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Involvement of CD45 in early thymocyte development Lai, Jacqueline Cheuk-Yan 2008

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INVOLVEMENT OF CD45 IN EARLY THYMOCYTE DEVELOPMENT by JACQUELINE CHEUK-YAN LAI B.Sc., The University of British Columbia, 2002  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES (Microbiology and Immunology)  THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  December 2008 © Jacqueline Cheuk-Yan Lai, 2008  ABSTRACT CD45 is a protein tyrosine phosphatase that is expressed on all nucleated hematopoietic cells. The major substrates of CD45 in thymocytes and T cells are the Src family kinases Lck and Fyn. The role of CD45 in thymocyte development and T cell activation via its regulation of Src family kinases in T cell receptor signaling has been studied extensively. However, the role of CD45 in processes that affect thymocyte development prior to the expression of the T cell receptor has not been explored. The overall hypothesis of this study was that CD45 is a regulator of spreading, migration, proliferation, and differentiation of early thymocytes during development in the thymus and the absence of CD45 would alter the outcome of thymocyte development. The first aim was to determine how CD45 regulates CD44-mediated signaling leading to cell spreading. The interaction between CD44 and Lck was first examined. CD44 associated with Lck in a zinc-dependent and a zinc-independent manner. Mutation analysis localized the zinc-dependent interaction to the membrane proximal region of CD44, but did not involve individual cysteine residues on CD44. CD44 and Lck co-localized in microclusters upon CD44-mediated cell spreading. CD45 co-localized with Lck and CD44 in microclusters and with F-actin in ring structures. The recruitment of CD45 to microclusters may be a mechanism of how CD45 negatively regulates CD44-mediated spreading. The second specific aim was to determine the role of CD45 in migration, proliferation, and progression and differentiation of early thymocytes. CD45 negatively regulated CXCL12-mediated migration, and positively regulated the proliferation and progression of CD117- DN1 thymocytes. Absence of CD45 led to an altered composition of thymic subsets. The CD45-/- thymus contained decreased numbers of ETPs and an aberrant CD117- DN1 population that lacked CD24, TCRβ, and CCR7 expression. There were also increased thymic NK and γδ T cells, but decreased NKT cells. In addition, a novel intermediate between DN1 and DN2 that required Notch for progression was identified. Overall, this study identified new roles for CD45 in early thymocytes and provided a better picture of how the development of T cells, a central component of the immune system, is regulated.  ii  TABLE OF CONTENTS ABSTRACT.............................................................................................................................. ii TABLE OF CONTENTS......................................................................................................... iii LIST OF TABLES................................................................................................................. viii LIST OF FIGURES ................................................................................................................. ix LIST OF ABBREVIATIONS.................................................................................................. xi ACKNOWLEDGEMENTS................................................................................................... xiv  CHAPTER 1: INTRODUCTION .............................................................................................1 1.1 Overview of the immune system ................................................................................... 2 1.2 T cell development......................................................................................................... 3 1.2.1 T cell progenitors from the bone marrow ........................................................... 3 1.2.2 Stages of αβ T cell development ........................................................................ 4 1.2.3 β-selection........................................................................................................... 7 1.3 Intrathymic signals for thymocyte development............................................................ 9 1.3.1 Thymic epithelial cells........................................................................................ 9 1.3.2 c-Kit signaling................................................................................................... 10 1.3.3 IL-7 signaling.................................................................................................... 11 1.3.4 Notch signaling ................................................................................................. 13 1.3.5 Interplay between Notch, IL-7 and CD117 signaling....................................... 14 1.3.6 In vitro differentiation of thymocytes ............................................................... 15 1.4 Cell migration during thymic development ................................................................. 16 1.4.1 Adhesion molecules involved in thymic seeding and intrathymic migration... 16 1.4.2 Chemokines involved in intrathymic migration ............................................... 17 1.5 Unconventional thymocytes......................................................................................... 19 1.5.1 Thymic γδ T cells.............................................................................................. 21 1.5.2 NK cells ............................................................................................................ 22 1.5.3 NKT cells .......................................................................................................... 23  iii  1.6 CD44 ............................................................................................................................ 24 1.6.1 CD44 structure and expression ......................................................................... 24 1.6.2 CD44-mediated migration, cell spreading and signaling.................................. 25 1.6.3 CD44 binding to hyaluronan and the actin cytoskeleton .................................. 26 1.6.4 Role of CD44 on hematopoietic progenitors and early thymocytes ................. 27 1.7 CD45 ............................................................................................................................ 28 1.7.1 CD45 expression and structure ......................................................................... 28 1.7.2 The Src family kinase Lck, a substrate of CD45 .............................................. 28 1.7.3 CD45 in TCR signaling .................................................................................... 32 1.7.4 CD45 in cell spreading and migration .............................................................. 33 1.7.5 Regulation of CD45 .......................................................................................... 33 1.8 Thesis objectives.......................................................................................................... 34 CHAPTER 2: MATERIALS AND METHODS ....................................................................36 2.1 Materials ...................................................................................................................... 37 2.1.1 Antibodies ......................................................................................................... 37 2.1.2 Reagents............................................................................................................ 38 2.1.3 Mice .................................................................................................................. 39 2.2 Methods........................................................................................................................ 39 2.2.1 Cell isolation and culture .................................................................................. 39 2.2.2 Cloning of CD44 constructs and transfection................................................... 42 2.2.3 Cell spreading assay and immunoprecipitation ................................................ 43 2.2.4 Western blotting................................................................................................ 45 2.2.5 Flow cytometry ................................................................................................. 46 2.2.6 Labeling for confocal microscopy and cell measurements............................... 47 2.2.7 Image collection and processing....................................................................... 48 2.2.8 In vivo proliferation assay................................................................................. 49 2.2.9 In vitro migration assay .................................................................................... 50 2.2.10 Fluorescence activated cell sorting ................................................................. 50 2.2.11 Co-culture of progenitor and stromal cells ..................................................... 51 2.2.12 Statistical analysis........................................................................................... 51  iv  CHAPTER 3: REGULATION OF CD44-MEDIATED CELL SPREADING BY CD45 ....................................................................................................................52 3.1 Introduction and rationale ............................................................................................ 53 3.2 Results.......................................................................................................................... 55 3.2.1 CD44 and Lck are recruited to microclusters upon CD44-mediated cell spreading ........................................................................................................... 55 3.2.2 The cytoplasmic domain of CD44 is required for the association of CD44 with Lck, but not Fyn........................................................................................ 57 3.2.3 Zinc-dependent association of CD44 with Lck requires the membrane proximal cytoplasmic domain of CD44............................................................ 60 3.2.4 Zinc-dependent association of CD44 with Lck does not involve key cysteine residues ............................................................................................................. 62 3.2.5 The cytoplasmic domain of CD44 is important for hyaluronan binding .......... 64 3.2.6 CD44-induced Pyk2 phosphorylation is dependent on Src family kinase activity but not microtubule rearrangement...................................................... 66 3.2.7 CD45 is recruited to microclusters and ring structures upon CD44-mediated spreading ........................................................................................................... 68 3.2.8 Phospholipase C is required for CD44-induced F-actin fiber formation and elongated cell spreading in CD45- cells............................................................ 72 3.2.9 CD45 affects the extent of cell spreading and cell polarization in primary cells ................................................................................................................... 74 3.3 Discussion .................................................................................................................... 79 3.3.1 Data summary ................................................................................................... 79 3.3.2 CD44 and Src family kinase interaction ........................................................... 81 3.3.3 CD44 and hyaluronan interaction ..................................................................... 84 3.3.4 CD45 regulation of the CD44 signaling complex upon spreading ................... 86 3.3.5 Translocation of CD45 and reorganization of the actin cytoskeleton upon CD44 spreading ................................................................................................ 87 3.3.6 CD44-mediated cell spreading of primary cells ............................................... 88 3.3.7 CD44 and cell migration................................................................................... 89  v  CHAPTER 4: REGULATION OF EARLY THYMOCYTE DEVELOPMENT BY CD45 ....................................................................................................................91 4.1 Introduction and rationale ............................................................................................ 92 4.2 Results.......................................................................................................................... 92 4.2.1 Altered DN populations in the thymus of CD45-/- mice ................................... 92 4.2.2 The absence of CD45 does not affect the amount of bone marrow or blood LSK cells........................................................................................................... 99 4.2.3 CD45 is a positive regulator of proliferation in vivo ...................................... 102 4.2.4 CD45 is required for optimal CXCL12 migration in the CD117- DN1 population ....................................................................................................... 102 4.2.5 CD45 is required for CCR7 expression in the CD117- DN1 population........ 104 4.2.6 Identification of an intermediate stage between DN1 and DN2 ..................... 108 4.2.7 The absence of CD45 alters the distribution of both CD117+ and CD117cells within the DN1 populations.................................................................... 114 4.2.8 CD45 regulates the progression and maturation of CD117- DN1 cells .......... 118 4.2.9 Lack of CD24 and TCRβ expressing DN1 cells in CD45-/- mice................... 121 4.2.10 CCR7 is exclusively expressed on the CD117- DN1.5 population............... 121 4.3 Discussion .................................................................................................................. 124 4.3.1 Data summary ................................................................................................. 124 4.3.2 CD45 is a positive regulator of proliferation .................................................. 127 4.3.3 The lack of CD45 favors the generation of non αβ T lineage cells in adult thymus............................................................................................................. 129 4.3.4 DN1.5 is a novel intermediate between the traditional DN1 and DN2 that requires Notch for progression........................................................................ 130 4.3.5 CD45 is required for CCR7 expression in CD117- DN1 thymocytes ............ 132 4.3.6 CD45 is a positive regulator of CXCL12 migration....................................... 133 4.3.7 CD45 does not affect the hematopoietic progenitor pool ............................... 134 4.3.8 Decreased ETP in CD45-/- mice is due to decreased proliferation ................. 135 4.3.9 CD45 is required for the maturation of atypical T cells ................................. 136  vi  CHAPTER 5: SUMMARY AND FUTURE PERSPECTIVES ...........................................140 5.1 Summarizing the involvement of CD45 in early thymocyte development................ 141 5.2 Involvement of CD45 in signaling pathways affecting lineage commitment............ 142 5.2.1 CD45 and IL-7 signaling ................................................................................ 142 5.2.2 CD45 and Notch signaling.............................................................................. 144 5.2.3 CD45 and TCR signaling................................................................................ 145 5.3 CD44 signaling pathway and regulation by CD45 .................................................... 146 5.4 Future directions ........................................................................................................ 149 FOOTNOTES ........................................................................................................................152 REFERENCES ......................................................................................................................154 APPENDIX A: CO-AUTHORSHIP STATEMENT............................................................186 APPENDIX B: BIOSAFETY AND ANIMAL CARE CERTIFICATES............................188  vii  LIST OF TABLES Table 4.1 Numbers and percentage of thymic subsets from CD45+/+ and CD45-/- mice ........95  viii  LIST OF FIGURES Figure 1.1 Thymocyte progenitor subsets and differentiation ................................................8 Figure 1.2 Schematic overview of thymocyte migration and development in the thymus..................................................................................................................20 Figure 1.3 CD45 structure.....................................................................................................29 Figure 3.1 Co-localization of CD44 and Lck to microclusters upon CD44-mediated spreading ..............................................................................................................56 Figure 3.2 Amino acid sequence of the wild type mouse CD44 cytoplasmic tail and mutant CD44........................................................................................................58 Figure 3.3 CD44 association with Lck but not Fyn requires the cytoplasmic domain of CD44................................................................................................................59 Figure 3.4 Effect of the zinc chelator 1,10-phenanthroline on the association of Lck and Fyn with CD44..............................................................................................61 Figure 3.5 CD44 interaction with Lck is independent of key cysteine residues on CD44 ....................................................................................................................63 Figure 3.6 Membrane proximal region of CD44 cytoplasmic domain is required for optimal binding to hyaluronan .............................................................................65 Figure 3.7 Pyk2 phosphorylation upon CD44-mediated spreading is dependent on Src family kinase activity.....................................................................................67 Figure 3.8 Pyk2 phosphorylation upon CD44-mediated spreading is not dependent on microtubule reorganization .............................................................................69 Figure 3.9  CD45 form distinct microclusters and ring structures upon CD44mediated spreading ..............................................................................................70  Figure 3.10 Lck co-localizes with CD45 in microclusters upon CD44-mediated spreading ..............................................................................................................71 Figure 3.11 Inhibition of PLC leads to F-actin ring formation...............................................73 Figure 3.12 Increased CD44 expression in CD45-/- primary cells..........................................75 Figure 3.13 CD44-mediated cell spreading and F-actin rearrangement in activated thymocytes ...........................................................................................................76 Figure 3.14 CD44-mediated cell spreading and F-actin rearrangement in activated lymph node T cells...............................................................................................78 ix  Figure 3.15 CD44-mediated signaling pathway leading to cell spreading .............................80 Figure 3.16 Schematic model of CD44 association with Lck and Fyn ..................................83 Figure 4.1 Flow cytometric analysis of CD45+/+ and CD45-/- thymocytes ex vivo...............94 Figure 4.2 Lack of CD45 alters the generation of NK and NKT cells in the thymus...........97 Figure 4.3 Lack of CD45 affects maturation of γδ T cells in the thymus.............................98 Figure 4.4 Reduced ETPs in CD45-/- mice .........................................................................100 Figure 4.5 LSK population is not altered in CD45-/- mice..................................................101 Figure 4.6 Reduced proliferation of CD45-/- cells in vivo...................................................103 Figure 4.7 Reduced CXCL12-mediated migration in CD45-/- thymocytes ........................105 Figure 4.8 CXCR4 expression is not affected in CD45-/- thymocytes................................106 Figure 4.9 Lack of CCR7 expression on DN1 thymocytes from CD45-/- mice..................107 Figure 4.10 Identification of a novel intermediate between DN1 and DN2.........................109 Figure 4.11 Progression of DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro............111 Figure 4.12 Requirement for IL-7 for survival of DN1.0 and DN1.5 thymocytes in vitro ....................................................................................................................112 Figure 4.13 Progression of DN1.0 and DN1.5 on OP9 and OP9-DL1 cells in vitro............113 Figure 4.14 Altered CD117+ and CD117- DN1 populations in CD45-/- mice ......................115 Figure 4.15 Progression of CD117+ DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro ....................................................................................................................117 Figure 4.16 Distribution of CD117+ cells between CD44+ DN populations ........................119 Figure 4.17 Progression of CD117+ DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro ....................................................................................................................120 Figure 4.18 Altered DN1a-e populations in CD45-/- mice....................................................122 Figure 4.19 Absence of TCRβ+ cells in the CD117- DN1 population of CD45-/- mice........123 Figure 4.20 CCR7 is predominantly expressed on CD117- DN1.5 thymocytes of CD45+/+ mice .....................................................................................................125 Figure 4.21 Involvement of CD45 in migration, proliferation, and progression of early thymocytes ................................................................................................126 Figure 4.22 Growth curves of CD45+ and CD45- cell lines .................................................128 Figure 4.23 Involvement of CD45 in non-canonical thymocyte development.....................139  x  LIST OF ABBREVIATIONS 7AAD  7-amino-actinomycin  A  alanine  Ab  antibody, antibodies  APC  allophycocyanin  ATCC  American Type Culture collection  BM  bone marrow  BrdU  5-bromo-2-deoxyuridine  BSA  bovine serum albumin  C  cysteine  CLP  common lymphoid progenitor  CMJ  cortico-medullary junction  CTP  circulating T cell progenitors  DC  dendritic cell  DL1  Delta-like 1  DMEM  Dulbecco’s modified Eagle medium  DMSO  dimethyl sulfoxide  DN  CD4 and CD8 double negative  DP  CD4 and CD8 double positive  ECM  extracellular matrix  EDTA  ethylene-diamine-tetra acetic acid  ERM  ezrin/radixin/moesin  ETP  early thymic/T cell progenitor  FAK  focal adhesion kinase  FCS  fetal calf serum  FITC  fluorescein isothiocyanate  FL  fluorescein  Flt3L  Fms-like tyrosine kinase receptor-1 ligand  G-CSF  granulocyte colony-stimulating factor  GFP  green fluorescent protein  xi  H  histidine  H+L chain  Heavy and light chain  HA  hyaluronan  hr  hour(s)  HRP  horseradish peroxidase  HSC  hematopoietic stem cell  Ig  immunoglobulin  IL  interleukin  IL-7R  interleukin-7 receptor  ITAM  immunoreceptor tyrosine-based activation motif  JAK  Janus kinase  Lin-  lineage negative  LSK  lineage-negative Sca-1+ c-Kit(CD117)+  mAb  monoclonal antibody  MEM  minimal essential medium  min  minute(s)  MPP  multipotent progenitor  N32  N-terminal, Src homology 3 and 2 domains  NK  natural killer  PBS  phosphate-buffered saline  PCR  polymerase chain reaction  PE  phycoerythrin  PFA  para-formaldehyde  PI3K  phosphoinositide 3-kinase  PLC  phospholipase C  PMA  phorbol myristate acetate  PMSF  phenyl methyl sulfonyl fluoride  PSGL-1  platelet-selectin glycoprotein ligand-1  PVDF  polyvinylidene difluoride  Pyk2  proline-rich tyrosine kinase 2  RAG  recombination-activating gene  xii  RBC  red blood cells  RT  room temperature  SCF  stem cell factor, c-Kit ligand  SDF-1  stromal cell-derived factor-1, CXCL12  SDS  sodium dodecyl sulfate  SEM  standard error of the mean  SFK  Src family kinase  SH2  Src homology 2  SMAC  supramolecular activation cluster  SP  single positive  STAT  signal transducer and activator of transcription  TBST  Tris-buffered saline Tween-20  TCR  T cell receptor  TCS  tissue culture supernatant  TEC  thymic epithelial cell  VCAM-1  vascular cell adhesion molecule-1  Y  tyrosine  xiii  ACKNOWLEDGEMENTS I would like to thank Dr. Pauline Johnson for her guidance, insight and support. I would also like to thank the members of my supervisory committee, Dr. Ninan Abraham, Dr. Michael Gold and Dr. Robert Kay for their intellectual contribution to this work. I wish to acknowledge the past and present members of the Johnson lab, the Abraham lab, and the UBC FACS Facility for useful discussions and technical support. Financial assistance from the Canadian Institutes of Health Research/Michael Smith Foundation for Health Research training program in transplantation, Canadian Society of Immunology, and the Robert Emmanuel and Mary Day Endowment was greatly appreciated.  xiv  CHAPTER 1 INTRODUCTION  1  1.1 Overview of the immune system  The immune system is a network of specialized tissue, cells and molecules that protects us from foreign pathogens in a highly coordinated manner. The immune system can also survey our tissues for cancer cells. In jawed vertebrates, the immune system has evolved into the innate and adaptive branches. The innate branch provides an immediate but non-specific response to foreign pathogens, whereas the adaptive branch generates an antigen-specific response and immunological memory to the pathogen. T cells play several central roles in the adaptive immune system, including direct effector functions such as killing virus-infected cells, as well as indirect effects of modulating immune responses via secreted cytokines. The importance of T cell function in humans can be seen in patients with acquired immunodeficiency syndrome (AIDS), in which human immunodeficiency virus (HIV) infection causes a gradual decrease in CD4 T cells, leading to a breakdown of the immune system and severe immunodeficiency. Different lymphocyte lineages share common progenitors. The generation of distinct cell types in the appropriate numbers requires the tight regulation of signals that promote or inhibit cell differentiation at developmental checkpoints. Since lymphocyte subsets coordinate with each other to generate an immune response, altering any signals involved in lymphocyte development and differentiation would affect the balance (homeostasis) that is needed for a proper response. Dysregulation of the development of lymphocytes can lead to autoimmunity, immunodeficiency or hypersensitivity. Therefore, factors regulating the development of lymphocytes must be tightly regulated.  2  1.2 T cell development  1.2.1 T cell progenitors from the bone marrow Hematopoietic stem cells (HSCs) that reside in the bone marrow (BM) can give rise to all hematopoietic lineages by undergoing a stepwise differentiation process in which the hematopoietic progenitors gradually lose multipotency and self-renewal potential. This generates a more lineage-restricted progenitor (1). The development of T cells occurs mainly in the thymus. However, progenitors within the thymus have limited self-renewal ability and can only maintain thymopoiesis for a short period (3 – 4 weeks) of time (2, 3). Hence, continual seeding of thymic progenitors from the bone marrow is necessary to maintain thymopoiesis throughout adult life. The thymus is only periodically receptive to progenitors from the circulation (4). It is suggested that thymic seeding and further colonization by progenitors is dictated by niche vacancy in the thymus. This is coordinated with the cyclic release of progenitors from the bone marrow (BM, 5). The most immature BM progenitor pool, termed LSK, are lineage negative (Lin-) and express Sca-1 and c-Kit (6, 7). This pool of LSK cells is very heterogeneous but can be divided into two major populations based on Fms-like tyrosine kinase receptor-1 (Flt3, CD135) expression: the population that lacks CD135 expression contains the self-renewing HSCs which have the potential to develop into all blood lineages. The other population is the CD135-expressing LSK cells. These CD135+ LSK cells are nonrenewing multipotent progenitors (MPPs) and can develop into cells of the lymphoid and myelo-erythroid lineages (8, 9). There is heterogeneity within these two major populations as well, which contain cells with varying degrees of self-renewing potential and multipotency  3  (reviewed in 9). Other lymphoid progenitors found in the BM also include the common lymphoid progenitors (CLPs). CLPs have a Lin-Sca-1loIL-7Rα(CD127)+c-Kit(CD117)lo CD135+ phenotype (10) and they arise from MPPs (11). There is evidence that BM HSCs, MPPs, as well as CLPs are able to initiate T cell development when injected intrathymically (10, 12, 13). However, it is still unclear which populations actually seed the thymus from the bone marrow and initiate T cell development. Thymic-seeding progenitors are thought to travel through the blood from the bone marrow to seed the thymus (14). There is evidence that HSCs, MPPs, and CLPs circulate the blood (14-17). One other blood circulating progenitor which has T cell potential is the circulating T cell progenitors (CTPs). These CTPs are Lin-CD127+CD117loCD135-Sca-1+ (18). The origin of these CTPs remain to be explored, but these cells are unique in that they are committed to the T cell lineage and have lost myeloid, erythroid, and B cell potential prior to entry into the thymus (18).  