A Critical Role for Gimap5 in CD4+ T cell Homeostasis and Maintenance of Peripheral Immune Tolerance A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfillment of the requirements for the degree of Doctor of Philosophy (Ph.D.) in the Immunobiology Graduate Program of the College of Medicine 2013 by Halil Ibrahim Aksoylar B.S., Middle East Technical University, Turkey, 2003 M.S., Sabanci University, Turkey, 2005 Committee Chair: Kasper Hoebe, Ph.D. Christopher Karp, M.D Edith Janssen, Ph.D. Julio Aliberti, Ph.D. David Plas, Ph.D. Abstract T cell lymphopenia is a condition which arises from defects in T cell development and/or peripheral homeostatic mechanisms. Importantly, lymphopenia is often associated with T cell-mediated pathology in animal models and in patients with autoimmune disease. In this thesis, using an ENU mutagenesis approach, we identified sphinx mice which presented severe lymphopenia due to a missense mutation in Gimap5. Characterization of Gimap5sph/sph mice revealed that Gimap5 is necessary for the development of NK and CD8+ T cells, and is required for the maintenance of peripheral CD4+ T and B cell populations. Moreover, Gimap5-deficient mice developed spontaneous colitis which resulted in early mortality. Gimap5sph/sph CD4+ T cells presented progressive lymphopenia-induced proliferation (LIP), became Th1/Th17 polarized, and mediated the development of colitis. Furthermore, Gimap5sph/sph FoxP3+ regulatory T cells became selectively reduced in the mesenteric lymph nodes and adoptive transfer of wild type regulatory T cells prevented colitis in Gimap5-deficient mice. Importantly, the expression of Foxo transcription factors, which play a critical role in T quiescence and Treg function, was progressively lost in the absence of Gimap5 suggesting a link between Gimap5 deficiency and loss of immunological tolerance. Using OT-II RAG-/- TCR transgenic model, we showed that treatment with cognate antigen under tolerizing conditions failed to induce a Treg population and resulted in the acquisition of LIP phenotype by Gimap5-deficient CD4+ T cells. Given that Gimap5 is expressed in lysosomes, we investigated whether Gimap5 is involved in lysosomal- autophagosomal pathways. Upon TCR activation, we observed larger autophagosomes that colocalize with mitochondria in Gimap5sph/sph CD4+ T cells suggesting an abnormal rate of mitochondrial turnover. Furthermore, TCR activated Gimap5sph/sph CD4+ T cells ii displayed elevated levels of reactive oxygen species (ROS) and oxygen consumption rate (OCR) indicating defects in mitochondrial function. Our results establish the critical role of Gimap5 in CD4+ T cell homeostasis and maintenance of peripheral tolerance. Importantly, our results provide a basis for further investigation of the molecular mechanisms how Gimap5 is involved in T cell homeostasis. iii iv Acknowledgements: I would like to express my sincere gratitude to my advisor, Dr. Kasper Hoebe, for his support and guidance during these past five years. His enthusiasm for science has been a motivation for me throughout my research training. I would also like to thank the members of my committee, Dr. Christpher Karp, Dr. Edith Janssen, Dr. David Plas, Dr. Julio Aliberti and Graduate Program Director Dr. David Hildeman for their valuable advice and support. I am also thankful to the past and present members of Hoebe and Janssen Labs, Kristin Lampe, Nate Harris, Rachel Reboulet, Dr. Hao Fang, Dr. Rob Tacker, Cassie Hennies and Maria Lehn for their help in my lab work. I would like to express my deepest gratitude to my family; my parents, Gulumser and Dr. Yasar Aksoylar; my sisters, Sevinc Ozturk and Dr. Gulcin Eken; and my wife Dr. Sema Kurtulus Aksoylar for their unconditional support and belief in me during my graduate education and throughout my life. v Table of contents: Page Abstract ii Acknowledgements v Table of Contents vi List of Abbreviations ix Chapter I: Introduction 1 1. Thesis Introduction 2 2. T cell development 2 3. Peripheral Homeostasis of T cells 6 3.1 Peripheral survival and homeostatic proliferation 6 3.2 Activation and effector differentiation of CD4+ T cells 7 3.3 Memory and memory phenotype T cells 10 3.4 T cell quiescence 11 4. Immune Tolerance mechanisms 12 4.1 Central Tolerance of T cells 13 4.2 Peripheral T cell Tolerance 15 4.2.1 T cell Anergy 16 4.2.2 Inhibitory Costimulatory Molecules 18 4.2.3 Regulatory T cells 19 4.2.3.1 Identification of Regulatory T cells 19 4.2.3.2 Regulatory T cell development 21 4.2.3.3 Tolerance Mechanisms Mediated by Regulatory T cells 24 5. Immune homeostasis and tolerance versus pathogenesis in the gut 26 5.1 Microbial flora 28 5.2 Pattern recognition receptors 29 5.3 Dendritic cells 29 5.4 Pro-inflammatory cytokines 31 5.5 Regulatory T cells 33 vi 5.6 T cell lymphopenia and lymphopenia induced proliferation 34 6. Forward genetics 36 7. Family of GTPase of Immunity Associated Proteins 38 7.1 Identification of Gimap GTPases in plants and vertebrates 38 