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Early T cell development and the pitfalls of potential
Review
Early T cell development and the pitfalls of potential Susan M. Schlenner1 and Hans-Reimer Rodewald2,3 1
Department for Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA Department for Cellular Immunology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 3 Institute for Immunology, University of Ulm, D-89081 Ulm, Germany 2
The long-standing model for hematopoiesis, which features a dichotomy into separate lymphoid and myeloid branches, predicts that progenitor T cells arise from a lymphocyte-restricted pathway. However, experiments that have detected myeloid potential in progenitor T cells have been reported as evidence to question this model. Mapping physiological differentiation pathways has now led to opposite conclusions, by showing that T cells and thymic myeloid cells have distinct origins and that, in vivo, T cell progenitors lack significant potential for myeloid lineages including dendritic cells. Here, we review the underlying experiments that have led to such fundamentally different conclusions. The current controversy might reflect a need to distinguish between cell fates that are possible experimentally from physiological fate choices, to build a map of immunological differentiation pathways. Controversy of T cell progenitor potential and implications for hematopoietic differentiation pathways Hematopoiesis is arguably the most-studied system in which stem cells give rise to progenitors that, in turn, yield mature and functionally diverse cells [1]. Much of what we know about the development of blood and immune cells is based on the isolation of phenotypically defined progenitor cell populations, followed by an assessment of their in vitro and in vivo developmental potential and deviations from the system in hematopoietic mutants [2]. Despite the information gained from these experiments, major routes within the hematopoietic tree have become increasingly vague [3,4]. Initially, relatively simple maps were drawn where long-term self-renewing multipotent hematopoietic stem cells (HSCs) were connected to short-term multipotent progenitors (MPPs). Downstream from the MPPs, a split into lymphocyte-restricted and myelo-erythroidrestricted progenitors was postulated. This dichotomy model was strongly supported by the identification of progenitors with distinct cell surface phenotypes that appeared to qualify as common lymphocyte progenitors (CLPs) [5] and common myeloid progenitors (CMPs) [6]. In the original model, the pathway from the bone marrow (BM) to the thymus belonged to the lymphoidrestricted branch of hematopoiesis. However, this model has recently been called into question [7–9]. This conceptual change was based on the detection of abundant Corresponding author: Rodewald, H.-R. ([email protected]).
myeloid potential in progenitor (pro) T cells [10,11]. These studies have implied that pro T cells give rise to myeloid cells, including dendritic cells (DCs), macrophages and granulocytes, in the thymus. The common T and myeloid potential of pro T cells seemed incompatible with a descent of pro T cells from a lymphoid-restricted progenitor, thus casting doubt on the fundamental lymphoid–myeloid division of the hematopoietic system. In the alternative, myeloid-based model, each lymphoid pathway retains myeloid potential downstream from the T versus B split [7–9]. In contrast to these reports, new experiments that have employed genetic tools to map physiological differentiation pathways in vivo have shown that T cells and thymic myeloid cells have distinct origins, and that T cell progenitors lack significant potential for myeloid lineages in vivo. We review the basic principles and results of these experiments, discuss their strengths and limitations, and their diverging conclusions in the broader context of hematopoietic differentiation. Plasticity of cell fate and vagaries in hematopoiesis Several developments in the field of hematopoiesis and lineage commitment have complicated the straightforward view of non-redundant differentiation pathways, such as MMPs giving rise to lymphoid-restricted CLPs or myeloidrestricted CMPs. The phenotypic resolution and isolation of progenitor populations has steadily improved because of the availability of vast numbers of antibodies for cell surface marker detection. Moreover, reporter mice can reveal expression of stage- and lineage-specific genes from transgenic, bacterial artificial chromosome (BAC), or targeted knock-in loci. These advances have been combined with major improvements in cell sorting. Consequently, additional progenitor subpopulations, partially overlapping with or distinct from MPPs [12–14], CLPs [15–17], CMPs [18] or lineage committed progenitor cells, have been identified. Are such populations redundant or do they represent different stages along the same pathway? With every new subset of functionally related progenitor cells that are identified, the overall importance of each individual population for the generation or maintenance of a given lineage becomes less certain. For the most part, quantitative information on specific developmental pathways during hematopoiesis is lacking, and the steps obligatory for and the possible alternative routes of development remain undefined. The sensitivity of readouts for hematopoietic potential has also increased greatly. In 2002, the induction of T cell
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Review Box 1. In vitro assays for early T cell development For many years, T cell development could not be recapitulated in 2D cell culture systems. Instead, T cell development has been studied successfully in intact fetal thymus organ culture (FTOC) [77], and in re-aggregated fetal thymus organ culture (RFTOC), assembled from epithelial and mesenchymal thymic stromal cells [78]. Entry of progenitors into the thymus via the normally impermeable capsule is enhanced by co-culturing of the thymic lobes with progenitors in hanging drops. Deoxyguanosine treatment purges the thymus of its endogenous thymocytes and thus reduces the competition with exogenous progenitors. FTOC, normally placed on filter discs, can also be kept in high oxygen submersion cultures that allow, in the presence of added cytokines, multi-lineage readouts from single progenitors [79]. FTOC or RFTOC experiments probably closely resemble the near-normal thymic environment, provided that the cells find their proper position in situ to receive the signals that fits their own stage of development [80]. Addition of exogenous growth factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF), which normally drives differentiation and proliferation of myeloid progenitors, to FTOC [11] is probably an unusual condition for the thymic environment. Although macrophages can be generated from pro T cells under these conditions [11], a plausible interpretation is that GM-CSF can impose a macrophage fate on pro T cells even in a thymic environment. Today, T cell development can also be studied in 2D co-cultures of progenitor cells with stromal cells. OP9 stromal cells were originally derived from the BM of a newborn op/op mouse that is deficient for macrophage colony-stimulating factor [81]. OP9 and other stromal cells that express Notch ligands, Jag1 [20], Dll1 [19], or Dll4 [10,11,21,23,57,60], efficiently support T cell commitment from uncommitted stem cells or progenitors, and induce proliferation of committed T cell progenitors in vitro. This requires addition of at least Il7 and Flk2 ligand. On mixtures of both T cell-supporting (OP9Dll1 or OP9Dll4) and non-supporting (OP9) stromal cells, supplemented with lymphoid and myeloid growth factors, single pro T cells can give rise to both T and non-T lineages in mixed colonies [10–12,23,82]. Collectively, sophisticated in vitro assays for hematopoietic lineage choice have been developed. As a result, both the cloning efficiency (the number of colonies growing per total number of cells plated), as well as multiple lineage readouts (the number of different lineages that can be elicited from single pro T cells in vitro), have been greatly increased.
