ANTIGEN-SPECIFIC EFFECTOR CD8 T CELLS REGULATE ALLERGIC RESPONSES VIA IFN-γ AND DENDRITIC CELL γ FUNCTION TANG YAFANG BSc (Honors), NUS A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY NUS GRADUATE SCHOOL FOR INTEGRATIVE SCIENCES AND ENGINEERING NATIONAL UNIVERSITY OF SINGAPORE 2012 Abstract While the pivotal role of CD4 T cells has been extensively studied in allergic asthma, the role of CD8 T cells remains controversial Previous studies have shown that CD8 T cells can both prevent and cause allergic responses However, the underlying mechanisms remain to be elucidated In this study, we aim to investigate the potential of CD8 T cells with different IFN-γ expressions to modulate the elicitation of allergic inflammation following ovalbumin (OVA) challenge and investigate the underlying mechanisms To study the role of IFN-γ in the effect of CD8 T cells, effector CD8 T cells from OT-I and IFN-γ-/-OT-I mice were transferred to OVA-sensitized mice one day before the consecutive challenges The effect on lung dendritic cells (DCs) exerted by CD8 T cells was studied with ex vivo culture of sorted DCs from treatment mice with either naïve or antigen-experienced CD4 T cells We found that effector OT-I, but not IFN-γ-/-OT-I CD8 T cells attenuated eosinophilia and mucus secretion in the lungs of sensitized mice in an antigen-specific manner Effector IFN-γ-/-OT-I CD8 T cells displayed a Tc2/Tc17-biased phenotype with weaker cytotoxicity and were able to both induce and exacerbate eosinophilia as well as neutrophilia OT-I CD8 T cells increased the ability of lung CD11b+CD103- DCs to both prime the differentiation of naïve CD4 T cells toward a Th1 phenotype and enhance IFN-γ production by antigen-experienced lung CD4 T cells In conclusion, effector CD8 T cells attenuate pulmonary inflammation and alter the ability of DCs within the allergic lung to polarize T cells to a Th1 phenotype during a Th2 response In the absence of IFN-γ, CD8 T cells assume a Tc2/Tc17-biased phenotype and potentiate inflammation Acknowledgements To Prof Kemeny You have been a wonderful supervisor: giving me enough freedom to explore my interest; willingly sacrificing your weekend to make time for appointment; encouraging me even when the result is absolutely negative; genuinely caring about my future career and life; generously allowing us to take leave to explore the world both scientifically and scenically The list can just go on Thank you for being more than merely a supervisor to me and guided me through issues and issues like an old wise friend I am grateful to all the help and encouragement that you gave me which meant a lot to me To Prof MacAry Thank you for giving me advices on my project and helping me edit my manuscript To the girls and boys in the cave It has been a pleasant experience with you guys around, making jokes and cheering each other up It is such a nice thing that we can share so many secrets and comments without worrying about being sabotaged To Shu Zhen and Pey Yng We have been in this lab together for years! I am lucky to have you gals with me to go through all the pains and share our joys We traveled to Europe, Japan and US together, laughed together, complained together, cursed together (maybe not so much for Pey Yng, you are just too nice)…You are truly great friends whom I am gonna really miss after we graduate! And I am so happy for you that both of you are now Mrs To Sophie I was supposed to be your mentor and help you with your project, but it seemed that you helped me more! Especially during my manuscript revision, you had to get up before sunrise in order to help me Besides work, you are a great friend as well We shared a lot of things and emotions and I wish that you stay happy and cheerful To Adrian You are such a happy person who brought lots of laughter to the cave You are also the lab encyclopedia Looking for something? Need help? Get Adrian Thanks for all the brain storming and advices regarding my project as well as the painful manuscript editing To Kenneth You never say no Even when you do, people