University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2004-09-13 The role of Janus Kinase 3 in CD4+ T Cell Homeostasis and Function: A Dissertation Shane Renee Mayack University of Massachusetts Medical School Let us know how access to this document benefits ou.y Follow this and additional works at: https://escholarship.umassmed.edu/gsbs_diss Part of the Amino Acids, Peptides, and Proteins Commons, Cells Commons, Enzymes and Coenzymes Commons, and the Hemic and Immune Systems Commons Repository Citation Mayack SR. (2004). The role of Janus Kinase 3 in CD4+ T Cell Homeostasis and Function: A Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/4qq7-k514. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/94 This material is brought to you by eScholarship@UMMS. It has been accepted for inclusion in GSBS Dissertations and Theses by an authorized administrator of eScholarship@UMMS. For more information, please contact [email protected]. THE ROLE OF JANUS KINASE 3 IN CD4+ T CELL HOMEOSTASIS AND FUNCTION A Dissertation Presented BY Shane Renee Mayack Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in partial llfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY September 13,2004 Interdisciplinary Graduate Program Department of Pathology Program in ImmunologyiVirology Copyright Information The content of this dissertation has appeared in the following publication: Mayack, S. Berg., L. (2004) Alternative CD4+ T cell differentiation in the absence of Jak3. Submitted for publication. Joonsoo Kang, Chair of Committee Alan Rothman, Member of Committee Robert Woodland, Member of Committee Katherine Luzuriaga, Member of Committee Gordon Freeman, Member of Committee Leslie J. Berg, Dissertation Mentor Anthony Carruthers, Dean of the Graduate School of Biomedical Sciences I would like to thank my longtime boyfnend David who has been by my side throughout my pursuit for higher education. It is him that I have looked to all of these years for advice and inspiration and as a model of honesty, integrity and ambition. It is him that has always, always provided me with a reason to smile. You have enriched every aspect of my life. Thank you for your love and support and the unwavering confidence you have had in me throughout the years. We are a team and I could not have done this without you. I would like to thank my parents. They are truly the most selfless people I have ever encountered. They have both, especially my Mother, sacrificed their entire lives for me and my siblings. For this my gratitude cannot be put into words. In addition, by way of example, they have taught me two very important lessons. The fist was to have an exemplary work ethic even in the face of seemingly little return. The second was to always have a positive attitude, be able to laugh and to persevere even under unpleasant circumstances. Emulating these traits have contributed greatly to my accomplishments in life and are particularly applicable to the many hurdles one faces in science. Thank you for instilling in me that I could achieve anything I wanted to in life. In addition, I am grateful to my mentor Dr. Leslie Berg for welcoming me into the lab without a second thought. Not only has she provided me with sound scientific guidance but also, by allowing me the freedom to pursue the questions of my choosing, has allowed me to gain the confidence in and passion for science that I had temporarily lost throughout my graduate career. Most remarkable though, is the positive and well rounded role model Leslie provides, in general, but particularly for women. She makes balancing a successful career and pursuing a family, or whatever else is important to you, not only seem obtainable but almost effortless. What truly sets Leslie apart as a mentor is her patience and professionalism, unwavering confidence in her students' abilities, and that she always seems to know exactly what to say and how to say it. Thank you for providing a stimulating and challenging yet supportive scientific environment which has allowed me to recognize my own abilities and grow even stronger as a scientist. I would like to acknowledge all of the members of the Berg lab and the entire Pathology department. The openness, collaborative and collegial nature of all of the investigators, post-docs, students and technicians in the Pathology department make it a great place to be a graduate student and significantly contributes to an environment conducive to learning and productivity. I would especially like to thank Sara Gozalo and Dr. Morgan Wallace for helping me get started in the lab. Regina Whitehead for ordering all of my reagents and dealing with any and every issue in a direct and timely fashion. Dr. Joe Maciaszek and Dr. SK Kim for assistance with tail vein injections. I