7.2 Genomic organization of Gimap family 38 7.3 Structural Features of Gimap proteins 39 7.4 Cellular localization of Gimap5 40 7.5 Gimap5 in autoimmunity 41 7.6 Gimap5 and T lymphocyte development 44 7.7 Gimap5 in peripheral T cell survival 46 7.8 Gimap5 in T cell homeostasis and immune tolerance 48 8. Summary 50 References 52 Chapter II: Loss of T Cell and B Cell Quiescence Precedes the Onset of Microbial Flora-Dependent Wasting Disease and Intestinal Inflammation in Gimap5-Deficient Mice 78 Abstract 79 Introduction 80 Materials and Methods 84 Results 89 Discussion 102 References 108 Figure Legends 114 Figures 118 Supplementary Figure Legends 126 Supplementary Figures 128 vii Chapter III: Loss of Immunological Tolerance in Gimap5-Deficient Mice is Associated with Loss of Foxo in CD4+ T Cells 135 Abstract 136 Introduction 137 Materials and Methods 140 Results 144 Discussion 151 References 156 Figure Legends 161 Figures 164 Supplementary Figure Legends 169 Supplementary Figure 171 Chapter IV: TCR Signaling in Gimap5-Deficient CD4+ T Cells is Associated with an Abnormal Bioenergetic Profile and Reduced Survival 175 Abstract 176 Introduction 177 Materials and Methods 180 Results 183 Discussion 189 References 194 Figure Legends 198 Figures 200 Chapter V: Summary and Discussion 207 1. Summary 208 2. Gimap5 and autoimmunity 209 2.1 Clinical implications 217 3. Role of Gimap5 in lymphocyte development and homeostasis 219 4. Molecular function of Gimap5 222 Summary Figures 228 References 229 viii List of abbreviations: APC Antigen Presenting Cell ATP Adenosine triphosphate DC Dendritic Cell EAE Experimental Autoimmune Encephalomyelitis ENU N-ethyl-N-nitrosourea Foxo Forkhead box, subgroup O FoxP3 Forkhead box, P3 Gimap GTPase of Immunity Associated Protein IBD Inflammatory Bowel Disease IL Interleukin IFN-γ Interferon gamma LIP Lymphopenia induced proliferation MHC Major Histocompatibility Complex NK Natural Killer OVA Ovalbumin PRR Pattern Recognition Receptor RAG Recombination Activating Gene Stat Signal transducer and activator of transcription TCR T cell receptor TNF-α Tumor Necrosis Factor alpha TGF-β Transforming Growth Factor beta Th T helper TLR Toll-like Receptor Treg Regulatory T cell ix Chapter I Introduction 1 1. Thesis Introduction Innate and adaptive arms of the immune system have evolved to discriminate between self and non-self. This system is crucial to protect the host from pathogens. However, the immune system also carries the potential to damage the host and requires strict regulation. Therefore, tolerance mechanisms have developed to counter-balance immune responses and ensure that they do not react against self. Central and peripheral tolerance mechanisms tightly regulate cells of the adaptive immune system throughout their peripheral lifespan. Breakdown of immune tolerance leads to autoimmune disorders. Therefore, understanding how immune tolerance is maintained is critical. In this thesis, we study a novel N-ethyl-nitrosurea germline mouse model that spontaneously develops colitis as a result of abnormalities in peripheral T cell survival and immune tolerance. In this introduction, we will discuss the processes that control T cell development, homeostasis and immune tolerance. 2. T cell development During T cell development, a large number of T cells with a wide repertoire of antigen specificity are generated. This clonal diversity of T cells allows the adaptive immune system to properly respond to a broad variety of foreign antigens. Generation of such diversity is achieved by the genomic rearrangement of the VDJ genes in the T cell receptor (TCR) loci. To ensure that only thymocytes with successful TCR rearrangements differentiate into mature T cells, several checkpoints exist during T cell development (1). 2 T cell development begins with the migration of progenitor cells from bone marrow to thymus. At this stage, the transcription factor Notch-1 promotes the commitment of progenitors into the T cell lineage (2). These progenitor cells are specified by the expression of interleukin-7 Receptor α (IL-7Rα), c-kit (receptor for stem cell factor) and the adhesion molecule CD44 and can give rise to all T cell lineages (3). In the thymus, thymocytes can be subdivided into four subsets based on the expression of CD4 and CD8 co-receptors. Among them, CD4-CD8- double negative (DN) thymocytes represent the earlier stage in the development. DN thymocytes are also subdivided into four sequential stages based on the expression of CD44 and Interleukin-2 receptor α (CD25). CD44+CD25- DN1 stage represents the initial stage and is followed by the CD44+CD25+ DN2 stage (4). Although CD25 serves as developmental marker for the DN thymocyte stages, the role of Interleukin-2 (IL-2) at these stages is not well established (5, 6). Survival and expansion of cells at DN1 and DN2 stages are largely regulated by SCF and IL-7 signals (7). Next, the cells down-regulate CD44 expression and proceed into the CD44-CD25+ DN3 stage.