IMPERIAL COLLEGE LONDON Regulatory B cells in seasonal allergic rhinitis and the influence of grass pollen immunotherapy JAMES EDWARD GERALD CHARLESWORTH Allergy and Clinical Immunology National Heart and Lung Institute Thesis submitted to Imperial College London in candidature for the degree of DOCTOR OF PHILOSOPHY (PhD) February 2015 Abstract Introduction: Interleukin (IL)-10-producing B cells (Bregs) regulate immune responses in autoimmune disease; however their role in allergy is unclear. Allergen exposure in predisposed atopic individuals results in the induction of IgE-secreting B cells, crucial in the immunopathophysiology of allergic rhinitis. Allergen-specific immunotherapy (AIT) is the only disease-modifying treatment for allergic rhinitis. AIT results in long-term clinical and immunological tolerance; however, whether Bregs contribute towards AIT-induced tolerance remains unclear. Hypotheses: 1. In vitro induced IL-10-producing B cells regulate allergen-driven Th2 inflammation, 2. Bregs are present in fewer numbers in seasonal grass pollen allergic (SAR) individuals compared with healthy controls, which is restored during AIT. Methods: B cells were isolated and subjected to flow cytometry to detect surface markers and IL-10 capacity following CpG stimulation. FluoroSpot, ELISA or qPCR were used to confirm IL-10. Suppression of T cell proliferation (by CFSE) and cytokine production (by ELISA) were carried out in co-cultures. Regulatory B cells in SAR (n=14), AIT (n=18) and healthy (n=14) donors were compared. Nasal allergen challenge (NAC) was carried out, with blood taken pre and post challenge for flow cytometry. Results: CpG significantly enhanced proportions of Bregs, with enrichment particularly within CD24hiCD27+, CD5hi, PD-L1+ and CD24hiCD38hi populations. Bregs suppressed both polyclonally- and grass pollen allergen-stimulated T cells. Ex vivo, proportions of IL-10+ B cells from SAR and healthy donors matched, but were significantly greater amongst AIT donors (particularly sublingual immunotherapy - SLIT) compared to SAR. Following NAC, proportions of B cells within CD24hiCD38hi, CD5hi, CD24hiCD27hi and CD25+ subsets were significantly increased amongst non-allergic and AIT groups, but not amongst SAR donors. Conclusion: Bregs are capable of suppressing allergen induced, Th2-driven inflammation in vitro and may be involved in the induction of tolerance during allergen immunotherapy in vivo, particularly following SLIT. 2 Declaration I confirm that the contents of this thesis are my own work; all laboratory-based experiments were performed by me. Clinical and diagnostic procedures were carried out by the persons acknowledged overleaf, for which I am extremely grateful. Experimental assistance, laboratory guidance and supervision are also acknowledged overleaf. A full list of references is given, with citing throughout the text where evidential assertions have been made. This work has not been submitted in application for any other higher degree. The copyright of this thesis rests with the author and is made available under a Creative Commons Attribution Non-Commercial No Derivatives licence. Researchers are free to copy, distribute or transmit the thesis on the condition that they attribute it, that they do not use it for commercial purposes and that they do not alter, transform or build upon it. For any reuse or redistribution, researchers must make clear to others the licence terms of this work. 3 Acknowledgements I am extremely grateful for this project to my supervisors, Professor Stephen Durham and Dr David Cousins. I owe the completion of this thesis to Prof Durham’s attention to detail and endlessly making time to read repeated drafts. Dr David Cousins was also able to coach me through a microarray, the figure included in the appendix was analysed by him. I am grateful to Dr Mohamed Shamji for the proposal of this project and for laboratory supervision. I am also extremely grateful to have been part of the MRC and Asthma UK PhD programme. The MRC centre has offered a hugely supportive peer and PI network and regular enjoyable seminars with world class researchers. I had the opportunity to present nationally and internationally with funding provided by the BSI and BSACI during my PhD, for which I am very grateful and has contributed to the project and my development. I wouldn’t have completed any experiments without the guidance, education, techniques and tips from those in the laboratory at the time I started. The research technicians, Mimi Poon and Delica Chung, were my lifeline when I first started. I am greatly indebted to the final year PhD students during the first year of my PhD, now Dr Bryony Stott and Dr Pascal Venn, who not only provided light relief and a friendly ear but also imparted their wealth of experience, taught me most of my techniques, ensured I was including