development in cultures of hematopoietic progenitor cells on OP9 stromal cells that expressed the Notch ligand delta-like-1 (Dll1) was reported [19]. Until March 2010, this method has been cited 384 times (source: Google Scholar), and has been applied in at least 66 publications (retrieved on pubmed.gov using the terms OP9 and T cell). This and other reports that have employed stromal cells that express Notch ligands [20,21] have paved the way to study early stages of T cell development in relatively simple cell culture systems (Box 1). The T cell development field, envious of their B cell colleagues who achieved the goal of in vitro differentiation years earlier [22], and limited to informative but tedious fetal thymus organ cultures (Box 1), enthusiastically welcomed the possibility to study T cell development in vitro. However, an as yet largely unrecognized pitfall of this experimental trend could be the exquisite sensitivity of the OP9 system, which, in combination with highly potent recombinant cytokines, might have reached a point at which these assays elicit potential that does not exist in vivo [23]. Finally, it has become possible to analyze global gene expression in rare cells that provide a glimpse of the homogeneity or heterogeneity of progenitors. Such experiments have shown that lineage-specific transcription 304
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factors are expressed in complex patterns along the path from HSCs to pro T cells [24]. Developments in the field of nuclear reprogramming, most prominently in the form of induced pluripotent stem cells, have revealed the capacity to reprogram the developmental potential of somatic cells [25]. One might consider the development of an adult mouse from a reprogrammed nucleus, derived originally from a mature lymphocyte [26], as an extreme case of this plasticity. However, if the nucleus of a mature B cell can be turned into a state of pluripotency, including germline competence, it might be of little surprise that progenitors committed within their hematopoietic environment, can be experimentally persuaded into alternative cell fates. T cell progenitors in BM, blood and thymus: where is the connection? The thymus is steadily colonized by progenitors of BM origin [27–31]. BM and blood contain ample progenitor populations that have shown T cell potential experimentally, but their importance as physiological T cell progenitors is not clear. The diversity and properties of progenitors at the interface between BM and thymus has been reviewed in depth [28–36]. Therefore, we outline progression of T cell development here only briefly. Long-term and short-term HSCs are included in the lineage marker (lin) Kit+Sca1+CD34 Flk2 and lin Kit+Sca1+CD34+Flk2 subsets. Downstream of HSCs are lin–Kit+Sca1+CD34+Flk2+ lymphoid-primed multipotent progenitors (LMPPs) [37], also termed MPPs [38], which lack self-renewal capacity. Based on expression of additional markers [Ccr9, L-selectin, B220, and recombination activating gene1 (Rag1)], subsets of MPPs exist [12–15,17,39]. All of these populations can generate T cells in the thymus following intravenous transfer but they are not T cell-committed. The second major progenitor group with T cell potential are interleukin 7 receptor a (Il7r)-expressing CLPs that exist as CLP-1 (lin KitloSca1loIl7r+Flk2+) [5,40] and CLP-2 (Kit Il7r+B220+CD19 ). CLP-2 cells were identified via expression of a transgenic pre T cell receptor a ( pTcra) gene reporter [16]. After intravenous transfer, CLPs have T and B cell potential, and lack sustained BM engraftment. As a result, CLPs give rise to T cells only transiently, and seed the thymus faster than MPPs [41,42]. CLPs have little, if any, myeloid potential in methylcellulose assays and in adoptive transfer experiments in vivo (Figure 1a) [5,40,43]. However, they do have myeloid potential in stromal cell cultures (Figure 1b) [17,23,43]. Particular BM progenitors might, in reality, not be released into the blood, thus making progenitor cells in the circulation more plausible thymic homing candidates than BM progenitors. The peripheral blood contains MPPs [14,42,44–46] and CLPs, according to some [14,42,45], but not others [46]. Committed T cell progenitors (KitlowThy1+Il7r+) were identified in fetal blood [44] and a corresponding population, termed circulating T cell progenitors (CTPs), has more recently been found in adult blood [47]. It was estimated that day 15.5 fetal mice have 4500 committed T cell progenitors in their blood [44], and that adult blood contains 300 MPPs [46], and 660 CTPs [47].
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Figure 1. Experimental approaches to determine hematopoietic progenitor potential and their conclusions. All diagrams show highly simplified, possible intermediate stages from HSCs (grey) to lymphoid (grey turning green) or myeloid (grey turning red) lineages. (a) Intravenous transplantation of CLP or CMP progenitor subsets, as well as stroma cell-free methylcellulose or liquid culture assays have overall supported a dichotomy model of hematopoiesis. Early after the megakaryocyte–erythrocyte branch point (not depicted), lymphoid and myeloid arms segregate, and give rise to T, B and NK cells, or macrophages, DCs, and granulocytes. The prediction from this model is that T- and B cell progenitors arise from a lymphoid-restricted pathway, and, consequently, should lack myeloid potential. (b) Single-cell stromal culture assays (such as the OP9 system), and modified thymic organ cultures, each supplemented with myeloid growth factors, have revealed abundant myeloid potential for macrophages and granulocytes in T cell progenitors. Together with earlier data on common B cell and macrophage progenitors, these findings support a new concept, the myeloid-based model of lymphopoiesis, in which myeloid potential is retained in T- and B cell progenitors. It postulates T/M and B/M progenitors (green and red) rather than separate lymphoid and myeloid pathways. (c) Recent genetic fate mapping studies have tracked the origin and potential of Il7r-mRNA-expressing cells in otherwise non-manipulated mice in vivo. These experiments have demonstrated quantitatively that the vast majority of all lymphoid cells arise from Il7r-positive progenitors, whereas most myeloid cells derive from Il7r-negative progenitors. Moreover, these experiments have found no evidence for the earlier claim that pro T cells realize potential for myeloid cells, including DCs, in the thymus in vivo. Hence, fate mapping provides in vivo support for a revived lymphoid–myeloid dichotomy model in which Il7r-positive lymphoid progenitors do not possess significant myeloid potential in central and peripheral lymphoid organs.
It is worthwhile to consider the cell numbers used in adoptive transfer experiments to study early T cell development. Considering MPPs and CTPs together as blood progenitor cells, intravenous transfer of 14 000 BM CLPs in adoptive transfer experiments [42] corresponds roughly to a 15-fold excess compared with normal numbers of circulating progenitor cells. Likewise, intravenous injection of 105 lin CD27+Flk2+ BM cells [42] is akin to placing 1.6107 total BM cells into the blood. This mimics a situation in which there are more BM cells than total blood leukocytes (107 per mouse) in the circulation. Similarly, in short-term homing assays, which reveal that CLP-2 cells more efficiently enter the thymus than CLP-1 cells, 107 lin BM cells were injected into mice, which corresponded to 5105 CLP-1 and CLP-2 cells [48]. It is possible that injection of such high numbers of BM progenitor cells into the blood generates undue pressure on the thymic entry niches. Thus, reminiscent of a hunter who uses buckshot to hit a target, this could result in intrathymic T cell development from non-physiological T cell progenitors. It has been estimated that BM CLPs seed the thymus at low frequencies (< 1/1000), while a subset of Vcam1 Ccr9 BM MPPs homes efficiently (1/19) to the thymus [14]. Hence, thymus colonization is not in principle rare for T cell progenitors. In the thymus, lin CD44+CD25 Kit+ pro T cells, also termed early thymic progenitors [41], or CD4 and CD8 double-negative (DN)1 cells [45], can be resolved into yet smaller subsets based on expression of CD24 [49], Flk2 [50] and Ccr9 [12]. Early stages of intrathymic T cell develop-
ment are controlled by cytokine receptor signaling, first via the receptor tyrosine kinases Kit [51] and Flk2 [52], and subsequently via the Il7r (reviewed in [53]). As discussed in more detail below, Notch1 function is essential to prevent pro T cells from aberrantly taking non-T lineage fates in vivo; notably towards B cell and DCs lineages [54] (reviewed in [55,56]). Which Notch1 ligand drives thymic T cell development has recently been addressed by deleting delta-like-4 (Dll4) in thymic epithelium [57,58], or using nude mouse blastocyst complementation [59] to construct a thymus that lacks Dll4 expression in the epithelium [54]. In all three cases, loss of Dll4 abrogated T cell development. Hence, Dll4 is the physiologically relevant thymic Notch1 ligand. Consequently, expression of Dll4 on stromal cells efficiently supports T cell development in vitro [10,11,23,57,60]. Collectively, comparative analysis of T cell progenitors isolated from the BM, blood and thymus has revealed no obvious precursor–product relationship between prethymic and intrathymic populations. This could be explained by phenotypic changes that might occur during BM exit, migration in the blood, or during thymic entry, as shown in vivo for Il7r mRNA which is transiently expressed along the pathway from HSCs to pro T cells [23]. Delineation of pathways leading to early T cell development based on Il7r expression The thymus might be seeded by MPPs [12,14,45,46], CLPs [5,16,42,48], common T and myeloid (TM) progenitors [10,11], or committed T cell progenitors [44,47]. These 305
