Intrinsic Mechanisms Regulating T Cell Tolerance in Autoimmune Diabetes by Qianxia Zhang B.Sc. Biological Science, Nankai University, 2012 Submitted to the Graduate Faculty of The School of Medicine in partial fulfillment of the requirements for the degree of Doctor of Philosophy University of Pittsburgh 2018 UNIVERSITY OF PITTSBURGH SCHOOL OF MEDICINE This dissertation was presented by Qianxia Zhang It was defended on January 11, 2018 and approved by Sarah L. Gaffen, PhD, Gerald P. Rodnan Professor, Department of Medicine Lawrence P. Kane, PhD, Professor, Department of Immunology Peter C. Lucas, MD, PhD, Associate Professor, Department of Pathology Jon D. Piganelli, PhD, Associate Professor, Department of Surgery Mark J. Shlomchik, MD, PhD, UPMC Endowed Professor, Department of Immunology Dissertation Advisor: Dario AA. Vignali, PhD, The Frank Dixon Chair in Cancer Immunology, Professor, Department of Immunology ii Copyright © by Qianxia Zhang 2018 iii Intrinsic Mechanisms Regulating T Cell Tolerance in Autoimmune Diabetes Qianxia Zhang, PhD University of Pittsburgh, 2018 Type 1 Diabetes (T1D) is a polygenic autoimmune disease characterized by immune cell infiltration into the islets of Langerhans, destruction of insulin-producing β cells, and uncontrolled hyperglycemia. Islet-antigen reactive CD4+ and CD8+ T cells, and regulatory T cells (Tregs) are the major players in T1D pathogenesis. Both cell-intrinsic and cell- extrinsic inhibitory mechanisms are required to maintain self-tolerance to islet-antigens. Lymphocyte Activation Gene 3 (LAG3, CD223), a co-inhibitory receptor highly expressed on T cells, is one of these essential mechanisms that intrinsically regulate T cell tolerance in autoimmune diabetes. I evaluate the role of LAG3 on T cells versus non- T cells, CD8+ T cells, and Tregs by generating mice in which LAG3 is specifically absent on these subsets in a murine model of T1D. In Chapter 3, I show that the predominant expression of LAG3 on insulitic CD4+ and CD8+ T cells is required to limit the pathogenesis of autoimmune diabetes in non-obese diabetic (NOD) mice. The loss of LAG3 on CD8+ T cells alone is sufficient to promote early onset of autoimmune diabetes by promoting islet-specific glucose-6-phophatase catalytic subunit-related protein (IGRP)-reactive CD8+ T cells to differentiate into effector T cells. In Chapter 4, I show that LAG3 is preferentially and constitutively expressed on a subset of insulitic Tregs, and loss of LAG3 on Tregs leads to delayed onset and reduced incidence of autoimmune diabetes. LAG3 may intrinsically limit Treg proliferation and functionality by repressing pathways that promote the maintenance of Tregs in the pancreas of NOD mice. iv In addition to dissecting the role of LAG3 in regulating T cell tolerance, I also show that removal of programmed death protein 1 (PD1) or overexpression of Neuropilin 1 (Nrp1) on Tregs protect NOD mice from autoimmune diabetes in Appendix A and Appendix B, respectively. In summary, my findings have advanced our understanding of cell-intrinsic mechanisms that regulate T cell tolerance in autoimmune diabetes. v Table of Contents Acknowledgement......................................................................................................... x Abbreviations .............................................................................................................. xii List of tables ............................................................................................................... xvi List of figures ............................................................................................................ xvii 1.0 Chapter 1: Introduction .................................................................................. 1 1.1 Type 1 diabetes ........................................................................................ 1 1.1.1 Epidemiology............................................................................................... 2 1.1.2 Etiology ....................................................................................................... 5 1.1.3 Pathology .................................................................................................... 6 1.2 The non-obese diabetic mouse model ...................................................... 8 1.2.1 Advantages and limitations of the NOD mouse model ................................. 8 1.2.2 The two-checkpoint hypothesis of autoimmune diabetes ............................10 1.3 T cell tolerance in the context of autoimmune diabetes .......................... 