Transcriptional control of tissue-resident memory T cell generation Filip Cvetkovski Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2019 © 2019 Filip Cvetkovski All rights reserved ABSTRACT Transcriptional control of tissue-resident memory T cell generation Filip Cvetkovski Tissue-resident memory T cells (TRM) are a non-circulating subset of memory that are maintained at sites of pathogen entry and mediate optimal protection against reinfection. Lung TRM can be generated in response to respiratory infection or vaccination, however, the molecular pathways involved in CD4+TRM establishment have not been defined. Here, we performed transcriptional profiling of influenza-specific lung CD4+TRM following influenza infection to identify pathways implicated in CD4+TRM generation and homeostasis. Lung CD4+TRM displayed a unique transcriptional profile distinct from spleen memory, including up-regulation of a gene network induced by the transcription factor IRF4, a known regulator of effector T cell differentiation. In addition, the gene expression profile of lung CD4+TRM was enriched in gene sets previously described in tissue-resident regulatory T cells. Up-regulation of immunomodulatory molecules such as CTLA-4, PD-1, and ICOS, suggested a potential regulatory role for CD4+TRM in tissues. Using loss-of-function genetic experiments in mice, we demonstrate that IRF4 is required for the generation of lung-localized pathogen-specific effector CD4+T cells during acute influenza infection. Influenza-specific IRF4−/− T cells failed to fully express CD44, and maintained high levels of CD62L compared to wild type, suggesting a defect in complete differentiation into lung-tropic effector T cells. This finding identifies IRF4 as an important regulator of CD4+TRM generation in response to respiratory infection. Furthermore, comparing whole transcriptome profiling of mouse and human lung memory T cell subsets, we define a lung CD4+TRM gene signature common to mice and humans. IRF4 protein was specifically up-regulated in lung CD4+TRM but not in circulating memory subsets, in both humans and mice previously infected with influenza. This result suggest that high expression of IRF4 contributes to a cross-species conserved molecular pathway of long term maintenance of CD4+TRM in the lung. Overall, our findings confirm lung CD4+TRM as a unique memory T cell subset regulated by tissue-specific transcription factors. These results have important implications in focusing future studies of tissue resident memory T cells to factors with translational potential. Importantly, by determining the lung CD4+TRM gene signature common to mice and humans, we motivate future genetic studies that could lead to the complete identification of the mechanisms of TRM maintenance in humans. TABLE OF CONTENTS List of figures ................................................................................................................................. iv List of tables ................................................................................................................................... vi Acknowledgements ....................................................................................................................... vii CHAPTER 1 : INTRODUCTION .................................................................................................. 1 The mammalian immune system .................................................................................................... 1 Innate immunity .............................................................................................................................. 1 Adaptive immunity ......................................................................................................................... 2 T lymphocyte development............................................................................................................. 4 The T cell receptor .......................................................................................................................... 5 T cell priming and activation ........................................................................................................ 10 Signaling pathways induced by T cell receptor stimulation ......................................................... 10 CD4+T helper subsets ................................................................................................................... 12 Immunological memory ................................................................................................................ 16 Heterogeneity of memory T cells ................................................................................................. 17 Memory homeostasis and persistence - role of cytokines and MHC............................................ 21 Influenza virus .............................................................................................................................. 21 Adaptive immune responses to influenza infection ...................................................................... 23 T cell homing to the lung .............................................................................................................. 25 Cytokine signals in Th1 differentiation ........................................................................................ 26 Lung TRM .................................................................................................................................... 27 TRM in nonlymphoid tissue sites ................................................................................................. 29 Phenotype of TRM ........................................................................................................................ 29 i Function of TRM .......................................................................................................................... 31 Mechanisms of TRM tissue retention ........................................................................................... 32 Generation of TRM ....................................................................................................................... 33 IRF4 regulates T cell differentiation ............................................................................................. 35 The role of IRF4 during in vivo development of effector and memory T cell subsets ................. 40 Thesis objectives ........................................................................................................................... 46 CHAPTER 2 : MATERIALS AND METHODS ......................................................................... 49 Mouse strains and genotyping ...................................................................................................... 49 Influenza virus infection ............................................................................................................... 50 T cell adoptive transfer ................................................................................................................. 50 Intravascular labeling for identification of tissue-resident T cells................................................ 50 Lymphocyte isolation from mouse tissues .................................................................................... 51 Immunostaining for flow cytometry ............................................................................................. 51 Sample preparation for confocal microscopy ............................................................................... 55 Human tissue acquisition .............................................................................................................. 55 RNA Isolation ............................................................................................................................... 55 RNA Sequencing .......................................................................................................................... 56 Differential gene expression analysis ........................................................................................... 56 Gene Set Enrichment Analysis ..................................................................................................... 57 Analysis of CD8+TRM gene signature ......................................................................................... 57 Generating lists of IRF4-regulated genes ..................................................................................... 60 T cell receptor repertoire analysis ................................................................................................. 62 Statistical tests ............................................................................................................................... 62 ii CHAPTER 3 : GENE EXPRESSION ANALYSIS OF TISSUE-RESIDENT MEMORY T CELLS ACROSS SPECIES UNCOVERS REQUIREMENT FOR IRF4 IN T CELL HOMING TO THE LUNG ............................................................................................................................ 63 Introduction ................................................................................................................................... 63 Results ........................................................................................................................................... 67 Discussion ..................................................................................................................................