- Email: [email protected]
T-sing progenitors to commit
Opinion
TRENDS in Immunology
Vol.27 No.3 March 2006
T-sing progenitors to commit Floor Weerkamp, Karin Pike-Overzet and Frank J.T. Staal Department of Immunology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
T-cell development in the thymus is a complex and highly regulated process. During the process of differentiation from multipotent progenitor cells to mature T cells, proliferation, restriction of lineage potential, TCR gene rearrangements and selection events occur, all accompanied by changes in gene expression. A comprehensive understanding of thymocyte differentiation remains to be established. Two related, key issues have received much attention recently: the nature of the thymus seeding cell and the regulation of T-cell lineage commitment. Here we review the perspectives of different researchers working both on murine and human T-cell development and argue that a true T-cell commitment factor might not be required because of the unique properties of the thymus.
T-cell commitment Leukocytes develop from pluripotent hematopoietic stem cells (HSCs) through a complex differentiation pathway involving several phenotypically distinct subpopulations [1–5] (Box 1). During lineage commitment and further differentiation into a certain lineage, unique regulatory programs are established that direct cell-type-specific patterns of gene expression. Thus, T-cell commitment minimally includes the following sequential processes: loss of alternative lineage gene expression, loss of alternative lineage potential, expression of a T-cell-specific gene program and initiation of T-cell-specific gene rearrangements [6–9]. Understanding T-cell commitment starts with the question of at what level the cells that seed the postnatal thymus are committed to the T-cell lineage. The difficulty in answering this question is that cells will immediately be influenced by the thymic microenvironment as soon as they enter the thymus. Progenitors that have T-cell developmental capacity but differ from HSCs have been identified in bone marrow and blood but it is unknown whether these cells seed the thymus physiologically. During the past decade, several groups have attempted to identify the nature of the first cells that enter the thymic anlage during fetal life [10–14]. Harman et al. [15] have elegantly addressed this question in the fetal thymus by isolating the cells that are present in the mesenchyme surrounding the fetal thymus but have not yet entered. These cells were shown to be committed T-cell precursors, which is in contrast to the situation in adult thymi where immature cells with broader lineage potential are found (see below). Indeed, the cells that initially populate the Corresponding author: Staal, F.J.T. ([email protected]). Available online 10 February 2006
fetal thymus and contribute to the establishment of the thymic microenvironment might be different from the few postnatal thymus seeding cells that originate from the bone marrow and predominantly serve to keep the thymocyte counts at a steady level. In the mouse, the identification of the thymus seeding cell has recently been approached by sorting minute double negative (DN) populations (Box 1) from the thymus and by studying their lineage potential and other characteristics, hoping to find the cells that have contacted the thymic stroma for a minimally short period [16–19]. These recent papers on the characterization of thymus seeding progenitors (TSPs) will be described in more detail below. A difficulty the field is facing is that various laboratories use different definitions of lymphoid progenitors and stem cell populations isolated from bone marrow, blood or thymus. How these subpopulations relate to each other is not always clear (Table 1), particularly for transgenic reporter mice [20,21]. It also is conceivable that the thymus is seeded by various different types of progenitors with different lineage potential and different quantitative capacity to generate T cells and commit to the T-cell lineage [22]. Progenitor cells in bone marrow In most strains of mice, HSCs can be readily identified as lin-negative, Sca-1C and c-kitC, a phenotype generally referred to as LSK cells [23,24]. The LSK cells can be subdivided on the basis of the expression of Flt3: Flt3K is the true HSC with abundant self-renewal activity and the Flt3C LSK cells are non-renewing multipotent progenitor cells that might have lost erythro-megakaryocytic potential (LMPP) [23,24]. A similar division can be made using the marker CD27 [25]. Interestingly, cells with an LSK phenotype can also be found as a minute population in the thymus and constitute a subpopulation that form so-called early T-cell progenitor cells (ETP, see below; reviewed in [26]). The identification of the lymphoid-restricted common lymphoid progenitor (CLP) in bone marrow by Kondo et al. [27] supported the widely held view that a common progenitor for B and T cells must exist, and that this cell seeds the thymus. CLPs were originally identified as LinK, IL-7RC, c-Kitlow and Sca-1low (Table 1), and indeed are highly responsive to interleukin-7 (IL-7). CLPs have also been identified using an alternative protocol in which c-Kit is replaced with AA4.1, a mAb that recognizes a protein homologous to the complement C1q receptor [28]. However, CLPs have never been shown to seed the thymus in a physiological setting (but do so when injected in the thymus or in fetal thymic organ cultures). Some
www.sciencedirect.com 1471-4906/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2006.01.006
Opinion
126
TRENDS in Immunology
Vol.27 No.3 March 2006
Box 1. T-cell development in the thymus: of mice and men In contrast to all other hematopoietic lineages, development of T cells from pluripotent hematopoietic stem cells takes place in the specialized microenvironment of the thymus. Several developmental stages can be distinguishes using cell surface markers. Primarily, thymocytes are subdivided into double negative (DN), double positive (DP) and single positive (SP) populations, referring to the expression of the co-receptors CD4 and CD8 (Figure I). The most immature thymocytes lack expression of both CD4 and CD8 and, therefore, are called double negative. Immature single positive cells arise when thymocytes express a co-receptor in the absence of high levels of CD3, as do mature SP cells. In humans, immature SP cells express CD4, whereas in most strains of mice they express CD8. Subsequently, both CD4 and CD8 are expressed and therefore these cells are referred to as double positive. The DP stage represents w85% of all thymocytes. After undergoing positive and negative selection, thymocytes that express a functional T-cell receptor (TCR) commit to either the CD4 or CD8 single positive lineage.
In both species, T-cell commitment towards the ab TCRC lineage takes place during the DN stages of development. Additionally, gd T cells also split off at the DN stages. In mouse and humans, additional but different surface markers are used to subdivide the DN stage further. For mouse, the markers CD25 and CD44 are used to divide the DN stage: CD44 C CD25 K cells are referred to as DN1 cells, CD44CCD25C cells are referred to as DN2, DN3 cells express CD25 but no CD44, and DN4 cells express neither CD25 nor CD44. The most immature human thymocyte population is characterized by the expression of CD34, but lacks CD1a and CD38 expression, and resembles the murine DN1 population. The next stage of differentiation is marked by the expression of both CD34 and CD38 and resembles the murine DN2 stage. The most mature human DN stage that can be discerned is made up of cells expressing CD34 and CD38 as well as CD1a. Important checkpoints and developmental decisions are indicated in Figure I.
CD44+ CD25–
CD44+ CD25+
CD44– CD25+
CD8+ CD3–
CD4+ CD8+ CD3–
CD4+ CD8+ CD3+
CD4+ or CD8+ CD3+
CD34– CD38– CD1a+
CD34– CD38+ CD1a+
CD34+ CD38+ CD1a+
CD4+ CD3–
CD4+ CD8+ CD3–
CD4+ CD8+ CD3+
CD4+ or CD8+ CD3+
αβ commitment
Positive and CD4 or CD8 negative selection commitment
β selection
Stem cell like UCB CD34+ Lin– Blood
DN1 CD117–
DN1 CD117+ DN2
SP CD8+ DN3
ISP/DN4
DP CD3–
DP CD3+ SP CD4+
Thymus
TRENDS in Immunology
Figure I. Comparison of the various stages of mouse and human T-cell development. The different phenotypic definitions are shown, as well as the main checkpoints during T-cell development. Human data are based on genome-wide microarray analyses and recently published TCR rearrangement studies [46]. Stages indicated mainly refer to human subpopulations, with names of their putative murine counterparts.
