T Cell Receptor Structure and Cytokine Responses in Lung Targeted Inflammatory Models

T Cell Receptor structure and cytokine responses in lung targeted inflammatory models A thesis submitted for the degree of Doctor of Philosophy University of London Catherine Jane Reynolds Lung Immunology Group National Heart and Lung Institute Sir Alexander Fleming Building Imperial College London SW7 2AZ Supervised by: Dr Rosemary Boyton Professor Daniel Altmann 2008 Acknowledgements Over the course of my PhD there have been many people without whom I would never have been able to get to this point. Whether it was the kind donation of reagents, training and advice in lab techniques, scientific input or simply morale support, all help has been invaluable and very much appreciated. I would particularly like to thank my supervisors, Rosemary Boyton and Danny Altmann. There is definitely no way that this thesis would exist without your unrelenting support, vision and enthusiasm. Your unwavering belief that I would get there in the end, even through the darker times of transgenic founder screening, and the enormous commitment, both in terms of your time and your energy, made my PhD an extremely rewarding and enjoyable experience. Many people contributed reagents, training and advice to me throughout my PhD and I've tried to list all of these people, to whom I am extremely grateful, below. There have also been lots of people who have contributed less specifically, but no less importantly, particularly in terms of moral support and maintaining my mental well- being, during the course of this thesis. I'd particularly like to thank all members past and present of the Boyton, Altmann and Dallman labs, especially Tracey and Xiaoming who were important extra pairs of hands in many big experiments! Finally I'd like to thank my partner, Matt. A massive thank you for your never ending support and patience including all the meals cooked, washing done and pep talks delivered! I couldn't have done it without you. 2 Reagents, help and support Transgenesis Helen Bodmer — HEL expression cassette Brian Sauer — floxed stop cassette Jeffrey Whitsett — CC10 promoter Diane Mathis and Christophe Benoist — TCR cassette vectors Maggie Dallman f3m70 Cre transgenic line Laurence Bugeon Colin Hetherington Pronuclear injections at Nuffield Department of Medicine Robert Sumner (University of Oxford) Karla Watson Zoe Webster — pronuclear injections at MRC Clinical Sciences Centre (Imperial College) Keith Murray Daniel Wells South Kensington campus animal facility technicians Darren Bilton Amy Wathen Technical help and support Kate Choy Lung function studies Sara Mathie Lorraine Lawrence — Lung histology Julia Llewellyn-Hughes — DNA sequencing Katlin Takacs — DNA preparation for pronuclear injection Yogesh Singh — Spectratyping Alan Ahern Molecular biology techniques Jennifer Rowland Gurman Kaur — Real time PCR 3 Abstract Thl or Th2 type immune responses are an important feature of many human diseases, including several lung pathologies. Multiple factors are involved in determining whether a naïve CD4+ T cell differentiates into a Thl or Th2 effector cell and several lines of evidence point towards an important role for the interaction between the T cell receptor (TCR) and its peptide ligand. A previous study showed that highly polarised Th2 CD4+ T cells selected under Th2 polarising conditions favour a structurally distinct TCR with an elongated TCR a chain complimentarity determining region 3 (CDR3), while those selected under Thl polarising conditions favour a shorter TCR CDR3 a chain. Molecular modelling predicted that this elongated TCR a chain results in a less optimal (lower avidity) interaction between the TCR and peptide/MHC complex. In this thesis, clonotypic analysis was used to identify dominant TCR chains from established Thl and Th2 polarised T cell lines. Dominant TCR a chains were cloned into TCR transgenic expression vectors and lines of mice with transgenic expression of these chains were identified. In vitro and in vivo studies were used to investigate T cell responses in these transgenic lines and the data suggests that expression of the Th2 derived TCR a chain can cause an inherent bias in CD4+ T cell phenotype. A long term aim of this work is to use the Thl and Th2 TCR transgenic animals to develop novel mouse models of lung disease and this thesis also describes the generation of transgenic mice that express the Thl and Th2 TCR cognate epitope, PLP 56-70, inducibly and specifically in the lung. Also implicated in the pathogenesis of many lung diseases is the CXC chemokine, interleukin 8. The final chapter of this thesis describes the generation of transgenic mice overexpressing this human protein constitutively in the lung and reports initial characterisation of protein expression and consequent lung inflammation in these animals. 