AN ASTHMA ALLERGEN SPECIFIC ANIMAL MODEL FOR THE STUDY OF RESPONSES TO MITE ALLERGEN INDUCED ASTHMA KENNETH WONG HOK SUM B.Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY YONG LOO LIN SCHOOL OF MEDICINE DEPARTMENT OF MICROBIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2013     ACKNOWLDGEMENTS       Acknowledgements First and foremost, I would like to express my gratitude to Prof. Kemeny for his patient guidance and support. When things did not work, and that happened a lot, you were always a calming influence and helped get me back on track. I also learnt so much more than just science from you and I really appreciate the discussions we had. To Dr Gijsbert Grotenbreg, thank you for taking me under your wing. I learnt a lot from you and your focus and drive is always a source of inspiration. Things certainly wouldn’t have gotten this far without your help on the project. To Dr Paul MacAry, thank you for all your advice and guidance throughout this project. To the members of the DMK lab, past and present, I am very grateful to have had the opportunity to work with you all. Most of you had been more than just good colleagues. You are great friends. I could not have asked for a better bunch of people to work with. Thank you especially to Yafang and Sophie for guiding me with the asthma studies. Thank you as well to Nayana for all the discussions and your help on those busy harvest days. So many people, so little time to acknowledge them. A big thank you to Benson as well. The lab would have been a mess without you handling the orders and the mice colonies. I am also very grateful to the people in the GMG lab. It had been great to work with you guys and we had more than our fair share of laughs. This is especially with Cynthia as we embarked on the big scary world of protein expression together, both knowing absolutely nothing to start with and making i     ACKNOWLDGEMENTS       every mistake in the book. To Joanna, Lionel and Michelle, I missed those weekends in the lab with you guys. Who says working overtime is not fun! And to Adrian Sim, Fatimah, Chien Tei, Michael, Lawrence and everyone else, thank you for your help and for simply adding color to my life. I also owe a debt of gratitude to my family in Malaysia. Thank you for your unconditional support in everything I do, no matter how dumb. Finally, to my long-suffering wife, the biggest thank you. You’ve endured the past few years with this grumpy ogre and bore the brunt of it when my experiments did not work. Hopefully I can make it up to you after this! I wouldn’t have made it through this without you. ii     SUMMARY       Summary T cells play a central role in the pathogenesis of allergic asthma. However, many studies into the roles of T cells in asthma had been performed using ovalbumin as a surrogate allergen. This is mainly due to the greater availability of research tools for use in the ovalbumin model. However, true asthma allergens had been shown to behave very differently than ovalbumin. In this study, we aim to expand the tools available for the study of mite allergen-induced asthma. Using a plasmid DNA immunization method, we induced T cell responses against allergens from Blomia tropicalis and Dermatophagoides pteronyssinus. We identified a number of epitopes recognized by allergenspecific CD4 T cells, including several novel epitopes for Blo t 5. We next demonstrated that the Blo t 5-specific CD4 T cells identified in this study were recruited into the lungs following Blo t inhalation. When administered intradermally, the peptides induced a tolerogenic response and attenuated the allergic airway inflammation induced by Blo t sensitization and challenge. The identification of CD4 T cell epitopes for Blo t would allow for the study of T cell responses to Blomia tropicalis, a major source of mite allergen in the tropics that remained poorly studied to date. Work done on the OVA model suggested a role for CD8 T cells in the attenuation of the allergic airway responses to allergen. In this study, we adoptively transferred Der p specific T cells into mice sensitized and challenged with