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Endogenous Retroviruses and the Immune System

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Endogenous Retroviruses and the Immune System Endogenous Retroviruses and the Immune System George Robert Young Submitted to University College London in part fulfilment of the requirements for the degree of Doctor of Philosophy 2012 Division of Infection and Immunity Division of Immunoregulation University College London National Institute for Medical Research Gower Street The Ridgeway London London WC1E 6BT NW7 1AA 2 I, George Robert Young, confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. 3 Abstract ABSTRACT 4 Initial sequencing of the human and mouse genomes revealed that substantial fractions were composed of retroelements (REs) and endogenous retroviruses (ERVs), the latter being relics of ancestral retroviral infection. Further study revealed ERVs constitute up to 10% of many mammalian genomes. Despite this abundance, comparatively little is known about their interactions, beneficial or detrimental, with the host. This thesis details two distinct sets of interactions with the immune system. Firstly, the presentation of ERV-derived peptides to developing lymphocytes was shown to exert a control on the immune response to infection with Friend Virus (FV). A self peptide encoded by an ERV negatively selected a significant fraction of polyclonal FV- specific CD4+ T cells and resulted in an impaired immune response. However, CD4+ T cell-mediated antiviral activity was fully preserved and repertoire analysis revealed a deletional bias according to peptide affinity, resulting in an effective enrichment of high-affinity CD4+ T cells. Thus, ERVs exerted a significant influence on the immune response, a mechanism that may partially contribute to the heterogeneity seen in human immune responses to retroviral infections. Secondly, a requirement for specific antibodies was shown in the control of ERVs. In a range of mice displaying distinct deficiencies in antibody production, products from the intestinal microbiota potentially induce ERV expression. Subsequent recombinational correction of a defective murine leukaemia virus (MLV) results in the emergence of infectious virus. In the long term, this leads to retrovirus-induced lymphomas and morbidity. ERVs, therefore, provide a potential link between the intestinal microbiota and a range of pathologies, including cancer. Finally, a new computational tool, REquest, was developed for use in the above studies. REquest allows the mining of retroelement (RE) and ERV expression data from the majority of commercially available human and murine microarray platforms and allows rapid hypothesis testing with publicly available data. 5 Acknowledgements ACKNOWLEDGEMENTS 6 I would like to express my thanks to my supervisor, George Kassiotis, without whom none of this work would have been possible. Thank you for having the faith to mentor an Oxford graduate with little but theoretical knowledge, for the discussion and guidance, and for encouraging the tangential research that our progress generated. Many others within the institute have given useful critique and analysis, specifically Jonathan Stoye and my thesis committee, Kate Bishop and Pavel Tolar. My thanks also to Anne O'Garra and the members of the Division of Immunoregulation. The National Institute for Medical Research provides an unparalleled support infra- structure, and I am indebted to members of the flow cytometry, electron microscopy, microarray, and large scale laboratory facilities, as well as the animal technicians and staff within and biological services. Underpinning both projects described here, Ula Eksmond has provided unfailing ef- fort and assistance, all whilst maintaining the smooth-running of the laboratory. I'm extremely appreciative of her contributions to my research. Over many years and in numerous ways, my parents have put my education and poten- tial above virtually all else. I am deeply grateful for their continued support and am extremely fortunate to be where I am today. Rachel has listened to, and sympathised with, the highs and the lows of experimental research and has provided the necessary motivation at the opportune moments. Now that this is complete, we can hopefully spend some more time together, and with our fantastic little William! He may be too young to understand this work, but perhaps I can read sections to him at bedtime. 7 Table of Contents TABLE OF CONTENTS 8 Abstract 3 Acknowledgements 5 Table of Contents 7 List of Figures 12 List of Tables 15 Abbreviations 17 1 Introduction 21 1.1 Retroviruses and retroelements . 22 1.1.1 A brief history of retrovirology . 22 1.1.2 The Retroviridae ........................... 22 1.1.3 Retroelements and endogenous retroviruses . 