1.2.2 Stages of αβ T cell development αβ T cell development in adult mice starts with seeding of the thymus by progenitors traveling from the blood. Multiple phenotypically distinct bone marrow populations can settle within the thymus after intravenous injection, suggesting several populations from the bone marrow and the blood may contribute to the thymic pool (reviewed in 19). However, the most physiological thymic-seeding progenitors have yet to be identified. The thymic-seeding progenitors enter the thymus at the cortico-medullary junction (CMJ, 20, 21). These progenitors lack CD4 and CD8 expression and are referred to as double negative (DN) cells. Four differentiation stages of DN thymocytes, DN1-4, have  4  been defined based on CD25 and CD44 expression (22). The earliest progenitors are found within the DN1 (CD44+CD25-) population and reside at the perimedullary-cortical zone in proximity to the site of thymic entry where initial differentiation and progression occurs. Further differentiation into DN2 (CD44+CD25+) and DN3 (CD44-CD25+) thymocytes is accompanied by the migration of the cells to the mid and outer cortex respectively. Differentiation into DN4 (CD44-CD25-) and the subsequent CD4 and CD8 double positive (DP) stage is accompanied by the inward migration of the cells from the cortex to the medulla, where maturation into single positive (SP) CD4 or CD8 T cells occur (20). Positively selected thymocytes in the cortex that escape the negative selection in the medulla mature into naïve αβ T cells and exit the thymus and enter the blood for peripheral circulation (reviewed in 23, 24). The putative thymic-seeding progenitors entering the thymus possess T, B, natural killer (NK), NKT, dendritic cell (DC), and myeloid potentials (25-28). B cell potential is lost almost immediately after entry into the thymus (19, 29-32). These thymic-seeding progenitors reside within the DN1 population, which is a heterogeneous population that can be divided into two subsets (DN1.1 and DN1.2) based on the presence or absence of CD135 expression. CD135-expressing DN1.1 cells can give rise to B cells and are thought to be the precursor of the CD135- DN1.2 cells, which have limited B cell potential (29). Alternatively, the DN1 population can be split into five subsets (DN1a, b, c, d and e) based on CD117 and CD24 (heat stable antigen, HSA) expression (33). Only the CD117+ DN1a and DN1b populations, which are collectively termed early T lineage/thymic progenitors (ETPs, 12), have a high proliferative capacity and can efficiently develop into T cells when co-cultured with OP9-DL1 cells that support thymocyte development in vitro (33). These Lin-CD44+  5  Sca-1+CD127lo/- ETPs are phenotypically similar to and are thought to be downstream of BM LSK cells (reviewed in 9, 19). ETPs include some cells that express low amounts of CD4 (CD4lo, 34). The CD4 expressed on these cells are thought to be passively acquired from other thymocytes (35). Depending on the stringency of anti-CD4 antibodies used, these CD4lo ETPs will not be depleted during the enrichment for DN thymocytes with anti-CD4 antibodies (reviewed in 9, 19). These ETPs have the potential to generate DCs and NK cells, but have minimal B cell or myeloid potential (reviewed in 19). They are highly proliferative and proliferate about 1000-fold (36) before differentiating into DN2 cells, which is marked by the expression of CD25 (IL-2Rα) while retaining CD44 and CD117 expression. It is unclear whether CLPs contribute to the CD117-/lo DN1 populations, denoted as DN1c, d and e (reviewed in 9). These non-canonical progenitors contain T cell lineage potential, although less efficient than DN1a and DN1b populations (33). For example, the DN1c and DN1d populations show a small amount of B cell potential, whereas DN1e cells show a small amount of NK potential (33). The CD117lo DN1c population shows a similar developmental profile to that of the DN1a and DN1b populations when co-cultured with OP9-DL1 cells. However, CD117- DN1d and DN1e populations show an atypical T cell developmental profile that do not seem to pass through the DN2 and/or DN3 stages (33). DN2 thymocytes retain limited potential for NK and DC lineage (31, 37), but B cell potential is lost (29, 33). This subset is characterized by active proliferation to expand the population for T cell receptor (TCR) gene rearrangements (reviewed in 38, 39). Recombination-activating gene (RAG)-1 and -2 expression are significantly upregulated and  6  RAG-mediated rearrangements of the TCRβ, γ and δ loci are initially detected in DN2 cells (40-42). The downregulation of the surface expression of CD117 and CD44 marks the DN3 stage, which is accompanied by the loss of NK and DC potential. DN3 cells are fully committed to the T cell lineage and undertake large scale rearrangement of the TCRβ, γ and δ gene loci. DN3 cells can be subdivided into two populations: cells that have not completed TCR gene rearrangement, express low levels of CD27, and are not as proliferative are defined as DN3a; and DN3b cells which are highly proliferative and have upregulated CD27 expression upon successful completion of the TCRβ or TCRγδ gene rearrangement (43, 44). Some DN3 cells express the γδ TCR and can differentiate into γδ T cells, whereas other DN3 cells progress down the αβ T cell lineage, expressing the pre-TCR complex which is comprised of a TCRβ chain associated with the invariant pre-Tα chain and CD3δ, ε, γ and ζ signaling molecules (45). The pre-TCR mediates the β-selection event (discussed in section 1.2.3) and induces the transition to the DP stage through a DN4 stage and a brief intermediate immature CD8 single positive (ISP) stage. Rearrangement of the TCRα gene locus is completed by the DP stage and DP thymocytes express the αβ TCR which is similar to the pre-TCR except the pre-Tα chain is replaced by the TCRα chain. DP cells that passed both positive and negative selection of the αβ TCR develop into SP CD4 and CD8 naïve T cells. A schematic overview of thymocyte development is shown in Figure 1.1.  1.2.3 β-selection β-selection of thymocytes expressing the pre-TCR complex is a critical T cell development checkpoint. Signals derived from the pre-TCR complex trigger a series of 7  Figure 1.1 Thymocyte progenitor subsets and differentiation. Thymic-seeding progenitors downstream of bone marrow and circulating progenitors enter the thymus as DN1 cells which do not express CD4 or CD8. DN cells go through four stages of development (DN1-4), which is marked by differential expression of CD44 and CD25. The DN4 thymocytes then progress to the DP stage where CD4 and CD8 is upregulated (upper right of the diagram). DP cells that passed positive and negative selection become either CD4 or CD8 SP cells that exit the thymus and become naïve αβ T cells. At different stages of differentiation, thymocytes can split off and develop into non-T lineage cells and unconventional T cells (curved arrows). Surface markers expressed are indicated in blue. 8  events including rescuing of the cells from apoptosis, inhibition of further recombination of the TCRβ gene locus (a process termed allelic exclusion) to preserve the clonality of the antigen receptor expressed, initiation of TCRα gene rearrangement, as well as the induction of proliferation and differentiation to the next developmental stage (reviewed in 46). Mice with deficiency in the RAG-1, RAG-2, CD3ε, TCRβ, or pre-Tα genes have a complete or partial block in development at the immature CD4-CD8- DN stage owing to the impairment of pre-TCR expression (47-51). Deficiencies in signaling molecules involved in pre-TCR signaling such as the Src family kinase (SFK) Lck, lead to a partial block of thymocytes at the DN3 to DN4 stage (52-54). The participation of Lck in pre-TCR signaling is evident because activated Lck can rescue developmental defects due to deficiency in RAG1 (55) or pre-Tα (56). Consistent with this, expression of a transgenic TCR in a RAG-2/Lck double knockout mouse does not rescue T cell development, suggesting that Lck is required for β-selection (57). Although there is no defect in β-selection in mice with Fyn deficiency, another SFK expressed in thymocytes (53, 54), Fyn can partially compensate for the requirement for Lck in pre-TCR signaling, as deletion of both Lck and Fyn leads to a complete halt of thymocyte development at the DN3 stage (53, 54).  1.3 Intrathymic signals for thymocyte development  1.3.1 Thymic epithelial cells The requirement of the thymus for T cell production was first demonstrated about 40 years ago in thymectomized mice (reviewed in 58). Thymic epithelial cells (TECs)  9  constitute a major component of the thymic stroma and are required for thymus organogenesis and the promotion of multiple stages of thymocyte differentiation (59, 60). Foxn1 is a transcription factor that regulates TEC differentiation and proliferation (61, 62). Deletion of the N-terminal domain of Foxn1 (Foxn1∆/∆) results in a thymus-specific phenotype where initial TEC differentiation occurs but the TECs fail to differentiate into mature epithelial cells that are required for T cell maturation. In the adult Foxn1∆/∆ thymus, there is an accumulation of thymocytes at the DN stage and an absence of CD117+ DN1 cells (63). The mutant thymus fails to support progenitor differentiation to DN2 and DN3 stages, leading to a loss of CD25+ DN thymocytes (63, 64). Interestingly, αβ T cells with an atypical phenotype are found in the periphery of the Foxn1∆/∆ mouse. These T cells are thought to develop from atypical CD117- DN1 progenitors via a non-canonical pathway (64, 65). TECs provide a network of receptor ligands and growth factors that can trigger T cell differentiation and support proliferation and survival of thymocytes (reviewed in 38, 66, 67). These factors include the growth factors stem cell factor (SCF) and interleukin-7 (IL-7), as well as Notch ligands, which affect lineage decisions and promote T cell development (reviewed in 68, 69). Mice deficient in SCF, IL-7, or Notch signaling all show reduced thymic cellularity, supporting a role for these signaling factors in thymocyte proliferation and differentiation (70-72).  1.3.2 c-Kit signaling Stem cell factor (SCF, c-Kit ligand) plays important roles in cell survival, proliferation, and differentiation. The protein-tyrosine kinase receptor for SCF (c-Kit,  10  CD117), is expressed on hematopoietic stem cells, myeloid progenitor cells, pro-B cells, and pro-T cells (22, 34, 73). Mast cells are the only mature cells that express CD117 (74). SCF induces dimerization and autophosphorylation of CD117 which then recruits downstream signaling molecules including Lck, phospholipase C-γ (PLCγ), and phosphoinositide 3kinase (PI3K, reviewed in 75). CD117 signaling is important in hematopoiesis. CD117-deficient mice (c-KitW/W) die around the first week after birth due to lethal anemia (76). The involvement of CD117 signaling in T cell development can be seen using a viable c-KitW/W mouse termed “Vickid” (77). Although CD4 and CD8 SP cells are found within the thymus of Vickid mice, absence of CD117 signaling leads to an accumulation of DN and a dramatic reduction of DP thymocytes. Within the DN population, cells accumulate at the DN1 stage. Although there are DN4 cells, there is almost a complete absence of DN2 and DN3 cells in the thymus of the Vickid mice (77). Intrathymic SCF is important in the proliferation of DN thymocytes (78). This is supported by in vitro analysis of responsiveness to the combination of SCF and IL-7 in CD25+ and CD25- DN thymocytes (79). Neutralization of SCF inhibits stem cell proliferation but accelerates differentiation, suggesting that SCF plays a role in maintaining progenitor cells in an undifferentiated state (80, 81).  1.3.3 IL-7 signaling IL-7 in the thymus is produced by stromal cells located primarily at the medulla and the CMJ (82). IL-7 has a non-redundant survival and proliferative function in early T cell development (83, 84). The receptor for IL-7 (IL-7R) is composed of an IL-7Rα chain  11  (CD127) associated with a common cytokine receptor gamma chain (γc, CD132), which is shared with the IL-2 and IL-15 receptors. IL-7R mediates cellular responses by signaling through the Janus kinase-3 (JAK-3, 85), signal transducer and activator of transcription-5 (STAT-5, 86), PI3K (87) in thymocytes, and Lck in human T cells (88). Mice deficient in IL-7 or any component of IL-7R signaling (CD127, CD132, or JAK-3) exhibit an inhibition of T cell differentiation at multiple stages of development (71, 85, 89, 90). Specifically, the absence of IL-7 signaling leads to a substantial reduction in the number of thymocytes and αβ T cells, and an absence of γδ T cells. Within the DN population, a developmental block occurs between the DN2 to DN3 stages (71). Responsiveness to IL-7 in developing thymocytes, as assessed by STAT-5 phosphorylation, is highest at the DN2 and DN3 stages but is quickly lost at the DN4 stage (83). ETPs, which are CD127- and are found within the DN1 population, show no responsiveness to IL-7 when cultured in vitro (12). This suggests that IL-7 plays stage specific roles during thymocyte development. One function of IL-7 signaling is to promote cell survival by up-regulating the antiapoptotic protein Bcl-2. Expression of Bcl-2 is dependent on IL-7 in DN2 to DN4 cells, but not in DN1 cells (91). Absence of IL-7 leads to increased amounts of apoptotic DN cells. However, this can be restored by overexpression of Bcl-2, supporting the role of Bcl-2 in enhancing cell survival (91). Expression of Bcl-2 can also rescue αβ but not γδ T cell development in IL-7-deficient mice. In addition to a possible role for IL-7 in the survival of γδ T cells (92, 93), IL-7 also promotes γδ T cell development by regulating TCRγ gene rearrangement (94-96).  12  1.3.4 Notch signaling Notch proteins (Notch 1-4) are receptors that activate an evolutionarily conserved signal transduction pathway and are widely expressed in a variety of tissues. Notch interacts with ligands of the Jagged family (Jagged 1 and 2) and the Delta-like family (Delta-like 1, 3 and 4). Interaction of Notch with its ligands leads to two sequential proteolytic cleavage events by the tumor-necrosis factor α-converting enzyme (TACE) and the γ-secretase presenilin, resulting in release of the cytoplasmic domain of Notch, which then translocates to the nucleus and interacts with the CBF1 transcription factor to activate gene transcription. Notch-mediated signaling leads to inhibition or promotion of cell differentiation at multiple stages along particular developmental pathways (reviewed in 69). The requirement for Notch1 signaling in T cell development can be observed in lossof-function and gain-of function analyses. Constitutive activation of Notch signaling abolishes B cell development and induces T cell development in the bone marrow (97). Mice with deficiencies in Notch1 show a severe block in T cell development at the DN stage, with concomitant development of B cells in the thymus (98). Notch signals have been shown to promote αβ over γδ T cell lineage (99-102). Further down the T cell developmental pathway, Notch has been implicated in promoting the CD8 over the CD4 lineage (103, 104). However, recent studies show that Notch modulates TCR signaling during positive selection, thereby affecting the generation of CD8 versus CD4 SP thymocytes (reviewed in 105). In the periphery, Notch has been shown to favor T helper 1 (Th1) cell differentiation when interacting with Delta-like family ligands and favor T helper 2 (Th2) cell differentiation when interacting with Jagged ligands (106, 107).  13  In the absence of Notch signaling, there is an accumulation of DN thymocytes. Most of the DN cells remain at the DN1 stage. Although DN4 cells are present, there is an absence of DN2 and DN3 cells (98). Constitutive activation of Notch allows thymocytes to bypass the β-selection checkpoint by inducing pre-TCR-like signaling (108). Notch also promotes the survival of DN3 cells at the β-selection checkpoint by regulating cellular metabolism in a PI3K-Akt dependent, but pre-TCR-independent pathway (109, 110). Furthermore, withdrawal of Notch signaling leads to cellular atrophy and apoptosis (109, 111).  1.3.5 Interplay between Notch, IL-7 and CD117 signaling Although both IL-7 and CD117-deficient mice exhibit some degree of impairment in T cell development, mice defective in both CD117 and CD132 have a complete block in T cell development. This suggests a partially overlapping role of CD117 and IL-7 signals in promoting the proliferation and survival of early progenitor thymocytes (70). In addition, mice deficient in IL-7 show decreased CD117 expression on DN1 thymocytes (112). This further suggests that there is interdependency between SCF and IL-7 signaling. Notch signaling is also involved in sustaining CD127 expression in proliferating human thymocytes, while IL-7 is required to maintain survival and proliferation signals in response to Notch signaling (113). The interplay between Notch, IL-7, and CD117 signaling in regulating proliferation, survival, and progression of developing thymocytes, although not completely characterized, has been studied extensively using in vitro culture systems that allow for T cell development. In vitro culturing of thymic progenitors revealed that DN1 and DN2 cells require both Notch and IL-7 signaling for efficient proliferation and differentiation into TCR-expressing  14  cells (114, 115). In addition, Notch induces CD127 expression as early thymocytes transit between DN1 and DN2 (113). Notch also mediates the expression of CD117 on DN1 and DN2 cells (116, 117). However, DN3 cells require Notch, but not IL-7 and CD117 for in vitro proliferation (114, 115, 118).  1.3.6 In vitro differentiation of thymocytes Until recently, in vitro cultures aimed at mimicking in vivo T cell development have utilized fetal thymus organ cultures (FTOCs), which have limitations such as low cellular yield and variable seeding efficiency (reviewed in 119, 120). Another in vitro model was later developed that supported T cell development from progenitor cells in a simple twodimensional tissue culture system involving co-culturing thymocyte progenitors with OP9 stromal cells (101). The OP9 cell line, derived from the bone marrow of macrophage colony stimulating factor (M-CSF)-deficient mice, supports the differentiation of all hematopoietic cells except the T cell lineage. Since Notch is involved in T cell development, OP9 cells expressing the Notch ligand, Delta-like-1 (OP9-DL1), were generated (101). Co-culturing of progenitor cells with OP9-DL1 cells in the presence of IL-7 and Flt3-L inhibited B cell development and induced differentiation to functionally-mature SP T cells. Utilizing the OP9-DL1 co-culture system, single progenitor cells can now be assayed for T cell progenitor-progeny relationships. With modifications of the growth factors added to the co-culture system, stage-specific gene expression and responses of developing cells can also be identified.  15  1.4 Cell migration during thymic development  Initiation of thymocyte development starts with the migration of thymic progenitors from the blood into the thymus. Although different hematopoietic progenitors circulate in the blood, the identity of the thymic-seeding progenitors and the mechanism of how progenitor cells migrate from the blood into the thymus are not well defined. It is implied that these progenitors must acquire the competence to migrate into the thymus (reviewed in 19, 24), which is thought to involve the chemokine receptors CCR9 (32, 121, 122) and CCR7 (123, 124), as well as the adhesion molecules selectins (125), CD44 (126, 127), and integrins (122, 128). The differentiation of thymocytes is accompanied by migration of the thymocytes through different thymic zones, allowing exposure of the developing thymocytes to different signals that drive T cell differentiation (reviewed in 24, 38, 129). The migration of these developing cells requires adhesion molecules interacting with ligands and is guided by chemokines produced by thymic stromal cells in specific regions of the thymus (reviewed in 130, 131).  1.4.1 Adhesion molecules involved in thymic seeding and intrathymic migration Adhesion molecules, such as selectins, integrins, and CD44 are important in mediating cell:cell and cell:extracellular matrix interactions. These interactions are involved in the migration of cells. Migration of thymic-seeding progenitors from the blood into the thymus is thought to be analogous to lymphocyte extravasation from the blood into tissues involving (i) the selectin-mediated rolling of the cell along the endothelium; (ii) integrin-  16  mediated tight binding to the endothelium; (iii) integrin-mediated movement through the endothelium; and (iv) chemokine-mediated migration into the thymus. In adult mice, thymic seeding is mediated by the P-selectins on thymic endothelial cells interacting with the platelet (P)-selectin glycoprotein ligand-1 (PSGL-1) expressed on thymic-seeding progenitors. Mice lacking P-selectin or PSGL-1 show a significant decrease in progenitors seeding the thymus (125). Although thymocyte development in CD44deficient mice appears to be normal (132), blocking of CD44 with antibodies leads to a reduction in the homing of intravenously injected thymocytes (127). Similarly, blocking antibodies against α4 or β2 integrins also reduce thymic homing (122). These results suggest that P-selectin, CD44, and integrins play a role in the migration of progenitors to the thymus during development. Adhesion molecules are not only important at the initial stage where thymic progenitors enter the thymus, but are also important at subsequent stages of differentiation where developing thymocytes move to specific zones within the thymus and localize to specific niches. For example, integrins expressed on DN2 and DN3 thymocytes are involved in the migration of the developing thymocytes across the cortex by interacting with the vascular cell adhesion molecule-1 (VCAM-1) expressed by cortical stromal cells (reviewed in 24, 38).  1.4.2 Chemokines involved in intrathymic migration In addition to adhesion receptors and their ligands, polarizing signals and/or directional cues are required to establish the directionality of cell migration into, and within the thymus. The exact role of chemokines in the initial seeding of the adult thymus is not  17  clear as neither the lack of a single gene nor a combination of genes has been demonstrated to completely abolish homing of hematopoietic precursors to the adult thymus (66). Stromal cell-derived factor-1 (SDF-1, CXCL12) is mainly expressed by thymic fibroblasts within the cortex (133). The CXCL12 receptor, CXCR4, plays a role in the migration, as well as proliferation and survival of early thymocytes (134, 135). Antibodies against CXCR4 reduce the number of progenitors homing to the thymus (136). In addition, CXCL12-mediated signaling is important for the outward migration of the DN2 thymocytes to the cortex for development (38). Supporting this, thymus-specific deletion of CXCR4 leads to an accumulation of thymocyte progenitors at the CMJ together with the arrest of development at the DN1 stage (133). CXCR4 is downregulated in DP and SP thymocytes, and the cells upregulate receptors for and become responsive to other chemokines (131). CCL25 is expressed at high levels in the thymic cortex and medulla (137, 138). Competitive reconstitution experiments showed that cells from CCR9-deficient mice are less efficient in repopulating the thymus compared to cells from wild type mice (121). Although there is normal T cell development in the CCR9-deficient mice, there is a mislocation of CD25+ DN thymocytes cells throughout the cortex instead of concentration at the subcapsular zone, where DN3 cells normally reside (139). CCR9 is also implicated in the trafficking of DP thymocytes across the cortex to undergo positive selection (137, 140). The chemokine receptor CCR7, and its ligands, CCL19 and CCL21, are implicated in coordinating the migration of thymocytes within the thymus (reviewed in 24, 129). CCL19 and CCL21 are expressed in the medulla and the CMJ area of the thymus (138). Within the DN population, CCR7 is predominantly expressed on a subpopulation of CD44+CD25lo cells. Deletion of CCR7 leads to an accumulation of CD44+CD25+ thymocytes at the CMJ (138).  18  CCR7 is also involved in attracting positively selected DP cells from the cortex to the medulla (141, 142). A schematic view of a developing thymocyte traveling within the thymus is shown in Figure 1.2. PSGL-1 can mediate enhanced chemotactic responses by directly binding to CCL19 and CCL21 in resting T cells (143). Similarly, human hematopoietic stem cells stimulated with CXCL12 show a spread and polarized morphology, and adhere to immobilized hyaluronan, the major ligand of CD44 (144). This demonstrates that adhesion molecules and chemokines work together to modulate directed cell migration. By directing the migration of thymocytes to specific zones within the thymus to receive survival and proliferation signals for differentiation, chemokines and adhesion molecules can modulate the outcome of the T cell differentiation process in the thymus.  1.5 Unconventional thymocytes  Other than the conventional αβ T cells, the thymus is also required for the generation of γδ T cells and NKT cells. These unconventional T cells, as well as NK cells, share common progenitors with the conventional αβ T cells. Hence, dysregulation of intrathymic signals would greatly affect the lineage choices made early in thymocyte development.  19  Figure 1.2 Schematic overview of thymocyte migration and development in the thymus. Thymocytes at specific stages occupy discrete regions within the thymus. Circulating thymic-seeding progenitors enter the thymus from the bloodstream at the cortico-medullary junction in a process which involves the adhesion molecules P-selectin, CD44, and integrins. The outward migration of DN thymocytes through the cortex to the subcapsular zone is mediated by CXCR4 and CCR7 signaling. DN3 cells at the subcapsular zone undergo TCR gene rearrangement and β-selection. Thymocytes are then guided by CCR9 signals to migrate through the cortex where positive selection occurs. Positively selected DP cells are attracted to the medulla, which expresses CCR7 ligands, for negative selection. The surviving SP cells re-enter the bloodstream as naïve T cells. Chemokines expressed in the thymus are indicated in blue.  20  1.5.1 Thymic γδ T cells γδ T cells are unconventional T cells that play a central role in bridging the innate and adaptive immune system (145). Most γδ T cells reside in the epithelial layers of tissues such as the skin, intestinal epithelium, lung and tongue, functioning as our first line of defense against foreign pathogens. As mentioned in the previous section, γδ T cells split from the conventional αβ T cell lineage at the DN3 stage. Productive pairing of in-frame TCRγ and TCRδ chains allows the differentiation of DN3 cells along the γδ T cell lineage, although αβ versus γδ T cell lineage choice depends on multiple factors including the strength of TCR, Notch, and IL-7 signaling (reviewed in 146). Unlike the αβ TCR, the γδ TCR repertoire is more limited and there is only one selection event. The selected γδ T cells remain CD4 and CD8 double negative (147-149). The different maturation stages of γδ TCR-expressing thymocytes can be further divided into three stages: CD25+, CD25-CD24+, and CD25-CD24-, through which the maturing γδ T cells gradually lose their potential to generate αβ T cells (43). Selected γδ T cells, which can be identified by the downregulation of CD24, undergo significant cell proliferation and upregulate CD27, similar to αβ T cells that passed βselection (43, 148, 150). The DN2 population can be divided into two populations based on CD127 expression, in which CD127+ cells have a tendency to develop into γδ T cells while CD127cells tend to develop into αβ T cells (151). This suggests that there is a TCR-independent lineage pre-commitment towards γδ T cells in CD127+ DN2 cells. However, there is also evidence suggesting that the γδ TCR is involved in directing lineage outcomes (152). Strong  21  γδ TCR signals lead to γδ T cell commitment, whereas weak γδ TCR signals direct αβ T cell development (153-155). γδ T cells can be divided into different subpopulations depending on whether development occurs in the fetal or adult thymus, intra or extrathymically, or in the resident tissue (reviewed in 156), each having differential requirements for development. γδ T cells that arise from the adult thymus require Lck, as well as a significant number of DP thymocytes for functional maturation (157).  