Review populations fall into two major groups with regards to Il7r expression. Most MPPs and presumably TMs are Il7r , whereas CLP subsets and committed T cell progenitors are Il7r+. Pro T cells lack Il7r mRNA expression at the population level [41,61]. Indeed, recent single-cell analyses have shown that 90% of pro T cells are negative for Il7r mRNA [23]. The absence of Il7r expression on pro T cells and their non-responsiveness to Il7 indicate that the thymus is colonized by Il7r-negative progenitors, and this has been taken as an argument to exclude lymphoidrestricted progenitors, such as CLPs, as relevant thymus-colonizing cells [41]. However, despite the lack of Il7r mRNA expression in pro T cells, new findings from Il7rCre-driven fate mapping experiments (Box 3) have showed that 85% of pro T cells have a previous history of Il7r expression. The pro T cell population can, in this way, be split into a major subset (85%) that is derived from Il7r+ progenitors, and a minor subset that arises from Il7r progenitors [23]. In other words, Il7r-marked progenitors are the predominant but not the exclusive route into the thymus. Are Il7r+ progenitor cells necessarily lymphoid restricted? An Il7-responsive fetal liver progenitor with Box 2. In vivo assays for early T cell development The potential of progenitors isolated from the BM, blood or thymus to home to the thymus, and to generate large numbers of progeny that follow all orderly stages of T cell differentiation [28], can be analyzed by intravenous cell transfer. The first question to consider is whether the injected cells have BM engrafting capability. If they do, then thymus seeding might occur not only rapidly (within the first day) after adoptive transfer, but also later and secondary to the BM engraftment. At least in irradiated recipients, this can be expected for long-term and short-term HSCs. Hence, these experiments do not readily resolve the question of whether HSCs have direct thymus-homing properties. In contrast, MPPs and CLPs lack BM engraftment potential, and are likely to seed the thymus directly. Irradiation of the host is a crucial factor [83]. Without irradiation, the injected progenitors have to conquer occupied niches in the entry sites of the thymus. This is almost impossible in wild-type mice, [84], but is possible in non-irradiated Il7r-deficient mice [42,84]. It is unknown whether BM progenitors that colonize the thymus following intravenous injection are also mobilized from BM to blood in normal mice, that is, cells that reach the thymus experimentally might not normally be destined to go there. In the thymus, and under the influence of chemokines, cytokines and Notch ligands, many progenitors are likely to enter the T cell pathway. Such progenitors would fulfill criteria of a T cell progenitor, that is, thymus colonization and generation of T cells; yet, the result might be determined by the experiment rather than an inherent property of the cell. An alternative to the strategy of testing the potential of positively isolated cells (one population at a time) has recently been reported. By injecting total BM cells depleted of certain phenotypes (for example total BM cells minus all Flk2+ BM cells), the T cell potential in BM might be narrowed down. Using this method, all T cell potential with the BM was found to reside in a Flk2+CD27+ population that included MPPs and CLPs [42]. Finally, by bypassing the necessity for thymus homing, T cell potential can be assessed by direct intrathymic injection [85], which, in non-irradiated recipients, reveals the precise intrathymic developmental kinetics of progenitor cells [86,87]. It should be emphasized that, in most adoptive transfer experiments, it cannot be excluded that progenitors alter their properties, which could include their potential when placed in a new environment. For example, the traditional lymphoid–myeloid dichotomy model (Figure 1a) is largely based on intravenous transfer experiments but it is unknown which the blood stream, a prerequsite to enter the T cell pathway.
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dual in vitro potential for B cells and macrophages was described many years ago [62]. Similarly, under in vitro conditions, CLPs, which are Il7r+, show significant potential for B cells and myeloid cells [43]. However, based on the history of Il7r expression, fate mapping has shown a clear segregation of marked lymphocytes [T, B and natural killer (NK) cells], and non-marked macrophages and neutrophilic granulocytes [23], which demonstrates that Il7r+ progenitors are lymphoid-restricted, and do not serve as myeloid progenitors under physiological conditions. It remains possible, however, that conditions that perturb steady-state hematopoiesis, for example, inflammation, infection or BM transplantation, lead to myeloid recruitment from myeloid and lymphoid pathways in vivo [63]. Myeloid potential of pro T cells: evidence for a myeloidbased model of hematopoiesis As an exception to the rule that pro T cells are expected to be lymphoid and not myeloid progenitors, DCs potential of pro T cells in vitro, and upon adoptive transfer was reported many years ago [64]. Although it has not been shown that DCs develop from pro T cells in the normal thymus in situ, a subset of thymic DCs has been considered lymphoid [65], and the DCs potential of pro T cells has become textbook knowledge [66]. The DCs potential of pro T cells is, under certain experimental conditions, a fact. However, fate mapping experiments in two different Cre mouse lines, Il7rCre [23] and Cpa3Cre [54], have provided evidence for a non-lymphoid origin of thymic and splenic DCs in vivo, and against the intrathymic DCs generation from pro T cells. These new findings contradict the concept of a common T cell and DCs progenitor. However, even though the DCs potential of pro T cells has been considered established, it does not seem to count towards myeloid potential of pro T cells [42], and, therefore, attempts have been made to measure true myeloid, that is, macrophage or granulocyte potential of pro T cells. Early experiments using methycellulose assays identified only one myeloid colony per 2500 pro T cells in response to Il3 and Epo [67] (Figure 1a). Although this low frequency might have come from contaminating HSCs or MPPs, pro T cells gave rise to macrophages at much higher frequencies (ranging from 1 in 14 [68] to almost 1 in 2 [69]) when cultured on stromal cells in the presence of myeloid cytokines. Finally, under conditions that promote both T cell and myeloid lineage development from single cells in culture (Box 1), between 7% and 60% of pro T cells generate mixed T and macrophage colonies, and thus behave in vitro as bipotent T-myeloid progenitors. The notion that pro T cells have myeloid potential seemed incompatible with the pro T cell descent from a lymphoid-restricted progenitor [10,11]. However, this rested on the assumption that only pro T cells have abundant myeloid potential, yet CLPs had not been analyzed in parallel. A recent direct comparison of pro T cells and CLPs has found comparably high frequencies of myeloid colonies from both types of progenitors [23]. This could mean: (1) that CLPs are not lymphoid-restricted because they have abundant myeloid potential in vitro; or (2) that the in vitro assay provokes non-physiological potential. The fact that neither CLPs nor pro T cells make significant numbers of myeloid cells in vivo [23,40,42,43]
