The Roles of Interleukin-27 in Tumor Immunity DISSERTATION Presented in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the Graduate School of The Ohio State University By Zhenzhen Liu Program: Ohio State Biochemistry Program (OSBP) * * * * * The Ohio State University 2012 Dissertation Committee: Professor Xue-Feng Bai, Advisor Professor Lai-Chu Wu, Co-advisor Professor Pravin Kaumaya Professor Qianben Wang i ii Abstract Interleukin-27 (IL-27) is a member of the IL-12 family of cytokines. IL-27 is a heterodimer consisting of an IL-12 p40-related protein subunit, EBV-induced gene 3 (EBI3) and a p35-related subunit, p28. IL-27 is mainly produced by activated antigen presenting cells. It functions through engaging IL-27 receptor, which is expressed on a variety of immune cell types, including CD4+ and CD8+ T cells. Overexpression of IL-27 by tumor cells exerts potent anti-tumor activity through diverse mechanisms, in which CD8+ T cells were considered to be the main effector cells. However, the exact mechanisms by which IL-27 enhances anti-tumor CD8+ T cell response and leads to tumor rejection remain unclear. The impacts of IL-27 on the differentiation and activation of CD8+ T cells were studied by stimulating naïve tumor antigen-specific CD8+ T cells (P1CTL) with cognate P1A peptide in the presence and absence of IL-27. First, T cell proliferation and apoptosis were examined by thymidine incorporation and Annexin V/7-AAD staining, respectively. Second, expressions of activation and differentiation markers of T cells were analyzed by Real time PCR and Western Blotting. Third, cytokine production was evaluated by ELISA and flow cytometry. To investigate the in vivo roles of IL-27 in tumor immunity, mouse models involving tumor cells (J558 plasmacytoma and B16 melanoma) expressing IL-27 and IL-27-deficient (EBI3-/-) mice were used. To delineate the mechanisms by ii which IL-27 enhances antitumor CTL responses, tumor antigen specific CD8+ T cells were adoptively transferred into various genetically engineered mice with established tumors, and their in vivo proliferation and apoptosis were studied by CSFE staining and flow cytometry analysis. To examine the role of IL-27 in response and function of T regulatory cells, Anti-CD25 antibody was used to deplete Treg cells; IL-27 deficient or WT CD4+CD25+ Treg cells were adoptively transferred together with effector T cells into tumor-bearing mice; tumor establishment and metastases were evaluated by monitoring s.c. tumor growth and weighing lungs, respectively. Overall, we have uncovered four novel findings that can explain why IL-27 boosts antitumor CD8+ T cell responses: 1) IL-27 enhances the survival of activated tumor antigen specific CD8+ T cells both in vitro and in vivo. 2) IL-27 induces a unique memory precursor cell (MPC) phenotype in activated tumor antigen specific CD8+ T cells, which is characterized by up-regulation of SOCS3, Bcl-6, Sca-1 and IL-10. 3) IL-27 robustly induces IL-10 production by tumor antigen specific CD8+ T cells, which contributes to IL-27-mediated tumor rejection in vivo. 4) IL-27 inhibits the expansion and immunesuppressive ability of CD4+FoxP3+ T regulatory cells, resulting in more potent anti-tumor CTL responses. iii Our findings suggest that: 1) IL-27 has the potential to be used as an adjuvant to boost the efficacy of antitumor vaccines; 2) IL-27 can be used to culture tumor antigen-specific CTLs for adoptive transfer therapy of cancer patients. iv Dedication To my Parents, Lierong Chen and Xiancheng Liu, who gave me life, taught me to be independent and grateful, and support me to pursuit my dreams. To my Advisor, Dr. Xue-Feng Bai, who gave me the chance to continue my Ph.D. study in the U.S., brought me into the exciting world of tumor immunology, and taught me how to employ the power of immunology to do cancer research. His unconditional support, enthusiasm, and encouragement have been instrumental to the success of this project. His style of decision-making, problem-solving and taking responsibility for his students will also serve as my role model for my future career. To my Boyfriend, Andreas Schick, who has been my rock, who has kept me going and always been positive and encouraging through the rough patches and the good ones as well. v Acknowledgments It is with sincerest gratitude that I would like to acknowledge the following people who have been instrumental in helping me to achieve my goal of Ph.D. Dr. Jin-Qing Liu, for her assistance with experiments and sharing her experience. Dr. Lai-Chu Wu, a special thank to you. For your support, your encouragement, and for truly being there for me. Dr. Fatemeh Talebian, my lab partner, for her friendship, lively discussions, and assistance with experiments and training. It was a much better journey since we were able to share it. Dr. Pravin Kaumaya, for everything you have done for me. Thank you for believing in me when I was going through