The T cell co-stimulatory molecule GITR in the control and treatment of a persistent viral infection

by

Derek Leonard Clouthier

A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Immunology

University of Toronto

© Copyright by Derek Leonard Clouthier 2015

 

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Abstract

The T cell co-stimulatory molecule GITR in the control and treatment of a persistent viral

infection. 2015. Derek Leonard Clouthier, Graduate Department of Immunology, University of

Toronto.

During persistent viral infections such as human immunodeficiency virus (HIV) in

human or lymphocytic choriomeningitis virus (LCMV) in mice, the immune response must

achieve a balance between immune control and pathology. CD4 T cell help and co-stimulatory

factors remain under-investigated in this context. This thesis explores the role of the

Glucocorticoid-Induced Tumour Necrosis Factor Receptor-Related Protein (GITR) and its

efficacy as a target for therapy in chronic infection. Mice that lack the T cell co-stimulatory

molecule GITR have impaired LCMV-specific CD8 T cell responses and control of chronic

LCMV infection. The effects of GITR were lost when mice were depleted of CD4 T cells.

GITR directly supports the accumulation of IL-2+ T helper type 1 (Th1) cells, thereby indirectly

facilitating early LCMV-specific CD8 T cell responses, late B cell responses, and viral control.

In vivo GITR-induced signals were detected at day 3 post-infection, and defects in CD4 T cell

accumulation in GITR-deficient T cells were apparent starting at day 5 post-infection. GITR-

Ligand (GITRL) is maximally induced on antigen presenting cells at day 2 post-infection, but is

downregulated to below baseline levels by day 8 post-infection, and remains so at the chronic

stage of infection (day 21 post-infection). GITR expression was highest on regulatory T cells

(Tregs) but was also detected on Th1 and LCMV-specific CD8 T cells at day 8 post-infection

and was maintained at low levels at day 21 post-infection. As GITRL was limiting at this late

time point, we investigated the potential of therapeutic stimulation of GITR using an anti-GITR

 

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agonist monoclonal antibody. Anti-GITR treatment at day 21 post-infection increased the

frequency and number of LCMV-specific CD8 T cells and improved viral control. These effects

of anti-GITR were CD8 T cell-intrinsic. Taken together, this thesis demonstrates that GITR

plays an important early role on CD4 T cells to support CD8 T and B cell responses to

persistent LCMV infection, but at later time points, anti-GITR therapy acts directly on the CD8

T cells to improve viral control. These studies may inform the development of novel immune

therapies for human persistent viral infections as well as malignancies.

 

 

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Acknowledgements

Most importantly, I want to thank all of the past and present members of the Watts Lab.

Tania, thank you for setting me up with a fantastic project; it has been a critical factor in my

success. Thank you also for always pushing me to think critically, and for letting me chase

ideas that excited me (and not being upset when they turned out to be wrong). I also appreciate

that you supported my decision to pursue law school. I thank Birinder Ghumman for always

making the lab a positive and efficient working environment, for technical assistance, and for

preparation of antibody and virus stocks. Of course, I also want to thank all of my lab mates

(past and present), some of whom I now consider to be good friends: Samia Afzal, Frank

Chang, William Chu, Maria Edilova, Adam Komorowski, Gloria Lin, Achire Mbanwi, Regina

Medvedev, Ann McPherson, Ali Abdul Sater, Laura Snell, Lisa Wagar, Chao Wang, Michael

Wortzman, and Angela Zhou.

I thank my committee members Mario Ostrowski and Pamela Ohashi, as well as

Jennifer Gommerman and Juan Carlos Zúñiga-Pflücker for helpful discussion and feedback on

my project. I thank Michael Buchmeier (UC Irvine) for the anti-LCMV antibody clone 1.1-3;

Michael Oldstone (Scripps Research Institute) and Pamela Ohashi (Princess Margaret Hosptial)

for LCMV Armstrong and clone 13; Carlo Riccardi (University of Perugia) and Pier Paolo

Pandolfi (Harvard Medical School) for the GITR-/- mice; Pamela Ohashi for the P14 and Smarta

mice, Pamela Ohashi and Tim Sparwasser (Hannover Medical School) for the DEREG mice;

Shimon Sakaguchi (Osaka University) for agonistic anti-GITR antibody clone DTA-1; the

National Institute of Allergy and Infectious Diseases NIH tetramer core facility for LCMV

MHC class I monomers; Stacy Nichols, Jenn Martin, and Kate Banks (CCBR) for veterinary

care; Dionne White (University of Toronto) for her guidance and expertise in flow cytometry.

I also thank all of my friends, family, and partner, Jamie. Your unremitting love and

encouragement have enabled me to achieve everything that I want. Mom and Dad: I hope that

this thesis reflects the hard work that I learned from both of you.

 

 

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Table of Contents

ABSTRACT ................................................................................................................................ II ACKNOWLEDGEMENTS ..................................................................................................... IV

TABLE OF CONTENTS ........................................................................................................... V LIST OF TABLES ................................................................................................................. VIII

LIST OF FIGURES .................................................................................................................. IX LIST OF APPEDICES ............................................................................................................. XI

LIST OF PUBLICATIONS AND PRESENTATIONS ........................................................ XII ABBREVIATIONS .................................................................................................................. XV

CHAPTER 1: INTRODUCTION .............................................................................................. 2

PART I: THE CO-STIMULATORY TNFRSF MEMBER GITR .......................................................... 2 1.1. AN INTRODUCTION TO T CELL CO-STIMULATION AND THE TNFR FAMILY MEMBER GITR .. 2 1.2. EXPRESSION, REGULATION, AND STRUCTURAL FEATURES OF GITR AND GITRL ................ 3 1.3. INTRACELLULAR SIGNALING BY GITR ................................................................................. 4 1.4. STRUCTURE-FUNCTIONAL IMPLICATIONS OF GITR-GITRL INTERACTION ........................... 8 1.5. EFFECTS OF GITR-GITRL INTERACTIONS ON INNATE CELL SUBSETS .................................. 8

1.5.1. GITR-GITRL in innate inflammatory responses in vitro ............................................. 8 1.5.2. GITR-GITRL in leukocyte adhesion and migration .................................................... 9 1.5.3. Reverse signaling through GITRL in innate cells ..................................................... 10 1.5.4. Effect of GITR on NK and NKT cells ......................................................................... 11

1.6. THE ROLE OF GITR-GITRL ON B CELLS ........................................................................... 11 1.7. SIGNALING BY GITR ON REGULATORY AND CONVENTIONAL CD4 AND CD8 T CELLS. ..... 12

1.7.1. GITR co-stimulation of murine T cells ...................................................................... 12 1.7.2. Cross-regulation between GITR and other co-stimulatory molecules on T cells ...... 14 1.7.3. GITR co-stimulation of human T cells ....................................................................... 14

1.8. CELL-TYPE SPECIFIC EFFECTS OF GITR IN CANCER ............................................................ 15 1.8.1. GITR-targeted therapies for cancer .......................................................................... 15 1.8.2. Mechanisms of enhanced tumour immunity with GITR-targeted therapies .............. 16 1.8.3. GITR-GITRL in cancer immune evasion: modulating of NK and tumour cells ........ 17

1.9. GITR-GITRL IN INFLAMMATION, TRANSPLANTATION, ALLERGY, AND AUTOIMMUNITY ... 18 1.9.1. GITR-GITRL in inflammatory diseases ..................................................................... 18 1.9.2. GITR-GITRL in allergy ............................................................................................. 19 1.9.3. GITR-GITRL in models of colitis and autoimmunity ................................................. 19 1.9.4. Autoimmune models with Treg-extrinsic effects of GITR-GITRL ............................. 22 1.9.5. GITR-GITRL in tissue transplant models .................................................................. 23 1.9.6. Correlating GITR and GITRL expression in human autoimmune diseases .............. 24

 

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1.10. ROLE OF GITR-GITRL IN INFECTIOUS DISEASE MODELS ................................................. 25 1.10.1. GITR-GITRL in immunity to parasitic infections .................................................... 25 1.10.2. GITR-GITRL in immunity to fungal infections ........................................................ 25 1.10.3. GITR-GITRL in anti-viral immunity ........................................................................ 26

1.11. GITR-GITRL IN ENHANCING VACCINE EFFICACY ........................................................... 27 1.12. SUMMARY OF THE STATE OF KNOWLEDGE ON GITR ........................................................ 28 PART II: LCMV CLONE 13 AS A MODEL OF PERSISTENT VIRAL INFECTION ................................ 30 1.13. LCMV AS A MOUSE MODEL FOR PERSISTENT VIRAL INFECTION ....................................... 30 1.14. BASIC VIROLOGY OF LCMV ............................................................................................ 30 1.15. LCMV CL 13 PERSISTENCE AND IMMUNE EVASION .......................................................... 31

1.15.1. Effects of LCMV cl 13 on the early (~48 hours post-infection) immune response .. 31 1.15.2. The T cell response to persistent LCMV infection ................................................... 36 1.15.3. NK cell responses to persistent LCMV infection ..................................................... 43 1.15.4. Follicular helper T cell and B cell responses to persistent LCMV infection .......... 43

1.16. SUMMARY OF THE INTRODUCTION TO PERSISTENT LCMV INFECTION ............................. 46

THESIS SYNOPSIS .................................................................................................................. 47

CHAPTER 2: COMBINED MATERIALS AND METHODS .............................................. 49

CHAPTER 3: GITR INTRINSICALLY SUSTAINS EARLY TH1 AND LATE TFH CD4 T CELL ACCUMULATION TO CONTROL A PERSISTENT LCMV CLONE 13 INFECTION IN MICE ............................................................................................................. 54

3.1. SUMMARY ...................................................................................................................... 54 3.2. INTRODUCTION ............................................................................................................ 55 3.3. RESULTS ......................................................................................................................... 57

3.3.1. GITR is required for CD8 T cell accumulation and function post-priming and control of chronic LCMV infection ...................................................................................... 57 3.3.2. GITR potentiates the CD8 T cell response through CD8 T cell-extrinsic effects ...... 61 3.3.3. GITR critically regulates early Th1 responses to LCMV .......................................... 61 3.3.4. IL-2 is necessary for the effect of GITR on CD8 T cell responses ............................ 62 3.3.5. GITR enhances follicular helper CD4 T cell responses to LCMV cl 13 and the production of LCMV-specific IgG ....................................................................................... 68 3.3.6. GITR acts intrinsically on CD4 T cells ..................................................................... 70 3.3.7. GITR affects the accumulation but not the initial rate of division or the differentiation of LCMV-specific CD4 T cells ..................................................................... 72 3.3.8. GITR induces NF-κB and mTORC1 activation in LCMV-specific CD4 T cells ........ 74

