CD200-CD200R Interaction 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 Fatemeh Talebian Program: Integrated Biomedical Science Graduate Program (IBGP) * * * * * The Ohio State University 2012 Dissertation Committee: Professor Xue-Feng Bai, Advisor Professor Amy Lovett-Racke Professor Ramish Ganju Professor Sujit Basu Abstract CD200 is a member of the Ig super family (IgSF) of proteins. It is expressed on cell surface of a variety of normal cells including lymphoid cells and some lineages of cancer cells such as melanoma and ovarian cancer cells. CD200 functions through engaging its specific receptor, CD200R. CD200R is also an IgSF protein, with an inhibitory intracellular NPXY signaling motif. CD200R has a restricted pattern of expression and is mainly detected on cells of the myeloid lineage. CD200-CD200R interaction inhibits the function(s) of myeloid cells. Myeloid cells are the first cells recruited by tumors and are essential in the regulation of tumor initiation, establishment, progression and metastasis. Tumor associated myeloid cells (TAMCs) are also known to inhibit activation and effector functions of T cells. Therefore, we hypothesized that CD200-CD200R interaction affects tumor formation, metastasis and tumor immunity via inhibiting TAMC functions. The goals of this dissertation thesis are three fold: 1) To investigate CD200/CD200R expression in the tumor compartments; 2) To determine the role of CD200-CD200R interaction in tumor formation and metastasis and 3) To determine if targeting CD200R is a feasible approach for cancer immunotherapy. We investigated CD200/CD200R expression on myeloid cells and T cells under various conditions. We have found that myeloid cells constitutively express CD200 and CD200R and upregulate both these cell surface molecules when activated. Naive T cells do not express CD200 and CD200R. When activated, T cells upregulate CD200 ii expression dramatically without upregulating CD200R. In the tumor microenvironment, myeloid cells express high levels of both CD200 and CD200R. Tumor infiltrating T cells express high levels of CD200, while their expression of CD200R is barely detectable. We next studied the impact of tumor expression of CD200 on tumor formation and metastasis, using the CD200-positive and CD200-negative B16 melanoma model. Subcutaneous injection of CD200-positive B16 melanoma cells inhibited tumor formation and growth in C57BL/6 mice but not in Rag1-/-C57BL/6 mice. However, i.v. injection of CD200-positive B16 melanoma cells dramatically inhibited tumor foci formation in the lungs of both C57BL/6 and Rag1-/-C57BL6 mice. Flow cytometry analysis revealed higher expression of CD200R in lung Gr1+ myeloid cells than in peripheral myeloid cells. In vivo depletion of Gr1+ cells dramatically inhibited tumor foci formation in the lungs. In addition, treatment with tumor antigen specific CD4 and CD8 T cells or their combination yielded a survival advantage for CD200 positive tumor bearing mice over mice bearing CD200-negative tumors. Analysis of microarray data from human cancer patients revealed that patients with CD200hi tumors have better prognosis and longer survival time. To test if CD200R is a suitable target for cancer immunotherapy, we first generated CD200R-/- mice and found that CD200-positive melanoma cells grow and metastasize progressively in CD200R-/- mice but not in WT mice. Stimulation of CD200R with an agonistic antibody dramatically inhibited lung metastasis of CD200- iii negative melanoma. Use of monoclonal agonistic CD200R antibodies inhibited tumor growth and improved survival time in established tumor models. Finally we found that many cancer cells derived from the myeloid lineage also express CD200R and they are more susceptible to CTL lysis. Blocking CD200 using antibody or knock down of CD200 gene expression in CTL significantly reduced CTL destruction of CD200R- positive cancer cells. Analysis of microarray data of human myeloma revealed that patients with CD200Rhigh cancer cells had lower relapse rates and a longer survival time. Taken together, we have found that in the tumor microenvironment there are highly significant expressions of CD200 and CD200R. CD200-CD200R interaction is broadly involved in regulating tumor formation, metastasis and tumor immunity. Targeting CD200R may be a novel approach for the immunotherapy of human cancer. iv Dedication To my Mother, Zahra Taghipour, who gave me life and taught me to have dreams and see the beauty in life. To her who was, is and will be my inspiration at every bend and curve I encounter in life. To my Father, Mohammad Ali Talebian, whose greatest dreams have always been the success of his children. Knowing the glowing happiness that would light up your faces when I finished this work was one of the strongest driving forces when times got tough. To My Husband, Bizhan Matin, who has been my rock, who has kept me going and always been positive and encouraging through the rough patches and the good ones too. It truly