laboratories have not been able to find these cells circulating in blood (reviewed extensively in [22,26]), a prerequisite for the thymus seeding cells, as they have to migrate from bone marrow to thymus. Various investigators in the field therefore regard CLPs as efficient and physiological progenitors for B but not for T lymphocytes [28,29]. These ideas are in line with seminal work done by Katsura and co-workers [30–32] who were the first to express doubts that CLPs are progenitors for T cells, albeit in fetal mice. In addition to CLPs, the bone marrow
contains other progenitors that will efficiently make T cells when put into contact with the thymus microenvironment. Kincade and colleagues [21] have identified a population of cells they termed early lymphoid progenitors (ELPs) on the basis of the expression of GFP under control of the RAG1 promoter. As the RAG genes are exclusively required for recombination of the immunoglobulin and T-cell receptor (TCR) loci, this reporter represents a lymphoid-specific marker. Unexpectedly, GFP expression was detected early on in the bone marrow. The expression
Table 1. Cell types that might seed the thymus – overview of terminology used to describe various progenitor and stem cell populations Common abbreviation HSC
Full name
Phenotype mouse
Phenotype human
Tissue
Refs
Hematopoietic stem cell
CD34CCD38K
[23–25]
ETP ELP
Long-term multi potent progenitor Early T-cell progenitor Early lymphoid progenitor
Bone marrow, cord blood (human) Bone marrow
[23,24]
LMPP
Lin- Sca-1hi c-kithi (Z LSK) Flt3K LSK Flt3C, CD27C
CD34CCD38K
Thymus Bone marrow
[17–19] [21]
CLP
Common lymphoid progenitor
CD34CCD10C
Bone marrow
[27,28]
www.sciencedirect.com
LSK, CD44C, IL7RaK RAG-1C, LSK,Flt3C, IL7RaK Lin-Sca1lo, c-kitlo, IL7RaC, AA4C
Not identified
Opinion
TRENDS in Immunology
of RAG by ELPs indicates that lymphoid specification might initiate earlier than previously thought. Expression of RAG is currently the earliest marker for hematopoietic progenitors undergoing lymphoid specification, because it precedes the expression of the IL-7 receptor, the marker used to identify CLPs. Why RAG would be expressed early on, before rearrangement initiates, is currently unclear. Upstream of both ELPs and CLPs are the LSK cells, containing the true HSCs. Because HSCs in mice and humans are known to circulate in the blood [33,34], and the thymus contains a small subpopulation of cells with an LSK phenotype, it is conceivable that HSCs seed the thymus. Most investigators do not believe this to be the case, as isolated cells from the thymus with the LSK phenotype (also known as ETPs) do not support full hematopoietic reconstitution in irradiated recipient mice [16,17]. However, it is possible that upon entry into the thymus cells rapidly lose their self-renewal capacity. Support from this notion stems from recent work using the human thymi [35], where T and B lymphoid, myeloid, erythroid and megakaryocytic potential was detected in early thymocytes. Progenitors in the thymus More than ten years ago, Shortman and colleagues [36] identified a subset of DN1 cells expressing c-Kit and low levels of CD4, hence their name, CD4lo cells. These cells have progenitor activity for T, B and NK cells, dendritic cells (DCs) and low levels of myeloid differentiation. More recent work from various laboratories has focused on ETPs as an alternative to CLPs for colonizing the thymus [16,17,26,28]. The thymus seeding cell is generally thought to lie within this ETP population. Classic ETPs are defined as being DN1, LinK and c-Kithi and are supposedly highly heterogeneous themselves. CD4 is usually excluded from the lineage cocktail for these cells, so as not to exclude the CD4lo cells, as ETP are DN1 cells at the transcriptional level but might express surface CD4 protein that is passively acquired. Additional markers, varying per research group, were included to subdivide ETPs and other DN populations into several fractions, which appeared to have differential T- and B-cell developmental capacities (which also depended on the in vivo and in vitro tests used to delineate lineage potential) [17–19]. These different developmental possibilities for the most immature thymocytes are controversial, with respect to B-cell lineage potential in the thymus. Although we and others found low but detectable B-cell differentiation in the adult thymus [16,17,37], others have found that B-cell potential is only marginal [19,38]. Nevertheless, all differentiation stages of B-cell development can be found in the thymus and mature B cells are present. The question remains whether these different subpopulations all arise from the same thymus seeding cell or whether diverse bone marrow precursors (some of which might not have T-cell potential) enter the thymus. In human studies, not all different lineages were definitively shown to have developed from a single precursor in the thymus [35]. Nevertheless, the full range of hematopoietic lineage potentials in the human DN1/2 (CD34CCD1aK) www.sciencedirect.com
Vol.27 No.3 March 2006
127
population, and that precursors for B and NK cells are found in the human thymus [5], suggested that the thymus is seeded by an HSC, or a mixture of progenitors with the collective potential to develop into all blood cell lineages. In the mouse, old but elegant studies also have indicated myeloid and low erythroid potential using c-kitC thymocytes in colony assays. [39]. Recent studies in the murine thymus, using CCR9–GFP as marker to track individual cells, have shown that clonal precursor cells with T, B and myeloid potential exist in the thymus [16]. These investigators have used a clever strategy of mixing stromal cell lines that permit B and myeloid development with those that induce T-cell development to generate a system that allows T, B and myeloid development in vitro [16]. Interestingly, granulocyte development was also observed, indicating that a precursor cell with broad lineage potential can be isolated from the thymus. It is clear that multilineage potential is present in the thymus and that full T-cell commitment occurs after entry into the thymus (Figure 1). To what extent the thymus seeding cells resemble HSCs or have a more limited lineage potential is not entirely clear. Signals inducing T-cell commitment If a quintessential T-cell commitment factor does exist, Notch would be the most likely candidate. Notch signaling, as measured by expression of the Hes1 target gene, is absent from bone marrow LSK cells, but present in the earliest thymocytes that can be identified, both in mice [17,37] and humans (F. Weerkamp, PhD Thesis, Erasmus University, Rotterdam, 2005). Loss of Notch1 function in lymphoid progenitors results in B lymphopoiesis in the thymus, at the expense of T-cell development [40], whereas overexpression of a constitutively active form of Notch induces ectopic T-cell development in the
Thymus HSC ?
TSC
? CLP ?
? ?
T/M T
Notch signaling
? T NK and DC Myeloid B E/M
Lineage potential
TRENDS in Immunology
Figure 1. Candidate populations that seed the thymus. Several types of thymus seeding cells (TSCs) have been proposed, ranging from HSCs or non-renewing multipotent progenitors to committed T-cell precursors (abbreviations: CLPs, common lymphoid progenitor; E/M, erythroid/megakaryocytic; T/M, T/myeloid bipotent progenitor; T, T-cell-committed progenitor). Cells that enter the thymus receive Delta-induced Notch signals and start developing into T cells. As thymocytes develop, lineage potential (either of individual cells or at the population level) declines. In our view,it is likely that a multipotent cell with broad lineage potential seeds the thymus, with rapid loss of non-T-cell potential upon thymic entry. However, it is possible that lineage potential is already reduced before cells arrive in the thymus.
128
Opinion
TRENDS in Immunology
bone marrow and inhibits B-cell development that would normally take place there [41,42]. The Notch signal transduction pathway regulates cell fate determination during developmental processes [43]. The Notch family of transmembrane receptor consists of four members, named Notch1 to Notch4, interacting with membrane-bound ligands on neighboring cells (Delta1, Delta3 and Delta4, and Jagged1 and Jagged2). The Delta1 and Delta4 ligands are particularly highly expressed on thymic epithelial cells [44]. The downstream mechanisms by which a Notch signal is translated into a T-cell-specific program are still largely unclear. The best known Notch target genes encode hairyenhancer of split (Hes)1 and Hes5 and Hes-related repressor protein (Herp), all basic-helix–loop–helix (bHLH) proteins that function as transcriptional repressors. Indeed, overexpression of Hes1 and Hes5 in bone marrow partly inhibits B-cell development [45]. However, mice deficient in Hes-1 show a proliferation defect of early DN thymocytes, but they are still present [46]. Furthermore, overexpression of Hes1 does not induce a T-cell fate in human hematopoietic progenitors, whereas overexpression of IC-Notch does [47]. Hes1 can therefore not be the sole target of Notch signaling in the thymus. In microarray studies aimed at identification of Notch target genes (F. Weerkamp, PhD Thesis, Erasmus University, Rotterdam, 2005), no clues were found that Notch signaling can directly induce a T-cell fate. This is in line with experiments by Taghon et al. [48] in which murine fetal liver progenitors were cultured on OP9-DL. This study showed that the T-cell-specific transcription factors GATA3, TCF1 and pre-T-cell receptor a gene (PTCRA) began to be expressed after three days of culture on OP9DL, whereas high HES1 transcription was detected after only one day. Therefore, either Notch signaling induces T-cell genes in already more differentiated thymocytes, or Notch signaling stimulates the expression of other transcription factors, which then in turn activate or repress lineage differentiation genes. Alternatively, the first function of Notch signaling in the thymus might be to induce stable metabolism and survival, as has been shown for DN3 thymocytes [49], whereas subsequent Notch signals induce differentiation. Two recent papers [17,37] have dealt with the role of Notch signaling in the most immature thymocytes. The paper from Cynthia Guidos’ laboratory [37] builds on earlier work using transgenic mice that express lunatic fringe, a Notch modifier that alters glycosylation thereby inhibiting Notch1 activation [50]. Using this and other strategies to change the levels of Notch activation it was shown that Notch1 is required for efficient production of ETPs and suppression of B cell development. Additionally, this report confirmed that Notch signals are continuously needed to promote survival or proliferation throughout the DN stages. The other paper from Warren Pear’s and Avinash Bhandoola’s laboratories [17] subdivided the ETPs on the basis of Flt3 expression. Flt3C ETPs were shown to have low potential to develop into B cells and Notch signals were required for development of ETPs, as shown by genetically inhibiting Notch signals via retrovirus mediated transduction of a potent dominant www.sciencedirect.com
Vol.27 No.3 March 2006
negative essential Notch co-factor. Although LSK cells in bone marrow and blood were normally present in mice reconstituted with HSCs expressing this Notch inhibitor, the numbers of ETPs and more mature DN2 thymocytes were low. The authors interpret these findings as follows: Notch signaling occurs after the thymus has been colonized by multipotent progenitors, before the generation of ETPs. Alternatively, it is possible that a subset of LSK cells in the bone marrow undergoes Notch signaling there and that these cells function as thymic seeding progenitors (TSPs). Together, these papers demonstrate that Notch signaling occurs quickly after entry of TSPs in the thymus at the levels of ETPs. It is obvious that TSPs need to undergo massive proliferation (10000- to 50000-fold), as the number of these cells is extremely low and they need to generate 100–200 million thymocytes [28]. It is estimated that only one cell a day enters the adult murine thymus [51] and indeed ETPs comprise less than 0.01% of all thymocytes; the corresponding human CD34CCD38K thymocytes are at least as rare (0.005% or less [5,52]). Before or concomitant to T-cell commitment the most immature thymocytes therefore receive proliferation-inducing signals by cytokines (SCF and later on IL-7) and also by Wnt proteins [53,54]. Wnt proteins are generally not considered to be T-cell commitment factors. The strong blocks seen in Tcf1(VII)K/K and Tcf1K/K/Lef1K/K mice demonstrated that Wnt signals are important for T-cell development, because of a failure in expansion of early subpopulations rather than a differentiation defect [55,56]. A predominantly proliferative function for Wnt signaling was confirmed by identification of the target genes of Wnt signaling in thymocytes [57]; these are mainly involved in proliferation. No T-cell-specific genes were found to be upregulated [57]. Inhibiting Wnt signaling in the most immature thymocytes therefore causes a block in differentiation by interfering with an essential proliferation step [58]. Various other transcription factors, most notably GATA3, E-box factors (HEB, E2A) and Ikaros family factors, have been shown to have more or less T-cellspecific expression patterns and are functionally required during T-cell development (for reviews see [6,59]). However, none of these can be regarded as a T-cell commitment factor because ectopic expression of these factors into HSCs neither induces T-cell commitment nor direct inhibition of other lineages. A comparison with B cells In contrast to the emerging picture of a highly complex regulation of T-cell development, the molecular regulation of B cell commitment appears to be relatively straight forward [60]. During B-cell development, lineage specification is induced by the transcription factors E2A and EBF, which also induce Pax5 expression. Pax5 is necessary for the induction of crucial B-cell genes and represses alternative lineage fates [61,62]. Interestingly, Pax5 inhibits T-cell development by active repression of Notch1 expression [63]. Thus a simple hierarchy from E2A via EBF to Pax5 seems to control early B-cell development.