3.4. DISCUSSION ................................................................................................................... 76

 

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CHAPTER 4: ANTI-GITR AGONIST THERAPY INTRINSICALLY ENHANCES CD8 T CELL RESPONSES TO CHRONIC LCMV, THEREBY CIRCUMVENTING LCMV-INDUCED DOWNREGULATION OF GITRL ..................................................................... 82

4.1. SUMMARY ...................................................................................................................... 82 4.2. INTRODUCTION ............................................................................................................ 83 4.3. RESULTS ......................................................................................................................... 85

4.3.1. GITRL is rapidly induced on macrophages and dendritic cells following LCMV cl 13 infection ............................................................................................................................... 85 4.3.2. GITR is highly expressed on Treg, Th1 and LCMV-specific CD8 T cells ................. 90 4.3.3. Agonistic DTA-1 improves control of chronic LCMV infection with no detectable immune pathology ................................................................................................................ 92 4.3.4. Agonistic DTA-1 augments CD8 T cell responses to LCMV with no apparent effects on CD4 T cell subsets .......................................................................................................... 94 4.3.5. Agonistic DTA-1 intrinsically enhances CD8 T cell responses to LCMV cl 13 ........ 98

4.4. DISCUSSION ................................................................................................................. 100

CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS ................................................ 105 5.1. SUMMARY AND MODEL OF THE ROLE OF GITR DURING PERSISTENT LCMV CL 13 INFECTION AND ITS USE AS AN IMMUNE MODULATORY THERAPY ............................................ 105 5.2. OUTSTANDING QUESTIONS .............................................................................................. 108

5.2.1. What are the factors that regulate GITRL expression during persistent LCMV cl 13 infection? ........................................................................................................................... 108 5.2.2. What are the APCs responsible for T cell GITR co-stimulation? ........................... 109 5.2.3. Are there different GITRL+ APC for ‘priming/imprinting’ and another for ‘sustaining/maintaining’ T cells? ...................................................................................... 111 5.2.4. Are there tissue-specific effects of GITR-GITRL? ................................................... 112

5.3. IMPLICATIONS FOR HIV AND OTHER HUMAN CHRONIC VIRAL INFECTIONS. ..................... 113 5.4. IMPLICATIONS FOR NON-VIRAL DISEASES ......................................................................... 114 5.5. ADDITIONAL UNDERSTANDING OF THE ROLE OF CD4 T CELLS DURING PERSISTENT VIRAL INFECTIONS ............................................................................................................................. 114

5.5.1. The Goldilocks effect of CD4 T cell help in persistent viral infections ................... 114 5.5.2. Understanding the complexities of co-stimulatory molecules on CD4 T helper cells during persistent LCMV clone 13 infection ....................................................................... 116 5.5.3. Decoding the complexities of CD4 T cell co-stimulation during persistent viral infections: final thoughts ................................................................................................... 121

CHAPTER 6: REFERENCES ............................................................................................... 123

APPENDIX I: LESSONS LEARNED FROM LITTERMATE CONTROLS .................. 157  

 

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List of Tables Table No. Title Page(s) 1-1 GITR and GITRL expression 29

 

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List of Figures

Figure Number

Title

Page

Chapter 1: Introduction to GITR and LCMV 1-1 Intracellular signaling by GITR 7 1-2 Context-dependent effects of GITR on Teff and Treg 21 1-3 Innate signaling induced by LCMV and inhibition of the

IFN-I response 35

1-4 The role of virus-specific CD4 T cell help in persistent LCMV infections

45

Chapter 3: CD4 T cell-intrinsic GITR sustains early type 1 and late follicular helper T cell responses to control a chronic viral infection 3-1 GITR-/- mice have impaired CD8 T cell responses and

compromised control of chronic LCMV cl 13 infection 58

3-2 CD8 T cell surface and intracellular stain gating strategy 59 3-3 GITR-/- LCMV-specific CD8 T cells express higher levels

of inhibitory receptors and are more functionally exhausted 60

3-4 The effect of GITR on the CD8 T cell response is CD8 T cell-extrinsic

63

3-5 A CD4 T cell population underscores the defective immunity in GITR-/- mice, and GITR-/- mice have fewer IL-2-producing Th1 cells

64

3-6 CD4 T cell surface and intracellular stain gating strategy 65 3-7 GITR-deficient Tregs do not play a critical role in the

impaired immunity of GITR-/- mice to LCMV cl 13 66

3-8 IL-2 is necessary for the effect of GITR on LCMV-specific CD8 T cell and CD4 Th1 responses

67

3-9 GITR-/- mice have defective follicular helper CD4 T cell responses following LCMV cl 13 infection

69

3-10 The effects of GITR-deficiency on immunity to LCMV are largely CD4 T cell-intrinsic

71

3-11 GITR co-stimulation does not affect early cell division or differentiation, but regulates CD4 T cell accumulation post-priming

73

3-12 GITR co-stimulation activates classical NF-κB and the Akt-mTORC1 signaling axis in LCMV-specific CD4 T cells

75

 

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Chapter 4: Agonist anti-GITR directly improves CD8 T cell responses and control of chronic LCMV cl 13 infection 4-1 Abbreviated gating strategy 87 4-2 GITRL is rapidly upregulated following LCMV cl 13 and

Armstrong infection, but later downregulated to below pre-stimulation levels in LCMV-infected macrophages and dendritic cells

88

4-3 GITRL is expressed by CD8, CD8, and NK cells and GITR is expressed on macrophages, DC, and B cells

89

4-4 GITR is transiently upregulated on activated Treg, Th1, LCMV-specific CD8 T cells, and NK cells following LCMV cl 13 infection

91

4-5 Agonist anti-GITR reduced chronic LCMV cl 13 viral burden in multiple organs without causing pathology

93

4-6 Agonist anti-GITR has little or no effect on CD4 T cell or B cell responses to LCMV cl 13 infection

95

4-7 Agonist anti-GITR results in durable antigen-specific CD8 T cell immunity to chronic LCMV infection

97

4-8 Agonist anti-GITR intrinsically enhances CD8 T cell responses to chronic LCMV infection

99

Chapter 5: Summary, Outstanding Questions, and Implications 5-1 The role and therapeutic efficacy of the GITR-GITRL

pathway during persistent LCMV cl 13 infection 107

5-2 The Goldilocks effect of CD4 T cell help during persistent LCMV infection

119

5-3 The variety of T cell co-signaling molecules and soluble factors that affect T cell mediated control of persistent viral infections

120

Appendix I: Lessons from littermate controls A1-1 The frequency of Foxp3+ Tregs from littermate and non-

littermate controls 158

 

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List of Appedices  

 

Appendix Title Page(s) 1-1 GITR and GITRL expression 28

 

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List of Publications and Presentations § work performed during my PhD studies but not as a part of my research requirements * presenting author Published Articles, excluding published abstracts (2011-2015)

1. Abdul-Sater, A, M Edilova, DL Clouthier, E Kremmer, and TH Watts. 2015. TRAF1 suppresses TLR signaling through limiting linear ubiquitination of NEMO. Manuscript submitted.

2. § Clouthier DL, CA Harris, RA Harris, C Martin, MC Puri, and N Jones. 2015. Requisite role for Nck adaptors in cardiovascular development, endothelial-to-mesenchymal transition and directed cell migration. Molecular and Cellular Biology 35(9): 1573-87. Highlighted as a Spotlight article in Molecular and Cellular Biology.

3. Clouthier DL, AC Zhou, ME Wortzman, O Luft, GA Levy, TH Watts. 2015. GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection. PLoS Pathogens 11(1):e1004517.

4. Clouthier DL, AC Zhou, and TH Watts. 2014. Anti-GITR agonist therapy intrinsically enhances CD8 T cell responses to chronic lymphocytic choriomeningitis virus (LCMV), thereby circumventing LCMV-induced downmodulation of costimulatory GITR ligand on APC. The Journal of Immunology 193(10): 5033-43. Highlighted In this Issue of The Journal of Immunology.

5. Clouthier DL and TH Watts. 2014. Cell-specific and context-dependent effects of GITR in cancer, autoimmunity, and infection. Cytokine and Growth Factor Reviews 25(2):91-106.

6. Bridle BW, § DL Clouthier, L Zhang, J Pol, L Chen, BD Lichty, JL Bramson, and Y Wan. 2013. Oncolytic vesicular stomatitis virus quantitatively and qualitatively improves primary CD8(+) T-cell responses to anticancer vaccines. Oncoimmunology 2(8):e26013.

7. Wortzman ME, DL Clouthier, AJ McPherson, GH Lin, and TH Watts. 2013. The contextual role of TNFR family members in CD8(+) T-cell control of viral infections. Immunological Reviews 255(1): 125-48

8. Lin GH, LM Snell, ME Wortzman, DL Clouthier, and TH Watts. 2013. GITR-dependent regulation of 4-1BB expression: implications for T cell memory and anti-4-1BB-induced pathology. The Journal of Immunology 190(9): 4627-39.

 

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Oral and/or Poster Presentations and Scientific Meetings (2011-2015)

1. Clouthier DL*, AC Zhou, ME Wortzman, O Luft, GA Levy, and TH Watts. 2015. GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection. American Association of Immunologists. New Orleans, LA, USA.

2. Clouthier DL, YH Chang, TH Watts*. 2015. Differential expression of GITRL by APC subsets during viral infection: implications for T cell responses. Keystone Dendritic Cells and Macrophages Reunited. Montreal, QC, Canada.

3. Clouthier DL*, AC Zhou, ME Wortzman, O Luft, GA Levy, and TH Watts. 2015. GITR intrinsically sustains early type 1 and late follicular helper CD4 T cell accumulation to control a chronic viral infection. Keystone Viral Immunity. Breckenridge, CO, USA.

4. Zhou AC*, LE Wagar, ME Wortzman, DL Clouthier, and TH Watts. 2015. Boosting with 4-1BBL induces long-lived protection against respiratory influenza infection, associated with a persistent CD127hi effector memory CD8 T cell population. Keystone Viral Immunity. Breckenridge, CO, USA.

5. Clouthier DL*, AC Zhou, ME Wortzman, O Luft, GA Levy, and TH Watts. 2014. The co-stimulatory molecule GITR intrinsically enhances type 1 and follicular helper CD4 T cell responses to establish early control of a persistent viral infection and potentiate the late humoral response. American Association of Immunologists. Pittsburgh, PA, USA.