1.5.2 NK cells NK cells are another subset of lymphocytes that mainly participate in the innate immune response, but also affect outcomes of the adaptive immune response. There are multiple sites for NK cell development including bone marrow, liver, thymus, lymph nodes, blood and spleen (reviewed in 158). The thymic pathway of NK cell development has been characterized in mice only recently (159, 160). T cells and NK cells are presumed to share a common intrathymic precursor. In addition to the early thymic progenitors found within the DN1 and DN2 populations that have the ability to generate NK and T cells in vitro, NK progenitor cells with only NK potential have been identified in the fetal thymus (161). The ability of the NK/T bipotent cells to generate NK cells require transient Notch signal, as prolonged Notch signaling inhibits NK cell development and instead promotes T cell development (162, 163). Thymic NK cells represent 0.05% of thymic cellularity and can be identified by their dependence on the thymus, IL-7, and the transcription factor GATA-3 for development (159). Thymic NK cells express high levels of CD127 and are functionally distinct from the  22  CD127- NK cells found in the spleen, blood, and liver, and represent a substantial fraction of resident NK cells in the lymph nodes (159). Recently, a population of NK-like γδ T cells that are NK1.1+CD127+ and express low levels of surface γδ TCR and CD3 has been identified. This population can only be discriminated from NK cells using a TCRγ locus-histone 2Benhanced green fluorescent protein (GFP) reporter and would have been identified as NK cells using standard protocols (164, 165). Although the physiological role of thymic NK cells and their developmental origin remain unclear, there is evidence for a role of thymic NK cells in monitoring thymic precursors for uncontrolled proliferation (166) and in regulating T cell selection (167).  1.5.3 NKT cells NKT cells are a thymic-dependent T cell subset that have been implicated in a variety of functions including enhancing innate immunity, tumor rejection, suppression of autoimmune disease, and promoting tolerance. However, they have also been implicated in exacerbating atherosclerosis, allergy, and autoimmune disease. Hence the regulation of NKT development and hematopoiesis is important (reviewed in 168). There is evidence that NKT cells might be important for the selection and/or the generation of other T cell subsets (169). NKT cells are defined by co-expression of the NK1.1 and αβ TCR and are segregated from conventional T cells at the DP stage in the thymic cortex (170, 171). Similar to conventional T cells, NKT development depends on pre-TCR signaling (reviewed in 172). A subset of NKT cells depend on Fyn for development through a SLAM-SAP-Fyn signaling pathway (172) that is independent of TCR signaling (173).  23  NKT cells exhibit a limited TCR repertoire, which is positively selected by the nonpolymorphic class I-like molecule CD1d (174) presented by DP thymocytes (175-177). Along with CD1d-presenting dendritic cells in the thymus, the DP thymocytes also mediate negative selection of the NKT cells (178-180). The selected NKT cells can quickly downregulate CD8 expression to develop into CD4+ or DN NKT cells (reviewed in 181). There is evidence that DN NKT cells are derived from CD4+ NKT cells, but the time of the branching is unclear, since DN NKT cells can be in either immature (NK1.1-) or mature (NK1.1+) forms (182, 183). Interestingly, most NKT cells leave the thymus at the immature NK1.1- stage (182, 184), whereas NKT cells that remain in the thymus are mature NK1.1+ NKT cells (185). Mature γδ T cells, NK, and NKT cells express high levels of CD44, which often contaminate the CD44+CD25- DN1 population in the thymus (186).  1.6 CD44  1.6.1 CD44 structure and expression CD44 is a type I transmembrane adhesion molecule that is widely expressed on most cells. It exists as multiple isoforms due to alternative splicing of 10 variably expressed exons, with molecular weights ranging from 80 – 200 kDa, due to differential glycosylation and glycosaminoglycan addition (reviewed in 187, 188). CD44 is highly expressed on bone marrow hematopoietic progenitors (189), circulating LSK cells, (16) and ETPs in the thymus (12, 22). The 95-kDa standard isoform  24  of CD44 (CD44s) is found on early T cell precursors (190) and a variant CD44 isoform (CD44v) is expressed on ETPs (191). This CD44v is required for the initial interaction of hematopoietic progenitor cells with the thymic stroma (191).  1.6.2 CD44-mediated migration, cell spreading and signaling Migration of thymocyte progenitors into and within the thymus is essential for T cell development (reviewed in 38). CD44 may play a role in the migration of progenitors to the thymus during development (132, 192). Migration of cells over a substratum is thought to involve five steps. These are: (i) extension of the leading edge; (ii) adhesion to matrix contacts; (iii) contraction of the cytoplasm; (iv) release from contact sites; and (v) recycling of membrane receptors from the rear to the front of the cell (193). Migratory cells have been described as adopting a polarized and spread shape with an extended membrane at the leading (front) edge and a protrusion called the uropod at the back end of the cell where CD44 is sequestered. CD44-induced signals can result in actin- and microtubule-dependent spreading in T cells (194, 195). CD44 can also mediate spreading in B cells (196). It has been shown that CD44-mediated cell spreading requires the cytoplasmic domain of CD44 and lipid rafts (N. Wong, Ph.D. thesis, 2006, University of British Columbia and 194). The 72 amino acid cytoplasmic domain of CD44 has no intrinsic catalytic activity. However, the ability of CD44 to mediate signals has been demonstrated. For example, crosslinking of CD44 with antibodies can induce an increase in the kinase activity of Lck as well as tyrosine phosphorylation of ZAP-70 (197), leading to calcium influx (198) and protein kinase C activation (199). CD44-mediated cell spreading is dependent on SFK activity  25  (195). The focal adhesion kinase family of proteins, FAK and Pyk2, are also part of the signaling cascade involved in spreading (195). PI3K, PLCγ and calcium mobilization are also required for CD44-mediated spreading (N. Wong, Ph.D. thesis and 200).  1.6.3 CD44 binding to hyaluronan and the actin cytoskeleton The physiological ligand of CD44, hyaluronan (HA), is a polymer of a repeating disaccharide of D-glucuronic acid and D-N-acetylglucosamine, which is a large component of the extracellular matrix. Most leukocytes require activation before they will bind HA (reviewed in 188). Clustering of CD44 can induce the binding of CD44 to HA (201). In mutant forms of CD44 where the CD44 cytoplasmic domain deleted, HA binding can be restored by pre-treatment with anti-CD44 antibodies (201, 202), suggesting that CD44 clustering promotes ligand binding by increasing the avidity. There is also evidence for the involvement of the actin cytoskeleton in the binding of CD44 to HA (203). Ankyrin and the ezrin/radixin/moesin (ERM) family proteins have been implicated in linking CD44 to the cytoskeleton (204, 205). ERM proteins play an important role in cell polarization during T lymphocyte migration and they are involved in anchoring CD44 to the uropod (reviewed in 206). Confocal microscopy showed that CD44 and ERM proteins are co-localized at cell adhesion contact sites (205, 207). In vitro experiments identified a region of the CD44 cytoplasmic tail that is rich in basic resides as the ERM binding site (208, 209). The CD44:ERM interaction has recently been confirmed by crystal structure analysis (210). There is also evidence for ERM proteins interacting with SFKs. The SH2 domain of Src can bind to phosphorylated Y190 of ezrin. This binding is a prerequisite for the phosphorylation of Y145 on ezrin by Lck in T cells, and Src in an epithelial cell line (211, 212). The Y145  26  residue is conserved in all three ERM proteins and its phosphorylation is thought to enhance SFK activity, generating a positive feedback loop in an epithelial cell line. Mutation of Y145 to a phenylalanine leads to impaired focal adhesion assembly and delayed spreading on fibronectin (212).  1.6.4 Role of CD44 on hematopoietic progenitors and early thymocytes The adhesion of HSCs to bone marrow stromal niches is essential for their development. Both CD44 and hyaluronan have been shown to play an important role in the localization of HSCs to the bone marrow and their survival (213). Anti-CD44 blocking antibodies reduce the homing of precursors to the bone marrow and the thymus (127, 214). Consistent with this, CD44-deficient lymphocytes show impaired entry into the thymus compared to wild type lymphocytes in competition experiments (132, 192). These data suggest that CD44 plays a role in the homing of lymphocytes to different niches. Although CD44-deficient mice show normal thymic development, competition experiments show that there is a decrease percentage of CD44-/- DN2 cells in the thymus compared to their wild type counterparts (192). The fact that CD44 is only highly expressed on DN1 and DN2 suggests that CD44 may play a non-essential role at the initial stages of thymocyte development. However, ex vivo thymocytes do not show specific binding of CD44 to HA, suggesting that CD44 is binding to some other ligands in the thymus (127). Supporting this, it has been shown that the migration of developing thymocytes in the thymus is dependent on the interaction between integrins and the stromal cell matrix rather than the extracellular matrix (215).  27  1.7 CD45  1.7.1 CD45 expression and structure CD45 is a type I transmembrane protein tyrosine phosphatase that is expressed on the surface of all nucleated cells of the hematopoietic system, including T cells, B cells, NK cells, macrophages, and DCs. It is one of the most abundant cell surface glycoproteins, comprising up to 10% of the cell’s surface area (216). The large extracellular domain of CD45 consists of a heavily glycosylated alternatively spliced region, a cysteine-rich domain, and three fibronectin III-like domains. CD45 has a single transmembrane domain and a cytoplasmic tail containing two tandem PTPase domains, D1 and D2. The D1 PTPase domain contains phosphatase activity, whereas the D2 domain is catalytically inactive (Figure 1.3). Depending on the cell type, developmental stage, and activation state, CD45 is expressed as one or more isoforms generated by alternate splicing of exons 4, 5, and 6 of a single gene, resulting in isoforms designated as A, B, and C in the extracellular domain (217219).  1.7.2 The Src family kinase Lck, a substrate of CD45 The best characterized substrates for CD45 are members of the non-receptor protein tyrosine Src family kinase (SFK) family. SFKs play important roles in antigen receptor signaling by initiating the signaling cascade upon antigen receptor activation (reviewed in 220, 221, 222). The major SFKs expressed in thymocytes and T cells are Lck (p56lck) and to a lesser extent, Fyn (p59fyn).  28  Figure 1.3 CD45 structure. CD45 can be expressed as multiple isoforms (A, B, and C) due to alternative splicing of the extracellular region that is O-linked glycosylated. The remaining extracellular domain is heavily N-linked glycosylated, consisting of a cysteinerich region and three fibronectin type III repeats. The cytoplasmic domain consists of two tandem protein tyrosine phosphatase domains (D1 and D2), with only D1 having phosphatase activity.  29  Expression of the lck gene is driven by two alternative promoters; the proximal promoter that is 5’ adjacent to the first coding exon and the distal promoter, located approximately 20 kb 5’ to the proximal promoter (223). The proximal promoter is active only in thymocytes, suggesting a role in T cell development, but the distal promoter is active in both thymocytes and peripheral T cells. Although lck mRNAs are found at equivalent levels in DN1-4 stages, Lck protein is only detected at the DN3 stage and onwards, suggesting a post-transcriptional regulation of Lck expression in immature thymocytes (224). However, DN1 is a heterogeneous population. If Lck is only expressed in a small subset of cells within the DN1 population, then the Lck protein may become undetectable in total DN1 cells. Using a GFP-reporter for the proximal lck promoter, lck promoter activity is observed in NK cells but not B cells or macrophages (224). Using the same approach, Shimizu et al. subdivided DN1 into two groups based on the presence or absence of proximal lck promoter activity (225). The presence of lck promoter activity corresponds to the loss of B cell and DC potential. This difference in lck promoter activity also supports the idea that Lck may be expressed in a subpopulation of DN1. In T cells, upon TCR ligation, Lck and possibly Fyn is required for the phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 chains of the TCR complex, which then leads to a series of signaling events by recruitment and activation of downstream signaling molecules such as ZAP-70, linker of activation in T cells (LAT), SLP-76, and PLCγ. The importance of Lck in thymocyte development can be seen in Lck-deficient mice which have a severe block in development from the DN to DP stage, and almost a complete block from the DP to SP stage, where Lck is required for pre-TCR and TCR signaling, respectively (52).  30  SFKs share similarities in domain structure. In addition to a unique N-terminal domain, each has a Src homology 3 (SH3) domain, a Src homology 2 (SH2) domain, a catalytic tyrosine kinase domain, and a short carboxyl-tail (reviewed in 226). The first three domains are collectively called N32. SFK activity is controlled by the balance between the phosphorylation of two key tyrosine (Y) residues. Autophosphorylation at a site within the kinase activation loop (Y394 in Lck) is necessary for kinase activity and this is counter-balanced by phosphorylation of a C-terminal tyrosine residue (Y505 in Lck) by the C-terminal Src kinase (Csk). Phosphorylation of the C-terminal tyrosine causes an intramolecular interaction between the phosphorylated tyrosine and SH2, causing a conformational change that prevents kinase activity. This inhibitory regulation of SFKs by C-terminal tyrosine phosphorylation affecting the conformation can be reversed by high affinity ligands that disrupt intramolecular interaction between the C-terminal tyrosine and the SH2 domain. CD45 can dephosphorylate both the regulatory tyrosine residues on SFKs, suggesting that CD45 is both a positive and negative regulator of SFK activity (reviewed in 227). In the absence of CD45, Lck and Fyn are hyperphosphorylated at the C-terminal negative regulatory tyrosine leading to abolishment of TCR signaling (228-230). However, there is evidence that hyperphosphorylation at both sites in the absence of CD45 can lead to increased SFK activity which suggests that phosphorylation at the positive regulatory tyrosine is dominant over phosphorylation at the negative regulatory tyrosine (231, 232). These data suggest that CD45 can modulate SFK signal transduction thresholds by affecting the balance between the two regulatory tyrosines of SFKs.  31  1.7.3 CD45 in TCR signaling The importance of CD45 in T cell development has been demonstrated in several CD45-deficient mice generated by targeted disruption of exons 6, 9 or 12 (233-235). There is a severe defect in T cell development in CD45-deficient mice which only have 5 – 10% of the normal numbers of T cells in the periphery (236). Within the thymus, development is partially blocked at the DN3 stage suggesting a partial defect in pre-TCR signaling, and a more profound block at the DP stage where αβ TCR signaling is required for the generation of SP cells (233-235). DP thymocytes from CD45-deficient mice undergo positive selection, but require stronger TCR signals for further development (233, 234) demonstrating that CD45 regulates T cell development by altering the signaling threshold. Although CD45 may be dispensable for β-selection shown using the RAG-2/CD45 double knockout mouse expressing a transgenic TCR (57), deletion of CD45 leads to a partial block in development at the DN3 to DN4 stage (233, 234) when the pre-TCR is expressed and β-selection occurs. In addition, the block in T cell development in CD45-deficient and RAG-1/CD45 double knockout mice can be rescued by expression of a constitutively active Lck mutant (237, 238), suggesting the involvement of CD45 in both pre-TCR and TCR signaling via Lck. Earlier findings show that CD45 can alter TCR selection at the DP stage. CD45deficient thymocytes do undergo positive and negative selection, but these cells require stronger TCR signals for further development (234). This is supported by the finding that more than half of the peripheral T cells in CD45-deficient mice express a self-reactive TCR (239). This shows that CD45 can alter the TCR signaling threshold.  32  1.7.4 CD45 in cell spreading and migration During extravasation of cells from the bloodstream into tissues, either upon entry of thymic progenitors into the thymus under homeostatic conditions, or the recruitment of leukocytes to inflammatory sites during an infection, the cell first assumes a polarized cell morphology involving redistribution of the chemokine receptors and adhesion molecules, as well as reorganization of the cytoskeleton (reviewed in 240). This polarization does not require the presence of a chemotactic gradient. Then the cell moves in cycles of five sequential steps (see section 1.6.2) during which adhesion molecules mediate attachment to the extracellular matrix, but also transduce signals leading to changes in the cytoskeleton, cell spreading, and eventually migration of the cell (193, 240). CD45 has been shown to regulate adhesion, spreading, and migration in various cell types. CD45 is required for sustained integrin-mediated adhesion in macrophages, yet it negatively regulates α5β1 integrin, but not α4β1 integrin-mediated adhesion in T cells (241, 242). This regulation of integrin-mediated adhesion by CD45 in macrophages was suggested to occur via the regulation of SFKs Hck and Lyn (241). Similarly, CD45 can negatively regulate CD44-mediated spreading that is dependent on SFK activity in a T cell line (195). Conversely, CD45 is a positive regulator of CXCL12-mediated migration in Jurkat T cells (243). Thus, CD45 is associated with both the positive and negative regulation of signaling, which may, at least in part, be due to its ability to positively and negatively regulate SFKs.  1.7.5 Regulation of CD45 Due to the pivotal role CD45 plays during T cell development, understanding the regulation of CD45 would help decipher how dysregulation of T cell development may  33  occur. Currently, little is known about how the tyrosine phosphatase activity of CD45 is regulated. With the ligand for CD45 yet to be identified, the possible regulation of the phosphatase activity of CD45 includes isoform expression, homodimerization to inhibit activity, temporal and spatial cellular localization, access to substrates, interactions with other proteins, and modulation by the non-catalytic domain (D2) of CD45 (reviewed in 227, 244).  1.8 Thesis objectives  CD45 is expressed on virtually all cells of the hematopoietic origin, from stem cells to memory cells, which are the building blocks of our immune system. The involvement of CD45 in T cell signaling has been studied mostly in more mature or terminally differentiated lymphocytes. Previous studies utilizing CD45-deficient mice focused mainly on the role of CD45 on the activation or effector functions of NK cells (245, 246) and B cells (220, 221, 247). The involvement of CD45 on pre-TCR and TCR selection events during T cell development has also been studied extensively (234, 239, 248). Although CD45 is expressed on all thymocytes, the role of CD45 on thymocyte development prior to pre-TCR expression has never been examined. Since multiple processes and signals affect the development and differentiation of lymphocytes, any alterations in these processes and signals would disrupt the balance (homeostasis) required for a functional immune system. CD45 has been implicated as a regulator of CD44-mediated cell spreading (195), chemokine-induced migration (243), and  34  cytokine (IL-3, IL-6, and IL-7) mediated proliferation (249-251). These processes are involved in thymocyte development, suggesting a role for CD45 in early T cell development. I therefore hypothesized that CD45 regulates multiple processes in early thymocyte development and that the absence of CD45 would alter the outcome of thymocyte development. Migration to specific niches within the thymus to receive stage-specific signals is important for the survival, proliferation, and differentiation of the developing thymocytes. In order to migrate, cells must first adopt a spread phenotype. CD44 can mediate cell spreading in T and B cells. Since CD44 is expressed on early thymocytes, I hypothesized that CD44 can also mediate cell spreading in thymocytes and that this may affect migration and development within the thymus. Because CD45 negatively regulates CD44-mediated cell spreading, the first major objective of this research was to determine how CD45 regulates CD44-mediated signaling leading to the spreading of thymocytes. The second major objective of this thesis was to determine the role of CD45 in proliferation, chemokine-induced migration, and the generation of early thymic progenitors. Together, these findings will provide a better picture of how the development of T cells, a central component of our immune system, is regulated.  35  CHAPTER 2 MATERIALS AND METHODS  36  2.1 Materials  2.1.1 Antibodies Antibodies (Ab) used for immunoprecipitation, Western blotting and confocal microscopy include the CD44 monoclonal antibodies (mAb) KM81 (252), IM7.8.1 (IM7, (190) and KM201 (253) from P.W. Kincade; CD45 mAb I3/2 from I.S. Trowbridge (254); rabbit antisera J1WBB raised against the C-terminal 32 residues of the CD44 cytoplasmic domain (195); rabbit antisera RO2.2 raised against the cytoplasmic domain of CD45 (255); rabbit antisera R54-3B raised against residues 34-150 of Lck (256); anti-Fyn (FYN3) and anti-Pyk2 (N-19) mAb from Santa Cruz Biotechnology (Santa Cruz, CA); antiphosphotyrosine mAb 4G10 from Upstate biotechnology (Lake Placid, NY); Alexa Fluor 568-conjugated goat-anti-rabbit Ab from Invitrogen (Burlington, ON). Protein A conjugated to horseradish peroxidase (HRP) was purchased from Bio-Rad Laboratories (Mississauga, ON) and goat-anti-rabbit IgG Heavy and Light (H+L) chain-HRP was from Jackson Immunoresearch Laboratories (West Grove, PA). Goat-anti-mouse IgG (H+L chain)-HRP and goat-anti-rat IgG (H+L chain)-HRP were from Southern Biotechnology (Birmingham, AL). The following reagents and Ab were used for flow cytometry: anti-CCR7 (4B12), CD3ε (145-2C11), CD4 (GK1.5), CD8α (53-6.7), CD11b (M1/70), CD19 (MB19-1), CD24 (M1/69), CD25 (PC61.5), CD45.2 (104), CD117 (ACK2), CD127 (A7R34), γδ TCR (UC713D5), Gr-1 (RB6-8C5), NK1.1 (PK136), Sca-1 (D7), TCRβ (H57-597), and Ter-119 (Ter119) conjugated to fluorescein isothiocyanate (FITC), phycoerythrin (PE), PE-Cy5, PE-Cy7, allophycocyanin (APC), Alexa Fluor 647, APC-Cy7, and Pacific Blue from BD Pharmingen  37  (San Diego, CA), eBioscience (San Diego, CA) or the Biomedical Research Centre antibody facility (Vancouver, BC). Purified anti-CD45 (I3/2) and anti-CD44 (IM7) mAb were conjugated to Alexa Fluor 488, Alexa Fluor 647 or Pacific Blue (Molecular Probes) as per manufacturer’s instructions. Fc receptor blocking mAb (2.4G2) was from (American Type Culture collection (ATCC) Manassas, VA). Biotinylation of purified anti-CD3ε, anti-CD4 and anti-CD8α mAb (ATCC) were performed with EZ-Link Sulfo-NHS-Biotin Reagents (Pierce, Rockford, IL) as per manufacturer’s instructions. Anti-biotin MicroBeads were from Miltenyi Biotec (Auburn, CA). Anti-BrdU-FITC and isotype control were from BD Pharmingen. Polyclonal anti-CXCR4 (G-19) Ab was from Santa Cruz Biotechnology. FITC- and PE-conjugated goat-anti-rat Ab was from Southern Biotech (Burlington, ON). Alexa Fluor 488-conjugated donkey-anti-goat and Alexa Fluor 568-conjugated goat-antirabbit Ab were from Molecular Probes. Ab to CD11b, CD19, γδ TCR, Gr-1 and NK1.1 were used to deplete lineage-positive thymocytes while Ab to CD3ε, CD4, CD8α, CD11b, CD19, Gr-1 and Ter-119 were used to deplete lineage-positive BM cells. Rooster comb hyaluronan (HA) from Sigma-Aldrich (Oakville, ON) was conjugated to fluorescein (FL-HA) as previously described (257).  2.1.2 Reagents Recombinant mouse stromal cell-derived factor-1 (SDF-1, CXCL12), interleukin-7 (IL-7), and Fms-like tyrosine kinase receptor-1 ligand (Flt3L) were from Peprotech (Rocky Hill, NJ). Recombinant mouse IL-2 was from R&D Systems, (Minneapolis, MN). Taxol, U73343, U73122, and PP2 were from Calbiochem (La Jolla, CA). Geneticin 418 was from  38  Invitrogen. 1,7-phenanthroline and 1,10-phenanthroline, L-histidinol, ionomycin and phorbol myristate acetate (PMA) were from Sigma-Aldrich.  2.1.3 Mice Congenic CD45.2 C57BL/6 (hereafter referred to as CD45+/+ mice) and CD45 exon 9 deficient mice (233) were purchased from the Jackson Laboratories (Bar Harbor, ME). The CD45-deficient mice were backcrossed onto the C57BL/6 background three more times to generate a ninth generation backcross (hereafter referred to as CD45-/- mice). Mice were housed and maintained by homozygous matings in the Wesbrook Animal Unit at the University of British Columbia. Animal experimentation was conducted in accordance with protocols approved by the University Animal Care Committee and Canadian Council of Animal Care guidelines. All mice were sex and age matched and used between 6 to 12 weeks of age.  2.2 Methods  2.2.1 Cell isolation and culture Spleens, lymph nodes and thymi harvested from mice euthanized by CO2 asphyxiation were passed through a strainer in FACS buffer (phosphate-buffered saline (PBS), 4% v/v fetal calf serum (FCS, Invitrogen), 2 mM ethylene-diamine-tetra acetic acid (EDTA)) or MACS buffer (PBS, 0.5% w/v bovine serum albumin (BSA, ≥96% pure, SigmaAldrich), 2 mM EDTA) to generate a single-cell suspension. Total BM cells were prepared  39  by flushing the tibiae and femurs from the epiphysis with wash buffer (Hank’s balanced salt solution, 5% v/v FCS, 5 mM EDTA) using a 26 gauge 3/8 inch needle. Splenocytes, lymphocytes and BM cells were treated with red blood cell (RBC) lysis buffer (0.83% w/v NH4Cl; 10 mM Tris-HCl, pH 7.25; 5 ml per mouse) for 10 min on ice before being washed with 30 ml of wash buffer. Blood (500 – 700 µl) was obtained from euthanized mice by heart puncture using a 26 gauge 1/2 inch needle and collected in tubes containing 1 ml of 20 mM EDTA/PBS and was subjected to two successive rounds of RBC lysis. Cells were centrifuged at 342 × g for 5 min at 4oC. For enrichment of T lymphocytes, CD4-CD8- double negative (DN) thymocytes, and lineage negative (Lin-) BM cells, lymphocytes from spleens or lymph nodes were labeled with biotinylated anti-CD19 (1 µg/106 cells), CD11b (1 µg/106 cells), NK1.1 (0.5 µg/106 cells) and Ter-119 (from BD lineage panel, 6.5 µl/107 cells) mAb; thymocytes were labeled with biotinylated anti-CD4 and CD8 mAb (0.95 µg/107 cells); BM cells were labeled with biotinylated anti-CD11b, Gr-1, Ter-119, (BD lineage panel, 6.5 µl/107 cells), CD3ε, CD19 (5 µg/107 cells), CD4 and CD8 mAb respectively. Cell were then washed with FACS buffer and labeled with anti-biotin MicroBeads (20 µl/107 CD45+/+ and CD45-/- lymphocytes, 4 µl/107 CD45+/+ thymocytes, 6 µl/107 CD45-/- thymocytes, 6 µl/107 CD45+/+ and CD45-/- BM) before passing through MS, LS columns or the AutoMACS (Miltenyi Biotec, Auburn, CA) according to the company’s protocols for enriching T lymphocytes, DN thymocytes or LinBM cells. CD19 was used as the B cell marker because the conventional B cell marker, B220, is an isoform of CD45 and is not expressed in CD45-/- mice. Typical yields for DN CD45+/+ and CD45-/- thymocytes were 3.1 and 3.8% of total thymocytes, whereas the yields for CD45+/+ and CD45-/- Lin- BM cells were 16 and 24% of total BM cells respectively.  