < 0.001). Grafts were fully protected from rejection as long as FGL2 was administered, but after discontinuation of treatment grafts were rejected within 9 days. The enhanced survival of skin grafts by FGL2 was equivalent or greater than that seen using cyclosporine (CsA) when a similar dosing regimen was employed.

Treatment with fibrinogen or Fc-isotype control failed to prolong survival of skin grafts as compared to the untreated group (Figure 4-1 and Table 4-1).

123

Figure 4-1. FGL2 prolongs the survival of fully mismatched skin allografts. Survival of skin grafts from donor BALB/cJ to C57BL/6J recipients, which were either untreated or treated with four IV injections of 20 μg FGL2, fibrinogen or isotype control on days 0, 3, 6 and 9. Cyclosporine A (CsA) (0.5 mg) was administrated to recipient mice by a subcutaneous injection on days 0, 3, 6 and 9 for protocol CsA-1 or every two days until day 20 for protocol CsA-2. Transplants of skin grafts from donor BALB/cJ to BALB/cJ recipients were used as a positive control. Graft survival was monitored daily by visual inspection until the time of rejection. Rates of animal survival were calculated using the Kaplan-Meier method and compared between groups with the log rank test.

124 4.3.2. FGL2 treatment improves the histology of skin allografts

Skin grafts harvested from untreated recipients on day 6 post-transplantation were

totally rejected. The grafted skin was shrunken, and totally necrotic with an absence of

skin appendages. Underlying the rejected graft is a layer of dense fibrous tissue with marked inflammation. These findings are typical of graft rejection and graft loss. In contrast, grafts obtained from FGL2-treated recipients all survived. All layers of the skin graft were fully intact. The epidermis was slightly thickened and skin appendages are seen in the mid dermis. A mild inflammatory infiltrate is present (Figure 4-2).

Figure 4-2. FGL2 treatment improves the histology of skin allografts. Graph shows representative H&E staining of normal, untreated or FGL2-treated skin grafts on day 6 following transplantation. Skin graft from untreated animal shows a complete rejection. The dark blue area is the residual full thickness skin graft, showing a shrunken, totally necrotic and desiccated residuum. Note the absence of skin appendages. Full thickness

125 skin graft from FGL2-treated animal is successful. Micrograph shows good preservation of the grafted skin. The graft is in place, intact and all layers of the skin are easily identifiable. There is mild thickening of the epidermis, hair follicles and sweat glands are all present. There is a mild inflammatory infiltrate present. Skin from normal animal is shown as a normal control for comparison of histological details. Note the similarity of the skin in the successful graft to what is shown here (magnification x 200).

4.3.3. Lymphocytes from FGL2-treated recipients show inhibition of proliferation in response to donor alloantigens compared to untreated recipients

Lymphocytes were isolated from FGL2-treated or untreated C57BL/6J recipients on day 6 post-transplantation, and were cultured for 4 days with irradiated splenocytes obtained from BALB/cJ mice. Proliferation rates of lymphocytes from FGL2-treated or untreated recipients were assessed by incorporation of tritiated thymidine added during the last 18 hours of culture. Lymphocytes from FGL2-treated recipients showed a significant inhibition of proliferation in response to donor alloantigens compared to lymphocytes from untreated recipients (P = 0.01) (Figure 4-3). These and the histological results are consistent with the increased survival of allograft in the FGL2-treated group, demonstrating the potent immunosuppressant activity of FGL2 in preventing allograft rejection.

126

Figure 4-3. Lymphocytes from FGL2-treated recipients show inhibition of proliferation in response to donor alloantigens compared with untreated recipients. Proliferation rates of lymphocytes isolated from FGL2-treated or untreated recipients on day 6 post-transplantation. Lymphocytes were cultured for 4 days with irradiated splenocytes of BALB/cJ mice, and their proliferation was assessed by incorporation of tritiated thymidine added during the last 18 hours of culture. A one-way ANOVA was used to compare statistical differences between groups. Graph shows the mean ± SD of 3- 4 mice in each group; *, P ≤ 0.05.

127 4.3.4. FGL2 binding to the inhibitory FcγRIIB receptor leads to inhibition in the maturation of BM-DC

Previously, we reported that recombinant FGL2 generated in CHO cells binds to DC and B cells through binding to the inhibitory FcγRIIB receptor (201). To examine the biological significance of the binding of FGL2 to the receptor, BM-DC were isolated from wild-type and FcγRIIB-/- mice (C57BL/6 background), and cultured in the presence of GM-CSF and IL-4 for 7 days. Immature DC were stimulated for 48 hours with LPS

(200 ng/ml) in the presence of fibrinogen (negative control) or FGL2 (10 μg/ml).

