The Critical Role of Cd4+ Th Cells in Cd8+ Ctl Responses

THE CRITICAL ROLE OF CD4+ TH CELLS IN CD8+ CTL RESPONSES AND ANTI-TUMOR IMMUNITY A Thesis Submitted to the College of Graduate Studies and Research In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy In the Department of Pathology and Laboratory Medicine University of Saskatchewan Canada By Channakeshava Sokke Umeshappa © Copyright Channakeshava Sokke Umeshappa. April 2012. All rights reserved. PERMISSION TO USE In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Department of Pathology and Laboratory Medicine Room 2841, Royal University Hospital, 103 Hospital Drive, University of Saskatchewan Saskatoon, Sask., S7N 0W8 i ABSTRACT The goal of this body of research was to elucidate the mechanism by which CD4+ T cells provide help for CD8+ cytotoxic T lymphocyte (CTL) responses in different immunization types. The establishment of diseases, such as chronic infections and cancers, is attributed to severe loss of or dysfunctions of CD4+ T cells. Even in acute infections, CD4+ T cell deficiency leads to poor memory responses. While the role of CD4+ T cells is being increasingly appreciated in these diseases, the timing and nature of CD4+ T help and associated molecular mechanisms are not completely understood. Growing evidence suggests that, depending on the type of infections or immunizations, the requirements of CD4+ T cells can vary for optimal CD8+ CTL responses. In order to understand the modulatory effects of CD4+ T cells for optimal CD8+ CTL responses, two distinct immunization types were chosen. These include: 1) non-inflammatory dendritic cell (DC) immunization, which fails to provide inflammatory/danger signals; and 2) inflammatory adenovirus (AdV) immunization, which provides profound inflammatory/danger signals. This allowed us to study CD4+ T cell’s participation under different inflammatory conditions. The studies described in Chapters 2 and 3 of this thesis were performed to further understand the concept of how CD4+ T cells mediate optimal CD8+ CTL responses. This has been called the “new dynamic model of CD4+ T helper – antigen (Ag)-presenting cells (Th- APCs),” proposed in 2005 by our laboratory. The study described in Chapter 2 shows that Th- APCs participate not only in augmenting CTL-mediated immune responses, perhaps during early phase, but also in regulating cellular immunity, perhaps during a later phase. Through enhanced IL-2, CD80 and CD40L singnaling, and weaker peptideMHC I (pMHC) signaling, Th-APCs stimulated naïve CD8+ T cells to differentiate into effector CTLs, capable of developing into, central memory CTLs. Th-APC-stimulated CD4+ T cells behaved like Th cells in function, augmenting the overall magnitude of CTL responses. In contrast, Th-APCs were able to kill DCs and other Th-APCs, predominantly through perforin-mediated pathway. The experiments described in Chapter 3 revealed a novel co-operative role of cognate Th-CTL interactions, contrary to previously known immune-regulatory mechanisms among Th-Th or CTL-CTL interactions. In our experiments, Th cells, via CD40L, IL-2, and acquired pMHC-I signaling, enhanced CTL survival and transition into functional memory CTLs. Moreover, RT-PCR, flow cytometry and western blot analysis demonstrate that increased survival of Th cell-helped CTLs is matched with enhanced Akt1/NF-κB activation, down-regulation of FasL and TRAIL, and ii altered expression profiles with up-regulation of prosurvival (Bcl-2) and down-regulation of proapoptotic (NFATc1, Bcl-10, Casp-3, Casp-4, Casp-7) genes/ molecules. Finally, helped CTLs were also able to induce protection against highly metastasizing tumor challenge, explaining why memory CTLs generated under cognate Th1’s help show survival and recall advantages. The studies in Chapter 4 showed how the precursor frequency (PF) of CD8+ T cells impacts CD4+ T helper requirements for functional CTL responses. At endogenous PF, CD4+ T helper signals were necessary for both primary and memory CTL responses. At increased PF, CD4+ T help, and its CD40L but not IL-2 signal became dispensable for primary CTL responses. In contrast, memory CTL responses required CD4+ T cell signals, largely in the form of IL-2 and CD40L. Thus, these results could impact the development of novel immunotherapy against cancers, since their efficacy would be determined in part by CD4+ T help and CD8+ T cell PF. Finally, the study showed the importance of CD4+ T cells for multiple phases of AdV transgene product-specific CTL responses. These include: a) cognate CD4+ T cells enhanced CTL responses via IL-2 and CD40L signaling during primary, maintenance and memory phases; b) polyclonal CD4+ T