Opinion
TRENDS in Immunology
Vol.27 No.3 March 2006
129
Thymus
Bone marrow Mono/mϕ
DC
Potential Developmental path taken
HSC NK
Mono/mϕ
DC
Developmental path actively inhibited
B
Mono/mϕ
DC
T
No (G)M-CSF present
(G)M-CSF present CLP
DN1
NK
B
NK
B DL Wnt Cytokines Adhesion molecules
No DL present T
T TRENDS in Immunology
Figure 2. Hypothetical model of B-cell and T-cell commitment. As multiple lineages develop in the bone marrow (left), non-B-cell lineages have to be actively suppressed in common lymphoid progenitors (CLPs). T cells will never develop in the bone marrow because of the absence of Delta, and therefore do not need active suppression. Conversely in the thymus (right), T-cell oriented microenvironmental signaling ensures that only T cells develop. No active repression of alternative lineages is necessary. Abbreviations: mono/4, monocyte/macrophage.
For T-cell development, a master regulator comparable to Pax5 has not yet been identified. Notch signaling appears to be able to repress a B-cell fate, as overexpression of IC-Notch induces T-cell development in the bone marrow, at the expense of B cells [32]. However, direct inhibition of Pax5 by Notch signaling has not been described. Furthermore, Notch signaling is not specific for the induction of a T-cell fate: it is also involved in selfrenewal of HSCs [64], in cell fate choice in a myeloid progenitor cell line [65], and in the choice between erythroid and megakaryocytic differentiation [66]. Also, although B cells develop more readily in the thymus in absence of Notch signals in the thymus [40], the number of B cells is still small and at least ten times lower than thymocyte numbers, indicating that indirect mechanisms such as proliferative factors preferentially acting on T cells strongly favor the T-cell lineage, rather than merely repressing B cell development. Thus, although Notch signals are required for the development of T lineage cells, Notch signaling cannot be the only signal responsible for setting up the full T-cell-specific gene program. Moreover, current experimental evidence does not indicate that Notch signaling suffices to directly suppress other lineages (e.g. Pax5 expression for B cells), thereby not strictly complying with the definition of a commitment factor. No need for a T-cell commitment factor? It is a tantalizing thought that there is no need for active suppression of alternative lineages in the thymus, as the www.sciencedirect.com
thymic microenvironment only supports T-cell development (Figure 2). This is exemplified by the rapid increase of T-cell precursors and the almost complete absence of alternative lineages when hematopoietic progenitors are cultured on OP9-DL [67,68]. Many lineages develop in the bone marrow: B cells, NK cells, various myeloid lineages, erythrocytes and platelets. The bone marrow stroma therefore provides growth and survival factors for all these lineages. The thymus provides an excellent environment for the development of early thymocytes: a high density of the Notch ligand Delta, an abundance of Wnt proteins, presence of cytokines such as IL-7, SCF and Flt3L, and appropriate adhesion molecules. It is our view that development of any other lineage than T cells simply does not stand a chance (Figure 2) and does not require active transcriptional repression. But does the thymic microenvironment actively induce T-cell commitment? Binding of the Notch ligand Delta is important for T-cell development but might support other functions, such as proliferation or survival, rather than T-cell commitment. Studies in human progenitor cells suggested that Notch signaling also inhibits a myeloid cell fate [42], but the molecular mechanism is unknown. Perhaps stringent inhibition of myeloid development on the molecular level is not so strictly necessary during thymocyte differentiation (Figure 2). Development of myeloid lineages is strongly dependent on myeloid cytokines such as macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating
130
Opinion
TRENDS in Immunology
factor (GM-CSF) [69]. These cytokines are expressed at low levels in the thymus, and myeloid development is thereby automatically not favored. In contrast to the earliest thymocytes, CLPs in bone marrow completely lack myeloid potential [27], although some myeloid developmental capacity was detected in a related population [70]. Because of the abundant presence of myeloid growth factors in the bone marrow, myeloid fates have to be actively repressed in CLP and B cell precursors (Figure 2). Similar reasoning explains why T-cell potential is still present in CLPs. As T cells will normally not develop in the bone marrow because of the absence or low expression of Delta ligands (they will develop as soon as Delta1 or Delta4 are ectopically expressed in bone marrow [71]), T-cell development does not have to be actively repressed. This model makes several testable predictions. For instance, ectopic expression of G-CSF or GM-CSF in thymic stroma should lead to a dramatic increase in the number of myeloid cells in the thymus. This could, for instance, be tested with virally transduced stromal cells in reaggregate cultures. Similar experiments could be done with erythropoietin (EPO) for erythrocytes. Also, murine ETPs, stringently purified while not using Mac1 or Ter119 but including all other markers (positive and negative), are expected to show erythroid and myeloid development. It might also be that transplantation of large numbers of such ETPs leads to low, but detectable self-renewal in irradiated recipients.
Future challenges The question about the nature of early T-cell progenitors has still not been answered satisfactorily. The thymus seeding cell and its immediate progeny must be identified by detailed immunophenotyping and subsequently characterized in terms of lineage potential. Difficult but elegant studies along these lines have already been undertaken and need to be expanded. Next, the different features of the unique thymic microenvironment [72] should be further characterized. For instance, studies have been initiated to establish the migration pattern of thymocytes through the microenvironmental niches and how this correlates with their differentiation [73]. Future research should also focus less on individual factors and pathways and more on the integration of the different signals. How do they all cooperate to set up a T-cell program? Studies on the establishment of lineagespecific gene programs will increasingly concentrate on novel regulatory mechanisms such as microRNAs, epigenetic phenomena and chromatin accessibility regulation by acetylation and methylation of histone proteins. Such studies should lead to a better understanding of T-cell development and help translate such knowledge to medical problems: development of better diagnostics and therapies for diseases involving the early stages of T-cell development (T-ALL, SCID), and improvement of T-cell reconstitution after bone marrow transplantation and acquired immune deficiencies, such as those underlying HIV infection. www.sciencedirect.com
Vol.27 No.3 March 2006
Acknowledgements We thank C.J.M. Loomans and A.W. Langerak and J.J.M. van Dongen for critically reading the manuscript. T. van Os and M. Comans-Bitter are acknowledged for expert help with illustrations. F.J.T.S. is supported in part by funds from the 5th and 6th EU Framework program [Contract Nrs QLK3-CT-2001-0427 (INHERINET) and LSHB-CT-2004-005242 (CONSERT)], as well as by the Translational Gene Therapy Research Programme of NOW-ZonMw – the Netherlands Organization for Health Research and Development, ICES-KIS ‘Virgo’ program and the Dutch Heart Foundation.
