A Study of T Cell Receptor Usage in Allergic Asthma and Analysis of a Humanised T Cell Receptor Transgenic Model of Immunity to Allergen A thesis submitted for the degree of Doctor of Philosophy by Tracey Elizabeth Pollard at Imperial College, London Lung Immunology Group, National Heart and Lung Institute, Faculty of Medicine, Sir Alexander Fleming Building, South Kensington campus, Imperial College, London SW7 2AZ Supervisors: Dr Rosemary J. Boyton Prof. Daniel M. Altmann June 2009 Abstract It has been previously shown that the length of complementarity-determining region 3 (CDR3) loops of a T-cell receptor (TCR) may determine T-cell phenotype, with elongated CDR3s being favoured in T helper-type 2 (Th2) cells. Here, in a human system of Th2-mediated allergic asthma, TCR CDR3 usage has been studied in T-cell lines derived from Human Leukocyte Antigen (HLA)-DR1-positive, cat-allergic asthmatic subjects, and generated against peptide 4, a T-cell epitope of the major cat allergen Fel d 1. Populations of T-cells expanded that expressed particular TCRs carrying elongated CDR3a or p loops. A system was developed to transduce the immortalised TCR-negative murine thymoma line BW7 with the human TCR genes identified here and also murine Thl and Th2 associated TCR genes identified from previous work in our laboratory to allow further study of the influence of TCR structure on T-cell phenotype. A TCR/MHC class II transgenic model of allergic asthma has been developed by others in our laboratory. As part of the initial characterisation of this model, to establish if the TCR was expressed, the impact of the presence of the TCR transgenes on thymic selection and the expression of costimulatory molecules was studied. A block at the double negative stage in thymocyte development is seen as well as differences in expression of the costimulatory molecules CD44, CD62L, ICOS and OX40. Allergic inflammatory lung disease was induced in this model using a sensitisation and challenge protocol with Fel d 1 and cat dander, and changes usually associated with an asthma phenotype studied. Increased airway hyperreactivity, eosinophilic lung influx, raised serum IgE, Th2-associated cytokines in BAL and lung, mild lung inflammation and increased mucus secretion in the airways were observed, all changes that would be expected in a good model of allergic asthma. 2 Statement of declaration I performed all of the experiments and data analysis contained in this thesis except where otherwise stated in the text. Tracey Pollard 2009 3 Acknowledgments I would like to thank my supervisor Rosemary Boyton for all her help and support, for her guidance and ideas throughout the course of my project, for her help and suggestions when I was having difficulties, and for her encouragement and advice throughout my time working in her lab. I am very grateful to her for providing me with this project as well as such a friendly and welcoming environment in which to work. I would also like to thank my co-supervisor Danny Altmann for his guidance throughout my PhD, particularly for all his help and suggestions when I encountered difficulties in my work, and for giving up his time to help me, particularly during my writing up. I would like to thank Catherine Reynolds for her help and advice throughout my time in the lab. In particular, I thank her for her advice in designing the Southern strategy for the P4TCRTg mice and her help with the allergic disease induction experiments - doing lung inflations, sectioning frozen lung, differential cell counting, inflammation and mucus scoring, and helping me with measuring Penh. Thank you to Kate Choy for doing resistance/compliance measurements and Eleanor Raynsford for doing differential cell counts and for her help with the disease induction experiments. Thanks to Xiaoming Cheng for devising the PCR strategy to determine correct splicing of TCR genes in the P4TCRTg mice, and for all her help in setting up the disease induction experimental model used here. Thanks to Rebecca Beavil for supplying our lab with recombinant Fel d 1, and Laboratorios Leti for supplying us with cat dander extract for use in these experiments. Last but not least, I am extremely grateful to the CBS staff for their care of the animals used in these experiments. Thank you to Alexander Annenkov for help setting up the retroviral transduction system used here, Diane Mathis and Christophe Benoist for supplying the pTcass TCR expression vectors and Hongyan Wang for her much-appreciated advice and help with this system. Thanks also to Julian Dyson for supplying me with EcoPhoenix cells. Many thanks to Mark Larche for supplying human HLA-DR1-restricted peptide 4- specific T-cell lines