The Role of Natural Killer Cells in the Context of Oncolytic Herpes Simplex Virotherapy for Glioblastoma Dissertation Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Christopher Allen Alvarez-Breckenridge Graduate Program in Integrated Biomedical Science Program The Ohio State University 2011 Committee: Dr. E. Antonio Chiocca MD, PhD “Co-Advisor” Dr. Michael Caligiuri, MD “Co-Advisor” Dr. Balveen Kaur, PhD Dr. Susheela Tridandapani, PhD Copyright by Christopher Allen Alvarez-Breckenridge 2011 Abstract It is controversial as to whether the host immune response hinders or improves the efficacy of oncolytic Herpes Simplex viral (oHSV) therapy of glioblastoma (GBM). Natural killer cells (NK) limit viral infections, and previous work suggests they may similarly attenuate virotherapy. Using both xenograft and syngeneic intracranial GBM tumor models, we used flow cytometry to evaluate the temporal pattern and phenotypic characteristics of NK cells present in the periphery and recruited to the site of oHSV infection. Within hours after infection and continuing through 72 hours following oHSV inoculation, NK cells were rapidly recruited to tumor bearing hemispheres. Moreover, these NK cells exhibited an activated phenotype, including enhanced CD69, CD62L, CD27, NKG2D, and Ly49D staining compared to vehicle treated mice. However, neither the number nor phenotype of peripheral NK cells was altered following oHSV infection. This robust NK response was confirmed to be detrimental to OV efficacy through the enhanced survival of NK depleted mice inoculated with oHSV compared to oHSV treated mice possessing NK cells. Interestingly, oHSV treated mice exhibited robust macrophage recruitment and activation at the site of infection. This was accompanied by the induction of macrophage/microglial associated inflammatory gene and protein expression, including CXCL9, CXCL10, CXCL11, iNOS, and TNF-α. However, when ii mice were depleted of their NK cells or IFN-γ knockout mice were used, their expression was abrogated. In vitro, human NK cells preferentially lysed oHSV-infected GBM in a cell contact, perforin, and DNAM-1 dependent manner. Fusion proteins were used to detect currently unknown ligands for the NK natural cytotoxicity receptors (NCR) and decipher the critical NK activating ligands that mediate this response. Following oHSV infection of a panel of GBM stem cells and cell lines, we detected robust up-regulation of ligands for NKp46 and NKp30. GFP expression was used to discriminate oHSV infected GBM cells and preferential NKp30/NKp46 ligand expression was found in the GFP+ population of cells. Additionally, blocking antibodies against either NKp30 or NKp46 abrogated NK mediated clearance of oHSV infected GBM, while antibodies against NKp44 did not inhibit killing. We have previously shown that immunomodulation with cyclophosphamide (CPA) and valproic acid (VPA) enhances oHSV efficacy. CPA administered prior to virus inoculation abrogated the oHSV induced NK and macrophage recruitment into the tumor at all time points tested compared to oHSV alone. Similarly, VPA treatment resulted in a decline in NK and macrophage recruitment at 6 and 24 hours post-oHSV; however, a robust increase at 72 hours-post-oHSV was seen, resembling the response seen with oHSV alone. VPA was also found to have a profound immunosuppressive effect on human NK cells in vitro. NK cytotoxicity was abrogated following exposure to VPA iii through down-modulation of cytotoxic gene expression and granzyme B protein levels. In addition, IFN-γ was suppressed in a Stat5/T-bet dependent manner. Collectively, these findings demonstrate that oHSV therapy for GBM is limited in part by a robust NK cell response mediated by specific NCRs, uncovering novel potential targets to enhance cancer virotherapy. Moreover, pharmacological co-therapies, such as CPA and VPA with oHSV, alter the host immune response to the virus albeit in differing ways. Future work will be needed to further define the nature of the innate immune response, how it coordinates downstream anti-tumor immunity, and how pharmacological agents can be optimized to modulate the host response to oHSV. iv Dedication This document is dedicated to those who have stood by my side on this long journey. In particular, I would like to thank my undergraduate advisor, Dr. Charis Eng, for her planting the idea of an MD/PhD; Dr. Gregory Jusdanis for the encouragement and support he has given me for so many years and ultimately on this quest to γνῶθι σεαυτόν; and to Dr. Lauren Walters—thank you for sticking with me! I would certainly not be here without the devotion of my parents, Carmen Alvarez- Breckenridge and David Breckenridge. This dissertation is a testament to their