Review makes it most likely that the myeloid readout of these populations is an in vitro phenomenon. Analyses of the precursor–product relationship between intrathymic progenitors and thymic macrophages in mixed BM chimeras suggested that 30% of thymic macrophages arise from intrathymic progenitors that also generate T cells [11]. It remains to be determined why this frequency is higher than that of marked thymic macrophages in Il7rCre fate mapping experiments. In a transgenic V(D)J recombination reporter mouse, in which a fluorescent reporter was permanently expressed in a Rag1- and Rag2-dependent manner [70], about 50% of pro T cells and almost half of the thymic granulocytes were marked [10]. Based on a reporter mouse that visualizes expression of the Rag1 locus [Rag1 green fluorescent protein (Gfp) reporter], pro T cells do not transcribe Rag1 mRNA [13], which suggests that pro T cells were labeled before the pro T cell stage. Thus, Rag expressing progenitors can give rise to granulocyte progeny but such progenitors are not necessarily pro T cells in the thymus. Expression of lymphoid markers and V(D)J recombination has been reported in a subset of neutrophilic granulocytes [71], which might be related to reporter marking of thymic granulocytes. The conclusions drawn from these studies were not limited to the potential of pro T cells, in the sense of a possible fate choice, akin to what can happen under artificial conditions such as ectopic expression of cytokine receptors (Il2rb) [72], transcription factors driving myeloid development (CEBPa, PU.1, Gata3) [73–75], or by interference with Notch signaling [54,55]. Instead, these studies have gained significance because of their broader implications for the in vivo origin of T cells, and hence for the hematopoietic tree in general. Based on these data, the fundamental division of the hematopoietic system into separate lymphoid and myeloid branches has been amended with a myeloid-based model in which each lymphoid pathway (T and B) retains myeloid potential (M) in the form of T/M and B/M but not T/B pathways [7–9] (Figure 1b). This model is, however, inconsistent with the aforementioned fate mapping experiment that has shown that most pro T cells descend from Il7r+ progenitors whereas thymic macrophages, DCs, neutrophils and granulocytes are generally the progeny of Il7r progenitors. These findings exclude the majority of, if not all pro T cells as the physiological progenitors of myeloid cells in the thymus [23], and provide strong support for a revived lymphoid–myeloid dichotomy model (Figure 1c). Availability of Notch ligands in in vitro and in vivo assays for progenitor potential How can this discrepancy between in vitro and in vivo measurements of T cell progenitor potential be explained? A key difference between the thymus in situ and the in vitro assays that reveal pro T cell myeloid potential is the local availability of Notch ligands. Within the thymic microenviroment, T cell progenitors are probably in constant contact with Notch ligand Dll4-expressing thymic epithelial cells [54,57,58], which provides continuous Notch1 signaling (reviewed in [55]). Deletion of Notch1 in HSCs, BM progenitors, or pro T cells blocks T cell development (reviewed in [55]), and results in cell-intrinsic fate conver-
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sion of pro T cells into immature B cells, as well as into conventional and plasmacytoid DCs [54]. It is unknown whether loss of Notch1 expression in pro T cells also leads to the generation of macrophages or granulocytes from pro T cells in the thymus. In any case, the thymic microenvironment itself is permissive for non-T cell fates because Notch1 mutated pro T cells can give rise to, at least, B cells and DCs in the thymus. All in vitro assays that support T cell development take advantage of the T cell lineage-promoting activity of Notch1 signaling by culture of progenitors on feeder cells that express Notch ligands, such as OP9-Dll1 or OP9-Dll4 (Box 2). In contrast, the development of myeloid cells from pro T cells occurs only when pro T cells are cultured with myeloid growth factors, and, at the same time, are deprived of Notch ligands, such as in OP9 cultures, or when Notch ligands are limiting, such as in cultures with mixtures of OP9 and OP9-Dll4. Even transdifferentiation of T cell progenitors by forced expression of Cebpa into macrophages [73] and by Gata3 into mast cells [75] was strongly diminished by Notch1 ligands in vitro. Collectively, Notch1 expression keeps pro T cells on their T cell track in vivo and in vitro by preventing lineage diversion towards non-Tlineage fates [56]. It is plausible that, in in vitro assays in which Notch ligands are limiting, the effects on T cell
Box 3. Fate mapping of early T cell development Fate mapping experiments can identify in vivo the developmental origin of cells. The principle is simple: lineage- or stage-specific gene expression is used to tag cells and their progeny, which belong to a particular lineage or pass though a certain stage of differentiation. Fate mapping is possible in an engineered manner by using Cre recombinase and its recognition sequences; a tool widely used to delete or flip genomic sequences in vivo [88]. Cre/loxP and Flp/ FRT are the most established recombination systems [89] but others exist (Dre and Tre recombinase) [90,91]. The goal is to bring Cre under control of an endogenous promoter, which can be achieved most reliably by knock-in strategies in embryonic stem cells. Engineered BAC transgenes are also used, but the results might differ from knock-in loci because BACs are usually present in multiple copies, and integrate at random sites in the genome leaving the possibly for position effects on expression patterns. The specificity of Cre expression might thus differ between BAC transgenes and knock-in loci. Once expressed, Cre turns on a Credependent reporter. Reporter genes are usually inserted in the ubiquitously expressed ROSA26 locus and permanently drive, after Cre-mediated recombination, expression of green, yellow, cyan [92] or red [93] fluorescent proteins. Ideally, Cre expression labels all cells of a given lineage (inclusiveness), and few other cells (specificity). Reporter onset becomes visible within 24 h and continues to increase its intensity as a result of reporter protein maturation and accumulation [23,94]. To trace the origin of hematopoietic lineages, it is crucial to choose informative loci. Examples include B cellspecific Mb-1Cre [95], early pan-lymphocyte alleles Rag1Cre [63, 96] and Il7rCre [23]. While Cre-driven fate mapping can be a powerful technique, shortcomings of this approach exist. The absence of marking by a particular Cre knock-in locus does not necessarily prove that Cre is not expressed in this cell population because expression thresholds must be crossed in order for marking to occur. Moreover, the Cre lines employed must be very carefully validated to demonstrate that they recreate the endogenous expression pattern with fidelity. Finally, it is important to note that neomycin resistance cassettes, if not removed from the targeted loci [96], can affect Cre expression, and that genotoxicity of Cre in the mouse genome can be a concern [97].
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Review potential are similar to blocking Notch1 signals in vivo. Finally, based on in vitro differentiation experiments, the concept that T-lineage commitment only occurs in DN3 cells, and not in DN1 and DN2 cells [10,11,76], might be viewed as Notch1-dependent (DN1 and DN2) and Notch1independent (DN3) phases of T cell commitment. However, important aspects of T cell development, such as pTa expression, remain Notch1-dependent in DN3 cells. Concluding remarks A hallmark of traditional models of hematopoiesis is the early separation into lymphoid and myeloid branches. Pathways from HSCs to the thymus are considered lymphocyte-restricted. Highly sensitive in vitro assays for T cell development have revealed abundant myeloid potential in pro T cells. Dual T cell and myeloid potential seemed incompatible with the idea that pro T cells arise from a lymphocyte-restricted pathway, and a new model of hematopoiesis, where the thymus is seeded by a common T and myeloid progenitor, has been proposed. However, in vivo marking of cells that transiently or permanently express Il7r has revealed separation of lymphoid and myeloid pathways. This lineage tracing has shown that the thymus is predominantly, but not exclusively, colonized by lymphoid-restricted progenitors, whereas the vast majority of myeloid cells in the thymus arise from a different, non-lymphoid restricted pathway. Moreover, in contrast to in vitro findings, T cell progenitors lack significant potential for myeloid lineages including DCs in the thymus. These discrepancies suggest that potent in vitro assays can impose myeloid potential onto lymphoidrestricted progenitors such as thymic pro T cells, and BM CLPs. Under particular conditions, it seems possible to override the normal developmental potential of progenitors. Thus, more sensitive assays for hematopoiesis do not necessarily produce physiological results. The cells that migrate from the BM to the thymus require further investigation to understand their identity, migration characteristics, turnover, release and homing mechanisms into thymic entry niches. Further challenging questions will address how pathways from HSCs to the thymus are disturbed under pathological conditions such as infection, myeloablation followed by BM transplantation, and under myelotoxic and thymotoxic conditions such as steroid treatment, ionizing radiation, or chronic diseases associated with catabolic stress. Acknowledgements We thank Hans Jo¨rg Fehling for collaborations on underlying fate mapping studies, and Carmen Blum, Hans Jo¨rg Fehling, Thorsten Feyerabend and Vera Martins for critical reading of the manuscript. SMS is supported by a fellowship from the DFG (Schl-1897/1-1). HRR is supported by the DFG through SFB 497-B5 and KFO 142-P8, and receives funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/20072013) / ERC grant agreement no. 233074.
