OX40 Promotes Differentiation of CD4+ T cells to Effector Cells by Cortny Ann l!uddleston A Dissertation Presented to the Department of Molecular Microbiology and Immunology Oregon Health & Sciences University School of Medicine In partial fulfillment of the requirements for the degree of Doctor of Philosophy September 2006 School of Medicine Oregon Health & Science University CERTIFICATE OF APPROVAL This is certify that the Ph.D. dissertation thesis of Cortny A. Huddleston Member Member Member '_) Table of Contents Table of Contents 1-11 Acknowledgements m-1v Preface v Abstract v1-vn Chapter 1. Introduction 1 1.1 Development of effector and memory CD4 T cells 1 1.2 Regulation of the immune response, tolerance induction 6 1.3 OX40 and its ligand 11 1.4 Other TNFR family members that regulate immunity 13 1.5 OX40 Signal transduction 16 1.6 OX40 in T cell expansion, survival, and memory 18 1.7 OX40 in T cell differentiation 19 1.8 OX40 in disease 20 1.9 OX40 in persistent versus transient antigen stimulation 24 Chapter 2. Manuscript #1: A Signal Through OX40 (CD134) Allows Anergic, 28 Autoreactive T Cells to Acquire Effector Cell Functions and Kill their Hosts Chapter 3. Manuscript #2: OX40 (CD134) Engagement Drives Differentiation 61 of CD4 + T Cells to Effector Cells Chapter 4. Manuscript #3: OX40-Mediated Differentiation to Effector Function 95 Requires IL-2 Receptor Signaling but not CD28, CD40, IL-12R~2, or T-bet Chapter 5. Conclusions, Perspectives 122 Appendix 135 Literature Cited 142 II Acknowledgements I would first like to thank the scientists who have inspired me over the years. I am thankful for the guidance of my mentor, David Parker, who has been excited about my research and supportive in my scientific development. He has taught me to be precise in my work and has inspired me to be well read in my field. David also provided me with an excellent research topic, and always made time to discuss the intricacies of the project, which taught me how to think critically about my work and that of others. I would also like to thank Dr. Scott Lapatra for taking the time to introduce me to research science as a high school student, which inspired me to pursue a Ph.D. I would like to thank Dr. Jerri Bartholomew and Dr. Mark Leid for providing research experiences and intellectual guidance during college that motivated me to continue my pursuit of a Ph.D. The celebrations and hardships of graduate school could not have been endured without the support, laughter, and tears shared with my friends. I am grateful for my friendship with Ezhilkani Subbian, who shared so much more than an apartment and the graduate school experience with me. Her understanding and compassion for science, and her commitment to friendship and fun continues to inspire me in my own life. I am also thankful for the loyal support of my friend Thuy Vo, who always has time to make me smile. I am fortunate to rely upon the support and fellowship of so many other friends, and I thank each of you for enriching my life. My family deserves the most credit for any happiness and success in my life. My parents, Terry and Nancy, have provided a happy and supportive environment for me to pursue any dream, and they gladly work hard to provide opportunities for me. They motivate me to always strive for my best because they are proud of all my Ill accomplishments. My brother, Seth, always reminds me of my roots when pride sweeps me away, and never fails to make me laugh. I am happy to have my husband Jay by my side, to hear first-hand about all the daily issues and joys, to patiently solve problems, and to encourage celebration of good news. I am grateful for Jay's support during graduate school, for encouraging me to do my best every day, and for taking extra time to celebrate the little things. I love my family with all my heart. IV Preface I have prepared my dissertation in accordance with the guidelines set forth by the Graduate Program of the School of Medicine, Oregon Health & Science University. This manuscript consists of a general introduction, three chapters of original data, and a section with summary and general conclusions. The references cited for all chapters are listed together at the end of the text and follow the format of the Journal of Immunology. Chapter two contains data, figures, and text as they appear in the original paper published in the Journal oflmmunology (1). Stephanie Lathrop and I contributed most of the work to this manuscript and David Parker wrote the manuscript. Chapter three contains data, figures, and text as they appear in the original paper published in the European Journal of Immunology (2). Chapter four is a manuscript that has been submitted for publication to the Journal oflmmunology. v Abstract CD4 T cells play an important role in protection against viruses, bacteria, parasites, and cancers, but can also contribute to undesired immune responses such as