love, prayers, and support. I would like to thank my wife, Jennifer Alvarez-Breckenridge, for her love, unceasing support of my academic pursuits, and for always showing me that the glass is half full. Lastly, I would never have embarked on this road had I not experienced the call to medicine during my time in Lourdes. I dedicate this work and my future endeavors to Our Lady of Lourdes, and her son, Jesus Christ. v Acknowledgments I would like to thank my mentors Dr. E. Antonio Chiocca and Dr. Michael Caligiuri for all of their help and support during my PhD. In particular, I would like to thank them for the opportunity to pursue this project; for constantly challenging me on every step along the way in order to achieve my goal of becoming an independent scientist; and for empowering me to exceed beyond my self-constructed limits. I would also like to thank Dr. Jianhua Yu for being by my side throughout this journey and never showing the slightest bit of frustration despite the numerous questions I have asked him over the years. Without your help, I could have never taken this project off the ground—thank you! The entire Dardinger Laboratory, especially: Dr. Balveen Kaur, Dr. Sean Lawler, Rick Price, Kazue Kasai, Dr. Hiroshi Nakashima, Dr. Kazuo Okemoto, Amy Hasely, Jason Pradarelli, Jayson Hardcastle, Dr. Nina Dmitrieva, Dr. Kazuhiko Kurozumi. In addition, a special thank you to the Dardinger support staff, in particular Melinda Akins, Erica Chambers, and Louvenia Broadnax. To the entire Caligiuri Laboratory, especially: Susan McClory, Min Wei, Charlene Mao, Dr. Tom Liu, Dr. Rossana Trotta, Tiffany Hughes, Nick Zorko, Edward Briercheck, and Dr. Jason Chandler. vi To the Integrated Biomedical Graduate Program and the Medical Scientist Program for their support and guidance, particularly Dr. Alan Yates, Dr. Larry Schlesinger, Dr. Lawrence Kirschner, and Ashley Bertran. I would like also like to thank my collaborators for their help in this project: Dr. Soledad Fernandez, Dr. Susheela Tridandapani, Dr. Alessandro Moretta, Dr. Ofer Mandelboim, and Dr. Eric Vivier. I would also like to thank my committee members for constantly challenging me and fostering my scientific acumen. Lastly, I thank the NIH, AMA Foundation, and Ohio State’s Center for Clinical and Translational Science for their financial support. vii Vita 2001................................................................St. Charles Preparatory High School 2005................................................................B.S. Biology, The Ohio State University 2005................................................................B.A. Classics, The Ohio State University 2005-present...................................................Graduate Research Associate, Department of Neurological Surgery, The Ohio State University Publications 1. Alvarez-Breckenridge, C., Waite K.A., Eng, C. (2007) PTEN Regulates Phospholipase D and Phospholipase C. Hum. Mol. Genet., 16, 1157-1163 2. Haseley, A., Alvarez-Breckenridge, C., Chaudhury A.R., Kaur, B. (2009) Advances in oncolytic virus therapy for glioma. Recent Pat. CNS Drug Disc., 4 (1): 1-13 3. Alvarez-Breckenridge, C., Kaur, B., Chiocca, E.A. (2009) Pharmacologic and chemical adjuvants in tumor virotherapy. Chem Rev., 109 (7): 3125-40 viii 4. Yu, J., Mitsui, T., Wei, M., Mao, H., Butchar, J.P., Shah, M.V., Zang, J., Mishra, A., Alvarez-Breckenridge, C., Liu, X., Liu, S., Yokohama, A., Trotta, R., Marcucci, G., Benson, D.M., Loughran, T.P., Tridandapani, S., Caligiuri, M.A. (2011) NKp46 identifies an NKT cell subset susceptible to leukemic transformation in mouse and human. J Clin Invest., Epud ahead of print. Field of Study Major Field: Integrated Biomedical Science Program ix Table of Contents Abstract............................................................................................................................... ii Dedication........................................................................................................................... v Acknowledgments.............................................................................................................. vi Vita...................................................................................................................................viii List of Tables ................................................................................................................... xiv List of Figures................................................................................................................... xv Chapter 1: Introduction....................................................................................................... 1 Section 1—Current treatment approaches for malignant