The development of an in vivo humanized mouse model to investigate epstein barr virus infection and tumorigenesis

THE DEVELOPMENT OF AN IN VIVO HUMANIZED MOUSE MODEL TO INVESTIGATE EPSTEIN-BARR VIRUS INFECTION AND TUMORIGENESIS MIN ZIN OO (M.B., B.S.) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN COMPUTATION AND SYSTEMS BIOLOGY (CSB) SINGAPORE-MIT ALLIANCE NATIONAL UNIVERSITY OF SINGAPORE 2013 DECLARATION I hereby declare that this thesis is my original work and it has been written by me in its entirety I have duly acknowledged all the sources of information which have been used in the thesis This thesis has also not been submitted for any degree in any university previously MIN ZIN OO 25th March 2013 ACKNOWLEDGEMENT I am highly indebted to my thesis advisors A/P Paul MacAry (Immunology Program, NUS) and Prof Jianzhu Chen (SMART ID-IRG; Department of Biology, MIT) for their advice, funding support, and guidance throughout the course of the project from its conception until its end I would also like to thank my mentor Dr Maroun Khoury (Universidad de los Andes, Faculty of Medicine, Santiago, Chile) for his wonderful guidance during the initial stages of my project during his tenure at SMART-ID IRG as a research scientist The success of this research project and thesis would not be possible without the help and support of each and every one of my colleagues and lab mates in both the PAM and SMART labs I would like to express special thanks to Ms Zhenying Song who taught me the basic research skills; Dr Adrian Sim who has consistently been a good friend, a comrade, and an academic counsel; Mr Wei Jian Tan who has never turned down my call for help during the hectic period of the final stages of my project; Ms Fatimah Bte Mustafa and Ms Chien Tei Too for their administrative support and relentless efforts to make everybody happy during the troughs of our mood; Ms Siew Chin Loh for her friendly technical support with flow cytometry; Ms Lan Hiong Wong for taking excellent care of our laboratory mice; and Ms Hooi Linn Loo for her technical and administrative assistance i I would like to express my gratitude to Singapore-MIT Alliance and SMART ID-IRG for funding support; Emeritus Prof Chan So Ha and Ms Nalini Srinivasan for providing us with the B95-8 strain of the Epstein-Barr virus and quantitative PCR viremia assessment system; A/P Jerry Chan (DukeNUS) and his research team who kindly provided us with human fetal livers; A/P Chng Wee Joo for his time for discussion and expert comments and opinion on my project; and lastly Ms Carol Cheng, Ms Juliana Chai, Ms Hong Yanling and Ms Chua Lay Peng for their administrative support throughout my PhD candidature at SMA ii TABLE OF CONTENTS Acknowledgement i Table of Contents iii Summary vii List of Tables ix List of Figures x List of Abbreviations xii Chapter : INTRODUCTION 1.1 Epstein-Barr virus life cycle 1.2 EBV and the immune system 1.2.1 1.2.2 CD4+ helper T cell responses .6 1.2.3 EBV and NK cells 1.2.4 EBV and the human cytokine system 1.2.5 Anti-EBV antibody responses 11 1.2.6 1.3 CD8+ cytotoxic T cell (CTL) responses .5 Immune evasion mechanisms of EBV .12 Latent EBV infection 15 1.3.1 1.3.2 Latency I or ‘EBNA1 only’ program .18 1.3.3 Latency II or the ‘Default’ program .19 1.3.4 Latency III or the ‘Growth’ program .20 1.3.5 Other latency programs 21 1.3.6 Regulation of EBV latency 22 1.3.7 1.4 EBV-encoded RNAs (EBERs) 18 The persistence of EBV 23 EBV-associated malignancies and the role of EBV in tumorigenesis 27 1.4.1 1.4.2 EBV and Hodgkin’s lymphoma .29 1.4.3 1.5 EBV and Burkitt’s lymphoma 27 EBV and Immunodeficiency-related lymphoproliferative disease 30 EBV-associated Post-transplant Lymphoproliferative disease (PTLD) 32 iii 1.5.1 1.5.2 The role of lytic EBV infection .33 1.5.3 The role of cytokines .34 1.5.4 The role of anti-EBV antibodies 35 1.5.5 1.6 Post-transplant lymphoproliferative disease 32 The role of T cell immunity .36 Humanized mouse models of Epstein-Barr virus infection 38 1.6.1 Early mouse models of EBV-associated lymphoproliferative disease 39 1.6.2 Humanized mouse models of EBV infection and lymphoproliferative disease .40 1.6.3 Differences in the humanized mouse strains in previous studies 43 1.6.4 Differences in virus strains used in previous humanized mouse studies 44 1.6.5 Controversy of the published models 45 1.6.6 Current concepts about the role of human B cells in EBVassociated lymphoproliferative diseases based on animal models 46 CHAPTER : MATERIALS AND METHODS .51 2.1 Isolation of hemopoietic stem and progenitor cells (HSCs) from human fetal liver 51 2.2 Establishment of the humanized mouse .52 2.3 Epstein-Barr virus culture, virus titer assay and in vivo infection .52 2.4 Flow cytometric immunophenotyping 53 2.5 Histopathology .56 2.6 Viremia assay .57 2.7 Multiplex cytokine assay .60 2.8 Treatment of EBV-infected mice with Rituximab .61 2.9 Secondary adoptive transfer tumor model 61 2.10 Statistical analysis 62 CHAPTER : RESULTS AND INTERPRETATION .64 3.1 Establishment of the humanized mouse model 64 3.1.1 Experimental scheme for development of a humanized