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Early T cell development and the pitfalls of potential Susan M. Schlenner1 and Hans-Reimer Rodewald2,3 1
Department for Cancer Immunology and AIDS, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA Department for Cellular Immunology, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, D-69120 Heidelberg, Germany 3 Institute for Immunology, University of Ulm, D-89081 Ulm, Germany 2
The long-standing model for hematopoiesis, which features a dichotomy into separate lymphoid and myeloid branches, predicts that progenitor T cells arise from a lymphocyte-restricted pathway. However, experiments that have detected myeloid potential in progenitor T cells have been reported as evidence to question this model. Mapping physiological differentiation pathways has now led to opposite conclusions, by showing that T cells and thymic myeloid cells have distinct origins and that, in vivo, T cell progenitors lack significant potential for myeloid lineages including dendritic cells. Here, we review the underlying experiments that have led to such fundamentally different conclusions. The current controversy might reflect a need to distinguish between cell fates that are possible experimentally from physiological fate choices, to build a map of immunological differentiation pathways. Controversy of T cell progenitor potential and implications for hematopoietic differentiation pathways Hematopoiesis is arguably the most-studied system in which stem cells give rise to progenitors that, in turn, yield mature and functionally diverse cells [1]. Much of what we know about the development of blood and immune cells is based on the isolation of phenotypically defined progenitor cell populations, followed by an assessment of their in vitro and in vivo developmental potential and deviations from the system in hematopoietic mutants [2]. Despite the information gained from these experiments, major routes within the hematopoietic tree have become increasingly vague [3,4]. Initially, relatively simple maps were drawn where long-term self-renewing multipotent hematopoietic stem cells (HSCs) were connected to short-term multipotent progenitors (MPPs). Downstream from the MPPs, a split into lymphocyte-restricted and myelo-erythroidrestricted progenitors was postulated. This dichotomy model was strongly supported by the identification of progenitors with distinct cell surface phenotypes that appeared to qualify as common lymphocyte progenitors (CLPs) [5] and common myeloid progenitors (CMPs) [6]. In the original model, the pathway from the bone marrow (BM) to the thymus belonged to the lymphoidrestricted branch of hematopoiesis. However, this model has recently been called into question [7–9]. This conceptual change was based on the detection of abundant Corresponding author: Rodewald, H.-R. ([email protected]).
myeloid potential in progenitor (pro) T cells [10,11]. These studies have implied that pro T cells give rise to myeloid cells, including dendritic cells (DCs), macrophages and granulocytes, in the thymus. The common T and myeloid potential of pro T cells seemed incompatible with a descent of pro T cells from a lymphoid-restricted progenitor, thus casting doubt on the fundamental lymphoid–myeloid division of the hematopoietic system. In the alternative, myeloid-based model, each lymphoid pathway retains myeloid potential downstream from the T versus B split [7–9]. In contrast to these reports, new experiments that have employed genetic tools to map physiological differentiation pathways in vivo have shown that T cells and thymic myeloid cells have distinct origins, and that T cell progenitors lack significant potential for myeloid lineages in vivo. We review the basic principles and results of these experiments, discuss their strengths and limitations, and their diverging conclusions in the broader context of hematopoietic differentiation. Plasticity of cell fate and vagaries in hematopoiesis Several developments in the field of hematopoiesis and lineage commitment have complicated the straightforward view of non-redundant differentiation pathways, such as MMPs giving rise to lymphoid-restricted CLPs or myeloidrestricted CMPs. The phenotypic resolution and isolation of progenitor populations has steadily improved because of the availability of vast numbers of antibodies for cell surface marker detection. Moreover, reporter mice can reveal expression of stage- and lineage-specific genes from transgenic, bacterial artificial chromosome (BAC), or targeted knock-in loci. These advances have been combined with major improvements in cell sorting. Consequently, additional progenitor subpopulations, partially overlapping with or distinct from MPPs [12–14], CLPs [15–17], CMPs [18] or lineage committed progenitor cells, have been identified. Are such populations redundant or do they represent different stages along the same pathway? With every new subset of functionally related progenitor cells that are identified, the overall importance of each individual population for the generation or maintenance of a given lineage becomes less certain. For the most part, quantitative information on specific developmental pathways during hematopoiesis is lacking, and the steps obligatory for and the possible alternative routes of development remain undefined. The sensitivity of readouts for hematopoietic potential has also increased greatly. In 2002, the induction of T cell
1471-4906/$ – see front matter ß 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.it.2010.06.002 Trends in Immunology 31 (2010) 303–310
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Review Box 1. In vitro assays for early T cell development For many years, T cell development could not be recapitulated in 2D cell culture systems. Instead, T cell development has been studied successfully in intact fetal thymus organ culture (FTOC) [77], and in re-aggregated fetal thymus organ culture (RFTOC), assembled from epithelial and mesenchymal thymic stromal cells [78]. Entry of progenitors into the thymus via the normally impermeable capsule is enhanced by co-culturing of the thymic lobes with progenitors in hanging drops. Deoxyguanosine treatment purges the thymus of its endogenous thymocytes and thus reduces the competition with exogenous progenitors. FTOC, normally placed on filter discs, can also be kept in high oxygen submersion cultures that allow, in the presence of added cytokines, multi-lineage readouts from single progenitors [79]. FTOC or RFTOC experiments probably closely resemble the near-normal thymic environment, provided that the cells find their proper position in situ to receive the signals that fits their own stage of development [80]. Addition of exogenous growth factors such as granulocyte–macrophage colony-stimulating factor (GM-CSF), which normally drives differentiation and proliferation of myeloid progenitors, to FTOC [11] is probably an unusual condition for the thymic environment. Although macrophages can be generated from pro T cells under these conditions [11], a plausible interpretation is that GM-CSF can impose a macrophage fate on pro T cells even in a thymic environment. Today, T cell development can also be studied in 2D co-cultures of progenitor cells with stromal cells. OP9 stromal cells were originally derived from the BM of a newborn op/op mouse that is deficient for macrophage colony-stimulating factor [81]. OP9 and other stromal cells that express Notch ligands, Jag1 [20], Dll1 [19], or Dll4 [10,11,21,23,57,60], efficiently support T cell commitment from uncommitted stem cells or progenitors, and induce proliferation of committed T cell progenitors in vitro. This requires addition of at least Il7 and Flk2 ligand. On mixtures of both T cell-supporting (OP9Dll1 or OP9Dll4) and non-supporting (OP9) stromal cells, supplemented with lymphoid and myeloid growth factors, single pro T cells can give rise to both T and non-T lineages in mixed colonies [10–12,23,82]. Collectively, sophisticated in vitro assays for hematopoietic lineage choice have been developed. As a result, both the cloning efficiency (the number of colonies growing per total number of cells plated), as well as multiple lineage readouts (the number of different lineages that can be elicited from single pro T cells in vitro), have been greatly increased.
