Unravelling the role of the microenvironment in chronic lymphocytic leukaemia: T helper cell support of cancer cells Doctoral thesis by Anna Maria Parente Ribes Centre for Immune Regulation, Department of Immunology, Institute of Clinical Medicine, University of Oslo, Norway 2018 © Anna Maria Parente Ribes, 2018 Series of dissertations submitted to the Faculty of Medicine, University of Oslo ISBN 978-82-8377-230-2 All rights reserved. No part of this publication may be reproduced or transmitted, in any form or by any means, without permission. Cover: Hanne Baadsgaard Utigard. Print production: Reprosentralen, University of Oslo. TABLE OF CONTENTS ACKNOWLEDGEMENTS ....................................................................................................... 5 ABBREVIATIONS .................................................................................................................... 7 LIST OF PUBLICATIONS ....................................................................................................... 8 INTRODUCTION .................................................................................................................... 10 B cells .................................................................................................................................... 10 B cell development ............................................................................................................ 10 Central and peripheral tolerance ................................................................................... 11 Germinal centre reaction .............................................................................................. 12 B cells as APC ................................................................................................................... 14 The BCR and the BCR pathway ........................................................................................ 14 T cells .................................................................................................................................... 16 Th cells .............................................................................................................................. 16 B cell malignancies ............................................................................................................... 19 Chronic Lymphocytic Leukaemia ..................................................................................... 21 The CD38 marker ......................................................................................................... 23 Staging and risk assessment ......................................................................................... 24 Treatment decision ....................................................................................................... 26 BCR pathway inhibitors ........................................................................................... 26 Multiple myeloma (MM) ................................................................................................... 27 Epstein-Barr Virus (EBV) ..................................................................................................... 28 EBV and malignancy. ........................................................................................................ 30 EBV and CLL .................................................................................................................... 31 AIMS OF THE STUDY ........................................................................................................... 32 SUMMARY OF THE INDIVIDUAL PAPERS ...................................................................... 33 METHODOLOGICAL CONSIDERATIONS ......................................................................... 38 Samples ................................................................................................................................. 38 T cell lines ............................................................................................................................. 39 Flow cytometry ..................................................................................................................... 39 Proliferation assays ............................................................................................................... 40 The T18 clone ....................................................................................................................... 41 The microenvironment .......................................................................................................... 41 Pros and cons in xenografting ............................................................................................... 41 EBV-GFP .............................................................................................................................. 43 Other statistical considerations ............................................................................................. 43 3 GENERAL DISCUSSION ....................................................................................................... 45 Immunosurveillance in CLL ................................................................................................. 45 Recognition of cancer neo-epitopes by T cells ..................................................................... 46 T cell support of B cell malignancies in the context of infection ......................................... 47 Neo-epitopes in B cell malignancies ..................................................................................... 48 Genesis of malignant B cells ................................................................................................. 49 T cells and B cell malignancies, specificities and evidence for Th mediated propagation of malignant B cells. .................................................................................................................. 50 CLL cells are malignant B cells that have active BCR signalling and autoreactive BCR .... 53 Do Th cells drive the gradual accumulation of mutations in malignant B cells? ................. 53 The immune (T cell) deficiency model ................................................................................. 54 The (autoimmune) inflammatory signature of CLL ............................................................. 55 Main hypothesis from the thesis ........................................................................................... 56 CLL and EBV ....................................................................................................................... 58 REFERENCE LIST .................................................................................................................. 60 4 ACKNOWLEDGEMENTS The work of this thesis has been carried out at the Centre for Immune regulation (CIR), Department of Immunology and transfusion medicine, Oslo University Hospital (OUS) and Institute of Clinical Medicine at the University of Oslo (UIO), during 2010-2017. Financial support was provided by the Norwegian Kreftforeningen (Cancer society) and various UNIFOR grants. I would like to acknowledge my supervisor Ludvig A. Munthe for believing in me since the very first second. You have guided me through a very nice and fruitful project, with amazing ideas and against the dogma. I admire your enthusiasm and I am looking forward to see the new Centre for B cell malignancies. I would like to thank Geir E. Tjønnfjord for your supervision and motivating discussions, as well as for supplying all the CLL patient samples. Also I am grateful to Bjarne Bogen for good conversations and for giving me another inspirational point of view to think about. A sincere thanks goes to Harald Jr. Holte who gave me my first job in Norway and suggested me to apply for this PhD, as well as for supplying follicular lymphoma samples, together with Arne Kolstad and colleagues at Radiumhospitalet. Next I would like to thank all the members in the Munthe group and Bogen group that have supported me along my PhD, to all my co-authors and collaborators, and to the ones continuing the projects. A special and big big thank you to my friend and close collaborator Simone, for teaching me all what I needed in the lab in a proficient and practical way. Thanks for all the fun and hardworking times in the lab! I would also like to thank Audun for starting this amazing CLL project. You had really good ideas and I am happy that some of them could be reflected in the articles. A very special thanks goes to Keith, for all his amazing tips and tricks in the lab, you always had some words full of truth and advice. A sincere thanks goes to Wang for your collaboration in my projects and for extending our findings into Multiple Myeloma. Another thanks to Sigrid and Kjetil for the good work we did together. Since 2016 I left the research lab and started to work as a medical doctor in the same department. I met a lot of fantastic people and got a lot of support and possibilities, thanks to all my colleagues and bosses for making this possible. Getting through my PhD required more than academic support. I would like to thank all the members of the pink and black lab and the ones sharing office with me, my “Spanish girls in Oslo”, my friends from Barcelona, and my colleagues in my actual work for your support and de-stressing dinners. 5 It is with immense gratitude I acknowledge my parents Antonio Parente and Antonia Ribes, both holding a PhD. Thanks for raising