would like to thank Min Shi and Amanda Prince for generating the RT-PCR data presented in this dissertation and for their support and confidence in me. In addition, I would like to thank them both for the opportunity to act (partially) as their mentor when they first joined the lab. I have learned as much from the both of you as I can only hope you have learned from me. I am indebted to the UMASS Sorting Facility, especially Tammy Krumpoch, for isolation of CD4+ CD44-high T cell populations and the UMASS Affyrnetrix Core Facility for performing the microarray hybridizations. I would like to thank Dr. Maria Zapp and Dr. Ellie Kittler for investing their time and resources in me early in my graduate career. During this time, I learned a great deal about science and life. I would like to thank all of the members of my thesis defense committee---Dr. Robert Woodland, Dr. Alan Rothrnan, Dr. Joonsoo Kang, Dr. Katherine Luzuriaga, and Dr. Gordon Freeman for participating in my thesis examination and for all of the time and energy a number of you have spent training me to be a competent scientist. I would especially like to thank Dr. Robert Woodland. At the lowest point in my graduate school career you were there for me and handled a somewhat sensitive matter with the utmost professionalism and concern for my well-being. Your faith in me during that time is the reason I remained in graduate school. Thank You. And, finally, a special thanks to Dr. Joonsoo Kang who has been extremely supportive of all of my research and provided the STATSab-deficient mice for experiments described in ths thesis. In addition, his high expectations provide an extra challenge for all of the students and in the end I think we are all stronger for it. He is an excellent addition to the UMASS community. vii Abstract This dissertation addresses the role for Janus Kinase 3 (Jak3) in CD4+ T cell homeostasis and function. Jak3 is a protein tyrosine kinase whose activity is essential for signals mediated by the yc dependent cytokines IL-2, -4, -7,-9,-15, and -21. Previous data have demonstrated that peripheral CD4+ T cells from Jak3-deficient mice have a memory phenotype and are functionally impaired in both proliferative and IL-2 responses in vitro. Interestingly, Jak3/yc activity has been previously shown to play a role in the prevention of T cell anergy. These studies were initiated to more precisely define the role for Jak31yc cytohnes in the prevention of T cell anergy and the maintenance of functional CD4+ T cell responses. We began to address this question by assessing global gene expression changes between wild type and Jak3-I- CD4+ T cells. These data indicate that Jak3-I- CD4+ T cells have an increase in gene expression levels of inhibitory surface receptors as well as immunosuppressive cytokines. Further analyses confirmed that Jak3-deficient T cells express high levels of PD-1, secrete a Trl-type cytokine profile following direct ex vivo activation, and suppress the proliferation of wild type T cells in vitro. These characteristics indicate that CD4+ Jak3-/- T cells share properties with regulatory T cell subsets that have an important role in peripheral tolerance and the prevention of autoimmunity. We next addressed whether these regulatory characteristics were T cell intrinsic or rather the result of expanding in a Jak3-deficient microenvironment characterized by a number of immune abnormalities and a disrupted splenic architecture. Jak3-I- CD4+ T cells proliferate in vivo in a lymphopenic environment and selectively acquire regulatory T cell characteristics in the absence of any additional activation signals. While the precise mechanism by which Jak3-deficient T cells acquire these characteristics remains unclear, our data indicate that one important component is a T cell-intrinsic requirement for Jak3 signaling. These findings indicate several interesting aspects of T cell biology. First, these studies, demonstrate that the homeostatic proliferation of CD4+ T cells is not dependent on signaling via yc-dependent cytokine receptors. And, second, that the weak activation signals normally associated with homeostatic expansion are sufficient to drive Jak3-1- T cells into a non- conventional differentiation program. Previous data indicate that, for wild type T cells, signaling through both the TCR as well as yc-dependent cytokine receptors promote the homeostatic proliferation of T cells in lymphopenic hosts. Since Jak3-I- T cells are unable to receive these cytokine signals, their proliferation is likely to be wholly dependent on TCR signaling. As a consequence of this TCR signaling, Jak3-I- T cells proliferate, but in addition, are induced to up regulate PD-1 and to selectively activate the IL-10 locus while shutting off the production of IL-2.