26 1.1.4 Retroelements in the genome { mutagenesis and disease . 28 1.1.5 Retroelements in the genome { co-option and function . 30 1.2 The immune system . 32 1.2.1 A high-level perspective . 32 1.2.2 Variable antigen receptors { form and function . 33 1.2.3 The T cell . 34 1.2.4 The B cell . 36 1.2.5 Importance and immunodeficiency . 38 1.3 Aims of this work . 39 1.3.1 A word on structure . 39 2 Methods 40 2.1 Mice . 41 2.1.1 Generation of Emv2 -/- congenics . 45 2.1.2 Ethics statement . 46 TABLE OF CONTENTS 9 2.2 Primers . 46 2.3 RNA expression assays . 49 2.4 DNA extraction and assays . 50 2.5 Sequencing and sequence analysis . 51 2.6 Media and culture conditions . 51 2.7 Isolation of primary cells . 52 2.8 Fluorescence Activated Cell Sorting . 53 2.9 Isolation and analysis of infectious MLVs . 54 2.10 Histology . 55 2.11 Electron microscopy . 55 2.12 Friend Virus . 55 2.13 FV-neutralising and cell-binding antibody titre assays . 56 2.14 Hybridoma production . 56 2.15 Stimulation assays . 57 2.16 Tra gene usage determination . 57 2.17 Computational and statistical methods . 58 2.18 REquest: determining retroelement expression with microarrays . 58 3 T cell development 64 3.1 Introduction . 65 3.1.1 Friend virus . 65 3.1.2 CD4+ cells in Friend virus infection . 66 3.1.3 T cell development and the prevention of autoimmunity . 67 3.1.4 Endogenous retroviruses in T cell selection . 68 3.1.5 Emv2 ................................. 68 3.1.6 Aims . 69 3.2 Results . 71 3.2.1 Characterisation of Emv2 expression and generation of Emv2 null congenics . 71 TABLE OF CONTENTS 10 + 3.2.2 Deletion of env122-141L-specific CD4 T cells by Emv2 ..... 72 3.2.3 Antiviral activity is retained despite Emv2 -mediated selection . 73 3.2.4 Emv2 -mediated selection increases the avidity of the CD4+ T cell response . 77 + 3.2.5 Presentation of env122-141Y causes the removal of non-Vα2 CD4 T cells . 77 + 3.2.6 Emv2 shapes the depth of the env124-138L-responsive CD4 T cell repertoire . 80 3.3 Discussion . 88 3.3.1 Derived work . 89 3.3.2 Implications of this work . 91 4 Retroelement expression 93 4.1 Introduction . 94 4.1.1 Activating retroelement expression . 94 4.1.2 Controlling retroelement expression . 94 4.1.3 The microbiome in health and disease { structure and function . 95 4.1.4 Aims . 96 4.2 Results . 98 4.2.1 eMLV transcripts are increased in immunodeficiency . 98 4.2.2 eMLV transcripts are increased in antibody deficiency . 101 4.2.3 Cell-surface MLV glycoprotein can be stained in antibody defi- cient mice . 102 4.2.4 Infectious virus can be isolated from Rag1-/- mice . 103 4.2.5 RARV infection occurs by vertical transmission to neonates . 108 4.2.6 The implications of RARV infection . 111 4.2.7 Antibodies are required for the control of systemic microbial products . 116 4.2.8 The frequency of RARV generation . 122 4.3 Discussion . 125 TABLE OF CONTENTS 11 5 Conclusion 130 References 132 Publications 159 12 List of Figures LIST OF FIGURES 13 1.1 Retroviral replication and structure . 23 1.2 Retroviral phylogenetic tree . 24 1.3 Retroelements and endogenous retroviruses . 27 1.4 The T and B cell receptors . 33 1.5 Antigen presentation to T cells . 35 1.6 CD4+ T cell subsets . 36 2.1 Emv2 typing by PCR . 46 2.2 RE-specific probe identification . 62 3.1 Thymocyte development . 67 3.2 Sequence and contact residues of the immunodominant H2-Ab-restricted F-MLV epitope . 69 3.3 Emv2 expression in B6 and Emv2-/- mice . 71 3.4 Clonal and polyclonal responses to env124-138 .............. 72 + 3.5 Emv2 -mediated deletion of env124-138L-reactive CD4 T cells . 73 3.6 Control of FV in Emv2 -sufficient and -deficient mice . 74 3.7 Differences in FV infection are not explained by loss of `helper' functionality 75 3.8 Equal effector activity in Emv2 -selected and -non-selected CD4+ T cells 76 3.9 Emv2 -mediated selection removes non-Vα2 CD4+ T cells . 78 3.10 Avidity for env124-138L is unchanged by Emv2 -mediated selection . 79 3.11 Deletion of non-Vα2 env124-138L-reactive cells is a thymic event . 80 + 3.12 Avidity for env124-138Y in Emv2 -selected and -non-selected EF4.1 CD4 T cells . 81 3.13 Selection by Emv2 reduces the depth of the TCR repertoire . 82 3.14 TCR cross-reactivity in Emv2 -selected and -non-selected Vα2+ hybrido- mas ...................................... 85 3.15 TCR cross-reactivity in Emv2 -selected and -non-selected non-Vα2 hy- bridomas . 86 3.16 Order of amino acid preference at defined TCR contact residues . 87 3.17 The contribution of H2-Ab to high-avidity F-MLV responses . 90 LIST OF FIGURES 14 3.18 The contribution of Trav to high-avidity F-MLV responses . 91 4.1 eMLV expression is elevated in Rag1-/- mice . 99 4.2 ERV expression across B6 and Rag1-/- tissues . 100 4.3 eMLV expression in selectively immunodeficient mice .
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