take it as yes You are such a nice person! Thanks for staying late and sometimes coming back on weekends to help me make tetramers You are also very helpful on whatever matter not related to experiment and I am so used to turn around and ask “Hey, can me a favor?” To Moyar When you are in the mood, you can light up the room Stay enthusiastic and energetic, we need the spirit! It was great fun in Europe especially Austria with your company, the hailstone, the strawberries etc To Isaac We have been lab mates for years as well! We have been lunch buddies, photo taking buddies, KTV buddies and OT buddies (for a short while) It is great to have your company To Richard Thanks for helping me a lot with my manuscript and also being the only one who stayed back and helped me with my experiment after the chemical explosion incident I wish you best luck with your mandarin study To Nayana We got to really connect to each other during our SF trip You are fun and comfortable to be with Thank for being so sisterly and caring To Fei Chuin and Paul Thanks for all the time you spent sorting cells for me Special thanks to Fei Chuin for being a caring elder sister to me To Kok Loon Thanks for all the fun moments we shared and the effort you put in in my project and manuscript I wish your MD journey smooth and fulfilling To DMK lab I am grateful to all your help in various ways, Benson for the all the mice and ordering and the rest for making the lab such a pleasant place To my dear family Thank you for bearing with me and supporting me for such a long time, for all my mood swings and being far away from home I shall spend more time with you while making you proud of me To Li Chao You have been there for me all the time, for all the happiness, sadness and frustrations Thank you for providing me a safe harbor where I can hide myself when I am not brave enough You mean a lot to me Table of Contents CHAPTER 1: Introduction 17 1.1 Asthma 17 1.1.1 Asthma prevalence and economic burden 18 1.1.2 Clinical symptoms of asthma and pathology 19 1.1.3 Animal models of asthma 20 1.2 The immune system and asthma 24 1.2.1 Innate immune responses in asthma 28 1.2.1.1 Macrophages in asthma 28 1.2.1.2 Mast cells in asthma 30 1.2.1.3 Eosinophils and neutrophils in asthma 31 1.2.1.4 Basophils in asthma 32 1.2.1.5 Epithelial cells in asthma 34 1.2.2 DCs in asthma – bridging innate and adaptive immunity 37 1.2.3 Adaptive immune responses in asthma 41 1.2.3.1 Humoral immune responses in asthma 41 1.2.3.2 Cell-mediated immune responses 42 1.2.3.2.1 CD4 T cells in asthma 43 1.2.3.2.2 CD8 T cells in asthma 48 1.3 Aims of the study 53 CHAPTER 2: Materials and Methods 55 2.1 Media and buffers 55 PBS buffer 55 MACS buffer 55 FACS buffer 55 Permeabilization buffer for intracellular staining 55 Optiprep density centrifugation media for lung DC isolation 56 Liberase for tissue digestion 56 Complete medium for cell culture 57 Complete DMEM for cell culture 57 Buffers for ELISA 58 RBC lysis buffer 58 Paraformaldehyde (4%) 58 2.2 Mice …………………………………………………………….…… ….… 59 2.3 Asthma model: Sensitization and airway challenges 59 2.3.1 Precipitation of OVA-alum 59 2.3.2 Sensitization and challenge protocol 60 2.4 CD8 T cell isolation, activation, CFSE labeling and adoptive transfer 61 2.4.1 Isolation and activation of CD8 T cells 61 2.4.2 CFSE labeling and adoptive transfer 63 2.5 Bronchoalveolar lavage analysis 64 2.6 Ex vivo assay of lung parenchymal dendritic cells 65 2.7 Lung histology 67 2.7.1 Preparation of lung tissue 67 2.7.2 Processing and sectioning of lung tissue 69 2.7.3 Mount tissue in a cassette 69 2.7.4 Staining 70 2.7.4.1 Materials: 70 2.7.4.2 Procedures: 71 2.8 Ex vivo CFSE proliferation assay 73 2.9 Intracellular staining of cells for FACS analysis 73 2.10 Assessment of airway function 74 2.11 Measurement of cytokines 75 2.12 Measurement of serum immunoglobulins 77 2.13 CTL killing assays 78 2.13.1 51 Cr release assay 78 2.13.2 CD107α degranulation assay 79 2.14 Genotyping………………………………………………………….