Treatment with FGL2 resulted in reduced expression of CD80, CD86 and MHCII in DC from wild-type mice (Figure 4-4A), whereas FGL2 treatment had no inhibitory effect on

DC from FcγRIIB-/- mice (Figure 4-4B).

128

Figure 4-4. FGL2 binding to the inhibitory FcγRIIB receptor leads to inhibition in the maturation of BM-DC. BM cells from C57BL/6 and FcγRIIB-/- mice were prepared and cultured for 7 days in the presence of GM-CSF and IL-4 to derive immature DC as described in Materials and Methods. Immature DC were stimulated with LPS (200 ng/ml) to reach final maturation for 2 days in the presence of fibrinogen (negative control) or FGL2 (10 µg/ml). The expression of surface maturation markers including CD80, CD86, and MHCII were measured by flow cytometry. Gray histograms show fluorescence signals of cells treated with FGL2 and stained with the specific Abs. Black histograms represent cells treated with fibrinogen. The blue shaded areas are the appropriate isotype- matched Ig control. (A) The expression of CD80, CD86, and MHCII on BM-derived DC from C57BL/6 mice was decreased by FGL2 treatment. (B) The expression of maturation markers of BM-derived DC from FcγRIIB-/- mice was not affected by FGL2 treatment.

129 4.3.5. FGL2 induces apoptosis in B cells through binding to the inhibitory FcγRIIB receptor

To study the effect of FGL2 on B cells, the A20 B cell line was cultured for 12 hours in the presence of FGL2 (500 nM) or fibrinogen used as a negative control. Recombinant

FGL2 bound to A20 cells, which express FcγRIIB (Figure 4-5A), resulting in apoptosis.

A20 cells treated with FGL2 contained 43.4% positive cells for FAM-VAD-FMK (poly caspases activation marker), indicative of cells undergoing apoptosis, and 18.5% PI positive cells, indicative of dead cells (Figure 4-5C). In contrast, no apoptosis was observed in cells incubated in the presence of fibrinogen (Figure 4-5C). A20IIA1.6 cells, which do not express FcγRIIB, did not bind FGL2 and did not undergo apoptosis (Figure

4-5B and 5D). Staurosporine (500 nM) was used as a positive control to induce apoptosis in both A20 and A20IIA1.6 cell lines.

130

Figure 4-5. FGL2 induces apoptosis in B cells through binding to the inhibitory FcγRIIB receptor. (A) Left panel, A20 cells stained with anti-FcγRIIB/III Ab (red histogram) but not with isotype Ab control (black histogram). Right panel, biotinylated FGL2 (2 µg) bound to A20 cells (red) and anti-FcγRIIB/III Ab blocked its binding (black). (B) In A20IIA1.6 cells, there is neither positive staining with anti-FcγRIIB/III Ab (left panel) nor positive binding with FGL2 (right panel). (C) Induction of apoptosis in A20 cells. Cells were treated with negative control (first row), 500 nM staurosporine (second row), or FGL2 (third row) for 12 h, stained with either PI or FAM-VAD-FMK (poly caspases activation marker) and analyzed by flow cytometry. Dot plot in the left panel displays the forward scatter against side scatter properties. Dead cells with low forward scatter and high side scatter were counted in gate H. PI and FAM-VAD-FMK staining is shown in middle and right panel, respectively. Both staurosporine and FGL2 showed apoptosis induction effects. (D) Staurosporine also induced apoptosis in A20IIA1.6 cells but FGL2 failed to induce apoptosis in A20IIA1.6 cells. Results are representative of three experiments.

131 4.3.6. FGL2 treatment fails to prolong skin allograft survival in FcγRIIB-/- recipient

mice

In order to evaluate whether the protective effect of FGL2 on skin allografts

(Figure 4-1) was mediated through the inhibitory FcγRIIB receptor, fully mismatched

allografts from donor BALB/cJ mice were transplanted onto FcγRIIB-/- recipients in the

presence of FGL2 treatment (20 μg IV on days 0, 3, 6 and 9). Survival of skin allografts from BALB/cJ donors was not prolonged in FGL2-treated FcγRIIB-/- recipients compared

to untreated FcγRIIB-/- recipients (Figure 4-6 and Table 4-1). These results together with

the in vitro studies indicate that FGL2 mediates its immunomodulatory effects through binding to the inhibitory FcγRIIB receptor expressed on APCs.

