LINEAGE RELATIONSHIP ANALYSIS OF LYMPHOID PROGENITOR SUBSETS IN THE BONE MARROW OF NAÏVE MICE AND DURING INFLAMMATION REBECCA JANE LEYLAND RESEARCH THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY AT UNIVERSITY COLLEGE LONDON SUPERVISOR: DR. ALEXANDRE J. POTOCNIK DIVISION OF MOLECULAR IMMUNOLOGY MRC NATIONAL INSTITUTE FOR MEDICAL RESEARCH MILL HILL, LONDON 2011 1 I, Rebecca Jane Leyland, 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. R J Leyland 2 ABSTRACT ABSTRACT During haematopoiesis multipotent stem cells generate all cellular components of the blood including lymphocytes. Despite great progress in the isolation of lineage restricted progenitors, the exact precursor-product relationship of these subsets remains poorly understood. In particular the exact branch point of T- and B-cell development in the bone marrow has not been unequivocally mapped. The aim of my project was to investigate the developmental relationship of various progenitor subsets in normal mice and during an acute inflammation. In order to permanently identify all cells which emanated from early lymphoid compartments we generated a mouse model in which a Cre recombinase was inserted into the Rag1 locus and functional Cre activity would result in activation of an eYFP reporter. Expression of the reporter was found in all T- and B-cells and in a significant subset of NK-cells and dendritic cells. Furthermore this model allowed the prospective isolation of an ‘ELP analogue’ and two subsets of CLPs on the basis of their reporter expression. Functional analysis of these subsets in vivo demonstrated comparable developmental properties with slightly different kinetics. Furthermore, in vitro analysis of isolated progenitors established that reporter-positive CLPs were significantly more advanced in their commitment to the B-cell lineage. We extended our studies by investigating the impact of an acute systemic inflammation on the size and composition of early haemato-lymphoid subsets. Administration of LPS or heat-inactivated E. coli to mice in vivo resulted in a complete halt of bone marrow lymphopoiesis. In addition, a marked decrease in the number of myeloid progenitors accompanied by upregulation of Sca-1 on haematopoietic progenitors was observed. These inflammation-induced changes were found to be mainly caused by IFNγ and to a 3 ABSTRACT lesser extend by TNFα, thus identifying these cytokines as key mediators for the infection-induced regulation of haematopoiesis. 4 ACKNOWLEDGEMENTS ACKNOWLEDGEMENTS I would primarily like to thank Dr. Alexandre Potocnik for the opportunity to work in his laboratory. During the last three and a half years, detailed discussions on haematopoietic development with Dr. Potocnik have enabled me to gain an in-depth understanding of the subject and has encouraged me to critically analyse experiments and the literature, which has certainly lead to the finalisation of this thesis and the results herein. In addition, Dr. Potocnik has provided me with scientific guidance and feedback which I am sure will be valuable in the next scientific position I undertake. I would also like to thank Dr. Douglas Brown for introducing me to flow cytometry and cell sorting and Dr. Nikolai Belyaev for help carrying out molecular biology techniques. Both of the aforementioned post-docs have also contributed to scientific discussions with myself which has subsequently broadened my knowledge of immunology as a whole. Ana-Isabel Garcia-Diaz has assisted me in both the preparation and analysis of infection experiments and I am very grateful for this. Judit Biro has provided sound advice, not exclusively related to science, and I am thankful for her company. I would also like to thank Dr. Dimitris Vasilakos and Dimitris Karamitros for general scientific advice. 5 CONTENTS CONTENTS ABSTRACT..............................................................................................................3 ACKNOWLEDGEMENTS ......................................................................................5 CONTENTS..............................................................................................................6 LIST OF FIGURES ................................................................................................10 LIST OF TABLES ..................................................................................................16 ABBREVIATIONS ................................................................................................17 CHAPTER 1: INTRODUCTION..........................................................................20 1.1 The discovery of haematopoietic stem cells ...............................................21 1.2 The classical model of haematopoiesis.......................................................22 1.3 Lineage-restricted progenitors of the lymphoid and myelo-erythroid pathways......................................................................................................27 1.4 Early progenitors of lymphopoiesis ............................................................28 1.5 Cellular pathways of T-cell fate specification ............................................34 1.6 Cellular pathways of B-cell fate specification ............................................40 1.7 Transcription factors important for lymphoid specification .......................41 1.8 The effect of aging and gender on haematopoiesis.....................................44 1.9 Infection-induced changes of haematopoiesis ............................................47 1.10 Aims of study ..............................................................................................50 CHAPTER 2: MATERIALS AND METHODS...................................................52 2.1 Mouse strains ..............................................................................................53 2.2 Isolation of progenitor subpopulations........................................................54 2.2.1 Cell preparation.............................................................................................................. 54 2.2.2 Magnetic mediated depletion of cells ............................................................................ 54 2.2.3 Analytical and preparative flow cytometry.................................................................... 54 6 CONTENTS 2.3 RNA Extraction and Complementary DNA Synthesis ...............................58 2.4 Semi-quantitative Polymerase Chain Reaction and Agarose Gel Electrophoresis............................................................................................58 2.5 Quantitative Reverse Transcription Polymerase Chain Reaction ...............59 2.6 In vitro culture of lymphoid progenitors.....................................................62 2.7 In vivo transplantation assays......................................................................62 2.8 Infection models..........................................................................................63 2.8.1 LPS-induced infection ................................................................................................... 63 2.8.2 E. coli-induced infection................................................................................................ 64 2.9 Statistical tests.............................................................................................65 CHAPTER 3: INVESTIGATION OF LYMPHOID PROGENITOR DEVELOPMENT UTILISING A NOVEL LINEAGE TRACING REPORTER ...................................................................................................................................66 3.1 Introduction.................................................................................................67 3.2 Defining haematopoietic progenitors in the adult BM................................70 3.3 Analysis of mature haematopoietic splenic subsets in Rag1 reporter mice 82 3.4 Expression of eYFP in mature and progenitor cell populations of the BM 91 3.5 Investigation of the frequency of eYFP+ cells in the thymic compartment of the haematopoietic system ........................................................................104 3.6 Comparison of cellularity of haematopoietic populations in Rag1wt/Cre x Rosa26wt/eYFP mice with C57BL/6 mice ....................................................108 3.7 Expression of eYFP in haematopoietic populations compared in different Cre reporter mouse strains ........................................................................114 3.8 The effect of gender and aging on haematopoiesis...................................118 7 CONTENTS 3.9 Expression of GATA-family transcription factors in haematopoietic progenitor populations ..............................................................................131 3.10 Discussion .................................................................................................139 CHAPTER 4: ANALYSIS OF THE FUNCTIONAL POTENTIAL OF LYMPHOID PROGENITOR POPULATIONS IN VIVO AND IN VITRO....152 4.1 Introduction...............................................................................................153 4.2 Analysis of the functional potential of the ‘ELP analogue’ in vivo ..........155 4.3 Analysis of the functional potential of the CLP population in vivo..........169 4.4 Analysing the functional potential of the ELP, CLP eYFP– and CLP eYFP+ pools in vivo ..............................................................................................178