mouse model of EBV infection 64 iv 3.1.2 3.1.3 3.2 Isolation of hemopoietic progenitor cells from the human fetal liver 65 In vivo infection of the humanized mouse model with EpsteinBarr Virus .68 Characterization of the humanized mouse model of EBV infection 71 3.2.1 Demonstration of virus replication in the plasma 72 3.2.2 Evidences of human cell responses in the peripheral blood 76 3.2.3 Peripheral blood activation marker profile 81 3.2.4 Gross pathological examination of fatal systemic lymphoproliferative lesions in EBV-infected humanized mice 85 3.2.5 Histopathological study of the systemic lesions 87 3.2.6 Significant T cell expansion with higher CD8:CD4 ratios in the spleens of infected mice 90 3.2.7 Activation marker profile in the spleens of infected mice .92 3.2.8 Marked expansion of CCR5+ Th1 cells in infected mice with poor memory T cell development 95 3.2.9 Human cytokine response in infected humanized mice 99 3.2.10 Distribution of human B cell subsets in the spleen 105 3.2.11 Clonality of splenic B cells 107 3.3 Demonstration of a pivotal role of human B cells in the pathogenesis of disease in EBV-infected mice 111 3.3.1 3.3.2 Complete clearance of viremia after B cell depletion 115 3.3.3 Immune correlates of rituximab-treated mice 117 3.3.4 3.4 Experimental scheme of Rituximab treatment of EBV-infected mice 112 Survival benefit of rituximab treated mice – Evidence that B cells are the major driving force in the pathogenesis of the disease 119 Experimental proof of the malignant nature of human B cells in EBVinfected mice 122 3.4.1 Experimental scheme of secondary adoptive transfer experiments 123 3.4.2 Life span of secondary mice 124 3.4.3 Appearance of systemic fatal tumors in secondary mice which received unfractionated splenocytes 126 v 3.4.4 Activated human cells in the spleens of secondary mice which received unfractionated splenocytes 128 3.4.5 The B cell population alone is both necessary and sufficient for tumorigenesis 131 3.4.6 Tissue lesions in secondary B cell tumor mice 134 3.4.7 Histopathological examination of secondary tumors .136 3.4.8 Demonstration of latency III pattern of EBV gene expression in secondary tumors 139 3.4.9 Demonstration of cellular proteins in secondary tumors .140 CHAPTER : DISCUSSION 144 4.1 Disease phenotype of the EBV infection in our humanized mouse model 145 4.2 Human immune responses in our mouse model 146 4.3 Proof of tumorigenicity of human B cells in EBV-infected mice .152 4.4 Future directions 155 REFERENCES .158 vi SUMMARY Epstein-Barr virus (EBV) is a human-specific B lymphotrophic gammaherpesvirus which causes life-long latent infection Although latent infection is mainly asymptomatic and thus inconsequential in the majority of cases, EBV latency is also consistently associated with many aggressive hematological and solid tissue malignancies of diverse tissue origins in different populations throughout the world The ubiquitous presence of the virus in more than 90% of the adult human population and the benign nature of latent infection make the pathogenic role of EBV questionable and thus many critics claim that the virus is simply an innocent bystander in most of the associated malignancies and does not play a direct causal role in tumorigenesis Clinical evidences from post-transplant lymphoproliferative diseases have implied a direct causative role of EBV in pathogenesis However, requirement for additional factors including signaling components and cytokine support from T lymphocytes were also identified as essential in the rapidly fatal spectrum of lymphoproliferative disease In this study, we describe a humanized mouse model that incorporates the clinical, virological and immunological features of latent EBV infection We employ a secondary adoptive transfer lymphoproliferative disease model to prove the tumorigenic potential of EBV-infected B lymphocytes in severely immunocompromised mice These data strongly suggest that B lymphocytes in infected hosts are both necessary and sufficient for the development of fatal systemic lymphoproliferative disease Treatment of EBV-infected humanized mice vii with the B lymphocyte depleting antibody Rituximab further highlighted the pivotal role of B lymphocytes in the pathogenesis of the disease Taken together, these data provide the first unequivocal evidence for a causal link between Epstein-Barr virus infection of human B cells with lymphoproliferative disease and tumorigenesis viii Humanized mouse model of EBV infection 55 56 57 58 59 60 61 62 63 64 65 66 References Strockbine LD, Cohen JI, Farrah T, et al The Epstein-Barr virus BARF1 gene encodes a novel, soluble colony-stimulating factor-1 receptor J Virol 1998;72(5):4015-4021 Prepublished on 1998/04/29 as DOI Cohen JI, Lekstrom K Epstein-Barr virus BARF1 protein is dispensable for B-cell transformation and inhibits alpha interferon secretion from mononuclear cells J Virol 1999;73(9):7627-7632 Prepublished on 1999/08/10 as DOI Thorley-Lawson DA, Gross A Persistence of the Epstein-Barr virus and the origins of associated lymphomas N 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