Figure 4-6. FGL2 treatment fails to prolong skin allograft survival in FcγRIIB-/- recipient mice. Survival of skin grafts from donor BALB/cJ to FcγRIIB-/- recipients, which were either untreated or treated with four IV injections of 20 μg FGL2 on days 0, 3, 6 and 9. Graft survival was monitored daily by visual inspection until the time of rejection. Rates of animal survival were calculated using the Kaplan-Meier method and compared between groups with the log rank test.

132 4.3.7. A proposed model of FGL2 immunoregulatory activities

Based on these studies, we propose a model of the mechanism by which FGL2

exerts its immunoregulatory effects (Figure 4-7). Regulatory T cells produce FGL2,

which binds to the inhibitory FcγRIIB receptor expressed on DC. FGL2 binding to

FcγRIIB during the activation of immature DC results in inhibition of their maturation

associated with reduced expression of the maturation markers CD80, CD86 and MHCII.

This suppressive effect of FGL2 on DC is mediated through inhibition of NF-κB nuclear

translocation as was previously reported (170). DC that are exposed to FGL2 would be

therefore less effective in inducing proliferation and effector function of helper and

cytotoxic T lymphocytes. Suppression of helper T cell activation and DC maturation by

FGL2 could lead indirectly to inhibition of T-dependent and T-independent B cell

responses, respectively. As demonstrated by our in vitro studies, FGL2 can also directly induce apoptosis in B cells upon binding to the inhibitory FcγRIIB receptor. In allotransplantation, the indirect and direct suppressive activities of FGL2 result in inhibition of the immune response against the allograft, leading to prolongation of skin graft survival.

133

Figure 4-7. A proposed model of FGL2 immunoregulatory activities. Regulatory T cells produce FGL2, which binds to the inhibitory FcγRIIB receptor expressed on DC, resulting in inhibition of DC maturation. Following exposure to FGL2, DC would be therefore less effective in inducing proliferation and effector function of helper and cytotoxic T lymphocyte. Suppression of helper T cell activation and DC maturation by FGL2 could lead indirectly to inhibition of T-dependent and T-independent B cell responses, respectively. FGL2 can also directly induce apoptosis in B cells upon binding to the inhibitory FcγRIIB receptor. In allotransplantation, these indirect and direct suppressive activities of FGL2 result in inhibition of the immune responses against the allograft, leading to prolongation of skin graft survival.

134

Summary of skin graft survival

Group Donor Recipient N Treatment Rejection day (H2) (H2) (POD) 1 BALB/cJ BALB/cJ 2 No No rejection (d) (d) 2 10 No 7.8 ±1.99 3 4 CsA, 0.5 mg on days 0, 3, 13.7 ± 0.58 * BALB/cJ C57BL/6J 6 and 9 4 (d) (b) 4 CsA, 0.5 mg on days 0, 2, 23 ±1* 4, 6, 8, 10, 12, 14, 16, 18 and 20 5 8 FGL2, 20 μg on days 0, 3, 15 ± 2.56 * 6 and 9 6 7 Isotype control, 20 μg on 8 ± 2 days 0, 3, 6 and 9 7 7 Fibrinogen, 20 μg on days 8.4 ± 1.13 0, 3, 6 and 9 8 BALB/cJ FcγRIIβ-/- 6 No 8.5 ± 1.76 9 (d) (b) 6 FGL2, 20 μg on days 0, 3, 8.3 ± 1.63 6 and 9

Table 4-1. Survival of fully mismatched skin grafts with different treatments in FcγRIIβ-/- and FcγRIIβ+/+ recipient mice. FGL2 treatment leads to prolonged skin graft survival in FcγRIIβ+/+ recipients compared with untreated, isotype control- or fibrinogen- treated recipients. FGL2 treatment fails to prolong skin graft survival in FcγRIIβ-/- recipients. Survival data show the mean ± SD. * P < 0.01 when compared with group 2.

135 4.4. Contributions

Skin allograft transplants were performed by Dr. Wei He. Treatment of graft recipient mice with FGL2 and other reagents was performed by Dr. Wei He and Dr. Hao

Liu in the laboratory of Dr. Gary Levy. Justin Manuel produced and provided recombinant FGL2 for the in vitro and in vivo studies. Paraffin-embedding, sectioning and staining for histological analysis were performed by the Department of Pathology at the Hospital for Sick Children. Expert histological analysis and pathological guidance were provided by Dr. M. James Phillips of the Department of Pathology at the Hospital for Sick Children. The in vitro studies of FGL2 effects on DC and B cells were performed by Dr. Hao Liu (Figure 4-4 and 4-5). Some figures and related text in this chapter have been adapted from Shalev et al. J Immunol 180:249-260 (copyright © 2008 by The American Association of Immunologists, Inc.), and from Liu and Shalev et al. Eur

J Immunol 38:3114-3126 (copyright © 2008 by The European Association of

Immunologists, Inc.) with written permission from both journals.