6. Clouthier DL*, ME Wortzman, and TH Watts. 2013. GITR potentiates anti-viral immunity by limiting regulatory T cells. American Association of Immunologists. Honolulu, HI, USA.

7. Clouthier DL* and TH Watts. 2012. Determining the role of GITR during chronic viral infection and evaluating its efficacy as a target for therapy. The Canadian Society for Immmunology. St. John’s, NF, CA.

8. Clouthier DL* and TH Watts. 2012. Determining the role of GITR during chronic viral infection and evaluating its efficacy as a target for therapy. The Canadian Student Health Research Forum, hosted by CIHR and The University of Manitoba. Winnipeg, MB, CA.

9. § Clouthier DL*, RA Harris, MC Puri, and N Jones. 2011. Investigating the role of the Nck adaptor proteins in endothelial cells during cardiovascular development and angiogenesis. The Great Lakes Mammalian Development Meeting. Toronto, ON, CA.

 

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10. Bridle BW*, L Zhang, S Koshy, § DL Clouthier, J Pol, R Dunn, JD Basset, BD Lichty, JL Bramson, Y Wan. 2011. Rapid and massive boosting of tumour-specific T cells by targeting antigen presentation to follicular B cells. The 14th Annual Meeting of the Translational Research Cancer Consortium. Seven Springs, PA, USA.

11. Bridle BW*, L Zhang, S Koshy, § DL Clouthier, J Pol, R Dunn, JD Basset, BD Lichty, JL Bramson, Y Wan. 2011. Rapid and massive boosting of tumour-specific T cells by targeting antigen presentation to follicular B cells. Annual Scientific Meeting of the Ontario Institute for Cancer Research. Alliston, ON, CA.

Invited Commentaries

1. Watts TH and DL Clouthier: F1000Prime Recommendation of Evaluation [Kim MS et al., Nat Immunol 2015, 16(5):525-33]. In F1000Prime, 06 May 2015; DOI: 10.3410/f.725424550.793506347. F1000Prime.com/725424550#eval793506347

2. Watts TH and DL Clouthier: F1000Prime Recommendation of [Utzschneider DT et al., Nat Immunol 2013, 14(6):603-10]. In F1000Prime, 24 Jul 2013; DOI: 10.3410/f.718013241.793480185.

3. Watts TH, DL Clouthier, and AN Mbanwi: F1000Prime Recommendation of [Lang PA et al., Proc Natl Acad Sci U S A 2012, 109(4):1210-5]. In F1000Prime, 20 Aug 2012; DOI: 10.3410/f.717952761.793458241.

4. Watts TH, DL Clouthier, and AN Mbanwi: F1000Prime Recommendation of [Waggoner SN et al., Nature 2012, 481(7381):394-8]. In F1000Prime, 20 Aug 2012; DOI: 10.3410/f.13414956.793457808.

 

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Abbreviations

αDG α-Dystroglycan

αGalCer α-Galactosylceramide

AdV5 Adenovirus type 5

Ag Antigen

AICD Activation-Induced Cell Death

AIDS Acquired Immune Deficiency Syndrome

AITD Autoimmune Thyroid Disease

AITR/L Activation-Induced TNFR-Related Protein / Ligand

Akt Ak Thymoma (aka Protein Kinase B, PKB)

ALT Alanine Aminotransferase

AML Acute Myeloid Leukemia

AP-1 Activator Protein-1

APC Antigen Presenting Cell

BATF Basic Leucine Zipper Transcription Factor, ATF-like

BCL B Cell Lymphoma

BLIMP-1 B Lymphocyte-Induced Maturation Protein 1

BM Bone Marrow

BMDC Bone Marrow-derived Dendritic Cells

BrdU 5-Bromo-2-Deoxyuridine

BSA Bovine Serum Albumin

CCR C-C Chemokine Receptor

CCL C-C Chemokine Ligand

CD Cluster of Differentiation

cDC Conventional Dendritic Cell

CDK Cyclin-Dependent Kinase

CFSE Carboxyfluorescein Succinimidyl Ester

CIA Collagen-Induced Arthritis

cIAP Cellular Inhibitor of Apoptosis Protein

CMP Common Myeloid Progenitor

 

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CLL Chronic Lymphocytic Leukemia

CLP Common Lymphoid Progenitor

COX-2 Cyclic Oxygenase-2

CpG ODN CpG Oligodeoxynucleotides

CTLA-4 Cytotoxic T Lymphocyte Antigen-4

CXCR C-X-C Chemokine Receptor

CXCL C-X-C Chemokine Ligand

DC Dendritic Cell

DD Death Domain

DEREG Depletion of Regulatory T cells (mouse strain)

DT/R Diphtheria Toxin / Receptor

EAE Experimental Autoimmune Encephalomyelitis

Erk Extracellular Signal-Regulated Kinase

Fab Fragment, Antibody Binding

FACS Fluorescence Activated Cell Sorting

Fc/R Fragment, Crystallizable / Receptor

FCS Fetal Calf Serum

FFU Focus Forming Unit

FLT3/L Fms-Like Tyrosine Kinase 3 / Ligand

FMO Fluorescence Minus One

FOXO1/3 Forkhead Box O1/3

FOXP3 Forkhead Box P3

FRC Fibroblastic Reticular Cell

FV Friend Virus

GC Germinal Centre

GCR Glucocorticoid Receptor

GFP Green Fluorescent Protein

GITR/L Glucocorticoid-Induced TNFR-Related Protein / Ligand

GM-CSF Granulocyte Monocyte Colony Stimulating Factor

GP Glycoprotein

GVHD Graft-Versus-Host Disease

 

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h Human, prefix

HA Hemagglutinin

HCV Hepatitis C Virus

HIV Human Immunodeficiency Virus

HPV Human Papilloma Virus

HRP Horseradish Peroxidase

hrs Hours

HSC Hematopoietic Stem Cell

HSV Herpes Simplex Virus

IBD Inflammatory Bowel Disease

ICAM Intercellular Adhesion Molecule

ICOS Inducible T Cell Costimulator

IDO Indoleamine 2,3-Dioxygenase

IFN-I/R Interferon α/β / Receptor (aka type I IFN)

IFNγ Interferon γ (aka type II IFN)

Ig Immunoglobulin

IκBα Nuclear Factor of κ Light Polypeptide Gene Enhancer in B-cells Inhibitor α

IKKα/β/γ IκB Kinase Complex (IKKγ aka NF-κB Essential Modulator, NEMO)

IL Interleukin

i.n. Intranasal

iNOS Inducible Nitric Oxide Synthase

i.p. Intraperitoneal

IRAK Interleukin-1 Receptor-Associated Kinase

IRF Interferon Regulatory Factor

i.v. Intravenous

JAK Janus Kinase

JNK Jun N-terminal Kinase

Kd Equilibrium Dissociation Constant

LAG-3 Lymphocyte Activation Gene-3

LCMV Arm Lymphocytic Choriomeningitis Virus, Armstrong isolate

LCMV cl 13 Lymphocytic Choriomeningitis Virus, clone 13 isolate

 

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LN Lymph Node

LP Lamina Propria

LPS Lipopolysaccharide

LTβ/R Lymphotoxin-β / Receptor

m Murine, prefix

MAPK Mitogen-Activated Protein Kinase

MAVS Mitochondrial Antiviral-Signaling Protein (aka Virus-Induced Signaling

Adapter, VISA; IPS-1; Cardif)

MDA-5 Melanoma Differentiation-Associated Protein 5

mDC Myeloid Dendritic Cell

MEM Minimum Essential Media

MHC Major Histocompatibility Complex (aka Human Leukocyte Antigen, HLA;

human)

MKK/K MAPK Kinase / Kinase

MMP-9 Matrix Metalloproteinase-9

mRNA Messenger Ribonucleic Acid

MS4A Membrane Spanning 4A

mTORC Mammalian Target of Rapamycin

MyD88 Myeloid Differentiation 88

nAb Neutralizing Antibody

NF-1 Nuclear Factor-1

NFAT Nuclear Factor of Activated T Cells

NIK NF-κB-Inducing Kinase

NK-κB Nuclear Factor-κB

NK Natural Killer Cell

NKT Natural Killer T Cell

NOD Non-Obese Diabetic

NP Nucleoprotein

OVA Ovalbumin

(p) Phospho-, prefix

PBMC Peripheral Blood Mononuclear Cells

 

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PBS Phosphate Buffered Saline

PD-1/L1 Programmed Cell Death Protein-1 / -Ligand 1

pDC Plasmacytoid Dendritic Cell

p.i. Post-Infection

PI3K Phosphoinositide-3 Kinase

PMA Phorbol 12-myristate 13-acetate

PMN Polymorphonuclear Cells

ppDC Precursor Plasmacytoid Dendritic Cell

PRR Pattern Recognition Receptor

RA Rheumatoid Arthritis

RAG Recombination-Activating Genes

RdRp RNA-Dependent RNA Polymerase

RIG-I Retinoic Acid-Inducible Gene 1

RING Really Interesting New Gene

RLR RIG-I-Like Receptor

RNA Ribonucleic Acid

RIP Receptor-Interacting Protein

RPE Retinal Pigment Epithelial Cells

RPMI Roswell Park Memorial Institute (medium)

s Soluble, prefix

SCID Severe Combined Immune Deficiency

SIV Simian Immunodeficiency Virus

SLE Systemic Lupus Erythematosus

SLO Secondary Lymphoid Organ

SP-D Surfactant Protein-D

STAT Signal Transducer and Activator of Transcription

TAB-1, 2, 3 TAK-Binding Protein-1, -2, -3

TAK-1 Transforming Growth Factor β-Activated Kinase 1

T-bet (aka T box Transcription Factor 21)

TBK TANK-Binding Kinase

TBST Tris Buffered Saline with Triton X-100

 

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TCR T Cell Receptor

Tfh T Follicular Helper Cells

Tfr T Follicular Regulatory Cells

Tg Transgenic

TGFβ/R Transforming Growth Factor β / Receptor

Th1, 2, 17 T helper cell type 1, 2, 17

TIM-3 T Cell Immunoglobulin and Mucin Domain-Containing Protein 3

TIRAP TIR Domain-Containing Adaptor Protein

TLR Toll-like Receptor

TNBS 2,4,6-Trinitrobenzenesulfonic Acid

TNF/R Tumour Necrosis Factor / Receptor

TNFR/SF Tumour Necrosis Factor Receptor / Superfamily

TRAF TNFR-Associated Factor

TRIF TIR Domain-Containing Adaptor-Inducing Interferon β

Teff Effector T cell

Treg Regulatory T cell

VCAM Vascular Cell Adhesion Molecule

WT Wild-type

 