the relevant controls and helped me plan my project properly. To Janice Layhadi, who started as a technician at the same time as me in the lab; I’m grateful for her support as we learnt our way around the lab together. A great debt of gratitude goes to Dr Mikila Jacobson, with whom it has been a real pleasure to work and is a font of all knowledge on immunohistochemistry and teaches a great deal of common (scientific) sense when planning, carrying out and interpreting experiments. I have greatly enjoyed working with Natalia Couto-Francisco who always brought very thoughtful discussions to the lab, as well as so much enthusiasm! I also can’t fail to mention Dr Tomokazu Matsouka, who brought a great deal of experience and wisdom to the lab as well as fantastic tales of a career as a physician scientist! I also owe a debt of gratitude to the supervisor of my master’s project, Dr Alistair Noble, who taught me flow cytometry, skills that have been essential and built on throughout my PhD. This work relied on the ability to use human samples, obtained by the clinical team, and the ability to recruit and support the blood donors was crucial to the progress of this project. I can’t fail to thank the donors for their essential contribution to this project. A huge debt of thanks for this goes to Shireen Quli Khan, Rachel Yan and Andrea Goldstone for arranging and bleeding volunteers for me throughout this project. I would also like to thank Andrea for her support, advice and direction when I was making the decision to apply to medicine and for very kindly (and expertly) training me in phlebotomy. When 4 at the clinic, Dr Moises Calderon was always a pleasure to work with, cheerful regardless of how early in the morning or late in the evening I bumped into him and always concerned that I was able to get hold of the samples I needed to complete my studies. The clinical administrators, Natalia Kimowska- Nassar and latterly Mimi Poon, were of immense help to me in navigating forms and finding my way around the clinic. They have also been a friendly ear for a world of advice throughout, who always made trips to the clinic a pleasure. Key to this thesis is the cross sectional study of participants who were recruited as part of Dr Guy Scadding’s PhD. Guy, as well as being a font of allergy knowledge and the most cheerful and pleasant colleague one could hope for, has been extremely kind by incorporating into the study protocol a dedicated blood collection to isolate B cells and for phenotyping of B cells within the nasal allergen challenge. I would like to acknowledge the contributions to this thesis made by Dr Guy Scadding and Dr Arif Eifan who performed the intradermal allergen challenges and nasal allergen challenges for the cross sectional study. The clinical data, as a result of these challenges, is presented here processed by Mimi Poon, who also processed participant data including age, gender, screening results and treatment received. Participants were recruited and screened by the extremely dedicated research nurses, Rachel Yan and Andrea Goldstone. To my friends in Leukocyte Biology and at King’s, thank you for getting me though, for your company, conversation and including me in your PhD journal club! Especially Bex, Kate, Jess, Maryam, Natasha, Thanu, Pallavi, Cheryl and Andia. To my family Mum, Dad, Caroline and Lawrence, thank you for never pressuring me towards anything but letting me follow my interests and indulging my curiosity. Especially the parents for encouraging me in whatever route I took. And of course Chantal for things too numerous to mention and without whom I couldn’t have got through these many years! Clearly I’ve been lucky to have a B cell biologist for a fiancée, who not only understands my experiments and the stress but also asks me the best questions about my work. This project has often kept us apart but I’m incredibly lucky to receive patience and understanding when I’ve returned from the lab late at night or, on occasion, in the early morning and for extensive proof reading of this thesis. Chantal’s given me enormous support and encouragement when making the decision to apply for medicine following this long slog for which I can only hope to repay, thank you! 5 Abstracts relating to this thesis J.E.G. Charlesworth, S.M. Quli Khan, G.W. Scadding, D.J. Cousins, S.R. Durham and M.H. Shamji IL-10- producing regulatory B cells suppress allergen-specific T cell proliferation and are elevated ex vivo following specific immunotherapy - - Clinical and Experimental Allergy, 2012, vol.42(12):p1817 James E. G. Charlesworth, Andrea Goldstone, Moises A. Calderon, David Cousins, Stephen R. Durham, Mohamed H. Shamji Grass Pollen Allergics Have Fewer IL-10-Producing B Cells Than Non-Atopic Controls -– J. Allergy and Clinical Immunology, 2013, vol.131(2 supplemental):pAB204 J. Charlesworth, A.