environment enhanced the survival of AdV-specific CTL survival, partially explaining protracted CTL contraction phase; and c) during the recall phase, the CD4+ T environment, particularly memory CD4+ T cells, considerably enhanced not only helped, but also unhelped, memory CTL expansion. Thus, these results suggest the participation of both cognate and polyclonal CD4+ T cells for multiple phases of AdV-specific CTLs. Taken together, the current work delineated the critical roles of CD4+ T cells in different stages of CTL responses and in the development of anti-tumor immunity. The results presented here will significantly advance our current understanding of immunity to cancers, autoimmunity and chronic infections, since pathogenesis of these diseases is largely determined by CD4+ T helper functions. As most immunization procedures use the principle that is based on functions of memory cells, the knowledge gained from this work will also have a major impact on designing vaccines against intractable diseases, including cancers and chronic infections. Moreover, in advanced tumors, vaccines developed using this knowledge may act synergistically with other cancer treatments such as irradiation, chemotherapy and microsurgery, minimizing their side effects and prolonging the lives of patients. iii ACKNOWLEDGEMENTS It would have been impossible to complete my doctoral thesis work without the help and support of the kind people around me, to only some of whom it is possible to give particular mention here. I would like to express sincere acknowledgement to my supervisor, Dr. J Xiang, whose mentorship allowed me to develop not only independent research skills, but also organizational and laboratory discipline skills required for every researcher. The good advice and encouragement of graduate chair and committee member, Prof. M Qureshi, have been invaluable my career, for which I am extremely grateful. Earnest thanks to all my supervisory committee, Drs. J Blondeau (Dept Head), A Saxena, Q Xu and Q Liu for their timely guidance throughout my program. Sincere thanks also to former Dept Head, Dr. J Krahn, for his timely guidance. Special thanks also to all my course instructors, Drs, V Gerdts, V Misra and P Pawha, and my Master’s supervisor, Dr. KP Singh, whose influences further moulded my research career. Thanks to all the past and present members of my laboratory and Saskatchewan Cancer Agency (SCA) for creating good working atmosphere. Special thanks to vice president of SCA, Dr. S Carlsen, for providing excellent research facilities at the center. Sincere thanks to Mrs. Roopa, who came helpful in most of the tedious experiments. Her personal support in all my endeavors as my wife at all times is unforgettable. I also express my gratitude to Dr. A Freywald for helping with manuscript editing and M Boyd for technical help with the flow cytometry. I sincerely acknowledge the financial and academic support of the U of S. Sincere thanks to the CGSR for Dean’s, Herb R and Marian H Clark and John Larson Cancer Research scholarships, the CIHR for Vanier CGS, and UCACS for reviewing animal protocols. I sincerely thank B Trost, Drs. R Cooley and R Chibbar for helping with Vanier CGS application. Sincere gratitudes also to Drs. T Vendan, D Tannis and S Parker, and Mr. J Eley and Mrs. L Hargreaves for their invaluable help with the development of my professional and leadership skills. The years spent in Saskatoon would not have been as feel-at-home without my friends, including Naveen, Kiran, Srinivas, Prabhakar, Srikanth, Naidu, Manju, Shankar, Sunitha, Vijay, Sudhakar, and/or their families. I am also thankful to Drs. RK Sharma, R Chibbar, Sujatha and R Prasad for their kind support. I sincerely thank Sateesh and family for their support and encouragement throughout my doctoral program. Last, but by no means least, I thank sincerely also my family, especially my mother Jayamma, who instilled the principle of hard work, moral values and ethics throughout my life and laid the foundation for my career. iv DEDICATED TO My family members for their selfless love and continuous encouragement And All the animals which sacrificed their valuable lives for the welfare of mankind v TABLE OF CONTENTS PERMISSION TO USE………………………………………………………………….………...i ABSTRACT……………………………………………………………………………….……...ii ACKNOWLEDGEMENTS……………………………………………………………….….......iv DEDICATION…………………………………………………………………………….………v TABLE OF CONTENTS………………………………………………………………….……...vi LIST OF TABLES………………………………………………………………………….……xii LIST OF FIGURES………………………………………………………………………….….xiii LIST OF ABBREVIATIONS………………………………………………………………........xv 1.

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