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Common lymphoid progenitor-independent pathways of innate and T lymphocyte development Ghaedi, Maryam 2015

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COMMON LYMPHOID PROGENITOR-INDEPENDENT PATHWAYS OF INNATE AND T LYMPHOCYTE DEVELOPMENT by Maryam Ghaedi D.V.M., The Shahid Chamran University of Ahvaz, 2005  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF SCIENCE in The Faculty of Graduate and Postdoctoral Studies (Interdisciplinary Oncology)   THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  April 2015 © Maryam Ghaedi, 2015 ii	  	  Abstract All lymphocytes are thought to develop from a single population of committed lymphoid progenitors termed common lymphoid progenitors (CLPs). However, upstream progenitors termed lymphoid-primed multi-potent progenitors (LMPPs) are known to be more efficient than CLPs in differentiating into T cells and group 2 innate lymphoid cells (ILC2s), suggesting alternative pathways of their development. Here, we have divided LMPPs into CD127- (LMPP-s) and CD127+ (LMPP+s) subsets and compared them with CLPs. Adult LMPP+s are the most efficient progenitors for T cells and ILCs in transplantation assays, and lineage tracking by the recombinase expression also suggests that most ILC2s and NK cells develop from LMPPs independent of CLPs. In the neonatal period CLPs are rare and, unlike prominent neonatal LMPP+s, incapable of differentiating into ILC2s and T cells while their development is highly active. These results suggest non-linear pathways of innate and T lymphocyte development from LMPP+s with limited CLP contributions.   iii	  	  Preface A part of the introduction is a version of an invited review entitled “Development of Group 2 Innate Lymphoid Cells” by Ghaedi, M., and Takei, F. that is submitted for potential publication. Chapter 3 of this thesis has been submitted for potential publication: Ghaedi, M., Steer, C., Halim, T., Abraham, N., and Takei, F.: “Common lymphoid progenitor-independent pathways of innate and T lymphocyte development”. I carried out all the experiments presented in Chapter 3 with the following exceptions. In Figure 2B, the analysis of CD127-null mice was carried out by Tim Halim. In Figure 5, Catherine Steer provided neonatal mice of different ages and also analyzed lung ILC2s.  Over all, for the submitted manuscript, I designed and performed the experiments and wrote the paper. Steer, C. performed the experiments and wrote the paper. Halim, T. performed the experiments and reviewed the paper. Abraham, N. reviewed the paper. Takei, F. supervised the project, designed the experiments and wrote the paper. All animal use was approved and performed in accordance with the guidelines of the animal care committee of the University of British Columbia and the Canadian Council on Animal Care. Canadian Council on Animal Care approval was granted under the certificate number: # A14-0182.     iv	  	  Table of contents Abstract	  ....................................................................................................................................................	  ii	  Preface	  ...................................................................................................................................................	  iii	  Table	  of	  contents	  .................................................................................................................................	  iv	  List	  of	  tables	  ........................................................................................................................................	  vii	  List	  of	  figures	  .....................................................................................................................................	  viii	  List	  of	  abbreviations	  ...........................................................................................................................	  ix	  Acknowledgements	  ..........................................................................................................................	  xii	  Chapter 1 Introduction	  .........................................................................................................................	  1	  1.1	  The	  immune	  system	  ................................................................................................................................	  1	  1.2	  ILCs	  ...............................................................................................................................................................	  2	  1.2.1	  Cytotoxic	  ILCs	  .........................................................................................................................................................	  2	  1.2.2	  Cytokine-­‐producing	  ILCs	  ...................................................................................................................................	  3	  1.3	  Lymphopoiesis	  ..........................................................................................................................................	  5	  1.4	  The	  development	  of	  ILCs	  .......................................................................................................................	  6	  1.4.1	  BM	  NK	  progenitor	  (NKP)	  ..................................................................................................................................	  6	  1.4.2	  BM	  ILC2	  progenitors	  ...........................................................................................................................................	  6	  1.4.3	  Common	  developmental	  programs	  to	  all	  ILCs	  and	  a	  common	  progenitor	  to	  all	  cytokine-­‐producing	  ILCs	  ..................................................................................................................................................................	  7	  1.4.4	  Transcription	  factors	  regulating	  ILC2	  differentiation	  .......................................................................	  10	  1.4.5	  CHILP	  and	  ILC	  development	  .........................................................................................................................	  13	  1.5	  Thesis	  objectives	  ....................................................................................................................................	  13	  Chapter	  2	  Materials	  and	  methods	  ................................................................................................	  14	  v	  	  2.1	  Mice	  ............................................................................................................................................................	  14	  2.2	  BM	  transplantation	  ................................................................................................................................	  14	  2.3	  Rag-­‐1/Red	  and	  Rag-­‐1/GFP	  ..................................................................................................................	  14	  2.4	  Primary	  leukocyte	  preparation	  ........................................................................................................	  14	  2.5	  Flow	  cytometry	  and	  cell	  sorting	  .......................................................................................................	  15	  2.6	  RNA	  isolation	  and	  microarray	  ...........................................................................................................	  15	  2.7	  Methylcellulose	  culture	  .......................................................................................................................	  15	  2.8	  Limiting	  dilution	  cultures	  for	  in	  vitro	  T	  cell	  differentiation	  ...................................................	  16	  2.9	  Accession	  numbers	  ...............................................................................................................................	  16	  2.10	  Statistics	  .................................................................................................................................................	  16	  Chapter3	  Results	  ................................................................................................................................	  17	  3.1	  LMPPs	  in	  adult	  mouse	  BM	  can	  be	  divided	  into	  two	  subsets	  by	  CD127	  expression	  .........	  17	  3.2	  Adult	  BM	  LMPP+s	  are	  potent	  progenitors	  for	  T	  cells	  and	  ILCs.	  .............................................	  20	  ß3.3	  LMPP+s	  differentiate	  into	  CLP	  and	  CHILPs	  ................................................................................	  23	  3.4	  LMPPs	  are	  the	  predominant	  neonatal	  lymphoid	  progenitors	  when	  ILC2	  and	  T	  cell	  development	  is	  highly	  active	  ....................................................................................................................	  27	  3.5	  Neonatal	  LMPP+s	  are	  potent	  T	  cell	  and	  ILC2	  progenitors	  .......................................................	  30	  Chapter	  4	  Discussion	  ........................................................................................................................	  32	  Chapter	  5	  General	  summary	  ..........................................................................................................	  38	  5.1	  General	  summary	  ..................................................................................................................................	  38	  5.2	  Significance	  ..............................................................................................................................................	  39	  5.3	  Future	  directions	  ...................................................................................................................................	  41	  5.3.1	  Affymetrix	  microarray	  analysis	  of	  CLPs	  ..................................................................................................	  41	  5.3.2	  Assessment	  of	  clonality	  ...................................................................................................................................	  41	  5.3.3	  Further	  analysis	  of	  the	  neonatal	  development	  of	  lymphocytes	  ....................................................	  42	  vi	  	  5.3.4	  ILC2s	  similarities	  with	  T	  cells	  ......................................................................................................................	  43	  References	  ...........................................................................................................................................	  44	  Appendices	  ..........................................................................................................................................	  52	  	  	     	    vii	  	  List of tables Table A-1.Defining mouse ILCs ................................................................................................ 56 Table A-2.Genes encoding the listed transcription factors ..................................................... 57  	   	  viii	  	  List of figures Figure 1 Lineage map for the development of cytotoxic and cytokine-producing ILCs. ...... 