References 1 Scollay, R. and Shortman, K. (1985) Identification of early stages of T lymphocyte development in the thymus cortex and medulla. J. Immunol. 134, 3632–3642 2 Scollay, R. (1991) T-cell subset relationships in thymocyte development. Curr. Opin. Immunol. 3, 204–209 3 Vanhecke, D. et al. (1997) Human thymocytes become lineage committed at an early postselection CD69C stage, before the onset of functional maturation. J. Immunol. 159, 5973–5983 4 Blom, B. et al. (1999) TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation. Blood 93, 3033–3043 5 Weerkamp, F. et al. (2005) Age-related changes in the cellular composition of the thymus in children. J. Allergy Clin. Immunol. 115, 834–840 6 Rothenberg, E.V. and Dionne, C.J. (2002) Lineage plasticity and commitment in T-cell development. Immunol. Rev. 187, 96–115 7 Radtke, F. et al. (2004) Notch regulation of lymphocyte development and function. Nat. Immunol. 5, 247–253 8 Collins, A. and Littman, D.R. (2005) Selection and lineage specification in the thymus: commitment 4-stalled. Immunity 23, 4–5 9 Garcia-Ojeda, M.E. et al. (2005) Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus. J. Immunol. 175, 4363–4373 10 Rodewald, H.R. et al. (1994) Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization. EMBO J. 13, 4229–4240 11 Peault, B. (1996) Hematopoietic stem cell emergence in embryonic life: developmental hematology revisited. J. Hematother. 5, 369–378 12 Carlyle, J.R. and Zuniga-Pflucker, J.C. (1998) Requirement for the thymus in alphabeta T lymphocyte lineage commitment. Immunity 9, 187–197 13 Douagi, I. et al. (2000) Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation. Eur. J. Immunol. 30, 2201–2210 14 Masuda, K. et al. (2005) Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors. EMBO J. 24, 4052–4060 15 Harman, B.C. et al. (2005) B lineage choice occurs prior to intrathymic notch signalling. Blood 16 Benz, C. and Bleul, C.C. (2005) A multipotent precursor in the thymus maps to the branching point of the T versus B lineage decision. J. Exp. Med. 202, 21–31 17 Sambandam, A. et al. (2005) Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat. Immunol. 6, 663–670 18 Porritt, H.E. et al. (2004) Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages. Immunity 20, 735–745 19 Balciunaite, G. et al. (2005) The earliest subpopulation of mouse thymocytes contains potent T, significant macrophage, and natural killer cell but no B-lymphocyte potential. Blood 105, 1930–1936 20 Gounari, F. et al. (2002) Tracing lymphopoiesis with the aid of a pTalpha-controlled reporter gene. Nat. Immunol. 3, 489–496 21 Igarashi, H. et al. (2002) Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17, 117–130 22 Petrie, H.T. and Kincade, P.W. (2005) Many roads, one destination for T cell progenitors. J. Exp. Med. 202, 11–13 23 Adolfsson, J. et al. (2001) Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(C)c-kit(C) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15, 659–669
Opinion
TRENDS in Immunology
24 Adolfsson, J. et al. (2005) Identification of Flt3C lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295–306 25 Wiesmann, A. et al. (2000) Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity 12, 193–199 26 Bhandoola, A. et al. (2003) Early T lineage progenitors: new insights, but old questions remain. J. Immunol. 171, 5653–5658 27 Kondo, M. et al. (1997) Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 28 Allman, D. et al. (2003) Thymopoiesis independent of common lymphoid progenitors. Nat. Immunol. 4, 168–174 29 Borghesi, L. et al. (2004) B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors. J. Exp. Med. 199, 491–502 30 Kawamoto, H. et al. (1999) Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver. J. Immunol. 162, 2725–2731 31 Ohmura, K. et al. (1999) Emergence of T, B, and myeloid lineagecommitted as well as multipotent hemopoietic progenitors in the aorta-gonad-mesonephros region of day 10 fetuses of the mouse. J. Immunol. 163, 4788–4795 32 Ikawa, T. et al. (1999) Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190, 1617–1626 33 Udomsakdi, C. et al. (1992) Characterization of primitive hematopoietic cells in normal human peripheral blood. Blood 80, 2513–2521 34 Wright, D.E. et al. (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294, 1933–1936 35 Weerkamp, F. et al. (2005) The human thymus contains multipotent progenitors with T/B-lymphoid, myeloid and erythroid lineage potential. Blood DOI 10.1182/blood-2005-08-3412 (http://www.bloodjournal.org) 36 Wu, L. et al. (1991) CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Nature 349, 71–74 37 Tan, J.B. et al. (2005) Requirement for Notch1 signals at sequential early stages of intrathymic T cell development. Nat. Immunol. 6, 671–679 38 Lu, M. et al. (2005) The earliest thymic progenitors in adults are restricted to T, NK, and dendritic cell lineage and have a potential to form more diverse TCRbeta chains than fetal progenitors. J. Immunol. 175, 5848–5856 39 Matsuzaki, Y. et al. (1993) Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation. J. Exp. Med. 178, 1283–1292 40 Wilson, A. et al. (2001) Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194, 1003–1012 41 Pui, J.C. et al. (1999) Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11, 299–308 42 De Smedt, M. et al. (2002) Active form of Notch imposes T cell fate in human progenitor cells. J. Immunol. 169, 3021–3029 43 Artavanis-Tsakonas, S. et al. (1999) Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 44 de La Coste, A. et al. (2005) In vivo and in absence of a thymus, the enforced expression of the Notch ligands delta-1 or delta-4 promotes T cell development with specific unique effects. J. Immunol. 174, 2730–2737 45 Kawamata, S. et al. (2002) Overexpression of the Notch target genes Hes in vivo induces lymphoid and myeloid alterations. Oncogene 21, 3855–3863 46 Tomita, K. et al. (1999) The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev. 13, 1203–1210 47 Hoebeke, I. et al. (2005) Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells. Blood
www.sciencedirect.com
Vol.27 No.3 March 2006
131
48 Taghon, T.N. et al. (2005) Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev. 19, 965–978 49 Ciofani, M. and Zuniga-Pflucker, J.C. (2005) Notch promotes survival of pre-T cells at the beta-selection checkpoint by regulating cellular metabolism. Nat. Immunol. 6, 881–888 50 Koch, U. et al. (2001) Subversion of the T/B lineage decision in the thymus by lunatic fringe-mediated inhibition of Notch-1. Immunity 15, 225–236 51 Lepault, F. and Weissman, I.L. (1981) An in vivo assay for thymushoming bone marrow cells. Nature 293, 151–154 52 Dik, W.A. et al. (2005) New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J. Exp. Med. 201, 1715–1723 53 Staal, F.J. and Clevers, H.C. (2003) Wnt signaling in the thymus. Curr. Opin. Immunol. 15, 204–208 54 Staal, F.J. and Clevers, H.C. (2005) WNT signalling and haematopoiesis: a WNT-WNT situation. Nat. Rev. Immunol. 5, 21–30 55 Okamura, R.M. et al. (1998) Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity 8, 11–20 56 Schilham, M.W. et al. (1998) Critical involvement of Tcf-1 in expansion of thymocytes. J. Immunol. 161, 3984–3991 57 Staal, F.J. et al. (2004) Wnt target genes identified by DNA microarrays in immature CD34C thymocytes regulate proliferation and cell adhesion. J. Immunol. 172, 1099–1108 58 Staal, F.J. et al. (2001) Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription. Eur. J. Immunol. 31, 285–293 59 Staal, F.J. et al. (2001) Transcriptional control of T lymphocyte differentiation. Stem Cells 19, 165–179 60 Hardy, R.R. (2003) B-cell commitment: deciding on the players. Curr. Opin. Immunol. 15, 158–165 61 Nutt, S.L. et al. (1999) Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 62 Urbanek, P. et al. (1994) Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79, 901–912 63 Souabni, A. et al. (2002) Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1. Immunity 17, 781–793 64 Radtke, F. et al. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547–558 65 Schroeder, T. et al. (2003) Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. J. Immunol. 170, 5538–5548 66 Lam, L.T. et al. (2000) Suppression of erythroid but not megakaryocytic differentiation of human K562 erythroleukemic cells by notch1. J. Biol. Chem. 275, 19676–19684 67 Schmitt, T.M. and Zuniga-Pflucker, J.C. (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 68 Schmitt, T.M. et al. (2004) Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5, 410–417 69 Watowich, S.S. et al. (1996) Cytokine receptor signal transduction and the control of hematopoietic cell development. Annu. Rev. Cell Dev. Biol. 12, 91–128 70 Balciunaite, G. et al. (2005) B220C CD117C CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential. Eur. J. Immunol. 35, 2019–2030 71 de La Coste, A. and Freitas, A.A. (2006) Notch signaling: Distinct ligands induce specific signals during lymphocyte development and maturation. Immunol. Lett. 102, 1–9 72 Boyd, R.L. et al. (1993) The thymic microenvironment. Immunol. Today 14, 445–459 73 Witt, C.M. et al. (2005) Directed migration of positively selected thymocytes visualized in real time. PLoS Biol. 3, e160
TRENDS in Immunology
Vol.27 No.3 March 2006
T-sing progenitors to commit Floor Weerkamp, Karin Pike-Overzet and Frank J.T. Staal Department of Immunology, Erasmus Medical Center, Dr. Molewaterplein 50, 3015 GE Rotterdam, The Netherlands
T-cell development in the thymus is a complex and highly regulated process. During the process of differentiation from multipotent progenitor cells to mature T cells, proliferation, restriction of lineage potential, TCR gene rearrangements and selection events occur, all accompanied by changes in gene expression. A comprehensive understanding of thymocyte differentiation remains to be established. Two related, key issues have received much attention recently: the nature of the thymus seeding cell and the regulation of T-cell lineage commitment. Here we review the perspectives of different researchers working both on murine and human T-cell development and argue that a true T-cell commitment factor might not be required because of the unique properties of the thymus.