40  For activation of cells for spreading assays, DN thymocytes enriched from 2×108 total thymocytes were cultured at 2×106 cells/ml in stimulation medium (RPMI-1640 medium (Invitrogen), 10% v/v heat-inactivated FCS, 0.055 mM β-mercaptoethanol, 10 mM HEPES, 2 mM L-glutamine, 2 mM sodium pyruvate, 50 U/ml of penicillin/streptomycin) containing 12.5 ng/ml PMA, 250 ng/ml ionomycin, and 20 U/ml recombinant mouse IL-2. On the 4th day, the medium was removed and replaced with fresh medium containing IL-2 only. Activated thymocytes were used for cell spreading assays on the 7th day. T cells enriched from 1.5 - 2×107 lymph node cells were activated with 2.5 ng/ml PMA and 500 ng/ml ionomycin for 48 hr in stimulation medium at 106 cells/ml, then used for spreading assays. CD45+ and CD45- BW5147 murine thymic lymphoma cells transfected with CD3ζ and δ chains for T cell receptor expression (258) were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Invitrogen), 10% heat-inactivated horse serum (Hyclone), 2 mM Lglutamine, and 2 mM sodium pyruvate along with 3 mM L-histidinol to maintain CD3 expression. Murine AKR.1G.1 T lymphoma cells (AKR) from ATCC transfected with various CD44 constructs in eukaryotic expression vectors were cultured in DMEM with 10% v/v heat-inactivated horse serum, 2 mM L-glutamine, and 2 mM sodium pyruvate with 1.5 mg/ml Geneticin 418 (50-75% active) to maintain CD44 expression. AKR cells expressing full-length CD44 (CD44wt) in pBCMGSneo were made by (A. Maiti). cDNA clones encoding CD44 with only two amino acids of the cytoplasmic domain (CD44∆Cyto) in pRc/RSV was from R. Hyman (259). OP9 cells transfected with the GFP-containing retroviral vector MigR1 or this vector containing cDNA for the Notch ligand Delta-like-1 (OP9-DL1) were obtained from J.C.  41  Zuniga-Pflucker (101). Cells were cultured in Minimal essential medium (MEM) alpha medium without ribonuclease and deoxyribonuclease (Invitrogen) containing 20% v/v heatinactivated FCS and 50 U/ml of penicillin/streptomycin. Cells were maintained at no more than 4.5×105 cells per 55 mm tissue culture dish. Cells were removed from the dish by rinsing once with PBS, then the cells were exposed to 0.25% trypsin/EDTA for 5 min at 37oC. Cells were then washed once with culture medium and re-plated at 1×105 cells per 55 mm tissue culture dish. Cells between passages 6 and 10 were used for co-culturing. CD45+ and CD45- Jurkat human T cells (clones E6.1 and J45.01 respectively) were cultured in RPMI-1640 with 10% v/v FCS. All cells were maintained at 37oC with 5% CO2.  2.2.2 Cloning of CD44 constructs and transfection A cDNA clone encoding the hematopoietic form of mouse CD44.1 (260) in pBluescript SK (Invitrogen) was used as the template for site directed mutagenesis. The CD44 Cysteine 297 to Alanine (C297A) mutation was made by Jennifer L. Cross using the forward oligonucleotide 5’ – CC CGC ACT GTG ACT CAT GGA TCC GAA TTA G - 3’ (UBC Nucleic Acid Protein Service Unit) and reverse oligonucleotide 5’ – GCC ACC GTT GAT CAC CAG CTT TTT CTT CTG CCC AGC CCT TCG TCG ACT ATT – 3’ (encoding the cysteine to alanine mutation). The polymerase chain reaction (PCR) product generated was then subcloned into a BamHI-BclI cut CD44.1 in pBluescript SK then into the eukaryotic expression vector pBCMGSneo (261) as a XhoI-NotI fragment. The CD44 mutant with membrane proximal 292NSRRRCGQKKKLV304 residues deleted (CD44∆13) was generated using the above forward oligonucleotide and reverse oligonucleotide 3’ – GCC ACC GTT GAT CAC CGC GAT GCA GAC GGC AAG – 5’.  42  The 181 base pair PCR product was subcloned into CD44.1 pBluescriptSK as a BamHI-BclI fragment before cloning into the eukaryotic expression vector pRc/RSV (Invitrogen) as a HindIII-XbaI fragment. All mutations were verified by sequencing. Eukaryotic expression vectors were electroporated into AKR1 cells as follows: 1×107 cells were washed twice with 20 ml of Ca2+/Mg2+ free PBS, resuspended in 850 µl of the same PBS containing 10 µg of DNA prepared using the QIAgen Endofree Plasmid Maxi Kit. Samples were placed on ice for at least 5 min before the cells were electroporated at 950 µF and 250 volts with the Bio-Rad Gene Pulser II. Cells were placed on ice for 5 min prior to culturing in 30 ml of culture medium without selection. After 24 hr, Geneticin 418 was added to the culture medium and cells were plated into flat-bottom 96-well tissue culture plates for identification of single clones. Two individual clones expressing the same construct were selected in each case. CD44 expressing clones were sometimes sorted for similar levels of CD44 expression in different cell lines.  2.2.3 Cell spreading assay and immunoprecipitation Anti-CD44 mAb (KM81, 500 µl at 40 µg/ml in PBS) was immobilized on 6-well plates (tissue culture-treated, Falcon) shaking at 4oC overnight. Wells were then blocked with 500 µl of 2% w/v BSA (Sigma-Aldrich) in PBS at 37oC for 2 hr, washed three times with 1 ml of PBS and equilibrated at 37oC for at least 10 min before cells were added. CD45BW5147 cells were washed once with pre-warmed binding medium (DMEM, 2 mM Lglutamine, 2 mM sodium pyruvate) and 5×106 cells/ml were pre-incubated in binding medium alone, plus dimethyl sulfoxide (DMSO, carrier), 1 or 5 µM Taxol, or 20 µM PP2 for 30 min at 37oC. Cell suspension (1 ml) was then transferred to wells immobilized with  43  KM81/BSA or BSA alone. Cells were incubated at 37oC with 5% CO2 for various periods. Cells were checked under a light microscope to ensure spreading was inhibited by Taxol or PP2 but not DMSO prior to lysing with 250 µl of ice-cold 5X lysis buffer (5% v/v Triton X100, 50 mM Tris-HCl pH 7.2, 700 mM KCl, 10 mM EDTA pH 8.0, 1 mM phenyl methyl sulfonyl fluoride (PMSF), 5 mg/ml leupeptin, 5 mg/ml pepstatin, 2.5 mM sodium orthovanadate, and 1 mM sodium molybdate). Cell lysates were kept on ice for 10 min, scraped and transferred to an eppendorf tube, then centrifuged at 16,100 × g at 4oC for 10 min to remove insoluble material. Protein G beads (10 µl, pre-incubated with 1 µg of antiPyk2 mAb for 1.5 hr rotating at 4oC, and then washed twice with 1X lysis buffer) were added to the lysate to immunoprecipitate Pyk2. Samples were rotated end-over-end at 4oC for 2 hr. Immunoprecipitates were then washed three times with 1 ml of ice-cold 1X lysis buffer. Samples were eluted from the beads with 25 µl of 3X reducing sample buffer (0.375M TrisHCl pH 6.8, 30% v/v glycerol, 6% w/v sodium dodecyl sulfate (SDS), 15% v/v βmercaptoethanol, 0.03% w/v bromophenol blue), heated at 95oC for 5 min, resolved on a 7.5% SDS-polyacrylamide gel and transferred to a polyvinylidene difluoride (PVDF) membrane (Immobilon P, Millipore). Immunoprecipitation controls included cell lysate only (1.2×105 cell equivalent), cell lysate with Protein G beads only, and Ab/Protein G beads with no cell lysate. For immunoprecipitation of CD44, 1×107 AKR T cells were spun down and lysed with 530 µl of Brij lysis buffer (1% v/v Brij-58, 10 mM Tris HCl pH 7.2, 140 mM KCl, 0.2 mM PMSF, 1 µg/ml aprotinin, 1 µg/ml leupeptin, and 1 µg/ml pepstatin) and incubated at 4oC for 10 min. Cell lysates were centrifuged at 16,100 × g for 10 min at 4oC to remove insoluble material. Thirty microliters of cleared lysate was saved as the lysate control. The  44  remaining lysate was added to 25 µl of pre-washed Sepharose or IM7-conjugated CNBr beads and rotated at 4oC for 2 hr. Immunoprecipitates were then washed three times with 1 ml of ice-cold lysis buffer. Samples were eluted from the beads with 25 µl of 3X nonreducing sample buffer (0.375M Tris-HCl pH 6.8, 30% v/v glycerol, 6% w/v SDS, 0.03% w/v bromophenol blue), heated at 95oC for 5 min, resolved on a 7.5% SDS-polyacrylamide gel and transferred to a PVDF membrane. In experiments examining zinc-mediated CD44:Lck associations, cells were resuspended in 1 ml of DMEM/10% horse serum containing 5 mM of 1,7- or 1,10phenanthroline or the equivalent amount of ethanol (carrier) and pre-incubated at 37oC for 20 min before lysis. Lysis buffer used at subsequent steps contained equivalent amounts of phenanthroline or ethanol and were at room temperature (RT) to prevent precipitation of phenanthroline.  2.2.4 Western blotting The air or methanol dried PVDF membrane was incubated with 1/5000 R54-3B antisera, 1/200 anti-Fyn, 1/200 anti-Pyk2, 1/1000 J1WBB, or 1/10 KM201 tissue culture supernatant in 5% w/v skim milk in TBST (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.1% v/v Tween 20) or 1/5000 4G10 in 0.5% w/v BSA/TBST shaking for 1 hr. The membrane was rinsed three times with TBST and incubated for 1 hr with HRP-conjugated goat-antimouse IgG, goat-anti-rat IgG, goat-anti-rabbit IgG or Protein A in 5% w/v skim milk or 0.5% w/v BSA in TBST buffer. The membrane was washed several times with TBST for 30 min before visualizing the bands with ECL or ECL plus (Amersham Pharmacia Biotech). Blots were scanned and spot densitometry was carried out using AlphaImager software (Alpha  45  Innotech Corp.) or ImageJ (NIH Image). If reprobing of the same membrane was required, the membrane was stripped by incubating with stripping buffer (50 mM glycine, 150 mM NaCl, 0.1% v/v Nonidet P-40 pH 2.5) for 30 min at RT and washed three times with TBST for 30 min.  2.2.5 Flow cytometry BW5147, AKR or Jurkat cells (1×105 in round bottom 96-well plates) or 1×106 3×107 primary cells (in V-bottom 96-well plates or FACS tubes) were incubated with 50 – 300 µl of Ab diluted in FACS buffer on ice for 20 min. For staining of lymph nodes, spleen and bone marrow cells, cells were first blocked with 100 µl – 1 ml of 2.4G2 tissue culture supernatant (TCS) for 15 min before labeling with Ab. Cells were washed once with FACS buffer in between different Ab incubations. In some cases, cells were stained with 1 µg/ml 7-amino-actinomycin (7AAD) for 5 min before to exclude non-viable cells. Cells were then washed twice before being resuspended with FACS buffer or fixed with 1% paraformaldehyde (PFA) in PBS. Samples were analyzed on a FACScan (BD Biosciences, San Jose, CA) using CellQuest software (Becton Dickinson, Mississauga, ON) or a LSRII flow cytometer using FACSDiva software (BD Biosciences). Data analysis was done using FlowJo (TreeStar, Ashland, OR). For determining HA binding to CD44, AKR cells were double labeled with 100 µl of IM7 TCS, then 100 µl of PE-conjugated goat-anti-rat Ab (1 in 100 dilution) and 10 µg/ml FL-HA. Labeling cells for both CD44 and HA allows the mean FL-HA binding to be expressed relative to CD44 cell surface expression.  46  Unlabeled and singly-labeled samples for each fluorochrome or dye were used for compensations settings. Negative controls were samples labeled with all Ab except the marker of interest. Titrations were performed to determine optimal concentrations of Ab to use. All labeling steps were carried out on ice, except for the CCR7-PE staining, which was performed at 37oC for 20 min.  2.2.6 Labeling for confocal microscopy and cell measurements Anti-CD44 mAb (KM81, 150 µl at 40 µg/ml in PBS), was immobilized on 8-chamber glass slides (Lab-Tek®, Naperville, IL) at 4oC overnight. Chambers were then blocked with 150 µl of 2% w/v BSA (Sigma-Aldrich) in PBS at 37oC for 2 hr, washed three times with 1 ml of PBS and equilibrated at 37oC for at least 10 min before incubation of cells. BW5147 cells (5×104) were washed twice and resuspended in 150 µl of spreading medium (DMEM with 0.1% v/v heat-inactivated FCS, 2 mM L-glutamine, 2 mM sodium pyruvate) at 37oC or first pretreated with 0.5 µM U73343, or 0.5 µM U73122 for 30 min at 37oC prior to being added to the slide (in the presence of the inhibitor) for various times (5, 30, or 120 min). Cells were then fixed at RT by adding 50 µl of 16% PFA for 20 min, permeabilized with 150 µl of 0.1% Triton X-100 in PBS for 10 min, and then incubated with 1% w/v BSA/PBS for 30 min to prevent non-specific binding. Cells were incubated in 150 µl with a 1/100 dilution of primary Ab in 1% BSA/PBS for 1 hr at RT and/or with 10 U/ml of Alexa Fluor 488conjugated phalloidin in PBS at 4oC overnight. Cells were then washed three times, incubated with corresponding Alexa Fluor-conjugated secondary Ab for 1 hr at RT, and then washed three times and mounted onto the slide in 90% v/v glycerol/2.5% w/v 1,4diazabicyclo-(2,2,2)-octan (DABCO, Sigma-Aldrich) in PBS. Slides were protected from  47  light and stored at 4oC before data collection on confocal microscopes within 24 hr. Controls included labeling for the protein of interest individually and without the primary Ab to ensure no cross-reactivity occurred between the secondary Ab. For F-actin labeling of activated primary cells, cells were washed twice with RPMI1640 spreading medium. Activated thymocytes (1.5×105) or activated T cells (5×104) were resuspended in 150 µl of medium and equilibrated at 37oC before incubation on the prewarmed slide for 30 min as mentioned above. F-actin labeling was done by incubation with 100 µl of 10 U/ml of Alexa Fluor 488-conjugated phalloidin for 1.5 hr at RT. Cell length for activated primary thymocytes and T cells was measured from confocal images of F-actinlabeled cells imported into ImageJ. Polarized cells were identified as cells showing asymmetrical, elongated phenotype and were distinguished from non-polarized cells, which were predominantly round.  2.2.7 Image collection and processing Confocal images of labeled BW5147 cells were captured with a Bio-Rad Radiance 2000 Plus confocal unit coupled to a Zeiss Axiovert microscope with a 60x oil immersion objective. Images were collected using a Kalman collection filter (2x) with a step size of 0.3 µm. Image size was 512×512 pixels covering 162×162 µm. Confocal images of labeled primary cells or BW5147 cells were captured using an Olympus FluoView FV1000 microscope. Images were collected with a 60x or 100x oil immersion objective covering 212×212 or 127×127 µm, respectively, with a step size of 0.19 µm. For double labeling experiments, images were collected with the same settings in a sequential manner. For any given experiment, the same laser power and gain controls settings were utilized to ensure  48  consistent signal intensities. Cells from random fields were imaged and analyzed from at least three independent experiments. Images were first processed using ImageJ. One slice or a stack of five slices close to the interface between the cells and the slide was first selected. The “Image/Adjust/Brightness&Contrast” command was sometimes utilized to adjust image contrast and all adjustments were applied to the whole image. Re-coloring and merging of images for co-labeling experiments was done using the “Image/Color/RGB merge” command. The representative image was then opened in Adobe Photoshop or Deneba Canvas (ACD Systems) and saved with the resolution of 600 pixels per inch. Light images of cells were taken with a Nikon coolpix 950 camera mounted on a Nikon inverted microscope (Eclipse TS100) with a 40x objective after confocal microscopy. Measurement of the length was calibrated with a microscope stage ruler. Images were opened in Canvas and saved with the resolution of 600 pixels per inch.  2.2.8 In vivo proliferation assay BrdU (1 mg in 100 µl of PBS) was administered to mice each day for two days by intraperitoneal injection and BrdU (1 mg/ml) was also included in their drinking water. After 48 hr, mice were sacrificed and their thymi were harvested. CD4 and CD8 DN enriched from one thymus or Lin- cells enriched from BM of one tibia and one femur were stained with surface markers (as mentioned above) before being fixed and permeabilized with 1% v/v PFA/0.1% v/v Tween-20/PBS for 1 hr at RT. Cells were then washed twice with FACS buffer before being incubated at 37oC for 30 min with 2 µl of 50 Kunitz/ml DNase (Roche, Laval, QC) in 4.2 mM MgCl2, 0.15 M NaCl, pH 5. Cells were then stained with 25 µl of  49  anti-BrdU-FITC or isotype-FITC control (BD Pharmingen) for 30 min, washed twice with FACS buffer before being analyzed by flow cytometry on a LSRII flow cytometer.  2.2.9 In vitro migration assay Migration assays were performed using 6.4 mm diameter Transwell filters with 5 µm pore size polycarbonate membranes (Costar Corning). A total of 3 – 8×108 thymocytes pooled from 3 or more mice were used to enrich for CD4 and CD8 DN thymocytes. 1×106 DN enriched thymocytes were added to the upper chamber of each well with 100 µl of chemotaxis medium (RPMI-1640 medium containing 0.1% w/v BSA). Chemotaxis medium (600 µl) containing 0 or 50 ng/ml recombinant mouse CXCL12 in was used in the lower chamber. Each condition was done in triplicate. Cells were allowed to migrate at 37oC for 3 hr to the lower chamber and then harvested for counting and analysis by flow cytometry. Thymocytes in the lower chamber were labeled with thymocyte lineage markers-FITC, CD4FITC, CD8-FITC, CD44-Alexa Fluor 647, CD25-PE, CD117-APC and 7AAD before being analyzed a LSRII flow cytometer. Percent migration for the various DN populations was calculated by dividing the number of cells migrated to the lower chamber by the number of cells added to the upper chamber.  2.2.10 Fluorescence activated cell sorting CD4 and CD8 DN-enriched thymocytes pooled from ≥3 mice (6 – 9 wks old) were labeled with thymocyte lineage markers-FITC, CD4- and CD8-PECy5, CD25-PE, CD44Alexa Fluor 647 and CD117-PECy7 or lineage depleted BM cells from pools of ≥5 mice were labeled with lineage markers-FITC, Sca-1-PE and CD117-APC before sorting on a  50  FACS Vantage or FACSAria (BD Biosciences) for DN1.0, DN1.5, CD117+ and CD117DN1.0 and DN1.5 populations from thymocytes; and LSK population from BM cells. Total thymocytes from one CD45+/+ mouse (~1.2×108) and one CD45-/- mouse (~0.8×108) yielded a minimum of 900 and 500 cells, respectively, of the rarest DN1 population. Total BM cells from one CD45+/+ mouse (4.4×107) and one CD45-/- mouse (3.4×107) respectively yielded approximately 1.7×104 purified BM LSK cells. Cell populations were shown to be ≥95% pure by flow cytometry.  2.2.11 Co-culture of progenitor and stromal cells Sorted cell subsets (1-5×103) were seeded into 12-well tissue culture plates containing a near-confluent monolayer of OP9 or OP9-DL1 stromal cells (101) in the presence or absence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. Cells were transferred to new wells containing a fresh monolayer of stromal cells on days 4, 8 and 12. Co-cultures were harvested by forceful pipetting at the indicated time points and passed through a 70 µm strainer to remove monolayers of stromal cells before staining with CD44-Alexa Fluor 647, CD25-PE, CD117PECy7, and 7AAD for flow cytometry. OP9 cells were excluded using a GFP negative gate.  2.2.12 Statistical analysis Unless indicated otherwise, results were expressed as the mean ± standard error of the mean (SEM) of at least three independent experiments and analyzed for statistical significance using an unpaired two-tailed Student t-test. p values ≤ 0.05 were considered statistically significant and are indicated as follows: *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001.  51  CHAPTER 3 REGULATION OF CD44-MEDIATED CELL SPREADING BY CD45  52  3.1 Introduction and rationale  CD44 is highly expressed on bone marrow hematopoietic progenitors, circulating progenitors, and early thymocytes. It has been suggested that CD44 may play a role in the homing of progenitors to the thymus during T cell development (127, 132, 192, 214). Inside the thymus, CD44 is expressed specifically on the DN1 to DN2 populations. However, the function of CD44 on these thymocytes and its involvement in thymocyte development is not clear. Integrins are another class of adhesion molecules involved in thymic homing (122). The interaction of integrins on DN2 and DN3 thymocytes with VCAM-1 expressed on thymic stromal cells is important for the migration of thymocytes across the cortex (215). This integrin-mediated migration is independent of the extracellular matrix. Interestingly, ex vivo early thymocytes expressing CD44 do not bind hyaluronan (127), suggesting that CD44 may be interacting with a stromal cell matrix in the thymus instead of an extracellular matrix, similar to integrins. Avigdor et al. demonstrated a role for CD44 in the CXCL12-dependent migration of human hematopoietic stem cells and anchorage within specific bone marrow niches (144). In the thymus, CXCL12 is responsible for attracting developing DN2 thymocytes to the cortex and the deletion of CXCR4 leads to a block in T cell development at the DN1 stage (133). This suggests that CD44 may be involved in the CXCL12-dependent intrathymic migration of DN2 cells across the cortex during early thymocyte development. Since a migratory cell must first adopt a spread phenotype and CD44-mediated signals can induce cell spreading, I  53  was interested in determining if CD44-mediated signaling leads to cell spreading in thymocytes. It was shown earlier that CD44 can be co-immunoprecipitated with Lck (197) and in vitro binding assays showed that there is a direct interaction between Lck and CD44 (D. Lefebvre, Ph.D. thesis, 2003, University of British Columbia). However, the exact molecular mechanism of the association between CD44 and Lck is still unclear. Therefore, the mechanism of CD44 interacting with Src family kinases (SFKs) was first examined here. Observations from our laboratory showed that BW5147 and AKR cells can be induced to spread upon incubation on immobilized CD44 mAb. CD44-mediated spreading depends on SFK activity and induces cytoskeletal changes (194, 195). The SFK Lck is a substrate of CD45 and SFK-dependent phosphorylation signals induced upon spreading are transient in CD45+ cells, compared to the sustained or enhanced signal in CD45- cells (200). In the presence of CD45, BW5147 cells show round spreading accompanied by the formation of F-actin rings, whereas absence of CD45 leads to SFK-mediated elongated cell spreading and the formation of fiber-like F-actin structures (195, 200). SFKs are also implicated in the activation of Pyk2 (proline-rich tyrosine kinase 2). Pyk2 activation requires two steps: autophosphorylation at Y402, which acts as a docking site for the recruitment of SFKs to further phosphorylate Y579, Y580, and Y881 for full activation of Pyk2 (262). As Pyk2 phosphorylation is enhanced in CD45- cells upon CD44mediated cell spreading (195), this suggest that during CD44-mediated spreading, CD45 plays a role in regulating F-actin formation and cell shape by influencing the outcome of the SFK-mediated signal. The exact mechanism of how CD45 exerts its negative regulatory role on CD44-mediated cell spreading remains to be explored.  54  Due to difficulties in isolating sufficient CD44-expressing DN1 and DN2 thymocytes from both wild type (CD45+/+) and CD45-deficient (CD45-/-) mice or primary peripheral T cells from the CD45-/- mice, I first chose to examine the mechanism of CD44-mediated signaling and cell spreading using two cell lines of thymic origin. Using BW5147 and AKR cells as model cell lines for thymocytes and T cells, the aim of study in this chapter was to determine how CD45 can affect CD44-mediated spreading leading to differential outcomes of F-actin organization and cell spreading, as well as further decipher the molecular mechanism of the CD44 interaction with SFKs.  3.2 Results  3.2.1 CD44 and Lck are recruited to microclusters upon CD44-mediated cell spreading Since CD44 can signal through Lck and that CD44-mediated spreading is dependent on SFK activity, I first set out to examine the spatial relationship of Lck with CD44 upon CD44-mediated spreading in BW5147 cells. Confocal microscopy revealed that CD44 formed microclusters after the cells were spread on immobilized CD44 mAb (KM81) for 30 min (Figure 3.1 right panel). Co-labeling of the cells for CD44 and Lck revealed that Lck was recruited to the CD44 microclusters upon spreading, suggesting an association between CD44 and Lck. This CD44:Lck co-localization occurred in both the CD45+ and CD45BW5147 cells, suggesting that the formation of CD44 microclusters and the presence of Lck in CD44 clusters were independent of CD45 expression. No microclusters were observed when the cells were incubated on immobilized BSA (Figure 3.1 left panel). In addition, goat-  55  Figure 3.1 Co-localization of CD44 and Lck to microclusters upon CD44-mediated spreading.1 Confocal microscopy of CD45+ and CD45- BW5147 cells upon 30 minutes of incubation on immobilized CD44 mAb (KM81) or BSA. Cells were fixed and permeabilized prior to labeling for CD44 with IM7-Alexa Fluor 647, and for Lck with R54-3B antisera and goat-anti-rabbit IgG Alexa Fluor 568. The images are a stack of five 0.19 µm slices close to the interface between the cells and immobilized mAb and are representative of over 100 cells observed over three independent experiments. The scale bar represents 10 µm. Copyright 2008. The American Association of Immunologists, Inc.  56  anti-rat IgG labeled the KM81-coated slide evenly (data not shown), suggesting that the CD44 clusters observed in spread cells were not a staining artifact.  3.2.2 The cytoplasmic domain of CD44 is required for the association of CD44 with Lck, but not Fyn To further examine the mechanism of association between CD44 and Lck, various CD44 constructs (Figure 3.2) were generated and expressed in the AKR cell line which does not express endogenous CD44. Since expression of CD44 lacking the cytoplasmic domain abolishes CD44-mediated cell spreading (N. Wong, Ph.D. thesis), the dependency of CD44:Lck association on the cytoplasmic domain of CD44 was first examined. CD44 was immunoprecipitated from AKR cells expressing full-length CD44 (CD44wt) or CD44 with the cytoplasmic domain deleted (CD44∆Cyto). In the absence of the cytoplasmic domain, CD44 was still able to associate with Lck and Fyn, suggesting that there is an indirect association between CD44 and the SFKs, possibly via lipid rafts (Figure 3.3). However, the deletion of the cytoplasmic domain of CD44 decreased the amount of Lck co-immunoprecipitated with CD44, suggesting that CD44 also associates with Lck via its cytoplasmic domain. Association of Fyn with CD44 was not affected by the deletion of the cytoplasmic domain of CD44. This suggests that CD44 interacts with Lck and Fyn in a different manner. The interaction of CD44 with Lck has been shown to occur in low density sucrose fractions (195). This fraction has been equated with the lipid rafts of the plasma membrane of cells, which are typically enriched in glycolipids, sphingomyelin, and cholesterol and are not well solubilized by detergents such as Brij (reviewed in 263, 264). The sucrose gradient  57  Figure 3.2 Amino acid sequence of the wild type mouse CD44 cytoplasmic tail and mutant CD44 constructs. The various CD44 constructs cloned into a eukaryotic expression vector were stably expressed in AKR cells (see Experimental procedures for details). The Putative ERM binding sites are outlined in the boxes. Dashes represent deleted residues. Point mutation is underlined.  58  Figure 3.3 CD44 association with Lck but not Fyn requires the cytoplasmic domain of CD44. Co-immunoprecipitation of SFKs with CD44. AKR cells transfected with full length CD44 (CD44wt) or CD44 with the cytoplasmic domain deleted (CD44∆Cyto) were lysed with 1% Brij-58. CD44 was immunoprecipitated and resolved by SDS-PAGE and analyzed by Western blotting. CD44 was detected with KM201 (top panel), Lck with R54-3B antisera (middle panel), and Fyn with FYN3 Ab (bottom panel). One representative of ≥3 independent experiments is shown.  59  fractions from cells expressing CD44wt and CD44∆Cyto were analyzed to ensure that CD44 was present in low density sucrose fractions (N. Maeshima, unpublished results). The reduced interaction of Lck with CD44∆Cyto was not due to the absence of CD44 from this membrane fraction.  