9 Figure 2 Adult mouse BM LMPPs can be divided into LMPP- and LMPP+. ...................... 19 Figure 3 LMPP+ are more efficient than LMPP-s and CLPs at generating T cells and ILCs. ............................................................................................................................................... 22 Figure 4 LMPP+s are the upstream lymphoid progenitors to both CLPs and CHILPs. .... 26 Figure 5 LMPP+s are prominent lymphoid progenitors in neonatal BM and spleen, when ILC2 and T cell development is highly active. .................................................................. 29 Figure 6 Neonatal LMPP+s are more efficient than CLPs at generating T cells and ILC2s. ............................................................................................................................................... 31 Figure 7 Schematic representation of non-linear model of lymphocyte development. ........ 36 Figure A-1 Transcription factors required for early lymphoid lineage development. ......... 52 Figure A-2 BM LMPP+s are potent progenitors for T cells and ILCs. ................................. 53 Figure A-3 Normal numbers of ILC2s were found in the lungs of Flt3l−/− mice. ................. 54 Figure A-4 LMPPs are the predominant neonatal lymphoid progenitors when ILC2 and T cell development is highly active. ....................................................................................... 55 	  	   	   	  ix	  	   List of abbreviations Ab                    antibody APC                 allophycocyanin B6                    C57Bl/6 BM                  bone marrow CLP                 common lymphoid progenitor GFP                 green fluorescent protein FACS              florescence activated cell sorting Flt3L               Fms-like tyrosine kinase ligand GATA3           GATA-binding protein 3 HSC                haematopoietic stem cell IFN                 interferon IFNγ               interferon gamma IL                    interleukin IL-17RB IL-25 receptor  ILC                 innate lymphoid cell x	  	  ILC1               innate lymphoid cell group 1 ILC2               innate lymphoid cell group 2 ILC3               innate lymphoid cell group 3 Lin                  lineage LMPP             lymphoid primed multipotent progenitor LSK                lineage-negative, Sca-1-positive, c-Kit-high LTi                  lymphoid tissue inducing Ly49               lymphocyte antigen 49 complex MHC              major histocompatibility complex NK                  natural killer NKT               natural killer T cell NOD/SCID    non obese diabetic/severe combined immunodeficient NSG               NOD/SCID IL-2 receptor gamma chain knockout PE                  phycoerythrobilin PI                   propidium iodide RAG              recombinase activating gene xi	  	  ROR              retinoic acid receptor orphan receptor Sca-1             stem cell antigen 1 SCF               stem cell factor TCR              T cell receptor TGFβ            transforming growth factor-beta Th                 T helper TNF-α           tumour necrosis factor-alpha TSLP            thymic stromal lymphopoietin    	     xii	  	  Acknowledgements Special thanks to my supervisor Dr. Fumio Takei, for all his exceptional mentorship and patience. I would like to thank my supervisory committee members, Dr. Kelly M. McNagny, Dr. Andrew P. Weng and Dr. Pauline Johnson for their helpful comments, advices and encouragements throughout my MSc studies.  I would also like to acknowledge the coop student Nesim Lichy who provided invaluable help for my project in the laboratory. Lastly I would like to acknowledge the present and past Takei Laboratory members for their advice and great friendship, including Dr. Itziar Martinez-Gonzalez, Dr. Grace Poon, Catherine Steer, Laura Mathae and Dr. Tim Halim and also core facility staff including David Ko, Wenbo Xu, Gayle Thornbury and the ARC facility staff. I would also like to thank Dr. Paul Kincade for providing us with the BM from RAG1-GFP mice and RAG1-Cre x Rosa26-FloxStop-TdRFP mice.       	   1	  Chapter 1 Introduction 1.1 The immune system The immune system can be divided into adaptive and innate immune systems, each of which is composed of multiple specialized cell types [1]. The adaptive immune system confers a highly antigen specific response through recombination activating gene (RAG) mediated V(D)J recombination and generation of receptors of diverse specificities. This compartment of the immune system consists of T and B lymphoid lineages [2]. T cells are generated in the thymus and they can be divided into two types based on the T cell receptor (TCR) expression, namely TCRαβ- and TCRγδ-expressing T cells. The latter population mostly develops in the perinatal period and seeds various tissues. Adult T cells are predominantly TCRαβ+ and they are further sub-grouped to CD8+ cytotoxic T cells and CD4+ T helper (Th) cells. CD8+ cytotoxic T cells destroy infected or transformed cells [2] whereas CD4+ T helper (Th) cells secrete cytokines that influence (stimulate) other immune functions. Th cells can be further divided into distinct subsets based on the cytokines they produce, namely Th1, 2, 9, 17 and follicular Th subsets. T cells also include regulatory T cells that inhibit other lymphocytes and Natural Killer T (NKT) cells which share properties with innate Natural Killer (NK) cells [3], [4]. B cells are generated in the bone marrow (BM) and confer antibody mediated humoral immunity [2]. They express cell surface Ig, CD19, CD20 and B220 and can be divided into B2 (classical B cells) and B1 subsets, based on their differences in genetical program, function and anatomical location. B1 cell subset is an innate-like B cell population and source of innate-Abs that is protective against commonly encountered pathogens. B1 cells are also a source of Abs directed against self-antigens that play an important role in homeostasis [5]. 	   2	  The innate immune system provides a primary line of defence, and is mediated by non-lymphoid cells including granulocytes, monocytes and mast cells as well as innate lymphoid cells (ILCs). Identification of several subsets of the ILCs over the past few years has further revealed the significant contribution of the innate immune system in immune regulation.  1.2 ILCs ILCs, that lack the expression of antigen-specific receptors and can develop in RAG-deficient mice, are mainly found at mucosal barriers and respond to factors derived from the epithelium in an antigen non-specific manner. ILCs are important in the regulation of tissue homeostasis and protection against pathogens [6]. The ILC family includes two separate types, cytotoxic and cytokine-producing ILCs [7]. 1.2.1 Cytotoxic ILCs Cytotoxic ILCs are conventional NK cells that resemble the adaptive cytotoxic CD8+ T cells. NK cells express NK1.1 (in C57BL/6 mice), NK cell p46-related protein (NKp46), class I major histocompatibility complex (MHC)-specific inhibitory molecules (NKG2A and Ly49 receptors) and the stimulatory receptor NKG2D. They express the T-box transcription factors Eomes and T-bet and require Eomes, but not T-bet, for their development. They respond to the stimulating cytokines interleukin-18 (IL-18), IL-12 and IL-15 and are dependent on IL-15 for their development and maintenance. NK cells express granzymes and perforin that mediate cell contact dependent cytotoxicity. They also produce interferon-γ (IFN-γ) and tumor necrosis factor (TNF) and are involved in the tumor immunosurveillance and protection against viral infections [8], [9]. 	   3	  1.2.2 Cytokine-producing ILCs Cytokine-producing ILCs are divided into three subsets, namely group 1 (ILC1), group 2 (ILC2) and group 3 (ILC3), based on the expression of distinct transcription factors and cytokines parallel to that of helper CD4+ T cells (Table A-1). LC1s are similar to NK cells.  They express the NK cell receptors NK1.1 and NKp46 as well as the transcription factor T-bet, but not Eomes. T-bet is required for their development. ILC1s like NK cells respond to IL-12 and IL-15 and produce IFNγ and TNFα, but IL-15 is not required for their development or maintenance. ILC1s are less cytotoxic and produce more cytokines than NK cells. ILC1s were initially considered to be a subset of NK cells. However, the finding of the distinct developmental requirements of ILC1s has led to their re-classification as one of the cytokine-producing ILC lineages different from the cytotoxic NK cell lineage. Intestinal ILC1s are involved in the protection against the intracellular protozoan Toxoplasma gondii [10]. Based on the expression of the transcription factor T-bet and the production of the type 1 cytokines (IFN-γ and TNF), ILC1s are considered to be the innate counterparts of the adaptive T helper 1 (Th1) cells [7], [10]. ILC2s are the innate counterparts of the adaptive Th2 cells as both express the transcription factor GATA-3 and produce IL-5 and IL-13 [11]. Characterization, function and development of ILC2s are discussed in detail below. ILC3s include two distinct subsets, CCR6hi lymphoid tissue inducer (LTi) cells that emerge prenatally and are important for the development of lymph nodes, Peyer’s patches and intestinal lymphoid clusters, and CCR6lo/− ILC3s that emerge during the first 2-4 weeks after birth and are dependent on the signals from the aryl hydrocarbon receptor (AHR), a transcription factor activated by the environmental toxins, for their expansion. All ILC3s 	   4	  express the transcription factor RORγt, on which they are dependent for their development, and produce IL-22 and IL-17. ILC3s are the innate counterparts of Th17 cells. IL-22 produced by ILC3s is involved in the defense against attaching-and-effacing intestinal pathogens such as Citrobacter rodentium [7], [12]. 1.2.2.1 Characterization of ILC2s ILC2s are activated by the epithelial cell-derived IL-25, IL-33 or thymic stromal lymphopoietin (TSLP) and produce the signature type 2 cytokines IL-5 and IL-13. They are involved in the immunity against helminth infections and also implicated in the allergic airway inflammation [13]. The existence of ILC2s was first reported after the discovery that IL-25 administration induces IL-5 and IL-13 production in RAG2 deficient mice that lack B and T cells [14]. Subsequently, a similar population was reported to provide a crucial source of type 2 cytokines in the initial stages of helminth infection [15]–[18].  Later on ILC2s were further characterized and shown to be present in the lungs, intestines, liver, spleen, lymph nodes and fat-associated lymphoid clusters (FALC) [19]–[24]. ILC2s were also shown to be important in the lung epithelial repair after influenza virus infection as they produce amphiregulin, a member of the Epidermal Growth Factor (EGF) family of growth factors [25], and in the initiation of type 2 adaptive immunity [26], [27]. ILC2s, like other ILCs, lack the expression of cell surface markers for mature hematopoietic and lymphoid cells (Lin−). They express CD127 (IL-7Rα), CD25 (IL-2Rα) and the receptors for the cytokines IL-33 (ST2) and IL-25 (IL-17RB). Thus, mouse ILC2s are defined by Lin−Id2+IL7Rα+Thy1+CD25+ST2+/−Sca-1+IL-17RB+ [28]. Human ILC2s, identified by Lin−IL-7Ra+CD25+CD161+CRTH2+, have been found in the lung, intestinal tissues and in elevated numbers in the nasal polyps of the patients with chronic rhinosinusitis [29]. 	   