University of Pennsylvania ScholarlyCommons Publicly Accessible Penn Dissertations 2018 Understanding Metabolic Adaptation Of T Cells During Activation And Nutrient Limitation Christopher Ecker University of Pennsylvania, [email protected] Follow this and additional works at: https://repository.upenn.edu/edissertations Part of the Allergy and Immunology Commons, Immunology and Infectious Disease Commons, and the Medical Immunology Commons Recommended Citation Ecker, Christopher, "Understanding Metabolic Adaptation Of T Cells During Activation And Nutrient Limitation" (2018). Publicly Accessible Penn Dissertations. 3110. https://repository.upenn.edu/edissertations/3110 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/edissertations/3110 For more information, please contact [email protected]. Understanding Metabolic Adaptation Of T Cells During Activation And Nutrient Limitation Abstract Immune cells employ a diverse array of metabolic programs upon stimulation that have far-reaching consequences outside of energy production. Activated T cells require glycolysis to generate the biosynthetic intermediates for proliferation and to enhance effector functions. Due to the disorganized vasculature of solid tumors and the highly glycolytic nature of tumor cells, T cells must compete for glucose with tumor cells in a nutrient-depleted environment. We hypothesized that cells that traffico t inflamed, nutrient-limiting environments in the periphery (effector memory T cells, TEM) may have enhanced abilities to adapt to nutrient limitation compared to cells that largely reside in nutrient-rich lymphoid organs (naïve and central memory T cells, TN and TCM respectively). We demonstrate that TN and TCM rely on fatty acid metabolism to survive and proliferate when glucose is limiting, whereas TEM do not. Furthermore, we find the reliance on fatty acid metabolism in limiting glucose by TN and TCM cells regulates IFN-γ production. Thus the first section of my thesis identifies a novel regulatory interaction between fatty acid synthesis and effector function. Other byproducts of metabolic pathways can also affect immune cell function. Recent work has suggested that reactive oxygen species are released following activation, which promote proliferative signals in T cells such as IL-2 production. The second section of my thesis investigates how T cells increase reactive oxygen species (ROS) production following activation. Using transmission electron microscopy we observe dramatic alterations to mitochondrial morphology following T cell activation. Mitochondria significantly increase in size and their cristae lose parallel patterning during the first 48 hours of T cell activation. Mitochondrial swelling and cristae disturbance are glucose and mTORC1 dependent, and highly reversible. Interestingly, we find that mitochondrial swelling does not correlate to oxidative phosphorylation rate, but strongly correlates to ROS production. We speculate that these mitochondrial changes are required to create the ROS necessary for subsequent IL-2 production and T cell proliferation. Together these data demonstrate novel relationships between cellular metabolism and cytokine production in CD4 T cells. By identifying how metabolites specifically affect immune function, we hope to exploit these discoveries in future cancer immunotherapies. Degree Type Dissertation Degree Name Doctor of Philosophy (PhD) Graduate Group Immunology First Advisor James L. Riley Keywords effector, glycolysis, IFN-y, memory, metabolism, mitochondria Subject Categories Allergy and Immunology | Immunology and Infectious Disease | Medical Immunology This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/edissertations/3110 UNDERSTANDING METABOLIC ADAPTATION OF T CELLS DURING ACTIVATION AND NUTRIENT LIMITATION Christopher Michael Ecker A DISSERTATION In Immunology Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy 2018 Supervisor of Dissertation ____ _________ James L. Riley Associate Professor of Microbiology Graduate Group Chairperson ________________ David M. Allman Professor of Pathology and Laboratory Medicine Dissertation Committee Taku Kambayashi, M.D., Ph.D., Associate Professor of Pathology and Laboratory Medicine E. John Wherry, Ph.D., Richard and Barbara Schiffrin President's Distinguished Professor of Microbiology Kathryn E. Wellen, Ph.D., Associate Professor of Cancer Biology Daniel J. Powell, Ph.D., Associate Professor of Pathology and Laboratory Medicine UNDERSTANDING METABOLIC ADAPTATION OF T CELLS DURING ACTIVATION AND NUTRIENT LIMITATION COPYRIGHT 2018 Christopher Michael Ecker This work is licensed under the Creative Commons Attribution- NonCommercial-ShareAlike 3.0 License To view