Role of the CTLA-4 Receptor in Regulatory T Cell Development, Homeostasis and Function By Emily Marta Schmidt A thesis submitted to The University of Birmingham For the degree of DOCTOR OF PHILOSOPHY School of Immunity and Infection College of Medical and Dental Sciences The University of Birmingham 2009 University of Birmingham Research Archive e-theses repository This unpublished thesis/dissertation is copyright of the author and/or third parties. The intellectual property rights of the author or third parties in respect of this work are as defined by The Copyright Designs and Patents Act 1988 or as modified by any successor legislation. Any use made of information contained in this thesis/dissertation must be in accordance with that legislation and must be properly acknowledged. Further distribution or reproduction in any format is prohibited without the permission of the copyright holder. Abstract Autoimmunity can occur when self-reactive lymphocytes of the adaptive immune system are activated upon encounter with antigen. This can lead to the development of debilitating and potentially life-threatening autoimmune diseases such as type-1 diabetes, rheumatoid arthritis and multiple sclerosis. Regulatory T cells (Tregs) are a subset of CD4+ T cells that express the lineage-specific transcription factor Foxp3 and exert dominant peripheral tolerance to maintain immune homeostasis. It is therefore important to fully understand the underlying mechanisms of Treg development, homeostasis and function due to the positive and negative effects that therapeutic manipulation could have on this essential T lymphocyte population. Many effector molecules have been proposed to have a central role in regulatory T cell function, and it is now clear that Tregs are equipped with multiple mechanisms by which to exert suppressive function. It has been reported that the cytotoxic T lymphocyte antigen-4 (CTLA-4) receptor is constitutively expressed by regulatory T cells and a role for this molecule in Treg suppression has been suggested. This investigation revealed a role for CTLA-4 in maintaining homeostasis of the peripheral regulatory T cell compartment. In addition, using a transgenic mouse model that permitted the development of antigen-specific Ctla-4-deficient Tregs, a role for the CTLA-4 receptor in Treg suppressive function was identified. The data obtained suggest that the CTLA-4 receptor may function on regulatory T cells by modulating CD80/CD86 co-stimulatory molecule expression by antigen-presenting cells, and hence their capacity to activate conventional T cells to generate effector T cells and instigate an effective immune response. Acknowledgements I am grateful for the opportunity to work in Dr. Lucy Walker’s group and I am thankful for the help and support provided throughout this project. I would also like to say a big thank you to Chunjing Wang as many experiments could not have been carried out without her additional help in the lab! Similarly, all past and present members of the Walker lab have provided invaluable technical assistance and very useful guidance in the development of the project over the years. Many thanks are also due to all those on the 4th floor of the IBR for their technical expertise and help provided when required. I am also thankful to all those at BMSU for looking after the mouse colonies, I would particularly like to thank Karen and Julie for the early typing of the Ctla-4-/- mice which was a big help in facilitating the study of these mice! Additionally, I am grateful to all those that have come for lunch over the years (particularly Omar Qureshi and Jen Baker – there you go, I have mentioned you specifically by name!) for the short break from work this provided :0) Outside work I would like to say cheers to Kerry, Nicola, Pidge et al. for making my time living in Birmingham most enjoyable! I would also like to say thank you very much for a lovely game of PhD fives to all members of Edgbaston (la la la) Hockey Club that have provided much fun and laughter on and off the pitch, applauwse applauwse! Last, but not least, I would like to thank all my family for their continued support over the years. Contents List of Figures…………………………………………………………………..4 List of Tables……………………………………………………………………8 List of Abbreviations…………………………………………………………...9 1.0 Introduction……………………………………………………………….12 1.1 Immunity…………………………………………………………………………12 1.1.1 The Innate Immune System…………………………………………….12 1.1.2 The Adaptive Immune System………………………………………….13 1.1.3 T Lymphocytes…………………………………………………………14 1.1.4 B Lymphocytes…………………………………………………………15 1.2 Autoimmunity……………………………………………………………………17 1.2.1 Immunological Tolerance………………………………………………17 1.2.2 Type-1 Diabetes………………………………………………………...20 1.2.3 Transgenic, Adoptive Transfer Mouse Model of