5	  1.3 Lymphopoiesis Mature blood cells derive from haematopoietic stem cells (HSC) that are located in the BM. HSCs that are multipotent and have self-renewal ability, differentiate into all types of blood cells via a stepwise loss of lineage potentials. During this process, HSCs lose their self-renewal capacity and differentiate into multipotent progenitors. HSCs are identified in mice as a rare population among the BM Lin−Sca-1+c-KithiFlt3−. These cells are further defined as EPCR+CD48-CD150+ (ESLAM) cells (Benz et al. 2012). HSCs differentiate into common myeloid progenitors (CMPs) and lymphoid primed multipotent progenitors (LMPPs). CMPs that are defined as Lin−Sca-1−c-Kit+CD34+CD16/32+ BM cells give rise to granulocyte/macrophage and megakaryocyte/erythrocyte progenitors. LMPPs that are defined as Lin−cKithiSca-1hiFlt3hiCD127− BM cells represent a population of progenitors that are primed for the lymphoid lineage development but retain residual myeloid potential [31].  In the classical model of lymphopoiesis LMPPs give rise to common lymphoid progenitors (CLPs) and all lymphocytes develop from the CLPs in the BM [32]. They lack any myeloid potential and equally give rise to all lymphocytes including T, B and NK cells.  CLPs are functionally defined by their clonal output of all lymphoid lineages in in vitro cultures and transplantation assays. They were initially identified by a combination of cell surface markers, namely Lineage marker-negative (Lin−) cKitloSca-1loCD127hi (IL-7Rαhi), which has been refined to include other markers, including high expression of Fms-related typrosin kinase 3 (Flt3, CD135) [33]. In the classical model, CLPs are the obligatory intermediate progenitors between LMPPs and any mature lymphocytes. However, the discovery and characterization of early thymic progenitors (ETPs) suggested that T cell development might not follow this classical pathway. ETPs were identified as the most immature and efficient T cell progenitors in the thymus. ETPs have residual myeloid 	   6	  potential and their development is mostly independent of the transcription factor Ikaros that is required for the development of CLPs, suggesting that ETPs can develop via a CLP-independent pathway [34], [35]. Furthermore, LMPPs or a subset of LMPPs termed Early Lymphoid Progenitors (ELPs) that was defined by the expression of recombination-activating gene 1 (Rag1) are much more efficient than CLPs in differentiating into T cells in transplantation assays [36]. It is evident that CLPs have the potential to differentiate into T cells, but they are barely detectable in the peripheral blood and seem to mostly remain in the BM. In contrast, circulating LMPPs can readily be detected [37].  The T cell potential of CLPs may be realized only when they are isolated from the BM and transplanted into irradiated recipients. A compelling amount of evidence supports a CLP-independent pathway of T cell development in normal steady state.  1.4 The development of ILCs 1.4.1 BM NK progenitor (NKP)  The first stage to the generation of NK cells occurs when lymphoid progenitors differentiate into NK progenitors (NKPs). According to the classical model, the lymphoid progenitors upstream of NKPs are CLPs. NKPs have acquired the IL-15 receptor complex, composed of IL-15 receptor α (IL-15Rα), IL-2Rβ (CD122), and IL-2 receptor common γ (IL-2Rγc), and are responsive to IL-15, which is required for NK cell development and survival. NKP has lost the ability to differentiate into T cells, B cells. These cells first acquire NK1.1 and NKG2D expression, and later acquire other NK lineage markers such as Ly49 family members and DX5 and become functionally mature [38]–[41].  1.4.2 BM ILC2 progenitors Adult BM contains ILC2-like cells [42], [43]. They were shown to be closely related to 	   7	  ILC2s by the genome-wide transcriptome analysis, have very low levels of cytokine production upon in vitro stimulation with IL-33 and TSLP and were suspected to be the lineage-specified precursors to ILC2s. Upon transplantation into alymphoid Rag2−/−IL2rg−/− mice, they only gave rise to mature ILC2s in the mucosal tissues such as lungs and intestines and were subsequently named ILC2 progenitors (ILC2Ps). ILC2Ps are defined by Lin−Id2+α4β7+IL-7Rα+Thy1+CD25+ST2+ (Hoyler et al. 2012; Halim et al. 2012). 1.4.3 Common developmental programs to all ILCs and a common progenitor to all cytokine-producing ILCs ILCs are thought to develop from CLPs (defined by Lin−cKitloSca-1loFlt3hiIL-7Rα+ that also give rise to T and B cells [45]. All ILCs share the requirement for the transcriptional repressor, inhibitor of DNA-binding 2 (ID2) for their development (Diefenbach et al. 2014; Benz et al. 2012). ID2 is a member of the Helix-Loop-Helix (HLH) family of transcriptional regulators, which lacks DNA binding domain and its hetero-dimerization with E proteins inhibits DNA binding of E proteins and represses their transcriptional activity. E proteins belong to the class I basic HLH (bHLH) family of transcription factors and are strictly required in the T and B cell development. ID2 suppression of the T and B cell fates is crucial in the development of all ILCs and mice lacking this transcription factor lack all ILCs [47]. Notch and IL-7 signaling pathways are also required for the development of cytokine-producing ILCs [7]. The shared developmental requirements of ILCs, led to the speculations that an ID2-expressing common progenitor to all or some ILCs should exist [11]. Examination of the fetal liver and adult BM of the ID2 reporter mice, in which green fluorescent reporter protein is expressed under the control of the Id2 promoter and ID2-expressing cells are readily identified, confirmed the existence of such an ID2-expressing progenitor. Upon transfer into alymphoid mice, the ID2+ progenitors gave rise to ILC1 (T-	   8	  bet+Eomes−), ILC2 (GATA-3+), ILC3 (RORγt+ including LTi cells), but not to NK (T-bet+Eomes+), T or B cells [10]. Therefore, the ID2+ progenitors were committed common progenitors for all the cytokine-producing (helper-like) ILCs and were named CHILPs (common progenitors to all helper-like ILCs). CHILPs are Lin−Id2+PLZF+/-α4β7+IL-7Ra+Thy1+Flt3−CD25−ST2−. The expression of ID2 and α4β7 and the lack of Flt3 expression distinguish CHILPs from CLPs. A subset of CHILPs expresses the transcription factor promyelocytic leukemia zinc finger (PLZF), encoded by the Zbtb16 gene. PLZF is required for the development of NKT cells and confers their innate properties [48], [49]. PLZF+ CHILPs highly express Gata3, Rora, Tcf7 (encoding TCF-1) and low level of Rorct (encoding RORγt). While ILCs do not express PLZF, lineage tracing by the expression of Zbtb16, a genetic fate mapping mouse model, in which Cre recombinase is expressed under the control of Zbtb16 promoter and permanently activates the yellow fluorescent reporter protein (YFP), has revealed that cytokine-producing ILCs, but not NK cells or LTi, show a history of substantial Zbtb16 expression during their development. In agreement with the lineage tracing data, PLZF+ CHILPs had the potential to give rise to ILC1, ILC2 and ILC3s but not to NK or LTi cells upon transfer to alymphoid mice [45],  showing their restricted developmental potential in comparison with CHILPs. As PLZF transcription is thought to be controlled by ID2 [50], the PLZF+ CHILPs might be the downstream progeny of the PLZF− CHILPs (Figure 1). Compelling evidences indicate that NK cell specification differ from cytokine-producing ILC specification. NK cells are not the downstream progeny of CHILPs [10], [45], NKPs are mainly ID2− and ID2 is only required after their lineage specification and further differentiation into NK cells [41], [51]. It has also been shown that while GATA-3 is required for the development of all cytokine-producing ILCs, it is dispensable for the 	   9	  development of NK cells [7]. Therefore, NK cells are now considered to constitute the lineage of cytotoxic ILCs separate from all the cytokine-producing ILC lineages.   Figure 1 Lineage map for the development of cytotoxic and cytokine-producing ILCs.  All lymphoid lineages are thought to be the downstream progenies of CLPs. The existence of an ILC-restricted progenitor (CILP) to the two main ILC lineages, cytotoxic and cytokine-producing ILCs, is speculated. NK cells are the only cytotoxic ILCs whereas cytokine-producing ILCs are composed of ILC1s, ILC2s and ILC3s. All cytokine-producing ILCs (but not cytotoxic ILCs) differentiate from the CHILPs. A subset of CHILPs is PLZF+ and has more restricted developmental potential in compare to CHILPs. PLZF+ CHILPs have the potential to give rise to ILC1, ILC2 and ILC3s but not to LTi cells. CLP, common lymphoid progenitor; CILP, a speculated common ILC progenitor of both cytotoxic ILCs and cytokine-producing ILCs; CHILP, common helper-like ILC progenitor; NKP, NK- restricted progenitor.  	  1.4.3.1 All ILCs require the transcription factor, nuclear factor interleukin-3 (IL-3) regulated (NFIL3) for their development  Previously it was shown that the differentiation of NK cells requires the transcription factor 	   10	  NFIL3 (also known as E4BP4) [52], [53] . One recent report has shown that Nfil3−/− mice lack CHILPs and have reduced numbers of ILC2s and ILC3s. These mice were also compromised in immune responses related to ILC subsets [54], [55]. Suggesting that NFIL3 might be required for the generation of both cytotoxic and cytokine-producing ILCs.  1.4.4 Transcription factors regulating ILC2 differentiation  CHILPs are directed toward the specific ILC lineages by the expression of the lineage-specifying transcription factors.  RORα, GATA-3, TCF-1 and growth factor independent 1 (Gfi1) have been shown to be the key transcription factors in ILC2 development, maintenance and specification.  1.4.4.1 RORα ILC2s highly express the transcription factor RORα (Table A-2), a member of the retinoic-acid-receptor-related orphan nuclear receptor (ROR) family, which also include the transcription factor RORγt. Using the Rora mutant staggerer (Rorαsg/sg) mice that are functionally RORα-deficient due to a spontaneous deletion in the DNA-binding domain of RORα, our lab demonstrated that RORα is specifically required for the ILC2 development. Although Rorα transcript is expressed to a lower degree in other ILC populations [56], RORα-deficiency specifically leads to ILC2 deficiency and does not affect the frequencies of other ILCs or adaptive lymphocytes (Halim et al. 2012). A genome-wide association study comparing asthmatic patients with healthy individuals showed that single-nucleotide polymorphisms (SNPs) within the Rorα gene were associated with asthma [57]. RORα-deficient mice also failed to develop allergic asthma [58]. Therefore, RORα may be a potential target for therapeutic interventions in allergy and asthma. 	   11	  1.4.4.2 GATA-3 The double zinc-finger transcription factor GATA-3 is required for the transcriptional regulation of the genes encoding the signature Th2 cytokines including Il5 and Il13, and Th2 differentiation of naïve CD4+ T cells [59]. Gata3 deletion resulted in impaired development of both Th1 and Th2 cells, but not that of CD8+ T cells [60]. Recently, several groups have shown that ILC2Ps in the BM and mature ILC2s in the mucosal surfaces express high amounts of GATA-3 [19], [21], [43].  