that tough time. Dr. Qianben Wang, for all your support and understanding, for the knowledge and encouragement for making a better future for myself. I am deeply grateful and appreciative for making me open my mind to my future capacity. Dr. Shulin Li, for his generousness providing me with Ad-IL-27, for his guidance on my Ph.D. study, and for his unhesitant sharing life experience with me. Dr. Kevin Foy and Megan Miller, for their helps and kindness that walked me through the darkness. I will always remember the time we were together in Dr. Kaumaya’s lab. Dr. Zhong Chen and Dr. Wen Yi, for their academic comments and suggestions on my Ph.D. research, for I might not have gone the distance without your guys support. Dr. Ying An and Dr. Li Ma, for their support and friendship that carried me through some tough times. It would have been much more difficult without them. I reserve my deepest appreciation and gratitude to my family, friends and loved ones who gave their unlimited stores of love and support. Thank them for reminding me “I Can Do It”. Last but certainly not least, I thank my mother and father, Lierong and Xiancheng, for everything they have given me, all they have done for me, and all they still do. Thank them for all the meaning they have infused into my life and all that they keep on giving. vi VITA Born: May 14th, 1983 Shiyan, Hubei Province, China 2001-2005: China Agriculture University, Beijing, China College of Animal Science and Technology Bachelor’s of Science Major: Animal Science and Technology 2005-2007: China Agriculture University, Beijing, China Department of Animal Genetics and Reproduction Master’s of Science Major: Animal Genetics 2007-2010: The Ohio State University, Columbus, Ohio USA Ohio State Biochemistry Program Master’s of Science Major: Biochemistry 2007-2012: The Ohio State University, Columbus, Ohio USA Department of Pathology Doctor of Philosophy Fields of Study: Cancer Immunology, Molecular and Cellular biology vii RESEARCH EXPERIENCE Research Lab Work Experience The Ohio State University, Department of Pathology/OBGYN Columbus, OH Graduate Research Associate in Cancer Immunology 2008 – Present Project 1: The roles of Interleukin-27 in tumor immunity Generated IL-27-overexpression tumor cell lines Conducted a variety of immuno-based and biochemical in vitro assays to test the effects of IL- 27 in the differentiation and function of tumor specific CD8+ cells Performed adoptive transfer of tumor specific CD8+ T cells to tumor bearing mice Analyzed the IL-27-induced immune responses to established local plasmacytoma (J558) and metastatic melanoma (B16) on various transgenic mouse models. Investigated the mechanisms by which IL-27 enhances antitumor CTL responses and leads to tumor regression Project 2: The interaction of CD200-CD200R in tumor immunity Examined CD200/CD200R expression on myeloid cells and T cells under various conditions Studied the impact of tumor expression of CD200 on tumor formation and metastasis, using the CD200-positive tumor cells, various transgenic mouse models, in vivo cell depletion, and adoptive cell therapy Discovered that high CD200 expression on human cancer cells may lead to long survival time through analyzing microarray data Project 3: Th17 and Treg responses in EBI3-deficient mice Characterized the dynamic changes of CD4+ T cell subsets in EBI3-/- mice with EAE disease Conducted in vitro and in vivo T cell proliferation assay to evaluate the suppressive effect of EBI3-/- Treg Project 4: Combination treatment with HER-2 and VEGF peptide mimics induces potent anti-tumor and anti- angiogenesis responses Designed and synthesized novel peptide mimics: HER2 and VEGF Evaluated the efficacy of the two peptide mimics as a combination treatment for breast cancer both in vitro and in vivo. China Agriculture University, Department of Animal Genetics Beijing, China Graduate Research Associate in Animal Genetics and Reproduction 2004 – 2007 Project 1: Molecular Mechanism of Chromium in Promoting Chicken Growth In order to understand the molecular mechanism by which chromium enhances the action of insulin and subsequently promotes chicken growth, I examined carcass traits, insulin and glucose in the serum, and mRNA expression of genes in the insulin signaling pathway, such as insulin, IR, IRS and IGF-1. Project 2: Sex-reversed Chickens Induced by Aromatase Inhibitor The purpose of this study is to understand the mechanism of avian sex determination and differentiation. Female chicken treated with aromatase inhibitor can ejaculate fully fertile spermatozoa. In addition, W sperms are present in sex-reversed chicken and they are able to reproduce healthy baby chickens. Project 3: Analysis of SNP Markers for Blue-shelled Gene in Chicken by PCR-SSCP In order to understand the cause of eggshell pigmentation biosynthesis, a bioinformatics approach was first used to blast the genes for enzymes involved in porphyrin pathway.