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UCSF UC San Francisco Electronic Theses and Dissertations Title Dynamic Tuning of CD4+ T cells by Tonic Signals Permalink https://escholarship.org/uc/item/6r11z6ft Author Myers, Darienne Ross Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Dynamic Tuning of CD4+ T cells by Tonic Signals by Darienne Myers DISSERTATION Submitted in partial satisfaction of the requirements for the degree of DOCTOR OF PHiLOSOPFTY in Biomedical Sciences in the GRADUATE DIVISION of the UNIVERSITY OF CALIFORNIA, SAN FRANCISCO Copyright 2017 by Darienne R. Myers ii DEDICATION AND ACKNOWLEDGEMENTS Chapter 1 of this dissertation is a reprint of material published in Myers, D.R., Roose, J.P., 2016. Kinase and Phosphatase Pathways in T Cells. Ratcliffe, M.J.H. (Editor in Chief), Encyclopedia of Immunobiology, Vol. 3, pp. 25-37. Oxford: Academic Press. Chapter 6 is a reprint of Daley et al, Rasgrp1 mutation increases naïve T-cell CD44 expression and drives mTOR-dependent accumulation of Helios+ T cells and autoantibodies. eLIFE 2013; 2:e01020. The research presented in this dissertation is the result of hard work, and it could not have been done without the support of many people. First, I would like to thank my mentor, Jeroen Roose. Jeroen, thank you for everything. You have taught me so much about being a scientist: how to think critically and strategically about experimental design and analysis, how to write, how to give clear and interesting presentations, and how to share my knowledge with my own mentees. I appreciate how you set me up with three fascinating projects and gave me the freedom to explore the questions I found most interesting, always providing me with guidance when I needed it. Thank you for always telling me to dream big. And, most importantly, thank you for encouraging me to have fun while doing all of that! I feel incredibly fortunate to have had the chance to work with you – your intellect, strong leadership, and positivity are admirable, and I hope I can channel these qualities as I move forward in my career. Additionally, thank you for building a lab full of smart, kind, and fun people – it was always a pleasure coming to work and I greatly value the friendships I have made as a member of the Roose lab. I would also like to thank every member of the Roose lab whom I’ve had the chance to work with over these past several years, and I’d like to specifically acknowledge a few people here. Olga Ksionda, thank you for being an incredible friend and mentor. You always answered my questions no matter how busy you were, and I had great fun with you swapping recipes and chatting in our bay. Yvonne Vercoulen, I admire your energy and organizational skills so much, thank you for teaching me how to handle big experiments and for contributing your interesting iii data on basal Rasgrp1 signaling to my Anaef manuscript. And, thanks for being the BEST conference buddy! Andre Limnander, thanks for getting me set up in the lab with the Anaef project and for being the ultimate SF restaurant guru. Marsilius Mues, thanks for teaching me flow cytometry, for always being willing to lend a hand, and for all the laughs! Philippe Depeille and Jesse Jun, you have both been in the lab with me the entire time, and I am so happy I had you both as friends and mentors these past few years. Jesse, thanks for answering my endless questions about signaling assays, and Philippe, thanks for being my friend despite me never listening to you about why researching the intestine is the best! Emilia Norlin, it has been so wonderful working with you on the Anaef project these past couple years – thanks for your energy and hard work, and for your warmth and friendship. Tannia Lau, thanks for working with me on the HDAC story and for being such a fun presence in the lab. I would also like to thank my rotation students, Jorge Ortiz-Carpena, Casey Beppler, Ian Boothby, and Anthony Venida for their hard work and creative thinking on the Rasgrp1-metabolism project presented in Chapter 8. I also received invaluable mentorship from several UCSF faculty members. Thanks to Mark Ansel, Julie Zikherman, and Davide Ruggero for being on my qualifying exam and thesis committees – your ideas and feedback at our meetings were incredibly helpful as I drove my projects forward. Thank you to Art Weiss and the Weiss lab for your comments and suggestions at our joint lab meetings. Prior to starting my Ph.D. at UCSF, I received fantastic research training as an undergraduate at Amherst College by Dr. Dominic Poccia and Dr. David Ratner. Following that, I was first exposed to the exciting world of immunology research in Dr. Frederick Alt’s lab. Thanks to Fred for giving me the opportunity to work in his lab, to Roberto