Mature ILC2s required GATA-3 for the production of type 2 cytokines after N. brasiliensis infection in mice [61], and human ILC2s were shown to require GATA-3 for their cytokine production in vitro [56]. It should be noted that Gata3 deletion early in all hematopoietic cells blocked the development of all cytokine-producing ILCs but not cytotoxic ILCs (NK cells) [62]. However, Gata3 deletion in all ILCs (all Id2+ cells) led to the selective loss of ILC2Ps in the BM and mature ILC2s in vivo while other ILCs developed normally [21]. Therefore, the role of GATA-3 in ILC development and function is complex and stage-specific, resembling that in T cell development.  1.4.4.3 Notch and TCF-1 ILC2 development in vitro requires Notch signalling [63]. Retroviral transduction of dominant-negative Mastermind like-1 (dnMAML), a pan-Notch inhibitor, into BM multipotent progenitors resulted in impaired ILC2 development in vivo [64], suggesting that Notch signaling is also required for the ILC2 development in vivo.  The high mobility group (HMG)-box transcription factor TCF-1 is essential in T cell lineage specification. TCF-1, encoded by the Tcf7 gene, is directly up-regulated by Notch signaling during T cell development [65]. Lung ILC2s express Tcf7, and Tcf7−/− mice lack ILC2Ps in the BM and mature ILC2s in mucosal barriers, suggesting that TCF-1 is required 	   12	  for ILC2 development. Also Tcf7−/− mice were compromised in immune responses to the nematode N. brasiliensis and protease allergen papain that requires ILC2s [64]. Interestingly, transduction of TCF-1 into dnMAML-expressing BM progenitors partially restored ILC2 generation from the progenitors, indicating that Notch regulation of ILC2 development is at least in part TCF-1 dependent [64]. Notch signaling pathway and the downstream TCF-1 in ILC2 development resemble that of T cell development. TCF-1 up-regulates the expression of Il17rb and Il2ra through up-regulation of Gata3, and directly up-regulate the expression of Il7r in ILC2s through binding to a conserved binding site upstream its transcription initiation site [64]. Notch signaling is also required for the development of ILC1s and ILC3s [7]. It was also shown that Tcf7−/− mice have reduced numbers of ILC3s [64]. 1.4.4.4 Gfi1 The transcription factor Gfi1 has broad roles in different hematopoietic lineages [66]–[69]. One of the roles of Gfi1 is the reciprocal regulation of Th2 and Th17 cell fates by promoting Th2 and antagonizing Th17 polarization in naïve T cells undergoing Th2 differentiation [70]. Similarly Gfi1 is required for the proper specification of ILC2s. Gfi1-deficient ILC2s had impaired GATA-3 expression, and expressed Rorct and Il17a and the cytokine receptors IL-23R and TGF-βR3 that promote the differentiation of ILC3s. It is notable that the ILC3 lineage is the innate counterpart of the adaptive Th17 lineage. In addition to the suppression of the ILC3 lineage, Gfi1 directly targets and activates Il1rl1 gene encoding the IL-33 receptor (ST2). Accordingly, Gfi1-deficient mice were impaired in mounting an effective type 2 immunity to N.brasiliensis infection and allergic inflammation in response to the protease allergen papain [71]. 	   13	  1.4.5 CHILP and ILC development According to the classical model the newly found ILCs and CHILPs are considered to develop from CLPs, however the upstream developmental processes of CHILPs have never been elucidated. ILCs can be generated from CLPs [45], [63]. However, we and others have previously reported that LMPPs are much more efficient than CLPs in generating BM ILC2 Progenitors (ILC2Ps) and lung ILC2s in transplantation assays (Yang et al. 2011; Halim et al. 2012). To further elucidate the differentiation pathways of ILCs, we now have extended our studies and characterized various lymphoid progenitors in both adult and neonatal mice and compared their capacities to differentiate into ILCs and other lymphoid lineages. Our results suggest that most T cells, CHILPs and ILCs develop from a subset of LMPPs defined by CD127 expression via CLP-independent pathways. This indicates that the CLP stage is not compulsory for the development of all lymphocytes. Based on our findings, we propose an alternative model of lymphocyte development, in which lymphoid progenitors upstream of CLPs have direct contributions to the production of T cells and ILCs.  1.5 Thesis objectives  The overall objective of this thesis is to elucidate the developmental processes of ILCs and CHILPs, common progenitors of all ILCs. Our first goal is to determine if all lymphocytes develop from CLPs and if there are CLP-independent developmental pathways for innate lymphocytes. We characterize lymphoid progenitors in the BM and their potential for ILCs as well as other lymphocytes by transplantation assays, in vitro differentiation cultures and in vivo lineage tracing analysis. Our second goal is to characterize lymphoid progenitors and their development into ILC2s and T cells in neonatal mice. We analyze different lymphoid progenitors in neonatal hematopoietic organs including spleen and BM and investigate their capacities to differentiate into ILC2s and T cells. 	   14	  Chapter 2 Materials and methods 2.1 Mice C57BL/6 (B6), B6.Ly5SJL and NOD.SCID.Il2rg-/- (NSG) mice were maintained in the British Columbia Cancer Research Centre (BCCRC) animal facility, under specific pathogen free conditions. B6.129P2(Cg)-Rorctm2Litt/J and B6.129S(Cg)-Id2tm2.1Blh/ZhuJ (Id2Gfp/+) mice were purchased from the Jackson Laboratories. C57BL/6-flt3Ltm1Imx mice were purchased from Taconic Farms. Adult mice were used at 6-8 weeks of age. All animal use was approved and performed in accordance with the guidelines of the animal care committee of the University of British Columbia and the Canadian Council on Animal Care. 2.2 BM transplantation B6.Ly5SJL (CD45.1) mice were lethally irradiated (10 Gy) and intravenously transplanted with highly purified progenitors from C57BL/6 mice (CD45.2) and 105 helper BM cells from NSG mice (CD45.1). Drinking water of the irradiated mice was supplemented with Ciprofloxacin and HCl for 4 weeks.  2.3 Rag-1/Red and Rag-1/GFP BM from Rag-1/Red and Rag-1/GFP was received from Dr. Paul Kincade’s laboratory, Department of Microbiology and Immunology, University of Oklahoma Health Sciences Center. Cell suspensions were prepared from the BMs and analyzed by flow cytometry. Also 5-8×106 BM cells were intravenously transplanted into lethally irradiated (10 Gy) B6.Ly5SJL. Transplanted mice were used after 2 months for analysis.   2.4 Primary leukocyte preparation Cell suspensions were prepared from the BM, lungs and spleen as described [73]. Cell 	   15	  suspensions from small and large intestines were prepared as described (Halim et al. 2012). 2.5 Flow cytometry and cell sorting All antibodies were purchased from eBioscience unless otherwise specified. Lineage (Lin) marker mABs conjugated with either FITC or eFluor® 450 included anti-CD3ε, TCRαβ, TCRγδ, CD19, B220, NK1.1, Mac-1, Gr-1, Ter119 and CD11c. Additional Abs used included FITC-conjugated anti-CD45.1, PE-conjugated anti-CD127, PerCP-Cy5.5-conjugated anti-CD19, CD25, ST2, PE.Cy7-conjugated anti-Sca-1, PE.Cy5-conjugated anti-CD25, allophycocyanin (APC)-conjugated anti-CD117 (cKit), NK1.1, APC-eFluor780®-conjugated anti-B220, Alexa Fluor® 700-conjugated anti-CD45.1, and eFluor® 605NC-conjugated Thy1.2. BD Horizon V500-conjugated anti-CD45 and CD45.2 were purchased from BD Bioscience. FITC-conjugated anti-ST2 was purchased from MD Bioproducts. Propidium iodide (PI) or eFluor® 780 (eBioscience) were used to exclude non-viable cells. The BD LSRFortessa™ cell analyzer was used for phenotypic analysis; BD FACSAria III was used for cell sorting. Flowjo v. 8.7 (Treestar) was used for data analysis. 2.6 RNA isolation and microarray Total RNA from highly purified sorted progenitors was extracted using Trizol (Invitrogen). Agilent Bioanalyzer 2100, RNA amplification, and microarray services were performed by Sick Kids®, The Centre for Applied Genomics. Hybridization was carried out on the Affymetrix Mouse GeneChip ST 2.0. All data analysis was performed with FlexArray 1.6.3 (Genome Quebec).  2.7 Methylcellulose culture Sorted progenitor cells (1,000 cells per plate) were cultured in Iscove’s MDM-based methylcellulose medium (MethoCult GF 3434; StemCell Technologies) supplemented with 	   16	  10 ng/ml of recombinant mouse (rm) IL-3, 50 ng/ml of rm stem cell factor, 10 ng/ml of recombinant human (rh) IL-6, and 3 units of rh erythropoietin to support the growth of BFU-E, CFU-GM, and CFU-GEMM. After 2 weeks, colonies were counted and classified. 2.8 Limiting dilution cultures for in vitro T cell differentiation  Limiting dilution assays were performed by culturing CLPs, LMPP-s and LMPP+s in serial dilutions (30–1 cells/well) on OP9-DL1 cells for three weeks in complete RPMI and in the presence of IL-7, and SCF (20 ng/ml each), after which they were analyzed by flow cytometry. 2.9 Accession numbers The microarray data is submitted to the Gene Expression Omnibus (GEO) database and the accession number is to be obtained. 2.10 Statistics Data were analyzed using GraphPad Prism version 6 (GraphPad Software). A Student’s t test was used to determine statistical significance between groups.     	   17	   Chapter3 Results 3.1 LMPPs in adult mouse BM can be divided into two subsets by CD127 expression We first divided Lin− cells in adult mouse BM into subpopulations based on the expression of cKit and Sca-1. Most cKithiSca-1hi (LSK) cells (Figure 2, red gate) did not express Thy1 (CD90) or ST2 (IL-33R) and contained the conventional LMPPs defined by high levels of Flt3 expression. Interestingly a minor fraction of LMPPs was positively stained for CD127. CLPs were found in the cKitloSca-1lo population (blue gate), which were mostly Thy1−ST2−, as Flt3hiCD127+ cells, whereas ILC2Ps (Halim et al. 2012) were cKitlo/−Sca1hi (green gate), expressed high levels of Thy1, ST2 and CD127 but were negative for Flt3. In addition, cKit− cells included a population of Sca1lo/− cells (yellow gate), often termed LSK- cells [74], [75]. This population had very little capacity to differentiate into lymphocytes in transplantation assays (data not shown) and was not further analyzed in this study. To confirm the expression of CD127 on LMPPs, we stained LMPPs from wild type (WT) and CD127-null mice with anti-CD127. Flow cytometric analysis showed clear expression of CD127 on a subset of WT LMPPs (Figure 2B). Therefore, LMPPs were divided into the CD127− and CD127+ subsets and termed LMPP- and LMPP+, respectively, hereafter. ELPs are previously characterized Rag1+ subset of the adult mouse BM LMPPs. As ELPs were reported to be CD127−, we examined Rag1 expression in LMPP-s and LMPP+s in Rag1-GFP knock-in mice [36]. Whereas the majority of Lin−GFP+ cells were found in the cKitlo population (Figure 2C, top left, blue gate), the cKithi population also expressed GFP albeit at lower levels and lower frequencies (red gate). When the LMPP population was divided into CD127− and CD127+, most GFP+ cells were in the latter while only a small fraction of the former expressed GFP at a low level. Most CLPs highly expressed GFP. 	   18	  We further compared LMPP- and LMPP+ by Affymetrix microarray analysis (Figure 2D). The expression of the lymphoid lineage-associated genes, including Il7r, Ets1, Rag1, Dntt and Lck were higher in LMPP+ than LMPP-. LMPP+ also highly expressed the myeloid cell lineage-associated genes Mpo, Fcer1g, Ctsg, Fes and Prtn3 although the expression of myeloid genes was generally lower in LMPP+ than LMPP-. Both populations expressed the key transcription factors Ikaros (Ikzf1), PU.1 (Sfpi1 in mouse), and E protein family member E2A (Tcf3) required for early lymphocyte developmental programs [76] while neither populations expressed the signature genes of the earliest T (Ptcra) and B (Pax5) cell restricted progenitors (Figure A-1). In addition, the expression of the gene encoding the chemokine receptor CCR9 that is important for trafficking into the thymus [35] was significantly higher in LMPP+s than LMPP-s. The expression of GFP by LMPP+s and their distinct gene profiles compared to LMPP-s support the notion that LMPP-s and LMPP+s are two distinct subsets.   	   