for the TCR CDR3 analysis, and to Adrienne Verhoef and Betty Lo for their help growing the lines. Thank you also to Julia Llewellyn-Hughes and her colleagues at the Natural History Museum sequencing facility for carrying out DNA sequencing. Finally, I would like to thank Catherine, Eleanor and Xiaoming for being good friends and colleagues to me, and making the experience a really enjoyable one. Also, thank you to Charis for her support and for not minding when my thesis seemingly took over the flat whilst I was writing up! Thanks also to my parents for worrying about me! Without the support of these people I don't think I'd have made it this far... 4 Table of contents Page number Title page 1 Abstract 2 Statement of declaration 3 Acknowledgments 4 Table of contents 5 List of figures 16 List of tables 25 List of abbreviations 27 List of appendices 37 Chapter 1 Introduction 40 1.1 Clinical and epidemiological aspects of allergic 40 asthma 1.1.1 Prevalence of allergic asthma 40 1.1.2 The 'hygiene hypothesis' 40 1.1.3 The clinical manifestations of asthma 42 1.2 Immune mechanisms of allergic asthma 43 1.2.1 Antigen-presenting cells (dendritic cells) in allergic 43 asthma 1.2.2 Immunoglobulin E in allergic asthma 45 1.2.3 Mast cells in allergic asthma 46 1.2.4 Eosinophils in allergic asthma 47 1.2.5 Basophils in allergic asthma 48 1.2.6 Neutrophils in allergic asthma 49 1.2.7 Invariant natural killer T (iNKT) cells in allergic 50 asthma 1.2.8 CD8+ T-cells (cytotoxic T-cells) in allergic asthma 52 1.2.9 The role of T helper type 17 (Th17) cells in allergic 55 asthma 1.2.10 The role of Thl cells in allergic asthma 56 5 1.2.11 The role of regulatory T-cells (Tregs) in allergic 59 asthma 1.2.12 The role of Th2 cells in allergic asthma 61 1.3 Mouse models of allergic asthma 64 1.3.1 The principles of molecular cloning and their use in 64 animal models of disease 1.3.2 The use of animals in human disease modelling 66 1.3.3 The use of ovalbumin (OVA) as an allergen in 69 mouse models of allergic asthma 1.3.4 Adoptive transfer of OVA-pulsed APCs or OVA- 72 specific T-cells as a means of inducing allergic airway disease in mice 1.3.5 Use of the OVA model in investigating disease 73 pathogenesis in allergic asthma 1.3.6 The use of house dust mite allergens in a mouse 76 model of allergic asthma 1.3.7 Uses of the HDM mouse model of allergic asthma to 79 investigate lung remodelling processes 1.3.8 Other uses of the HDM mouse model of allergic 81 asthma 1.3.9 Other mouse models of allergic asthma 81 1.3.10 TCR transgenic models of allergic asthma 82 1.3.11 Human T-cell responses in allergic asthma caused 87 by the major allergen in cat dander, Fel d 1 1.4 T-cell receptor structure 90 1.4.1 Identification of the T-cell antigen receptor (TCR) 90 1.4.2 The interaction between TCR, peptide and MHC 91 molecule 1.4.3 T-cell receptor structure 92 1.4.4 V(D)J recombination and T-cell receptor diversity 92 1.5 T-cell development in the thymus 95 1.5.1 Precursor entry into the thymus and the earliest 95 stages of T-cell development 1.5.2 TCRa gene rearrangement 97 6 1.5.3 Positive and negative selection 97 1.5.4 The CD4 vs. CD8 lineage decision 99 1.6 T-cell signalling, activation and differentiation 99 1.6.1 Proposed mechanisms of TCR triggering 99 1.6.2 T-cell signalling through the TCR 101 1.6.3 The immunological synapse 104 1.6.4 The signalling pathways involved in differentiation 106 of naive T helper cells into effector T helper lineages 1.6.5 Factors affecting the differentiation of naive T-cells 108 into effector phenotypes upon antigen stimulation 1.6.6 The serial triggering model 112 1.6.7 The role of TCR structure in determining the 113 affinity of the interaction with peptide-MHC 1.6.8 Clonotypic expansion of T-cells in response to 115 antigen stimulation and functional maturation of the T-cell response 1.6.9 Can TCR structure influence the fate of a naive T 117 helper cell upon stimulation with antigen? 1.7 Aims of this thesis 121 Chapter 2 Materials and Methods 122 2.1 Molecular biology techniques 122 2.1.1 Human TCR CDR3 analysis 122 2.1.1.1 RNA isolation from human T-cell lines 122 2.1.1.2 cDNA preparation from human T-cell RNA 122 2.1.1.3 Polymerase chain reaction (PCR) 123 2.1.2 Cloning of TCRa and 13 chains into MFG and 123 pTcass expression vectors 2.1.2.1 RNA isolation from murine T-cells 126 2.1.2.2 cDNA preparation from murine T-cell RNA 126 2.1.2.3 PCR using high-fidelity DNA polymerase 127 2.1.3 General DNA cloning techniques 127 2.1.3.1 Preparation of competent cells 127 7 2.1.3.2 Restriction digests 128 2.1.3.3 Oligonucleotide preparation 129 2.1.3.4 Nuclease treatment 129 2.1.3.5 Cloning ligations 129 2.1.3.6 TA cloning ligations 130 2.1.3.7 E.