4 Table of contents page number Title page 1 Acknowledgements 2 Abstract 4 Table of contents 5 List of figures 10 List of tables 15 List of appendices 16 List of abbreviations 18 Chapter 1. Introduction 1.1 The ar3 T cell receptor 24 1.2 T cell receptor gene rearrangement 25 1.3 T cell receptor sequence diversity 28 1.4 T cell receptor assembly 29 1.5 Thymic selection 34 1.6 Dual TCR expression 36 1.7 TCR editing and revision 37 1.8 Structure of the TCR/CD3 signalling complex 38 1.9 pMHC binding by TCR 44 1.10 TCR binding affinity and conformational change 46 1.11 TCR triggering 49 1.12 CD4+ T cell phenotype 53 1.13 Thl and Th2 cell differentiation 56 1.14 The TCR and CD4+ T cell differentiation 61 1.15 Thl and Th2 responses in human lung disease 64 1.16 Mouse models of human lung disease 70 1.17 Approaches to transgenesis 74 1.18 Interleukin 8 80 1.19 Neutrophil function 83 5 1.20 Interleukin 8 and human lung disease 86 1.21 Aims of this thesis 90 Chapter 2. Materials and methods 2.1 Genomic DNA extraction 94 2.2 RNA extraction 94 2.3 DNase treatment of RNA samples 95 2.4 cDNA synthesis 95 2.5 Polymerase Chain Reaction (PCR) 96 2.6 Restriction enzyme digest 96 2.7 Preparation of XL-Gold competent cells 96 2.8 E-coli transformation 97 2.9 Standard cloning and ligation protocol 97 2.10 Blunt end generation 98 2.11 Oligonucleotide annealing 99 2.12 TA cloning of PCR products 99 2.13 Preparation and screening of plasmid DNA 99 2.14 T cell receptor PCR analysis 100 2.15 Sequencing and T cell receptor sequence analysis 100 2.16 TCR CDR3 spectratyping 101 2.17 Real-time PCR 101 2.18 Preparation of DNA for oocytes pronuclear injection 102 2.19 Genotyping 102 2.20 Southern blotting 103 2.21 Southern blot Digoxigenin (DIG) probe labelling, hybridisation 103 and detection 2.22 Tamoxifen induction 105 2.23 Footpad/flank peptide immunisation 105 2.24 Immediately ex vivo T cell proliferation assay 105 2.25 Culture of T cell lines 106 2.26 Measurement of bronchial hyperreactivity (BHR) 106 6 2.27 Measurement of Resistance (R) and Compliance (Cdyn) 107 2.28 Collection of Bronchoalveolar lavage (BAL) and serum 107 2.29 Cell extraction from whole lung lobe 108 2.30 Differential cell counting 108 2.31 Lung homogenate 108 2.32 Lung histology — tissue preparation 109 2.33 Haematoxylin and Eosin (H+E) staining 109 2.34 Periodic Acid Schiff (PAS) staining and scoring 109 2.35 Immunohistochemistry — tissue fixation 110 2.36 Peroxidase based immunohistochemistry 110 2.37 Immunofluorescence tissue staining 111 2.38 Inflammatory cell scoring 112 2.39 Induction of allergic airway disease using ovalbumin (OVA) 112 2.40 Enzyme Linked ImmunoSorbant Assay (ELISA) 112 2.41 Flow cytometric analysis 113 Chapter 3. Generation of Thl and Th2 TCR a chain transgenic lines Results 3.1 Determination of a and p TCR gene usage in Thl and Th2 lines 114 specific for PLP 56-70 3.2 Construction of Thl and Th2 TCR a chain transgenes 124 3.3 Identification of Thl and Th2 TCR a chain transgenic founder 132 animals 3.4 Determination of TCR a chain transgene expression 136 Discussion 146 Chapter 4. TCR 13 chain selection and cytokine phenotype in Thl and Th2 a chain transgenics Results 4.1 Analysis of TCR p chain usage in unprimed Th2 TCR a chain 153 transgenics 7 4.2 Analysis of TCR 13 chain usage in Th2 TCR a chain transgenic 157 in the context of antigen specificity 4.3 Analysis of PLP (56-70) specific Th2 TCR a chain transgenic 160 T cell lines 4.4 Construction of a Th2 TCR (3 chain transgene 171 4.5 Identification of a Th2 TCR p chain transgenic founder animal 174 4.6 Analysis of a PLP (56-70) specific Thl TCR a chain transgenic 176 T cell line 4.7 Altered cytokine phenotype of an in vivo memory response to antigen 178 in Th2 TCR a chain transgenic animals Discussion 182 Chapter 5. Generation of transgenic lines for inducible, lung targeted, antigen expression Results 5.1 Design and construction of transgenes for inducible, lung targeted, 192 expression of the antigen PLP 56-70 5.2 Identification of MIPLP transgenic founder animal 203 5.3 Determination of stop sequence excision and MIPLP transgene 205 expression 5.4 Re-design of the SIPLP transgene 209 5.5 Identification of SIPLP transgenic founder animals 212 Discussion 216 Chapter 6. Generation and initial characterisation of transgenic lines with lung targeted expression of human interleukin 8 Results 6.1 Design and construction of a transgene for lung targeted expression 222 of human interleukin 8 6.2 Identification of lung targeted hIL-8 transgenic founder animals 226 6.3 Determination of lung specific hIL-8 transgene expression 228 8 6.4 Lung function and lung cell infiltrate in lung targeted hIL-8 233 transgenics 6.5 Investigation of the impact of IL-8 expression in the murine lung 239 during ovalbumin induced lung inflammation Discussion 246 Overview 256 Bibliography 260 Appendices 307 9 List of Figures Page number Chapter 1.

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