a copy of this license, visit https://creativecommons.org/licenses/by-nc-sa/3.0/us/ ACKNOWLEDGMENTS First, I would like to thank my mentor and thesis advisor Jim Riley. This work would not have been possible without his support. Jim constantly challenged my ideas and made me a better thinker, scientist and writer. He gave me the freedom to be creative, and believed in me as a scientist before I did. Jim taught me how to take informal discussions on ideas and problems into testable questions and experiments. My thesis committee have been instrumental to the development of my thesis projects and have been extremely generous with their time and guidance. I would like to also thank all of the Riley lab members (former and current). Jim fostered a laboratory environment in which we all talk and learn from each other. Rachel Leibman and Colby Maldini were/are both brilliant graduate students that provided constant scientific and life advice and helped me create better experiments (aka the “electric factory”). Gavin Ellis, Jan Pawlicki, Kevin Tosh, and Max Richardson are incredible scientists who offered guidance, insight and support over my entire thesis. Hong Kong, Chui Lau, Kevin Gayout, Delaine Winn have also generously given their time. Whether it was lunch time conversations or random complaints about our problems, everyone has always made the lab a great place work. I would like to additionally thank Hong, Chui, and Kevin for providing me the numbers of cells needed to do many of the experiments. Andrew Medvec was the first person in the lab I got to know well who mentored me during my rotation. Our early work fostered a lot of the conversations, ideas, and collaborations that were needed to do this thesis. Irene Su, iii Stefana Voicu, and Luis Cortina were all undergraduate students who I was fortunate enough to work with. They all performed many of the experiments throughout my thesis, and are each creatively brilliant in their own right. Our collaborations with Thermofisher Scientific and Ian Blair’s group were essential for this work. In particular Angel Varela-Rohena, Jackie Padja, Lili Guo, Luis Gil- de-Gómez, and Clementina Mesaros for their generous support, time, and performed a large amount of HPLC and mass spectrometry that made my thesis possible. I would like to thank Douglas Wallace, Kevin Foskett for offering their technical support and knowledgeable insight into mitochondria and calcium dynamics. Graduate school cannot be survived without great friendships. I would like to specifically thank Sarah Sneed, Caroline Bartman, Walter Mowel, and Scarlett Yang who have been here with me since the beginning. I would like to thank my friends inside and outside the Immunology Graduate Group for being there when I needed someone. Kilson Lima has been a pivotal rock during my thesis. We drank, danced, and laughed our way around Philadelphia. You all listened to my constant complaints and late night ramblings, and pushed me when I thought I had nothing left to give. Last but certainly not least, I’d like to thank my family for their support and encouragement over the years. My parents have always allowed me to chase my dreams and taught me to find a job that I would be happy with even when the work was hard. My aunts, uncles, grandparents, cousins have all helped shape me into who I am today. iv ABSTRACT UNDERSTANDING METABOLIC ADAPTATION OF T CELLS DURING ACTIVATION AND NUTRIENT LIMITATION Christopher M. Ecker James L. Riley Immune cells employ a diverse array of metabolic programs upon stimulation that have far-reaching consequences outside of energy production. Activated T cells require glycolysis to generate the biosynthetic intermediates for proliferation and to enhance effector functions. Due to the disorganized vasculature of solid tumors and the highly glycolytic nature of tumor cells, T cells must compete for glucose with tumor cells in a nutrient-depleted environment. We hypothesized that cells that traffic to inflamed, nutrient-limiting environments in the periphery (effector memory T cells, T EM ) may have enhanced abilities to adapt to nutrient limitation compared to cells that largely reside in nutrient-rich lymphoid organs (naïve and central memory T cells, TN and T CM respectively). We demonstrate that T N and T CM rely on fatty acid metabolism to survive and proliferate when glucose is limiting, whereas TEM do not. Furthermore, we find the reliance on fatty acid metabolism in limiting glucose by TN and T CM cells regulates IFN-γ production. Thus the first section of my thesis identifies a novel regulatory interaction between fatty acid synthesis and effector function. Other byproducts of metabolic pathways can also affect