… …… 80 2.15 Statistical analysis 82 2.16 List of Antibodies Used 84 CHAPTER 3: OVA immunization and challenge induced robust allergic responses 85 3.1 Introduction 85 3.2 OVA immunization and challenge protocol 87 3.3 Increased serum immunoglobulin production in OVA immunized and challenged mice 87 3.4 Increased eosinophil and neutrophil infiltration in the airway in OVA immunized and challenged mice 89 3.5 Enhanced cell infiltration and mucus production in the lung in OVA immunized and challenged mice 93 3.5 Enhanced cell infiltration and mucus production in the lung in OVA immunized and challenged mice 93 3.6 Increased airway hyperresponsiveness (AHR) in OVA immunized and challenged mice 95 3.7 Discussion 97 CHAPTER 4: Phenotypic characterization of in-vitro activated CD8 T cells and their recruitment into OVA-immunized mice 100 4.1 Introduction 100 4.2 The generation of IFN-γ-/-OT-I mice 101 4.3 Effector phenotype of activated CD8 T cells 104 4.4 Differential transcription factor expression of activated CD8 T cells 107 4.5 Cytokine production by activated CD8 T cells 109 4.6 Distinct cytotoxicity properties of activated CD8 T cells 111 4.7 Similar proliferation index of CD8 T cells 114 4.8 CD8 T cell transfer protocol 116 4.9 Efficient recruitment of effector CD8 T cells into the lungs of sensitized mice………………… 117 4.10 Similar in vivo proliferation rate of transferred CD8 T cells 120 4.11 Discussion 122 CHAPTER 5: Effector OT-I but not IFN-γ-/-OT-I CD8 T cells attenuate Th2 γ responses 126 5.1 Introduction 126 5.2 Unchanged serum immunoglobulin production in response to CD8 T cell transfer………… 128 5.3 Altered cytokine and chemokine production in the bronchoalveolar lavage in response to CD8 T cell transfer 129 5.4 Inhibition of eosinophil infiltration by OT-I CD8 T cells and enhancement of eosinophil and neutrophil infiltration by IFN-γ-/-OT-I CD8 T cells 131 5.6 Persistent airway hyperresponsiveness following CD8 T cell transfer 136 5.7 Discussion 138 CHAPTER 6: Effector OT-I CD8 T cells interact with DCs and condition them for Th1 priming 141 6.1 Introduction 141 6.2 Unaffected cell infiltration in the airway following wild type CD8 T cell transfer ……………………………………………………………………………143 6.3 Increased antigen presenting cells in the lung in response to CD8 T cell transfer………… 146 6.4 Purification of CD11b+CD103- and CD11b-CD103+ DCs from the lung 148 6.5 Conditioning of naïve CD4 T cells by lung DCs in response to CD8 T cell transfer………… 151 6.6 Conditioning of antigen-experienced lung CD4 T cells by DCs in response to CD8 T cell transfer 155 6.7 Discussion 157 CHAPTER 7: IFN-γ neutralization restores allergic inflammation inhibited by γ OT-I CD8 T cells while IFN-γ-/-OT-I CD8 T cells induce asthma-like pathology γ 161 7.1 Introduction 161 7.2 Eosinophil and neutrophil infiltration in the airway in response to CD8 T cell transfer and IFN-γ neutralization 163 7.3 Mucus secretion in the lung in response to CD8 T cell transfer and IFN-γ neutralization 166 7.4 Conditioning of naïve CD4 T cells by lung DCs in response to CD8 T cell transfer and IFN-γ neutralization 169 7.5 Conditioning of antigen-experienced lung CD4 T cells by DCs in response to CD8 T cell transfer and IFN-γ neutralization 171 7.6 Eosinophil and neutrophil infiltration in the airway in response to CD8 T cell transfer in IFN-γR-/- mice 173 7.7 Discussion 175 CHAPTER 8: Effector IFN-γ-/-OT-I CD8 T cells induce an asthma-like pathology γ while OT-I CD8 T cells not 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CD4 T cells in asthma CD4 T cells are recognized as helper T cells as opposed to cytotoxic CD8 T cells CD4 T cells are not directly involved in phagocytic or cytotoxic activities, but rather... for other cells such as B cells (for affinity maturation and isotype switching), CD8 T cells and macrophages, either through cell- cell interaction or through the cytokines they produce CD4 T cells, ... institute T- bet T- box expressed in T cells TCR T- cell receptor TGF-β Transforming growth factor beta TLR Toll-like receptor TNF-α Tumor necrosis factor α TSLP Thymic stromal lymphopoietin WT Wild