T-cell commitment Leukocytes develop from pluripotent hematopoietic stem cells (HSCs) through a complex differentiation pathway involving several phenotypically distinct subpopulations [1–5] (Box 1). During lineage commitment and further differentiation into a certain lineage, unique regulatory programs are established that direct cell-type-specific patterns of gene expression. Thus, T-cell commitment minimally includes the following sequential processes: loss of alternative lineage gene expression, loss of alternative lineage potential, expression of a T-cell-specific gene program and initiation of T-cell-specific gene rearrangements [6–9]. Understanding T-cell commitment starts with the question of at what level the cells that seed the postnatal thymus are committed to the T-cell lineage. The difficulty in answering this question is that cells will immediately be influenced by the thymic microenvironment as soon as they enter the thymus. Progenitors that have T-cell developmental capacity but differ from HSCs have been identified in bone marrow and blood but it is unknown whether these cells seed the thymus physiologically. During the past decade, several groups have attempted to identify the nature of the first cells that enter the thymic anlage during fetal life [10–14]. Harman et al. [15] have elegantly addressed this question in the fetal thymus by isolating the cells that are present in the mesenchyme surrounding the fetal thymus but have not yet entered. These cells were shown to be committed T-cell precursors, which is in contrast to the situation in adult thymi where immature cells with broader lineage potential are found (see below). Indeed, the cells that initially populate the Corresponding author: Staal, F.J.T. ([email protected]). Available online 10 February 2006
fetal thymus and contribute to the establishment of the thymic microenvironment might be different from the few postnatal thymus seeding cells that originate from the bone marrow and predominantly serve to keep the thymocyte counts at a steady level. In the mouse, the identification of the thymus seeding cell has recently been approached by sorting minute double negative (DN) populations (Box 1) from the thymus and by studying their lineage potential and other characteristics, hoping to find the cells that have contacted the thymic stroma for a minimally short period [16–19]. These recent papers on the characterization of thymus seeding progenitors (TSPs) will be described in more detail below. A difficulty the field is facing is that various laboratories use different definitions of lymphoid progenitors and stem cell populations isolated from bone marrow, blood or thymus. How these subpopulations relate to each other is not always clear (Table 1), particularly for transgenic reporter mice [20,21]. It also is conceivable that the thymus is seeded by various different types of progenitors with different lineage potential and different quantitative capacity to generate T cells and commit to the T-cell lineage [22]. Progenitor cells in bone marrow In most strains of mice, HSCs can be readily identified as lin-negative, Sca-1C and c-kitC, a phenotype generally referred to as LSK cells [23,24]. The LSK cells can be subdivided on the basis of the expression of Flt3: Flt3K is the true HSC with abundant self-renewal activity and the Flt3C LSK cells are non-renewing multipotent progenitor cells that might have lost erythro-megakaryocytic potential (LMPP) [23,24]. A similar division can be made using the marker CD27 [25]. Interestingly, cells with an LSK phenotype can also be found as a minute population in the thymus and constitute a subpopulation that form so-called early T-cell progenitor cells (ETP, see below; reviewed in [26]). The identification of the lymphoid-restricted common lymphoid progenitor (CLP) in bone marrow by Kondo et al. [27] supported the widely held view that a common progenitor for B and T cells must exist, and that this cell seeds the thymus. CLPs were originally identified as LinK, IL-7RC, c-Kitlow and Sca-1low (Table 1), and indeed are highly responsive to interleukin-7 (IL-7). CLPs have also been identified using an alternative protocol in which c-Kit is replaced with AA4.1, a mAb that recognizes a protein homologous to the complement C1q receptor [28]. However, CLPs have never been shown to seed the thymus in a physiological setting (but do so when injected in the thymus or in fetal thymic organ cultures). Some
www.sciencedirect.com 1471-4906/$ - see front matter Q 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2006.01.006
Opinion
126
TRENDS in Immunology
Vol.27 No.3 March 2006
Box 1. T-cell development in the thymus: of mice and men In contrast to all other hematopoietic lineages, development of T cells from pluripotent hematopoietic stem cells takes place in the specialized microenvironment of the thymus. Several developmental stages can be distinguishes using cell surface markers. Primarily, thymocytes are subdivided into double negative (DN), double positive (DP) and single positive (SP) populations, referring to the expression of the co-receptors CD4 and CD8 (Figure I). The most immature thymocytes lack expression of both CD4 and CD8 and, therefore, are called double negative. Immature single positive cells arise when thymocytes express a co-receptor in the absence of high levels of CD3, as do mature SP cells. In humans, immature SP cells express CD4, whereas in most strains of mice they express CD8. Subsequently, both CD4 and CD8 are expressed and therefore these cells are referred to as double positive. The DP stage represents w85% of all thymocytes. After undergoing positive and negative selection, thymocytes that express a functional T-cell receptor (TCR) commit to either the CD4 or CD8 single positive lineage.
In both species, T-cell commitment towards the ab TCRC lineage takes place during the DN stages of development. Additionally, gd T cells also split off at the DN stages. In mouse and humans, additional but different surface markers are used to subdivide the DN stage further. For mouse, the markers CD25 and CD44 are used to divide the DN stage: CD44 C CD25 K cells are referred to as DN1 cells, CD44CCD25C cells are referred to as DN2, DN3 cells express CD25 but no CD44, and DN4 cells express neither CD25 nor CD44. The most immature human thymocyte population is characterized by the expression of CD34, but lacks CD1a and CD38 expression, and resembles the murine DN1 population. The next stage of differentiation is marked by the expression of both CD34 and CD38 and resembles the murine DN2 stage. The most mature human DN stage that can be discerned is made up of cells expressing CD34 and CD38 as well as CD1a. Important checkpoints and developmental decisions are indicated in Figure I.
CD44+ CD25–
CD44+ CD25+
CD44– CD25+
CD8+ CD3–
CD4+ CD8+ CD3–
CD4+ CD8+ CD3+
CD4+ or CD8+ CD3+
CD34– CD38– CD1a+
CD34– CD38+ CD1a+
CD34+ CD38+ CD1a+
CD4+ CD3–
CD4+ CD8+ CD3–
CD4+ CD8+ CD3+
CD4+ or CD8+ CD3+
αβ commitment
Positive and CD4 or CD8 negative selection commitment
β selection
Stem cell like UCB CD34+ Lin– Blood
DN1 CD117–
DN1 CD117+ DN2
SP CD8+ DN3
ISP/DN4
DP CD3–
DP CD3+ SP CD4+
Thymus
TRENDS in Immunology
Figure I. Comparison of the various stages of mouse and human T-cell development. The different phenotypic definitions are shown, as well as the main checkpoints during T-cell development. Human data are based on genome-wide microarray analyses and recently published TCR rearrangement studies [46]. Stages indicated mainly refer to human subpopulations, with names of their putative murine counterparts.
laboratories have not been able to find these cells circulating in blood (reviewed extensively in [22,26]), a prerequisite for the thymus seeding cells, as they have to migrate from bone marrow to thymus. Various investigators in the field therefore regard CLPs as efficient and physiological progenitors for B but not for T lymphocytes [28,29]. These ideas are in line with seminal work done by Katsura and co-workers [30–32] who were the first to express doubts that CLPs are progenitors for T cells, albeit in fetal mice. In addition to CLPs, the bone marrow
contains other progenitors that will efficiently make T cells when put into contact with the thymus microenvironment. Kincade and colleagues [21] have identified a population of cells they termed early lymphoid progenitors (ELPs) on the basis of the expression of GFP under control of the RAG1 promoter. As the RAG genes are exclusively required for recombination of the immunoglobulin and T-cell receptor (TCR) loci, this reporter represents a lymphoid-specific marker. Unexpectedly, GFP expression was detected early on in the bone marrow. The expression
Table 1. Cell types that might seed the thymus – overview of terminology used to describe various progenitor and stem cell populations Common abbreviation HSC
Full name
Phenotype mouse
Phenotype human
Tissue
Refs
Hematopoietic stem cell
CD34CCD38K
[23–25]
ETP ELP
Long-term multi potent progenitor Early T-cell progenitor Early lymphoid progenitor
Bone marrow, cord blood (human) Bone marrow
[23,24]
LMPP
Lin- Sca-1hi c-kithi (Z LSK) Flt3K LSK Flt3C, CD27C
CD34CCD38K
Thymus Bone marrow
[17–19] [21]
CLP
Common lymphoid progenitor
CD34CCD10C
Bone marrow
[27,28]
www.sciencedirect.com
LSK, CD44C, IL7RaK RAG-1C, LSK,Flt3C, IL7RaK Lin-Sca1lo, c-kitlo, IL7RaC, AA4C
Not identified
Opinion
TRENDS in Immunology