would not have been possible without you. And to my Babies, Mehraneh and Mohammad, who make me realize how valuable life is every morning and how blessed I am every evening. v Acknowledgments It is with profound gratitude that I would like to acknowledge the following people who have been instrumental in my achieving my goal of PhD. Dr. Xue-Feng, My mentor and PI, for giving me a place in his lab to learn and refine my skills, and become a better scientist. Jin-Qing Liu for her assistance with experiments and sharing her experience. Zhenzhen Liu, my lab partner, for her friendship, lively discussions, and assistance with experiments and training. It was a much better journey because we were able to share it. Christine Kerr, for her support and friendship that carried me through some tough times. It would have been much more difficult without her. Dr. Virginia Sanders, for her support, her encouragement and for truly being there for us as students when we needed an advocate. Dr. Amy Lovett-Racke for her advice and her support. You have been Immensely helpful and hopefully will be a lifelong collaborator and colleague. Dr.Sujit Basu, a special thanks to you. Because of everything you have done for me and your unwavering support. Thank you for believing in me and thank you for your time and your refreshing attitude toward a scientist’s life. Dr. Ramesh Ganju, Thank you for your time and purposeful scrutiny. I reserve my deepest appreciation and gratitude for my family, friends and loved ones who gave of their unlimited stores of love and support. Thank you for reminding me “I Can Do It”. Thank you Hassan and Atefeh for bieng the best siblings anyone ever had. For always believing in me. Thank you Bizhan, for I might not have gone the distance without your support. Last but certainly not least, I thank my mother and father, Zahra and Mohammad, for everything you have given me, all you have done for me, and all you still do. Thank you for all the meaning you have infused into my life and all that you keep on giving. vi Abbreviations cDNA Complementary DNA IgSF Immunoglobulin Super Family Kb Kilo bases TLR Toll-like receptor NLR NOD-like receptor ROS Reactive Oxygen Species RNS Reactive Nitrogen Species ITIM Immunoreceptor Tyrosine-based Inhibitory Motif ERK Extrsacellular signal regulated Kinase JAK Janus Kinase/Just another kinase JNK C-Jun N-terminal Kinase DOK Downstream of Kinase CNS Central Nervous System PNS Peripheral Nervous System NOS Nitric Oxide Synthase AD Alzheimer’s Disease MHC Major Histocompatibility Complex ICAM Intracellular Adhesion Molecule CIA Collagen Induced Arthritis RA Rheumatoid Arthritis vii vCD200 Viral CD200 B-CLL B cell Chronic Lymphoproliferative Leukemia Hu-SCID Human Severe Combined Immunodefeciency NK Natural Killer AML Acute Myeloid Leukemia mRNA messenger RNA DLN Draining Lymph node TAMC Tumor Associated Myeloid Cells MDSC Myeliod Derived Suppressor Cells TAM Tumor Associated Macrophages GM-CSF Granulocyte Macrophage Colony Stimulating Factor STAT Signal Transducers and activators of Transcription IMC Immature Myeloid Cells TIL Tumor Infiltrating Lymphocytes CTL Cytotoxic T Lymphocyte TCR T-Cell Receptor ELISA Enzyme Linked Immunosorbent Assay LPS Lipopolysaccheride IL Interleukin TNF-α Tumor Necrosis Factor- α IFN-γ Interferon-γ viii ALL Acute Lymphocytic Leukemia PCM Plasma Cell Myeloma MCL Mantle Cell Lymphoma BCLD B cell Lymphoproliferative Disease ix VITA Born: September 1979 Kansas City, Kansas 1996-1998: University of Illinois at Chicago (UIC), Chicago Illinois Bachelor’s of Art: Major 1: Biochemistry Major 2: English Literature 1998-2002: Tehran University, College of Sciences, Tehran Iran Department of Cellular and Molecular Biology; Bachelor’s of Science: Cellular & Molecular Biology 2003-2007: MS Society of Iran Research Assistant in Project: Immunotherapy with Mesenchymal Stem Cells in MS patients 2003-2006: Tehran University of Medical Sciences (TUMS), Tehran, Iran Department of Immunology Master’s of Science: Medical Immunology No.1 in MSc Entrance Exam 2007-2012: Ohio State University, Columbus Ohio USA Department of Pathology Doctor of Philosophy: Integrated Biomedical Sciences Fields of Study: Cancer Biology, Immunology, Molecular Biology, Bioinformatics, immunohistochemistry Publications ¾ F. Talebian, J.Q. Liu, Z. Liu M. Khattabi, Y. He, R. Ganju, X.F. Bai. Melanoma cell expression of CD200 inhibits tumor formation and lung metastasis via inhibition of myeloid cell functions. PLoS One, 2012, 7(2):e31442. ¾ Fatemeh Talebian and Xue-Feng Bai. The role of tumor expression of CD200 in tumor formation, metastasis and susceptibility to T lymphocyte adoptive transfer therapy. Oncoimmunology, 2012 In Press ¾ L.Wang, F. Talebian, J.Q. Liu, M. Khattabi, L.Yu, X.F. Bai Tumor Associated Myeloid Cell-Derived IL-10 Mediates Evasion of Immunotherapy by Cytotoxic T Lymphocytes. Scandinavian Journal of Immunology. 2012, 75: 273-281. x ¾ L. Wang, JQ Liu, F. Talebian, H.Y. El-Omrani, M. Khattabi, L.Yu, X.F.