136

Chapter 5: Discussion and Conclusions

The ability of the immune system to differentiate self from non-self is primarily

achieved through negative selection of autoreactive T cells in the thymus (226). However,

some cells do escape central tolerance, therefore an alternative mechanism of peripheral

tolerance exists to keep autoreactive T cells under control. A population of naturally

occurring CD4+CD25+ regulatory T (Treg) cells has been identified with an important

role in the maintenance of self-tolerance in the periphery. Depletion or functional

alteration of this subset in normal animals results in the development of autoimmune

diseases (227). In athymic nude mice, transfer of syngeneic splenic cells depleted in

CD4+CD25+ T cells produces autoimmune disease that is preventable by the co-transfer

of small numbers of CD4+CD25+ T cells (141). In addition to their role in the control of

self-tolerance and autoimmune diseases, Treg cells are also involved in the regulation of

T cell homeostasis, as well as in the modulation of immune responses to cancer,

pathogens, alloantigens and prevention of allograft rejection (144). Furthermore, Treg

cells have been implicated in suppressing T cell immune responses and appear to play a

role in viral persistence by suppressing HCV- and HBV-specific T cell responses leading

to chronic infection (206,207). It was demonstrated that depletion of Treg cells in mice

+ enhances the HBV-specific CD8 T cell response (209), and that circulating and liver

resident Treg cells actively influence the anti-viral immune response and disease progression in patients with Hepatitis B (208).

In recent years, there has been an intensive investigation to define the molecules that

are involved in the activity of Treg cells, which may be logical targets in the development

of novel therapeutic strategies for various diseases. Fibrinogen-like protein 2 (fgl2) has

137 been recently identified by microarray analysis screening as a putative candidate gene for

Treg cell function. Herman et al. were the first to report an increased expression of fgl2 in

CD4+CD25+ Treg cells in an insulities model (176). Following this report, Rudensky and

colleagues showed high levels of fgl2 transcripts in freshly isolated Foxp3+CD4+ Treg

cells (148,177,178,191,192). These findings are consistent with earlier studies by

Marazzi et al. (164,165), who demonstrated preferential expression of fgl2 in CD45R0+ memory T cells; in both animals and humans, circulating Treg cells have been consistently defined as belonging to the memory T cell compartment (227).

FGL2, also known as fibroleukin, is a member of the fibrinogen-related protein superfamily. Members of this family contain the fibrinogen-related domain (FRED) (179), a highly conserved region found in the carboxyl terminus of the β and γ domains of fibrinogen. This is a functionally diverse superfamily that includes the extracellular

matrix proteins tenascin, angiopoietin and ficolin. These proteins have been demonstrated

to have immunomodulatory properties, in addition to their roles in coagulation and angiogenesis (179,180,195). This activity has been mapped to the FRED region of fibrinogen, tenascin and angiopoietin (195,228). The gene fgl2 was originally cloned

from human cytotoxic T cells (CTL) using a subtractive cDNA library. The encoded

protein shares a 36% homology to the fibrinogen β and γ chains and a 40% homology to

the FRED of tenascin (162,180). Membrane-associated FGL2 (mFGL2) expressed by

activated reticuloendothelial cells (macrophages and endothelial cells) has been shown to

play an important role in innate immunity as an immune coagulant that cleaves

prothrombin to thrombin (167). This activity of mFGL2 was implicated in the

pathogenesis of several inflammatory disorders, including human and murine viral

138 hepatitis (167), xenograft rejection (175,186) and murine and human Th1 cytokine-

induced fetal loss syndrome, in which fibrin deposition is a prominent feature (188,189).

Marazzi et al. have also demonstrated that T cells isolated from human peripheral

blood express a secreted form of FGL2. He suggested that T cell-generated FGL2

possesses immunoregulatory activity, but has no coagulation function (164). Kohno et al.

found that fgl2 expression was down-regulated in both acute and chronic adult T cell leukemia/ lymphoma (171). Furthermore, proteins in the fibrinogen-related protein

superfamily have been shown to regulate immune activation. Based on these observations, it was postulated that FGL2 might play a role in acquired immune responses. In vitro

studies by Chan et al. from our laboratory have provided the first evidence that FGL2 has

immunomodulatory activity (170). Recombinant FGL2, which was generated in a

baculovirus expression system, inhibited T cell proliferation in response to alloantigens, anti-CD3/anti-CD28 monoclonal antibody and Con A in a dose dependent manner, whereas it had no direct inhibitory effect on CTL activity. The inhibitory effect of FGL2

was blocked by the use of anti-FGL2 antibody. Addition of FGL2 to allogeneic cultures

caused the polarization towards a Th2 cytokine profile with increased levels of IL-4 and

