Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2019 Determination of Transforming Growth Factor-β Signaling Targets in Activated T Cells Ramiah Jacks Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Immunology of Infectious Disease Commons Recommended Citation Jacks, Ramiah, "Determination of Transforming Growth Factor-β Signaling Targets in Activated T Cells" (2019). Dissertations. 3341. https://ecommons.luc.edu/luc_diss/3341 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2019 Ramiah Jacks LOYOLA UNIVERSITY CHICAGO DETERMINATION OF TRANSFORMING GROWTH FACTOR-β TYPE I RECEPTOR (TGF-βRI) SIGNALING TARGETS IN ACTIVATED T CELLS A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN MICROBIOLOGY AND IMMUNOLOGY BY RAMIAH D. JACKS CHICAGO, ILLINOIS AUGUST 2019 ACKNOWLEDGMENTS I would like to first acknowledgment my parents, Greg and Regina Jacks, for their love, encouragement, commitment to my success, and, most importantly, their sacrifices. I will be forever grateful for their unwavering dedication to ensuring that their children have the education and resources required to achieve anything that we desire. I am extremely appreciative of the opportunities afforded to me because of the hard work, strength and perseverance of my parents, extended family members and ancestors. I recognize that I am living the dreams of my ancestors and, inspired by their efforts, I strive to create an even better future for the next generation. I also would like to thank my sister Rakeva, brother Greg, and my boyfriend Joseph for their unconditional love and support, as well as regular therapeutic fun and laughs. I am also extremely appreciative for the leadership and mentorship of Dr. Makio Iwashima. Makio was instrumental in the development of my ‘scientific mind’. He is an incredible scientist and taught me that the drivers of science are data, even if they do not support your hypotheses. We’ve had regular enriching conversations and meetings about my data and his feedback was invaluable for my development as an immunologist. I’ve thoroughly enjoyed my time in Makio’s lab and I look forward to continue to collaborate with him the in future. I must also thank my friends/labmates in the Iwashima lab, both old and new. Not only did we have great conversations about the directions of our projects and how to improve presentations, we also had a lot of fun together. We were very collaborative iii and had no problem helping each other with whatever was needed, in the lab or in our personal lives. Their support and friendship are things that I will cherish forever. Finally, I cannot thank my committee enough. Drs. Phong Le, Katherine Knight, Clodia Osipo and Jose Guevara-Piñto always provided critical and constructive feedback that kept my project moving forward in meaningful ways. Their mentorship also allowed me to become a better scientist and communicator. In addition, this work would not be possible without: funding support from the T32 Immunology Training Grant to Dr. Knight; funding support from the Predoctoral Ruth L. Kirchstein National Research Service F31 Diversity Award from the from the National Institute of General Medical Sciences (NIGMS), the guidance and expertise of Pat Simms, Ashley Hess and Steven Kirk at the flow cytometry core; and the continued support and encouragement from the students, faculty, and staff of the Department of Microbiology and Immunology. iv TABLE OF CONTENTS ACKNOWLEDGEMENTS ................................................................................................ iii LIST OF FIGURES ......................................................................................................... viii ABSTRACT ...................................................................................................................... x CHAPTER ONE: REVIEW OF LITERATURE .................................................................. 1 T lymphopoiesis ............................................................................................................ 1 Positive and Negative selection .................................................................................... 4 T cell Subsets ................................................................................................................ 6 CD4+ T cell Subsets ................................................................................................... 6 CD8+ T cell Subsets ................................................................................................. 11 Perinatal vs Adult T cells……………………………… .................................................. 11 Transforming Growth Factor-β (TGF-β) ...................................................................... 13 Processing of TGF-β ............................................................................................... 14 Activation of Latent TGF-β ....................................................................................... 16 TGF-β Signal Propagation ........................................................................................... 19 Canonical TGF-β Signaling ...................................................................................... 22 Non-canonical TGF-β Signaling ............................................................................... 24 TGF-β Signaling in T cells ........................................................................................... 25 TGF-β Signaling in T cell Differentiation ................................................................... 25 TGF-β Signaling in T cell Suppression ..................................................................... 29 c-Jun Amino-Terminal Kinase (JNK) Activation in T cells………… ............................. 31 Upstream Kinases in JNK Activation ........................................................................ 31 Adaptor Molecules, CARMA1 and BCL10, in JNK Activation .................................. 34 JNK Substrates: The Jun Family of Transcription Factors .......................................... 39 c-Jun ......................................................................................................................... 40 Jun B ........................................................................................................................ 41 Jun D ........................................................................................................................ 42 The Function of JNK1 and JNK2 in T cells .................................................................. 43 JNK1 and JNK2 Signaling in T cell Activation .......................................................... 43 JNK1 and JNK2 Signaling in T cell Differentiation ................................................... 45 JNK1 and JNK2 Signaling in T cell Apoptosis .......................................................... 46 Purpose of Dissertation ............................................................................................... 47 CHAPTER TWO: MATERIALS AND METHODS ........................................................... 49 Cell Preparation and Reagents ................................................................................... 49 Phenotype Analysis and Cytokine Profile of Naïve CD4+ T cells ................................ 50 Th1 and Th2 Differentiation Assays ............................................................................ 51 Generation of a TGF-βRI Deficient Jurkat T cell Line using CRISPR/cas9 Genome Editing ........................................................................................................ 51 Stimulation of the TGF-βRI Deficient Jurkat T cell Line ............................................. 52 Stimulation of the Primary Human 1383I TCR T cell Line ........................................... 53 Western Blot Analysis ................................................................................................. 54 Flow Cytometry............................................................................................................ 54 v Reporter Assays in the TGF-βRI Deficient Jurkat T cell Line and Primary Human T cells........................................................................................................... 55 RNA Sequence Analysis of the TGF-βRI Deficient Jurkat T cell Line ......................... 56 Activation of Primary Mouse T cells ............................................................................ 57 Statistical Analysis ...................................................................................................... 57 CHAPTER THREE: RESULTS ....................................................................................... 58 SECTION 1: Characterization of Naïve CD4+ T cells from Umbilical Cord Blood vs Peripheral Adult Blood ............................................................................ 58 CD26 is Highly Expressed by Naïve CD4+ T cells from Umbilical Cord Blood ..................................................................................................................... 60 IL-7 Induced