Chapter 1

Introduction

Parts of this chapter are modified from published work in Cytokine and Growth Factor Reviews: Figures 1-1 to 1-3 are published in: Clouthier DL and TH Watts. (2014). Cell-specific and context-dependent effects of GITR in cancer, autoimmunity, and infection. Cytokine & Growth Factor Reviews 25: 91-106. http://dx.doi.org/10.1016/j.cytogfr.2013.12.003 Figure 1-4 is modified from: Homann D and RG Gill. (2004). To help and help not. Nature Immunology 5: 878-79. http://dx.doi.org/10.1038/ni0904-878

1

2

 

Chapter 1: Introduction

Part I: The Co-stimulatory TNFRSF member GITR

1.1. An introduction to T cell co-stimulation and the TNFR family member GITR

Upon viral infection, T cells recognize cognate antigen in the form of peptides presented

on major histocompatibility complex (MHC) molecules. T cell co-inhibitory and co-stimulatory

molecules play important roles in T cell biology by fine-tuning the fate of T cells following

activation. CD28 is the prototypical ‘co-stimulatory’ molecule, and provided the first evidence

for the two-signal model of T cell activation, which was posited in the late 1970s (Lafferty and

Woolnough, 1977). This model was validated when CD28, a T cell co-stimulatory molecule,

prevented anergy and was critical for the induction of IL-2 (June et al., 1987; Mueller et al.,

1989).

The two-signal model has since been revised; there are dozens of T cell co-signaling

molecules that act beyond the initial T cell priming events to modulate T cell subset

differentiation, cytokine production, effector function, proliferation and survival (Chen and

Flies, 2013). These co-signaling molecules and their ligands are spatiotemporally regulated.

The tight regulation of the numerous positive and negative T cell co-signaling molecules has

given rise to the ‘tidal’ model of T cell co-signaling, whereby successive waves of stimulatory

co-signaling molecules are upregulated with distinct functions in controlling T cell activation,

prior to the gradual upregulation of regulatory co-signaling molecules later in the response (Zhu

et al., 2011). This is in contrast to the binary ‘on/off’ state of T cells associated with the two-

signal model. Most T cell co-signaling molecules belong to the immunoglobulin superfamily,

the TIM family, the SLAM family, or the TNFR superfamily. This thesis largely deals with

those of the latter.

Members of the TNFR superfamily play critical roles in immune regulation (Watts,

2005; Croft, 2009). Constitutive signaling by co-stimulatory TNFR family members generally

results in pathology. Thus, the expression of ligands and receptors is highly regulated such that

these pathways rarely signal on resting immune effector cells. These rapidly inducible signals

generally come into play after initial CD28-B7 interactions and are important for sustaining the

ensuing T cell response rather than for initial priming. This thesis discusses the role of GITR, a

member of the TNFR superfamily that is largely expressed on T cells. Evidence to date

3

 

suggests that GITR is a positive modulator of T cells. However, GITR-GITRL interactions are

far more complex than initially described, with diverse functions in several immune cell

subsets.

1.2. Expression, regulation, and structural features of GITR and GITRL

Murine (m)GITR (also referred to as TNFRSF18; activation-induced TNFR, AITR;

CD357) is a 228 residue type I transmembrane protein (Nocentini et al., 1997). Human

(h)GITR is a 241 residue type I transmembrane protein that shares ~60% sequence identity with

mGITR (Gurney et al., 1999; Kwon et al., 1999). The murine and human Tnfrsf18 loci are

found on chromosomes 4 and 1, respectively, clustered with TNFRs Ox40 and 4-1bb. GITR is

selectively activated by its ligand, GITRL (also referred to as TNFSF18; AITR-ligand, AITRL).

mGITRL is a 173-residue type II transmembrane protein (Kim et al., 2003; Tone et al., 2003).

hGITRL is a 177-residue protein with 51% sequence identity with mGITRL (Gurney et al.,

1999; Kwon et al., 1999). Both m and h Gitrl are found on chromosome 1, clustered with

TNFSF members Fasl and Ox40l (Gurney et al., 1999; Kim et al., 2003; Tone et al., 2003). The

homology and clustering of TNF/R superfamily members is likely a result of several gene

duplication events.

GITR was identified in 1997 (Nocentini et al., 1997) and initially received attention

owing to its constitutive high-level expression on Foxp3+ Tregs (McHugh et al., 2002; Shimizu

et al., 2002). GITR is also expressed at low to moderate levels on conventional naïve and

memory T cells, though is rapidly upregulated upon activation. GITR and GITRL are also

expressed on a number of other immune cell types and, on most cell types, increase upon

activation (Table 1-1). In contrast to GITR, which exhibits more sustained expression during

activation and inflammation, GITRL expression increases with activation (4-24 hrs) followed at

followed by a reduction to below pre-stimulation levels (48-96 hrs) both in vitro and in vivo

(Tone et al., 2003; Stephens et al., 2004; Suvas et al., 2005; Clouthier et al., 2014). The wide

expression pattern of GITR and GITRL increase the complexity of delineating the direct roles

of GITR-GITRL interactions in vivo.

GITR was first identified by differential display following treatment of a T cell

hybridoma with dexamethasone. GITR is also upregulated following anti-CD3, concanavalin A,

or PMA/ionomycin treatment (Nocentini et al., 1997), with similar findings in human T cells

4

 

(Kwon et al., 1999). The glucocorticoid-induced TNFR is somewhat of a misnomer;

glucocorticoid receptor (GCR)-deficient mice do not have altered development of GITRlo or

GITRhi T cell subsets, and GITR is induced by TCR signaling equivalently in GCR+/+ and GCR-

/- mice (Zhan et al., 2004). Additionally, dexamethasone treatment in vitro does not impact

GITR expression in primary mouse T cells, though there is a modest increase in GITR

expression on CD4+CD25+ cells following dexamethasone treatment in vivo (Gurney et al.,

1999; Chen et al., 2004; Zhan et al., 2004).

In Tregs, Foxp3 modulates GITR expression. Ectopic Foxp3 expression in initially

GITRlo cells confers both suppressive activity and high-level GITR expression (Khattri et al.,

2003; Wu et al., 2006). In Teff, GITR expression is reciprocally regulated by AP-1 and

canonical NF-κB (inducing) and NFAT (repressing) downstream of the TCR (Zhan et al.,

2008). In agreement with positive regulation of GITR by NF-κB, the 5’ region of the Tnfsf18

locus contains a NF-κB p65 consensus site (Nocentini et al., 2000). JNK inhibition impairs

TCR-mediated GITR upregulation (Zhan et al., 2008; Chattopadhyay and Chakraborty, 2009).

GITR is also upregulated by CD28 signaling in both conventional and regulatory T cells

(Stephens et al., 2004; Kohm et al., 2005). In bone marrow, but not spleen, IL-15 acts locally to

upregulate GITR to higher-than-baseline levels on memory phenotype CD8 T cells, perhaps

contributing to survival of these cells (Snell et al., 2012). Foxo3a may also impair GITR

upregulation (Zhan et al., 2004).

Less is known about the signals that control GITRL expression. The transcription factor

nuclear factor-1 (NF-1) positively modulates Gitrl in the context of TLR4 signaling on primary

(bone marrow dendritic cells) BMDC as well as RAW264.7 cells (Tone et al., 2003).

1.3. Intracellular signaling by GITR

Like other co-stimulatory TNFRs, GITR lacks intrinsic enzymatic activity. GITR

recruits TNFR-associated factor (TRAF) signaling adapters, of which there are six in mammals:

TRAF1-6 (reviewed in (Xie, 2013)). TRAF proteins share a conserved C-terminal TRAF

domain, which can be subdivided into a TRAF-C and TRAF-N domain (Aggarwal, 2003; Xie,

2013). The TRAF-C domain is a 7-8 stranded anti-parallel β-sandwich structure which

associates with assembled TNFR complexes (Park et al., 1999; Ye et al., 1999), while the

TRAF-N domain is responsible for oligomerization and the formation of a coiled-coil structure

5

 

(Aggarwal, 2003; Zheng et al., 2010). The N-termini of TRAFs 2-6 contain a RING domain

with a variable number of Zinc-finger domains. The RING domain of TRAFs 2 and 6 have E3

ligase activity that mediates K63-linked polyubiquitylation and the generation of signaling

platforms (Deng et al., 2000; Shi and Kehrl, 2003). While mGITR has been shown to signal via

TRAFs 1, 2, 3, 4, and 5 (Kwon et al., 1999; Esparza and Arch, 2004, 2005b; Esparza et al.,

2006; Snell et al., 2010), most of these interactions were deduced using yeast 2-hybrid screens

or over-expression systems. TRAF1-/- primary T cells respond normally to GITR, and maximal

signaling downstream of GITR in T cells requires TRAFs 2 and 5 (Snell et al., 2010). TRAF2

binds several other TNFRs (Aggarwal, 2003) but also has a role in suppressing the non-

canonical NF-κB pathway(Grech et al., 2004; Vallabhapurapu et al., 2008; Zarnegar et al.,

2008). GITR activates canonical and non-canoncial NF-κB as well as MAPK signaling (Fig. 1-

1). I have found that GITR also directly activates Akt (see Chapter 3).

The NF-κB family of transcription factors include NF-κB1 (p50), NF-κB2 (p52), RelA,

RelB, and c-Rel (p65) (Bonizzi and Karin, 2004; Vallabhapurapu and Karin, 2009). Generally

the canonical pathway is associated with RelA:p50 or p65:p50 heterodimers, and the non-

canonical pathway generally results in nuclear translocation of RelB with p100 or its

cleaved/active form p52 (Vallabhapurapu and Karin, 2009). The canonical NF-κB pathway

involves TRAF2 recruiting the cIAP proteins, which subsequently mediate K63-

polyubiquitylation of RIP1, which leads to the recruitment and activation of the IKK complex.

The IKK complex K48-polyubiquitylates IκB, which leads to its proteasome-dependent

degradation, and subsequent relief of inhibition of NF-κB dimers. cIAP proteins also have E3

ubiquitin ligase activity and may directly modify RIP (Yin et al., 2009; Zhang et al., 2010).

Basal non-canonical NF-κB signaling is suppressed by the constitutive degradation of NIK.

