Regulation of cellular metabolism in tumor infiltrating CD8+ T cells and its role in their dysfunction Lelisa Fikru Gemta Nejo, Ethiopia A.S., Community College of Aurora, Colorado, 2004 B.S., University of Colorado Denver, Colorado, 2006 A Dissertation presented to the Graduate Faculty of the University of Virginia in Candidacy for the Degree of Doctor of Philosophy Experimental Pathology University of Virginia May, 2019 i Abstract Tumor infiltrating CD8+ T cells (CD8+ TIL) play pivotal role in fighting cancers. Their accumulation within tumors has been associated with better tumor control in mouse models of cancers and favorable clinical outcomes in human patients. However, it has been well-understood that these cells undergo progressive loss of function and often fail to eradicate tumor cells. While significant progress has been made in understanding and targeting the mechanisms underlying CD8+ TIL dysfunction, the molecular details of the dysfunction remain to be fully elucidated. It has been widely appreciated that elevated glycolysis and oxidative phosphorylation (OXPHOS) are essential to support the generation and function of effector T cells. Here, we demonstrate the perturbation of energy metabolism as a biochemical basis of CD8+ TIL dysfunction. We found that both glycolytic metabolism and OXPOHS were attenuated in melanoma CD8+ TIL. Glycolysis was repressed by impaired activity of enolase 1, a key glycolytic enzyme responsible for the synthesis of phosphoenolpyruvate that is essential for the effector function of T cells. While this enzyme is highly expressed in CD8+ TIL, its activity was post- translationally regulated by mechanism that involved immune checkpoint signals from PD-1, CTLA-4, and TIM-3. The details of this mechanism remain to be elucidated, and we have developed a robust reporter of enolase activity to aid future investigation in this area. We also found impaired enolase activity in the CD8+ TIL that infiltrated human melanoma tumors and different types of murine tumor models. In addition to glycolysis, impaired enolase activity also limited the OXPHOS capability of CD8+ TIL. This was at ii least partly mediated by inability of glycolysis to produce sufficient pyruvate to feed into the mitochondrial metabolism. However, CD8+ TIL also had low mitochondrial mass and membrane potential that may be a major contributor to the OXPHOS deficiency of these cells. Importantly, we demonstrated that bypassing the enolase inactivity through provision of metabolites produced downstream of it significantly improved the glycolytic metabolism, OXPOHS, and effector function of CD8+ TIL. Furthermore, we showed that a combination of immune checkpoint blockade therapy that slowed tumor growth in mouse model generated CD8+ TIL with stronger enolase active that was essential for their function. Our studies demonstrated that metabolic dysfunction mediated by impaired enolase activity is a major contributor to the functional impairment of CD8+ TIL, and that reactivating this enzyme may reinvigorate antitumor immunity. iii Acknowledgements I would first like to thank my mentor Dr. Timothy Bullock, who went above and beyond what is expected of him as a mentor to support me as a person and to make sure that I make the most out of the opportunities that graduate school provides. He continually pushed my limits and encouraged me to do things that are important not only to fulfill the requirements of my training, but are also beneficial to my future career as a scientist. As a result of such mentorship, I have obtained four merit-based travel awards to present my research at national and international scientific conferences and produced a manuscript that has been accepted for publication in a prestigious journal. Dr. Bullock has always been there for me throughout my time at UVA. Whenever I requested extra meetings with him to obtain advice on personal and professional matters, his responses were usually “for you, I will make time.” I would also thank Dr. Janet Cross, Dr. Victor Engelhard, Dr. David Kashatus, and Dr. Adam Goldfarb for serving on my thesis committee and for their extraordinary contribution to the development my project into the work presented in this dissertation. When I asked each of them to serve on my committee, my intention was to assemble a group of exceptional scientists who would give me the best guidance with my project. However, I never expected that they would be invested as much as they have been in my personal growth as a scientist. I truly appreciate all the time and effort they put into guiding, encouraging, and challenging me. Especially thank you to Dr. Janet iv Cross for serving as chair of my thesis committee and for all the personal and professional supports she provided me to overcome the challenges I faced while in graduate school. I am also indebted to Dr. Kyle Hoehn, who was instrumental in the establishment of the project described in this work. Thanks to all the members of Bullock lab and Engelhard lab who taught me, encouraged me, and supported me throughout my time at UVA. This work was financially supported by Immunology Training Grant and CRCHD Diversity Supplements (R01CA166458-03S1). Finally, I need to thank all the members of my family who have been supportive of my long professional journey. Thank you does not come close to expressing my deepest appreciation for my father Fikru Gemta, whose loss I am morning as I am writing this dissertation, and my mother Mulunesh Abdissa. Their endless love, support, and guidance made me who I am as a person today. They always put my best interest before their own even when that required sacrificing a lot and working extra hard to meet my needs. Especially, I will never forget how hard they worked from dawn to dusk to put my siblings and me through school in a small town in Ethiopia. Lastly, thank you to my amazing wife Biftu without whose love, support, understanding, and dedication to my career, I simply could have never been able to make it through graduate school. v Table of Contents Abstract .................................................................................................................................. i Acknowledgements............................................................................................................... iii Table of Contents ................................................................................................................... v List of Figures ........................................................................................................................ ix Abbreviations ....................................................................................................................... xi Chapter One: General Introduction......................................................................................... 1 The importance of antitumor immunity ...................................................................................... 1 Dysfunctional state of TIL............................................................................................................. 2 The link between immunity and cellular metabolism ................................................................. 4 Glucose metabolism ..................................................................................................................... 7 Role of glycolytic metabolism in T cell ....................................................................................... 10 Metabolism of TIL ...................................................................................................................... 14 Immunopathological consequence of altered glucose metabolism in T cell ............................. 15 THESIS RATIONALE ............................................................................................................... 16 Chapter Two: Materials and Methods ................................................................................... 19 Tumor cell line, mice, and tumor injection ................................................................................ 19 Human Samples ......................................................................................................................... 19 Checkpoint blockade treatment ................................................................................................ 20 vi Adoptive T cell transfer .............................................................................................................. 21 Glucose uptake .......................................................................................................................... 23 Analysis of enolase activity in in vitro and ex vivo T cell samples .............................................. 24 Enolase reporter, MitoTracker Green (MTG), and tetramethylrhodamine, ethyl ester (TMRE) staining ....................................................................................................................................... 25 Metabolic assay ......................................................................................................................... 25 Metabolomics ............................................................................................................................ 26 Development of Enolase reporter ............................................................................................. 27 Enolase 1 post-translational modification analysis ...................................................................