The Role of CD1d-Mediated Lipid Presentation by Regulatory B Cells in Invariant Natural Killer T Cell Suppression of Autoimmunity by Kristine Oleinika Supervisor Professor Claudia Mauri UCL Immunology I, Kristine Oleinika, 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. Kristine Oleinika Acknowledgements To Professor Claudia Mauri, my supervisor and mentor Throughout my PhD years I have so admired your energy and passion, which has made me fall in love with science over and over again. Thank you for looking after me, your guidance has been invaluable in my journey in both science and life. I am incredibly grateful for being part of the wonderful team that you have gathered around you, scientific debate that you have fostered in the group, and for encouraging the friendships within it. To all my colleagues You have made this such an adventure, sharing my enthusiasm for science, politics and footwear, with you I have always felt like a member of an incredibly, almost insultingly, over-educated and over-loving family. And I apologise for the many times you have had to endure listening to my idea for “PhD: the musical” and late night immunology aphorisms, or being on the receiving end of the impromptu enforced hugs. Lizzy, Diego and Paul – thank you for making me think about immunology, socialism, feminism, literature and art (..) ad infinitum. And for your helpful comments on the thesis. Madhvi, overachieving mama bear, thanks for being a fantastic listener. Diana, my Romanian sister, thank you for all your help and always being so overwhelmingly positive. Marsilio, thank you for the advice, particularly when walking together to King’s X after long hours in the lab; and for providing so much positivity with statements about the “package” or stories about your boys giving you dad achievement stickers. Hannah, you discussing Cinnamon and his birthday cakes during writing kept me very happy. Scott, you have provided much banter and super energy. Laura, the lab cheerleader, thank you for always humming along and all valuable advice that you provided. Ale, thanks for keeping me company with conversations about signalling at 2am doing experiments. Professor Isenberg, your music and all our warm interactions have made the fourth floor more sunny, and time with you in the clinic re- iterated why it is important to do science. And particularly Chris(-py) – I could not have been able to survive, if not for your level-headedness, when I have been losing my mind, for the countless occasions you have saved me, all the fun we have had together and I want to simply thank you for being such a great person. To friends My Triumfeminate of SuperFemales – Inga, Ksenia, Marta (in alphabetical order), you have been my safe harbour and ideas-incubator, and I colossally appreciate just how badass you are. Mahdi, my little panda, you have always been there for me, with solutions to my science problems, when I am experiencing “technical difficulties”. I can only aspire to be as knowledgeable, hard working, and overwhelmingly kind. Dion, my most inspiring and supportive partner, you have loved me, entertained my eccentric needs, and taught me about social interactions during these years. You have been the sunshine to many of my days, and thank you for mirroring my uncontainable excitement about experiments done in 1950s-1970s, watching all those documentaries with me, and taking me to lectures, museums and abroad. You are the best human I have ever met and I have so much fun with you. The Family Grandmothers and grandfather, Elžuk! Mani labie, mīļie, Jums visvairāk veltīts šis darbs, paldies par gēniem, grāmatām, padomu un atbalstu it visā. And finally, my incredible superhuman mother, the original feminist, you have been the greatest role model, my inspiration in hard work, perseverance, love for others, pursuit of knowledge and from time to time enjoying the fruits of ones labour. You have the biggest heart and I love you more than you will ever know. Abstract Regulatory B cells (Bregs) express high levels of CD1d that presents lipid antigens to invariant natural killer T (iNKT) cells. The role of CD1d in Breg biology and the specific contribution to iNKT cell function and in suppressing inflammation remains unknown. Combining chimeric mice, cell depletion and adoptive transfer strategies, we show that CD1d lipid presentation by B cells to iNKT cells is critical for the induction of iNKT cells that down-regulate Th1 and Th17 adaptive immune responses and arthritis, whilst dispensable for Breg development. Mice lacking CD1d- expressing B cells developed exacerbated arthritis compared to wild-type mice and failed to respond to α-GalCer treatment. Absence of lipid presentation by B cells led to altered activation of iNKT cells, with disruption of regulatory pathways including those involved in metabolism and cytokine responses. Thus, we have identified a novel mechanism by which Bregs via CD1d, in an IL-10 independent manner, control and restrain excessive inflammation. Table of contents Table of figures ................................................................................................... 9 List of abbreviations ......................................................................................... 12 Chapter I: Introduction ................................................................................... 16 B cells ......................................................................................................................... 16 B cell ontogeny in the bone marrow and central tolerance .................................... 16 Generation of B cell antigen receptors ........................................................................... 17 B cell tolerance checkpoints ........................................................................................... 18 Microenvironmental cues in B cell development ........................................................... 19 A role for the BCR during B cell development, maturation and survival .............. 20 B cell heterogeneity and functional maturation in the periphery ........................... 23 B-1 cells .......................................................................................................................... 23 B-2 cells .......................................................................................................................... 23 Characteristics of mature B cell subsets ......................................................................... 25 MZ versus Fo B cell fate decision .................................................................................. 28 Generation of humoral responses within GCs ........................................................ 30 Molecules important in GC reaction .............................................................................. 31 B cell roles in addition to antibody production ...................................................... 34 Regulatory B cells ..................................................................................................... 36 Historical perspective ............................................................................................. 36 Breg Phenotype ...................................................................................................... 40 T2-MZP B cells .............................................................................................................. 40 B10 cells ......................................................................................................................... 43 B-1 cells .......................................................................................................................... 45 Other Breg phenotypes ................................................................................................... 45 IL-10-independent mechanisms of Breg function .................................................. 48 Signals in Breg induction ....................................................................................... 52 CD40 ............................................................................................................................... 52 TLRs and inflammation .................................................................................................. 53 BCR and antigen-specificity ........................................................................................... 55 Bregs in humans ..................................................................................................... 57 iNKT cells .................................................................................................................. 59 iNKT cell ontogeny ................................................................................................ 60 iNKT cell ligands ................................................................................................... 63 α-GalCer and synthetic analogues .................................................................................. 63 Exogenous ligands .......................................................................................................... 64 Endogenous ligands ........................................................................................................ 65 CD1d and lipid presentation ................................................................................... 69 6 iNKT cell activation