IL-10 and decreased levels of IL-2 and IFN-γ. In addition, FGL2 abrogated the LPS-

induced maturation of BM-derived DC by inhibiting NF-κB nuclear translocation,

resulting in reduced expression of CD80 and MHCII and the ability to induce alloreactive

T cell proliferation. The immunosuppressive activity was localized to the C-terminal region containing FRED (170).

Based on these immunomodulatory properties of FGL2 and the increased expression

of fgl2 in Treg cells, we proposed that FGL2 acts as an immunoregulatory cytokine of

139 Treg cells. To investigate the role of FGL2 in Treg cells, the expression levels of fgl2 in

Treg cells were measured, and the ability of anti-FGL2 antibody to inhibit Treg cell

activity was examined. Additionally, the percentage/number, Foxp3 expression and

activity of Treg cells of fgl2-/- mice were assessed (Chapter 2). In a separate study, the importance of FGL2 as an effector cytokine of Treg cells was evaluated in a murine model of fulminant viral hepatitis (FH) induced by murine hepatitis virus strain 3 (MHV-

3) (Chapter 3). Lastly, the immunosuppressive activity of FGL2 was tested in vivo in a

fully mismatched allotransplant model and the mechanism of FGL2 activity was studied

(Chapter 4).

In agreement with previous studies, high levels of fgl2 mRNA expression were

detected in Treg cells by real-time PCR. In fgl2-/- mice, increased number and percentage

of Treg cells were found with a greater expression of Foxp3 compared with wild-type

Treg cells; however, the suppressive activity of Treg cells isolated from fgl2-/- mice was

significantly impaired, and antibody to FGL2 completely inhibited wild-type Treg cell

activity in vitro. Consistent with the importance of FGL2 to Treg cell activity, targeted

deletion of fgl2 resulted in increased immune reactivity of DC, T cells and B cells, as

well as the development of an autoimmune kidney disease. T cells from fgl2-/- mice were

skewed towards a Th1 cytokine profile, and showed increased proliferation in response to

Con A and alloantigens. Increased numbers of antibody-producing B cells were observed

in fgl2-/- mice following LPS and NP-Ficoll stimulation. DC from fgl2-/- mice had higher

expression of CD80 and MHCII and an increased migration rate into the periarteriolar

lymphoid sheath (PALS) following stimulation. Six-month-old fgl2-/- mice were smaller

in size and developed autoimmune glomerulonephritis, characterized by renal cellular

140 infiltration, fibrin deposition, hemorrhage and abnormal glomerulus and tubule structure.

Fgl2-/- mice had normal proportions of T cells, B cells and macrophages, but there was an

increase in DC that might be explained by a decrease in cells undergoing apoptosis

compared to DC from littermate controls. These results are consistent with Hancock et al.

who showed that fgl2-/- mice had an intact Th1 immune response (190).

In our studies on experimental viral FH, we showed that prior to infection with MHV-

3, susceptible BALB/cJ mice had increased numbers of Treg cells and expression of

FGL2 compared to resistant A/J mice. Following MHV-3 infection, plasma levels of

FGL2 in BALB/cJ mice were significantly increased, which correlated with increased

percentage of Treg cells. Treatment with anti-FGL2 antibody completely inhibited Treg

cell activity and protected susceptible BALB/cJ mice against MHV-3-induced liver injury

and mortality. Furthermore, adoptive transfer of wild-type Treg cells into resistant fgl2-/- mice increased their mortality to MHV-3 infection.

To study the immunosuppressive role of FGL2 in vivo we utilized a fully mismatched allotransplant model. Skin grafts from donor BALB/cJ mice (H-2d) were transplanted

onto recipient C57BL/6J mice (H-2b) and treated with recombinant FGL2. Survival of

skin grafts was markedly prolonged with FGL2 treatment from 7.8 ± 1.99 to 15 ± 2.56

days (P < 0.001). Treatment of mice with fibrinogen failed to prolong survival of skin

grafts compared to the untreated group. Histological analysis of skin grafts from FGL2-

treated recipients on day 6 post-operative day (POD) revealed well preserved skin

structure with minimal cellular infiltration and hemorrhage compared with marked

cellular rejection and complete necrosis of the skin graft in untreated recipients. In

addition, lymphocytes from recipients treated with FGL2 in contrast to lymphocytes from

141 untreated recipients showed inhibition of proliferation in response to donor alloantigens.