development in cultures of hematopoietic progenitor cells on OP9 stromal cells that expressed the Notch ligand delta-like-1 (Dll1) was reported [19]. Until March 2010, this method has been cited 384 times (source: Google Scholar), and has been applied in at least 66 publications (retrieved on pubmed.gov using the terms OP9 and T cell). This and other reports that have employed stromal cells that express Notch ligands [20,21] have paved the way to study early stages of T cell development in relatively simple cell culture systems (Box 1). The T cell development field, envious of their B cell colleagues who achieved the goal of in vitro differentiation years earlier [22], and limited to informative but tedious fetal thymus organ cultures (Box 1), enthusiastically welcomed the possibility to study T cell development in vitro. However, an as yet largely unrecognized pitfall of this experimental trend could be the exquisite sensitivity of the OP9 system, which, in combination with highly potent recombinant cytokines, might have reached a point at which these assays elicit potential that does not exist in vivo [23]. Finally, it has become possible to analyze global gene expression in rare cells that provide a glimpse of the homogeneity or heterogeneity of progenitors. Such experiments have shown that lineage-specific transcription 304
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factors are expressed in complex patterns along the path from HSCs to pro T cells [24]. Developments in the field of nuclear reprogramming, most prominently in the form of induced pluripotent stem cells, have revealed the capacity to reprogram the developmental potential of somatic cells [25]. One might consider the development of an adult mouse from a reprogrammed nucleus, derived originally from a mature lymphocyte [26], as an extreme case of this plasticity. However, if the nucleus of a mature B cell can be turned into a state of pluripotency, including germline competence, it might be of little surprise that progenitors committed within their hematopoietic environment, can be experimentally persuaded into alternative cell fates. T cell progenitors in BM, blood and thymus: where is the connection? The thymus is steadily colonized by progenitors of BM origin [27–31]. BM and blood contain ample progenitor populations that have shown T cell potential experimentally, but their importance as physiological T cell progenitors is not clear. The diversity and properties of progenitors at the interface between BM and thymus has been reviewed in depth [28–36]. Therefore, we outline progression of T cell development here only briefly. Long-term and short-term HSCs are included in the lineage marker (lin) Kit+Sca1+CD34 Flk2 and lin Kit+Sca1+CD34+Flk2 subsets. Downstream of HSCs are lin–Kit+Sca1+CD34+Flk2+ lymphoid-primed multipotent progenitors (LMPPs) [37], also termed MPPs [38], which lack self-renewal capacity. Based on expression of additional markers [Ccr9, L-selectin, B220, and recombination activating gene1 (Rag1)], subsets of MPPs exist [12–15,17,39]. All of these populations can generate T cells in the thymus following intravenous transfer but they are not T cell-committed. The second major progenitor group with T cell potential are interleukin 7 receptor a (Il7r)-expressing CLPs that exist as CLP-1 (lin KitloSca1loIl7r+Flk2+) [5,40] and CLP-2 (Kit Il7r+B220+CD19 ). CLP-2 cells were identified via expression of a transgenic pre T cell receptor a ( pTcra) gene reporter [16]. After intravenous transfer, CLPs have T and B cell potential, and lack sustained BM engraftment. As a result, CLPs give rise to T cells only transiently, and seed the thymus faster than MPPs [41,42]. CLPs have little, if any, myeloid potential in methylcellulose assays and in adoptive transfer experiments in vivo (Figure 1a) [5,40,43]. However, they do have myeloid potential in stromal cell cultures (Figure 1b) [17,23,43]. Particular BM progenitors might, in reality, not be released into the blood, thus making progenitor cells in the circulation more plausible thymic homing candidates than BM progenitors. The peripheral blood contains MPPs [14,42,44–46] and CLPs, according to some [14,42,45], but not others [46]. Committed T cell progenitors (KitlowThy1+Il7r+) were identified in fetal blood [44] and a corresponding population, termed circulating T cell progenitors (CTPs), has more recently been found in adult blood [47]. It was estimated that day 15.5 fetal mice have 4500 committed T cell progenitors in their blood [44], and that adult blood contains 300 MPPs [46], and 660 CTPs [47].
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Figure 1. Experimental approaches to determine hematopoietic progenitor potential and their conclusions. All diagrams show highly simplified, possible intermediate stages from HSCs (grey) to lymphoid (grey turning green) or myeloid (grey turning red) lineages. (a) Intravenous transplantation of CLP or CMP progenitor subsets, as well as stroma cell-free methylcellulose or liquid culture assays have overall supported a dichotomy model of hematopoiesis. Early after the megakaryocyte–erythrocyte branch point (not depicted), lymphoid and myeloid arms segregate, and give rise to T, B and NK cells, or macrophages, DCs, and granulocytes. The prediction from this model is that T- and B cell progenitors arise from a lymphoid-restricted pathway, and, consequently, should lack myeloid potential. (b) Single-cell stromal culture assays (such as the OP9 system), and modified thymic organ cultures, each supplemented with myeloid growth factors, have revealed abundant myeloid potential for macrophages and granulocytes in T cell progenitors. Together with earlier data on common B cell and macrophage progenitors, these findings support a new concept, the myeloid-based model of lymphopoiesis, in which myeloid potential is retained in T- and B cell progenitors. It postulates T/M and B/M progenitors (green and red) rather than separate lymphoid and myeloid pathways. (c) Recent genetic fate mapping studies have tracked the origin and potential of Il7r-mRNA-expressing cells in otherwise non-manipulated mice in vivo. These experiments have demonstrated quantitatively that the vast majority of all lymphoid cells arise from Il7r-positive progenitors, whereas most myeloid cells derive from Il7r-negative progenitors. Moreover, these experiments have found no evidence for the earlier claim that pro T cells realize potential for myeloid cells, including DCs, in the thymus in vivo. Hence, fate mapping provides in vivo support for a revived lymphoid–myeloid dichotomy model in which Il7r-positive lymphoid progenitors do not possess significant myeloid potential in central and peripheral lymphoid organs.