19	   Figure 2 Adult mouse BM LMPPs can be divided into LMPP- and LMPP+.  (A) Adult B6 mouse BM cells were analyzed by flow cytometry.  Live (PI−) CD45+ cells were gated for Lin− (black gate) and then for cKithiSca-1+ (red gate), cKitloSca-1lo (blue gate), cKit−Sca1lo/− (yellow gate) and cKitlo/−Sca1hi (green gate). CKithiSca-1+ cells were further gated for Thy1−ST2− (red gate, middle left) and divided into Flt3hi CD127− LMPPs (LMPP-s) and Flt3hiCD127+ (LMPP+s) (bottom left). CKitloSca-1lo cells were gated for Thy1−ST2− (blue gate, middle center) and Flt3hiCD127+ (CLPs) (bottom center).  CKitlo/−Sca1hi were gated for Thy1+ST2+ (green gate, middle right) and Flt3−CD127+ (ILC2Ps) (bottom right). Numbers in plots indicate mean percentages of the gated cells in each plot.   (B) BM cells from WT and CD127-null mice were analyzed by flow cytometry. Lin− cKithi Sca-1+ cells were first gated and the expression of Flt3 and CD127 were analyzed. The expression of CD127 on WT (red) and CD127-null (grey) LMPPs were compared.   (C) Lin−cKithiSca-1+ (red gate) and cKitloSca-1lo cells (blue gate) from adult RAG-1/GFP mouse BM were analyzed for GFP expression by flow cytometry (top left). The cKithi population was divided by Flt3 expression and the Flt3hi subset (red gate, top middle) was subdivided by CD127 expression into LMPP-s and LMPP+s (top right). Bottom histograms show the expression of GFP by LMPP-s (red), LMPP+s (red) and CLPs (blue) from RAG-1/GFP and WT control (gray). 	   20	  (D) RNA was extracted from purified LMPP-s and LMPP+s and analyzed for global gene expression by Affymetrix microarray. The expression of the genes associated with lymphoid and myeloid lineages in LMPP+s was compared to those of LMPP-s in triplicate experiments and shown in heat maps.  The results are representative of more than 6 experiments for A, 4 experiments for B, and 3 experiments for C. See also Figure A-1.  3.2 Adult BM LMPP+s are potent progenitors for T cells and ILCs.  To determine the differentiation capacities of LMPPs, we purified LMPP-s, LMPP+s and CLPs from adult C57BL/6 mouse BM (CD45.2) by cell sorting and transplanted 2,000 cells each into lethally irradiated congenic B6.Ly5SJL mice (CD45.1) together with helper BM cells from the lymphocyte-deficient NOD.SCID.Il2rg-/- (NSG) mice (CD45.1). LMPP+ transplantation resulted in strikingly robust thymic engraftment, and much higher numbers of thymocytes were generated from LMPP+s than LMPP-s and CLPs 3 weeks post-transplantation (Figure 3A). We also investigated their in vitro T cell differentiation by limiting dilution cultures on OP9-DL1 stroma cells [77].  CD4+ and CD8+ double positive T cells were generated from the plated progenitor populations after three weeks. The frequency of the cells that could generate CD4+ and CD8+ double positive T cells was not significantly different among the three progenitor populations (Figure A-2A). These results suggested that the efficient thymus engraftment of LMPP+s might be due to their ability to circulate and settle in the thymus. It was reported that BM LMPPs were the main T lymphoid progenitors that have access to the thymus through the blood [34], [37]. Therefore, we analyzed the progenitors in the peripheral blood and found that the circulating LMPPs were uniformly CD127+, phenotypically resembling BM LMPP+s (Figure A-2B).  In contrast to T cells, the numbers of spleen B cells (CD19+B220+) derived from the three donor progenitor populations were not significantly different at 3 weeks post 	   21	  transplantation (Figure 3B). At one week post transplantation, more B cells were generated from CLPs than LMPP-s and LMPP+s although the total numbers of B cells were very low and less than 0.2% of those at the 3 weeks time point (Figure A-2C). Significantly (p<0.05) more spleen NK cells (TCR−CD19− NK1.1+) were generated from LMPP+s than CLPs (Figure 3C). In addition to those lymphocytes, small numbers of myeloid cells (CD11b+TCR−B220−CD19−NK1.1−) were also generated in the spleen from LMPP-s and LMPP+s but not CLPs (Figure 3D). The myeloid lineage potentials of LMPP-s and LMPP+s were also compared by granulocyte/monocyte (G/M) colony formation in vitro. More than 30% of the plated LMPP-s and less than 15% of plated LMPP+s formed G/M-colonies in vitro (Figure A-2D).  We also detected donor-derived ILC2s in the lung of the recipient mice 3 weeks post transplantation. Very few (less than 100 cells per mouse) ILC2s were generated from CLPs whereas the number of LMPP+ derived ILC2s was significantly (p<0.01) higher (Figure 3E). To test the capacities of the progenitors to differentiate into ILC3s, we purified CLPs, LMPP-s and LMPP+s from the RORγt reporter allele (Rorct GFP/+) mouse BM [78] and transplanted them into lethally irradiated B6.Ly5SJL mice. Donor-derived ILC3s were detected in the small intestine of the recipients as CD45.2+CD45.1−Lin−CD127+GFP+ cells. At 3 weeks after the transplantation, only small numbers (less than 50 cells per mouse) of ILC3s were detected. Nevertheless, significantly more ILC3s were generated from LMPP+s than CLPs or LMPP-s (Figure 3F). Thus, all three progenitor populations were capable of differentiating into T, B, NK, ILC2 and ILC3 cells. However, they were significantly different from each other in the numbers of progenies they generated in the transplanted mice. LMPP+s were potent progenitors for all the lymphoid lineages tested, particularly T cells, NK cells, ILC2s and ILC3s. In contrast, much smaller numbers of lymphocytes were 	   22	  generated from CLPs, with the exception of B cells.  Figure 3 LMPP+ are more efficient than LMPP-s and CLPs at generating T cells and ILCs. (A) CLPs, LMPP-s, or LMPP+s were purified by cell sorting from C57BL/6 mice (CD45.2+), 	   23	  and 2,000 cells of each population were intravenously injected with 105 helper BM cells from NSG mice (CD45.1+) into lethally irradiated congenic B6.Ly5SJL recipients (CD45.1+). Three weeks post-transplantation, the cells in the thymuses of the recipient mice were analyzed by flow cytometry. Live (PI−) donor-derived cells (CD45.1‒ CD45.2+) were gated (top row) and the expression of CD4 and CD8 were plotted (bottom row). The bar graph shows the absolute numbers of donor-derived cells in the thymus of the recipient mice. (B) Absolute numbers of donor-derived B cells (TCRαβ−TCRγδ−NK1.1−CD19+B220+) in the spleen of the transplanted mice 3 weeks post-transplantation were calculated from the frequency of  donor-derived B cells as determined by flow cytometry and the total numbers of spleen cells. (C) Absolute numbers of donor-derived NK cells (TCRαβ−TCRγδ−CD19−NK1.1+) in the spleen of the transplanted mice 3 weeks post-transplantation were calculate as in B. (D) Absolute numbers of donor-derived myeloid cells (TCRαβ−TCRγδ−CD19−NK1.1− CD11b+) in the spleen of the transplanted mice one week post-transplantation were calculated as in B. (E) The lungs of the recipient mice were analyzed 3 weeks post-transplantation for donor-derived ILC2s by flow cytometry. Live (PI−) CD45.1‒CD45.2+Lin− cells were first gated and ILC2s were gated by Thy1+ST2+ (top) and CD127+ (bottom). The bar graph (right) shows the absolute number of donor-derived ILC2s in the lungs of transplanted mice.  (F) The intestines of the recipient mice were analyzed 4 weeks post-transplantation for donor-derived ILC3s. Donor-derived live CD45.1‒ CD45.2+ cells were further gated for Lin−GFP+ (ILC3s). The bar graph (right) shows the absolute numbers of donor-derived ILC3s cells in the intestines of transplanted mice.  The absolute number of donor-derived cells in tissues of recipient mice was calculated by multiplying cell frequency by the total number of cells. Mean ± S.E.M., * = p ≤ 0.05, ** = p ≤ 0.01 and ***= p ≤ 0.001 (two-tailed Student’s t-test). Data are representative of at least three independent experiments with 4 mice in each condition. See also Figure A-2.   ß3.3 LMPP+s differentiate into CLP and CHILPs To investigate the relationship between LMPP-s, LMPP+s and CLPs, we transplanted them into irradiated recipients and analyzed donor-derived cells in the BM one week later. LMPP-s differentiated into both cKithi and cKitlo Lin− cells expressing CD127 and Flt3, resembling LMPP+s and CLPs. In contrast, LMPP+s differentiated into Lin−Sca-1locKitloFlt3hiCD127+ CLPs but not LMPP-s (Figure 4A). These data suggest that LMPP-s are the most immature 	   24	  whereas LMPP+s are intermediate between LMPP-s and CLPs, and CLPs are the most differentiated lymphoid progenitors. Recently, common progenitors for all helper-like ILCs or CHILPs in the adult mouse BM have been identified by the expression of the transcription factor Id2 [10], [45]. As the above results showed that LMPP+s are more efficient than CLPs in differentiating into ILC2s, we compared their potential to give rise to CHILPs. We purified LMPP+s and CLPs from the Id2 reporter Id2GFP/+ mouse BM, transplanted them into irradiated B6.Ly5SJL mice and analyzed BM cells for donor-derived CHILPs (CD45.2+CD45.1−Lin−Thy1.2+CD25−CD127+ST2−GFP+). Significantly (p<0.05) more CHILPs were generated from LMPP+s than CLPs one week post-transplantation although the numbers of donor-derived CHILPs were low (Figure 4B).  