Conditioning Murine B Cells for. Immunoglobulin A Production In vitro

Transcription

1 Conditioning Murine B Cells for Immunoglobulin A Production In vitro by Wilford Goh BSc Biomedical Science This thesis is presented for the Honours degree in Biomedical Science at Murdoch University School of Veterinary and Biomedical Sciences Murdoch University Western Australia November 2012

2 Declaration I declare this thesis is my own account of my research and contains as its main content, work which has not been previously submitted for a degree at any tertiary educational institution. Wilford Goh ii

3 Abstract The respiratory mucosa is continuously exposed to a myriad of pathogens and allergens that are inhaled during respiration. B cells of the immune system produce immunoglobulin A (IgA) that protects the respiratory mucosa from inhaled pathogens and allergens. IgA is produced after naïve B cells undergo activation, proliferation and differentiation, developing into antibody producing plasma cells. The process that facilitates IgA production as B cells develop into plasma cells is known as class switch recombination (CSR). Factors favouring IgA have been determined by in vitro studies of CSR. These established two cytokines, IL-21 and TGF-β1, to be important factors for IgA production. Prior studies with human naïve B cells that were cultured with IL-21 and TGF-β1, in addition to anti-igm, anti-cd40, IL-2 and CpG, developed into precursors of IgA producing plasma cells (plasmablasts) that possessed mucosal homing capabilities: a process termed B cell conditioning. The aim of this project was to establish whether the same factors were active on murine naïve B cells in order to eventually test the in vivo therapeutic potential of B cell conditioning. A protocol was optimised for CD19 + CD27 - naïve B cell isolation from mouse spleen and cell division, which is crucial for IgA CSR, by CFSE labelling and flow cytometry. The number of divisions predicts the generation of IgA mucosal homing plasmablasts in vitro. Cells were found to have undergone 5 divisions after 7 days in culture suggesting that cells had undergone a sufficient number of divisions for CSR and IgA production. Attempts were made to quantitate IgA production in culture supernatants, however evidence for the generation of these cells by IgA production remains to be confirmed. The results of this project provide iii

4 preliminary evidence that murine naïve B cells can be conditioned in vitro for IgA production, with potential for homing to, and protection of, mucosal surfaces in vivo. iv

5 Table of Contents Declaration Abstract Index to Figures List of Abbreviations Acknowledgements ii iii x xii xiv Chapter 1. Literature Review and Introduction Organisation of the Respiratory Tract Respiratory Mucosal Protection From Infections and Allergens Physical and Chemical Protection of the Respiratory Mucosa Immunological Protection of the Respiratory Mucosa Innate Mucosal Responses Adaptive Mucosal Immune Responses Immune Sites of the Respiratory Mucosa Inductive Sites Effector Sites Naïve B Cell Development and Maturation into Mucosal Plasma Cells Development of Naïve B Cells Naïve B Cell Activation T Cell Independent Activation T Cell Dependent Activation 16 v

6 1.6.3 Germinal Centre Reactions Generate Plasmablasts Somatic Hypermutation Class Switch Recombination Plasmablasts Migrate to Effector Sites and Terminally Differentiate into Plasma Cells IgA is Crucial for Respiratory Mucosal Immunity Physical and Structural Properties of IgA Secretion of Dimeric IgA onto the Respiratory Mucosa Mechanisms of Pathogen Removal by Dimeric and Secretory IgA at the Respiratory Mucosa Immune Exclusion Intraepithelial Neutralisation Excretion of Immune Complexes Conditioning of B cells for Immunoglobulin A Production 24 Chapter 2. Aims and Hypothesis 26 Chapter 3. Materials and Methods Animal Agistment Euthanasia and Tissue Collection Naïve B cell Extraction and Enrichment Preparation of Splenocyte Suspension Enrichment of Splenocyte Suspension for Naïve B Cells Naïve B Cell Labelling and In vitro Conditioning 34 vi

