Discovery of Novel Epigenetic Regulators of CD8+ T Cell Effector Function The Harvard community has made this article openly available. Please share how this access benefits you. Your story matters Citation Tay, Rong En. 2019. Discovery of Novel Epigenetic Regulators of CD8+ T Cell Effector Function. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences. Citable link http://nrs.harvard.edu/urn-3:HUL.InstRepos:42029527 Terms of Use This article was downloaded from Harvard University’s DASH repository, and is made available under the terms and conditions applicable to Other Posted Material, as set forth at http:// nrs.harvard.edu/urn-3:HUL.InstRepos:dash.current.terms-of- use#LAA Discovery of Novel Epigenetic Regulators of CD8+ T Cell Effector Function A dissertation presented by Rong En Tay to The Department of Medical Sciences in partial fulfilment of the requirements for the degree of Doctor of Philosophy in the subject of Immunology Harvard University Cambridge, Massachusetts April 2019 © 2019 Rong En Tay All rights reserved. Dissertation Advisor: Kai Wucherpfennig Rong En Tay Discovery of Novel Epigenetic Regulators of CD8+ T Cell Effector Function Abstract CD8+ cytotoxic T lymphocytes (CTLs) play a key role in acquired immunity by killing infected or cancerous cells. Upon antigen recognition and activation, the majority of naïve CD8+ T cells differentiate into potent but short-lived effector CTLs, while a small fraction generates long-lived memory cells that are poised to proliferate rapidly upon antigen re-encounter. While transcriptional control of CD8+ T cell differentiation and effector function has been extensively studied, little is known about epigenetic regulation of these processes. Here we use two screening approaches to uncover novel epigenetic regulators of CD8+ T cell effector function. We first engineered a CRISPR-Cas9 genetic screening platform to reveal epigenetic regulators in tumour-infiltrating CD8+ T cells that could potentially be targeted to improve CD8+ anti- tumour function. This approach identified CARM1 as a strong candidate epigenetic regulator suppressing the accumulation of tumor-specific CD8+ T cells in the pre-clinical B16 melanoma tumour model. We also used a functional pharmacological approach to screen for epigenetic regulators of CD8+ T cell effector function during in vitro T cell activation. We thus identified a novel role of the histone deacetylase HDAC3 as an epigenetic regulator of CD8+ T cell cytotoxicity and persistence. Hdac3-deficient CD8+ T cells transiently acquired augmented cytotoxicity that was associated with early resistance to chronic LCMV infection but did not confer durable disease protection. Mechanistically, HDAC3 inhibited a gene programme of cytotoxicity-associated genes and terminal effector differentiation beginning early during CD8+ T cell activation, and required the transcription factors Runx3 and Blimp-1 as key downstream iii mediators for its regulation of CD8+ T cell effector function. Targeting HDAC3 activity to epigenetically modulate the balance between a short-lived, potent cytotoxic effector state and a durable, long-lived response could potentially lead to the development of new approaches in adoptive T cell immunotherapy and in therapeutic vaccine development. iv Table of Contents Acknowledgements vi Dedication vii Chapter 1 – Introduction 1 Chapter 2 – Materials and Methods 7 Chapter 3 – Discovery of CARM1 as a suppressor of CD8+ T cell accumulation in 23 tumours using an in vivo CRISPR-Cas9 genetic screening platform Chapter 4 – Discovery of HDAC3 as an inhibitor of CD8+ T cell cytotoxicity in an in 28 vitro functional pharmacologic screen Chapter 5 – HDAC3 is required during T cell activation for persistence of antigen- 40 experienced CD8+ T cells Chapter 6 – Effects of loss of HDAC3 activity in CD8+ T cells in settings of chronic 51 antigen burden Chapter 7 – Transcriptional and epigenetic signatures of HDAC3 60 Chapter 8 – Uncovering potential mechanisms of HDAC3-mediated regulation of 71 CD8+ T cell effector function using genetic approaches Chapter 9 – Summary and Discussion 87 Appendix 99 References 106 v Acknowledgements I reverently thank the LORD my God for sustaining me through this long and difficult journey. By His grace have I come thus far while keeping my honour and integrity intact. I thank my PhD advisor, Kai Wucherpfennig, for his supervision and direction and for giving me the opportunity to work on an interesting project. I am deeply and forever grateful to Hye-Jung Kim for her unwavering support of me, both scientifically and personally. Thank you for being a true friend and mentor to me and for believing in me. I thank my collaborators Paloma Cejas, Henry Long, and Clifford Meyer (DFCI) for their