Modulation of NK Cell Function with Agonistic -CD137 Antibodies during MCMV Infection Dahn Hahm A thesis submitted to the Faculty of Graduate and Postdoctoral Studies in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Microbiology and Immunology Department of Biochemistry, Microbiology and Immunology Faculty of Medicine University of Ottawa © Dahn Hahm, Ottawa, Canada, 2017 ABSTRACT The Tumor Necrosis Factor Receptor Superfamily (TNFR) is responsible in regulating a myriad of physiological function including the regulation of the immune system. Among the members include CD137 (4-1BB), an inducible costimulatory receptor known for its potent activation, proliferation, and survival effects on T cells. Stimulation of NK cells with agonistic -CD137 antibodies are known to increase IFN- production and proliferation in NK cells as well as increase efficacy of anti-tumor responses. However, NK cell death has also been seen in certain circumstances, although the mechanism remains to be determined. In vitro stimulation of NK cells revealed that -CD137 induced NK cell death occurs through both TNFR1 and TNFR2, although the action of TNF- and TNF- remain uncertain. Death was independent of other cytotoxic mechanisms such as granzyme/perforin, Fas-Fas ligand, and TRAIL. During MCMV infection, -CD137 induces NK cell death during the early phase of infection reducing viral resistance. This causes increased viral proliferation which drives NK cell proliferation, likely through Ly49H-m157 interactions, to high levels by day 4 of infection. The use of -CD137 as a tumor therapeutic is promising with several applications undergoing clinical trials. However, my results raise concern of other effects including the depletion of NK cells. This may cause a temporary impairment in immune function against pathogenic infections and a compensatory reaction of NK cell proliferation, both of which may cause damage to the host. However, with proper co- stimulation or co-treatments, this impairment may be overcome and prevent adverse effects in patients. ii ACKNOWLEDGEMENTS I would like to thank Dr. Seung-Hwan Lee for his guidance and amazing patience. He has always provided generous amounts of time to meet, ask questions and concerns. His supportive and encouraging approach to teaching has always been a great help and I have learned a great deal under his supervision. Thanks to my Thesis Advisory Committee members Dr. Subash Sad and Dr. Paul MacPherson for their advice and suggestions. I would like to thank all my laboratory members for all their help. Alaa, Saeedah, and Jun have helped tremendously with some of the more laborious experiments. They were always willing to offer help, suggestions, and exchange ideas, for which I am very grateful. Special thanks to Dr. Tania Watts and Dr. Robert Mittler for providing the -CD137 agonistic antibody 3H3 to use in my project. Finally, thanks to my family that have always encouraged me to pursue my studies. I am grateful for their continuing support. iii TABLE OF CONTENTS ABSTRACT .......................................................................................................................................... ii ACKNOWLEDGEMENTS .................................................................................................................. iii LIST OF ABBREVIATIONS.............................................................................................................. vii LIST OF FIGURES .............................................................................................................................. ix LIST OF TABLES ................................................................................................................................ xi 1. INTRODUCTION ......................................................................................................................... 1 1.1 NK cells ..................................................................................................................................... 1 1.1.1 NK Cell Function ................................................................................................................... 3 1.1.2 Tumor Therapy using NK Cells ............................................................................................ 5 1.2 TNF and TNFR Superfamily ..................................................................................................... 9 1.2.1 TNF and TNFR Function....................................................................................................... 9 1.2.2 TNFR Signaling pathway .................................................................................................... 12 1.2.3 CD137 (4-1BB) ................................................................................................................... 13 1.2.4 Agonistic Antibodies as Tumor Therapies .......................................................................... 20 1.3 Cytomegalovirus ...................................................................................................................... 24 2. HYPOTHESIS ............................................................................................................................. 29 3. MATERIALS AND METHODS ................................................................................................ 30 3.1 Mice ......................................................................................................................................... 30 3.2 Preparation of Agonistic anti-CD137 antibodies ..................................................................... 30 3.3 MCMV ..................................................................................................................................... 31 3.4 MCMV Infection with -CD137 stimulation .......................................................................... 32 3.5 Poly(I:C) .................................................................................................................................. 32 3.6 Cell Isolation ............................................................................................................................ 32 3.6.1 Splenocyte Isolation............................................................................................................. 32 3.6.2 Liver Lymphocyte Isolation ................................................................................................ 33 3.6.3 PBMC Isolation ................................................................................................................... 33 3.7 MCMV Plaque Assay .............................................................................................................. 33 iv 3.8 Cell Staining and Flow Cytometry .......................................................................................... 34 3.9 RNA Isolation .......................................................................................................................... 34 3.10 cDNA Synthesis ...................................................................................................................... 35 3.11 qPCR ........................................................................................................................................ 35 4. RESULTS .................................................................................................................................... 36 4.1 CD137 Expression on NK cells ............................................................................................... 36 4.1.1 CD137 expression on NK cells is induced upon cytokine activation with IL2, 15, 12, and 18 ............................................................................................................................................. 36 4.1.2 CD137 expression on NK cells is dependent on cytokine concentration ............................ 39 4.2 NK cells have increased IFN- production on day 1 and day 2 post -CD137 stimulation .... 39 4.3 -CD137 mediated NK cell death ........................................................................................... 45 4.3.1 -CD137 stimulation induces NK cell death on day 2 in vitro ........................................... 45 4.3.2 -CD137 induced NK cell death is independent of ADCC and intrinsic to NK cells ......... 48 4.3.3 -CD137 induced NK cell activation and death have identical thresholds ......................... 52 4.3.4 -CD137 induced NK cell death is not dependent on TNF-, TRAIL, Fas ligand, or activating cytokines ............................................................................................................................. 55 4.3.5 TNFR1 and TNFR2 double knockout NK cells are resistant to -CD137 induced death .. 59 4.4 In vivo -CD137 stimulation on NK cells during poly(I:C) induced inflammation ............... 62 4.4.1 TNF- and TNF- mRNA levels are unchanged in the spleen at 18 and 48 hours post injection with poly(I:C) ....................................................................................................................... 62 4.4.2 -CD137 stimulation decreases NK proportions in spleen, liver, and blood ...................... 65 4.4.3 -CD137 treatment increases TNFR2 expression on NK cells upon treatment with poly(I:C) ............................................................................................................................................. 68 4.5 Effects of in vivo -CD137 stimulation on the resistance against