7 3.4.1 CFSE Labelling for Tracking Cell Division In vitro B Cell Conditioning Sample Collection and Storage Flow Cytometric Analysis of Labelled Cells Instrumentation and Analysis Software Optimisation of FACS Staining by Titration Fluorochrome-conjugated Monoclonal Antibodies Biotin-conjugated Monoclonal Antibody LIVE/DEAD Near-IR Fixable Cell Stain CellTrace CFSE Cell Proliferation Stain Staining Panels Enrichment Staining Proliferation Staining Controls and Compensation Early Cell Gating Strategy IgA Enzyme-linked Immunosorbent Assay ELISA Optimisation and Controls Coating and Blocking of Microplates Standard, Sample and Control Incubation IgA Detection and Colour Development Absorbance Measurement and Data Analysis Statistical Analysis and Graphing Software 45 vii

8 Chapter 4. Results I: Experimental Optimisation Titrations Monoclonal Antibody Titres Viability Dye Titre CFSE Titre Compensation Matrix for Enrichment and Proliferation Panels ELISA Optimisation LPS Titration 57 Chapter 5. Results II: Experimental Findings Magnetic Depletion Yielded High Naïve B Cell Purity of 93% Modified Separation Protocol Removed CD27 + Subpopulations to Different Extents Trends in Cell Viability Revealed Extent of Cell Proliferation Comparing Cell Divisional Responses Between Different Conditions Majority of Conditioned Cells Underwent 3 Rounds of Division 72 Chapter 6. Discussion Magnetic Isolation for Obtaining High Naïve B Cell Purity Naïve B Cell Activation TD Activation TI Activation Cell Proliferation IgA Production 82 viii

9 6.5 Immunophenotyping for Better Resolution of B Cell Populations Refining CFSE Labelling for Better Determination of Cell Division Future Directions Conclusion 87 References 88 Appendix A: Reagent List and Preparatio 97 Appendix B: Animal Monitoring Sheet 101 ix

10 Index to Figures Figure 1.1 Mucosal surfaces of the body Figure 1.2 Mucociliary escalator of the respiratory tract Figure 1.3 CD4+ T cell subsets Figure 1.4 Inductive and effector site of mucosa-associated lymphoid tissue Figure 1.5 Structural organisation of a lymph node Figure 1.6 Structural organisation of the mucosa-associated lymphoid tissue Figure 1.7 Structure of IgA Figure 1.8 Structure of dimeric IgA Figure 1.9 Release of secretory IgA (SIgA) into the airway Figure 3.1 Extraction procedure of splenocytes from mouse spleen Figure 3.2 Isolation of naïve B cells by magnetic depletion Figure 3.3 Naïve B cell isolation protocol Figure 3.4 Cell and culture supernatant collection protocol Figure 3.5 Early cell gating strategy Figure 4.1 PE-CD19 MAb titration Figure 4.2 APC-CD27 titration Figure 4.3 Secondary control for APC-CD Figure 4.4 Titration of viability dye Figure 4.5 CFSE titration Figure 4.6 Standard curves for identical mouse IgA standards generated before and after ELISA optimisation Figure 4.7 Cross reactivity with IgG1 and media interference Figure 4.7 LPS titration Figure 5.1 Naïve B cell purity before and after isolation x

11 Figure 5.2 Gating based on CD19 and CD27 FMO control Figure 5.3 The average naïve B cell purity before and after enrichment Figure 5.4 Removal of CD27+ cells after enrichment Figure 5.5 Viability of cells cultured in different conditions for 12 days Figure 5.6 Viability of cells cultured in different conditions for 7 days Figure 5.7 Estimation of cell division by analysis of CFSE fluorescence intensity.. 69 Figure 5.8 Comparison of % cell divided and proliferation index for different culture conditions at day Figure 5.9 Changes in the number of cell divisions for cells cultured over 12 days and 7 days in condition xi