3.2.3 Zinc-dependent association of CD44 with Lck requires the membrane proximal cytoplasmic domain of CD44 The co-stimulatory molecule CD4 has been shown to associate with Lck via the chelation of a zinc ion, with contribution from two cysteines from CD4 and two cysteines from Lck (265, 266). This association can be disrupted by addition of the zinc chelator, 1,10 phenanthroline (267, 268). To determine if CD44 also associates with Lck via zinc, AKR cells were pre-treated with the membrane permeable zinc chelator 1,10-phenanthroline, its inactive analog 1,7-phenanthroline, or ethanol (carrier) prior to CD44 immunoprecipitation. Figure 3.4 (left panel) shows that 5 mM of 1,10-phenanthroline significantly reduced Lck binding to CD44wt down to ~50%. The non-chelating 1,7-phenanthroline did not affect Lck:CD44 binding. This suggests that CD44 partially associates with Lck in a zincdependent manner. CD44∆Cyto co-precipitated much less Lck and the residual binding was not affected by zinc chelation (Figure 3.4 middle panel). The amount of zinc-independent binding of Lck to full-length CD44 was similar to that of CD44∆Cyto, indicative of a zincdependent association of Lck requiring the cytoplasmic domain of CD44. In support of this, in vitro binding studies with recombinant proteins of Lck N32 and CD44 cytoplasmic domains demonstrated a direct CD44:Lck interaction that is enhanced in the presence of zinc (D. Lefebvre, Ph.D. thesis).  60  Figure 3.4 Effect of the zinc chelator 1,10-phenanthroline on the association of Lck and Fyn with CD44.2 AKR cells transfected with various CD44 constructs were pre-treated with media alone, media containing ethanol (-), 5 mM 1,7-phenanthroline (1,7) or 5 mM 1,10phenanthroline (1,10) for 20 minutes at 37oC. Cells were then lysed with 1% Brij-58 and CD44 was immunoprecipitated. (A) Western blot analysis of a CD44 immunoprecipitation blotted for Lck with R54-3B antisera (top panel) and CD44 with KM201 (bottom panel). (B) Graphical representation of the average ± standard error of the mean (SEM) amount of Lck per unit CD44 that co-precipitated with CD44 determined by spot densitometry. Data is an average of at least three independent experiments. The amount of Lck per unit CD44wt in media alone was normalized to 100% in each experiment. *, p ≤ 0.05; **, p ≤ 0.01  61  3.2.4 Zinc-dependent association of CD44 with Lck does not involve key cysteine residues Both CD4 and CD8 have two cysteines in close proximity (CxCP) in the membrane proximal region that are involved in chelating zinc with Lck (267, 268). While the membrane proximal region of CD44 has an arginine-rich sequence in common with CD4 and CD8, it does not possess a CxCP motif. CD44 has one cysteine residue located in the transmembrane region (C288) and one in the membrane proximal cytoplasmic domain (C297). Mutation of either one of these cysteine residues to an alanine (C288A or C297A) did not affect the ability of CD44 to co-precipitate Lck (Figure 3.5A). In addition, the C297A mutation did not have an effect on the zinc-dependent binding of CD44 with Lck, seen when 1,10-phenanthroline was added (Figure 3.5B). Together, these data suggest that the zinc-dependent interaction between CD44 and Lck is not mediated by these two individual cysteines in CD44. The most well known protein:protein interaction with a zinc molecule are the zincfinger proteins which chelate zinc in a tetrahedral manner through cysteine or histidine residues (reviewed in 269). The cytoplasmic domain of CD44 does contain a conserved histidine residue (H332). Double mutation of C297 and H332 to alanines (C297A/H332A) did not affect the zinc-dependent or the zinc-independent binding of CD44 to Lck (Figure 3.5C). This suggests that the zinc-dependent association of CD44 with Lck is also not dependent on the histidine residue in the cytoplasmic domain CD44.  62  Figure 3.5 CD44 interaction with Lck is independent of key cysteine residues on CD44. (A) AKR cells transfected with wild type CD44 (CD44wt), CD44 with cysteine 288 or 297 mutated to an alanine (C288A and C297A, respectively) were lysed with 1% Brij-58 and CD44 was immunoprecipitated. CD44 was detected by Western blotting with J1WBB and Lck with R54-3B antisera.2,3 One representative of seven independent experiments is shown. (B and C) Cells were pre-treated with media containing ethanol (-), 5 mM 1,7-phenanthroline (1,7) or 5 mM 1,10-phenanthroline (1,10) for 20 minutes at 37oC before CD44 was immunoprecipitated and analyzed as in (A). One representative of four independent experiments is shown.  63  To determine if the membrane proximal region of CD44 is at all responsible for the zinc-dependent association of CD44 with Lck, the 13 membrane proximal residues (292 – 304) were deleted from the cytoplasmic domain of CD44 (CD44∆13). The amount of Lck co-precipitated with CD44∆13 was not significantly affected by the addition of 1,10phenanthroline (Figure 3.4 right panel). This is supported by in vitro binding assays indicating that the zinc-dependent interaction is abolished between the Lck N32 domains and CD44∆13 cytoplasmic domain (D. Lefebvre, Ph.D. thesis). This suggests that the zincdependent association of Lck with CD44 lies within residues 292 – 304 of CD44.  3.2.5 The cytoplasmic domain of CD44 is important for hyaluronan binding Interestingly, the 13 membrane proximal residues of CD44 that were deleted in the CD44∆13 mutant encompass an ERM binding site (outlined in Figure 3.2). ERM proteins have been implicated in linking the cytoplasmic domain of CD44 to the actin cytoskeleton, which is important for the binding of CD44 to HA (reviewed in 270). Phosphorylation of ezrin Y415 by SFKs is involved in cell spreading (212). This suggests that a SFK may be phosphorylating the CD44-associated ERM proteins, leading to changes in the cytoskeleton that are involved in the binding of CD44 to HA. To determine if the membrane proximal region of CD44, which is involved in the zinc-dependent association of CD44 with Lck, is affecting HA binding, cells were double labeled with CD44 and HA conjugated to fluorescein (FL-HA). Figure 3.6A shows the FLHA binding and CD44 expression levels of AKR cells transfected with CD44wt, CD44∆Cyto or CD44∆13. Since HA binding requires a threshold level of CD44 expression and that increased expression of CD44 increased HA binding (Figure 3.6B and 259), it was important  64  Figure 3.6 Membrane proximal region of the CD44 cytoplasmic domain is required for optimal binding to hyaluronan.4 Flow cytometric analysis of CD44 expression and FL-HA binding of AKR cells expressing various CD44 constructs (CD44wt, CD44∆13, CD44∆Cyto) doubly labeled with CD44 and FL-HA. Negative controls were cells labeled with secondary Ab only. One representative experiment of three is shown. (A) Histogram plot of CD44 expression. (B) Plot of FL-HA binding relative to CD44 expression levels. (C) Graph of FLHA binding to equivalent amounts of CD44. CD44wt was normalized to 100% in each experiment. Data is an average ± SEM of three independent experiments. *, p ≤ 0.05  65  to compare HA binding in cells expressing similar, intermediate levels of CD44. Therefore, the mean FL-HA was expressed as a function of CD44 expression, then an intermediate level of expression of CD44 was selected and the mean FL-HA binding was compared between different transfected cells. Figure 3.6C shows that for cells expressing equivalent levels of CD44, there was reduced FL-HA binding in cells expressing CD44∆Cyto and CD44∆13 compared to CD44wt. This suggests that the membrane proximal region of CD44 is important in CD44:HA binding.  3.2.6 CD44-induced Pyk2 phosphorylation is dependent on Src family kinase activity but not microtubule rearrangement Similar to Lck, Pyk2 has been shown to co-localize with CD44 in microclusters upon spreading on immobilized CD44 mAb (N. Wong, Ph.D. thesis). Although Pyk2 phosphorylation is only detected in CD45- but not CD45+ cells in microclusters by confocal microscopy (N. Wong, Ph.D. thesis), Western blotting shows that Pyk2 is phosphorylated in CD45+ cells and this phosphorylation is enhanced in CD45- cells (195). As one of the main functions of CD45 is to regulate Lck in T cells and that Lck is also found in CD44 microclusters, it was of interest to determine if the enhanced Pyk2 phosphorylation observed in CD45- BW5147 cells upon CD44-mediated spreading was dependent on SFK activity. Pyk2 was immunoprecipitated from CD45- cells treated with 20 µM of the SFK inhibitor, PP2, prior to spreading on immobilized CD44 mAb. Tyrosine phosphorylation of Pyk2 induced upon spreading for 30 min was abolished when PP2 was added (Figure 3.7). This indicates that the phosphorylation and thus activation of Pyk2 was dependent on SFK activity.  66  Figure 3.7 Pyk2 phosphorylation upon CD44-mediated spreading is dependent on Src family kinase activity. CD45- BW5147 cells were pre-treated with media alone (-), media containing DMSO (carrier), or 20 µM of PP2 (Src family kinase inhibitor) for 20 min prior to incubation on immobilized CD44 mAb (KM81) for 30 min at 37oC. Cells were then lysed with 1% Triton X-100, Pyk2 immunoprecipitated and blotted for phosphotyrosine (top panel) and Pyk2 (bottom panel).  67  Using Taxol, which inhibits microtubule depolymerization, it has been shown that elongated CD44-mediated spreading in CD45- cells was dependent on microtubule rearrangement (N. Wong, Ph.D. thesis). Since Pyk2 can associate with the microtubule cytoskeleton (271), it was of interest to determine if microtubule rearrangement had a causative role in CD44-induced Pyk2 phosphorylation leading to CD44-mediated cell spreading. Pyk2 was immunoprecipitated from CD45- BW5147 cells which had been treated with 1 or 5 µM of Taxol and induced to spread on immobilized CD44 mAb. CD44-induced tyrosine phosphorylation of Pyk2 was not affected by the addition of Taxol (Figure 3.8). This indicates that microtubule reorganization is either downstream of, or independent of Pyk2 activation.  3.2.7 CD45 is recruited to microclusters and ring structures upon CD44-mediated spreading As Pyk2, Lck, and CD44 co-localized in microclusters upon spreading independently of CD45 (N. Wong, Ph.D. thesis and Figure 3.1), this suggests that CD45 does not affect CD44-mediated signaling by affecting the recruitment of signaling molecules to CD44 microclusters. Localization of CD45 has been suggested as a mechanism to regulate CD45 phosphatase activity (reviewed in 227, 244). To investigate how CD45 negatively regulates CD44 and downstream signaling molecules upon spreading, the spatial organization of CD45 in cells was monitored upon incubation on immobilized CD44 mAb. Surprisingly, CD45 was also recruited to microclusters within 5 min and was still associated at around 30 min (Figure 3.9A). Double labeling confirmed that CD45 co-localized with Lck in microclusters (Figure 3.10). This co-localization of CD45 with Lck/CD44 microclusters was consistent with the reduced total tyrosine phosphorylation and the absence of Lck Y394  68  Figure 3.8 Pyk2 phosphorylation upon CD44-mediated spreading is not dependent on microtubule reorganization. CD45- BW5147 cells were pre-treated with media containing 1 or 5 µM of Taxol (microtubule depolymerization inhibitor) or DMSO (carrier) for 30 min prior to incubation on anti-CD44 mAb (KM81) for 30 min at 37oC. Cells were then lysed with 1% Triton X-100, Pyk2 immunoprecipitated and blotted for phosphotyrosine (top panel) and Pyk2 (bottom panel).  69  Figure 3.9 CD45 form distinct microclusters and ring structures upon CD44-mediated spreading. Confocal microscopy of BW5147 cells upon 5 and 30 minutes of incubation on immobilized CD44 mAb (KM81). Cells were fixed and permeabilized prior to labeling for CD45 with RO2.2 and goat-anti-rabbit IgG Alexa Fluor 568, then for F-actin with phalloidinAlexa Fluor 488. (A) CD45 staining showing microclusters and ring structures.1 (B) Colocalization of CD45 and F-actin in rings. The images are a 0.3 µm slice close to the interface between the cells and are representatives of over 100 cells observed over three independent experiments. The scale bar represents 10 µm. Copyright 2008. The American Association of Immunologists, Inc.  70  Figure 3.10 Lck co-localizes with CD45 in microclusters upon CD44-mediated spreading.1 Confocal microscopy of BW5147 cells upon 5 and 30 minutes of incubation on immobilized CD44 mAb (KM81). Cells were fixed and permeabilized prior to labeling for CD45 with I3/2-Alexa Fluor 488, and for Lck with R54-3B antisera and goat-anti-rabbit IgG Alexa Fluor 568. The images are a stack of five 0.17 µm slices close to the interface between the cells and immobilized mAb and are representative of over 100 cells observed over three independent experiments. The scale bar represents 10 µm. Copyright 2008. The American Association of Immunologists, Inc.  71  phosphorylation detected in CD44 microclusters upon spreading of CD45+ BW5147 cells compared to CD45- cells (200). This suggests that CD45 may be negatively regulating CD44-induced signaling events by recruitment to microclusters and inducing the dephosphorylation of Lck. In addition to localizing to CD44 microclusters, CD45 also formed a ring around the spread cell, similar to that previously observed for F-actin (200). Co-labeling confirmed that CD45 and F-actin co-localized in ring structures but not microclusters at both 5 and 30 min (Figure 3.9B).  3.2.8 Phospholipase C is required for CD44-induced F-actin fiber formation and elongated cell spreading in CD45- cells CD45+ BW5147 cells form F-actin rings and show round spreading. In contrast, CD45- cells form F-actin fibers and show elongated spreading upon incubation on immobilized CD44 mAb (195, 200). As CD44-mediated spreading leads to SFK-dependent activation of PLCγ1 in CD45- but not CD45+ cells (200), it was of interest to determine if PLC activity accounted for the difference in F-actin organization and cell spreading observed between CD45+ and CD45- BW5147 cells. Cells were treated with the PLC inhibitor, U73122, for 30 min before incubation on immobilized CD44 mAb, and stained for F-actin. Treatment of cells with 0.5 µM of U73122 but not the inactive analog, U73343, resulted in the formation of F-actin rings in the CD45- cells, similar to that observed in the CD45+ cells but in contrast to CD45- cells not treated with the PLC inhibitor (Figure 3.11). This indicates that PLCγ activation is important for polarized F-actin rearrangement and elongated cell spreading. The inhibitor did not affect F-actin ring formation in the CD45+ cells, although  72  Figure 3.11 Inhibition of PLC leads to F-actin ring formation. BW5147 cells were pretreated with 0.5 µM of the PLC inhibitor, U731222, or its inactive analog, U73343 for 30 min before incubation on immobilized CD44 mAb (KM81). Cells were then fixed and labeled with Alexa Fluor 488-conjugated phalloidin to stain for F-actin. The confocal images are 0.3 µm slices close to the interface between the cells and immobilized mAb and are representative of over 100 cells observed in three independent experiments. The scale bar represents 10 µm.  73  the F-actin rings were smaller in the presence of the PLC inhibitor. This suggests that CD45 may be inhibiting F-actin fiber formation and elongated spreading by inhibiting PLC activity.  3.2.9 CD45 affects the extent of cell spreading and cell polarization in primary cells To determine if the effects of CD45 on CD44-mediated cell spreading observed in the BW5147 cells was also observed in primary cells, the spreading between thymocytes and peripheral T cells isolated from CD45+/+ and CD45-/- mice were compared. As it has previously been demonstrated that CD44-mediated spreading only occurs with activated T cells (272), CD4 and CD8 double negative (DN) thymocytes and T cells were activated with PMA and ionomycin prior to spreading on the immobilized CD44 mAb. Analysis of DN populations ex vivo indicated that CD44 was expressed at significantly higher levels on CD45-/- DN1 cells compared to CD45+/+ cells (Figure 3.12). This was also true for the BM Lin-Sca-1+c-Kit+ (LSK) cells and peripheral CD3+ T cells, suggesting that CD45 influences the expression level of CD44. However, this difference in CD44 expression level between CD45+/+ and CD45-/- cells was lost upon activation of DN enriched thymocytes with PMA and ionomycin for 7 days. Although only the DN1 and DN2 populations of the DN thymocytes expressed CD44 ex vivo (Figure 3.12), the majority of activated DN thymocytes at day 7 were CD44+ and expression levels determined by flow cytometry were comparable between CD45+/+ and CD45-/- cells (Figure 3.13A). Activated DN thymocytes were incubated on immobilized CD44 mAb or BSA for 30 min. Both CD45+/+ and CD45-/- thymocytes spread on CD44 mAb, however, CD45-/- thymocytes spread more extensively than CD45+/+ thymocytes (Figure 3.13B). The CD45+/+ activated DN thymocytes spread round or round with a slight extension or polarization. Quantitation of the  74  Figure 3.12 Increased CD44 expression in CD45-/- primary cells. Flow cytometric analysis of CD44 expression (upper panels) and cells size (bottom panels) of ex vivo (A) bone marrow LSK cells, (B) DN thymocytes and (C) CD3+ splenic T cells. Data represents an average ± SEM of ≥9 mice over at least three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001  75  Figure 3.13 CD44-mediated cell spreading and F-actin rearrangement in activated thymocytes.1 (A) CD44 expression on activated CD45+/+ and CD45-/- DN-enriched thymocytes. Thymocytes pooled from 3 mice were labeled with IM7-Alexa Fluor 647 and analyzed by flow cytometry. Data is graphed as the average percentage of CD44-expressing cells and mean fluorescence intensity (MFI) ± SEM of six independent experiments. (B) Phase-contrast images and graph of the average cell length ± standard deviation of activated DN thymocytes immobilized BSA or CD44 mAb (KM81) for 30 min prior to fixing with 4% PFA. Data represents at least 85 cells over three independent experiments. (C) Graph of the average percentage of polarized cells ± SEM of four independent experiments. (D) F-actin staining of spreading DN thymocytes by confocal microscopy. Cells induced to spread were fixed and permeabilized prior to labeling with phalloidin-Alexa Fluor 488. The images are a stack of five 0.19 µm horizontal slices close to the interface between the cells and the immobilized mAb and are representative of four independent experiments. Scale bars represent 10 µm. *, p ≤ 0.05; *** p ≤ 0.001 Copyright 2008. The American Association of Immunologists, Inc.  76  cell length shows that CD45-/- thymocytes spread significantly longer (Figure 3.13B graph) and were more extensively polarized than CD45+/+ thymocytes (Figure 3.13C). No elongated spreading was observed with cells immobilized on BSA (Figure 3.13B). To examine the effects of CD45 on F-actin formation in the spread thymocytes, cells were labeled with Phalloidin-Alexa Fluor 488 (Figure 3.13D). In keeping with the round spreading, F-actin was primarily localized at the periphery of the cell in the majority of CD45+/+ activated DN thymocytes. This contrasts with the CD45-/- cells where the majority of cells had localized Factin accumulation at one or both ends of the cell. Similar to thymocytes, lymph node T cells enriched for by negative selection and activated with PMA and ionomycin for two days also spread on CD44 mAb but not BSA (Figure 3.14A). Activated CD45-/- T cells spread more extensively and were more polarized compared to CD45-/- T cells (Figure 3.14A graph and Figure 3.14B). All T cells expressed CD44 ex vivo and the levels were enhanced upon activation with PMA and ionomycin. Activated CD45+/+ and CD45-/- T cells expressed comparable levels of CD44 (Figure 3.14C). F-actin labeling in the activated T cells also revealed distinct differences between the CD45+/+ and CD45-/- T cells (Figure 3.14D). Similar to the CD45+ BW5147 cells and activated CD45+/+ DN thymocytes, CD45+/+ T cells showed the cortical staining of F-actin, forming a ring around the periphery of the cell. In contrast, F-actin was localized to specific areas of the CD45-/- cell and was often enriched in cell protrusions at one end of the cell. Small filamentous spikes of actin were also often observed protruding from one end of the cell. Together, these data establish a role for CD45 in regulating CD44-mediated cell spreading and F-actin rearrangement in activated primary CD44+ DN thymocytes and peripheral T cells.  77  Figure 3.14 CD44-mediated cell spreading and F-actin rearrangement in activated lymph node T cells.1 Activated CD45+/+ and CD45-/- T cells enriched from lymph nodes pooled from 3 mice were incubated on immobilized BSA or CD44 mAb (KM81) for 30 min prior to fixing with 4% PFA. (A) Phase-contrast images and graph of the average cell length ± standard deviation of at least 221 cells over two independent experiments. (B) Graph of the average percentage of polarized cells ± SEM of two independent experiments. (C) CD44 expression on activated T cells labeled with IM7-Alexa Fluor 647 and analyzed by flow cytometry. Data is graphed as the average MFI ± SEM of two independent experiments. (D) F-actin staining of spreading T cells by confocal microscopy. Cells induced to spread were fixed and permeabilized prior to labeling with phalloidin-Alexa Fluor 488. The images are a stack of five 0.19 µm horizontal slices close to the interface between the cells and the immobilized mAb. Scale bars represent 10 µm. **, p ≤ 0.01; *** p ≤ 0.001 Copyright 2008. The American Association of Immunologists, Inc.  78  3.3 Discussion  3.3.1 Data summary In this study, the mechanism of CD44 interacting with Src family kinases was investigated. CD44 was shown to associate with Lck via both a zinc-dependent and zincindependent mechanism. The zinc-dependent mechanism required the cytoplasmic domain of CD44. Unlike interaction of the CD4 and CD8 co-stimulatory molecules with Lck, the zinc-dependent association of CD44 with Lck did not require a specific cysteine residue within the 292 – 304 segment of CD44 that mediated zinc-dependent association of CD44 with Lck. These findings suggest that CD44 interacts with Lck via a novel mechanism. CD44 signaling had a different outcome on F-actin formation and cell spreading depending on the presence or absence of CD45 in activated primary thymocytes and T cells. This difference in F-actin organization and spreading was similar to that observed in CD45+ and CD45- BW5147 cells (195, 200), validating the work in T cell lines. Using BW5147 cells, the spatial and temporal localization of CD45 was monitored to determine the mechanism of how CD45 exerts its negative regulatory role on CD44-mediated spreading. CD45 was recruited to CD44/Lck microclusters and co-localized with F-actin in rings. Inhibition of PLC activity in CD45- cells reverted F-actin rearrangement and spreading similar to that observed in CD45+ cells. As well, inhibition of SFK abolished the enhanced Pyk2 phosphorylation observed in CD45- cells. A schematic model of CD44-mediated signaling leading to cell spreading in CD45+ and CD45- cells is summarized in Figure 3.15.  79  Figure 3.15 CD44-mediated signaling pathway leading to cell spreading.  80  3.3.2 CD44 and Src family kinase interaction Despite the lack of any intrinsic catalytic activity in CD44, clustering of CD44 is known to induce an intracellular signaling cascade (reviewed in 188). Here, it was shown that expression of CD44 lacking the cytoplasmic domain (CD44∆Cyto) in AKR cells abolished the zinc-dependent association with Lck. This coincided with the CD44 cytoplasmic domain, zinc, and SFKs being required for CD44-mediated cell spreading (N. Wong and D. Lefebvre, Ph.D. theses and 195), suggesting a requirement for zinc-mediated Lck interaction with CD44 during spreading. Further examination of the mechanism of the CD44:Lck interaction revealed both a zinc-dependent and zinc-independent component in cells. The zinc-dependent CD44:Lck association required the membrane proximal region. This is supported by results from in vitro binding assays where the N32 region of Lck was found to interact with the membrane proximal region of CD44 in a zinc-dependent manner and the kinase domain of Lck interacts with CD44 in a zinc-independent manner (D. Lefebvre, Ph.D. thesis). Unlike the conventional protein:protein interaction involving zinc (reviewed in 269), the zinc-dependent CD44:Lck association did not involve individual cysteine residues, C288A or C297A, of CD44. It is possible that either cysteine can substitute for one another if one of them was mutated to an alanine. Therefore a double cysteine mutant (C288A/C297A) should be generated to see if these cysteine residues were truly not mediating the zinc-dependent association of CD44 with Lck. It should be noted that there is another distal cysteine (C345) in the cytoplasmic domain of CD44. However, this cysteine is not conserved in human CD44, making its significance questionable.  81  Interestingly, when the two cysteines in the unique, amino-terminal region of Lck (C20 and C23) were mutated to serine residues, Lck was still capable of mediating a zincdependent interaction with the CD44 cytoplasmic domain in vitro (N. Maeshima, unpublished results). These two cysteine residues on Lck have been shown to mediate the interaction with CD4 and CD8 via a zinc molecule (265, 266). This shows that the mechanism of the zinc-dependent association of Lck with CD44 is different from that of the interaction with CD4 and CD8 in that it does not involve cysteine residues. The association of Fyn with CD44 did not require the cytoplasmic domain of CD44. As CD44 cross-linking causes the translocation of CD44 and SFKs to low-density sucrose fractions (194) where the CD44:SFK association exclusively occurs (195, 273), this CD44 cytoplasmic domain-independent association may be occurring through lipid rafts. Previously, in vitro binding assays showed no direct interaction or zinc-dependent binding between the cytoplasmic tail of CD44 and Fyn (D. Lefebvre, Ph.D. thesis). Together, these data suggest that Fyn association with CD44 is indirect and is likely through co-localization in lipid rafts. The significant decrease in the amount of Lck co-immunoprecipitated with CD44 without the cytoplasmic domain and the dependency on zinc for optimal CD44:Lck association suggested that Lck interaction with CD44 occurs via a different mechanism than that of Fyn and CD44. As deletion of the cytoplasmic domain of CD44 did not alter the distribution of CD44 in lipid raft fractions (N. Maeshima, unpublished data), the small amount of Lck co-immunoprecipitated with CD44∆Cyto may be due to the co-localization of Lck and CD44 in lipid rafts, as is thought to be the case for Fyn. A schematic model of CD44 association with Lck and Fyn is shown in Figure 3.16.  82  Figure 3.16 Schematic model of CD44 association with Lck and Fyn. The interaction of CD44 with Lck and Fyn is thought to occur in the lipid raft fraction of the cytoplasmic membrane. Dotted arrows represent lipid raft dependent associations, which may involve a second transmembrane protein. The red arrow represents the zinc-dependent association between the membrane proximal region (residues 292 – 304) of CD44 with the N32 domains of Lck. The solid arrow represents zinc-independent association between CD44 and Lck kinase domain.  83  Further testing is needed to determine if the zinc-independent and CD44 cytoplasmic domain-independent CD44:SFK association is mediated by lipid rafts or not. This would include determining if a non-relevant lipid raft protein, for example, cholesterol-binding protein prominin-1 (CD133) also co-immunoprecipitates with CD44∆Cyto. It is also possible that CD44 is associating with Lck via a third protein. However, this would involve the transmembrane domain of CD44, as the cytoplasmic domain of CD44 has been shown in vitro to directly associate with Lck (D. Lefebvre, Ph.D. thesis). It would require the transmembrane of CD44 interacting with another transmembrane protein, which then interacts with Lck. This transmembrane protein would most likely be associated with lipid rafts. A similar scenario may also occur with CD44:Fyn association.  