autoimmunity, graft rejection, and allergic reactions. Understanding the mechanisms that control CD4 T cell effector function will lead to more effective vaccine design and the management of aberrant immune responses. The tumor necrosis factor receptor (TNFR) family member OX40 (CD134) is a costimulatory protein expressed exclusively on activated T cells that augments clonal expansion and survival of antigen-specific CD4 T cells, as well as enhancing the generation of effector and memory T cells. Mechanistically, it has been proposed that OX40 enhances CD4 T cell survival and memory cell generation by enhancing anti-apoptotic protein expression, as well as enhancing effector cytokine production. However, blocking OX40 signaling in vivo specifically reduces inflammation induced by cytokines, suggesting that OX40 may directly influence differentiation to effector function. I was interested in how OX40 regulates effector function in CD4 T cells, so I hypothesized that OX40 signaling could promote differentiation independent ofT cell survival. We have developed a model in which a peptide antigen covalently bound to MHC class II is expressed at low levels on all MHC class II positive cells in mice. Upon transfer of small numbers of antigen specific T cell receptor transgenic CD4 T cells, rapid expansion and infiltration of tissues is observed, but the T cells are tolerant and the animals remain healthy. Addition of an agonist antibody to OX40 at the time ofT cell transfer induces accumulation of large, granular effector CD4 T cells that express the IL- VI 2 receptor alpha chain, CD25, and secrete interferon-y directly ex vivo or in response to cytokine stimulation, and the animals die within one week. We have also developed a polyclonal model in which a small percentage ofB6 CD4 T cells transferred into MHC class II disparate mice behave similarly to the monoclonal T cells described above. These adoptive transfer systems provide useful models in which to examine the immune consequences of OX40 signaling pathways. I found that OX40 signaling induces effector cytokine production early in T cell priming, before changes in anti-apoptotic proteins could be detected. I also showed that genetically altered CD4 T cells with enhanced survival do not acquire effector function independent of OX40 costimulation, and OX40 deficient CD4 T cells can acquire effector function in the presence of OX40 sufficient cells. These experiments suggest that OX40 directly influences differentiation, but may also require cooperation with other factors. I tested the requirement for additional costimulation in supporting OX40 signaling, and found that OX40 costimulation induces differentiation independent of CD28 and CD40 signaling. I also showed that OX40 signaling does not depend upon T­ bet expression for differentiation, but enhances responsiveness to cytokine stimulation to promote effector function. However, I found that OX40 is dependent on IL-2 receptor signaling to promote effector cytokine production. While the mechanism of OX40 signaling is not completely understood, this evidence indicates that OX40 signaling can promote differentiation via induction of cytokine and cytokine receptor expression. VII Chapter 1-Introduction The broad goal of my research is to understand how CD4 T cell effector function is regulated during an immune response. Specifically, I am interested in how engagement of the tumor necrosis factor receptor (TNFR) family member CD134 (OX40) regulates survival and differentiation during CD4 T cell activation. To appreciate the influence of OX40 on CD4 T cells, it is important to first understand that CD4 T cells play a central role in coordinating the host's innate and adaptive immune response to infectious agents. CD4 T cells enhance both innate and adaptive immune cell effector function to destroy pathogens, and are conversely able to inhibit effector function when the pathogen has been cleared. CD4 T cells in tum receive activation, survival, and differentiation signals at each stage of an immune response that influence the decision to respond, and how to respond, to a foreign agent. Members of the TNFR family are emerging as key mediators of effector CD4 T cell development. In this thesis, I will address the role of OX40 in promoting accumulation of effector CD4 T cells, and will discuss how OX40 influences survival and differentiation during effector cell development. 1.1 Development ofeffector and memory CD4 T cells The CD4 T helper cell compartment of the immune system plays an important role in the adaptive immune response to infectious agents, as well as contributing to autoimmune disease and anti-tumor immunity. Activated antigen-specific CD4 T cells release cytokines or directly interact with phagocytic cells such as macrophages to help destroy intracellular pathogens. Similarly, CD4 T cells also help B cells and CD8 T cells in their responses to antigen (3, 4).