Molecular mechanisms regulating CD8 T cell differentiation following external cues Jolie Cullen orcid.org/0000-0001-7780-0629 November 2018 Department of Microbiology and Immunology, The Peter Doherty Institute The University of Melbourne Submitted in fulfilment of the requirements of the degree of Doctor of Philosophy 1 Table of Contents ABSTRACT ................................................................................................................................. 5 DECLARATION ........................................................................................................................... 7 COMMUNICATIONS .................................................................................................................... 8 PREFACE .................................................................................................................................. 10 ACKNOWLEDGMENTS .............................................................................................................. 11 ABBREVIATIONS ...................................................................................................................... 14 LIST OF FIGURES ...................................................................................................................... 20 CHAPTER 1: INTRODUCTION ............................................................................................ 21 1.1 ADAPTIVE IMMUNE RESPONSE ........................................................................................... 21 1.1.1 CD8 T cells ............................................................................................................. 22 1.2 RECOGNITION OF INFECTED CELLS BY T CELLS ................................................................ 22 1.2.1 The T cell receptor and antigen recognition .......................................................... 22 1.2.2 Major histocompatibility complex (MHC) ............................................................. 23 1.3 ANTIGEN PROCESSING AND PRESENTATION ...................................................................... 23 1.3.1 Endogenous processing and presentation pathway ............................................... 24 1.3.2 Cross presentation .................................................................................................. 24 1.4 CD8 T CELL MEDIATED IMMUNITY .................................................................................... 24 1.4.1 Activation of naïve CD8 T cells .............................................................................. 24 1.4.2 Homing and migration of CD8 T cells ................................................................... 28 1.5 EFFECTOR FUNCTIONS OF CD8 T CELLS ............................................................................ 28 1.5.1 Acquisition and expression of cytolytic molecules within effector CTL ................. 28 1.5.2 Cytotoxicity ............................................................................................................. 29 1.5.3 Effector Cytokines .................................................................................................. 29 1.6 DIFFERENTIATION OF MEMORY CD8 T CELLS ................................................................... 31 1.6.1 Effector/memory fate decisions .............................................................................. 31 1.7 THE MEMORY PHASE .......................................................................................................... 31 1.7.1 Memory cell subsets ............................................................................................... 31 1.7.2 The foundation of memory T cells .......................................................................... 33 1.8 THE ROLE OF CD4 T CELL HELP DURING CD8 MEMORY DIFFERENTIATION ..................... 37 1.8.1 Dependence on CD4 T cell help ............................................................................. 37 1.8.2 Delivery of CD4 T cell help .................................................................................... 38 1.8.3 Timing of CD4 T cell help ...................................................................................... 38 1.8.4 CD4 T cell help and its role in exhaustion ............................................................. 39 1.8.5 Memory cells are a heterogeneous population: heterogeneous requirement for CD4? 40 1.9 T CELL ENERGY PRODUCTION ............................................................................................ 42 1.9.1 Modes of energy production .......................................................................... 42 1.9.2 Metabolism during the memory phase ................................................................... 43 1.10 TRANSCRIPTIONAL REGULATION OF CD8 MEMORY T CELLS .......................................... 45 1.10.1 T-bet and eomesodermin ...................................................................................... 45 1.10.2 Blimp-1 and Bcl-6 ................................................................................................ 46 1.10.3 ID2 and ID3 ......................................................................................................... 46 1.10.4 STAT family .......................................................................................................... 47 1.11 THESIS AIMS ..................................................................................................................... 49 CHAPTER 2: MATERIALS & METHODS .......................................................................... 50 2.1 MATERIALS ........................................................................................................................ 50 2.1.1 Mice ........................................................................................................................ 50 2.1.2 Antibodies ............................................................................................................... 50 2.1.3 Primers ................................................................................................................... 51 2.1.4 Buffers and Media .................................................................................................. 52 2.1.5 Seahorse Instrument (Seahorse Bioscience) .......................................................... 52 2.2 METHODS ........................................................................................................................... 54 2.2.1 Infection of mice ..................................................................................................... 54 2.2.2 Tissue sampling, processing and counting ............................................................. 54 2.2.3 Adoptive transfer .................................................................................................... 55 2 2.2.4 OT-I T cell enrichment ........................................................................................... 56 2.2.5 CD8 T cell purification by cell sorting ................................................................... 56 2.2.6 Violet tracker labelling ........................................................................................... 56 2.2.7 In vitro T cell activation ......................................................................................... 57 2.2.8 Determination of lung viral titres ........................................................................... 57 2.2.9 Plaque Assay .......................................................................................................... 57 2.2.10 Purification of cells by ficoll gradient ................................................................. 57 2.2.11 Antibody staining ................................................................................................. 58 2.2.12 Intracellular staining ........................................................................................... 58 2.2.13 Cytometric Bead Array ........................................................................................ 58 2.2.14 Flow cytometry ..................................................................................................... 58 2.2.15 Measuring Bioenergetics in T Cells by Seahorse Bioscience .............................. 59 2.2.16 RNA extraction and cDNA synthesis .................................................................... 60 2.2.17 TaqMan gene expression assay ............................................................................ 61 2.2.18 Sybr green real-time PCR .................................................................................... 61 2.2.19 Mathematical modelling ...................................................................................... 61 2.2.20 ATAC-seq: Assay for Transposase-Accessible Chromatin with high throughput sequencing ........................................................................................................................... 63 2.2.21 Bioinformatic analyses ......................................................................................... 65 2.2.22 Statistical Analyses .............................................................................................. 66 CHAPTER 3: THE MOLECULAR BASIS OF CD4 T CELL HELP DURING THE PRIMING OF CD8 T CELL MEMORY ................................................................................ 67 3.1 INTRODUCTION .................................................................................................................