A COMPREHENSIVE FRAMEWORK FOR SYSTEMS IMMUNE MONITORING IN PATIENTS WITH CANCER By Allison Rae Greenplate Dissertation Submitted to the Faculty of the Graduate School of Vanderbilt University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY in Microbiology and Immunology May 11, 2018 Nashville, Tennessee Approved: Luc Van Kaer, Ph.D. Jeffrey Rathmell, Ph.D. Ann Richmond, Ph.D. Earl Ruley, Ph.D. Jonathan Irish, Ph.D. To Jason, for joining me in this adventure. Wherever I’ve gone and whatever I’ve accomplished, it is only because of you. ii ACKNOWLEDGEMENTS There is neither enough room nor enough time for me to accurately and appropriately express my gratitude. To borrow from Tolkien, five and a half years is far too short a time to live among such excellent and admirable scientists. I am a different, and I hope better, person than I was when I started this journey. Here I do my best to acknowledge and thank those of you who shaped me, supported me, and promised me that there was a light at the end of this graduate school tunnel. I must thank my mentor, Dr. Jonathan Irish, for allowing me to call your lab “home” for these 5 odd years. Thank you for the freedom to be creative and to direct my own project. Thank you for providing a space that encouraged deep, sometimes heretical, scientific conversations. Under your guidance, I have learned how to ask big questions and fine-tune small details. You have taught me how to defend my ideas, while still remaining open to criticism. Thank you for fostering independence and teaching me when it was ok to ask for help. I would not be the scientist I am today without your mentorship. I would also like to thank my thesis committee, Dr. Luc Van Kaer, Dr. Jeffrey Rathmell, Dr. Ann Richmond, and Dr. Earl Ruley for their hard work and mentorship. You all provided guidance and encouragement while simultaneously pushing me to become the best scientist I could possibly be. I’d like to especially thank Earl for teaching me how to write grants and for always opening his door to me in my (many) moments of crisis. Earl, you helped me become a better scientist and a tougher person. I am forever indebted to the members of the Irish and Ihrie labs. To Dr. Rebecca Ihrie, your thoughtful comments and keen eye for detail have helped me improve everything I’ve created. Thank you for the joint lab meetings. And thank you, Jonathan and Rebecca, for assembling two fantastic teams. In your labs, I have found best friends and for that I am forever iii grateful. Justine Sinnaeve, Nalin Leelatin, and Caroline Roe, thank you for the 11am lunches, morning coffee, and the endless scientific and emotional support. The most important thing I have gained during my time at Vanderbilt is your friendship. Finally, I’d like to thank Dr. Brent Ferrell for the support, encouragement, and for helping me get my first paper published. I must acknowledge my good friends who have helped me, at Vanderbilt and elsewhere. Thank you Lisa Poole, Bethany Carbonaeu, Becca Bluett, Theresa Barke, and Marilyn Holt for potlucks, happy hours, and loving support. To Sam Moxely and the members of Nashville Ultimate Machine, I wouldn’t have survived my first few years as a graduate student without you. Thank you for providing an outlet and community that didn’t involve science. Special thanks to Tim Noftzger who has been a friend for almost 10 years. Thank you for moving here and for always being excited about freezing cold camping trips. Zach Shunk, thank you for appearing when I needed a friend the most and for sticking around ever since. Thank you for being the company my misery needed. Finally, I’d like to thank Trey Fitch, Lacee Strickland, Allie Mikels, Jarrod Arnold, Matt Poppert, and all the members of CrossFit HDub. Without those early morning workouts, I would not have been motivated to make it in to lab during that last year. None of my success would have been possible without the love and encouragement of my family. Mom, your support has been unwavering. Without your unconditional love and weekly phone calls, I would have never made it this far in life, let alone graduate school. You are selfless, wise, hilarious, beautiful, and inspiring. I am beyond lucky to be your daughter. Dad, thank you for always believing in me and encouraging me to “rock the ball”. Thank you for raising me to embrace my weirdness. Most importantly, thank you for throwing me a life raft while I was drowning in science. Those months of working on EMD together were, by far, my favorite of graduate school. This PhD is only a reality because of you. Maddy, thank you for 4 years of sister dates. You made Nashville feel like home. Those runs and breakfasts made the hardest weeks not just bearable, but enjoyable. Your strength and drive pushed me to be better. Corrine, thank iv you for loving me even when I was distant. You have understood me, always, and for the last 27 years I have considered you my very best friend. Your creativity and motivation inspire me daily. Thank you and Tom for bringing Dorothy Lucia into this world. I love her more than I can describe. By far, the person I am most indebted to is my husband, Jason. You have made my life so inexplicably, undeservedly Good (with a capital G). Though I begrudgingly agreed to take your last name when we got married, I am so glad I did. Now, every grant, paper, and award will have your name attached as proof that none of it would be possible without you. Your patience and wisdom guided me through times of existential crisis. Your unconditional love reminded me that I am worthy and whole, even when I felt broken. Thank you for never giving up on me and always running out in the middle of the night to buy ice cream. When I married Jason, I was lucky enough to marry into a second, wonderful family. To my in-laws, Cindy and Paul, thank you for your constant prayers and love. And to my siblings-in- law, Andrew, Bekah, Josh, Monique, Emily, Isaac, Melissa, Vito, and Soriya, thank you for accepting me and loving me like a sister. I have cherished our crazy adventures and look forward to a life time of more. The work in this thesis has been made possible in part by grants F31 CA199993-01. Finally, I would like to thank all of the patients and their families who donated their biospecimens to make this work possible. Their sacrifice is what pushes science forward. v TABLE OF CONTENTS Page DEDICATION…………………………………………………………………………………………...…ii ACKNOWLEDGMENTS…………………………………………………………………………………iii LIST OF TABLES ...................................................................................................................... ix LIST OF FIGURES .................................................................................................................... x LIST OF ABBREVIATIONS ..................................................................................................... xiv Chapter I. Background and research objectives .................................................................................... 1 Introduction .......................................................................................................................... 1 T cell development and function ........................................................................................... 3 Generation of the T cell receptor .................................................................................... 3 Antigen-specific T cell response ..................................................................................... 5 Chronic antigen stimulation and T cell exhaustion ........................................................ 11 Melanoma and Tumor Immunity ......................................................................................... 15 Immune surveillance in cancer ..................................................................................... 15 Immune evasion and T cell dysfunction in the tumor microenvironment ....................... 19 Development and success of checkpoint blockade ....................................................... 21 Systems Immune Monitoring .............................................................................................. 25 Rationale and techniques ............................................................................................. 26 Single-cell biology in clinical practice ............................................................................ 28 Machine learning for human immunology ..................................................................... 30 Research Objectives and Preface ...................................................................................... 33 II. Multiparameter analysis of stimulated human peripheral blood mononuclear cells: a comparison of mass and fluorescence cytometry ............................................................... 37 Introduction .................................................................................................................. 37 Materials and Methods ................................................................................................. 38 Results ......................................................................................................................... 41 Fluorescence and mass cytometry panels to track T cell identity-panel design .. 41 vi Values derived from fluorescence and mass cytometry were closely correlated 47 viSNE identified similar frequencies of activated CD4+ T cell populations ......... 51 Dimensionality reduction