This electronic thesis or dissertation has been downloaded from the King’s Research Portal at https://kclpure.kcl.ac.uk/portal/ Tissue-specific butyrophilin-like proteins are TCR selecting ligands distinct from antigens Zlatareva, Iva Awarding institution: King's College London The copyright of this thesis rests with the author and no quotation from it or information derived from it may be published without proper acknowledgement. END USER LICENCE AGREEMENT Unless another licence is stated on the immediately following page this work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International licence. https://creativecommons.org/licenses/by-nc-nd/4.0/ You are free to copy, distribute and transmit the work Under the following conditions: Attribution: You must attribute the work in the manner specified by the author (but not in any way that suggests that they endorse you or your use of the work). Non Commercial: You may not use this work for commercial purposes. No Derivative Works - You may not alter, transform, or build upon this work. Any of these conditions can be waived if you receive permission from the author. Your fair dealings and other rights are in no way affected by the above. Take down policy If you believe that this document breaches copyright please contact [email protected] providing details, and we will remove access to the work immediately and investigate your claim. Download date: 10. Oct. 2021 Tissue-specific butyrophilin-like proteins are gdTCR selecting ligands distinct from antigens Iva Ivanova Zlatareva King’s College London PhD Supervisors: Professor Adrian C. Hayday and Dr James N. Arnold A thesis suBmitted for the degree of DoCtor of Philosophy King’s College London January 2020 1 Declaration I Iva Zlatareva confirm that the work presented in this thesis is my own. Where information has been derived from other sources, I confirm that this has been indicated in the thesis. The majority of the data presented in chapters III, IV and V were published in Melandri, et al., 2018 and Willcox et al., 2019. 2 Abstract Tissue-resident gd T cells are a population of unconventional lymphocytes able to mount rapid immune responses following tissue damage. It has long been debated whether selecting elements for gd T cells exist, akin to MHC for ab T cells. In mice, gd T lymphocytes show tissue- restricted T cell receptor (TCR) repertoires, which are dependent, at least in the epidermis and gut, on the tissue-specific expression of butyrophilin-like proteins (Btnl). Hence, we hypothesised that the Btnl family of proteins are physiological ligands modulating gd T cell selection and function. We have previously established in vitro that BTNL3 and BTNL8, specifically expressed in the human intestinal epithelium, induce selective TCR-dependent responses in a subset of colonic gd T cells. The aim of this project was to investigate the mechanism of interaction between BTNL3 plus BTNL8 and the TCR of responding cells. To this end, responding, human colonic intraepithelial lymphocytes (IELs) co-cultured with BTNL3 plus BTNL8-expressing cells were single cell-sorted and sequenced. Majority of responding cells were clonally diverse Vg4+ lymphocytes and several paired gdTCR sequences were expressed in TCR-deficient Jurkat cells. In co-culture assays, Vg4-expressing Jurkats downregulated their TCR and upregulated CD69 when exposed to BTNL3 plus BTNL8. Site- directed mutagenesis showed unexpectedly, that the hypervariable loop 4 (HV4) of the Vg4 chain was critical for BNTL3-specific recognition. Intriguingly, CD1c-phosphatidylcholine- reactive Vg4Vd1 and endothelial protein C receptor (EPCR)-reactive Vg4Vd5 cells were capable of mounting a response against BTNL3 plus BTNL8 deeming them dually reactive. Importantly, CD1c and EPCR recognitions were CDR3-mediated. Thus, human Vg4+ TCRs appear to have two spatially distinct regions involved in antigen recognition. The adaptive, non-germline encoded gdCDR3 loops are required for binding to clonally-restricted ligands whereas the innate, germline-encoded HV4g loop is required for interaction with the tissue-specific complex BTNL3/8. The same phenomenon is true for murine intestinal Vg7+ cells which require Btnl1/4/6 for their development and tissue retention. Although these molecules are not direct human orthologues, an evolutionarily conserved mechanism emerges. BTNL3/8 are perhaps selecting elements of intestinal Vγ4+ IELs and future studies should investigate the biological significance of these interactions in humans. Of note, early studies presented in this thesis suggest that BTNL3 and BTNL8 heteromeric complex is partially depleting the Vg4 TCR from the lymphocyte surface, suggesting an immunoregulatory role for this interaction. 