These findings were consistent with the prolonged survival of FGL2-treated skin grafts, and suggest that FGL2 has a potent immunosuppressant activity capable of preventing allograft rejection. Enhanced survival of skin grafts by FGL2 was equivalent or greater than that seen using the immunosuppressive calcinueurin inhibitor cyclosporine A (CsA)

when a similar dosing regimen was used. However, upon cessation of treatment of FGL2

or CsA grafts were rejected within a few days (Figure 4-1). Whether a longer treatment

course or higher dose of FGL2 can induce indefinite graft survival or tolerance is now being examined. Recent generation of fgl2-transgenic mice, which ubiquitously over- express FGL2, will allow overcoming the technical difficulties of FGL2 treatment to further evaluate the effect of FGL2 as an immunosuppressant in the setting of

allotransplantation. Fgl2-transgenic mice will be used as donors or recipients of a skin

graft or vascularized solid organ, such as heart and kidney, to further evaluate the immunosuppressive effects of FGL2.

We have also shown that FGL2 mediates its immunoregulatory effects through

binding to the inhibitory FcγRIIB receptor expressed on APCs (Chapter 4). This receptor

is a member of the Fcγ receptor (FcγRs) family, which plays a key role in both innate and

acquired immune responses, including phagocytosis, antibody dependent cell mediated

cytotoxicity, inflammatory mediator release and autoimmune diseases (229). The high

diversity, redundancy and adjustability of FcγRs have enabled the host to finely modify

the immune responses to different antigenic challenges while maintaining homeostasis

(229). In mice, there are four classes of Fcγ receptors, high affinity FcγRI, moderate

affinity FcγRIV and low affinity FcγRIIB and FcγRIII (230). The activating FcγRI,

142 FcγRIII and FcγRIV receptors consist of two subunits, the ligand binding α-chain and the

γ-chain containing the immunoreceptor tyrosine-based activating motif (ITAM). The

inhibitory FcγRIIB receptor is comprised of a monomeric α-chain, which contains the

immunoreceptor tyrosine-based inhibitory motif (ITIM). As activating and inhibitory

FcγRs can bind to the same ligand leading to opposing signals, the expression level ratio

of the two class types and their ligand affinities will determine the outcome of the

immune response. The expression levels of activating/inhibitory FcγRs have been shown

to be regulated by Th1 and Th2 cytokines (231). Different mouse strains, expressing

different levels of the inhibitory FcγRIIB receptor due to polymorphism of the promoter, developed autoimmune disease (232,233). The expression of the inhibitory receptor was shown to control the balance of efficient clearance of streptococcus pneumoniae and the cytokine-mediated consequences of sepsis (234). Targeted deletion of FcγRIIB resulted in

the development of autoimmune disease, whereas deletion of the activating FcγRIII led to

impaired IgG-dependent anaphylaxis (204,235,236).

We also showed that FGL2 binding to the inhibitory low affinity FcγRIIB receptor

resulted in inhibition of DC maturation and induction of B cell apoptosis. These effects

were abrogated when DC from FcγRIIB-/- mice or B cells with mutated FcγRIIB were

used. FGL2 treatment, therefore, failed to prolong allograft survival in FcγRIIB-deficient

recipients. In agreement with our studies, it was previously reported that selective

blocking of FcγRIIB increased the activity of DC and downstream activity of T cells

(237-240), and that homo aggregation of the inhibitory receptor triggered B cell apoptosis

(241,242). Additionally, targeting FcγRIIB in B cells led to a loss of peripheral tolerance

(242) and increased activation state of B cells in response to soluble antigens (243-245).

143 The identification of the novel FGL2-FcγRIIB immunoregulatory pathway may provide better understanding of the molecular and cellular mechanisms involved in the pathogenesis of various diseases, including viral hepatitis, autoimmunity and transplant rejection, and may also have therapeutic implications. Preliminary experimental studies of ischemic-reperfusion (I/R) liver injury demonstrated that targeting FGL2 or FcγRIIB with neutralizing antibodies or by deletion had similar ameliorating effects (unpublished data), implicating a key role for FGL2-FcγRIIB in the pathogenesis of I/R injury.

Moreover, in our studies fgl2-/- mice developed autoimmune glomerulonephritis similar to that observed in FcγRIIB-deficient mice (204). As renal tubular cells express high levels of FcγRs (246), the loss of FGL2 may account for the loss of an important negative pathway leading to the development of the autoimmune disease. Thus, the FGL2-

FcγRIIB interaction could also have a direct role in prevention of autoimmunity.