3.3.3 CD44 and hyaluronan interaction Although a variety of ligands for CD44 have been identified including collagen, fibronectin, osteopontin, serglycin and hyaluronan (HA), most studies looking at downstream effects of CD44 engagement have been performed using immobilized CD44 antibody or immobilized HA, the principal ligand of CD44. It has been observed by Fanning et al that activated peripheral human T lymphocytes incubated on HA failed to show the firm adhesion or polarized morphology that are observed when the lymphocytes were incubated on CD44 antibody (199). Compared to the physiological situation, where HA is presented in a threedimensional extracellular matrix and HA-binding proteins that can enhance CD44:HA interactions are present (274), presentation of immobilized HA on a two-dimensional planar surface in vitro may not be sufficient to provide enough stimulation to reach the CD44 signaling threshold leading to cell spreading. This is further supported by the observation  84  that activated peripheral human T lymphocytes display an elongated cell spreading morphology on immobilized HA only when cytokines or chemokines were added (275), suggesting that these cytokines and chemokines may help enhance signals generated upon CD44 engagement with HA. For work done in this thesis, immobilized CD44 mAb was used to mimic CD44 engagement. As previously suggested by N. Wong (Ph.D. thesis), the high affinity interaction between CD44 and the antibody can provide enough stimulation to reach a signaling threshold without the need for additional stimulation in vitro. Here, it was shown that the cytoplasmic domain was required for the binding of CD44 to HA. The CD44 cytoplasmic domain consists of 72 amino acids, for which 70 residues were deleted in the CD44 cytoplasmic deletion mutant used here. It has been shown that deletion of the N-terminal 57 residues of the cytoplasmic domain of CD44 abolished CD44:HA binding in Jurkat T cells (203). ERM proteins can associate with the membrane proximal region of CD44. The significant decrease in HA binding in cells expressing CD44∆13 suggests that CD44:ERM interaction is important for HA binding. However, in vitro binding assays and coimmunoprecipitation experiments will be required to show that CD44∆13 does not bind to ERM. It cannot be ruled out that reduced binding of CD44∆13 to HA may also be caused by the loss of other proteins binding to the membrane proximal region of CD44. It has been shown that the clustering of CD44 can induce binding of CD44 to HA (201). In mutants with the cytoplasmic domain of CD44 deleted, the impairment in HA binding can be restored by pre-treating with anti-CD44 antibodies (201, 202), suggesting clustering is important for ligand binding by increasing the avidity. There is also evidence for the involvement of the actin cytoskeleton in binding of CD44 to HA (203). Since ERM  85  proteins link CD44 to the actin cytoskeleton, these membrane proximal 13 amino acid residues may be important in the actin-mediated clustering of CD44, which is needed to enhance binding to HA. Here, the membrane proximal region of CD44 was also found to be important for the zinc-dependent binding with Lck. Src can phosphorylate ezrin at Y145 which generates a positive feedback loop to enhance SFK activity and phosphorylation at Y145 is important for cell spreading (212). These data suggest that this positive feedback loop between ezrin and Lck may play a part in CD44-mediated cell spreading. It would be interesting to determine of CD44-mediated cell spreading is abolished or impaired in CD44∆13 cells.  3.3.4 CD45 regulation of the CD44 signaling complex upon spreading Previous studies showed that CD45 is a negative regulator of CD44-mediated signaling leading to cytoskeletal actin rearrangement and cell spreading that is dependent on SFK activity (195, 200). However, the mechanism of how CD45 exerts its negative role on CD44 and downstream signaling is not clear. Here, it was shown that immobilization of cells on CD44 mAb led to the co-clustering of CD44 and Lck independent of the presence or absence of CD45. Similarly, Pyk2 co-clustered with CD44 upon antibody-mediated spreading independently of CD45 (N. Wong, Ph.D. thesis). Since CD45 was also recruited to these CD44/Lck clusters, CD45 may be regulating CD44-initiated signaling by recruitment to the CD44 signalsomes. In the absence of CD45, there is a SFK-dependent induction of PLCγ phosphorylation (200). Here, it was found that enhanced Pyk2 phosphorylation in CD45- cells (195) was also dependent on SFK activity. Along with the absence of Lck Y394 phosphorylation and  86  reduced protein tyrosine phosphorylation observed in CD44 microclusters in CD45+ BW5147 cells compared to CD45- cells (200), the recruited CD45 may be exerting its negative regulatory role by dephosphorylating Lck within the CD44 microclusters, thereby decreasing signaling. This is consistent with the ability of CD45 to directly dephosphorylate Y394 of Lck in vitro (276) and with the hyperphosphorylation of Y394 in CD45- T cells (231). CD45 may be regulating CD44-mediated signaling thresholds in these signaling clusters via dephosphorylation of Y394 and inactivation of Lck. This would lead to a transient CD44-mediated phosphorylation signal, leading to F-actin ring formation and round spreading in the presence of CD45. And in the absence of CD45, CD44-mediated phosphorylation is sustained, leading to F-actin rearrangement along the sides of the elongated spread cell.  3.3.5 Translocation of CD45 and reorganization of the actin cytoskeleton upon CD44 spreading The shape of a given cell is largely dictated by the cytoskeleton, which is composed of actin filaments, microtubules and the intermediate filaments. The cytoskeleton also provides a scaffold for signaling molecules upon receptor engagement. CD45 has been shown to bind to the actin-binding proteins, spectrin and ankyrin (277, 278). Here, it was shown that CD45 co-localized with F-actin in ring structures upon CD44-mediated spreading. As noted earlier, CD44 can also interact with the cytoskeleton via ERM proteins. This suggested that the actin cytoskeleton may be involved in the translocation or recruitment of CD44 and CD45 to specific structures and therefore mediating or modulating signaling.  87  Depending on the presence or absence of CD45, CD44 signaling induced different phosphorylation intensities of signaling molecules leading to a different outcome of F-actin formation and cell spreading in BW5147 cells (200). Upon CD44-mediated spreading, Lck activation (as assessed by Y394 phosphorylation) is transient and no PLCγ phosphorylation is observed in CD45+ cells. In contrast, Lck activation is sustained and there is a transient SFK-dependent induction of PLCγ phosphorylation and PI3K activation in CD45- cells (200). Here, it was shown that PLC activity was required for F-actin fiber rearrangement and elongated cell spreading in CD45- cells. Inhibition of PLC activity led to F-actin ring formation and round spreading, similar to that observed with CD45+ cells. This suggests that CD45 may be negatively regulating PLCγ activation in CD45+ cells leading to F-actin ring formation. CD45 may be exerting its regulatory role by inactivating Lck, which is required for the subsequent activation of PLCγ (Figure 3.15).  3.3.6 CD44-mediated cell spreading of primary cells In this study, activated primary thymocytes and T cells also showed similar morphologies to that of BW5147 cells upon CD44 signaling. Signaling in the presence of CD45 resulted in peripheral F-actin ring formation and predominantly round spreading, whereas in the absence of CD45, CD44 ligation led to a directed or localized F-actin rearrangement and elongated, polarized cell spreading in both activated thymocytes and T cells. As with the BW5147 cells, CD44 clustering was also observed in spread CD45+/+ and CD45-/- thymocytes (data not shown), suggesting that the difference in CD44 signaling outcome in activated primary cells may be due to the transient versus sustained nature of CD44-induced signaling in the CD45+ and CD45- cells, respectively.  88  3.3.7 CD44 and cell migration In order for cells to migrate, cell elongation and polarization has to occur. It has been reported that activated human lymphocytes, which exhibit polarized cell spreading, do migrate on immobilized CD44 antibody (199). The requirement for PLC activity in CD44mediated BW5147 cell spreading shown here is similar to the observation that PLCγ1 is important for integrin-mediated cell spreading and elongation in endothelial cells, which is required for subsequent cell motility (279). Given that CD45 negatively regulated CD44mediated thymocyte and T cell polarized spreading, it is possible that CD45 may also modulate thymocyte and T cell migration. A certain amount of cell adhesion is needed for migration. However, this has to be finely regulated as either too little or too much adhesion can prevent migration. N. Maeshima and A. Suo (unpublished results) tested the migration of CD45+ and CD45BW5147 cells in response to the chemokine CXCL12 and found that the CD45- cells did not migrate as efficiently as the CD45+ cells. This shows that elongated spreading is not a good indicator of migration. In this case, the absence of CD45 may lead to increased cell adhesion, as is the case in a CD45- T cell line (242), thereby preventing migration. Bone marrow progenitors and early thymic populations express high levels of CD44. CD44 isoform expression on early human fetal thymocytes is developmentally regulated (280). CD44-deficient cells are less competitive in repopulating the thymus compared to wild type cells. These data suggest that CD44 has a role on these progenitor cells and early thymic populations. In this study, it was observed that ex vivo expression levels of CD44 were always higher in CD45-/- cells than CD45+/+ cells, suggesting that CD45 may also influence the expression of CD44. As CD44 play a role in the homing of thymic progenitors  89  to the thymus or intrathymic migration of developing thymocytes, in addition to CD45 negatively regulating CD44-mediated cell spreading and polarization, CD45 may also modulate thymocyte migration by down regulating the expression of the CD44 adhesion molecule. This increased CD44 expression may be a compensatory mechanism to increase adhesion if CD45 is positive regulator of CD44-mediated adhesion, similar to the role of CD45 in integrin-mediated adhesion in macrophages (241). The increased CD44 expression may also be due to increased phosphotyrosine signals caused by the absence of CD45. Either way, the negative regulatory effect of CD45 on CD44-mediated spreading of thymocytes would possibly affect early thymocyte development. The involvement of CD45 in early thymocyte development will be explored in detail in the next chapter.  90  CHAPTER 4 REGULATION OF EARLY THYMOCYTE DEVELOPMENT BY CD45  91  4.1 Introduction and rationale  In the previous chapter, CD45 was found to negatively regulate thymocyte spreading upon incubation on CD44 mAb. In addition, the absence of CD45 led to increased CD44 expression in bone marrow LSK cells and early thymocytes. Therefore, the absence of CD45 may lead to increased adhesion of hematopoietic progenitors and early thymocytes, affecting the migration of these cells. Since Lck is involved in CD44-mediated cell spreading, this suggests that CD45 may also alter signaling pathways that involve Lck and thereby affect thymocyte development. In this chapter, it was hypothesized that the absence of CD45 affects multiple processes leading to altered development of thymocytes. The role of CD45 in thymocyte development was investigated using wild type and CD45-deficient mice. The frequency of different thymic populations was first compared. In vivo proliferation, in vitro chemokinemediated migration and cytokine-mediated survival were also examined in early thymic populations. The progression of early thymic populations was then examined using the OP9DL1 in vitro co-culture system.  4.2 Results  4.2.1 Altered DN populations in the thymus of CD45-/- mice To evaluate the role of CD45 in early thymic development, the numbers and populations of thymocytes were compared between wild type C57Bl/6 and the exon 9 CD45-  92  deficient mice (233) backcrossed nine times onto the C57Bl/6 background (hereafter referred to as CD45+/+ and CD45-/- mice respectively). Flow cytometric analysis of total thymocytes was first performed to confirm that CD45-/- thymocytes do not express CD45 on the surface (Figure 4.1A). Similar to the original report (233), there was a significant two-fold decrease in the total number of thymocytes from CD45-/- mice compared to CD45+/+ mice (see Table 4.1). Likewise, CD45-/- mice showed a significant reduction in the percentage and number of single positive (SP) CD4 and CD8 thymocytes (Figure 4.1B and Table 4.1). There was no significant difference in the numbers of CD4-CD8- double negative (DN) cells in the CD45-/thymus compared to the CD45+/+ thymus. The DN population is known to be heterogeneous and contains the developing conventional αβ T cells, mature non-conventional T cells (γδ T and NKT), as well as non-T lineage cells. To gate out the mature non-conventional T and non-T lineage cells from the DN population, a cocktail of lineage markers (Gr-1, CD19, NK1.1, CD11b, and γδ TCR) was used. Although there was no statistical difference in the numbers of lineage negative (Lin-) DN thymocytes from CD45+/+ and CD45-/- mice, this equated to a 1.5-fold increase in the percentage of Lin- DN cells in the CD45-/- thymus, as it has almost half the number of total thymocytes compared to the CD45+/+ thymus. There was a significant increase in the percentage of DN3 CD45-/- thymocytes and a corresponding decrease in the percentage of DN4 CD45-/- thymocytes compared to their CD45+/+ counterparts within the Lin- DN population. This mirrored what was previously reported for the CD3- DN population (233) and was consistent with the hypothesis that CD45-/- thymocytes have a partial defect in preTCR signaling at the DN3 stage which is required for progression to the DN4 stage (237).  93  Figure 4.1 Flow cytometric analysis of CD45+/+ and CD45-/- thymocytes ex vivo. (A) CD45 and (B) CD4 and CD8 expression of total thymocytes. (C) CD44 versus CD25 profile of Lin- (Gr-1, CD11b, NK1.1, γδ TCR, CD19-negative) DN thymocytes with the four developmental stages of DN thymocytes (DN1-4) indicated.  94  Table 4.1 Numbers and percentage of thymic subsets from CD45+/+ and CD45-/- mice a  Cells/thymusa  Percent of thymus Population  CD45+/+  CD45-/-  CD45+/+ 1.5 ± 0.1 ×10  Total CD4 SP CD8 SP DP DN  8  6.2 ± 0.3  1.1 ± 0.3 ×10  7  2.0 ± 0.1  0.6 ± 0.1 ***  3.8 ± 1.0 ×10  6  88 ± 0.5  92 ± 0.5 ***  1.6 ± 0.3 ×10  8  2.3 ± 0.2  5.4 ± 0.4 ***  3.6 ± 0.6 ×10  6  CD45 c  Lin DN  +/+  88 ± 0.6  CD45  Mice  *** 5 ** 5 6.0 ± 1.1 ×10 **  7.5 ± 0.6 ×10  0.5 ± 0.1 ***  Percent of DN -  CD45-/7  b  5.3 ± 1.2 ×10 9.7 ± 1.7 ×10  7  5.9 ± 1.2 ×10  6  85 ± 0.6  CD45  +/+  7 7 7  4.6 ± 0.6 ×10  CD45-/-  6  3.1 ± 0.5 ×10  6  7  Lin DN1  1.9 ± 0.1  1.3 ± 0.2 *  8.4 ± 1.0 ×10  Lin- DN2  1.4 ± 0.1  1.1 ± 0.1 ***  6.2 ± 0.7 ×10  4  Lin DN3  45 ± 1.0  71 ± 0.9 ***  2.1 ± 0.3 ×10  6  2.2 ± 0.3 ×10  6  Lin- DN4  51 ± 0.9  26 ± 0.7 ***  2.3 ± 0.3 ×10  6  8.0 ± 1.5 ×10  5  -  7  Cells/thymus -/-  4  -  ≥75  4.0 ± 1.0 ×10  4  3.3 ± 0.5 ×10  4  ** **  7 7 7  ***  7  a  Data represents average ± SEM of multiple mice over at least three independent experiments. b *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001 c Lineage markers include Gr-1, CD11b, NK1.1, γδ TCR, and CD19  95  Independent analysis of the lineage positive cells using Gr-1, CD19, NK1.1, CD11b, γδ TCR, and CD3ε antibodies, showed a significant increase in the amount of NK cells and a significant decrease in NKT cells in both the DN and total CD45-/- thymic populations compared to the CD45+/+ thymus (Figure 4.2). This mirrored a previous study where an increase in NK cells and a decrease in NKT cells was observed in the exon 9 CD45-deficient spleen (281). On the other hand, there was no difference in the number of γδ T cells in the CD45-/- thymus compared to the CD45+/+ thymus (Figure 4.3), as was previously reported in the fetal thymus (282). However, there was an increase in the percentage of γδ T cells within the DN population. Using the downregulation of CD24 expression as an indicator of γδ T cell maturation (43), there was an increase in the percentage of immature, CD24+ γδ T cells in the CD45-/- thymus, suggesting a possible defect in the maturation of γδ T cells in the absence of CD45 (Figure 4.3). A similar increase in immature γδ T cells was also observed by others in the CD45-deficient fetal thymus (fetal day 16, 282). There was no detectable difference in the minute amounts of B cells and granulocytes/macrophages found in the CD45+/+ and CD45-/- thymi (data not shown). As some lineage positive cells also express high levels of CD44 and no CD25, similar to that of DN1 cells, removal of these non αβ T lineage cells allowed further examination of T cell precursors in the DN population in more detail. There was a significant decrease in the percentage of both DN1 and DN2 thymocytes from CD45-/- mice when Gr-1, CD19, NK1.1, CD11b, and γδ TCR expressing cells were excluded from the DN population. There was also a significant decrease in the number of DN1, DN2, and DN4 cells from the CD45-/- thymus compared to the CD45+/+ thymus, but an equivalent number of DN3 cells (see Table 4.1).  96  Figure 4.2 Lack of CD45 alters the generation of NK and NKT cells in the thymus. Flow cytometric analysis of NK, NKT and T cell populations ex vivo. (A) Graph of the average percentage ± SEM and (B) the average cell number ± SEM of NK, NKT, and T cell populations within the DN and total thymic populations. Data is an average of 8 individual mice over three independent experiments. *, p ≤ 0.05; ***, p ≤ 0.001  97  Figure 4.3 Lack of CD45 affects maturation of γδ T cells in the thymus. Flow cytometric analysis of γδ T cell populations ex vivo. (A) Graph of the average percentage ± SEM and (B) the average cell number ± SEM of total, immature CD24+, and mature CD24γδ T cell populations within the DN population. Data is an average of 6 individual mice over two independent experiments. **, p ≤ 0.01; ***, p ≤ 0.001  98  To further examine the early thymic progenitors found within the DN1 population, the CD117(c-Kit)+ DN1 subpopulation was analyzed (33). These cells, termed ETPs, are the canonical T cell progenitors and express negligible levels of CD127 (IL-7Rα chain). The remaining majority of DN1 cells are CD127+CD117- (Figure 4.4A). Comparison of the ETP and CD117- populations within the DN1 population between CD45+/+ and CD45-/- mice revealed that the percentage of ETPs was significantly reduced in the CD45-/- DN1 population (Figures 4.4A and 4.4B) from 12±1.2% to 7.1±0.9% (Figure 4.4C, left panel), representing an approximate 5-fold drop in the number of ETPs in the CD45-/- thymus (Figure 4.4C, right panel). Although the percentage of CD117- cells was slightly increased in the CD45-/- DN1 population, it was not significant. However, due to a 2-fold decrease in the total number of CD45-/- thymocytes, the number of CD117- DN1 cells in the CD45-/- thymus was significantly reduced compared to the CD45+/+ thymus (Figure 4.4D).  4.2.2 The absence of CD45 does not affect the amount of bone marrow or blood LSK cells Progenitors which have the potential to enter the thymus and develop into T cell progenitors are found within the Lin-Sca-1+CD117+ (LSK) population in the bone marrow (reviewed in 19). To investigate whether the reduced ETP numbers in the CD45-/- mice were due to reduced progenitors from the bone marrow and/or in the blood, the percentage and number of LSK cells were examined. There was no significant difference in the frequency and number per tibia of LSK cells in the bone marrow or the frequency of LSK cells in the blood between the CD45+/+ and CD45-/- mice (Figure 4.5). This suggests that the reduced ETPs in the CD45-/- mice are not due to reduced progenitors in the bone marrow or blood, and that the reduction is occurring upon entry or within the thymus.  99  Figure 4.4 Reduced ETPs in CD45-/- mice. Flow cytometric analysis of the DN1 population from CD45+/+ and CD45-/- thymi ex vivo. (A and B) CD127 and CD117 expression on DN1 cells. ETPs are outlined in the CD117+ gate. One representative of ≥7 individual mice over three independent experiments is shown. (C and D) Graph of the average percentage ± SEM (left panels) and the average cell number ± SEM (right panels) of CD117+ and CD117- DN1 populations. *, p ≤ 0.05; **, p ≤ 0.01  100  Figure 4.5 LSK population is not altered in CD45-/- mice. Flow cytometric analysis of the bone marrow (BM) and blood LSK populations ex vivo. (A) Sca-1 versus CD117 plot of Lin- BM cells. LSK cells are outlined in the Sca-1+CD117+ gate. One representative of 15 individual mice over five independent experiments is shown. (B) Graph of the average percentage and cell number ± SEM of BM LSK cells from one tibia. (C) Graph of the average percentage ± SEM of blood LSK cells of ≥6 individual mice over three independent experiments.  101  4.2.3 CD45 is a positive regulator of proliferation in vivo The decreased amount of ETPs observed in the CD45-/- mice could be due to decreased entry of progenitors into the thymus, decreased survival or decreased proliferation within the thymus. Since ETPs have a high proliferative capacity and can expand by approximately 1000-fold at the DN1 stage prior to progression to the DN2 stage (36), the proliferation of early thymic populations was first examined. Mice were administered with 5-bromo-2-deoxyuridine (BrdU) and 48 hours later, the percentage of BrdU-positive (BrdU+) cells in the Lin- DN populations was measured by flow cytometric analysis (Figure 4.6). Significantly reduced proliferation, as indicated by the decreased percentage of BrdU+ cells, was observed in the CD45-/- CD117- DN1, CD117+ DN1, and DN2 populations compared to the CD45+/+ populations (Figure 4.6B). This was consistent with the reduced numbers of CD117- DN1, CD117+ DN1, and DN2 cells in the CD45-/- thymus (Table 4.1). Both CD45+/+ and CD45-/- CD117+ DN1 cells proliferated more than their CD117- DN1 counterparts, consistent with CD117+ DN1 populations having a higher proliferative capacity. However, significantly reduced proliferation was also observed in the DN3 and DN4 thymic populations, as well as bone marrow LSK cells in the CD45-/- mice (Figure 4.6B). This suggests that CD45 has a positive regulatory effect on proliferation.  4.2.4 CD45 is required for optimal CXCL12 migration in the CD117- DN1 population Chemokine signaling is necessary for the development of early thymocytes, as evidenced in the CXCR4-knockout mice (133). Thymic-specific deletion of CXCR4 by CRE activation with the lck proximal promoter led to the accumulation of DN1 cells at the corticomedullary junction (CMJ) together with a developmental arrest, suggesting a close  102  Figure 4.6 Reduced proliferation of CD45-/- cells in vivo. Flow cytometric analysis of BrdU incorporation in thymocytes and BM LSK cells 48 hr post BrdU administration. (A) Histogram of anti-BrdU (open histogram) and isotype control (shaded histogram) staining of one representative of 5-8 individual mice over three independent experiments. (B) Graph of the average percentage ± SEM of BrdU+ cells. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001  103  correlation between localization and development (133). Since CD45 has been shown to positively regulate CXCL12 migration in Jurkat T cells (243), the response of Lin- DN thymocytes to CXCL12 was examined. Transwell migration was assessed using DNenriched thymocytes (Figure 4.7). Lineage positive cells were gated out during analysis. In the absence of the chemokine CXCL12, some migratory activity was observed in the CD117DN1 population, with a tendency of CD45-/- cells to show reduced migration. In the presence of CXCL12, migration of all the DN populations was significantly increased (p ≤ 0.024) in the CD45+/+ thymocytes. CD45-/- DN1, DN2, and DN4 populations showed less CXCL12induced migration compared to the CD45+/+ populations. This difference between CD45+/+ and CD45-/- thymocytes was most dramatically seen in the CD117- DN1 and, to a lesser extent, DN4 populations where they were statistically significant. Measurement of the CXCR4 expression levels by flow cytometric analysis (Figure 4.8) in the CD117- and CD117+ DN1 populations showed equivalent levels of expression in the CD45+/+ and CD45-/cells. This suggests that CXCL12-induced migration of DN thymocytes can still occur in the absence of CD45, but the migration is reduced compared to CD45+/+ DN thymocytes.  4.2.5 CD45 is required for CCR7 expression in the CD117- DN1 population CXCL12 is not the only chemokine involved in early thymic migration. Previous studies showed that CCL19 and CCL21 are important for the intrathymic migration of thymocytes (reviewed in 38, 129). Examination of the CCL19/21 receptor, CCR7, on the DN1 populations showed that CCR7 was expressed at low levels on CD117+ DN1 cells and expressed at high levels on a subset (22±2.6%) of CD117- DN1 cells. Strikingly, CCR7 expression was completely absent on CD45-/- DN1 cells (Figure 4.9A). CCR7 expression on  104  Figure 4.7 Reduced CXCL12-mediated migration in CD45-/- thymocytes. Migration of DN thymocytes pooled from ≥3 mice per experiment in response to CXCL12 for 3 hr in a Transwell system. Migrated cells were labeled with lineage markers, CD117, CD44, and CD25 Ab and analyzed by flow cytometry. Graph of the average percentage of migration ± SEM of the indicated Lin- DN populations in response to 0 or 50 ng/ml of CXCL12 is shown. Data an average of four independent experiments. *, p ≤ 0.05; **, p ≤ 0.01  105  Figure 4.8 CXCR4 expression is not affected in CD45-/- thymocytes. Flow cytometric analysis of CXCR4 expression of Lin- DN populations ex vivo. (A) Graph of the average MFI ± SEM of ≥6 individual mice over three independent experiments. *, p ≤ 0.05 (B) Histogram plots of CXCR4 expression on CD117+ and CD117- DN1 populations from CD45+/+ and CD45-/- thymi. Samples were enriched for DN thymocytes pooled from ≥3 mice prior to labeling for flow cytometry.  106  Figure 4.9 Lack of CCR7 expression on DN1 thymocytes from CD45-/- mice. Flow cytometric analysis of CCR7 expression on DN thymocytes ex vivo. Histogram plots of CCR7 expression on (A) CD117+ and CD117- DN1 populations and (B) CD4 SP cells from CD45+/+ and CD45-/- thymi. Samples were enriched for DN thymocytes pooled from 4 mice prior to labeling for flow cytometry.  107  the CD4 SP population was also analyzed to show that CD45-/- thymocytes can express CCR7 and the effect of CD45 on CCR7 expression was stage-specific (Figure 4.9B). This suggests that CD45 may positively affect the migration of the CD117- DN1 population to two different chemokines by two different mechanisms: one presumably by affecting the signaling pathway induced by CXCL12 and the other by regulating the expression of the chemokine receptor CCR7.  4.2.6 Identification of an intermediate stage between DN1 and DN2 Closer analysis of the DN1 compartment of the CD45-/- thymus revealed a distinct CD44+CD25lo population that was not apparent in the CD45+/+ thymus (Figure 4.10A). This population was also previously noted in the exon 9 CD45-deficient mice (283). The DN1 population in the CD45-/- mice was divided into two populations based on CD25 expression, which was defined here as DN1.0 (CD44+CD25-) and DN1.5 (CD44+CD25lo). Comparison with the CD45+/+ mice revealed that both the number and percentage of DN1.0 cells were significantly decreased in the CD45-/- DN1 population with a concomitant and significant increase in the percentage of DN1.5 cells, but not a significant increase in numbers (Figure 4.10B). This suggested that DN1.5 may be an intermediate between DN1.0 and DN2 and that the absence of CD45 not only reduced the number of progenitors in the DN1 population but favors their progression and accumulation at a CD44+CD25lo DN1.5 stage. To determine if DN1.5 was an intermediate between DN1.0 and DN2, equal numbers of sorted DN1.0 and DN1.5 cells from CD45+/+ and CD45-/- mice were plated on OP9 stromal cells expressing the Notch ligand Delta-like-1 (OP9-DL1) in the presence of IL-7 and Flt3-ligand (Flt3L), which has previously been shown to support thymocyte development  108  Figure 4.10 Identification of a novel intermediate between DN1 and DN2. (A) Flow cytometric analysis of CD44 and CD25 expression in Lin- DN thymocytes ex vivo. CD44+CD25- DN1.0 and CD44+CD25lo DN1.5 populations are outlined. One representative of 7 individual mice over three independent experiments is shown. (B) Graph of the average numbers and percentage ± SEM of DN1 population and the subpopulations, DN1.0 and DN1.5. *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001  109  in vitro (101). Figure 4.11 (left panel) shows that CD45+/+ and CD45-/- DN1.0 cells had the ability to make DN1.5 and DN2 cells, while DN1.5 cells made DN2-3, but not DN1.0 cells. This suggests that DN1.5 is an intermediate between DN1.0 and DN2. When sorted DN1.0 and DN1.5 thymocytes were plated on OP9 cells not expressing the Notch ligand in the presence of IL-7 and Flt3L (Figure 4.11 right panel), DN1.0 cells from both CD45-/- and CD45+/+ mice progressed to the DN1.5 stage, but neither DN1.0 nor DN1.5 populations progressed beyond the DN1.5 stage in the absence of DL1. This demonstrates that Notch signaling is required for the progression of DN1.5 cells to DN2, but not for the progression of DN1.0 cells to the DN1.5 stage. To determine the importance of IL-7 in the survival and progression of DN1.0 and DN1.5 populations, sorted DN1.0 and DN1.5 cells were cultured with OP9 or OP9-DL1 cells in the presence or absence of IL-7. IL-7 increased the survival of both DN1.0 and DN1.5 populations (Figure 4.12A) and IL-7 together with DL1 further enhanced cell survival or induced proliferation (Figure 4.12A) and promoted progression (Figure 4.12B). In the absence of a Notch signal, IL-7 is a survival factor that allowed the progression of DN1.0 to DN1.5. However, both IL-7 and Notch signaling are required for the progression beyond the DN1.5 stage. While both CD45+/+ and CD45-/- DN1.0 cells progressed to DN2 on OP9-DL1 at approximately the same rate in the presence of IL-7 and Flt3L, or IL-7 alone, the progression of CD45-/- DN1.5 cells to DN2 was significantly slower than the CD45+/+ DN1.5 cells (Figure 4.11 and 4.12B). A quantitative analysis of the percentage of DN1.0 and DN1.5 cells progressing to DN2 on OP9-DL1 in the presence of IL-7 and Flt3L is shown in Figure 4.13.  