3 Acknowledgements I would like to thank my supervisor Prof Adrian Hayday for giving me the opportunity to work on this exciting project, for his insight, support, contagious enthusiasm for this field and for his critical feedback on all my work, reports, presentations and this thesis. I would also like to thank my second supervisor, Dr James Arnold, and my thesis committee members, Dr Patricia Barral and Dr Susan John, for their support and advice throughout my studies. A special thanks to Dr Susan John in particular, for also providing me with moral support and the occasional shoulder to cry on. I have thoroughly enjoyed our long discussions about life and science. I would like to acknowledge the National Institute for Health Research (NIHR) Biomedical Research Centre based at Guy’s and St Thomas’ NHS Foundation Trust and King’s College London who funded this PhD. This work would not have been possible without our collaborators: Dr Salah Mansour and Dr Andrew Chancellor from the University of Southampton; Dr Daniela Wesch and Professor Dieter Kabelitz from Kiel; Dr Vivian Li and Dr Laura Novellasdemunt from the Francis Crick institute; and Dr Oxana Polyakova and Dr Oliver Nussbaumer from Gamma Delta Therapeutics, all of whom provided critical reagents or protocols for this study. Special thanks are due to Prof Benjamin Willcox, Dr Carrie Willcox, Dr Mahboob Salim and the rest of the Willcox laboratory from the University of Birmingham for conducting all the biochemical studies which helped prove a direct interaction; to Dr Peter Irving from Guy’s and St Thomas’ NHS foundation Trust who provided access to patient samples; and Raphael Chaleli and Prof Paul Bates from the Francis Crick institute who did the BTNL3 and BTNL8 modelling and docking experiments. Several members of the Hayday laboratory have been responsible for my scientific training. Particularly, I would like to thank Dr Pierre Vantourout for his mentorship and constant guidance throughout my PhD. Thank you for putting up with all my questions and teaching me the wonders of molecular biology. Thank you for being not only my teacher, but also a friend, supporting me in time of crisis, nervous breakdowns and self-doubt – I would not have survived this PhD without your support! I would also like to thank Efstathios Theodoridis, Dr Fernanda Kyle, Dr Shraddha Kamdar, Dr Abhishek Das, Dr Magdalen Joseph, Dr Adam Laing, Dr Robin Dart, Dr Yin Wu and Dr Yasmin Haque all of whom have helped me numerous times with scientific discussions, data analysis and thought me new techniques or were just there to brightened my day. It has been a pleasure working alongside you every day. I thank Daisy Melandri who conducted parallel studies in mice which helped generalize the findings in this thesis across species. Finally, I am grateful to all members of the Hayday laboratory, past and present, for 4 creating such a stimulating research environment in which I had the opportunity to develop as a scientist. Outside of the laboratory, I would like to thank my friends and family for their constant support and love throughout the years. Special thanks are due to my mother who has always supported me even though she never quite understood what I was doing and why I chose this career path. Thank you for always believing in me! Finally, I am grateful to all the brave patients who gifted pieces of their gut in the name of science, despite undergoing an incredibly uncomfortable procedure! 5 Dedication This thesis is dedicated to the strongest woman that I know - my mother, Kristina Zlatareva. Thank you for all your love and support in the pursuit of my dreams. 6 Table of contents DECLARATION ......................................................................................................................... 2 ABSTRACT................................................................................................................................ 3 ACKNOWLEDGEMENTS ........................................................................................................... 4 DEDICATION ............................................................................................................................ 6 TABLE OF CONTENTS ............................................................................................................... 7 TABLE OF FIGURES ..................................................................................................................12 LIST OF TABLES .......................................................................................................................13 ABBREVIATIONS .....................................................................................................................14 CHAPTER I. INTRODUCTION....................................................................................................19