Autoimmune Diabetes…………………………………………………………………21 1.3 Regulatory T Cells………………………………………………………………24 1.3.1 Development of Natural Regulatory T cells in the Thymus……………25 1.3.2 Adaptive Regulatory T Cells in the Periphery………………………….27 1.3.3 Peripheral Homeostasis of Regulatory T Cells…………………………28 1.3.4 Mechanisms of Regulatory T Cell Function……………………………29 1.4 The Cytotoxic T Lymphocyte Antigen-4 (CTLA-4) Receptor………………..35 1.4.1 The CD28 Co-Stimulatory Receptor and CD80/CD86 Ligands………..35 1.4.2 CTLA-4 Receptor Function…………………………………………….36 1.4.3 Mechanisms of CTLA-4 Receptor Function……………………………38 1.4.4 CTLA-4 and Regulatory T Cells………………………………………..39 1.5 Aims and Objectives…………………………………………………………….42 2.0 Materials and Methods…………………………………………………...43 2.1 Mice………………………………………………………………………………43 2.1.1 Mice…………………………………………………………………….43 2.1.2 Adoptive T Cell Transfers………………………………………………44 2.1.3 Bone Marrow Chimeras………………………………………………...44 2.1.4 In Vivo Anti-CTLA-4 / Anti-ICOSL Blocking Antibody Treatment…..44 2.1.5 In Vivo OVA Treatment………………………………………………...44 2.1.6 In Vivo BrdU Treatment………………………………………………...44 2.1.7 Diabetes Monitoring……………………………………………………45 2.2 Immunohistology………………………………………………………………...46 2.2.1 Section Preparation……………………………………………………..46 2.2.2 Immunochemistry………………………………………………………46 2.2.3 Immunofluorescence……………………………………………………46 2.2.4 H & E Staining………………………………………………………….47 2.2.5 Antibodies………………………………………………………………47 2.3 Flow Cytometry………………………………………………………………….48 2.3.1 Cell Isolation……………………………………………………………48 2.3.2 Staining Protocol………………………………………………………..48 2.3.3 Antibodies………………………………………………………………49 2.4 Cell Separation / Sorting………………………………………………………..52 2.4.1 Magnetic Cell Separation (MACS)……………………………………..52 2.4.2 Fluorescent Activated Cell Sorting (FACS)……………………………52 2.5 In Vitro Cell Culture…………………………………………………………….53 2.5.1 Overnight Cultures……………………………………………………...53 2.5.2 CTLA-4 Cycling………………………………………………………..53 2.5.3 TGF-β Induction………………………………………………………..53 2.5.4 Suppression Assays……………………………………………………..54 2.5.5 Anergy Assay…………………………………………………………...54 2.5.6 T Cell / Treg and B Cell / DC Co-cultures……………………………..54 2.6 Molecular Biology……………………………………………………………….55 2.6.1 RNA Isolation…………………………………………………………..55 2.6.2 cDNA Preparation………………………………………………………55 2.6.3 qPCR……………………………………………………………………55 2.6.4 TaqMan Gene Expression Assays………………………………………56 2.7 Statistics………………………………………………………………………….57 3.0 Role of CTLA-4 in Regulatory T Cell Development and Homeostasis………………………………………………………………..58 3.1 Introduction……………………………………………………………………...58 3.2 Results……………………………………………………………………………59 3.2.1 Cellular expression of the CD28 and CTLA-4 receptors, and their ligands CD80 and CD86…………..........................................................59 3.2.2 CTLA-4 receptor endocytosis…………………………………………..65 3.2.3 CTLA-4 normally functions to restrict the proliferation of Tregs in the periphery……………………………………………………………67 3.2.4 Tregs deficient in Ctla-4 undergo less cell death……………………….86 3.2.5 Induction of the Treg-specific transcription factor FOXP3…………….92 3.2.6 Ctla-4-deficiency and Treg pathogenicity…………………………….103 3.2.7 Role of CTLA-4 in T cell help for B cells…………………………….113 3.3 Discussion……………………………………………………………………….122 4.0 Role of CTLA-4 in the Suppressive Function of Regulatory T Cells…129 4.1 Introduction…………………………………………………………………….129 4.2 Results…………………………………………………………………………..131 4.2.1 In vivo evidence for a role of the CTLA-4 receptor in Treg suppressive function…………………………………………………...131 4.2.2 Ctla-4-deficient Tregs exert suppressive function in vitro....................138 4.2.3 Effect of Ctla-4-deficiency on the generation and function of antigen-specific regulatory T cells…………………………………….143 4.2.4 Induction of anergy in CD4+ CD25- conventional T cells following co-culture with Ctla-4-deficient Tregs………………………………...150 4.2.5 Modulation of CD80/CD86 expression on antigen-presenting cells by CTLA-4 on Tregs…………………………………………………..154 4.3 Discussion……………………………………………………………………….160 5.0 Role of Additional Accessory Pathways in Regulatory T Cell Homeostasis………………………………………………………………165 5.1 Introduction…………………………………………………………………….165 5.2 Results…………………………………………………………………………..166 5.2.1 Mechanistic analysis of regulatory T cell proliferation resulting from a lack of CTLA-4………………………………………………166 5.2.2 Role of the secondary co-stimulatory molecule ICOS in CTLA-4 modulation of regulatory T cell homeostasis………………………….172 5.2.3 Role of the TNF receptors OX40 and CD30 in CTLA-4 modulation of regulatory T cell homeostasis………………………………………185 5.2.4 Role of the TNF receptor RANK in regulatory T cell development and homeostasis……………………………………………………….189 5.3 Discussion……………………………………………………………………….208 6.0 General Discussion………………………………………………………212 Appendix……………………………………………………………………..216