This occurs when TRAF2 and TRAF3 constitutively form a complex, with TRAF3 recruiting

NIK and TRAF2 recruiting cIAP. In this complex, cIAP constitutively cause the K48-

polyubiquitylation of NIK, resulting in NIK proteasomal degradation (Grech et al., 2004;

Vallabhapurapu et al., 2008; Zarnegar et al., 2008). Upon engagement of TNFRs, TRAF2 and

TRAF3 are degraded, allowing for NIK accumulation and autophosphorylation, resulting in

activation of the non-canonical NF-κB pathway (Grech et al., 2004; Vallabhapurapu et al.,

2008; Zarnegar et al., 2008).

6

 

Some studies imply a regulatory role for GITR. Relative to GITR+/+ T cells, GITR-/- T

cells hyper-proliferate in response to anti-CD3 (Ronchetti et al., 2002). GITR does not contain a

death domain (DD), but the pro-apoptotic SIVA-1 interacts with the cytoplasmic tails of GITR

and CD27 (Prasad et al., 1997; Spinicelli et al., 2002; Py et al., 2004). The DD homology

region of SIVA-1 is dispensable for apoptotic induction; instead, SIVA-1 directly binds to Bcl-

xL and prevents its pro-survival function (Xue et al., 2002). How SIVA-1 binds to GITR and

subsequent SIVA-1/Bcl-xL interaction regulates apoptosis is unclear.

7

 

GITRL

GITR

TRAF2

agonistic anti-GITRmAb

TRAF2TRAF5 cIAP1/2

RIP-1

K63Ubn

NEMO

IKKα IKKβ

(p)IκBα

(p)K48Ubn-(p)IκBα

proteasome

Akt

mTORC1

rpS6

Erk JNKp38

MKKKs

c-Jun c-Fos

PROTEINTRANSLATION

cytokines,Bcl-xl, Bcl-2cytokines

SURVIVAL

constitutive NIKdegradation

GITRengaged

TRAF2 TRAF2

cIAP1/2TRAF1 TRAF3

NIK

TRAF2 TRAF2

cIAP1/2

TRAF1 TRAF3NIK

NIKNIK

NIK(p)NIK

(p)IKKα

(p)

(p)

p100

p52 RelBprocessing

p52 RelBcytokines

CYTOKINES

p65/c-Rel

p65/c-Rel

RelA p50

or

p50

p50

Derek Clouthier, 2015

T cell

Antigen Presenting Cell

Cytosol

Nucleus

MKKs

Figure 1-1. Intracellular signaling by GITR. GITR positively modulates Erk, JNK and p38 MAPK as well as NF-κB signaling (Kanamaru et al., 2004; Ronchetti et al., 2004; Esparza and Arch, 2005b, a; Esparza et al., 2006; Ronchetti et al., 2007; Snell et al., 2010). TRAFs 2 and 5 are required downstream of GITR for maximal activation of the canonical NF-κB pathway and upregulation of the anti-apoptotic molecule Bcl-xL (Esparza and Arch, 2005b; Esparza et al., 2006; Snell et al., 2010). There is also evidence that GITR weakly activates the non-canonical NF-κB pathway (Hauer et al., 2005). Overexpression studies have shown that TRAF2 is a positive modulator of GITR-dependent NF-κB activation (Gurney et al., 1999; Kwon et al., 1999) where another report that used a dominant negative TRAF2 reported augmented NF-κB signaling downstream of GITR (Esparza and Arch, 2005b). However, this discrepancy is due to the fact that GITR likely also activates the non-canonical NF-κB pathway, and while TRAF2 positively modulates the canonical NF-κB pathway, TRAF2 is important for restricting NIK (as discussed above). The studies using the DN-TRAF2 used a luciferase reporter system that does not distinguish between canonical and non-canonical signaling and so the readouts may be difficult to interpret because NF-κB signaling may be affected by the balance between these two pathways and the amount of TRAF2 or DN-TRAF2 in the cell (Snell et al., 2011). In vitro stimulation of Tregs with GITRL also results in the nuclear translocation of cRel, p50, and p65 and activates JNK, but not Erk or p38 MAPK. The role of TRAF3 signaling downstream of GITR has been investigated in human T cells. In these studies, TRAF3 was found to have an inhibitory role (Kwon et al., 1999), consistent with its role in inhibiting the classical and alternative NF-κB pathways (Vallabhapurapu et al., 2008; Zarnegar et al., 2008).

8

 

1.4. Structure-functional implications of GITR-GITRL interaction

There are important differences in the models of GITR stimulation that have been

studied, including endogenous GITRL, transfected GITRL, forced trimeric GITRL multimers,

and agonistic anti-GITR antibodies. Importantly, mGITRL does not engage hGITR and vice

versa (Bossen et al., 2006). hGITRL can form stable dimers, trimers, and higher-order

superclusters in solution (Zhou et al., 2008b; Zhou et al., 2008c). hGITRL has shorter TNF

homology domains and a more loosely packed structure with a 450 angle between protomers

(compared to 20-300 between typical TNF superfamily members) (Chattopadhyay et al., 2007;

Zhou et al., 2008b). In keeping with this, hGITRL has only 10 residues buried between

protomers, compared to other members of the family that have up to 40 residues buried between

protomers. This reduced surface interaction may result in an inherently less stable hGITRL

oligomer, perhaps in part explaining the higher Kd for hGITR-GITRL (Kd ≈ 560nM) compared

to other TNFSF ligands (Kd ≈ 0.1-10nM) (Chattopadhyay et al., 2007; Chattopadhyay et al.,

2009a). Agonistic anti-GITR antibodies, recombinant forced-trimeric surfactant protein D (SP-

D)-mGITRL (Stone et al., 2006a; Kanagavelu et al., 2012), coiled-coil hGITRL

(Chattopadhyay et al., 2007) or Ile-zipper motif-linked hGITRL (Cui et al., 2010) trimers may

circumvent the issue of weak GITRL trimer formation because the oligomers are already pre-

formed and stable. Indeed, forced-trimeric hGITRL has a higher binding affinity (Kd ≈ 4 nM)

and greater co-stimulatory potency (Chattopadhyay et al., 2007; Chattopadhyay et al., 2009a),

with similar results obtained using stabilized superclusters (Zhou et al., 2008b). Interestingly,

forced trimeric mGITRL (Stone et al., 2006a; Kanagavelu et al., 2012) is a potent activator of

mGITR despite mGITRL crystalizing as a dimer in three independent studies, with additional

evidence for dimer formation in solution (Chattopadhyay et al., 2008; Zhou et al., 2008c;

Chattopadhyay et al., 2009b). It is not clear whether differences in oligomerization states of m

and hGITRL exist at the cell surface and whether this confers functional differences between m

and hGITR.

1.5. Effects of GITR-GITRL interactions on innate cell subsets

1.5.1. GITR-GITRL in innate inflammatory responses in vitro

GITR agonists induce NF-κB-dependent expression of matrix metalloprotease-9 (MMP-

9), TNF, and IL-6 in macrophage cell lines and primary macrophages (Kim et al., 2006c; Bae et

9

 

al., 2007). In the same cell types, GITRL reverse signaling with anti-GITRL or GITR-Fc

induces cyclic oxygenase 2 (COX-2) (Shin et al., 2002a), inducible nitric oxide synthase

(iNOS) (Shin et al., 2002b; Shin et al., 2003b), MMP-9 (Lee et al., 2003), ICAM-1 and pro-

inflammatory cytokines (Bae et al., 2008). Similar findings were observed in microglia (Hwang

et al., 2010). While all of the studies clearly implicate GITR or GITRL in the upregulation of

pro-inflammatory cytokines and MMP-9, readouts were determined at >15 hrs, making it is

difficult to distinguish between direct effects of GITR-GITRL interaction or secondary effects

due to induction of cytokines or increased cell-cell contact, resulting in engagement of

additional receptor-ligand pairs.

1.5.2. GITR-GITRL in leukocyte adhesion and migration

GITRL agonist (GITR-Fc) revealed rapid phosphorylation of STAT1 and induction of

VCAM-1 and ICAM-1, whereas blocking GITRL or GITR with antibodies decreased leukocyte

adhesion and extravasation (Lacal et al., 2013). Similarly, GITR-/- splenocytes exhibit impaired

adherence to endothelium, and this defect was restored with GITR-Fc treatment, suggesting a

role for reverse signaling through GITRL on endothelium (Lacal et al., 2013). In vivo, GITR-/-

mice or GITRL antagonist treated mice have reduced upregulation of ICAM-1, P- and E-

selectin in response to inflammation in epithelial tissue and endothelium (Cuzzocrea et al.,

2004; Cuzzocrea et al., 2006; Galuppo et al., 2011b). Consistent with these findings, GITRL-/-

RAG-/- mice develop less severe colitis than GITRL+/+ RAG-/- mice in a model of

CD4+CD45RBhi transfer and anti-CD40-induced models of colitis (Liao et al., 2013). Decreased

colitis was associated with fewer macrophages in the Lamina Propria (LP) and mesenteric LN,

whereas more of these cells were found in the spleen, leading the authors to conclude that

GITRL regulates egress of monocytes/macrophages from the spleen during inflammatory

processes (Liao et al., 2013). Anti-GITRL, but not anti-GITR-treatment reduced the number of

splenic monocytes, suggesting that reverse signaling through GITRL may modulate monocyte

egress from the spleen. It is unclear if the VCAM-1 and ICAM-1 upregulation by GITRL as in

(Cuzzocrea et al., 2004; Cuzzocrea et al., 2006; Galuppo et al., 2011b) has a role or not in the T

cell transfer models of colitis (Liao et al., 2013), or whether the effects of GITRL reverse

signaling on monocyte egress are secondary to altered production of cytokines or chemokines.

There is some evidence that soluble (s)GITR treatment can also induce cyclin D2, cyclin A,

10

 

CDK2 and CDK4, resulting in reduced cell cycle arrest and apoptosis in macrophages (Shin et

al., 2004). Thus, it is possible that direct effects of GITRL reverse signaling on monocyte

inhibition of monocyte proliferation and survival are also important in the aforementioned

colitis models.

1.5.3. Reverse signaling through GITRL in innate cells

The upregulation of MMP-9 and ICAM-1 by sGITR in macrophages was shown in one

study to be mediated by protein kinase C δ and phospholipase D (Lee et al., 2004). However,

while the effects of protein kinase C inhibition have been verified, another study was unable to

verify a role for phospholipase D downstream of GITRL (Bae et al., 2008), perhaps due to

differences in genetic background of the mice used in these studies. The latter study (Bae et al.,

2008) also demonstrated that reverse signaling to induce MMP-9 and ICAM-1 was Erk-

dependent. Erk-dependent NF-κB p50 nuclear localization peaked at 40 min post-stimulation

and so is likely due to direct effects of GITRL signaling and not secondary to increased

cytokine production (Bae et al., 2008).