Chiarle for mentoring me on a challenging and engaging project, and to Chunguang Guo for his mentorship and friendship. iv Science is hard work, and I couldn’t have made it through graduate school without my amazing friends. Thank you for sharing so many meals, cups of coffee, movies, hikes and boxing classes with me! All my love to Kathryn Cubria, Stacey Frumm, my Cooley Dorm roomies, my SF roommate Bevin English, and my BMS and MSTP friends. Max, thank you for being my partner, friend, and the love of my life. I could not have done this without you. Whether we’re at the top of a mountain or dancing in the kitchen, every moment with you is full of love and joy and laughter. I can’t wait to go on more adventures with you for the rest of our lives. Thank you to my family – mom, dad, and Jenna, I love you more than words can express. Thank you for encouraging me every step of the way, for supporting me as I moved across the country to pursue grad school, and for all the fun and laughter when we are together! Mom, thanks for the daily walk-to-work phone calls, talking with you always brightens my day. Thank you for being my best friend, and for all of your love, always. v ABSTRACT Dynamic Tuning of CD4+ T cells by Tonic Signals Darienne R. Myers To maintain immunological tolerance and prevent autoimmunity, naïve CD4+ T cells need to be able to discriminate between self and nonself. In addition, naïve T cells need to remain quiescent and prevent spontaneous activation or differentiation, yet they also must be primed to efficiently respond when they do encounter foreign antigen. These features suggest that there must be active mechanisms to regulate the naïve state of CD4+ T cells. We have uncovered that tonic (basal) signaling pathways in peripheral CD4+ T cells dynamically maintain this primed-yet-quiescent state in three manners: transcriptional, translational, and metabolic programs. We identified a tonic pathway involving the adapter LAT that dynamically controls a transcriptional program in naïve CD4+ T cells. LAT perturbation in T cells leads to a spontaneous Th2-biased lymphoproliferative disease. We found that LAT is critical for tonic regulation of the repressor HDAC7. Tonic signals lead to phosphorylation and cytoplasmic localization of HDAC7, where it does not de-acetylate and repress its target genes. Without basal LAT function, HDAC7 is dephosphorylated and resides in the nucleus, where it represses target genes such as Nur77 and Irf4. Appropriate expression of these genes is important for preventing spontaneous proliferation of CD4+ T cells and differentiation to Th2. Second, we identified a connection between tonic Rasgrp1-mTOR signals and translational programs. CD4+ T cells with a point mutation in the Ras activator Rasgrp1, called Rasgrp1Anaef, exhibit increased basal mTOR signaling and as a result the mice develop autoimmune features. vi We found that Rasgrp1Anaef CD4+ T cells have an autoreactive TCR repertoire and show increased differentiation to the Tfh- and Th2- lineages, with concomitant increase in Gata3 protein levels that we hypothesize is caused by increased translation. We are currently investigating what global basal translation programs are regulated by tonic Rasgrp1-mTOR signals in naïve T cells. Additionally, we are following up on findings that tonic mTOR signals control the metabolic state of naïve T cells and allow for efficient switching of metabolic programs upon T cell activation. Overall, our results provide novel insights into the role of tonic signaling pathways in controlling naïve CD4+ T cells and how point mutations in signaling proteins can disrupt these tonic pathways, leading to perturbed immune function. vii TABLE OF CONTENTS Chapter 1: Kinase and Phosphatase Effector Pathways in T cells ........................................ 1 Abstract ..................................................................................................................................... 2 Introduction ............................................................................................................................... 3 Main text .................................................................................................................................... 4 Concluding remarks ................................................................................................................ 26 Figures .................................................................................................................................... 28 References .............................................................................................................................. 38 Chapter 2: Tonic Signals in T Cells and their Role in Immune Function ............................