+ LYSOPHOSPHATIDIC ACID SIGNALING VIA LPA5 INHIBITS CD8 T CELL ACTIVATION AND CONTROL OF TUMOR PROGRESSION by SHANNON KUNIKO ODA B.A., Linfield College, McMinnville, 2004 A thesis submitted to the Faculty of the Graduate School of the University of Colorado in partial fulfillment of the requirements for the degree of Doctor of Philosophy Immunology Program 2013 This thesis for the Doctor of Philosophy degree by Shannon Kuniko Oda has been approved for the Immunology Program by David Riches, Chair John Cambier Laurent Gapin John Kappler Dennis Voelker Raul Torres, Advisor Date 1/22/13 . ii Oda, Shannon Kuniko (Ph.D., Immunology) + Lysophosphatidic Acid Signaling via LPA5 Inhibits CD8 T Cell Activation and Control of Tumor Progression Thesis directed by Associate Professor Raul Torres. CD8+ T lymphocytes are an important component of adaptive immunity, capable of specific identification of a foreign antigen, rapid proliferation, and cytotoxic activity to kill cells expressing the targeted antigen. T cell activation occurs via engagement of the T cell receptor (TCR) by its cognate antigen within the context of the MHC molecule and is modulated by co-receptors that function to either support or inhibit activation. The adaptive immune system, and T cells in particular, are important for their ability to recognize and eliminate nascent tumors in cancer immunosurveillance and tumor rejection. However multiple inhibitory mechanisms within the tumor microenvironment have been described that protect the tumor from T cells, including increased signaling of inhibitory co- receptors. Lysophosphatidic acid (LPA) is a lysophospholipid that is present at low nanomolar concentrations in the plasma of healthy individuals. It has been well documented that many cancers aberrantly produce LPA and the increased level of LPA has been shown to be beneficial to the tumor, promoting tumorigenesis, invasion, metastases, and vascularization. However the effects of elevated levels of LPA on the adaptive immune response have not been addressed. We demonstrate here that LPA inhibits CD8+ T cell activation in vitro and in vivo. Specifically, we show that CD8+ T cells express LPA receptor 5 iii (LPA5) that, in the presence of LPA, is able to inhibit TCR signaling and subsequent cell activation and proliferation. These data document that LPA is able to negatively regulate T cell activation and that LPA5 functions as an inhibitory T cell co-receptor. Finally, we have shown that transfer of LPA5- deficient T cells is able to abate tumor progression. We propose that lysophospholipid signaling is an additional protective mechanism of the tumor microenvironment to avoid anti-tumor CD8+ T cell immunity. The form and content of this abstract are approved. I recommend its publication. Approved: Raul Torres iv ACKNOWLEDGEMENTS I would first like to thank my mentor, Dr. Raul Torres, for his guidance, enthusiasm, and tireless support for my research project and for my progression as a scientist. My committee members have also made major contributions to this project and served as a valuable source of mentors. I would like to thank the members of the R&R lab, especially Pamela Strauch, for technical assistance and helpful discussions. I have also been fortunate to have a variety of science experiences that influenced me to pursue a career in research, including work in the Lubchenco-Menge lab and the Mourich group at Sarepta Therapeutics. I would especially like to thank my parents, Stephen and Karen Oda, for their continued support throughout my lengthy education and wisdom for life events. I would also like to thank my sister, Adrienne Oda, for her support and motivation. Finally, I am indebted to my husband, Freddie Elchlepp, for his patience, love, and encouragement and I look forward to conquering new challenges with him. v TABLE OF CONTENTS CHAPTER I. INTRODUCTION AND BACKGROUND ........................................................ 1 The adaptive immune response ............................................................ 1 CD8+ T cells and cancer ........................................................................ 4 Lysophosphatidic acid ............................................................................ 5 Structure ....................................................................................... 6 Metabolism ................................................................................... 8 LPA signaling ......................................................................................... 10 LPA receptor 5 ....................................................................................... 12 LPA and cancer ..................................................................................... 13 LPA and immune cells ........................................................................... 15 Summary ................................................................................................. 18 II. MATERIALS AND METHODS ...................................................................... 20 Mice ........................................................................................................ 20 Calcium mobilization ............................................................................... 22 qPCR ...................................................................................................... 23 Flow cytometry ........................................................................................ 24 Lipid preparation ..................................................................................... 25 Transwell assay ...................................................................................... 27 In vitro T cell proliferation and activation assays .................................... 27 In vitro effector function assays .............................................................. 28 vi Generation of bone marrow-derived dendritic cells ................................ 30 In vivo SIINFEKL-BMDC stimulation ...................................................... 30 IL-2 ELISA ............................................................................................... 31 B16.cOVA tumor transfer ........................................................................ 31 In vivo cytotoxicity assay with tumor burden ........................................... 32 Statistical analyses ................................................................................. 33 III. LPA SIGNALING INHIBITS CD8+ T CELL ACTIVATION AND PROLIFERATION VIA LPA5 .............................................................................. 34 Introduction ............................................................................................. 34 Results .................................................................................................... 36 LPA signaling inhibits TCR-mediated calcium mobilization + in CD8 T cells via LPA5 ............................................................... 36 LPA does not influence chemotaxis in naïve T cells .................... 43 Increased LPA signaling inhibits proliferation, accumulation, and activation of OT-I T cells in vitro ............................................ 44 OTP treatment does not affect APC function and inhibits activation and proliferation to a lesser degree to a high affinity antigen .............................................................................. 52 LPA signaling modestly inhibits effector function in vitro ............. 54 Increased LPA signaling dampens proliferation and activation of CD8+ T cells in response to SIINFEKL-BMDC stimulation in vivo ......................................................................... 58 Conclusion .............................................................................................. 65 + IV. LPA5-DEFICIENT CD8 T CELLS EXHIBIT ENHANCED IMMUNE RESPONSES AND ANTI-TUMOR ACTIVITY ................................................... 69 Introduction ............................................................................................. 69 Results .................................................................................................... 70 vii Characterization of LPA5-deficient mice ....................................... 70 LPA5-deficient T cells show enhanced proliferation to SIINFEKL-BMDC in vivo .............................................................. 72 + LPA5-deficient CD8 T cells do not produce nor consume IL-2 differently .............................................................................. 76 –/– + Transfer of LPA5 OT-I CD8 T cells controls tumor growth in vivo ........................................................................................... 78 Single transfer of LPA5-deficient OT-I T cells is not sufficient to ablate B16.cOVA tumor ............................................ 83 –/– + LPA5 OT-I CD8 T cells accumulate within the tumor ............... 85 –/– LPA5 T cells kill target cells and produce intracellular cytokines similarly to wild-type T cells.......................................... 90 Conclusion .............................................................................................. 94 V. DISCUSSION AND FUTURE DIRECTIONS ................................................ 98 A model for LPA inhibition of CD8+ T cells ............................................. 98 LPA signaling in CD8+ T cells in vivo: implications for enhancing the CD8+ T cell response ......................................................................