of RAG by ELPs indicates that lymphoid specification might initiate earlier than previously thought. Expression of RAG is currently the earliest marker for hematopoietic progenitors undergoing lymphoid specification, because it precedes the expression of the IL-7 receptor, the marker used to identify CLPs. Why RAG would be expressed early on, before rearrangement initiates, is currently unclear. Upstream of both ELPs and CLPs are the LSK cells, containing the true HSCs. Because HSCs in mice and humans are known to circulate in the blood [33,34], and the thymus contains a small subpopulation of cells with an LSK phenotype, it is conceivable that HSCs seed the thymus. Most investigators do not believe this to be the case, as isolated cells from the thymus with the LSK phenotype (also known as ETPs) do not support full hematopoietic reconstitution in irradiated recipient mice [16,17]. However, it is possible that upon entry into the thymus cells rapidly lose their self-renewal capacity. Support from this notion stems from recent work using the human thymi [35], where T and B lymphoid, myeloid, erythroid and megakaryocytic potential was detected in early thymocytes. Progenitors in the thymus More than ten years ago, Shortman and colleagues [36] identified a subset of DN1 cells expressing c-Kit and low levels of CD4, hence their name, CD4lo cells. These cells have progenitor activity for T, B and NK cells, dendritic cells (DCs) and low levels of myeloid differentiation. More recent work from various laboratories has focused on ETPs as an alternative to CLPs for colonizing the thymus [16,17,26,28]. The thymus seeding cell is generally thought to lie within this ETP population. Classic ETPs are defined as being DN1, LinK and c-Kithi and are supposedly highly heterogeneous themselves. CD4 is usually excluded from the lineage cocktail for these cells, so as not to exclude the CD4lo cells, as ETP are DN1 cells at the transcriptional level but might express surface CD4 protein that is passively acquired. Additional markers, varying per research group, were included to subdivide ETPs and other DN populations into several fractions, which appeared to have differential T- and B-cell developmental capacities (which also depended on the in vivo and in vitro tests used to delineate lineage potential) [17–19]. These different developmental possibilities for the most immature thymocytes are controversial, with respect to B-cell lineage potential in the thymus. Although we and others found low but detectable B-cell differentiation in the adult thymus [16,17,37], others have found that B-cell potential is only marginal [19,38]. Nevertheless, all differentiation stages of B-cell development can be found in the thymus and mature B cells are present. The question remains whether these different subpopulations all arise from the same thymus seeding cell or whether diverse bone marrow precursors (some of which might not have T-cell potential) enter the thymus. In human studies, not all different lineages were definitively shown to have developed from a single precursor in the thymus [35]. Nevertheless, the full range of hematopoietic lineage potentials in the human DN1/2 (CD34CCD1aK) www.sciencedirect.com
Vol.27 No.3 March 2006
127
population, and that precursors for B and NK cells are found in the human thymus [5], suggested that the thymus is seeded by an HSC, or a mixture of progenitors with the collective potential to develop into all blood cell lineages. In the mouse, old but elegant studies also have indicated myeloid and low erythroid potential using c-kitC thymocytes in colony assays. [39]. Recent studies in the murine thymus, using CCR9–GFP as marker to track individual cells, have shown that clonal precursor cells with T, B and myeloid potential exist in the thymus [16]. These investigators have used a clever strategy of mixing stromal cell lines that permit B and myeloid development with those that induce T-cell development to generate a system that allows T, B and myeloid development in vitro [16]. Interestingly, granulocyte development was also observed, indicating that a precursor cell with broad lineage potential can be isolated from the thymus. It is clear that multilineage potential is present in the thymus and that full T-cell commitment occurs after entry into the thymus (Figure 1). To what extent the thymus seeding cells resemble HSCs or have a more limited lineage potential is not entirely clear. Signals inducing T-cell commitment If a quintessential T-cell commitment factor does exist, Notch would be the most likely candidate. Notch signaling, as measured by expression of the Hes1 target gene, is absent from bone marrow LSK cells, but present in the earliest thymocytes that can be identified, both in mice [17,37] and humans (F. Weerkamp, PhD Thesis, Erasmus University, Rotterdam, 2005). Loss of Notch1 function in lymphoid progenitors results in B lymphopoiesis in the thymus, at the expense of T-cell development [40], whereas overexpression of a constitutively active form of Notch induces ectopic T-cell development in the
Thymus HSC ?
TSC
? CLP ?
? ?
T/M T
Notch signaling
? T NK and DC Myeloid B E/M
Lineage potential
TRENDS in Immunology
Figure 1. Candidate populations that seed the thymus. Several types of thymus seeding cells (TSCs) have been proposed, ranging from HSCs or non-renewing multipotent progenitors to committed T-cell precursors (abbreviations: CLPs, common lymphoid progenitor; E/M, erythroid/megakaryocytic; T/M, T/myeloid bipotent progenitor; T, T-cell-committed progenitor). Cells that enter the thymus receive Delta-induced Notch signals and start developing into T cells. As thymocytes develop, lineage potential (either of individual cells or at the population level) declines. In our view,it is likely that a multipotent cell with broad lineage potential seeds the thymus, with rapid loss of non-T-cell potential upon thymic entry. However, it is possible that lineage potential is already reduced before cells arrive in the thymus.
128
Opinion
TRENDS in Immunology
bone marrow and inhibits B-cell development that would normally take place there [41,42]. The Notch signal transduction pathway regulates cell fate determination during developmental processes [43]. The Notch family of transmembrane receptor consists of four members, named Notch1 to Notch4, interacting with membrane-bound ligands on neighboring cells (Delta1, Delta3 and Delta4, and Jagged1 and Jagged2). The Delta1 and Delta4 ligands are particularly highly expressed on thymic epithelial cells [44]. The downstream mechanisms by which a Notch signal is translated into a T-cell-specific program are still largely unclear. The best known Notch target genes encode hairyenhancer of split (Hes)1 and Hes5 and Hes-related repressor protein (Herp), all basic-helix–loop–helix (bHLH) proteins that function as transcriptional repressors. Indeed, overexpression of Hes1 and Hes5 in bone marrow partly inhibits B-cell development [45]. However, mice deficient in Hes-1 show a proliferation defect of early DN thymocytes, but they are still present [46]. Furthermore, overexpression of Hes1 does not induce a T-cell fate in human hematopoietic progenitors, whereas overexpression of IC-Notch does [47]. Hes1 can therefore not be the sole target of Notch signaling in the thymus. In microarray studies aimed at identification of Notch target genes (F. Weerkamp, PhD Thesis, Erasmus University, Rotterdam, 2005), no clues were found that Notch signaling can directly induce a T-cell fate. This is in line with experiments by Taghon et al. [48] in which murine fetal liver progenitors were cultured on OP9-DL. This study showed that the T-cell-specific transcription factors GATA3, TCF1 and pre-T-cell receptor a gene (PTCRA) began to be expressed after three days of culture on OP9DL, whereas high HES1 transcription was detected after only one day. Therefore, either Notch signaling induces T-cell genes in already more differentiated thymocytes, or Notch signaling stimulates the expression of other transcription factors, which then in turn activate or repress lineage differentiation genes. Alternatively, the first function of Notch signaling in the thymus might be to induce stable metabolism and survival, as has been shown for DN3 thymocytes [49], whereas subsequent Notch signals induce differentiation. Two recent papers [17,37] have dealt with the role of Notch signaling in the most immature thymocytes. The paper from Cynthia Guidos’ laboratory [37] builds on earlier work using transgenic mice that express lunatic fringe, a Notch modifier that alters glycosylation thereby inhibiting Notch1 activation [50]. Using this and other strategies to change the levels of Notch activation it was shown that Notch1 is required for efficient production of ETPs and suppression of B cell development. Additionally, this report confirmed that Notch signals are continuously needed to promote survival or proliferation throughout the DN stages. The other paper from Warren Pear’s and Avinash Bhandoola’s laboratories [17] subdivided the ETPs on the basis of Flt3 expression. Flt3C ETPs were shown to have low potential to develop into B cells and Notch signals were required for development of ETPs, as shown by genetically inhibiting Notch signals via retrovirus mediated transduction of a potent dominant www.sciencedirect.com
Vol.27 No.3 March 2006
negative essential Notch co-factor. Although LSK cells in bone marrow and blood were normally present in mice reconstituted with HSCs expressing this Notch inhibitor, the numbers of ETPs and more mature DN2 thymocytes were low. The authors interpret these findings as follows: Notch signaling occurs after the thymus has been colonized by multipotent progenitors, before the generation of ETPs. Alternatively, it is possible that a subset of LSK cells in the bone marrow undergoes Notch signaling there and that these cells function as thymic seeding progenitors (TSPs). Together, these papers demonstrate that Notch signaling occurs quickly after entry of TSPs in the thymus at the levels of ETPs. It is obvious that TSPs need to undergo massive proliferation (10000- to 50000-fold), as the number of these cells is extremely low and they need to generate 100–200 million thymocytes [28]. It is estimated that only one cell a day enters the adult murine thymus [51] and indeed ETPs comprise less than 0.01% of all thymocytes; the corresponding human CD34CCD38K thymocytes are at least as rare (0.005% or less [5,52]). Before or concomitant to T-cell commitment the most immature thymocytes therefore receive proliferation-inducing signals by cytokines (SCF and later on IL-7) and also by Wnt proteins [53,54]. Wnt proteins are generally not considered to be T-cell commitment factors. The strong blocks seen in Tcf1(VII)K/K and Tcf1K/K/Lef1K/K mice demonstrated that Wnt signals are important for T-cell development, because of a failure in expansion of early subpopulations rather than a differentiation defect [55,56]. A predominantly proliferative function for Wnt signaling was confirmed by identification of the target genes of Wnt signaling in thymocytes [57]; these are mainly involved in proliferation. No T-cell-specific genes were found to be upregulated [57]. Inhibiting Wnt signaling in the most immature thymocytes therefore causes a block in differentiation by interfering with an essential proliferation step [58]. Various other transcription factors, most notably GATA3, E-box factors (HEB, E2A) and Ikaros family factors, have been shown to have more or less T-cellspecific expression patterns and are functionally required during T-cell development (for reviews see [6,59]). However, none of these can be regarded as a T-cell commitment factor because ectopic expression of these factors into HSCs neither induces T-cell commitment nor direct inhibition of other lineages. A comparison with B cells In contrast to the emerging picture of a highly complex regulation of T-cell development, the molecular regulation of B cell commitment appears to be relatively straight forward [60]. During B-cell development, lineage specification is induced by the transcription factors E2A and EBF, which also induce Pax5 expression. Pax5 is necessary for the induction of crucial B-cell genes and represses alternative lineage fates [61,62]. Interestingly, Pax5 inhibits T-cell development by active repression of Notch1 expression [63]. Thus a simple hierarchy from E2A via EBF to Pax5 seems to control early B-cell development.