GITRL has been proposed to reverse-signal through the non-canonical NF-κB pathway

to induce p52-RelB nuclear translocation and indoleamine 2,3-dioxygenase (IDO), and thereby

mediate immunosuppression. IFNα was necessary, but not sufficient for this effect (Grohmann

et al., 2007). Interestingly, the same group showed that CTLA-4-Ig induces IDO by reverse

signaling through B7 (Puccetti and Fallarino, 2008), effects that were abrogated B7-deficient

DC (Grohmann et al., 2002). Dexamethasone treatment increased IDO in mice, ostensibly via

upregulation of GITR on CD4 T cells (Grohmann et al., 2007). It is rather difficult to assess the

role of GITR during in vivo treatment of mice with dexamethasone; this steroid could have

effects on several immune cells. Further, several independent groups have suggested that

upregulation of GITR on T cells is glucocorticoid-independent (Gurney et al., 1999; Chen et al.,

2004; Zhan et al., 2004), yet in this study, dexamethasone increased the fraction of GITR+ of

total CD4+ T cells 2-fold (Grohmann et al., 2007). Thus the role of GITR in regulating IDO

expression needs further substantiation.

11

 

1.5.4. Effect of GITR on NK and NKT cells

Primary human natural killer (NK) cells express GITR and plasmacytoid dendritic cells

(pDC) isolated from human PBMC highly express GITRL after viral infection or TLR9

stimulation. GITRL enhances primary NK cell cytotoxicity and IFNγ production in the

presence of IFN-I in vitro. This effect was prevented by anti-GITRL blocking antibodies

(Hanabuchi et al., 2006). GITRL transfectants alone had no effect on the activation of primary

human NK cells; however GITRL acted in synergy with IL-2, IFNα, and NKG2D engagement

(Hanabuchi et al., 2006). Although this study implies a positive role for GITR on NK cells in

vitro, others have found that immobilized agonist anti-GITR, GITRL-Ig, or endogenous GITRL

on target cells negatively modulates NK cell NF-κB activation (Baltz et al., 2008; Liu et al.,

2008). Furthermore, the majority of studies suggest that GITR inhibits NK cell activation in

vivo in the context of malignancy (see section 1.8.3).

The role of GITR on NKT cells has not yet been extensively addressed. NKT cells

express GITR at low levels but expression increases upon activation (Kim et al., 2006a). While

it was initially thought that GITR co-stimulation was co-stimulatory for NKT cells (Kim et al.,

2006a), subsequent and more rigorous studies have arrived at the opposite conclusion. GITR

engagement on NKT cells by agonistic anti-GITR (DTA-1) suppressed the proliferation and

cytokine production by iNKT cells upon addition of αGalCer. Moreover, GITR-/- NKT cells

have enhanced proliferation and cytokine production upon αGalCer treatment (Chen et al.,

2008). These opposing results may be due to the former study using an NKT cell line DN32.D3

stimulated with αGalCer or anti-CD3, whereas the latter used primary CD1d-tetramer+ NKT

cells stimulated with αGalCer. The latter study (Chen et al., 2008) also employed GITR-/- mice

to more clearly define an inhibitory role for GITR on NKT cells.

1.6. The role of GITR-GITRL on B cells

Despite moderate expression of both GITR and GITRL on B cells, GITR is largely

dispensable for B cell development and function (Teodorovic et al., 2012). Early B cell

development is completely normal in GITR-/- mice, though there is a minor effect of GITR-

deficiency on the accumulation of pre-B cells in the bone marrow (BM) and follicular and

marginal zone B cells in the spleen. However, this deficit had no major impact on the antibody

response to model T-dependent and -independent antigens (Ag) (Teodorovic et al., 2012).

12

 

Despite normal B cell responses in GITR-/- mice, there is evidence that IL-10+ Tr1 cells

induce B cell IgG4 in a cell-contact-dependent manner, and this effect is dependent on GITR-

GITRL. Blockade of GITR or GITRL prevented IgG4 production, but could be rescued by

exogenous IL-10 treatment (Satoguina et al., 2008). It is possible that the GITR-GITRL

interaction in this model is triggering IL-10 production, which several groups have reported

upon GITR ligation (Kanamaru et al., 2004; Zhou et al., 2007; Igarashi et al., 2008).

In a tumour model, anti-GITR agonist (DTA-1) was abrogated in mature B-deficient

(JHD) mice (Zhou et al., 2010a). In this model, anti-GITR had effects on B cells, CD4, and

CD8 T cells; however the direct target cell for the anti-GITR activity cannot be ascertained

from these data. Another study using a different anti-GITR agonist (clone 2F8) showed

increased HA and OVA-specific IgG responses by strongly shifting toward a Th1 response with

increased IgG2a and IgG2b titers (Ponte et al., 2010). Taken together, it is clear that GITR is

dispensable for B cell development and activation; however, B cells may have a role in

supporting or augmenting GITR-GITRL-targeted therapies. However, it is unclear if this is due

to direct or indirect effects on B cells.

1.7. Signaling by GITR on regulatory and conventional CD4 and CD8 T cells.

1.7.1. GITR co-stimulation of murine T cells

GITR is co-stimulatory for CD4 and CD8

effector T cells. GITR ligation on T cells in

vitro with endogenous or recombinant sGITRL, mGITRL transfected cells, or agonist anti-

GITR antibodies enhances activation markers IL-2Rα/CD25 and CD69 as well as IL-2 and

IFNγ expression, cell proliferation, and cell survival—especially in the context of a sub-optimal

TCR signal (Tone et al., 2003; Kanamaru et al., 2004; Kohm et al., 2004; Ronchetti et al., 2004;

Stephens et al., 2004; Esparza and Arch, 2005a; Ronchetti et al., 2007; Igarashi et al., 2008).

GITR-/- or antisense Gitr mRNA-treated T cells are more sensitive to activation-induced cell

death (AICD), and GITR overexpression or ligation protects anti-CD3 treated T cells from

AICD (Nocentini et al., 1997; Gurney et al., 1999; Ronchetti et al., 2002). However, GITR

overexpression did not protect from Fas- or ultraviolet irradiation-mediated apoptosis

(Nocentini et al., 1997). Therefore, the induction of Bcl-xL downstream of GITR signaling in T

cells (Ronchetti et al., 2007; Snell et al., 2010) may be sufficient to prevent apoptosis by

intrinsic, but not extrinsic, programmed cell death pathways. Conversely, in the case of a strong

13

 

TCR stimulus or high Ag load, GITR co-stimulation may augment AICD in CD4 effector T

cells in vitro and in vivo (Tone et al., 2003; Kanamaru et al., 2004; Muriglan et al., 2004; Cho et

al., 2009).

GITR co-stimulation of naïve murine T cells was initially proposed to abrogate the

effects of CD4+CD25+ Tregs (McHugh et al., 2002; Shimizu et al., 2002). Treg are non-

responsive to IL-2, and GITR engagement independent of TCR signaling, allows Treg to gain

responsiveness to IL-2. Importantly, the anergic state of Tregs is often closely associated with

their suppressive function. sGITRL, together with effector T cell-derived IL-2, breaks Treg

anergy and results in Treg proliferation, similar to the effects of IL-6 and anti-CD28 on Tregs

(Ji et al., 2004). IL-2 is important in this model; the addition of neutralizing anti-IL-2

diminished the effects of sGITRL on breaking the anergic state of the Tregs (Ji et al., 2004).

Whereas some studies suggest that GITR co-stimulation abrogates Treg suppression, others

have shown that GITR engagement in vitro in fact increases Treg numbers, enhances IL-10

production, and augments their suppressive capacity (Kanamaru et al., 2004; Zhou et al., 2007;

Igarashi et al., 2008). IL-10 induction may counter-regulate the co-stimulatory effects of GITR.

In fact, when IL-10 is neutralized with anti-IL-10 in DTA-1 treated Teff/Treg co-cultures, Teff

proliferation was further enhanced (Kanamaru et al., 2004). Others have reported that GITRL-

Fc potently co-stimulates regulatory, and only minimally affects effector T cells in vitro, and

these expanded Tregs have increased suppressive ability (Liao et al., 2010). Interestingly

however, WT and GITR-/- Tregs are equally suppressive in vitro (Ronchetti et al., 2004;

Stephens et al., 2004). Therefore, high level GITR expression on naïve cells may mark

suppressive populations, but does not confer suppressive function per se.

The notion that GITR acts primarily through effects on Tregs began shifting in 2004.

Using combinations of WT and GITR-/- Treg and Teff, Stephens et al. found that DTA-1 acts on

Teff to make them refractory to Tregs (‘contrasuppression’) (Stephens et al., 2004). However,

another study found that DTA-1 acted on both Teff and Treg to mediate this effect (Ronchetti et

al., 2004). The co-culture study by Stephens et al. do not rule out a biological effect of GITR

engagement on Tregs; in fact, they also found that DTA-1 could induce proliferation of

CD4+CD25+ Tregs based on CFSE dilution (Stephens et al., 2004). Ephrem et al. also

demonstrated that Fc-GITRL expanded Tregs as measured by BrdU incorporation (Ephrem et

al., 2013). Additionally, GITRL expressing cells could co-stimulate effectors and abrogate Treg

14

 

suppression in vitro, with GITRL-specific antagonists ablating this effect (Stephens et al.,

2004). To date, the weight of the evidence suggests that GITR enhances proliferation by both

Teff and Treg. Several models discussed below provide evidence for effects of GITR on both

Teff and Treg, with different effects depending on the context of the immune response (Fig. 1-

2).

1.7.2. Cross-regulation between GITR and other co-stimulatory molecules on T cells

CD28 co-stimulation increases GITR levels on both Treg and Teff independent of IL-2

(Kohm et al., 2005). In response to sub-optimal anti-CD3, the absence of GITR on CD8 T cells

lowered CD28-induced activation, whereas a lack of CD28 did not affect the level of co-

stimulation in response to anti-GITR (Ronchetti et al., 2007). This effect was CD8 T cell-

specific and was not observed with CD4 T cells (Ronchetti et al., 2007). T cell-intrinsic effects

of GITR in these in vitro cultures are likely due to autocrine or paracrine GITR-GITRL,

because treatment of WT T cells (which are GITRLlo) with neutralizing GITRL-Fc

recapitulated the effects of GITR-deficiency (Ronchetti et al., 2007).

