Identification and characterization of tissue-resident memory T cells in humans Brahma Vencel Kumar Submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy under the Executive Committee of the Graduate School of Arts and Sciences COLUMBIA UNIVERSITY 2018 ©2017 Brahma Vencel Kumar All rights reserved ABSTRACT Identification and characterization of tissue-resident memory T cells in humans Brahma Vencel Kumar Memory T cells are critical for maintaining lifelong immunity by protecting against reinfection with previously encountered pathogens. In recent years, a subset of memory T cells termed tissue-resident memory T cells (TRM) has emerged as the primary mediator of protection at many tissue sites. Numerous studies in mice have demonstrated that TRM accelerate pathogen clearance compared with other subsets of memory T cells. The defining characteristic of TRM is that they are retained within tissues and do not circulate in the blood. The lack of TRM in blood has proved to be a barrier for investigating the role of TRM in healthy humans. As a result, there are many outstanding questions about TRM biology in humans, including which phenotypic markers identify TRM, if TRM represent a unique memory subset, as well as defining transcriptional and functional characteristics of this subset. Through a unique collaboration with the local organ procurement agency, we obtained samples from >15 tissue sites from healthy organ donors of all ages. We found that the surface marker CD69 was expressed by memory CD4 + and CD8 + T cells in multiple tissues including spleen and other lymphoid tissues, lung, and intestines, but not in blood, suggesting that this marker may identify TRM in human tissues. We identify a core transcriptional signature that distinguishes CD69+ memory T cells in tissues from CD69- memory T cells in tissues and blood with key homologies to the transcriptional profile of TRM in mice, suggesting that CD69 expression identifies TRM in humans. We show that human TRM have a distinct profile of adhesion and migration markers, and a unique dual functional capacity encompassing effector cytokine production but also the upregulation of inhibitory markers and the ability to produce IL- 10 upon stimulation. These results suggest unique adaptations for TRM to maintain long-term residence within tissues and carry out pathogen clearance. We found substantial heterogeneity within human TRM in lymphoid and mucosal tissue sites, including a substantial fraction (40-60%) of TRM in various human tissues with the ability to efflux fluorescent dyes. These efflux(+) TRM had phenotypic and transcriptional characteristics associated with quiescence, including expression of immunomodulatory markers, reduced expression of exhaustion markers, and reduced turnover at steady state. Upon TCR stimulation, efflux(+) TRM produced lower levels of proinflammatory cytokines and cytotoxic molecules but had a superior ability to proliferate compared with efflux(-) TRM. However, efflux(+) also had an enhanced capacity for IL-17 production along with transcriptional features of IL-17 signaling following stimulation. Overall, these studies establish universal properties of human TRM and hint at the function of distinct TRM subsets in mediating tissue immunity. TABLE OF CONTENTS LIST OF FIGURES ......................................................................................................................iii LIST OF TABLES ........................................................................................................................vi LIST OF ABBREVIATIONS .....................................................................................................vii ACKNOWLEDGEMENTS .......................................................................................................viii DEDICATION ...............................................................................................................................x CHAPTER 1: Introduction ...........................................................................................................1 Section 1.1: Overview of the T cell response to infection...................................................1 Section 1.2: Human T cell development and maintenance of naïve T cells .......................7 Section 1.3: T cell activation and subset differentiation....................................................13 Section 1.4: Memory T cells..............................................................................................17 Section 1.5: Tissue-resident memory T cells (TRM).........................................................21 Section 1.6: Thesis aims....................................................................................................40 CHAPTER 2: Materials and methods .......................................................................................44 Section 2.1: Acquisition and isolation of T cells from human tissues...............................44 Section 2.2: Flow cytometric analysis of human T cells...................................................49 Section 2.3: Functional Assays..........................................................................................53 Section 2.4: Whole transcriptome profiling by RNA Sequencing.....................................54 Section 2.5: Imaging of human lymphoid tissues..............................................................63 Section 2.6: Statistical tests................................................................................................64 CHAPTER 3: Human Tissue-Resident Memory T Cells Are Defined by Core Transcriptional and Functional Signatures in Lymphoid and Mucosal Sites ……………...65 Section 3.1: Introduction....................................................................................................66 i Section 3.2: Results............................................................................................................66 Section 3.3: Discussion....................................................................................................118 CHAPTER 4: Dye efflux capacity defines a functionally distinct subset of human CD8 + tissue resident memory cells. .....................................................................................................123 Section 4.1: Introduction..................................................................................................124 Section 4.2: Results..........................................................................................................126 Section 4.3: Discussion....................................................................................................164 CHAPTER 5: Conclusions........................................................................................................167 REFERENCES ...........................................................................................................................180 APPENDICES ............................................................................................................................199 Appendix A. The functional profile of naïve and memory T cells with aging................199 Appendix B. Functional properties of CD103+ vs CD103- human TRM.......................207 Appendix C. Imaging of frozen sections of human lymph nodes....................................210 Appendix D. Accepted Abstracts.....................................................................................216 Appendix E. Abstracts of contributing author manuscripts.............................................218 Appendix F. Curriculum Vitae........................................................................................222 ii LIST OF FIGURES Figure 1.1. Overview of the T cell development and response to infection. Figure 1.2. Distribution of TRM phenotype cells in human tissues. Figure 2.1. Tissue sites received from organ donors. Figure 2.2. RNA-Sequencing workflow used in this study. Figure 3.1. CD69+ memory T cells are prevalent in tissues and do not show features of activation. Figure 3.2. Sorting strategy for RNA Sequencing. Figure 3.3. CD69 expression defines a transcriptionally distinct memory subset in humans with features of tissue residency. Figure 3.4. PCA of randomly selected genes. Figure 3.5. A core gene signature defines tissue CD69+ memory T cells distinct from circulating CD69– cells in tissues and blood. Figure 3.6. Expression of CD49a, CD103, and CXCR6. Figure 3.7. Expression of PD-1, CD101, and CX3CR1. Figure 3.8. Comparison of the human and mouse TRM transcriptome. Figure 3.9. T-bet and Eomes expression by human memory T cells. Figure 3.10. TCR repertoire overlap. Figure 3.11. TCR clonal analysis, turnover, and function of CD69+ and CD69– cells. Figure 3.12. Transcript levels of cytokines in TRM and TEM Figure 3.13. Cytokine production by CD103+ and CD103- human CD69+ memory T cells. Figure 3.14. Lineage and tissue-specific transcription and phenotypic profiles in human CD69+ memory T cells. iii Figure 3.15. Tissue- and lineage- specific genes. Figure 3.16. CCR9 Expression in CD69+ and CD69- memory T cells. Figure 3.17. TRM are a phenotypically distinct subset across multiple tissues. Figure 3.18. t-SNE analysis of TRM and TEM from additional donor. Figure 3.19. A core signature of human TRM. Figure 4.1. Identification of Memory T cell subsets in Humans. Figure 4.2. A subset of memory CD8 + T cells across human tissues effluxes fluorescent dyes. Figure 4.3. Dye efflux yields populations