house dust mite (HDM) extract. CD8 T cell responses were tracked by class I MHC tetramers produced in-house. Our results showed that, iii     SUMMARY       unlike in the ovalbumin model, the CD8 T cells were unable to attenuate the allergic airway inflammatory responses to HDM. However, we showed that the Th2 airway inflammation was reduced following the adoptive transfer of Der p specific CD8 T cells when mice were sensitized and challenged by purified Der p protein. We proceeded to demonstrate in vivo and in vitro that exogenous HDM was a poor inducer of CD8 T cell responses. Finally, using peptide-pulsed BMDCs to induce a Der p 1-specific immune response, we observed that CD8 T cell responses exacerbated the allergic lung inflammation response to HDM by increasing the number of infiltrating immune cells and the production of IL-5 and IL-13. Therefore, our results suggested that the induction of a CD8 T cell response by HDM was markedly inefficient and it is unlikely that CD8 T cells could play a role in the acute phase of asthma development. However, our results showed also that a CD8 T cell response might actually be detrimental and exacerbate the inflammatory responses in the lung. Finally, we cloned and characterized the T cell receptor (TCR) gene from a Der p specific CD8 T cell. The TCR genes were cloned into expression cassettes for the generation of TCR transgenic mice with CD8 T cells specific for the HDM allergen, Der p 1. We believe that these mice could be useful in the study of chronic asthma, where CD8 T cells had been shown to play a role. iv     LIST  OF  PUBLICATIONS       List of publications 1. Wong KL, Tang LF, Lew FC, Wong HS, Chua YL, MacAry PA, Kemeny DM. CD44high memory CD8 T cells synergize with CpG DNA to activate dendritic cell IL-12p70 production. J Immunol. 2009 Jul 1; 183(1):41-50 2. Tang Y, Guan SP, Chua BY, Zhou Q, Ho AW, Wong KH, Wong KL, Wong WS, Kemeny DM. Antigen-specific effector CD8 T cells regulate allergic responses via IFN-γ and dendritic cell function. J Allergy Clin Immunol. 2012 Jun; 129(6):1611-20.e4 3. Ge MQ, Ho AW, Tang Y, Wong KH, Chua BY, Gasser S, Kemeny DM. NK cells regulate CD8+ T cell priming and dendritic cell migration during influenza A infection by IFN-γ and perforindependent mechanisms. J Immunol. 2012 Sep 1; 189(5):2099-109. 4. Prabhu N, Ho AW, Wong KH, Hutchinson PE, Chua YL, Kandasamy M, Lee DC, Sivasankar B, Kemeny DM. Gamma interferon regulates contraction of the influenza virus-specific CD8 T cell response and limits the size of the memory population. 87(23):12510-22 v J Virol. 2013 Dec;     TABLE  OF  CONTENTS       Table of Contents Chapter 1.   Introduction .   1.1   Asthma .   1.1.1   Prevalence and cost of asthma .   1.1.2   Causes of asthma   1.2   The immunology of allergic asthma   1.2.1   The innate immune response in allergic asthma   1.2.1.1   Dendritic  cells  and  their  role  in  allergic  asthma   .  8   1.2.1.2   Eosinophils   .  11   1.2.1.3   Mast  cells   .  12   1.2.1.4   Basophils   .  13   1.2.1.5   Neutrophils   .  15   1.2.1.6   Macrophages    16   1.2.1.7   Innate  lymphoid  cells   .  17   1.2.1.8   Natural  killer  T  (NKT)  cells  and  γδ  T  cells   .  17   1.2.1.9   The  airway  epithelium    18   1.2.2   The adaptive immune system and allergic asthma 19   1.2.2.1   CD4  T  cells  in  allergic  asthma    20   1.2.2.2   CD8  T  cells  in  allergic  asthma    26   1.2.2.3   The  humoral  response  in  allergic  asthma   .  28   1.3   Asthma allergens . 29   1.3.1   The mite allergens 30   1.3.2   Mite allergens and the immune system 32   1.4   Aims of the study . 34   Chapter 2.   Materials and Methods . 38   2.1   Media and buffers . 38   2.1.1   PBS buffer 38   2.1.2   MACS buffer . 38   2.1.3   FACS buffer . 38   2.1.4   Red Blood cell (RBC) lysis solution . 39   2.1.5   Complete RPMI for cell culture . 39   2.1.6   Buffers for ELISA 40   2.1.7   LB broth . 40   2.1.8   LB agar . 40   2.1.9   CaCl2 solution for preparation of competent cells . 40   2.1.10   SDS-PAGE gel electrophoresis buffers . 40   2.1.11   Buffers for protein purification from E.coli . 