110  Figure 4.11 Progression of DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro. DN thymocytes pooled from ≥3 mice and labeled with lineage markers, CD44, and CD25 were sorted into DN1.0 (CD44+CD25-) and DN1.5 (CD44+CD25lo) populations. Sorted DN1.0 and DN1.5 thymocytes were co-cultured with OP9-DL1 (left panel) or OP9 (right panel) stromal cells in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. Cells were harvested at the indicated times and analyzed by flow cytometry. Plots from one representative experiment of three are presented. Axes are in log scale.  111  Figure 4.12 Requirement for IL-7 for survival of DN1.0 and DN1.5 thymocytes in vitro. Sorted DN1.0 and DN1.5 thymocytes were co-cultured on OP9 or OP9-DL1 cells in the presence and absence of 1 ng/ml IL-7 but not Flt3L. Cells were harvested at the indicated times and analyzed by flow cytometry. (A) Graph of the number of cells on Day 6 of culturing in vitro, normalized by the initial number of cells that was plated. Data is an average ± SEM of three independent experiments using thymocytes pooled from ≥3 mice per experiment. (B) Representative plots from one experiment. Axes are in log scale.  112  Figure 4.13 Progression of DN1.0 and DN1.5 on OP9 and OP9-DL1 cells in vitro. Percentage of cells that progressed to DN2 from DN1.0 (top panel) and DN1.5 cells (bottom panel) on OP9-DL1 in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L plotted over time. Data is an average ± SEM of three independent experiments using thymocytes pooled from ≥3 mice per experiment. **, p ≤ 0.01  113  4.2.7 The absence of CD45 alters the distribution of both CD117+ and CD117- cells within the DN1 populations To further determine why the DN1.5 population from CD45-/- mice exhibited a lag phase in the progression to or proliferation at the DN2-3 stage, the distribution of ETP within the DN1.0 and DN1.5 populations was examined. These CD117+ DN1 cells are known to progress from DN1 through DN4. The number of CD45-/- CD117+ cells was decreased to roughly the same extent (about 4-fold) in both the DN1.0 and DN1.5 populations compared to their CD45+/+ counterparts (Figure 4.14 left panel). However, the percentage of CD117+ cells in the DN1.0 population was significantly increased by approximately 2-fold, whereas the percentage of CD117+ cells in the DN1.5 population was significantly decreased by approximately 7-fold in the CD45-/- thymus compared to the CD45+/+ thymus (Figure 4.14A right panel). The percentage changes were largely attributed to a decrease in CD117- cells in the DN1.0 population and an increase in the DN1.5 population in the CD45-/- thymus, which represent the majority of cells in the DN1 population. CD45-/- mice had approximately 9fold fewer cells in the DN1.0 population of the CD45+/+ mice and approximately 3-fold as many cells in the DN1.5 population (Figure 4.14B left panel). This was reflected as a slight decrease in the percentage of CD117- cells in the DN1.0 population and an increase in the percentage of CD117- cells in the DN1.5 population of the CD45-/- mice (Figure 4.14B right panel). Since the distribution of CD117+ and CD117- cells within the DN1.0 and DN1.5 populations was different between CD45+/+ and CD45-/- cells, it was estimated that there was approximately 3 times more ETPs in the CD45-/- DN1.0 population, and 7 times fewer ETPs  114  Figure 4.14 Altered CD117+ and CD117- DN1 populations in CD45-/- mice. Flow cytometric analysis of DN1.0 and DN1.5 populations from CD45-/- and CD45-/- thymi ex vivo. (A) Graph of the number and percentage of CD117+ cells within DN1.0 and DN1.5 populations. (B) Graph of the number and percentage of CD117- cells within DN1.0 and DN1.5 populations. Data is an average ± SEM of 7 individual mice over three independent experiments. *, p ≤ 0.05; **, p ≤ 0.01; and ***, p ≤ 0.001  115  in the CD45-/- DN1.5 population when equal numbers of DN1.0 and DN1.5 cells from CD45+/+ and CD45-/- mice were plated on OP9 and OP9-DL1 cells. This reduction in ETP in the CD45-/- DN1.5 population may relate to the longer lag time observed for this population before it progressed to DN2. To determine if this was the case, equal numbers of CD117+ DN1.0 and DN1.5 cells were plated on OP9-DL1. Interestingly, all populations rapidly progressed and by Day 6, the majority of the cells were at the DN2-3 stage and the cells had proliferated substantially (Figure 4.15 left panel). No obvious difference in the rate of progression was observed between the CD45+/+ and CD45-/- thymocytes (data not shown). Similar to findings with total DN1.0 and DN1.5 populations, the progression of CD117+ DN1.5 cells to DN2, but not DN1.0 cells to DN1.5, was dependent on Notch signaling, as cells did not progress past the DN1.5 stage when the cells were co-cultured on OP9 cells (Figure 4.15 right panel). This indicated that CD45 was required to generate optimal numbers of ETPs, but was not essential for their progression to the DN2-3 stage. Once the cells reached the DN2 stage, the cells underwent rapid expansion, more so than was observed with the total DN1.0 and DN1.5 populations (Figure 4.11). Since proliferation and survival of cells at the DN2-3 stage is dependent on IL-7 (284, 285), there may be a competition for IL-7 with the CD117- DN1 thymocytes, which are IL-7Rα(CD127)+ and forms a majority of the total DN1 population. This may lead to the slower progression and proliferation of total DN1.0 and DN1.5 thymocytes compared to when only CD117+ DN1.0 and DN1.5 cells were plated on OP9-DL1 cells. Since CD117 expression is maintained on ETP as they progress from DN1 to DN2 (186), the percentage of CD117+ cells distributed between the DN1.0, DN1.5 and DN2 populations (i.e., between different CD25 expressing CD44+ DN populations) were assessed  116  Figure 4.15 Progression of CD117+ DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro. DN thymocytes pooled from ≥3 mice and labeled with lineage markers, CD44, CD25 and CD117 were sorted into CD117+ DN1.0 (CD44+CD25-CD117+) and CD117+ DN1.5 (CD44+CD25loCD117+) populations. Sorted populations were co-cultured on OP9-DL1 (left panel) or OP9 (right panel) cells in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. Cells were harvested on Day 6 for flow cytometric analysis. Plots from one representative experiment of three are presented. Axes are in log scale.  117  ex vivo. The distribution of CD117+ cells between the three populations was similar for both CD45+/+ and CD45-/- thymocytes (Figure 4.16). This supports the in vitro data that there is no block or delay in the progression of CD45-/- ETPs to the DN2 stage.  4.2.8 CD45 regulates the progression and maturation of CD117- DN1 cells In CD45-/- mice, there were significantly fewer CD117- DN1 cells in the thymus and these cells showed significantly less migration and less in vivo proliferation than their CD45+/+ counterparts. In addition, there were fewer CD117- cells in the DN1.0 population and more in the DN1.5 population in the CD45-/- mice, suggestive of either advanced progression of CD117- cells from DN1.0 to DN1.5, or delayed progression beyond the DN1.5 stage. It has previously been shown that CD117- DN1 cells plated on OP9-DL1 show an atypical progression profile and progress directly to DN4, by passing the typical DN1-4 route used by the ETPs (33). Thus CD117- DN1.0 and DN1.5 populations from CD45+/+ and CD45-/- mice were plated on OP9 or OP9-DL1 cells in the presence of IL-7 and Flt3L and their progression was monitored. CD45+/+ CD117- DN1 cells followed this atypical route when co-cultured with OP9-DL1 but arrested at DN1.5 on OP9, again demonstrating the need for Notch signals for progression beyond the DN1.5 stage (Figure 4.17). Both CD117DN1.0 and DN1.5 populations in the CD45+/+ mice progressed to the DN4 stage. In contrast, the CD117- DN1 cells from the CD45-/- mice stayed primarily at DN1.5 and did not progress to DN4. This suggests a block in progression from the DN1.5 population in cells lacking CD45.  118  Figure 4.16 Distribution of CD117+ cells between CD44+ DN populations. Flow cytometric analysis of the distribution of CD117+ cells between DN1.0, DN1.5 and DN2 populations ex vivo. Graph of the average percentage ± SEM of CD117+ cells within the CD44+ DN population. Data is an average of 9 individual mice over three independent experiments.  119  Figure 4.17 Progression of CD117+ DN1.0 and DN1.5 on OP9-DL1 and OP9 cells in vitro. DN thymocytes pooled from ≥3 mice were labeled for lineage markers, CD44, CD25 and CD117 were sorted into CD117- DN1.0 (CD44+CD25-CD117-) and CD117- DN1.5 (CD44+CD25loCD117-). Sorted populations were co-cultured on OP9-DL1 (left panel) and OP9 (right panel) cells in the presence of 1 ng/ml IL-7 and 5 ng/ml Flt3L. Cells were harvested on Day 6 and Day 9 for flow cytometric analysis. Plots from one representative experiment of three are presented. Axes are in log scale.  120  4.2.9 Lack of CD24 and TCRβ expressing DN1 cells in CD45-/- mice It has previously been shown that DN1 can be subdivided into five distinct populations (DN1a – e) based on CD24 versus CD117 expression (33). Two CD117populations, DN1d and DN1e, were identified based on the presence or absence of CD24 expression. Both of these CD117- DN1 populations were capable of progressing to DN4 in an atypical way when co-cultured on OP9-DL1 in vitro, yet they were distinct populations as DN1e but not DN1d show evidence of TCRβ rearrangement (33). To further characterize the CD117- DN1 population in the CD45-/- mice, the five DN1 subpopulations were examined. As expected, CD45+/+ DN1 cells showed five distinct subpopulations DN1a – e. Surprisingly, the CD45-/- DN1 cells had one major population, DN1e, and showed a correspondingly reduced percentage of DN1a – d populations (Figure 4.18). There was virtually no DN1d population in the CD45-/- thymus. As DN1e but not other DN1 populations show TCRβ gene rearrangement (33), to further characterize this DN1e population, surface TCRβ expression on these cells was analyzed. Interestingly, while the CD45+/+ CD117- DN1 cells predominantly expressed TCRβ, which may be corresponding to the majority of CD117- DN1 cells being DN1e, there were almost no cells expressing TCRβ in the CD45-/- CD117- DN1 population, despite that fact that almost all the cells were DN1e (Figure 4.19).  4.2.10 CCR7 is exclusively expressed on the CD117- DN1.5 population It was observed earlier that a subset of CD45+/+ CD117- DN1 cells expressed CCR7 but this was absent in the CD45-/- cells. As CCR7-deficient mice show an accumulation of an early thymic population (CD44+CD25lo) that is phenotypically very similar to the DN1.5  121  Figure 4.18 Altered DN1a-e populations in CD45-/- mice. Flow cytometric analysis of CD24 versus CD117 expression of DN1 thymocytes ex vivo. (A) Representative plot of 7 individual mice over three independent experiments is shown. DN1a-e populations are outlined. (B) Table of the average percentage ± SEM of DN1a-e cells within the DN1 population of CD45+/+ and CD45-/- mice.  122  Figure 4.19 Absence of TCRβ+ cells the CD117- DN1 population of CD45-/- mice. Flow cytometric analysis of TCRβ expression in CD117- DN1 thymocytes ex vivo. (A) Representative plot of 6 individual mice over two independent experiments is shown. (B) Graph of the average percentage ± SEM of TCRβ expressing cells. ***, p ≤ 0.001  123  population described here (138), further analysis of the expression of CCR7 within the DN1 population of CD45+/+ mice was performed. Strikingly, CCR7 was found to be almost exclusively expressed on the CD117- DN1.5 population and not the DN1.0 population in the CD45+/+ mice (Figure 4.20). This difference in CCR7 expression clearly distinguishes the CD117- DN1.5 population from the CD117- DN1.0 population, consistent with our findings of different properties between the DN1.0 and DN1.5 populations. While this DN1.5 population was never characterized in the CCR7-deficient mouse, it raises the possibility that the lack of progression of this progenitor population in CD45-/- mice may be related to a lack of CCR7-dependent migration.  4.3 Discussion  4.3.1 Data summary In this study, a novel intermediate thymocyte progression stage between the traditional DN1 and DN2 stages which requires Notch for progression was identified. The absence of CD45 had an overall negative effect on the proliferation of thymocytes, consistent with the decreased numbers of DN1 cells. The lack of CD45 particularly affected the CD117- subset of the DN1 population, as ex vivo CD24, CCR7, and TCRβ expression, in vitro progression to DN4, and in vitro CXCL12-mediated migration were defective. This work therefore identified new roles for CD45 in early thymocyte development. A diagram summarizing the involvement of CD45 in early thymocyte development is shown in Figure 4.21.  124  Figure 4.20 CCR7 is predominantly expressed on CD117- DN1.5 thymocytes of CD45+/+ mice. Flow cytometric analysis of CCR7 expression on DN1 thymocytes ex vivo. Plot of CCR7 expression on CD117+ and CD117- DN1.0 and DN1.5 populations from CD45+/+ and CD45-/- thymi. Samples were enriched for DN thymocytes pooled from 4 mice prior to labeling for flow cytometry.  125  Figure 4.21 Involvement of CD45 in migration, proliferation, and progression of early thymocytes. The conventional DN1 population can be split into two subpopulations, DN1.0 and DN1.5, based on CD25 expression. Progression of DN1.0 to DN1.5 required IL-7, while progression of DN1.5 to DN2-3 or DN4 required IL-7 and Notch. CD45 is a positive regulator of the in vivo proliferation of CD117+ DN1, CD117- DN1, DN2, DN3, and DN4 populations, as well as the in vitro CXCL12-mediated migration of CD117+ DN1, CD117DN1, and DN4 populations. Surface markers expressed are indicated in brown.  126  4.3.2 CD45 is a positive regulator of proliferation The general trend of decreased proliferation observed in CD45-/- BM LSK cells and various thymic populations suggests that CD45 is required for optimal proliferation in vivo. Interestingly, it was observed that two CD45-deficient T cell lines (BW5147 cells and Jurkat cells) also proliferated slower than cells expressing CD45 (Figure 4.22). As growth factors signal to affect the metabolism of a cell, which closely correlates to cell proliferation (reviewed in 286), this suggests that CD45-deficient cells may be impaired in signaling that is involved in regulating the metabolism of the cell. Supporting this, there is evidence that many pathways involved in the proliferation and differentiation of early T cell progenitors signal through SFKs (115). For example, stem cell factor-1 signals through SFKs (reviewed in 287). IL-7 can induce the phosphorylation of Pyk2, which requires phosphorylation by the SFK Fyn for full activation, and this is implicated in the survival or proliferation of a mouse IL-7-dependent thymocyte cell line (288). In addition, IL-7 receptor ligation leads to an increase in kinase activity of the associated Lck and Fyn (88). Since CD45 is the central regulator of SFKs (289), these data suggest that CD45 may play a role in signaling pathways leading to cell proliferation. Interestingly, Notch signaling can enhance the glycolytic rate (109) which indirectly inhibits cell death (reviewed in 290). As Lck is involved in the Notch-mediated anti-apoptotic pathway (291) in a T cell line, it is possible that CD45 regulates Notch signaling by activating Lck. Collectively, these data suggest a regulatory role for CD45 in proliferation, possibly by affecting signaling pathways involving SFKs. Although CD45 is a positive regulator of proliferation, as there are multiple growth factors and cues involved in stimulating the proliferation of cells in vivo, it is difficult to determine the precise role of CD45 in each growth factor signaling pathway. CD45 can act  127  Figure 4.22 Growth curves of CD45+ and CD45- cell lines. Proliferation of CD45+ and CD45+ BW5147 mouse T cells and human Jurkat T cells. Cells were grown in duplicates and counted by flow cytometry in triplicates over five days. Cell counts were normalized to the initial number of cells plated on Day 0. Data is graphed as the average of the duplicates ± standard deviation. One representative of three independent experiments is shown.  128  as a negative regulator of IL-7 mediated proliferation in pro-B cells (292), as well as IL-3 mediated proliferation in bone marrow-derived mast cells (249). However, IL-6 mediated proliferation of myeloma cells has been shown to require CD45 (250, 251). This may be due to the different effector functions of the SFK being expressed. For example, Lck is an activator of TCR signaling by phosphorylation of the ITAMs (293), whereas Lyn phosphorylation of the immunoreceptor tyrosine-based inhibitory motifs (ITIMs) on the Fc receptor inhibits the mast cell response (294). These data suggest that depending on the cell type, which can express different members of SFKs, SFKs can act as either positive or negative regulators of proliferation. In addition, since CD45 can positively and negatively affect SFKs, this adds another layer of complexity when trying to determine the precise role of CD45 in these signaling pathways.  4.3.3 The lack of CD45 favors the generation of non αβ T lineage cells in adult thymus The DN1 population is a very heterogeneous population, having the ability to generate B, NK, DC, and myeloid cells (25-28). Notch is an important factor that drives commitment towards the T cell lineage at multiple stages along the T cell developmental pathway (reviewed in 69). Overexpression of Notch signaling can induce T cell development in the bone marrow where B cell development normally occurs (97). On the other hand, a decrease in Notch signaling can skew thymocyte development towards the NK and γδ T cell lineages, to the detriment of the conventional αβ T cell lineage (99, 163). In CD45-/- mice, the enhanced numbers of thymic NK cells also support the notion that there may be weakened Notch signaling, which would coincide with the hypothesis that  129  CD45 is a positive activator of Notch. Supporting this, more NK and B cells are observed in the spleen of CD45-deficient mice (233, 235). Although there was no difference in the number of γδ T cells found within the CD45+/+ versus CD45-/- thymi, there was an increased frequency of γδ T cells in the CD45-/thymus. As αβ and γδ T cells share the same progenitors, this increase in frequency may also be due to weakened Notch signaling in the absence of CD45 leading to a skewing towards γδ T cell lineage choice (99). Similar to peripheral γδ T cells (282), the majority of thymic γδ T cells were halted at an immature stage in the CD45-/- mice. Since Lck has been shown to play a role in the maturation of γδ T cells (282, 295), the defect in γδ T cell maturation in the CD45-/- mice may be due to the lack of Lck activation by CD45. NKT cells branch off from the conventional T cell development pathway at the DP stage (170, 171). The decreased number of NKT cells observed in the CD45-/- thymus may be due to the decreased generation of DP cells in the CD45-/- mice (233). However, similar to αβ T cells, selection for the TCR expressed on the NKT cells is a requirement for maturation (172). Due to the essential role CD45 plays in the TCR selection process, these NKT cells might fail in the selection for their TCR, leading to a decrease in NKT cell numbers in CD45-/- mice.  4.3.4 DN1.5 is a novel intermediate between the traditional DN1 and DN2 that requires Notch for progression. In addition to the CD45-deficient mice, a population with a CD44+CD25lo phenotype was also identified in the CCR7-deficient mice (138, 283). However, this population was never characterized. Here, using an in vitro OP9-DL1 co-culture system, it was shown that  130  the CD44+CD25lo population in the CD45-/- mice, referred to as DN1.5 here, is in fact a progression intermediate between the traditional DN1 and DN2 stages. It was also shown that the progression of cells from DN1.0 to DN1.5 was dependent on IL-7 and independent of Notch, while progression of DN1.5 to DN2 was dependent on both IL-7 and Notch. This added an additional Notch independent stage to thymocyte development as it has been shown that progression of the traditional DN1 to DN2 requires Notch and IL-7 signaling (114). The accumulation of cells at the DN1.5 stage in the CD45-/- thymus suggests that the absence of CD45 led to increased proliferation or delayed progression of DN1.5 cells. It was shown that the proliferation of CD45-/- thymocytes was less than the CD45+/+ thymocytes in vivo, suggesting that the accumulation of DN1.5 cells may be caused by a defect in progression. However, in vitro cultures of either BM LSK or DN1.0 cells from CD45-/- mice on OP9-DL1 (data not shown) did not show an accumulation of cells at the DN1.5 stage. This indicates that there is a difference between the in vitro and in vivo situations. One caveat of using the OP9-DL1 co-culture system is that is does not fully mimic the physiological situation. In vivo, the number of Notch ligands are limiting due to the limited number of niches within the thymus, which is thought to be a mechanism of regulating the amount of T cells generated (30). It has been shown that pre-TCR signaling synergizes with Notch signaling during TCR selection processes to promote progression during thymocyte development (296). If the effects of CD45 on the progression of DN1.5 is through a positive regulatory effect on Notch-signaling, then the requirement for CD45 in Notch-mediated progression of DN1.5 to DN2 may be overcome by excessive Notch ligands presented by the OP9-DL1 cells. As well, the accumulation of CD45-/- DN1.5 cells in vivo may be caused by defects in cues only found in the thymus, such as directional cues, which have been shown to  131  play a role in directing the developing thymocytes to microenvironments within the thymus that is required for proper development. In this study, the progression of CD117+ DN1.0 and DN1.5 cells to DN2-3 was shown to require Notch signaling. The progression of these two populations was not different between CD45+/+ and CD45-/- thymocytes suggesting that CD45 does not play a role in Notch-mediated progression. The progression of CD45+/+ CD117- DN1.0 and DN1.5 cells to DN4 also required Notch signaling. However, this progression of CD117- DN1.0 and DN1.5 to DN4 was not observed in CD45-/- thymocytes. This could be due to the lack of the progenitors that could generate DN4 (discussed in section 4.3.9), or it could be due to CD45 being a positive regulator of Notch signaling that is required for this progression, despite CD45 being dispensable for the Notch-mediated progression of CD117+ DN1.0 or DN1.5 to the DN2-3 stage. Notch can enhance thymocyte survival by affecting the glycolytic cycle (109) or by promoting αβ T cell lineage commitment (69). The decreased cell proliferation and increase in NK and γδ T cells observed in CD45-/- mice suggest that CD45 may be a positive regulator of Notch signaling affecting both survival via glycolysis and promotion of the αβ T cell lineage commitment. Since Notch can signal through Lck, so it is possible that CD45 may be affecting Notch signaling by activating Lck.  4.3.5 CD45 is required for CCR7 expression in CD117- DN1 thymocytes It has been shown, with the Foxn1∆/∆ mice that show an abnormal thymic architecture lacking the cortex and medulla domains, that CD117+ DN1 cells require interactions with the thymic epithelial cells (TECs) for development (63, 64). These CD117+ DN1 cells have been shown in vitro to develop via the DN2-3 stage (33) and are known to travel out to the cortex  132  for development (reviewed in 38). Due to the high concentrations of CCL19 and CCL21 expressed in the thymic medulla (138), it was hypothesized that CCR7 signals have a repulsive effect on early thymocytes, and together with the SDF-1 in the cortex that attracts the developing thymocytes, guide thymocytes to the cortex for further development (reviewed in 38, 297, 298). In this study, it was found that CD117+ DN1 cells expressed very low levels of CCR7, which suggests that it is unlikely that CCR7 plays a role in the chemorepulsion of this population away from the CMJ. On the other hand, it was the CD117- DN1 cells that expressed CCR7. As CD117- DN1 cells develop into atypical mature αβ T cells in the absence of a normal thymus (64, 65) and do not go through the typical DN2-3 stage when developed in vitro (33), one could speculate that the CD117- cells do not need to travel to the cortex for development. The expression of CCR7 on CD117- cells was restricted to the CD117- DN1.5 population, which accumulated in the CD45-/- thymus. Interestingly, CD45-/DN1 cells did not express CCR7 at all. This is consistent with the accumulation of a population with a DN1.5 phenotype in the CCR7-deficient mice (138), suggesting that CCR7 plays a role in the development and progression of CD117- DN1.5 thymocytes. It also suggests that CD45 is a positive regulator of CCL19/21-mediated chemotaxis by regulating CCR7 expression.  4.3.6 CD45 is a positive regulator of CXCL12 migration Localization to proper and specific niches within the thymus is important for thymocyte development, as seen in the CCR7- and CXCR4-deficient mice (133, 299). The reduced CXCL12-mediated migration of CD45-/- CD117- DN1 cells may in part lead to the  133  accumulation of cells at the DN1.5 stage in the CD45-/- thymus. Unlike CCR7 expression, there was no significant difference in the levels of CXCR4 expression between the CD45+/+ and CD45-/- thymic populations. As SFKs are involved in CXCL12-mediated migration of a T cell line (300), this suggests that CD45 may also regulate CXCL12 migration by affecting SFK-mediated signaling. Although the CD117- DN1 cells were the most migratory when stimulated with CXCL12 in vitro, some of the migration was contributed by random movements (chemokinesis) seen when no CXCL12 was added. Interestingly, the CD117+ DN1 cells were the most responsive to CXCL12 compared to other DN populations. These CD117+ DN1 cells are thought to progress through the DN2-3 stages as they travel across the cortex (38). In addition, the absence of CD45 led to a decrease in CXCL12-mediated migration of CD117- DN1 and CD117+ DN1 cells, but the decreased migration was only significant in CD117- DN1. This difference in migration and the effect of CD45 on CXCL12 stimulation raises the possibility that CD117+ and CD117- DN1 cells may require different chemotactic signals for development. This is supported by the difference in CCR7 expression between CD117+ and CD117- DN1 cells from CD45+/+ mice.  4.3.7 CD45 does not affect the hematopoietic progenitor pool Here, it was shown that CD45 had no effect on the number of LSK cells present in the bone marrow or blood compartment prior to entry into the thymus. Although the proliferation of BM LSK cells from CD45-/- mice was less than that of the CD45+/+ mice, since the homeostatic generation of progenitor cells is dependent on the availability of niches in the BM (301), it is not surprising that the amount of LSK cells found in the BM at steady-  134  state is not significantly different between CD45+/+ and CD45-/- mice. The amount of LSK cells in the blood was also not affected by the absence of CD45. These data suggest that CD45 is not affecting the LSK progenitor pool at steady state, prior to progenitor entry into the thymus.  