UNIVERSITÉ DE STRASBOURG ÉCOLE DOCTORALE des Sciences de la Vie et de la Santé IGBMC - CNRS UMR 7104 - Inserm U 964 THÈSE présentée par : Katarzyna POLAK soutenue le : 15 Octobre 2015 pour obtenir le grade de : !"#$%&'($')R%+,-$&.,# ' de Strasbourg Discipline : Sciences du vivant Spécialité : Aspects moléculaires et cellulaires de la biologie Role of the Eos and Helios transcription factors in regulatory T cell biology Rôle des facteurs de transcription Eos et Helios dans la biologie des cellules T régulatrices Thèse dirigée par: Dr. Susan Chan, DR, Inserm, IGBMC, Illkirch Rapporteur interne: Pr. Sylviane Muller DR, CNRS, IBMC, Strasbourg Rapporteurs externes: Dr. Maria-Cristina Cuturi DR, Inserm, ITUN, Nantes Dr. Jean-Christophe Bories DR, Inserm, IUH, Paris ACKNOWLEDGMENTS First of all, I wish to thank my PhD supervisors, Susan Chan and Philippe Kastner for accepting me in the lab and for their mentorship over the last four years. Susan, my direct PhD advisor, I want to thank for reading my thesis manuscript and always pointing in the good direction. I would like to thank my jury members, Pr. Sylviane Muller, Dr. Maria-Cristina Cuturi and Dr. Jean-Christophe Bories, for agreeing to evaluate my work and discuss it during the thesis defense. I wish to greatly acknowledge the HEM_ID network, for providing a great PhD training and possibility to attend international meetings and workshops, during which I could present my work. Special thanks are for my HEM_ID colleagues and fellows, for all the scientific contributions and social interactions. I am really happy I have met you all. Patricia, thank you for welcoming me warmly in the lab and helping to accommodate in Strasbourg, for helping me with so many things, teaching how to handle mice, and organize my work. R"# extremely grateful for all your help. I also wish to thank Claudine, responsible for the flow cytometry platform, for all the advice, patience, trusting with her precious machines in front of which I spent a considerable part of "$#%&'G#)*+#,&-#.//01-23 Sanjay, I am very grateful for helping me out to set up the colitis experiment and advising during the past few months. I would also like to thank everyone from IGBMC and ICS animal facilities, especially to Sylvie, William, Michael and Nadine for taking care of the mice and all the precious help they provided. I want to thank all the past and present members of the lab, Alejandra, Apostol, Deepika, Jérôme, Marie-Pierre, Clément, Stephanie, Ujjwal, Beate, Attila, Patricia, Isma, Peggy, Marie, Gaëtan , Célestine, Rose-Marie, Marie for creating a great atmosphere in the lab. I would like to thank especially Rose and Deepika who were my first teachers in the lab and also great friends, on whom I can always count. Marie and Gaëtan, thank you for your support and interesting discussions, Isma and Clement, for being the best bench neighbors and friends. Attila thanks for all the help and fun, peculiar discussions and for entertaining (!) during lunch time. Colleagues and friends, thank you for being there and making my PhD an amazing time: Anna, Iskander, Anastazja, Damien, Aurelia, Shilpy, Ira, Alexey, Claire, Nico, Léa, Isabelle, Anne-Sophie, Heena, Katerina, Vova, Firas, Pierre-Etienne, Grigory, Ismail, JoLi, Benjamin. Thank you guys! Anna and Iskander, we started together and we finish together, but the adventure will continue! Ana thanks for being a friend and for the distraction at the gym during the last months. Finally, I wish to thank my parents for all their encouragement and support. Ben, of course thank you for reading and ,&-*G# 4-)+1*5# )5)1* my thesis manuscript. For always being there for me and cheering me up, for making the four years of my PhD such a beautiful adventure. 1 TABLE OF CONTENTS LIST OF FIGURES AND TABLES ....................................................................................... 5 LIST OF ABBREVIATIONS ................................................................................................. 7 CHAPTER I INTRODUCTION AND LITERATURE REVIEW ................................... 10 I.1 Hematopoietic stem cells and their progeny ................................................................... 11 I.1.1 Identification and characterization of HSCs ............................................................ 12 I.1.2 Properties and choices of a hematopoietic stem cell ................................................ 15 I.1.3 Regulatory mechanisms ........................................................................................... 18 I.2 The T cell lineage............................................................................................................ 19 I.2.1 T cell development in the thymus ............................................................................ 19 I.2.2 Pathways and genes governing T cell development................................................. 21 I.2.3 T cell activation ........................................................................................................ 23 I.2.4 T cell lineage diversification .................................................................................... 24 I.3 Regulatory T cells M a brief history................................................................................. 26 I.3.1 Origins of Treg cells ................................................................................................. 27 I.3.1.1 Thymic Treg cells .............................................................................................. 28 I.3.1.2 Alloantigen specific Treg cells .......................................................................... 30 I.3.1.3 Induced Treg cells ............................................................................................. 30 I.3.2 Phenotypical and functional Treg subsets ................................................................ 32 I.3.2.1 CD4 + Treg cells ................................................................................................. 32 I.3.2.2 CD8 + Treg cells ................................................................................................. 36 I.3.3 Mechanisms of suppression ..................................................................................... 38 I.4. Ikaros family of transcription factors ............................................................................. 41 I.4.1 Discovery and evolution........................................................................................... 41 I.4.2 Protein structure ....................................................................................................... 41 I.4.4 Expression in the hematopoietic system .................................................................. 45 I.4.5 Mechanisms of transcriptional regulation ................................................................ 46 I.5. Regulation of the Treg cell lineage ................................................................................ 50 I.5.1 Crucial role of the Foxp3 transcription factor .......................................................... 50 I.5.2 Foxp3 mediated gene regulation in Treg cells ......................................................... 50 I.5.3 Is Foxp3 sufficient to define Treg cells? .................................................................. 53 I.6. Eos and Helios M biological functions ........................................................................... 56 2 I.6.1 Eos and Helios during hematopoiesis M a brief overview ........................................ 56 I.6.2 Role in Treg cells ..................................................................................................... 57 I.6.2.1 Eos ..................................................................................................................... 57 I.6.2.2 Helios ................................................................................................................. 59 1.7 Summary and aims of study ........................................................................................... 61 CHAPTER II MATERIALS AND METHODS................................................................. 62 II.1 Mice ............................................................................................................................... 62 II.2 DNA and RNA preparation ........................................................................................... 63 II.2.1 Genomic DNA extraction ....................................................................................... 63 II.2.2 RNA extraction ....................................................................................................... 64 II.3 PCR ................................................................................................................................ 65 II.3.1 Genotyping .............................................................................................................. 65 II.3.2 RT-PCR................................................................................................................... 66 II.3.3 RT-qPCR................................................................................................................. 66 II.4 Western blot ................................................................................................................... 67 II.4.1 Antibodies ............................................................................................................... 67 II.5 Flow cytometry .............................................................................................................