11 1.3.1 Recessive and dominant tolerance .............................................................12 1.3.2 Generation of the islet-antigen reactive T cell repertoire .............................13 1.3.3 Co-stimulatory and co-inhibitory receptors in autoimmune diabetes ...........15 1.3.4 An essential role for Tregs in self-tolerance ................................................21 1.4 LAG3 in T cell tolerance ......................................................................... 26 1.4.1 LAG3 structure, ligands, and signaling .......................................................27 1.4.2 Regulation of LAG3 expression ..................................................................29 1.4.3 Role of LAG3 in disease .............................................................................30 2.0 Chapter 2: Materials and methods .............................................................. 34 vi 2.1 Mice and study design ............................................................................ 34 2.2 Generation of Lag3L/L-YFP mice ................................................................ 35 2.3 Measurement of diabetes and insulitis .................................................... 36 2.4 Islet isolation and lymphocyte preparation .............................................. 37 2.5 Antibodies and flow cytometry ................................................................ 37 2.6 Micro-suppression assay ........................................................................ 39 2.7 Treg expansion and adoptive transfer .................................................... 40 2.8 Ikzf4 overexpression and knockdown in Tregs ....................................... 40 2.9 vi-SNE clustering .................................................................................... 41 2.10 Statistical analyses ................................................................................. 41 2.11 Low cell number T cell repertoire sequencing......................................... 42 2.12 Low cell number RNA sequencing .......................................................... 43 2.12.1 RNA-seq in Chapter 4.1 .............................................................................43 2.12.2 RNA-seq in Chapter 3.2 and 4.2 ................................................................44 2.13 Bioinformatics ......................................................................................... 44 2.13.1 Analyses in Chapter 4.1 .............................................................................44 2.13.2 Analyses in Chapters 3.2 and 4.2 ...............................................................46 3.0 Chapter 3: Role of LAG3 in regulating diabetogenic T cells .................... 47 3.1 A predominant role for LAG3 on T cells in autoimmune diabetes ........... 49 3.1.1 LAG3 is highly upregulated on T cells at inflammatory sites .......................49 3.1.2 The predominant function of LAG3 in autoimmune diabetes is limited to CD4+ and CD8+ T cells .......................................................................................................52 3.2 Impact of LAG3 on selecting diabetogenic CD8+ T cells ......................... 55 3.2.1 The absence of LAG3 on CD8+ T cells results in accelerated autoimmune diabetes 55 vii 3.2.2 LAG3 intrinsically limits IGRP-reactive CD8+ T cells to differentiate into pathogenic effector T cells ........................................................................................60 3.3 Summary and discussion ........................................................................ 66 4.0 Chapter 4: Role of LAG3 in Treg-mediated self-tolerance ........................ 70 4.1 Extrinsic and intrinsic impacts of LAG3 on Tregs .................................... 72 4.1.1 The absence of LAG3 on Tregs results in reduced autoimmune diabetes ..72 4.1.2 LAG3 intrinsically limits Treg proliferation ...................................................81 4.2 Transcription and clonotype differences between Lag3+ and Lag3– Treg subsets 91 4.2.1 Lag3 is preferentially expressed in a subset of Tregs but is not necessarily a Treg differentiation factor ..........................................................................................91 4.2.2 Lag3 is selectively and stably expressed on a proportion of intra-islet Treg clones 94 4.3 Summary and discussion ........................................................................ 96 5.0 Chapter 5: Hypotheses for future studies ................................................ 100 5.1 Relative contribution and transcriptional regulation of different co-inhibitory receptors 100 5.1.1 The overall impact of co-inhibitory receptors may depend on which immune population(s) play the dominant role .......................................................................101 5.1.2 Co-inhibitory receptors may function distinctively but cooperatively to regulate T cell tolerance .......................................................................................................102