To investigate the differentiation of LMPPs and CLPs into the innate lymphocyte lineages in steady state, we analyzed the RAG-1Cre/Rosa26 tandem dimer red fluorescent protein (tdRFP) mice (Rag-1/Red), in which RAG-1 expression during lymphocyte development results in irreversible expression of tdRFP [79]. LMPP-s were almost all negative for RAG/Red whereas ~25% of LMPP+s and more than 65% of CLPs were RAG/Red+ (Figure 4C). As expected, almost all T cells and a very small fraction of myeloid cells expressed RAG/Red. Only about 20% of splenic NK cells were RAG/Red+. The low percentages of RAG/Red+ NK cells suggest that the majority of NK cells may develop from LMPP-s or LMPP+s while only a minor fraction, if any, may develop from CLPs. About ~33% of BM ILC2Ps and 40% of lung ILC2s were RAG/Red+. These results suggest that the majority of BM ILC2Ps may directly develop from LMPP+s with minor CLP contribution. The small but significant (p<0.05) difference in the percentages of RAG/Red+ cells between 	   25	  lung ILC2s and BM ILC2Ps also suggests that not all lung ILC2s develop from BM ILC2Ps and there might be minor contributions from CLPs to lung ILC2s. To further investigate if the development of ILC2s could bypass the CLP stage of development, we analyzed Flt3-ligand deficient (Flt3l−/−) mice. In agreement with previous studies (Sitnicka et al., 2002; Sitnicka et al., 2007), our results showed that Flt3 ligand is necessary for the efficient generation of both LMPPs and CLPs. However, LMPP-s were less severely reduced than CLPs and LMPP+s in Flt3l−/− mice (Figure A-3A). ILC2Ps were also severely reduced, and yet near normal numbers of ILC2s were found in the lungs of Flt3l−/− mice (Figure A-3B-C). These results imply that the ILC2 development does not strictly require CLPs or LMPP+s as an intermediate stage and these cells can directly develop from the more immature LMPP-s. The results also suggest that some lung ILC2s may develop directly from LMPP-s bypassing the BM ILC2P stage. This is in contrast to the classically defined linear model of lymphopoiesis, which describes one pathway to lymphocyte development through CLPs.  	   26	   Figure 4 LMPP+s are the upstream lymphoid progenitors to both CLPs and CHILPs. (A) Lethally irradiated B6.Ly5SJL mice received intravenous injection of 2,000 cells each of purified LMPP-s (top row) or LMPP+s (bottom row) from adult B6 mice BM, along with 105 helper BM cells from NSG mice, and the BM were analyzed 1 week post-transplantation. Live donor-derived (PI‒ CD45.2+) leukocytes were gated for Lin− (left), and further gated for the LMPP (dashed line) and CLP (solid line) populations and analyzed for Flt3 and CD127 expression. (B) Lethally irradiated B6.Ly5SJL mice received intravenously injection of 2,000 cells each of 	   27	  LMPP+s and CLPs from BM of Id2Gfp/+ mice, along with 105 BM cells from NSG mice.  The BM of the recipient mice was analyzed 1 week post-transplantation.  The donor-derived (CD45.1‒CD45.2+) cells were gated for Lin−Thy1+ and further gated for CD127+ST2‒, and CHILPs were identified by the expression of Id2 (GFP). The bar graph shows the absolute numbers of donor-derived CHILPs. (C) Lethally irradiated B6.Ly5SJL mice received 106 BM cells from Rag-1/Red mice or normal B6 mice (control). Eight weeks later, cells in the BM, spleen and lung of the recipient mice were analyzed by flow cytometry. Donor-derived (CD45.2+) LMPP‒, LMPP+, CLPs and ILC2Ps were gated as in Figure 2A. T cells, myeloid cells and NK cells in the spleen and ILC2s in the lung were gated as in Figure 3.  Histograms show the expression of tdRFP in indicated populations from Rag-1/Red mice (shaded) and from control mice (dashed line). The numbers indicate the percentages of cells in the gates. The data are representative of 3 experiments. See also Figure A-3.  3.4 LMPPs are the predominant neonatal lymphoid progenitors when ILC2 and T cell development is highly active To further study the development of lymphocytes, we analyzed progenitors in the perinatal period when lymphocyte development is very active. As ILC2Ps and ILC2s were undetectable in the fetal liver and lung (data not shown), we analyzed neonatal tissues. In 1 day old mice, there were very few CD45+Lin− cells and almost undetectable numbers of CLPs and LMPP+s in the BM. Instead, lymphoid progenitors present in the 1 day old spleen were predominantly LMPP+ while CLPs were barely detectable (Figure 5A). Similarly, ILC2Ps were undetectable in the BM of 1 day old mice, but abundant in the spleen at this time point. In 10 days old mice, lymphoid progenitors were found in both the BM and spleen. Only ~300 ILC2s in the lung of 1 day old mice and more than 1,500 ILC2s in 5 day old mice were detected. The number of lung ILC2s rapidly increased to more than 15,000 in 10 day old mice, whereas there were around 5,000 in the adult lungs (Figure 5B). Thymopoiesis is also known to be very active in the early postnatal life [80]. In agreement with the published findings [81], the frequencies of ETPs, defined as LinlocKithiCD25- thymocytes, in the thymus were much higher in neonatal (3 day old) compared to adult mice (Figure A-4A). 	   28	  The total number of ETPs peaked in 10 day old mice to three times those of adults, 3 and 5 day olds (Figure 5C). Therefore, there appeared to be a wave of rapid expansion of both lung ILC2s and thymus ETPs between 5 days and 10 days after birth. To elucidate the pathways of neonatal ILC2 and ETP development, we then analyzed for LMPP+s, CLPs and CHILPs at different time points after birth. Neonatal BM contained small numbers of LMPP+s but barely detectable CLPs until 5 days after birth. In contrast, LMPP+s and CLPs were readily detectable in neonatal spleens as young as 1 day after birth. Unlike in adult BM, LMPP+s were much more abundant than CLPs in neonatal spleens (Figure 5D and Figure A-4B). CHILPs and ILC2Ps were also readily detected in the spleen of neonatal mice (Figure 5E). These observations suggested that LMPP+s, but not CLPs, in the spleen are the major progenitors for ETPs, ILC2s and CHILPs in neonatal (up to 5 days old) mice. The site of lymphopoiesis slowly shifts from the spleen to BM but CLPs remain a minor population until 10 days after birth. We also analyzed lymphoid progenitors in the peripheral blood of neonatal mice. Almost five times more LMPP+s than CLPs were detected in the neonatal blood (Figure A-4C). Interestingly, high frequencies of ILC2-like cells were also found in the neonatal blood. CHILPs or other lymphoid progenitors were not detected in neonatal lungs, and transplantation of different subsets of neonatal lung lymphocytes failed to generate donor-derived ILC2s or other lymphocytes in the recipients’ lungs (data not shown). Thus, in neonatal mice ILC2s seem to develop mainly in the spleen and migrate through the blood to the lung. 	   29	   Figure 5 LMPP+s are prominent lymphoid progenitors in neonatal BM and spleen, when ILC2 and T cell development is highly active.  (A) The BMs and spleens of 1 and 10 day old neonatal C57BL/6 mice were analyzed by flow cytometry.  Live (PI−) CD45+ cells were first gated for Lin− Thy1− (blue gate) and Lin−Thy1+ (brown gate) cells. Lin−Thy1− were then gated for cKithiSca-1+ (red gate) and cKitloSca-1lo (black gate). Flt3hiCD127−LMPP-s, Flt3hiCD127+LMPP+s, and CLPs were identified by the expression of Flt3 and CD127. ILC2Ps among Lin−Thy1+ cells were identified by ST2+CD127+cKitlo/−Sca1hi. Numbers in plots indicate mean percentages of gated cells among live CD45+ cells.  	   30	  (B) Absolute numbers of lung ILC2s were calculated and compared at different neonatal and adult time points.  (C) Absolute numbers of ETPs were calculated and compared at different neonatal and adult time points.  (D) Absolute numbers of LMPP+s and CLPs from spleen and BM were calculated and compared at different neonatal time points.  (E) Absolute numbers of CHILPs defined as CD45.2+CD45.1−Lin− Thy1.2+ CD25−CD127+ ST2−GFP+ (top) and ILC2Ps (bottom) from spleen and BM of Id2Gfp/+ mice were calculated and compared at different neonatal time points.  The data in A are representative of 4 independent analyses. The data in B – D are the mean of 4 independent analyses and error bars show the SEM. See also Figure A-4.  3.5 Neonatal LMPP+s are potent T cell and ILC2 progenitors  To examine the lymphoid differentiation capacities of neonatal LMPP+s and CLPs, they were purified from 3-6 days old mouse spleens, and transplanted into irradiated congenic B6.Ly5SJL recipients. In comparison with CLPs, LMPP+s showed robust thymic engraftment and generated significantly (<0.05) more T cells two weeks post-transplantation (Figure 6A). We then compared these neonatal spleen lymphoid progenitors and CHILPs for their ILC2 potential. In addition to producing T cells, neonatal LMPP+s efficiently gave rise to ILC2s in the lung (Figure 6B). Comparable numbers of ILC2s were generated from CHILPs. These results suggest that LMPP+s are the main source of T cells and ILC2s in the neonatal stage.   	   31	   Figure 6 Neonatal LMPP+s are more efficient than CLPs at generating T cells and ILC2s. (A) CLPs or LMPP+s were purified by cell sorting from pooled spleen cells from 3-6 day old C57BL/6 mice and 1,125 cells each, along with 105 BM cells from NSG mice, were intravenously injected into lethally irradiated congenic B6.Ly5SJL recipients. The thymuses of the recipient mice were analyzed 2 weeks post-transplantation. Donor-derived (CD45.1‒D45.2+) cells were gated (top) and analyzed for CD4 and CD8 expression. The bar graph shows the absolute numbers of donor-derived cells in the recipients’ thymus. Numbers in plots indicate the mean percentages of the cells in the gates. (B) The lungs of the recipient mice were analyzed 2 weeks post-transplantation. Live (eF780−) donor-derived (CD45.1‒CD45.2+) cells were gated for Lin‒ Thy1+ (top row) and ILC2s were further gated by CD127+ST2+ (bottom row). The bar graph shows the absolute numbers of donor-derived ILC2s (Mean ± S.E.M) in the lungs of transplanted mice.  * = p ≤ 0.05, ** = p ≤ 0.01 and ***= p ≤ 0.001 (two-tailed Student’s t-test). Data are representative of at least two independent experiments with 4 mice in each condition.    	   32	  Chapter 4 Discussion In the conventional scheme of lymphocyte differentiation, all lymphocytes develop from CLPs that develop from HSCs in a single linear pathway of HSC → LMPP → CLP → committed progenitors (pre-T, Pre-B, NKP, CHILP) → mature T, B, NK and ILCs. However, our current study suggests that many T cells and ILCs likely develop from LMPP+s bypassing the CLP stage, particularly in neonatal mice. LMPP+s significantly differ from the conventional LMPPs, termed LMPP-s in our study, in the capacities to differentiate into lymphocytes and myeloid cells as well as in their gene expression profiles. In transplantation assays, the LMPP+s are much more efficient than the LMPP-s in thymic engraftment and in differentiating into ILCs whereas LMPP+s are less efficient than LMPP-s in forming myeloid colonies in vitro. The genes associated with lymphoid development including Il7r and Rag2 are more highly expressed in LMPP+s than LMPP-s, however, the expression of myeloid-associated genes including Clec9a and Irak3 is lower in LMPP+s than LMPP-s. LMPP+s and CLPs are also very different from each other in their ability to differentiate into all lymphoid lineages examined with the exception of B cells. Our analyses of neonatal mice have also shown that CLPs are scarce compared to LMPP+s. In newborn mice (1 day old), the BM contains very few CD45+ cells whereas LMPP+s, CHILPs, ILC2Ps are readily detected in the spleen and a small number (less than 500) of ILC2s are found in the lung. Thus, in the first 3 days after birth, lymphopoiesis seems to take place in the spleen rather than BM. Although LMPP+s become detectable in the BM 3 days after birth, the numbers of CLPs remain much lower than LMPP+s in neonatal spleen and BM up to 10 days after birth when T cell development is very active and ETPs are found in large numbers in the thymus. ILC2 development is also very active for the first 10 days after birth as the number of ILC2s in the lung increases significantly. Transplantation experiments also confirmed that neonatal 	   33	  CLPs have very low capacity to differentiate into T cells and ILC2s. Therefore, most neonatal T cells and ILC2s likely develop from LMPP+s through a CLP-independent pathway.  