University of Massachusetts Medical School eScholarship@UMMS GSBS Dissertations and Theses Graduate School of Biomedical Sciences 2003-07-31 The Function of the Tyrosine Kinase, Itk, in CD4+ T Cell Differentiation and Death: a Dissertation Andrew Todd Miller 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, Animal Experimentation and Research Commons, Cells Commons, Enzymes and Coenzymes Commons, and the Hemic and Immune Systems Commons Repository Citation Miller AT. (2003). The Function of the Tyrosine Kinase, Itk, in CD4+ T Cell Differentiation and Death: a Dissertation. GSBS Dissertations and Theses. https://doi.org/10.13028/2jm2-2156. Retrieved from https://escholarship.umassmed.edu/gsbs_diss/58 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 FUNCTION OF THE TYROSINE KINASE, ITK, IN CD4+ T CELL DIFFERENTIATION AND DEATH A Dissertation Presented Andrew Todd Miler Submitted to the Faculty of the University of Massachusetts Graduate School of Biomedical Sciences, Worcester in parial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY JULY 31 t, 2003 IMMUNOLOGY AND VIROLOGY PROGRAM COPYRIGHT INFORMATION The information and data in this thesis has appeared in the following publications: Miler A.T. and Berg LJ. 2002. Defective Fas Ligand Expression and Activation- J. Immunol. Induced Cell Death in the Absence of 1L- Inducible T Cell Kinase. 168:2163. Miler A.T. and Berg L.J. 2002. New Insights into the Function and Regulation of Current Opinions in Immunology. Tec Family Kinases in the Immune System. 14:331-340. Lucas J. , Miler A.T. , Atherly L.O., Berg L.J. 2003. The Role ofTec Family Immunological Reviews. 191:119- Kinases in T cell Development and Function. 138. though Itk promotes T Miler A.T. , Wilcox, H.M., and Berg LJ. 2003. Signaling helper 2 differentiation via negative regulation of T -bet. Manuscript submitted. Approved as to style and content by: Dr. Raymond Welsh, Chair of Committee Dr. Cynthia Chambers, Member of Committee Dr. Joonsoo Kang, Member of Committee Dr. Aldo Rossini, Member of Committee Dr. Brigitte Huber, Member of Committee Dr. Leslie J. Berg, Dissertation Mentor Dr. Anthony Carruthers, Dean of the Acknowledgements I would first like to thank my parents, my sister Lauren, my grandparents, and the many members of the Miler and Salk families for their unwavering love and support not only during my graduate career, but also throughout life. Without you, I would not have made it where I am today! I owe a great deal of gratitude to my thesis advisor, Leslie Berg. At every stage of my thesis work, her guidance, direction, mentorship, as well as her confidence in me, has been extremely invaluable. I greatly appreciate the freedom and the environment that she has provided me with that has allowed my interest in T cell biology to flourish. Lastly, her enthusiasm and passion for understanding the immune system has been quite an inspiration. In addition, I am grateful to all the members of the Berg lab, past and present, whom I have had the opportunity to work with. Andrea, Heather, Julie, Katherine, Luana, Marin Michael, Min, Morgan, Regina, Sara, Shane, and Wen Chin, you have all in one way or another provided a very stimulating, interactive, educational, and of course entertaining environment throughout the years. In paricular, I have to thank Sara Gozalo for being a great friend and for putting up with me through all of our ups and downs during graduate school. I would also like to thank the members of my thesis. advisory committee, Drs. Cynthia Chambers, Joonsoo Kang, and Raymond Welsh for their direction, insightful opinions and thoughtful suggestions throughout my thesis work. Abstract The Tec family tyrosine kinase , Itk, plays an important role in signal transduction following T cell receptor engagement. Several prior studies have established the importance of Itk in immune system processes, such as T cell development and T cell activation. Additional biochemical studies have found that Itk specifically functions within a multi-molecular signalosome complex, which ultimately functions to provide a platform by which Itk can phosphorylate and activate PLC-y 1 , a crucial step in T cell activation. To further study how Itk regulates distinct immune outcomes via T cell effector processes within the peripheral immune system , and to further understand how Itk functions in T cells in response to a physiological ligand-receptor interaction, I crossed Itk-deficient mice to mice transgenic for a TCR specific for a moth cytochrome C peptide. My studies have established a unique role for Itk in several important aspects of T cell function. Following T cell activation, I identified an imperative role for Itk in activation-induced cell death via FasL, a mechanism