Opinion
TRENDS in Immunology
Vol.27 No.3 March 2006
129
Thymus
Bone marrow Mono/mϕ
DC
Potential Developmental path taken
HSC NK
Mono/mϕ
DC
Developmental path actively inhibited
B
Mono/mϕ
DC
T
No (G)M-CSF present
(G)M-CSF present CLP
DN1
NK
B
NK
B DL Wnt Cytokines Adhesion molecules
No DL present T
T TRENDS in Immunology
Figure 2. Hypothetical model of B-cell and T-cell commitment. As multiple lineages develop in the bone marrow (left), non-B-cell lineages have to be actively suppressed in common lymphoid progenitors (CLPs). T cells will never develop in the bone marrow because of the absence of Delta, and therefore do not need active suppression. Conversely in the thymus (right), T-cell oriented microenvironmental signaling ensures that only T cells develop. No active repression of alternative lineages is necessary. Abbreviations: mono/4, monocyte/macrophage.
For T-cell development, a master regulator comparable to Pax5 has not yet been identified. Notch signaling appears to be able to repress a B-cell fate, as overexpression of IC-Notch induces T-cell development in the bone marrow, at the expense of B cells [32]. However, direct inhibition of Pax5 by Notch signaling has not been described. Furthermore, Notch signaling is not specific for the induction of a T-cell fate: it is also involved in selfrenewal of HSCs [64], in cell fate choice in a myeloid progenitor cell line [65], and in the choice between erythroid and megakaryocytic differentiation [66]. Also, although B cells develop more readily in the thymus in absence of Notch signals in the thymus [40], the number of B cells is still small and at least ten times lower than thymocyte numbers, indicating that indirect mechanisms such as proliferative factors preferentially acting on T cells strongly favor the T-cell lineage, rather than merely repressing B cell development. Thus, although Notch signals are required for the development of T lineage cells, Notch signaling cannot be the only signal responsible for setting up the full T-cell-specific gene program. Moreover, current experimental evidence does not indicate that Notch signaling suffices to directly suppress other lineages (e.g. Pax5 expression for B cells), thereby not strictly complying with the definition of a commitment factor. No need for a T-cell commitment factor? It is a tantalizing thought that there is no need for active suppression of alternative lineages in the thymus, as the www.sciencedirect.com
thymic microenvironment only supports T-cell development (Figure 2). This is exemplified by the rapid increase of T-cell precursors and the almost complete absence of alternative lineages when hematopoietic progenitors are cultured on OP9-DL [67,68]. Many lineages develop in the bone marrow: B cells, NK cells, various myeloid lineages, erythrocytes and platelets. The bone marrow stroma therefore provides growth and survival factors for all these lineages. The thymus provides an excellent environment for the development of early thymocytes: a high density of the Notch ligand Delta, an abundance of Wnt proteins, presence of cytokines such as IL-7, SCF and Flt3L, and appropriate adhesion molecules. It is our view that development of any other lineage than T cells simply does not stand a chance (Figure 2) and does not require active transcriptional repression. But does the thymic microenvironment actively induce T-cell commitment? Binding of the Notch ligand Delta is important for T-cell development but might support other functions, such as proliferation or survival, rather than T-cell commitment. Studies in human progenitor cells suggested that Notch signaling also inhibits a myeloid cell fate [42], but the molecular mechanism is unknown. Perhaps stringent inhibition of myeloid development on the molecular level is not so strictly necessary during thymocyte differentiation (Figure 2). Development of myeloid lineages is strongly dependent on myeloid cytokines such as macrophage colony stimulating factor (M-CSF) and granulocyte macrophage colony stimulating
130
Opinion
TRENDS in Immunology
factor (GM-CSF) [69]. These cytokines are expressed at low levels in the thymus, and myeloid development is thereby automatically not favored. In contrast to the earliest thymocytes, CLPs in bone marrow completely lack myeloid potential [27], although some myeloid developmental capacity was detected in a related population [70]. Because of the abundant presence of myeloid growth factors in the bone marrow, myeloid fates have to be actively repressed in CLP and B cell precursors (Figure 2). Similar reasoning explains why T-cell potential is still present in CLPs. As T cells will normally not develop in the bone marrow because of the absence or low expression of Delta ligands (they will develop as soon as Delta1 or Delta4 are ectopically expressed in bone marrow [71]), T-cell development does not have to be actively repressed. This model makes several testable predictions. For instance, ectopic expression of G-CSF or GM-CSF in thymic stroma should lead to a dramatic increase in the number of myeloid cells in the thymus. This could, for instance, be tested with virally transduced stromal cells in reaggregate cultures. Similar experiments could be done with erythropoietin (EPO) for erythrocytes. Also, murine ETPs, stringently purified while not using Mac1 or Ter119 but including all other markers (positive and negative), are expected to show erythroid and myeloid development. It might also be that transplantation of large numbers of such ETPs leads to low, but detectable self-renewal in irradiated recipients.
Future challenges The question about the nature of early T-cell progenitors has still not been answered satisfactorily. The thymus seeding cell and its immediate progeny must be identified by detailed immunophenotyping and subsequently characterized in terms of lineage potential. Difficult but elegant studies along these lines have already been undertaken and need to be expanded. Next, the different features of the unique thymic microenvironment [72] should be further characterized. For instance, studies have been initiated to establish the migration pattern of thymocytes through the microenvironmental niches and how this correlates with their differentiation [73]. Future research should also focus less on individual factors and pathways and more on the integration of the different signals. How do they all cooperate to set up a T-cell program? Studies on the establishment of lineagespecific gene programs will increasingly concentrate on novel regulatory mechanisms such as microRNAs, epigenetic phenomena and chromatin accessibility regulation by acetylation and methylation of histone proteins. Such studies should lead to a better understanding of T-cell development and help translate such knowledge to medical problems: development of better diagnostics and therapies for diseases involving the early stages of T-cell development (T-ALL, SCID), and improvement of T-cell reconstitution after bone marrow transplantation and acquired immune deficiencies, such as those underlying HIV infection. www.sciencedirect.com
Vol.27 No.3 March 2006
Acknowledgements We thank C.J.M. Loomans and A.W. Langerak and J.J.M. van Dongen for critically reading the manuscript. T. van Os and M. Comans-Bitter are acknowledged for expert help with illustrations. F.J.T.S. is supported in part by funds from the 5th and 6th EU Framework program [Contract Nrs QLK3-CT-2001-0427 (INHERINET) and LSHB-CT-2004-005242 (CONSERT)], as well as by the Translational Gene Therapy Research Programme of NOW-ZonMw – the Netherlands Organization for Health Research and Development, ICES-KIS ‘Virgo’ program and the Dutch Heart Foundation.