It is worthwhile to consider the cell numbers used in adoptive transfer experiments to study early T cell development. Considering MPPs and CTPs together as blood progenitor cells, intravenous transfer of 14 000 BM CLPs in adoptive transfer experiments [42] corresponds roughly to a 15-fold excess compared with normal numbers of circulating progenitor cells. Likewise, intravenous injection of 105 lin CD27+Flk2+ BM cells [42] is akin to placing 1.6107 total BM cells into the blood. This mimics a situation in which there are more BM cells than total blood leukocytes (107 per mouse) in the circulation. Similarly, in short-term homing assays, which reveal that CLP-2 cells more efficiently enter the thymus than CLP-1 cells, 107 lin BM cells were injected into mice, which corresponded to 5105 CLP-1 and CLP-2 cells [48]. It is possible that injection of such high numbers of BM progenitor cells into the blood generates undue pressure on the thymic entry niches. Thus, reminiscent of a hunter who uses buckshot to hit a target, this could result in intrathymic T cell development from non-physiological T cell progenitors. It has been estimated that BM CLPs seed the thymus at low frequencies (< 1/1000), while a subset of Vcam1 Ccr9 BM MPPs homes efficiently (1/19) to the thymus [14]. Hence, thymus colonization is not in principle rare for T cell progenitors. In the thymus, lin CD44+CD25 Kit+ pro T cells, also termed early thymic progenitors [41], or CD4 and CD8 double-negative (DN)1 cells [45], can be resolved into yet smaller subsets based on expression of CD24 [49], Flk2 [50] and Ccr9 [12]. Early stages of intrathymic T cell develop-
ment are controlled by cytokine receptor signaling, first via the receptor tyrosine kinases Kit [51] and Flk2 [52], and subsequently via the Il7r (reviewed in [53]). As discussed in more detail below, Notch1 function is essential to prevent pro T cells from aberrantly taking non-T lineage fates in vivo; notably towards B cell and DCs lineages [54] (reviewed in [55,56]). Which Notch1 ligand drives thymic T cell development has recently been addressed by deleting delta-like-4 (Dll4) in thymic epithelium [57,58], or using nude mouse blastocyst complementation [59] to construct a thymus that lacks Dll4 expression in the epithelium [54]. In all three cases, loss of Dll4 abrogated T cell development. Hence, Dll4 is the physiologically relevant thymic Notch1 ligand. Consequently, expression of Dll4 on stromal cells efficiently supports T cell development in vitro [10,11,23,57,60]. Collectively, comparative analysis of T cell progenitors isolated from the BM, blood and thymus has revealed no obvious precursor–product relationship between prethymic and intrathymic populations. This could be explained by phenotypic changes that might occur during BM exit, migration in the blood, or during thymic entry, as shown in vivo for Il7r mRNA which is transiently expressed along the pathway from HSCs to pro T cells [23]. Delineation of pathways leading to early T cell development based on Il7r expression The thymus might be seeded by MPPs [12,14,45,46], CLPs [5,16,42,48], common T and myeloid (TM) progenitors [10,11], or committed T cell progenitors [44,47]. These 305
Review populations fall into two major groups with regards to Il7r expression. Most MPPs and presumably TMs are Il7r , whereas CLP subsets and committed T cell progenitors are Il7r+. Pro T cells lack Il7r mRNA expression at the population level [41,61]. Indeed, recent single-cell analyses have shown that 90% of pro T cells are negative for Il7r mRNA [23]. The absence of Il7r expression on pro T cells and their non-responsiveness to Il7 indicate that the thymus is colonized by Il7r-negative progenitors, and this has been taken as an argument to exclude lymphoidrestricted progenitors, such as CLPs, as relevant thymus-colonizing cells [41]. However, despite the lack of Il7r mRNA expression in pro T cells, new findings from Il7rCre-driven fate mapping experiments (Box 3) have showed that 85% of pro T cells have a previous history of Il7r expression. The pro T cell population can, in this way, be split into a major subset (85%) that is derived from Il7r+ progenitors, and a minor subset that arises from Il7r progenitors [23]. In other words, Il7r-marked progenitors are the predominant but not the exclusive route into the thymus. Are Il7r+ progenitor cells necessarily lymphoid restricted? An Il7-responsive fetal liver progenitor with Box 2. In vivo assays for early T cell development The potential of progenitors isolated from the BM, blood or thymus to home to the thymus, and to generate large numbers of progeny that follow all orderly stages of T cell differentiation [28], can be analyzed by intravenous cell transfer. The first question to consider is whether the injected cells have BM engrafting capability. If they do, then thymus seeding might occur not only rapidly (within the first day) after adoptive transfer, but also later and secondary to the BM engraftment. At least in irradiated recipients, this can be expected for long-term and short-term HSCs. Hence, these experiments do not readily resolve the question of whether HSCs have direct thymus-homing properties. In contrast, MPPs and CLPs lack BM engraftment potential, and are likely to seed the thymus directly. Irradiation of the host is a crucial factor [83]. Without irradiation, the injected progenitors have to conquer occupied niches in the entry sites of the thymus. This is almost impossible in wild-type mice, [84], but is possible in non-irradiated Il7r-deficient mice [42,84]. It is unknown whether BM progenitors that colonize the thymus following intravenous injection are also mobilized from BM to blood in normal mice, that is, cells that reach the thymus experimentally might not normally be destined to go there. In the thymus, and under the influence of chemokines, cytokines and Notch ligands, many progenitors are likely to enter the T cell pathway. Such progenitors would fulfill criteria of a T cell progenitor, that is, thymus colonization and generation of T cells; yet, the result might be determined by the experiment rather than an inherent property of the cell. An alternative to the strategy of testing the potential of positively isolated cells (one population at a time) has recently been reported. By injecting total BM cells depleted of certain phenotypes (for example total BM cells minus all Flk2+ BM cells), the T cell potential in BM might be narrowed down. Using this method, all T cell potential with the BM was found to reside in a Flk2+CD27+ population that included MPPs and CLPs [42]. Finally, by bypassing the necessity for thymus homing, T cell potential can be assessed by direct intrathymic injection [85], which, in non-irradiated recipients, reveals the precise intrathymic developmental kinetics of progenitor cells [86,87]. It should be emphasized that, in most adoptive transfer experiments, it cannot be excluded that progenitors alter their properties, which could include their potential when placed in a new environment. For example, the traditional lymphoid–myeloid dichotomy model (Figure 1a) is largely based on intravenous transfer experiments but it is unknown which the blood stream, a prerequsite to enter the T cell pathway.
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dual in vitro potential for B cells and macrophages was described many years ago [62]. Similarly, under in vitro conditions, CLPs, which are Il7r+, show significant potential for B cells and myeloid cells [43]. However, based on the history of Il7r expression, fate mapping has shown a clear segregation of marked lymphocytes [T, B and natural killer (NK) cells], and non-marked macrophages and neutrophilic granulocytes [23], which demonstrates that Il7r+ progenitors are lymphoid-restricted, and do not serve as myeloid progenitors under physiological conditions. It remains possible, however, that conditions that perturb steady-state hematopoiesis, for example, inflammation, infection or BM transplantation, lead to myeloid recruitment from myeloid and lymphoid pathways in vivo [63]. Myeloid potential of pro T cells: evidence for a myeloidbased model of hematopoiesis As an exception to the rule that pro T cells are expected to be lymphoid and not myeloid progenitors, DCs potential of pro T cells in vitro, and upon adoptive transfer was reported many years ago [64]. Although it has not been shown that DCs develop from pro T cells in the normal thymus in situ, a subset of thymic DCs has been considered lymphoid [65], and the DCs potential of pro T cells has become textbook knowledge [66]. The DCs potential of pro T cells is, under certain experimental conditions, a fact. However, fate mapping experiments in two different Cre mouse lines, Il7rCre [23] and Cpa3Cre [54], have provided evidence for a non-lymphoid origin of thymic and splenic DCs in vivo, and against the intrathymic DCs generation from pro T cells. These new findings contradict the concept of a common T cell and DCs progenitor. However, even though the DCs potential of pro T cells has been considered established, it does not seem to count towards myeloid potential of pro T cells [42], and, therefore, attempts have been made to measure true myeloid, that is, macrophage or granulocyte potential of pro T cells. Early experiments using methycellulose assays identified only one myeloid colony per 2500 pro T cells in response to Il3 and Epo [67] (Figure 1a). Although this low frequency might have come from contaminating HSCs or MPPs, pro T cells gave rise to macrophages at much higher frequencies (ranging from 1 in 14 [68] to almost 1 in 2 [69]) when cultured on stromal cells in the presence of myeloid cytokines. Finally, under conditions that promote both T cell and myeloid lineage development from single cells in culture (Box 1), between 7% and 60% of pro T cells generate mixed T and macrophage colonies, and thus behave in vitro as bipotent T-myeloid progenitors. The notion that pro T cells have myeloid potential seemed incompatible with the pro T cell descent from a lymphoid-restricted progenitor [10,11]. However, this rested on the assumption that only pro T cells have abundant myeloid potential, yet CLPs had not been analyzed in parallel. A recent direct comparison of pro T cells and CLPs has found comparably high frequencies of myeloid colonies from both types of progenitors [23]. This could mean: (1) that CLPs are not lymphoid-restricted because they have abundant myeloid potential in vitro; or (2) that the in vitro assay provokes non-physiological potential. The fact that neither CLPs nor pro T cells make significant numbers of myeloid cells in vivo [23,40,42,43]