Aside from a role for GITR in the context of TCR activation, GITR-GITRL is required

for surface expression of 4-1BB on a fraction of memory T cells in the BM and liver of naïve

mice (Lin et al., 2013). Treatment of naïve WT mice with agonistic anti-4-1BB can cause

severe splenomegaly and hepatitis; however, GITR-/- mice, which lack 4-1BB+ CD44hi CD8 T

cells, are resistant to anti-4-1BB-induced immunopathology.

1.7.3. GITR co-stimulation of human T cells

Studies on the role of GITR in human T cells remain scarce. Staphylococcus enterotoxin

B-stimulated human monocytes express hGITRL, and counteracted Treg suppression of Teff

(Cardona et al., 2006). However, whether this was due to Treg contrasuppression or Teff co-

stimulation was not clear. Two studies suggested that hGITR does not abrogate Treg

suppression (Levings et al., 2002; Tuyaerts et al., 2007); however, these studies were performed

in vitro using human Tregs from cancer patients and thus these finding may be a reflection of

an already-compromised immune system. In the context of HIV infection, anti-GITR was

shown to increase CD4 T cell IFNγ and TNF in vitro in response to HIV p55. TNF+ CD4 T

cells also had reduced active caspase 3, consistent with a pro-survival role for GITR (Lahey et

15

 

al., 2007). Effects on Tregs were not examined, and there was no effect of GITR on the CD8 T

cell responses in this study. However, CD8 T cells respond poorly to intact protein Ag, making

it difficult to assess a CD8 T cell-intrinsic role of GITR in this study (Lahey et al., 2007).

Multimeric macaque GITRL is immunostimulatory to human T cells in vitro; it co-stimulated

Teff and prevented suppression by Tregs, however it is unclear whether these effects were due

to GITR ligation on the Teff, Treg, or both (Stone et al., 2006b; Lahey et al., 2007; Cui et al.,

2010).

1.8. Cell-type specific effects of GITR in cancer

1.8.1. GITR-targeted therapies for cancer

Anti-GITR demonstrated efficacy as a cancer immune therapy in several mouse models,

including concomitant immunity to B16 melanoma (Turk et al., 2004), Meth-A sarcomas (Ko et

al., 2005), and CT26 colon carcinoma (Zhou et al., 2007). Stimulatory GITRL-Fc is also a

potent stimulator of anti-tumour immunity (Hu et al., 2008), with similar results from GITRL-

expressing tumours (Cho et al., 2009; Piao et al., 2009). Tumours transduced in vivo with

AdV5-GITRL (Calmels et al., 2005), or treatments that induce GITRL on DC (Tian et al.,

2012) also augment anti-tumour responses. DCs engineered to secrete GITRL-Fc or anti-GITR

agonist (DTA-1) also induce potent anti-tumour CD8 T cell response (Boczkowski et al., 2009).

Co-transfection with DNA for CMS5 antigens (mErk2) and GITRL together impaired tumour

progression (Nishikawa et al., 2008). CTLA-4 blockade and agonistic anti-GITR are synergistic

in cancer models (Ko et al., 2005; Mitsui et al., 2010). Similarly, transfection of DC with

tumour Ag together with anti-CTLA-4 (antagonist) and anti-GITR (agonist) antibodies had a

synergistic effect in both vaccine and tumour protection models (Pruitt et al., 2011).

A key concern with cancer immune-mediated therapy is the development of

autoimmunity. Although initial reports suggested that anti-GITR agonist (DTA-1) treatment of

Balb/c mice induced colitis (Shimizu et al., 2002), no effect on autoimmunity (Ko et al., 2005;

Boczkowski et al., 2009; Mitsui et al., 2010), or very mild autoimmunity (Ramirez-Montagut et

al., 2006; Cohen et al., 2010) was noted in the different murine tumour models.

16

 

1.8.2. Mechanisms of enhanced tumour immunity with GITR-targeted therapies

After anti-GITR agonist therapy, more CD4 and CD8 T cell produce IFNγ and

Granzyme B (Ko et al., 2005; Hu et al., 2008; Nishikawa et al., 2008; Cohen et al., 2010) and

multifunctional IFNγ+TNF+CD107a+ cells with GITR agonist therapy (Imai et al., 2009). The

effect of anti-GITR was lost in IFNγ-deficient mice (Ko et al., 2005), although this does not

distinguish between a direct effect of DTA-1 on IFNγ and a requirement for IFNγ in tumour

control that is required in addition to the effects of DTA-1.

In several of the aforementioned studies, depletion of CD4 T cells (Cohen et al., 2006)

or the absence of functional Tregs (Calmels et al., 2005; Ramirez-Montagut et al., 2006) did not

affect the outcome, but the therapeutic effect was lost after CD8 T cell depletion (Hu et al.,

2008; Nishikawa et al., 2008; Cho et al., 2009; Liu et al., 2009; Piao et al., 2009). In a B16

model, some have found that tumour rejection was dependent on CD4, CD8, and NK1.1+ cells

and required IFNγ and FasL, but was perforin-independent (Ramirez-Montagut et al., 2006).

Another study demonstrated that B cells are also required support the DTA-1-mediated

increases in CD8 T cell cytotoxicity and degranulation (Zhou et al., 2010a). However, it is

unclear if the B cells in this model are contributing to a humoral response, acting as APC, or a

source of cell-surface or soluble factors to co-ordinate the response. Several studies used subset

depletion to infer a role for GITR on those cell types, but these studies do not demonstrate an

intrinsic role for GITR on these cells.

Anti-GITR (DTA-1) treatment was shown to decrease Foxp3 expression or alter

intracellular localization of Foxp3 in intratumoural Tregs (Cohen et al., 2010). Others have

reported that the same reagent is an effective therapy for malignancy because it depletes Tregs

(Coe et al., 2010; Bulliard et al., 2013). Activating FcγRs are required for the anti-tumoural

effects of DTA-1; FcγR-/- mice or DTA-1 with an N296A mutation that abrogates FcγR binding

had no effect on Treg populations or tumour control (Bulliard et al., 2013). However, the

former study (Cohen et al., 2010) demonstrated that anti-GITR resulted in decreased Foxp3

expression, rather than depletion of Tregs. After transfer of CD45.1 congenic Foxp3+GFP+

Tregs (from Foxp3-gfp mice), DTA-1 treatment decreased the Foxp3+ Treg population.

However, CD45.1+ unstable ‘ex-Tregs’ could be identified in the tumour that had lost Foxp3

expression (Cohen et al., 2010). Whether the effect of DTA-1 on Foxp3 levels was due to a

direct effect of GITR signaling on the Tregs causing loss of Foxp3 or indirect due to an altered

17

 

cytokine environment was not clear. In the same model, the authors reconstituted RAG-/- mice

with mixtures of WT and GITR-/- Teff and Treg and monitored tumour size after treatment with

anti-GITR. Although the effects on tumour size were only transient, DTA-1 therapy was most

effective when both Teff and Treg were GITR-sufficient (Cohen et al., 2010), similar to in vitro

findings using combinations of WT and GITR-/- Teff and Treg (Ronchetti et al., 2004).

In contrast, Côté et al. demonstrated a CD8 T cell-intrinsic role for GITR by

reconstituting RAG-/- mice with WT or GITR-/- Treg and Teff (Cote et al., 2011). When GITR-

sufficiency was limited to the CD8 T cell compartment, DTA-1 had optimal effects and

intrinsically enhanced CD8 T cell functional avidity, whereas the absence of GITR on Tregs

had no impact on the efficacy of DTA-1. Thus, despite rigorous analysis of the cell types

required for cancer therapy in these two models (Cohen et al., 2010; Cote et al., 2011), there are

clearly differences depending on the model studied and the experimental techniques used. It is

likely that DTA-1 acts on, or at least through, effects on multiple immune subsets, with the

relative importance reflecting the different mechanisms of tumour clearance in different models

(Ramirez-Montagut et al., 2006; Cohen et al., 2010; Zhou et al., 2010a).

1.8.3. GITR-GITRL in cancer immune evasion: modulating of NK and tumour cells

GITRL is expressed on a number of primary human intestinal cancers as well as on

cancer cell lines of leukemic, melanoma, and prostate origin. During co-culture of tumour cell

lines with NK cells, blocking GITR-GITRL interactions led to increased NK cell cytotoxicity

(Baltz et al., 2007). In addition to increasing CLL survival factors (Buechele et al., 2012),

GITRL on CLL lines from patients also mediates resistance to Rituximab-induced NK

reactivity and IFNγ production. Anti-GITR blocking antibodies prevented GITR inhibition of

NK cells (Buechele et al., 2012), with similar findings in AML (Baessler et al., 2009).

Moreover, sGITRL-containing patient sera impaired human NK cell function in vitro (Baltz et

al., 2008). Tumours that do not express GITRL have adopted a unique approach to evade NK

cell killing: hematogenously disseminating tumour cells ‘pseudo-express’ GITRL via coating

by GITRL+ platelets (Placke et al., 2012). Interestingly, GITR engagement on primary human

NK cells in vitro by GITRL+ platelet-coated tumour cells reduced NK cell cytotoxicity and

IFNγ production, which was partially prevented by an anti-GITR blocking antibody (Placke et

al., 2012). Taken together, these studies further support the notion that GITR is inhibitory for

18

 

NK cells (see section 1.5.4) and that GITRL expression by human cancers allows evasion of

NK-mediated lysis. It remains to be determined if the stimulatory effect of anti-GITR on T cell

populations outweighs the inhibitory effects of GITR on NK cells. In 2007, a National Cancer

Institute workshop rated GITR 12th among promising strategies for cancer immunotherapy

(http://ncifrederick.cancer.gov; search term: Immunotherapy workshop). Trials of agonist anti-

GITR for stage III/IV melanoma and solid tumours are recruiting (see www.clinical trials.gov;

Search term: GITR).

1.9. GITR-GITRL in inflammation, transplantation, allergy, and autoimmunity

In contrast to what has been shown in cancer models (where the net effect of GITR

engagement appears to be therapeutically advantageous) models of inflammation,

autoimmunity, and tissue transplantation suggest that blocking GITR-GITRL interactions is

beneficial. Blockade or lack of GITR signaling mitigates pathological inflammation, anti-self,

or anti-transplant immune responses.