Overall, the data presented here demonstrate that the levels of FGL2, produced by

Treg cells, contribute to the outcome of the immune response. As summarized in figure

5-1, lack or low levels of FGL2 result in increased immune reactivity, which could lead to the development of autoimmunity as seen in fgl2-/- mice (Chapter 2). Immune hyperactivity due to low FGL2 levels also lead to an effective anti-viral immune response as shown in MHV-3-resistant A/J mice (Chapter 3), and to accelerated cellular graft rejection as reported by Mendicino et al. (175). On the other hand, increased levels of

FGL2 result in suppression of the immune response, leading to inability to mount an effective anti-viral immunity as seen in MHV-3-susceptible BALB/cJ mice (Chapter 3), and to prolonged allograft survival (Chapter 4). Recent functional studies of T cells from fgl2-transgenic mice (over-expressing FGL2) further support the concept that high levels

144 of FGL2 lead to immunosuppression. In these studies, CD4+ effector T cells isolated from fgl2-transgenic mice exhibited hypoproliferative responses in cell cultures. In addition, purified CD4+CD25+ Treg cells from fgl2-transgenic mice had increased suppressive

activity compared with wild-type Treg cells in vitro, further demonstrating the

immunoregulatory role of FGL2 as an effector of Treg cells.

Figure 5-1. Levels of FGL2, produced by Treg cells, contribute to the outcome of the immune response. A summary view of the effects of FGL2 levels on the outcome of the immune response.

Clinical implications

The results presented here implicate FGL2 as a logical target for the development of novel therapeutic approaches for the treatment and prevention of a wide spectrum of diseases. This might be achieved by the use of FGL2 or its analogs as an immunosuppressant therapy for patients with autoimmune syndromes and graft rejection.

Depleting and neutralizing FGL2 by antibody strategies may prove to be beneficial to increase anti-viral immune responses in patients with viral hepatitis.

Preliminary studies of transplant patients and patients with chronic HCV infection confirm the immunosuppressive effects of FGL2 (Chapter 3 and 4). Patients with

145 resolved rejection had high levels of circulating FGL2 in their plasma, while only low levels of circulating FGL2 were detected in plasma of patients with graft rejection

(unpublished data). Similarly, plasma from patients with HCV contained increased levels of circulating FGL2 compared with low levels in healthy individuals or patients who cleared HCV following treatment with rebetron (interferon α and ribavirin) (Figure 5-2A).

Also, increased Foxp3+ and FGL2+ lymphocytes were detected in liver biopsies of patients who were infected with HCV (Figure 5-2B). Extensive studies in transplant and

HCV patients are presently being performed to determine the applicability of targeting

FGL2 in clinical settings. In addition, clinical trials are now being conducted to assess whether FGL2 levels can predict disease progression in HCV patients. This will allow for an early and appropriate treatment of patients with HCV to improve the efficiency of current treatments, which will lead to better clinical outcomes.

146

Figure 5-2. Increased plasma levels of FGL2 and staining of Foxp3+FGL2+Treg cells in explanted liver of patients with chronic HCV infection (A) Levels of FGL2 in the plasma of patients with chronic HCV infection compared with healthy individuals (n=8 and n=22, respectively) (* P = 0.004 by student’s t-test). (B) Panels 1 and 3: FGL2 immunostain showing cytoplasmic reactivity (brown) in many lymphocytes within cirrhotic septae in the liver of a patient with HCV infection (original magnification 1 = x 50; 3 = x 100, inset = x 400). Panels 2 and 4: Foxp3 immunostain showing nuclear reactivity (brown) in many lymphocytes within cirrhotic septae in the liver of a patient with HCV infection (original magnification 2 = x 50; 4 = x 100, inset = x 400).

147 Future plans

Future work will focus on the contribution of FGL2 to other Treg cell subsets in

addition to CD4+CD25+ Treg cells. Gene expression profiling using microarray analysis

combined with real-time quantitative PCR showed significantly higher expression of fgl2

mRNA in mouse small intestine intraepithelial lymphocytes (IEL) that express TCRαβ

and CD8αα homodimers compared with effector CD8+ T cells in the spleen (120). It has

been suggested that TCRαβ+ CD8αα+ T cells have a regulatory function in the mucosal

immune system, but the mechanism by which they might regulate immune responses remains undefined (120). The authors in their report proposed that CD8αα+ T cells could

play a role in immune regulation in the intestine through a novel mechanism involving

FGL2. Interestingly, in the same report it was also shown that TCRγδ+ Treg cells and

CD8αβ+ T cells of the intestine highly express fgl2 transcripts compared to splenic

CD8αβ+ T cells, although to a lesser extent than the expression detected in CD8αα+ T

cells (120). Consistent with these findings, Marazzi et al. previously reported increased

expression of FGL2 in the extracellular matrix of the T lymphocyte-rich upper portion of

the human intestinal lamina propria (164). Together, these data suggest that FGL2 may

have an important function in immune tolerance at mucosal sites.