41   2.1.12   Buffers for protein refolding and purification . 41   2.2   List of antibodies used 42   2.3   Mice 43   2.4   Molecular biology protocols . 43   2.4.1   Preparation of chemically competent E.coli 43   2.4.2   Transformation of E.coli 44   2.4.3   General PCR protocol 45   2.4.4   Agarose gel extraction . 47   2.4.5   Subcloning into TOPO vector by TA cloning . 47   2.4.6   Plasmid miniprep . 49   2.4.7   Restriction enzyme digest 49   2.4.8   PCR purification 50   vi     TABLE  OF  CONTENTS       2.4.9   Ligation into plasmid vector 51   2.4.10   RNA extraction and purification 51   2.4.11   Reverse transcription of mRNA . 52   2.4.12   5’ RACE reaction . 52   2.5   Protein expression and purification protocols . 55   2.5.1   SDS-PAGE gel electrophoresis . 55   2.5.2   Production of class I MHC tetramers . 56   2.5.3   Production of recombinant Blo t . 60   2.6   Cell isolation protocols . 62   2.6.1   Processing of splenic and lymph node cells 62   2.6.2   Isolation of CD8 cells by magnetic separation 63   2.7   Cell culture 64   2.7.1   T cell line production and maintenance . 64   2.7.2   Bone-marrow derived Dendritic Cells . 65   2.8   Protocols for evaluation of cell functionality 66   2.8.1   IFN-γ ELISPOT . 66   2.8.2   ELISA 68   2.8.3   3H-thymidine proliferation assay 68   2.8.4   Chromium-51 release assay . 69   2.9   Flow cytometry 70   2.9.1   Cell surface marker staining for flow cytometry . 70   2.9.2   Intracellular cytokine staining for flow cytometry 70   2.9.3   Peptide exchange and class I MHC tetramer staining . 71   2.10   Intradermal immunization of mice with plasmid DNA by skin tattoo 72   2.11   Murine model of asthma 72   2.11.1   Intranasal sensitization or challenge of mice . 72   2.11.2   Bronchoalveolar lavage analysis 72   2.11.3   Analysis of lung cells . 73   2.11.4   Culture of lung-draining mediastinal lymph node (MLN) cells 74   2.11.5   Lung histology . 75   Chapter 3.   Expression and purification of recombinant Blo t 78   3.1   Introduction . 78   3.2   The Blo t gene . 79   3.3   Cloning of the Blo t gene into pET 28 expression vector 81   3.4   Expression of recombinant Blo t in E. coli BL 21 . 83   3.5   Purification of recombinant tBlo t 85   Chapter 4.   Production of class I MHC tetramers . 90   4.1 Introduction 90   4.1   Expression and purification of class I MHC proteins . 91   4.2   Testing the functionality of UV cleavable class I MHC tetramers 96   Chapter 5.   Mapping of mite allergen T cell epitopes 99   5.1   Introduction 99   5.2   DNA immunization constructs 102   5.3   DNA vaccination of mice . 109   5.4   Epitope mapping studies 112   5.1.1   Mapping of Blo t epitopes . 116   vii     TABLE  OF  CONTENTS       5.1.2   Mapping of Der p epitopes . 124   5.5   Further characterization of Blo t epitopes 128   5.6   Discussion 138   Chapter 6.   6.1   6.2   6.3   6.4   6.5   6.6   6.7   Der p specific CD8 T cells and HDM-induced asthma. 145   Introduction 145   Immunization of mice 147   Generation of a Der p specific CD8 T-cell line. 153   Characterization of T cell receptor gene 156   Cloning of T cell receptor gene into cassette vectors 162   Role of CD8 T cells in immune response to mite allergens . 173   Discussion 198   Chapter 7.   Final discussion . 203   7.1   Summary of findings 203   7.2   Limitation of current study 210   7.3   Future work . 212   T cells epitopes as tools for immunotherapy . 216   viii     73. 74. 75. 76. 77. 78. 79. 80. 81. 82. 83. 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Journal of Allergy and Clinical Immunology 127: 1562-1570.e1566. 240 [...]