12 List of Abbreviations 2-ME AID AMDCs APC ANOVA ARC BCR BP BER BALT CFSE CCR CD40L CSR CD CXCR CpG CTL DC DNA DMSO DPBS ELISA FMO FACS FAE FDC FSC Fab Fc GIT GC HI FBS C H HSC HRP FcαR Ig IL IL-2R IL-21R IEL kda LPS LN 2-mercaptoethanol Activation-induced deaminase Airway mucosal dendritic cells Allophycocyanin Analysis of variance Animal Resources Centre B cell receptor Band pass Base excision repair Bronchus-associated lymphoid tissue Carboxyfluorescein C-C chemokine receptor CD40 ligand Class switch recombination Cluster of differentiation CXCR4 Cytosine phosphate-guanosine dinucleotide motif Cytotoxic lymphocyte Dendritic cells Deoxyribonucleic acid Dimethylsulphoxide Dulbecco's phosphate buffered saline Enzyme-linked immunosorbent assay Fluorescence minus one Fluorescent activated cell sorting Follicle associated epithelium Follicular dendritic cells Forward scatter characteristic Fragment antigen-binding Fragment crystallisable gastrointestinal tract Germinal Heat inactivated fetal bovine serum Heavy chain Hematopoietic stem cell Horse radish peroxidase IgA Fc receptors Immunoglobulin Interleukin Interleukin-2 receptor Interleukin-21 receptor Intraepithelial lymphocytes Kilo dalton Lipopolysaccharide lymph node xii

13 MHC II MZ M cell MAb MALT NALT NK cells Near-IR NFκB PE PB pigr RPMI SC SIgA SSC SSB SHM SEM SA TD TI T FH T reg T H TMB TLR TβR TGF-β1 UNG V H V L αtp Major histocompatibility complex class II Marginal zone Microfold cell Monoclonal antibody Mucosal-associated lymphoid tissue Nasopharynx-associated lymphoid tissue Natural killer cells Near-infrared Nuclear factor kappa B Phycoerythrin Plasmablast Poly-immunoglobulin receptors Roswell Park Memorial Institute Secretory component Secretory IgA Side scatter characteristic Single-strand break Somatic hypermutation standard error of mean Streptavidin T cell dependent T cell independent T follicular helper cell T regulatory cell T helper cell Tetramethylbenzidine Toll-like receptor Transforming growth factor β receptor Transforming growth factor β1 Uracil-DNA glycosylase Variable binding site of heavy chain Variable binding site of light chain α tail-piece xiii

14 Acknowledgements What a journey it has been. I am now ready to embark on another journey, my holiday that is. Before this journey officially concludes, I would like to thank the many people who have made this journey bearable. My supervisors Sarah, Phil, Deb and John are top of the list. I have learnt so much from our interactions. They have granted me all the freedom I could ever ask for as a honours student, thus allowing me to enjoy the entire show. Besides the official supervisors, I also have many surrogate supervisors to thank. These people have seen the best and worst of me. We ve had good laughs at my best, although their ears bled and suffered at my worst. Nevertheless, I claim no part in their loss of hearing in the future. Tracey and Michelle the flow gurus, Jenny the jack of all trades, and Jay the wise man, have all imparted valuable skills and knowledge to me. Of course, there are the other surrogate supervisors who have had their fair share of ear bleeds and migraines due to my winging. These people have been so overwhelmingly positive and encouraging that my own pessimism (or in my defence, just being down to earth) had to abandon me. The outcome, a confident me who can make my own decisions (slowly getting there) and stand up for myself, which I have been doing since I officially became bipedal. These people, in no particular order, other than wonderous ways a sleep deprived brain works, are Mum, Ivan, Sian, Catherine, Rebecca, Kathryn, Narelle, Chelsea, Penny, Maria, John and Susan. Thanks guys, for being here with me on my journey. xiv