4.3.8 Decreased ETP in CD45-/- mice is due to decreased proliferation If the number of available niches in the thymus is regulating the number of ETPs found in the thymus, then one would predict that the number of ETPs found in the CD45-/thymus would be similar to the CD45+/+ thymus. However, this was not the case. Examination of the ETP population within the thymus showed that CD45-/- mice have decreased numbers of ETPs compared to CD45+/+ mice. It should also be noted that a majority of the DN1 population are CD117- cells. This population, which showed a migration defect in the absence of CD45, may be occupying the thymic niches and making the niches less available for ETPs, hence leading to decreased ETP numbers in the CD45-/thymus. Other possible factors that can affect the number of ETPs in the thymus are the ability of the progenitor cells to home to the thymus and the localization of cells to proper thymic niches to receive signals for proliferation and progression to the next developmental stage. 5-weeks competitive bone marrow reconstitution experiments between CD45+/+ and CD45-/cells showed that CD45-/- cells have a significant competitive advantage over CD45+/+ cells in repopulating the thymus at the DN and DP stages, suggesting there is no defect of CD45-/cells to home to the thymus (data not shown). However, this observed competitive advantage of the CD45-/- cells may be due to the blocks in CD45-/- thymocyte development at the DN3  135  and DP stages, leading to an increased portion of CD45-/- cells over CD45+/+ cells at the DN and DP stages in the reconstituted mice. Proper analysis must be done to determine if there is a defect in the entry of CD45-/- bone marrow cells or circulating progenitors into the thymus before further conclusions can be drawn. A recently published paper showed that CD45 expression was upregulated in BM leukocytes and hematopoietic progenitors during their release into the blood upon granulocyte colony-stimulating factor (G-CSF) stimulation (302). G-CSF-stimulated mobilization of white blood cells into the blood as well as CXCL12-mediated migration of bone marrow mononuclear cells was reduced in CD45deficient mice compared to wild type mice. These data suggest that CD45 plays a role in mobilization of progenitors into the blood upon G-CSF stimulation which may in turn affect the amount of circulating progenitors entering the thymus. ETPs are thought to expand by 1000-fold upon entry into the thymus and prior to progressing to the DN2 stage. Since ex vivo and in vitro observations of thymocytes from CD45-/- mice showed no evidence for accelerated progression of ETP to DN2, and that CD45-/- ETPs proliferated slower than CD45+/+ ETPs, this suggests that the decreased ETP numbers in the CD45-/- mice may be related to the general decrease in proliferative capacity of CD45-/- cells. Interestingly, the deletion of Notch1 also leads to decreased thymic ETP numbers (30). As Notch signaling is involved in the proliferation of early thymocytes, this further supports the idea that CD45 may be an activator of Notch-mediated signaling.  4.3.9 CD45 is required for the maturation of atypical T cells The absence of CD45 led to the loss of the DN1d population and the formation of an aberrant DN1e population that did not express surface TCRβ or CCR7 in the CD45-/- mice.  136  These two populations comprise the CD117- subpopulation of DN1 thymocytes. The origin of this CD117- DN1 population is still unclear. Due to the complexity of multiple progenitors and pathways being able to feed into the generation of T cells in the thymus (9), more detailed analysis is needed to determine whether the altered composition of the CD117DN1 population in CD45-/- mice is due to a defect in the generation of progenitors for CD117- DN1 in the bone marrow, or a defect in the entry of these progenitors into the thymus, or if there is a defect in the generation of the CD117- DN1 cells from an earlier population within the thymus. Interestingly, a majority of the CD117- DN1 population of CD45+/+ cells express surface TCRβ. Since NK1.1 antibodies were added to the lineage cocktail to gate out NK1.1+ cells, this population could not be NKT cells that expressed high levels of CD44 and no CD25. Since this TCRβ-expressing CD117- DN1 population can generate DN4 cells directly from DN1, it is tempting to speculate that the CD117- DN1 population are cells that have passed β-selection at DN3 the stage but down regulated CD25 and upregulated CD44. However, the CD117- DN1 cells could also be another T cell subset that follows an atypical pathway of development. Supporting this, the CD117- DN1 populations (i.e., DN1d and DN1e) show smaller proliferative capacity, accelerated progression, and do not follow a conventional differentiation pathway in vitro (33). It should be noted that it is not clear whether these TCRβ+ CD117- DN1 cells are expressing the pre-TCR or the αβ TCR. Determining which type of TCR the CD117- DN1 cells are expressing will help determine the origin and identity of these TCRβ-expressing CD117- DN1 cells. Unlike TCRβ, which was expressed on both CD117- DN1.0 and CD117- DN1.5 cells (data not shown), CCR7 was highly expressed on the more downstream CD117- DN1.5, but  137  not the CD117- DN1.0 population in the CD45+/+ mice. If the transition between DN1.0 and DN1.5 represents a checkpoint for the selection of the TCR expressed, then this may lead to the upregulation of CCR7 on these CD117- DN1.5 cells, similar to the upregulation of CCR7 on positively selected DP thymocytes (142). This upregulation of CCR7 on CD117- DN1.5 cells may lead to the attraction of these thymocytes directly to the medulla for further TCR selection, feeding back into the normal thymocyte developmental pathway. Supporting this, there is an accumulation of CD44+CD25+ cells (which would include DN1.5 and DN2 cells) at the CMJ of the thymus in CCR7-deficient mice, but CD44-CD25+ DN3 cells are abundant in the cortex and SP cells were still generated. In the absence of CD45, the inability of the CD117- DN1 cells from the CD45-/- mice to generate DN4 cells in vitro suggests that CD45 may be involved in the progression of this DN1 population to DN4, or it could also be due to the lack of DN1d within the CD117- DN1 population as DN1d has been shown to generate DN4 cells in vitro (33). A more detailed analysis must be performed to determine if this is the case. As well, RT-PCR should be performed on the CD117- DN1 population to see if the lack of TCRβ expression on these CD45-/- cells was due to the lack of TCRβ gene rearrangement, or impairment in the surface expression of TCRβ. In summary, a novel early thymocyte developmental stage that is Notch dependent was identified. The conventional DN1 population can now be split into two populations which showed different requirements for Notch to develop. This study also suggested new roles for CD45 in early thymocyte development affecting the proliferation and migration of thymocytes. A role for CD45 in the development of non-canonical thymic progenitors was also revealed (Figure 4.23).5  138  Figure 4.23 Involvement of CD45 in non-canonical thymocyte development. CD117+ DN1.0 and DN1.5 cells can progress to DN4 via DN2-3 stages. CD117- DN1.0 and DN1.5 cells can progress to DN4 directly. CD45 is required for the progression of CD117- DN1.5 cells to the DN4 stage. CD45 is also involved in the generation of the DN1d population and the expression of CCR7 and TCRβ within the CD117- DN1 population. Surface markers expressed are indicated in brown.  139  CHAPTER 5 SUMMARY AND FUTURE PERSPECTIVES  140  5.1 Summarizing the involvement of CD45 in early thymocyte development  In this study, novel roles of CD45 in spreading, migration, proliferation, and differentiation involved in thymocyte development were identified. The model is that CD45 can act as a positive or negative regulator of these processes through its regulation of SFKs. Alterations of signaling events involved in these processes during thymocyte development would affect the final outcome of the developing thymocyte. Since the balance between the different lymphocyte subsets that develop from the thymus is important for a functional immune system, studying how CD45 affected these processes would provide a better picture of how thymocyte development is regulated and how lymphocyte subsets are generated. Most of the work done in this study focused on characterizing the role of CD45 in early thymic developmental events, prior to the expression of the pre-TCR or TCR. Specifically, CD45 was found to be a negative regulator of CD44-mediated spreading and a positive regulator of CXCL12-mediated migration, in vivo proliferation, and in vitro progression in thymocytes. The overall effect of the loss of CD45 led to an altered composition of thymic subsets in vivo. The CD45-/- thymus was found to have decreased numbers of ETPs, an aberrant CD117- DN1 population, and increased non αβ T lineage cells. The mechanism of how CD45 regulated CD44 signaling leading to cell spreading was also investigated here. However, the mechanism of how CD45 regulates other processes involved in thymocyte development and the functional outcome of the loss of CD45 remains to be explored.  141  5.2 Involvement of CD45 in signaling pathways affecting lineage commitment  Hematopoietic cell development follows a hierarchal system and cells from different lineages share common progenitors. The balance in signals and cues directing lineage commitment at each developmental checkpoint is important for generating the right amount of cells in each lymphocyte subset for a functional immune system. These signals include Notch signaling, which is a major factor affecting several lineage decisions; IL-7 signaling, which synergizes with Notch at multiple stages of T cell development; and TCR signaling. Although it was not tested in this study, signaling events induced during migration, proliferation, and progression that were regulated by CD45, all involved SFKs. This suggests that CD45 may be regulating signaling events in these processes by regulating SFKs. Although the involvement of CD45 in Notch, IL-7, and TCR signaling was not specifically explored here, these signaling pathways also involve SFKs (291, 303, 304). In addition to the role of CD45 as a central regulator of SFKs (227), this raises the possibility that CD45 may play a role in affecting lineage commitment during thymocyte development by regulating SFKs. This may provide a possible functional outcome of CD45 regulating signaling events during thymocyte development. However, further experimentations are needed to verify this hypothesis.  5.2.1 CD45 and IL-7 signaling CD127+ DN2 cells have a higher tendency to develop into γδ T cells, whereas CD127- DN2 cells tend to develop into αβ T cells (151). In addition, Bcl-2 has the ability to rescue αβ but not γδ T cell development in IL-7-deficient mice. These data suggest that IL-7  142  plays a role in affecting lineage choices (93). Upon IL-7R engagement, the CD132associated JAK-3 phosphorylates CD127 and the associated JAK-1, creating docking sites for, and inducing phosphorylation of, the transcription factors STAT 1,2,3, and 5, leading to the activation of specific genes (reviewed in 303). CD45 can directly bind to, and dephosphorylate JAKs (249). Specifically, interferon α-induced phosphorylation of JAK-1 in CD45-deficient Jurkat T cells, and IL-4-induced phosphorylation of JAK-1 and JAK-3 in primary CD45-/- B cells is increased (249). These data demonstrate that CD45 is a negative regulator of JAKs and may therefore be involved in regulating IL-7 signaling. Lck and Fyn can associate with CD127 and become phosphorylated in response to IL-7 (88). In pre-B cells, IL-7 induces kinase activity of both Fyn and Lyn (305). Moreover, the proliferation of pre-B cells in response to IL-7 is impaired by a SFK inhibitor (306). In CD45-deficient pro-B cells, IL-7 stimulation leads to prolonged JAK/STAT activation, constitutively elevated levels of Lyn phosphorylation and prolonged survival (292). These data suggest that CD45 is a negative regulator of IL-7, possibly through negatively regulating SFKs associated with the IL-7 receptor. IL-7 signaling may be enhanced in developing CD45-/- thymocytes due to the loss of the negative regulation of CD45 through JAKs and SFKs. This may lead to increased survival and/or proliferation of CD127+ DN2 cells, which have the tendency to develop into γδ T cells (151). Then at the DN3 stage, enhanced IL-7 signaling promotes TCRγ rearrangement over TCRβ chain rearrangement (94-96). This is consistent with the observation that CD45-/- mice generated a higher percentage of γδ T cells, although there was an impairment in the maturation of these γδ T cells. This suggests that CD45 may negatively affect γδ T cell lineage commitment by being a negative regulator of IL-7 signaling.  143  5.2.2 CD45 and Notch signaling With findings from this study, it is hypothesized that CD45 is a positive regulator of Notch by activating Lck involved in the Notch signaling pathway. Notch is a major regulator of lineage choices at developmental checkpoints (reviewed in 69). Notch+/- mice tend to generate more γδ T cells compared to Notch+/+ mice (99). This is consistent with the higher percentage of γδ T cells in CD45-/- mice observed here. However, it should be noted that there is interplay between IL-7 and Notch (discussed in section 1.3.5), therefore it would be hard to assess if CD45 was directly affecting IL-7 and/or Notch signaling, thereby affecting lineage commitment. This is also the case at another thymocyte developmental checkpoint. Expression of thymic IL-7 and CD127 on DP thymocytes promotes CD8 lineage differentiation (307). Similarly, Notch signaling has been implicated in promoting the CD8 over CD4 lineage (103, 104). However, according to my hypothesis, there would be enhanced IL-7 signaling, but decreased Notch signaling in the CD45-/- mice. As there was a 20-fold decrease in CD4 SP thymocytes, whereas there was only a 6-fold decrease in CD8 SP thymocytes found in the CD45-/- mice compared to the CD45+/+ mice, this suggests that the lack of CD45 led to a skewing towards the CD8 over CD4 lineage, or that CD45 was more important for the generation of cells in the CD4 lineage. This supports the hypothesis that CD45 is a negative regulator of IL-7 signaling, but not the hypothesis that CD45 is a positive regulator of Notch signaling. It should also be noted that Notch modulates TCR signaling during positive selection and thereby affecting the generation of CD8 versus CD4 SP thymocytes (105). As TCR signaling is defective in the CD45-deficient mice (234), this may also mask the effects of CD45 on Notch signaling if this was assessed by CD4 versus CD8 lineage commitment.  144  Notch is also involved in making choices between B, NK, and T cell lineages. Notch signaling inhibits development of B cells over the NK/T lineage. It also inhibits NK cell differentiation and promotes T cell development (69). Consistent with the hypothesis that CD45 positively regulates Notch signaling, there were more B cells and NK cells observed in the CD45-/- mice (235, 281).  5.2.3 CD45 and TCR signaling CD45 is the central regulator of Lck, having the dual activity to dephosphorylate the activating and inhibitory tyrosines of Lck (Y394 and Y505). CD45 is expressed as a very abundant molecule, comprising up to 10% of the cell’s surface area (216). It was demonstrated by McNeill et al that the level of CD45 expression is correlated with the relative levels of Y394 and Y505 phosphorylation, thus altering Lck activity (308). Although decrease in CD45 expression below a threshold level leads to an increase in the relative Y505 to Y394 phosphorylation ratio leading to a decrease in Lck activity, decrease in CD45 expression to intermediate levels enhances Lck activity due to increased Y394 phosphorylation (308). Higher Lck activity has been shown to favor CD4 over CD8 differentiation (reviewed in 309). Similarly, an intermediate reduction in CD45 expression enhances CD4 lineage commitment (308). These data support the hypothesis that CD45 can alter lineage commitment by altering the TCR signaling threshold by regulating the tyrosine phosphorylation of Lck. The involvement of Lck in all of Notch, IL-7, and TCR signaling (291, 303, 304) suggests that CD45, the central regulator of Lck, may be involved in lineage commitment during early thymocyte development. To propose that CD45 is a negative regulator of IL-7  145  signaling, but a positive regulator of Notch signaling, a mechanism of how CD45 can differentially regulate Lck is needed. One explanation is that SFKs can themselves be positive or negative regulators of signaling pathways. Another mechanism of how CD45 can be a positive and negative regulator of Lck in two different signaling pathways will be discussed in the following section.  5.3 CD44 signaling pathway and regulation by CD45  In this study, the molecular mechanism of CD44 signaling leading to cytoskeletal rearrangement and cell spreading was investigated. Combining the findings from the literature, work from D. Lefebvre and N. Wong (Ph.D. theses), and this study, a working model of CD44 signaling is summarized as follows: At resting state, CD44 is associated with Lck via a zinc-dependent and zincindependent interaction in lipid rafts, where CD44:SFK association occurs (195, 273). The clustering of CD44 would then cluster Lck, which then triggers the activation of Lck leading to the autophosphorylation of Y394 on Lck (310). A signaling cascade is then initiated leading to actin cytoskeleton rearrangement. The activated Lck can also interact with ezrin via its SH2 domain and phosphorylate ezrin at Y145 (211, 212). Phosphorylation of ezrin at Y145 leads to cell spreading as well as sustains SFK activity in a positive feedback loop (212). The ERM proteins also link CD44 to the actin cytoskeleton (205). The actin cytoskeleton, along with lipid rafts, further aggregate CD44 and the associated Lck into microclusters which form the beginning of the CD44 signalsome.  146  Upon CD44 engagement, activation of SFK leads to the recruitment of Pyk2 to the CD44/Lck microclusters (N. Wong, Ph.D. thesis). This is required for the subsequent phosphorylation and full activation of Pyk2 (195). Consistent with the role of the actin cytoskeleton serving as a scaffold for signaling molecules, this SFK-dependent activation of Pyk2 is dependent on actin cytoskeleton rearrangement, PLC activity, extracellular calcium, and PI3K activity (N. Wong, Ph.D. thesis and 200), but is not dependent on the integrity of the microtubule cytoskeleton. The Pyk2 autophosphorylated at Y402 then serves as a docking site for the recruitment of Fyn, which can further recruit downstream signaling molecules that regulate actin cytoskeleton rearrangement (262). CD45 is involved in regulating the strength and duration of CD44-mediated signals. In the presence of CD45, CD44-mediated phosphorylation is transient, leading to F-actin ring formation and round spreading. However, in the absence of CD45, CD44-mediated phosphorylation is sustained, leading to a directional F-actin formation and elongated cell spreading (195, 200). As CD45 was recruited to the CD44/Lck clusters upon cell spreading, one would speculate that CD45 is involved in the priming of Lck during CD44-mediated spreading, similar to the priming of Lck by CD45 in early central supramolecular activation clusters (cSMAC) upon TCR stimulation (311). This priming of Lck by CD45 is essential for initiating TCR-mediated signals and suggests that CD45 is a positive regulator of TCRmediated signaling. However, CD45 is not essential for CD44-mediated signaling. In fact, CD45- BW5147 cells show enhanced CD44-mediated signals upon stimulation to spread, suggesting that CD45 is a negative regulator CD44-mediated signaling (195, 200). The difference between the two scenarios is that CD45 remains associated with the CD44/Lck microclusters throughout the duration of cell spreading whereas during TCR  147  stimulation, CD45 is later translocated to the distal SMAC (dSMAC) to prevent CD45 from downregulating signals generated at the cSMAC (311). This suggests that the duration of the recruitment or translocation of CD45, to or away from the signalsomes, may be a mechanism of how CD45 can be a positive and negative regulator of two different signaling pathways involving SFKs. Upon Lck activation mediated by CD44, a transient signal is observed in CD45+ cells versus the sustained signal observed in CD45- cells (200). This may be achieved by the recruitment of CD45 to microclusters. However, since it was observed here that CD45 was already present at the microclusters by 5 minutes and the CD44-mediated Pyk2 signaling has been shown to peak at 30 minutes (N. Wong, Ph.D. thesis), this suggests that the presence of CD45 in microclusters does not completely inhibit signals generated from the CD44 clusters. This might be due to a fraction of CD45 being translocated to the ring in the periphery. This effectively decreases the phosphatase activity of CD45 (by decreasing the number of CD45 molecules) at the microclusters, allowing some CD44-mediated signals to be generated. This is supported by the model that the equilibrium of Y505 and Y394 phosphorylation affecting Lck kinase activity is affected by CD45 expression levels (308). The fact that Lck can still be activated leading to cell spreading in the absence of CD45 further demonstrates a difference between CD44-mediated signaling and TCR signaling. CD45 is essential to activate the Lck associated with the TCR signaling complex, whereas CD45 is not required to activate the Lck associated with CD44. This suggests that there may be two different pools of Lck: one that requires priming by CD45 and one that does not. CD44 may be selectively associating with the pool of Lck residing in lipid rafts that does not require CD45 for activation. It has been shown that the kinase activity of SFKs  148  in the lipid raft fraction of the membrane is higher than that in the non-raft fraction (312, 313), although the reverse has been reported (314). Nonetheless, the difference in SFK activity in lipid raft versus non-raft fractions at resting state is thought to be due to the difference in the amount of CD45 found in the two fractions (314-316), affecting the equilibrium between Y505 and Y394 phosphorylation on Lck, thus altering SFK activity. It has been shown that the phosphorylation of Lck in lipid raft fractions is similar between CD45+ and CD45- cells (314). As there is dominance of Y394 phosphorylation over Y505 phosphorylation affecting Lck activity even when both tyrosines on Lck are phosphorylated (231), CD44-mediated Lck signaling can still be achieved. Another possibility is that Lck can be activated independently of CD45, similar to the mechanism of Lck activation by Unc119 (317, 318). This can be achieved if binding of CD44 to Lck disrupts the intramolecular interaction between the phosphorylated Y505 and SH2 domain of Lck, opening up and activating Lck.  5.4 Future directions  Since CD45 is involved in multiple processes that would affect the overall outcome of lymphocyte populations generated from the thymus, understanding the molecular mechanism of how CD45 regulates these processes would provide insights to treatment of diseases where the immune response is dysregulated, such as autoimmune diseases. Although the characterization of thymic populations in the CD45-/- mice in this study has provided some insights into how CD45 affects thymocyte development, there are  149  questions that remain to be addressed from this work. First, it needs to be determined if there is a defect in the homing of CD45-/- thymic progenitors to the thymus. This would help determine if the decrease in early thymic cell numbers in CD45-/- mice was due to a defect in entry of the progenitors, or a defect in the proliferation and/or survival of cells within the thymus. As the lack of CD45 affected cell spreading, chemokine-induced migration, and chemokine receptor expression of thymocytes, it was speculated that the intrathymic migration and localization to specific thymic zones of thymocytes would be affected. Therefore, it would be of interest to see if the absence of CD45 would lead to improper niche localization of these progenitors upon entry into the thymus. This can be achieved by confocal microscopy of thymic slices. This is important as specific niches within the thymus are required to provide stage-specific survival and differentiation signals for thymocyte development (38). The lack of CD45 led to the formation of an aberrant CD117- DN1 population. This suggests that CD45 is important for the generation or progression of this population. However, the origin or identity of these CD117- DN1 cells must first be determined. The OP9-DL1 co-culture system can be utilized. Then the effector function of the mature T cells generated from this CD117- DN1 population should also be examined. Other areas that remain to be explored include comparing the lineage potential of the CD45+/+ and CD45-/- DN1a-e progenitors by clonal assays using OP9-DL1 co-cultures, or intrathymic injection. Both in vitro and in vivo approaches are necessary since lineage choice, as discussed in previous sections, is affected by multiple factors which cannot be completely mimicked in vitro. However, the OP9-DL1 co-cultures does allow for single-cell  150  lineage analysis and titration/manipulation of different factors that affect lineage commitment. This would be needed to compare the lineage potential of progenitors between CD45+/+ and CD45-/- mice. These results would provide insights into the effects of CD45 on overall thymocyte development. Then finally, deciphering the signaling pathways that CD45 is involved in during early thymocyte development would build a framework for the mechanism of how CD45 affects these signaling processes.  151  FOOTNOTES  152  1  A version of Figures 3.1, 3.9A, 3.10, 3.13 and 3.14 has been published in: Lai, J.C.Y.*,  Wong, N.K.Y.*, Birkenhead, D., Shaw, A., and P. Johnson. 2008. CD45 down regulates Lck-mediated CD44 signaling and modulates actin rearrangement in T cells. J Immunol 181:7033-7043. (* Both authors contributed equally to this work) 2  A version of Figures 3.4 and 3.5A will be submitted for publication: Jacqueline C.Y. Lai*,  Dennis C. Lefebvre*, Nina Maeshima, Jennifer L. Ford, Nelson K. Y. Wong, Darlene Birkenhead, Andrea S. L. Wong, Jennifer L. Cross, and Pauline Johnson. Zinc facilitates the association of Lck with CD44. (* Both authors contributed equally to this work) 3  The left panel of Figure 3.5A was contributed by D. Lefebvre  4  A version of Figure 3.6 has been published in: Brown, K.L., Birkenhead, D., Lai, J.C.Y.,  Li, L., Li, R., and P. Johnson. 2005. Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells. Exp Cell Res 303:400-414. 5  A version of Chapter 4 has been submitted for publication: Jacqueline C.Y. Lai, Marta  Wlodarska, David J. Liu, and Pauline Johnson. CD45 regulates migration, proliferation and progression of early thymocyte progenitors.  153  REFERENCES  154  1.  Kondo, M., A. J. Wagers, M. G. Manz, S. S. Prohaska, D. C. Scherer, G. F. Beilhack, J. A. Shizuru, and I. L. Weissman. 2003. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol 21:759-806.  2.  Scollay, R., J. Smith, and V. Stauffer. 1986. 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Cross. Some of the experimental repeats for Figure 4.7 were performed by David Liu. Some of the experimental repeats for Figures 4.7, 4.9, 4.18 and 4.20, and all of the repeats for Figure 4.19 were performed by Marta Wlodarska. Darlene Birkenhead, Jennifer L. Cross and Katharine Kott helped with the bone marrow extraction in some experiments.  187  APPENDIX B BIOSAFETY AND ANIMAL CARE CERTIFICATES  188  189  190  191  192  193  194  195  196  197  198  199  200  201  THE UNIVERSITY OF BRITISH COLUMBIA  ANIMAL CARE CERTIFICATE Application Number: A07-0786 Investigator or Course Director: Pauline Johnson Department: Microbiology & Immunology Animals: Mice C57bl/6 (Thy1.2) 3 Mice C57 x 45KO 110 Mice EXON 9 45ko 364 Mice B6.SJL (BoyJ) 42 Rabbits 1 Mice IL-7RKO 36 Mice GFP/Ly5.2 18 Mice C57Bl/6 385 Mice 45RAGKO 65 Mice OT II 12 Mice RAGKO 65 Start Date:  Approval Date: June 10, 2008  October 1, 2005  Funding Sources: Funding Agency: Funding Title:  Canadian Institutes of Health Research (CIHR) Regulation of signaling and dendritic cell function by CD45  Unfunded title:  n/a  The Animal Care Committee has examined and approved the use of animals for the above experimental project. This certificate is valid for one year from the above start or approval date (whichever is later) provided there is no change in the experimental procedures. Annual review is required by the CCAC and some granting agencies. A copy of this certificate must be displayed in your animal facility. Office of Research Services and Administration 102, 6190 Agronomy Road, Vancouver, BC V6T 1Z3 Phone: 604-827-5111 Fax: 604-822-5093  202  

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