Allograft acceptance using CD40Ig treatment in rats is mediated by

CD8+CD45RClow Treg cells as was previously reported (112). Microarray analysis

revealed over-expression of fgl2 transcripts in CD8+CD45RClow Treg cells that were

isolated from tolerant rats. This increased expression of fgl2 was also confirmed by real-

time PCR. A preliminary in vitro study demonstrated that antibody to FGL2 can block

148 the activity of CD8+CD45RClow Treg cells (personal communication), further strengthening our hypothesis that FGL2 is important for the function of Treg cells.

CD3+CD4-CD8- (double negative (DN)) Treg cells, which have been implicated in the maintenance of immunologic self-tolerance, prevention of transplant rejection, graft-versus-host disease and tumor outgrowth (128), were also found to express high levels of fgl2 (Figure 5-3). Functional DN Treg cell lines (CN04 and L12.24) that maintained suppressive activity produced increased levels of fgl2 transcripts and protein following their stimulation in vitro with irradiated alloantigens. However, FGL2 expression was undetectable in non-functional DN Treg mutant cell lines (CN48 and

TN12.8), which had lost their ability to suppress (Figure 5-3A and 5-3B). Furthermore, abundant levels of fgl2 mRNA were also detected in isolated DN Treg cells that were stimulated in culture for 7 days with irradiated alloantigens in a complete media including recombinant IL-2 and IL-4. The expression of fgl2 was increased during the culture of the primary DN Treg cells in a specific and time dependent manner. This increase correlated with the known increase in the function of DN Treg cells following their activation in vitro (Figure 5-3C and 5-3D). These results suggest that FGL2 may also be important for the activity of DN Treg cells.

149

Figure 5-3. Increased expression of fgl2 by both primary and cloned DN Treg cells. (A) Fgl2 mRNA expression in functional DN T cell lines that have maintained their suppressor function (DNT) relative to non-functional DN Treg mutant cell lines (Mutant), in which this function has been lost, as measured by real-time PCR. (B) FGL2 protein levels in culture supernatants of functional DN Treg cell lines (DN) and non-functional DN Treg cell lines (Mut) as measured by a sandwich ELISA. (C) Expression levels of fgl2 mRNA by real-time PCR in isolated DN Treg cells during their stimulation in vitro. Levels of fgl2 expression were normalized to those of freshly isolated DN Treg cells (day 0). (D) Expression levels of fgl2 mRNA by real-time PCR in purified DN Treg cells (DN), CD4+ T cells and CD8+ T cells on day 1 and 3 following their stimulation in vitro. Levels of fgl2 expression were normalized to those of CD4+ T cells after 1 day in culture.

150 Future studies will also focus on characterization of the structure of the membrane

and secreted forms of FGL2, as well as the mechanisms that regulate the generation of

the two forms. Ghanekar et al. have previously reported of the existence of multiple mRNA isoforms of fgl2 (4.3kb and 1.5kb), which were derived from porcine tissues

(186). The carboxyl terminal containing the FRED domain was present in both transcripts,

while the 1.5kb mRNA of fgl2 lacked the 3’ untranslated region as shown by 3’ and 5’

RACE (186). These findings suggest that multiple transcripts produced by alternative splicing may give rise to the membrane and secreted forms of FGL2; however, additional studies are still required in order to further define the post-transcriptional/translational modifications that are involved in this process.

Conclusions

These studies indicate the importance of FGL2 to the function of Treg cells as an immunoregulatory cytokine. The loss of fgl2 results in significant immune dysregulation and manifestation of an autoimmune kidney disease in aged fgl2-/- mice. Additionally,

Treg-expressed FGL2 plays a key role in the outcome of MHV-3-induced FH. Treatment

with recombinant FGL2 prolongs the survival of skin graft in a fully-mismatched

allotransplant model. FGL2 exerts its activities through binding to the low affinity

FcγRIIB, an important immunoregulatory receptor expressed on APCs. The results presented here may provide a rationale for targeting FGL2 for the treatment of patients with acute and chronic viral hepatitis, autoimmunity and graft rejection.

151

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