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Yang2012.Pdf This thesis has been submitted in fulfilment of the requirements for a postgraduate degree (e.g. PhD, MPhil, DClinPsychol) at the University of Edinburgh. Please note the following terms and conditions of use: • This work is protected by copyright and other intellectual property rights, which are retained by the thesis author, unless otherwise stated. • A copy can be downloaded for personal non-commercial research or study, without prior permission or charge. • This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author. • The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author. • When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given. Characterization of bovine granzymes and studies of the role of granzyme B in killing of Theileria -infected cells by CD8+ T cells Jie Yang PhD by Research The University of Edinburgh 2012 Declaration I declare that the work presented in this thesis is my own original work, except where specified, and it does not include work forming part of a thesis presented successfully for a degree in this or another university Jie Yang Edinburgh, 2012 i Abstract Previous studies have shown that cytotoxic CD8+ T cells are important mediators of immunity against the bovine intracellular protozoan parasite T. parva . The present study set out to determine the role of granule enzymes in mediating killing of parasitized cells, first by characterising the granzymes expressed by bovine lymphocytes and, second, by investigating their involvement in killing of target cells. Experiments using the perforin inhibitor concanamycin A confirmed that CD8+ T cell killing of T. parva -infected cells is dependent on granule exocytosis, a process that involves release of granzymes into the target cell, resulting in activation of apoptotic pathways. Analysis of the bovine genome sequence identified orthologues of granzymes A, B, H, K and M, as well as another gene O, most closely related to granzyme A. The genes were found within 3 loci in the genome. Using specific PCR assays, all of these granzymes were shown to be expressed in Theileria -specific CD8+ T cells. Further studies were undertaken to study the role of granzyme B in killing. DNA constructs encoding functional and non-functional forms of bovine granzyme B were produced and the proteins expressed in COS cells were used to establish an enzymatic assay to detect and quantify expression of functional granzyme B protein. Using this assay, the levels of killing of different T. parva - specific CD8+ T cell clones were found to be significantly correlating with levels of granzyme B protein expression. Moreover, the granzyme B inhibitor III, Z-IETD- FMK was shown to inhibit killing by CD8+ T cell clones. ii Acknowledgements I would like to thank my supervisor, Prof. Ivan Morrison, for his patience, help, support and advice during this study, particularly at time of writing up. Special thanks must be given to Dr Timothy Connelley for all his guidance, help and encouragement during my PhD, especially at the beginning of this study. I wish to thank Dr Niall MacHugh for his helpful discussions and Dr Jane Hart for her technical advice and always being supportive. Thanks also need to go to Dr Darren Shaw for his statistical help and advice. Finally, I would like to thank family and friends for all their understanding and support. iii Contents Abstract……………………………………………………………………………...ii Acknowledgements…………………………………………………………………iii Contents…………………………………………………………………………......iv List of figures……………………………………………………………………….xi List of tables……………………………………………………………………....xvii Abbreviations…………………………………………………….………….…….xix Chapter 1 Introduction........................................................................................ 1 1.1 Theileria parva and East coast fever............................................................ 1 1.1.1 Life cycle of T. parva ........................................................................... 2 1.1.2 Clinical disease..................................................................................... 4 1.1.3 Control of T. parva ............................................................................... 5 1.2 Effector mechanism of immunity to T. parva .............................................. 7 1.2.1 The role of CD8+ T cells in protective immunity................................ 7 1.2.2 Parasite stain specificity and major histocompatability class I (MHC1) restriction.............................................................................................................. 9 1.2.3 Identification of antigens and epitopes .............................................. 10 1.2.4 Immunodominance............................................................................. 11 1.2.5 Subunit vaccine - Immunization with T. parva antigens ................... 12 1.3 CD8+ T cell- mediated effector mechanisms against intracellular pathogens... ............................................................................................................ 14 1.3.1 Cytokine-mediated mechanisms in Theileria ..................................... 15 1.3.2 Mechanisms of cell-mediated cytotoxicity ........................................ 15 iv 1.3.2.1 Death receptor pathway (Fas-Fas Ligand) ..................................... 15 1.3.2.2 Granule exocytosis pathway .......................................................... 17 1.4 Perforin....................................................................................................... 18 1.5 Granzymes.................................................................................................. 20 1.5.1 Synthesis and processing of granzymes............................................. 23 1.5.2 Expression of granule enzymes.......................................................... 24 1.5.3 Apoptotic function of granzymes....................................................... 26 1.5.3.1 Apoptotic function of Granzyme A................................................ 28 1.5.3.2 Apoptotic function of Granzyme B................................................ 30 1.5.3.3 Apoptotic function of orphan granzymes....................................... 33 1.5.3.4 Inhibitors of granzyme activity...................................................... 35 1.5.4 Other possible functions of granzymes.............................................. 37 1.6 Aims of the project..................................................................................... 39 Chapter 2 Materials and Methods.................................................................... 41 2.1 Experimental animals................................................................................. 41 2.2 General cell culture techniques.................................................................. 41 2.2.1 Lymphocyte preparation .................................................................... 41 2.2.2 Concanavalin A (ConA) -activated PBMC........................................ 42 2.2.3 Complement-mediated cell lysis........................................................ 42 2.2.4 Generation of CD8+ T cell line in vitro............................................. 43 2.2.5 Expansion of clones ........................................................................... 44 2.2.6 Cryopreservation of cellular samples................................................. 44 2.2.7 Flow cytometry .................................................................................. 45 2.2.8 Cytotoxicity assay.............................................................................. 46 2.3 General molecular techniques.................................................................... 49 v 2.3.1 Isolation of RNA................................................................................ 49 2.3.2 RNA and DNA quantification............................................................ 49 2.3.3 RNA reverse transcription.................................................................. 50 2.3.4 General RT-PCR assay ...................................................................... 50 2.3.5 Agarose gel electrophoresis ............................................................... 51 2.3.6 DNA purification ............................................................................... 51 2.3.7 Sub-cloning of PCR products ............................................................ 52 2.3.8 Plasmid DNA Preparation-(Mini-preps)............................................ 53 2.3.9 Identify the insert containing plasmid clones..................................... 54 2.3.10 Sequencing......................................................................................... 54 Chapter 3 Identification and characterisation of bovine granzymes ............ 55 3.1 Introduction................................................................................................ 55 3.2 Materials and methods ............................................................................... 57 3.2.1 Bovine granzyme genome analysis.................................................... 57 3.2.2 Sequence analysis............................................................................... 58 3.2.3 Chromosomal location analysis ......................................................... 58 3.2.4 Phylogenetic analysis........................................................................
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