1.9.1. GITR-GITRL in inflammatory diseases

In models of inflammation, GITR-/- mice exhibit reduced leukocyte infiltration,

inflammatory cytokines and mediators, and disease severity compared to WT mice (Cuzzocrea

et al., 2004; Cuzzocrea et al., 2005; Cuzzocrea et al., 2006; Cuzzocrea et al., 2007; Santucci et

al., 2007; Nocentini et al., 2008; Galuppo et al., 2011a; Galuppo et al., 2011b). WT and GITR-/-

Tregs from mice with collagen-induced arthritis had similar suppressive capacity in vitro;

however, GITR-/- Treg protected more than WT Treg when co-transferred with Treg-depleted

splenocytes from WT arthritic mice into SCID mice (Cuzzocrea et al., 2005). Treatment with

GITR-Fc mitigates inflammation in WT mice (Cuzzocrea et al., 2006; Cuzzocrea et al., 2007;

Santucci et al., 2007; Nocentini et al., 2008; Galuppo et al., 2011a; Galuppo et al., 2011b;

Takata et al., 2012). GITR-Fc had an effect in WT but not GITR-/- mice, suggesting that this

effect was due to blocking GITR, rather than inducing GITRL reverse signaling (Nocentini et

al., 2008). In contrast to the aforementioned studies, treatment of naïve mice with GITR-Fc

resulted in inflammation, which is thought to be due to reverse signaling via GITRL (Shin et

al., 2003a). Perhaps the aggregation state of different GITR-Fc preparations influences whether

19

 

it is blocking GITR-GITRL or inducing reverse signaling. These studies require further

investigation to substantiate their conclusions.

1.9.2. GITR-GITRL in allergy

In a model of allergic asthma induced by OVA sensitization, GITR engagement

increased airway hyperresponsiveness, serum IgE and Th2 cytokines after ex vivo restimulation

(Motta et al., 2009). This study also demonstrated a role for GITR co-stimulation of CD4 cells,

but there is a possible additional contribution in vivo by blocking GITRL reverse signaling as in

a previous study (Grohmann et al., 2007), but this was not evaluated. Whereas Tregs normally

suppress allergic responses in the lung, this effect was lost upon anti-GITR-induced JNK

activation in Tregs (Joetham et al., 2008; Joetham et al., 2012).

IL-4, -13, and TNF increase GITRL expression in keratinocytes in atopic dermatitis and

together with GITR-Fc can increase keratinocyte CCL17 that attracts skin-homing Th2 cells

(Byrne et al., 2012), as well as IL-8 and monocyte chemotactic protein-1 (Byrne et al., 2009).

Similarly, IL-4 can increase GITRL expression on LA4, an airway epithelial cell line, in vitro

(Albrecht et al., 2012). Taken together, there is clear evidence for cross talk between

inflammatory cytokines and non-immune cells that can up-regulate GITRL.

1.9.3. GITR-GITRL in models of colitis and autoimmunity

Transfer of CD4+CD45RBhi or CD4+CD25- conventional T cells in the absence of

CD4+CD45RBlo cells (which contain a mixture of memory, effector, and regulatory T cells) or

CD4+CD25+ into RAG-/- or SCID mice induces a severe wasting disease and colitis (Singh et

al., 2001). Accordingly, transfer of GITR-depleted or CD4+GITRlo cells also induced disease,

consistent with GITR expression on Tregs (Shimizu et al., 2002; Uraushihara et al., 2003). In

vivo administration of anti-GITR (DTA-1) into WT mice (Shimizu et al., 2002) or into SCID

mice reconstituted with CD4+CD45RBlo and CD4+CD45RBhi cells resulted in colitis

(Uraushihara et al., 2003). DTA-1 also increased disease severity and death in a TNBS-induced

colitis model (Lee et al., 2006) with similar results in experimental autoimmune thyroiditis

(Morris and Kong, 2006).

In naïve mice, GITRL-Fc increases Treg proliferation; however, in the context of a

CD4+CD45RBhi into RAG-/- transfer model of colitis, GITRL-Fc made disease worse and

20

 

increased Teff accumulation (Ephrem et al., 2013). When GITR-/- Teff and WT Treg were co-

transferred into RAG-/- mice, so that only Treg could respond to GITRL-Fc, the authors noted

dramatic Treg apoptosis and increased IBD severity (Ephrem et al., 2013). While it was

assumed by Ephrem et al. that this is a direct effect of GITR on Tregs, a caveat to this

interpretation is that other cells in RAG-/- mice also express GITR, so the effects on Tregs could

be indirect, via production of cytokines produced by RAG-/- recipient non-T cells. Interestingly,

transfer of purified Tregs into RAG-/- mice together with GITRL-Fc resulted in loss of Foxp3

expression; however, these cells did not cause pathology, perhaps because there remained a

fraction of Foxp3+ donor cells to suppress the putative ‘ex-Treg’ effectors (Ephrem et al., 2013)

(Fig. 1-2).

One paradoxical notion is that several studies report that GITR engagement decreases

Foxp3 expression and limits Foxp3+ Treg accumulation both in vitro and in vivo. Conversely,

others report GITRL overexpression or exogenous GITR agonists expand Foxp3+ Tregs both in

vitro and in vivo (Ji et al., 2004; Kanamaru et al., 2004; Stephens et al., 2004; Zhou et al., 2007;

Igarashi et al., 2008; Liao et al., 2010). Consistent with a role for GITRL in maintaining Tregs

in naïve mice, GITR-/- mice have slightly fewer Tregs (Ronchetti et al., 2002; Stephens et al.,

2004; Cuzzocrea et al., 2005) despite equivalent thymic output. Transgenic overexpression of

GITRL on MHC II+ APC (Carrier et al., 2012) or B cells (Cd19-Gitrl) (van Olffen et al., 2009)

results in a ~3-fold increase in Tregs and Cd19-Gitrl mice have delayed onset of EAE (van

Olffen et al., 2009). Conversely, B cell-deficient or -depleted mice exhibited an exacerbation of

the acute phase of EAE, and correction of this defect by addition of WT B cells was abrogated

when pre-incubated with a blocking anti-GITRL (Ray et al., 2012). Thus, under these

circumstances, GITRL enhances Treg accumulation and regulates recovery from the acute

phase of EAE. Others have reported that agonist anti-GITR (DTA-1) (Ramirez-Montagut et al.,

2006) or treatment with GITRL-Fc also enhances Treg proliferation in vivo (Liao et al., 2010;

Ephrem et al., 2013) and this enhances self-tolerance (Liao et al., 2010).

21

 

Figure 1-2. Context-dependent effects of GITR on Teff and Treg. In the naïve state, GITR co-stimulation sustains and may increase Foxp3+ Treg accumulation. However, in the inflamed state, GITR co-stimulation may limit Treg accumulation while simultaneously augmenting Teff responses. However, it is unclear if this is due to a direct cell-intrinsic effect of GITR or due to secondary effects from changes in other cell-associated receptors or soluble factors.  

TCR NFκB?

GITR

TNFSF18 (GITR)

Foxp3 pro-survival

genes?

Peripheral Treg Context: Naïve

Peripheral Treg Context: Immune challenge/inflammation

TCR NFκB

GITR other co-signals?

JNK others?

Immune regulatory

genes

TCR

GITR

TNFSF18 (GITR)

TRAF2/5 complex

NFκB

JNK NFAT

NFκB IκB

Effector T cell Context: TCR activation

GITRL, GITRL-Fc

agonist anti-GITR mAb

proliferation signals?

TNFSF18 (GITR)

Foxp3

cell death? role?

GITRL, GITRL-Fc

agonist anti-GITR mAb

GITRL, GITRL-Fc

agonist anti-GITR mAb

IκB degradation

Bcl-xl

NFκB

NFκB

T cell survival

?

Erk

JNK

p38

cytokines

activation markers

OTHERS?

22

 

1.9.4. Autoimmune models with Treg-extrinsic effects of GITR-GITRL

While Th1 and Th2-mediated models of autoimmunity are exacerbated by anti-GITR

agonist treatment (Patel et al., 2005), only a handful of studies attempted to dissect cell-intrinsic

effect of GITR. In NOD mice, anti-GITR (2F8) accelerated disease onset and increased the

number of activated diabetogenic CD4 and CD8 Teff, without affecting the number of Tregs.

Moreover, treatment with anti-GITRL blocking antibody had the opposite effect (You et al.,

2009). These findings were recapitulated in CD28-/- NOD mice, which lack Tregs, allowing the

authors to conclude that Treg-intrinsic GITR is not the target of anti-GITR and is dispensable in

this model. Similar findings were obtained in an EAE model, where anti-GITR (DTA-1)

increased severity of disease and prior functional inactivation of Tregs with anti-CD25 did not

influence the outcome (Kohm et al., 2004). A caveat to these studies is that anti-CD25 may act

on activated Teff as well as Tregs, and anti-CD25 treatment is transient, with rapid Foxp3+ Treg

repopulation (Couper et al., 2007). Although the absence of Tregs in these models did not

change the effect of anti-GITR, these data do not demonstrate Teff-intrinsic effects of anti-

GITR.

In another study, GITR-deficiency on Teff and Treg cells had no impact on colitis

induction following WT or GITR-/- CD4+CD45RBhi T cell transfer into RAG-/- mice. Instead,

GITR-/- RAG-/- recipients exhibited more severe colitis due to an imbalance between CD103+

tolerogenic DC and PDCA-1+ pDC, thereby skewing the ensuing T cell response towards the

more colitogenic Th1 fate equivalently whether the transferred T cells were WT or GITR-/-

(Liao et al., 2012). Interestingly, even non-fractionated CD4 T cells induced colitis in GITR-/-

RAG-/-, but not GITR+/+ RAG-/- mice. This study is in contrast to studies of T cell-induced

colitis (Ephrem et al., 2013) and autoimmune diabetes (You et al., 2009) models that

demonstrate GITR is important on the T cells for exacerbation of autoimmunity by GITRL-Fc

or agonistic anti-GITR. An important distinction is that the latter study (Liao et al., 2012) is in

the context of WT and GITR-deficient T cell transfer-induced colitis, whereas the previous

studies (You et al., 2009; Ephrem et al., 2013) involve GITR agonists. It is possible that upon

transfer into a naïve mouse, the level of GITRL is insufficient to trigger GITR on the

transferred populations, so the effect of T cell GITR-sufficiency or -deficiency is only realized

in the presence of exogenous GITR agonists.

23

 

1.9.5. GITR-GITRL in tissue transplant models

In tolerized transplant recipients, in which CD25- CD4 and CD8 T cells were transferred

into RAG-/- mice, anti-GIT