... layers of the epithelium and lamina propria of the airways These DCs are at an immature state Therefore, at steady state, uptake and presentation of antigen by these DCs would result in tolerance rather than induce an inflammatory response For example, the administration of ovalbumin (OVA) into the lung of mice had been shown to result in immunologic tolerance rather than an allergic lung response to the. .. immune system is the first component of the immune system to make contact with the allergen and hence a key determinant in the nature of the response mounted against the allergen In many cases, encounter with the allergen results in elimination of the allergen and maintenance of immune tolerance However, in allergic asthma, this may lead to the triggering of an immune response to the allergen 7    ... mediators can profoundly affect the permeability of the vascular endothelium, allowing the migration of circulating immune cells to migrate through the endothelium into the site of inflammation These mediators contribute to the symptoms of the immediate hypersensitivity reaction such as coughing, sneezing, bronchospasm and mucus secretion as well as mediate the recruitment of other immune cells to the. .. disease than in animal models The use of surrogate allergen like OVA in animal studies are likely to complicate matters as true asthma allergens are a complex mix of proteins that may induce responses not seen in the OVA model Current studies are beginning to focus on asthma allergens and their role in the induction of allergic responses and this may provide greater understanding of the role of Th2 cells... inflammatory mediators These include the cytokine TSLP TSLP receptors are found on DCs and bronchial epithelial cells and the presence of TSLP can induce DC activation and the production of IL-13 from epithelial cells TSLP also promote the development of basophils, believed to be a source of early IL-4 that enhances Th2 responses Epithelial cell cultures from asthma patients overproduce GM-CSF, another cytokine... treatment as the disease often varies from patient to patient in severity and causative agent Patients allergic to the same triggering factor 2     CHAPTER  1:  INTRODUCTION       differ in the severity of their responses and many patients were responsive to more than one triggering factor The most common form of asthma and the focus of most of the research in the field is allergic asthma Patients... process, these components can have a profound influence on the nature of the allergic immune response to allergen The airway epithelial cells represent the initial barrier encountered by infectious microbes or allergens Epithelial cells express many PRRs that could detect and respond to a variety of PAMPs and DAMPs Activation of epithelial cell PRRs results in the release of cytokines and chemokines and the. .. role for these cells in asthma pathogenesis Moreover, administration of the NKT ligand α-galactosylceramide (α-GalCer) to the airways induced the production of IL-13 and TSLP by NKT cells (87) γδ T cells can be found at the lung epithelium and the numbers were observed to be higher in asthma patients γδ T cells expressing the Vg1+ TCR were observed to produce IL-5 and IL13 and contribute to AHR Meanwhile,... expressed the Vg4+ TCR appeared to suppress AHR and allergic airway inflammation, a role linked to their secretion of IL-17A (87) 1.2.1.9 The airway epithelium For many years, the study of allergic asthma focused predominantly on the immune cells Now, recent studies have drawn attention to the structural components of the lung and have shown that, rather than being a passive bystander in the inflammatory... resistance 5     CHAPTER  1:  INTRODUCTION       This results in long-term changes to lung function that characterizes chronic asthma Figure 1.1 Overview of the immune responses involved in allergic asthma The protease activity of the allergen allows the allergen to penetrate through the epithelium Activation of PRRs on the dendritic cells lead to the maturation of the dendritic cells and antigen . AN ASTHMA ALLERGEN SPECIFIC ANIMAL MODEL FOR THE STUDY OF RESPONSES TO MITE ALLERGEN INDUCED ASTHMA KENNETH WONG HOK SUM B.Sc. (Hons), NUS A THESIS SUBMITTED FOR THE DEGREE. differ in the severity of their responses and many patients were responsive to more than one triggering factor. The most common form of asthma and the focus of most of the research in the field. asked for a better bunch of people to work with. Thank you especially to Yafang and Sophie for guiding me with the asthma studies. Thank you as well to Nayana for all the discussions and your