of immune homeostasis. Furthermore, I found Itk plays a unique role in the process of T cell differentiation , where Itk positively regulates the induction of cytokine genes , such as IL- , while negatively regulating the induction of T-bet, a transcription factor important for Thl differentiation. Lastly, following T cell differentiation , I found that Itk mRNA and protein are up- regulated during Th2 differentiation , while Rlk, a related Tec kinase, disappears rapidly fromTh2 cells, indicating a critical role for Itk in Th2 cell function. Collectively, my thesis work has more clearly defined an important function for Itk not only in TCR signaling, but also in immune processes such as T cell differentiation and activation- induced cell death that are required for proper immune function. Table of Contents Acknowledgements Abstract Table of Contents vii List of Figures and Tables vii Attributions Chapter I. Introduction Chapter II. The Function of Itk in CD4+ T Lymphocyte Activation- Induced Cell Death Introduction Results Discussion Chapter III. Aberrations in the Phenotype of CD4+ T Lymphocytes From Itk-Deficient Mice Introduction Results Discussion 112 Chapter IV. 115 The Function of Itk in CD4+ T Lymphocyte Differentiation Introduction 116 Results 120 Discussion 149 Chapter V. 161 Discussion and Future Directions Chapter VI. 179 Materials and Methods Chapter VII. 192 References Cited Appendix I. 214 Publications V11 List of Figures and Tables Chapter I: Introduction Figure 1: Domains of the Tec, Syk, and Src Family Kinases Figure 2: Positive Mechanisms Regulating Itk Function Figure 3: Mechanisms Down-Regulating the Activity ofltk Figure 4: TCR Signaling Downstream From Itk Figure 5: Relative Expression Levels ofTec Family Members in CD4+ T Cells Table I: T helper responses in Itk and Rlk- Itk- mice Chapter II: The Function of Itk in CD4+ T Lymphocyte Activation- Induced Cell Death Figure 6: NaIve Itk CD4+ T cells have defects in IL- production and proliferation in response to MHC/peptide stimulation Figure 7: Itk- CD4+ T cells proliferate more vigorously than control T cells upon secondar stimulation Figure 8: FasL upregulation is defective in Itk CD4+ T cells Figure 9: Decreased apoptosis correlates with defective FasL expression in Itk- CD4 + T cells Figure 10: Calcium, ERK, and JNK pathways are defective in previously-activated Itk CD4+ T cells Figure 11: Reduced Egr2, Egr3 , and FasL transcription after TCR stimulation ofltk- T cells Figure 12: CD4+ T cells in Itk mice are defective in AICD in vivo Figure 13: A model for the role of Itk in TCR-mediated FasL expression V11 Chapter III: Aberrations in the Phenotype of CD4+ T Lymphocytes from Itk- Deficient Mice Figure 14: Itk-deficient mice exhibit a 2- to 3-fold increase in the percentage and numbers of CD4+ memory cells hi cells Figure 15: Increase in the number of CD44 producing IL-2 in Itk- spleen Figure 16: Thl/Th2 cytokine imbalance in Itk-deficient memory cells Figure 17: Increased numbers of IFN-y and IL-4 producers in Itk spleen in both the CS7BL/l0 and Balb/c backgrounds Figure 18: SC.C7 Itk- CD4+ T cells differentiate into both Thl and Th2 subsets under non-skewing conditions 103 Figure 19: Normal dendrtic cell populations in Itk mice 107 Figure 20: Normal activation markers on dendrtic cells from Itk mice 110 Chapter IV: The Function of Itk in CD4+ T Lymphocyte Differentiation Figure 21: Itk- CD4+ T cells can efficiently differentiate into polarized Thl and Th2 cells 125 Figure 22: Low avidity TCR stimulation promotes Th2 differentiation by Itk+ but not by Itk CD4+ T cells 130 Figure 23: bet mRNA expression is aberrantly regulated by TCR signals in the absence of Itk 136 Figure 24: Itk negatively regulates T -bet upon low avidity TCR stimulation 139 Figure 25: Itk is upregulated and Rlk is downregulated during Th2 differentiation 143 Figure 26: Signaling deficiencies in Thl versus Th2 cells lacking Itk 147 Figure 27: A model for Itk in the process of CD4+ T lymphocyte differentiation and effector function 154 (A) Strong TCR signal 158 (B ) Weak TCR signal 159 (C) Thl and Th2 effector function 160 Chapter V: Discussion Table II: Similar phenotypes of mice deficient in T cell signaling proteins 171 Attributions Chapter II. Julie Lambert and Dr. Michael Brehm assisted me in the SEB experiments described in Figure 7. Chapter III. The dendrtic cell analysis in Figure 6 and Figure 7 was a collaborative effort between myself and Dr. Morgan Wallace. Chapter IV. The optimization of the quantitative PCR reactions for Itk and Rlk used to analyze expression levels in Figure 5 was a collaborative effort between myself and Martin Felices. The protein gels and immunoblotting in Figure 5 and Figure 6 were performed by Heather Wilcox.