LMPP+s and CLPs in our study likely overlap with All-Lymphoid Progenitors (ALPs) and B cell-biased Lymphoid Progenitors (BLPs), respectively, described as subsets of CLPs by the Weissman group [82]. Whereas CLPs are defined, as clonogenic common lymphoid progenitors with equal potential to give rise to all lymphocytes and no myeloid potential [32], [33], LMPP+s (and ALPs) have myeloid potentials as they differentiate into myeloid cells in transplantation assays as well as in vitro colony assays. Therefore, LMPP+s do not fit the classical definition of CLPs. Instead, LMPP+s are more similar to ELPs reported by the Kincade group [36]. ELPs were defined by the expression of RAG reporter gene. Although, ELPs and LMPP+s express the RAG reporter gene and have high capacities to differentiate into T cells in transplantation assays, ELPs were reported to be CD127−. As a small subset of LMPP-s also expresses the RAG reporter gene, the exact relationship between ELPs and LMPP+s is still unclear. LMPP+s might be the adult counterparts of the recently described fetal lympho-myeloid progenitors [31]. These fetal progenitors are Lin‒cKithiFlt3+CD127+Rag1+ cells and phenotypically resemble the LMPP+s in our study. They emerge at embryonic day (E) 9.5 in the yolk sac and E10.5 in the fetal liver.  Interestingly, these cells co-expressed the key lymphoid and myeloid genes and showed a robust contribution to both myeloid and lymphoid lineages. The differentiation capacities of these fetal progenitors have been tested by in vitro cultures, but not transplantation assays, and their relationship with postnatal LMPP+s is currently unclear. Although LMPP-s, LMPP+s and CLPs in adult mice are all capable of differentiating 	   34	  into the lymphoid lineages including T, B, NK cells and ILCs in transplantation assays, LMPP+s are far more efficient than the other progenitors in their capacity to repopulate the thymus. In vitro differentiation cultures on OP9-DL1 stroma, demonstrated that all three progenitors efficiently differentiated into immature T cells.  Thus, the highly efficient thymic engraftment of LMPP+s as compared to the other progenitors is likely due to their ability to migrate to the thymus. Consistent with this notion, our microarray analysis showed that the expression of the gene encoding the chemokine receptor CCR9, which is thought to be important for trafficking into the thymus [83], is significantly higher in LMPP+s than LMPP-s. Whereas LMPP-s are able to differentiate into LMPP+s in transplanted mice, they likely have to migrate into the BM first for the differentiation into LMPP+s. LMPP+s also seem to be the major T cell progenitors in steady state, not just in transplantation assays. They are more abundant than CLPs in neonatal mice when intrathymic T cell differentiation is most active. Furthermore, LMPP+s are readily detected in the peripheral blood of adult and neonatal mice, suggesting that LMPP+s emigrate from the adult BM and neonatal spleen, circulate and seed the thymus.  LMPP+s are also more efficient than CLPs in differentiating into innate lymphocytes including NK cells, ILC2s and ILC3s in transplantation assays. While the generation of more ILC2s in the lung [72] and ILC2Ps in the BM (Halim et al. 2012) from LMPPs than CLPs has previously been reported, our current transplantation assays have shown that LMPP+s are responsible for the efficient generation of the common ILC progenitors termed CHILPs and subsequent ILC2 and ILC3 development. The lineage tracking by RAG-Cre mediated RAG/Red expression also shows that the percentage of RAG/Red positive cells among BM ILC2Ps (~33%) is significantly (p<0.001) lower than those among CLPs (~65%) and slightly higher (p<0.05) than those among LMPP+s (~25%). Similarly, only about 20% of NK cells 	   35	  in adult mouse lung and spleen express RAG/Red. Therefore, most ILC2s and NK cells seem to develop from LMPP+s before they differentiate into CLPs, whereas CLPs and LMPP-s also seem to directly contribute to the ILC2 and the NK cell lineages, respectively, to lower degrees. Although CLPs are inefficient in differentiating into lung ILC2s in transplantation assays, normal adult mouse BM contains five times more CLPs than LMPP+s. Therefore, CLPs likely contribute to the ILC2 lineage to some extent in adult mice as suggested by the lineage tracking analysis by RAG/Red. However, our results with Flt3l−/− mice, which have greatly reduced CLPs but near normal numbers of lung ILC2s, indicate that the development of ILC2s can bypass the CLP stage. Furthermore, in the neonatal period when T cells and ILC2s actively develop, CLPs comprise a very minor population and they have very low capacities to differentiate into T cells and ILC2s in transplantation assays. In contrast, LMPP+s are prominent in neonatal mice and they have high capacities to differentiate into T cells and ILC2s. These results further support the existence of CLP-independent developmental pathways for these cells. Whereas our transplantation assays have shown the differentiation processes of LMPP- → LMPP+ → CLP, our data do not fit the conventional model of this linear single pathway of lymphocyte development. Instead, it is more likely that different lymphoid lineages start branching out at different stages as delineated in (Figure 7). The NK cell lineage seems to be the first to branch out from LMPP- and continues until the LMPP+ stage whereas the ILC lineage appears to start branching out mainly at the LMPP+ stage but continues until the CLP stage. Although all the progenitors have the capacities to differentiate into T cells and B cells, most B cells seem to develop from CLPs whereas most T cells likely develop from LMPP+s. CLPs retain the capacities to differentiate into all lymphoid lineages and seem to contribute to the T cell and the ILC2 lineages to some degree 	   36	  in adult mice. In contrast, CLPs are scarce in neonatal mice when T and ILC2 development is very active, and their contribution is likely insignificant. Our model of non-linear differentiation of lymphocytes from multiple progenitors with varying probabilities clarifies confusions in the field.  As the development of the immune system in the neonatal period is thought to impact immunity in later life, our results with neonatal lymphocyte development will also have important implications to our understanding of the development of the immune system.   Figure 7 Schematic representation of non-linear model of lymphocyte development. The histograms (top) depict the probability of LMPP-s, LMPP+s and CLPs differentiating into indicated cell lineages, whereas the bottom graph depicts the changes in the expression of CD127, RAG1 and cKit in these progenitors. LMPP-s have a low probability to differentiate into the myeloid lineage (green histogram) whereas the probability further declines as LMPP-s differentiate into LMPP+s and it is lost as they further differentiate into CLPs. The probability of differentiating into the NK cell lineage (black) is high in the LMPP- and LMPP+ stages and it declines to a very low level as they differentiate into CLPs. LMPP+s have high probabilities of differentiating into the ILC (blue) and T cell (red) lineages, whereas the probabilities of LMPP-s and CLPs differentiating into the ILC and T cell lineages are lower. In this model, LMPPs differentiate into multiple lymphoid lineages 	   37	  without differentiating into CLPs. CLPs also have the capacities to differentiate into the ILC, T and NK cell lineages, but with only low probabilities. Thus, individual lymphoid lineages can develop from multiple progenitors with varying probabilities.   	   38	  Chapter 5 General summary  5.1 General summary It is thought that all lymphocytes develop from a single population of committed lymphoid progenitors in the BM termed CLPs. CLPs derive from the upstream lymphoid progenitors termed LMPPs that have the capacities to differentiate into myeloid and multiple lymphoid lineages. Thus, the classical model of lymphocyte development implicates a single linear differentiation pathway HSC à LMPP à CLP à lineage committed progenitors (pre-T, pre-B etc.) à mature T, B and NK cells. However, CLPs are not efficient T cell progenitors and the identity of T cell progenitors in the BM has been debated.  In addition to T, B and NK cells, the lymphocyte population now includes ILCs, which are divided into three groups: ILC1, ILC2 and ILC3. Common progenitors for all ILCs, termed CHILPs or ILCPs, have also been identified. As the ILCPs can be generated from CLPs in vitro, the ILC lineages are thought to develop from CLPs. However, we have previously found that LMPPs are much more efficient than CLPs in the generation of BM ILC2 progenitors and mature lung ILC2s in transplantation assays (Halim et al. 2012). Our results are consistent with a report from the Bhandoola group. These results suggested that a CLP-independent pathway of ILC2 development might exist. To clarify the contradictory data, we now have carried out detailed analyses of lymphoid progenitors by transplantation assays and lineage tracking by RAG-1 expression. We have also analyzed lymphocyte development in neonatal mice.  The scientific questions being asked by our current study are:  § Do all lymphocytes develop from CLPs? § Is there a CLP-independent developmental pathway for innate lymphocytes? 	   39	  § From which lymphoid progenitors do neonatal ILC2s and T cells develop? The principal new findings are: ü The LMPP population can be divided into two subsets based on the expression of CD127 (IL-7Rα). The CD127‒ subset (termed LMPP‒) and the CD127+ subset (termed LMPP+) significantly differ in their capacities to generate T cells in transplantation assays as well as gene expression profiles. LMPP-s differentiate into LMPP+s and CLPs whereas LMPP+ differentiate into CLPs but not LMPP- in transplanted mouse BM. ü LMPP+s are much more efficient than CLPs in generating ILCs, CHILPs, NK and T cells in transplantation assays.  ü LMPP+s are detected in the peripheral blood of adult and neonatal mice whereas CLPs are very rare. ü Lineage tracking by RAG-1 reporter expression shows that most ILC2s and NK cells develop from the LMPP subsets. ü Flt3L-knockout mice have severely reduced CLPs, LMPP+s and ILC2 progenitors in the BM and yet near normal numbers of ILC2s in the lung. ü In the first 10 days after birth, CLPs are rare and, unlike prominent neonatal LMPP+s, incapable of differentiating into ILC2s and T cells while their development is highly active. 5.2 Significance  The work in this thesis challenges the conventional scheme of lymphocyte differentiation and the idea of “common lymphoid progenitors” as an obligatory stage for the development of all lymphocytes. We propose a model for the differentiation of lymphocytes within which different lymphoid lineages branch out from multiple progenitors with varying probabilities 	   40	  rather than all differentiating from CLPs. In this model LMPPs have very high probabilities to differentiate into NK, ILC and T lineages directly, without differentiating into CLPs. The data provided here significantly contribute to our better understanding of lymphopoiesis. BM transplantations are often preceded by lethal irradiation. Following BM transplantation, peripheral T cell pool, which is important in patients defense against infections, mainly recovers through thymus dependent maturation of the transplanted progenitor cells. Due to the radiosensitivity of thymic epithelial cells, irradiation can limit the chemokine signaling on thymic endothelium and slow down thymus settling of transplanted progenitors and T cell reconstitution. It is important to know which lymphoid progenitors are the most efficient progenitors in settling in and reconstituting the thymus in this condition. However, significant differences might also exist between mice and the human lymphopoiesis and further study is required to reveal the existence of such progenitors in human BM.   