invaluable help in performing the ChIP-seq analyses, and Peng Jiang (DFCI) for computational analysis of the in vivo screen. I am very grateful to my dissertation advisory committee members Ulrich von Andrian (HMS), Myles Brown (DFCI), and Nicholas Haining (DFCI) for their helpful guidance and constructive feedback. I thank Scott Hiebert (Vanderbilt), Christina Weng and David Fisher (MGH), Steven Elledge (HMS), and Ulrich von Andrian (HMS) for their kind gifts of tools and resources in support of my projects. Special thanks to Olamide Olawoyin for her incredible hard work and dedication during our time working together. It was an honour and privilege to teach one so deserving as yourself. I thank Sabrina Haag for her insightful comments and scientific input into my projects. I thank my fellowship sponsors, the Agency for Science, Technology, and Research (A*STAR, Singapore), for their generous financial support over the course of my doctoral work. I thank all other members of the Wucherpfennig lab for their collegiality. vi Dedication Dedicated to the LORD my God as a testimony of His faithfulness to me, to my loving family for their unwavering support and continual prayers for the safe return of their son and brother, to my dearest fiancée Kayla, for her patience and love during this period of separation, and to Kayla’s father Sam, for his faith that this day would come. Give thanks to the LORD, for He is good. His love endures forever. - Psalm 136:1 (NIV) vii Chapter 1 Introduction Introduction Upon antigen encounter during inflammation, naïve CD8+ T cells undergo phenotypic changes during activation to develop into effector cytotoxic T lymphocytes that mediate immunity via contact-dependent killing of infected or malignant transformed cells and by secreting effector cytokines such as IFN-γ and TNF-α. By the peak of the CD8+ T cell effector response to acute antigen exposure, activated CD8+ T cells are already committed to one of two cell fates – a short-lived terminally-differentiated state with potent effector function1,2, or a long-lived memory precursor phenotype with high proliferative potential but weak effector function3,4. After antigen clearance, most of the terminally-differentiated effector cells die off, resulting in a quantitative and qualitative contraction of the CD8+ T cell response, whereas memory precursor cells persist and subsequently differentiate into memory cells, which are poised to mount a robust and rapid defence upon secondary antigen encounter. All these differentiation processes require co-ordination between the molecular pathways integrating extracellular signalling inputs with cell- intrinsic programming for proper acquisition of full effector CD8+ T cell function as well as for proper control and homeostasis of the CD8+ T cell immune response5. The transcriptional regulation of the developmental processes following CD8+ T cell activation has been extensively described and studied. Advances in RNA-sequencing technology and analysis techniques have led to comprehensive profiling of the roles of such key transcription factors as T-bet, Eomes, Blimp-1, and Bcl-6 in regulating the acquisition of effector functions of CD8+ T cells, as well as in regulating the commitment of activated CD8+ T cells to one of the two phenotypically-distinct cell fates following acute antigen encounter5,6. These studies have been indispensable to our current understanding of CD8+ T cell differentiation as a process governed by interactions of key driver molecules within an intricately complex transcriptional network7. Each transcription factor governs a distinct suite of effector functions or 2 phenotypes (e.g. T-bet is required to potentiate IFN-γ secretion and cytotoxicity, as well as effector phenotype acquisition8), and the co-ordination of several of these transcription factors across time thus constitutes a fundamental molecular control mechanism governing the acquisition of effector function and cell fate commitment. Whereas control of CD8+ T cell function and differentiation is proximally mediated by transcription factors, it is the epigenetic landscape9 that provides the framework within which the gene regulatory network operates. Broadly speaking, epigenetic processes constrain the spectrum of potential phenotypes that a given cell can acquire by covalently modifying DNA bases or specific amino acid residues of chromatin- associated proteins such as histones. These modifications serve as beacons for ‘reader’ proteins that recognise and recruit specific transcription factors to the marked regions of chromatin. The patterns of epigenetic marks also generate regions of chromatin with differential conformational states and accessibility to transcriptional machinery (i.e. ‘open’ and ‘closed’