References 1 Scollay, R. and Shortman, K. (1985) Identification of early stages of T lymphocyte development in the thymus cortex and medulla. J. Immunol. 134, 3632–3642 2 Scollay, R. (1991) T-cell subset relationships in thymocyte development. Curr. Opin. Immunol. 3, 204–209 3 Vanhecke, D. et al. (1997) Human thymocytes become lineage committed at an early postselection CD69C stage, before the onset of functional maturation. J. Immunol. 159, 5973–5983 4 Blom, B. et al. (1999) TCR gene rearrangements and expression of the pre-T cell receptor complex during human T-cell differentiation. Blood 93, 3033–3043 5 Weerkamp, F. et al. (2005) Age-related changes in the cellular composition of the thymus in children. J. Allergy Clin. Immunol. 115, 834–840 6 Rothenberg, E.V. and Dionne, C.J. (2002) Lineage plasticity and commitment in T-cell development. Immunol. Rev. 187, 96–115 7 Radtke, F. et al. (2004) Notch regulation of lymphocyte development and function. Nat. Immunol. 5, 247–253 8 Collins, A. and Littman, D.R. (2005) Selection and lineage specification in the thymus: commitment 4-stalled. Immunity 23, 4–5 9 Garcia-Ojeda, M.E. et al. (2005) Stepwise development of committed progenitors in the bone marrow that generate functional T cells in the absence of the thymus. J. Immunol. 175, 4363–4373 10 Rodewald, H.R. et al. (1994) Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization. EMBO J. 13, 4229–4240 11 Peault, B. (1996) Hematopoietic stem cell emergence in embryonic life: developmental hematology revisited. J. Hematother. 5, 369–378 12 Carlyle, J.R. and Zuniga-Pflucker, J.C. (1998) Requirement for the thymus in alphabeta T lymphocyte lineage commitment. Immunity 9, 187–197 13 Douagi, I. et al. (2000) Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation. Eur. J. Immunol. 30, 2201–2210 14 Masuda, K. et al. (2005) Prethymic T-cell development defined by the expression of paired immunoglobulin-like receptors. EMBO J. 24, 4052–4060 15 Harman, B.C. et al. (2005) B lineage choice occurs prior to intrathymic notch signalling. Blood 16 Benz, C. and Bleul, C.C. (2005) A multipotent precursor in the thymus maps to the branching point of the T versus B lineage decision. J. Exp. Med. 202, 21–31 17 Sambandam, A. et al. (2005) Notch signaling controls the generation and differentiation of early T lineage progenitors. Nat. Immunol. 6, 663–670 18 Porritt, H.E. et al. (2004) Heterogeneity among DN1 prothymocytes reveals multiple progenitors with different capacities to generate T cell and non-T cell lineages. Immunity 20, 735–745 19 Balciunaite, G. et al. (2005) The earliest subpopulation of mouse thymocytes contains potent T, significant macrophage, and natural killer cell but no B-lymphocyte potential. Blood 105, 1930–1936 20 Gounari, F. et al. (2002) Tracing lymphopoiesis with the aid of a pTalpha-controlled reporter gene. Nat. Immunol. 3, 489–496 21 Igarashi, H. et al. (2002) Transcription from the RAG1 locus marks the earliest lymphocyte progenitors in bone marrow. Immunity 17, 117–130 22 Petrie, H.T. and Kincade, P.W. (2005) Many roads, one destination for T cell progenitors. J. Exp. Med. 202, 11–13 23 Adolfsson, J. et al. (2001) Upregulation of Flt3 expression within the bone marrow Lin(-)Sca1(C)c-kit(C) stem cell compartment is accompanied by loss of self-renewal capacity. Immunity 15, 659–669
Opinion
TRENDS in Immunology
24 Adolfsson, J. et al. (2005) Identification of Flt3C lympho-myeloid stem cells lacking erythro-megakaryocytic potential a revised road map for adult blood lineage commitment. Cell 121, 295–306 25 Wiesmann, A. et al. (2000) Expression of CD27 on murine hematopoietic stem and progenitor cells. Immunity 12, 193–199 26 Bhandoola, A. et al. (2003) Early T lineage progenitors: new insights, but old questions remain. J. Immunol. 171, 5653–5658 27 Kondo, M. et al. (1997) Identification of clonogenic common lymphoid progenitors in mouse bone marrow. Cell 91, 661–672 28 Allman, D. et al. (2003) Thymopoiesis independent of common lymphoid progenitors. Nat. Immunol. 4, 168–174 29 Borghesi, L. et al. (2004) B lineage-specific regulation of V(D)J recombinase activity is established in common lymphoid progenitors. J. Exp. Med. 199, 491–502 30 Kawamoto, H. et al. (1999) Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver. J. Immunol. 162, 2725–2731 31 Ohmura, K. et al. (1999) Emergence of T, B, and myeloid lineagecommitted as well as multipotent hemopoietic progenitors in the aorta-gonad-mesonephros region of day 10 fetuses of the mouse. J. Immunol. 163, 4788–4795 32 Ikawa, T. et al. (1999) Commitment of common T/Natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay. J. Exp. Med. 190, 1617–1626 33 Udomsakdi, C. et al. (1992) Characterization of primitive hematopoietic cells in normal human peripheral blood. Blood 80, 2513–2521 34 Wright, D.E. et al. (2001) Physiological migration of hematopoietic stem and progenitor cells. Science 294, 1933–1936 35 Weerkamp, F. et al. (2005) The human thymus contains multipotent progenitors with T/B-lymphoid, myeloid and erythroid lineage potential. Blood DOI 10.1182/blood-2005-08-3412 (http://www.bloodjournal.org) 36 Wu, L. et al. (1991) CD4 expressed on earliest T-lineage precursor cells in the adult murine thymus. Nature 349, 71–74 37 Tan, J.B. et al. (2005) Requirement for Notch1 signals at sequential early stages of intrathymic T cell development. Nat. Immunol. 6, 671–679 38 Lu, M. et al. (2005) The earliest thymic progenitors in adults are restricted to T, NK, and dendritic cell lineage and have a potential to form more diverse TCRbeta chains than fetal progenitors. J. Immunol. 175, 5848–5856 39 Matsuzaki, Y. et al. (1993) Characterization of c-kit positive intrathymic stem cells that are restricted to lymphoid differentiation. J. Exp. Med. 178, 1283–1292 40 Wilson, A. et al. (2001) Notch 1-deficient common lymphoid precursors adopt a B cell fate in the thymus. J. Exp. Med. 194, 1003–1012 41 Pui, J.C. et al. (1999) Notch1 expression in early lymphopoiesis influences B versus T lineage determination. Immunity 11, 299–308 42 De Smedt, M. et al. (2002) Active form of Notch imposes T cell fate in human progenitor cells. J. Immunol. 169, 3021–3029 43 Artavanis-Tsakonas, S. et al. (1999) Notch signaling: cell fate control and signal integration in development. Science 284, 770–776 44 de La Coste, A. et al. (2005) In vivo and in absence of a thymus, the enforced expression of the Notch ligands delta-1 or delta-4 promotes T cell development with specific unique effects. J. Immunol. 174, 2730–2737 45 Kawamata, S. et al. (2002) Overexpression of the Notch target genes Hes in vivo induces lymphoid and myeloid alterations. Oncogene 21, 3855–3863 46 Tomita, K. et al. (1999) The bHLH gene Hes1 is essential for expansion of early T cell precursors. Genes Dev. 13, 1203–1210 47 Hoebeke, I. et al. (2005) Overexpression of HES-1 is not sufficient to impose T-cell differentiation on human hematopoietic stem cells. Blood
www.sciencedirect.com
Vol.27 No.3 March 2006
131
48 Taghon, T.N. et al. (2005) Delayed, asynchronous, and reversible T-lineage specification induced by Notch/Delta signaling. Genes Dev. 19, 965–978 49 Ciofani, M. and Zuniga-Pflucker, J.C. (2005) Notch promotes survival of pre-T cells at the beta-selection checkpoint by regulating cellular metabolism. Nat. Immunol. 6, 881–888 50 Koch, U. et al. (2001) Subversion of the T/B lineage decision in the thymus by lunatic fringe-mediated inhibition of Notch-1. Immunity 15, 225–236 51 Lepault, F. and Weissman, I.L. (1981) An in vivo assay for thymushoming bone marrow cells. Nature 293, 151–154 52 Dik, W.A. et al. (2005) New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling. J. Exp. Med. 201, 1715–1723 53 Staal, F.J. and Clevers, H.C. (2003) Wnt signaling in the thymus. Curr. Opin. Immunol. 15, 204–208 54 Staal, F.J. and Clevers, H.C. (2005) WNT signalling and haematopoiesis: a WNT-WNT situation. Nat. Rev. Immunol. 5, 21–30 55 Okamura, R.M. et al. (1998) Redundant regulation of T cell differentiation and TCRalpha gene expression by the transcription factors LEF-1 and TCF-1. Immunity 8, 11–20 56 Schilham, M.W. et al. (1998) Critical involvement of Tcf-1 in expansion of thymocytes. J. Immunol. 161, 3984–3991 57 Staal, F.J. et al. (2004) Wnt target genes identified by DNA microarrays in immature CD34C thymocytes regulate proliferation and cell adhesion. J. Immunol. 172, 1099–1108 58 Staal, F.J. et al. (2001) Wnt signaling is required for thymocyte development and activates Tcf-1 mediated transcription. Eur. J. Immunol. 31, 285–293 59 Staal, F.J. et al. (2001) Transcriptional control of T lymphocyte differentiation. Stem Cells 19, 165–179 60 Hardy, R.R. (2003) B-cell commitment: deciding on the players. Curr. Opin. Immunol. 15, 158–165 61 Nutt, S.L. et al. (1999) Commitment to the B-lymphoid lineage depends on the transcription factor Pax5. Nature 401, 556–562 62 Urbanek, P. et al. (1994) Complete block of early B cell differentiation and altered patterning of the posterior midbrain in mice lacking Pax5/BSAP. Cell 79, 901–912 63 Souabni, A. et al. (2002) Pax5 promotes B lymphopoiesis and blocks T cell development by repressing Notch1. Immunity 17, 781–793 64 Radtke, F. et al. (1999) Deficient T cell fate specification in mice with an induced inactivation of Notch1. Immunity 10, 547–558 65 Schroeder, T. et al. (2003) Notch signaling induces multilineage myeloid differentiation and up-regulates PU.1 expression. J. Immunol. 170, 5538–5548 66 Lam, L.T. et al. (2000) Suppression of erythroid but not megakaryocytic differentiation of human K562 erythroleukemic cells by notch1. J. Biol. Chem. 275, 19676–19684 67 Schmitt, T.M. and Zuniga-Pflucker, J.C. (2002) Induction of T cell development from hematopoietic progenitor cells by delta-like-1 in vitro. Immunity 17, 749–756 68 Schmitt, T.M. et al. (2004) Induction of T cell development and establishment of T cell competence from embryonic stem cells differentiated in vitro. Nat. Immunol. 5, 410–417 69 Watowich, S.S. et al. (1996) Cytokine receptor signal transduction and the control of hematopoietic cell development. Annu. Rev. Cell Dev. Biol. 12, 91–128 70 Balciunaite, G. et al. (2005) B220C CD117C CD19- hematopoietic progenitor with potent lymphoid and myeloid developmental potential. Eur. J. Immunol. 35, 2019–2030 71 de La Coste, A. and Freitas, A.A. (2006) Notch signaling: Distinct ligands induce specific signals during lymphocyte development and maturation. Immunol. Lett. 102, 1–9 72 Boyd, R.L. et al. (1993) The thymic microenvironment. Immunol. Today 14, 445–459 73 Witt, C.M. et al. (2005) Directed migration of positively selected thymocytes visualized in real time. PLoS Biol. 3, e160