Technical limitations of my work include the requirement for transplantation of lymphoid progenitors into lethally irradiated hosts to investigate their potential. Irradiation can severely disrupt the steady state hematopoiesis, and only cells that are able to colonize a niche will be able to generate detectable progeny. Additionally considering the stress that transplanted cells tolerate during their isolation as well as engraftment, it is possible that the differentiation capacities of progenitors in transplantation assays may not mimic their differentiation in normal steady state lymphopoiesis. Therefore, we have extended our studies to include the lineage tracking mouse model of RAG-Cre mediated RAG/Red expression. The model provides a valuable index of the history of RAG-1 expression during lymphocyte development and relatedness of mature lymphoid cells to their upstream 	   41	  lymphoid progenitors. In this model, most ILC2s and NK cells seem to be more related to LMPP+s than CLPs by means of their RAG/Red expression.  Innate lymphocyte development in the neonatal period has not been extensively studied. My thesis study has shown that neonatal and adult mice significantly differ in lymphoid progenitors and the development of T cells and ILC2s is highly active in the neonatal period. As the development of immune system early in life is thought to impact adult immunity, this study has important implications to our understanding of the development of the immune system.  5.3 Future directions 5.3.1 Affymetrix microarray analysis of CLPs We have compared LMPP-s and LMPP+s by Affymetrix microarray analysis and realized that the expression of the lymphoid lineage-associated genes is generally higher in LMPP+s than LMPP-s. LMPP+s also expressed the myeloid cell lineage-associated genes albeit in lower levels than LMPP-s. We would like to carry out the same analysis for CLPs and compare the gene expression profile of these cells with the ones from LMPP+s. This comparison would allow us to investigate how exactly the expression of the lymphoid lineage associated genes are different between LMPP+s and CLPs. The differential expression of myeloid genes by these two populations may also explain the observed differences in their in vitro and in vivo myeloid potential.  5.3.2 Assessment of clonality  In order to track the clonality of cells within the LMPP+s we would like to do barcoding experiments. In cellular barcoding, integration of viral vectors with random sequence tags or barcodes will introduce a unique and heritable mark into the host cell genome. A sequencing-	   42	  based detection system can then be used to detect the clonal progeny of each cell (Gerrits et al., 2010). The barcoding experiments would enable us to assess the existence of different clones and heterogeneity within the LMPP+s. We will be able to investigate the existence of CILP as the ILC-restricted progenitor to both cytotoxic and cytokine producing ILCs among the LMPP+s. Also we will be able to investigate if the myeloid contribution of the LMPP+s is from the same clones that give rise to lymphocytes. Although the caveats associated with barcoding experiments such as in vitro manipulation of the progenitors as well as the required transplantation step in most of these experiments can compromise LMPP+s potentials in comparison with their steady state hematopoiesis.  5.3.3 Further analysis of the neonatal development of lymphocytes  Lymphocyte development is very active during neonatal period. We have carried out phenotypic analysis of the lymphoid progenitors in different hematopoietic tissues at this stage. LMPPs including both LMPP-s and LMPP+s were the predominant lymphoid progenitors during neonatal stage. We have compared the differentiation capacities of spleen LMPP+s with CLPs and realized that LMPP+s are much more efficient progenitors for ILC2s and T cells like their adult counterparts. We would like to extend our observations including LMPP-s in our comparison. We would also like to compare the differentiation potential of the same progenitors from neonatal spleen and BM to investigate if there is any difference regarding their lineage potential.  We would like to investigate the neonatal development of B cells. CLPs are the main B cell progenitors in adults. In the neonatal period CLPs are scarce and there are intrinsic differences between neonatal and adult B cells. I would like to investigate if different lymphoid progenitors would contribute to neonatal and adult stage B cell development.  	   43	  5.3.4 ILC2s similarities with T cells  ILC2s phenotypically resemble double-negative 3 (DN3) thymocytes that are committed to the T cell fate. DN1 and DN2 thymic progenitors can give rise to ILC2s in vitro. However, ILC2s can develop normally in nude mice, indicating that the thymus is not required for their development (Wong et al., 2012). 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Bhandoola, “CCR7 and CCR9 together recruit hematopoietic progenitors to the adult thymus.,” Blood, vol. 115, no. 10, pp. 1897–905, Mar. 2010.  	    	   52	  Appendices   Figure A-1 Transcription factors required for early lymphoid lineage development. Expression of the genes associated with lymphocyte developmental programs in LMPP+s was compared to those of LMPP-s by Affymetrix microarray analysis. The heat map shows the mean values from triplicate analyses.      Gata3Bcl11bEomesLMPP+LMPP-Sfpi1Ikzf1Tcf3Ikzf3Foxo1Tbx21 Id2Nfil311.4 3.8Pax5Ptcra	   53	  Figure A-2 BM LMPP+s are potent progenitors for T cells and ILCs. (A) The T cell differentiation potential of purified CLPs, LMPP-s, or LMPP+s was assessed by limiting dilution cultures on OP9-DL1. After 21 days in cultures, wells with growing cells were scored and the cells in every 6 wells were pooled and analyzed by flow cytometry for the expression of CD4 and CD8. The frequencies of T cell progenitors were 1/2 for CLPs, 1/3 for LMPP-s and 1/2 for LMPP+s. The differences between the slopes are not significant (P=0.5261). Numbers in plots indicate mean percentages of gated cells among live CD45+ cells.  (B) Phenotype of adult blood Lin−cKithiSca-1+.  The blood of 20 adult (6-8 weeks old) mice were pooled and red blood cells were lysed. Blood leukocytes and age matched BM cells were analyzed by flow cytometry. Live (PI−) CD45+Lin− were gated and cKithiSca1+ cells (red box) were analyzed for Flt3 and CD127 expression. The numbers in the plots show the percentages of the cells in the gates.   (C) Absolute numbers of B cells (TCRαβ−TCRγδ−NK1.1−CD19+B220+) derived from CLPs, LMPP-s and LMPP+s in the spleen of the transplanted mice one week post-transplantation were calculated.  (D) LMPP-s and LMPP+s were purified from adult BM and 1,000 cells per plate were cultured in Iscove’s MDM-based methylcellulose cultures for a monocyte/granulocyte colony forming assay for 14 days. Data are the mean of triplicate cultures in one of 2 independent experiments, both showing similar results.  	   54	   Figure A-3 Normal numbers of ILC2s were found in the lungs of Flt3l−/− mice. (A) Flow cytometry analysis of lymphoid progenitors in the BM of C57BL/6 and Flt3l−/− mice. Live (PI−) CD45+Lin− cells were first gated for Thy1− (black box) and Thy1+CD127+ (green box) cells. Thy1− cells were then gated for cKithiSca-1+ (red gate) and cKitloSca-1lo (blue gate), and cKithiSca-1+ cells were further gated for Flt3hiCD127− LMPPs (LMPP-s) and Flt3hiCD127+ (LMPP+s) (red boxes) while cKitloSca-1lo cells were gated for Flt3hiCD127+ (CLPs) (blue box). Thy1+CD127+ were gated for Flt3−ST2+cKitlo/−Sca1hi ILC2Ps (green gate). Numbers in plots indicate mean percentages of the cells in the indicated gates.   (B) Flow cytometry analysis of ILC2s in the C57BL/6 and Flt3l−/− mouse lung. Live (PI−) CD45+Lin− cells were gated for CD90+CD127+, ST2+ and cKitlo/−Sca1hi ILC2s. Numbers in plots indicate mean percentages of the parent gates.   (C) Absolute numbers of BM lymphoid progenitors (left) and lung ILC2s (right) from C57BL/6 and Flt3l−/− mice were calculated.  Mean ± S.E.M., * = p ≤ 0.05, ** = p ≤ 0.01 and ***= p ≤ 0.001 (two-tailed Student’s t-test). Data are representative of at least three independent experiments with 4 mice each condition.   	   55	   Figure A-4 LMPPs are the predominant neonatal lymphoid progenitors when ILC2 and T cell development is highly active.  (A) Flow cytometry analysis of ETPs in the thymus of 3 day old (left) and 45 day old (right) C57BL/6 mice. The plots were gated on live (PI−) CD45.2+Linlo cells and further gated for cKithiCD25− ETPs. Numbers in plots indicate mean percentages of gated cells among live CD45+ cells.    (B) The frequencies of BM LMPP+s and CLPs among CD45+ cells at different ages were calculated and compared. (C) Flow cytometry analysis of circulating lymphoid progenitors in the blood of neonatal mice. The blood from 16 neonatal (3 days old) mice was pooled and red cells were lysed.  Live (PI−) CD45+ cells were gated for Lin−CD90− (blue gate) and Lin−CD90+ (green gate) cells. Lin−CD90− were then gated for cKithiSca-1+ (red gate) and cKitloSca-1lo (black gate) while cKithiSca-1+ cells (red gate) were further gated for Flt3hiIL-7Rα− LMPPs (LMPP-s) and Flt3hiCD127+ (LMPP+s). CLPs among cKitloSca-1lo cells (black gate) were defined by Flt3hiCD127+.  ILC2Ps among Lin−CD90+ were identified by ST2+CD127+cKitlo/−Sca1hi. Numbers in plots indicate the percentages of the gated cells among CD45+ cells.    	   56	    Table A-1.Defining mouse ILCs  Lineage Surface Markers  Transcription factors Signature Cytokines  Cytotoxic ILCs NK cells Lin-CD122+ NK1.1+NKp46+Ly49+NKG2A+  NKG2D+ Eomes, E4BP4 (NFIL3) IFN-γ, TNF   Cytokine producing ILCs - ILC1 Lin-IL7Rα+/-Thy1+NK1.1+NKp46+  T-bet IFN-γ, TNF ILC2 Lin-IL7Rα+Thy1+CD25+ST2+/- Sca-1+IL-17RB+  RORα, GATA-3, TCF-1 IL-5, IL-13 ILC3 LTi cells Lin-IL7Rα+Thy1+NKp46-cKithiCCR6hi RORγt LTs, IL-17 IL-22 ILC3s LinIL7Rα+Thy1+ NKp46+cKitintCCR6lo/- RORγt, AHR IL-22 ILC, innate lymphoid cell; Lin, lineage markers (CD3, CD4, CD8, CD19, B220, GR1, CD11b, CD11c and TER119); -IL7Rα, IL7 receptor subunit-α; NKp46, NK cell p46-related protein; NKG2A and Ly49 receptors, MHC-1 specific NK receptors; NKG2D, NK cell stimulatory receptor; IL, interleukin; CD25, IL2 receptor subunit-α; ST2, IL33 receptor; IL-17RB; Il17 receptor B; CCR6, c-c chemokine receptor type 6; IFN, interferon; TNF, tumor necrosis factor;  LT, lymphotoxin. 	   57	  Table A-2.Genes encoding the listed transcription factors Genes Transcription factors Id2 Inhibitor of DNA-binding 2 (ID2) Zbtb16 Promyelocytic leukemia zinc finger (PLZF) Nfil3 Nuclear factor interleukin-3 (IL-3) regulated (NFIL3) also known as E4 promoter-binding protein 4 (E4BP4) Rora Retinoic-acid-receptor-related orphan receptor-α  (RORα) Gata3 GATA binding protein-3 (GATA-3) Tcf7 T cell factor 1 (TCF-1) Gfi1 Growth factor independent 1 (Gfi1) Rorct Retinoic-acid-receptor-related orphan receptor-γt (RORγt) Ikzf1 IKAROS family zinc finger 1 (Ikaros) Pax5 Paired box 5 Ptcra Pre T-cell antigen receptor alpha  	  

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