Review makes it most likely that the myeloid readout of these populations is an in vitro phenomenon. Analyses of the precursor–product relationship between intrathymic progenitors and thymic macrophages in mixed BM chimeras suggested that 30% of thymic macrophages arise from intrathymic progenitors that also generate T cells [11]. It remains to be determined why this frequency is higher than that of marked thymic macrophages in Il7rCre fate mapping experiments. In a transgenic V(D)J recombination reporter mouse, in which a fluorescent reporter was permanently expressed in a Rag1- and Rag2-dependent manner [70], about 50% of pro T cells and almost half of the thymic granulocytes were marked [10]. Based on a reporter mouse that visualizes expression of the Rag1 locus [Rag1 green fluorescent protein (Gfp) reporter], pro T cells do not transcribe Rag1 mRNA [13], which suggests that pro T cells were labeled before the pro T cell stage. Thus, Rag expressing progenitors can give rise to granulocyte progeny but such progenitors are not necessarily pro T cells in the thymus. Expression of lymphoid markers and V(D)J recombination has been reported in a subset of neutrophilic granulocytes [71], which might be related to reporter marking of thymic granulocytes. The conclusions drawn from these studies were not limited to the potential of pro T cells, in the sense of a possible fate choice, akin to what can happen under artificial conditions such as ectopic expression of cytokine receptors (Il2rb) [72], transcription factors driving myeloid development (CEBPa, PU.1, Gata3) [73–75], or by interference with Notch signaling [54,55]. Instead, these studies have gained significance because of their broader implications for the in vivo origin of T cells, and hence for the hematopoietic tree in general. Based on these data, the fundamental division of the hematopoietic system into separate lymphoid and myeloid branches has been amended with a myeloid-based model in which each lymphoid pathway (T and B) retains myeloid potential (M) in the form of T/M and B/M but not T/B pathways [7–9] (Figure 1b). This model is, however, inconsistent with the aforementioned fate mapping experiment that has shown that most pro T cells descend from Il7r+ progenitors whereas thymic macrophages, DCs, neutrophils and granulocytes are generally the progeny of Il7r progenitors. These findings exclude the majority of, if not all pro T cells as the physiological progenitors of myeloid cells in the thymus [23], and provide strong support for a revived lymphoid–myeloid dichotomy model (Figure 1c). Availability of Notch ligands in in vitro and in vivo assays for progenitor potential How can this discrepancy between in vitro and in vivo measurements of T cell progenitor potential be explained? A key difference between the thymus in situ and the in vitro assays that reveal pro T cell myeloid potential is the local availability of Notch ligands. Within the thymic microenviroment, T cell progenitors are probably in constant contact with Notch ligand Dll4-expressing thymic epithelial cells [54,57,58], which provides continuous Notch1 signaling (reviewed in [55]). Deletion of Notch1 in HSCs, BM progenitors, or pro T cells blocks T cell development (reviewed in [55]), and results in cell-intrinsic fate conver-
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sion of pro T cells into immature B cells, as well as into conventional and plasmacytoid DCs [54]. It is unknown whether loss of Notch1 expression in pro T cells also leads to the generation of macrophages or granulocytes from pro T cells in the thymus. In any case, the thymic microenvironment itself is permissive for non-T cell fates because Notch1 mutated pro T cells can give rise to, at least, B cells and DCs in the thymus. All in vitro assays that support T cell development take advantage of the T cell lineage-promoting activity of Notch1 signaling by culture of progenitors on feeder cells that express Notch ligands, such as OP9-Dll1 or OP9-Dll4 (Box 2). In contrast, the development of myeloid cells from pro T cells occurs only when pro T cells are cultured with myeloid growth factors, and, at the same time, are deprived of Notch ligands, such as in OP9 cultures, or when Notch ligands are limiting, such as in cultures with mixtures of OP9 and OP9-Dll4. Even transdifferentiation of T cell progenitors by forced expression of Cebpa into macrophages [73] and by Gata3 into mast cells [75] was strongly diminished by Notch1 ligands in vitro. Collectively, Notch1 expression keeps pro T cells on their T cell track in vivo and in vitro by preventing lineage diversion towards non-Tlineage fates [56]. It is plausible that, in in vitro assays in which Notch ligands are limiting, the effects on T cell
Box 3. Fate mapping of early T cell development Fate mapping experiments can identify in vivo the developmental origin of cells. The principle is simple: lineage- or stage-specific gene expression is used to tag cells and their progeny, which belong to a particular lineage or pass though a certain stage of differentiation. Fate mapping is possible in an engineered manner by using Cre recombinase and its recognition sequences; a tool widely used to delete or flip genomic sequences in vivo [88]. Cre/loxP and Flp/ FRT are the most established recombination systems [89] but others exist (Dre and Tre recombinase) [90,91]. The goal is to bring Cre under control of an endogenous promoter, which can be achieved most reliably by knock-in strategies in embryonic stem cells. Engineered BAC transgenes are also used, but the results might differ from knock-in loci because BACs are usually present in multiple copies, and integrate at random sites in the genome leaving the possibly for position effects on expression patterns. The specificity of Cre expression might thus differ between BAC transgenes and knock-in loci. Once expressed, Cre turns on a Credependent reporter. Reporter genes are usually inserted in the ubiquitously expressed ROSA26 locus and permanently drive, after Cre-mediated recombination, expression of green, yellow, cyan [92] or red [93] fluorescent proteins. Ideally, Cre expression labels all cells of a given lineage (inclusiveness), and few other cells (specificity). Reporter onset becomes visible within 24 h and continues to increase its intensity as a result of reporter protein maturation and accumulation [23,94]. To trace the origin of hematopoietic lineages, it is crucial to choose informative loci. Examples include B cellspecific Mb-1Cre [95], early pan-lymphocyte alleles Rag1Cre [63, 96] and Il7rCre [23]. While Cre-driven fate mapping can be a powerful technique, shortcomings of this approach exist. The absence of marking by a particular Cre knock-in locus does not necessarily prove that Cre is not expressed in this cell population because expression thresholds must be crossed in order for marking to occur. Moreover, the Cre lines employed must be very carefully validated to demonstrate that they recreate the endogenous expression pattern with fidelity. Finally, it is important to note that neomycin resistance cassettes, if not removed from the targeted loci [96], can affect Cre expression, and that genotoxicity of Cre in the mouse genome can be a concern [97].
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Review potential are similar to blocking Notch1 signals in vivo. Finally, based on in vitro differentiation experiments, the concept that T-lineage commitment only occurs in DN3 cells, and not in DN1 and DN2 cells [10,11,76], might be viewed as Notch1-dependent (DN1 and DN2) and Notch1independent (DN3) phases of T cell commitment. However, important aspects of T cell development, such as pTa expression, remain Notch1-dependent in DN3 cells. Concluding remarks A hallmark of traditional models of hematopoiesis is the early separation into lymphoid and myeloid branches. Pathways from HSCs to the thymus are considered lymphocyte-restricted. Highly sensitive in vitro assays for T cell development have revealed abundant myeloid potential in pro T cells. Dual T cell and myeloid potential seemed incompatible with the idea that pro T cells arise from a lymphocyte-restricted pathway, and a new model of hematopoiesis, where the thymus is seeded by a common T and myeloid progenitor, has been proposed. However, in vivo marking of cells that transiently or permanently express Il7r has revealed separation of lymphoid and myeloid pathways. This lineage tracing has shown that the thymus is predominantly, but not exclusively, colonized by lymphoid-restricted progenitors, whereas the vast majority of myeloid cells in the thymus arise from a different, non-lymphoid restricted pathway. Moreover, in contrast to in vitro findings, T cell progenitors lack significant potential for myeloid lineages including DCs in the thymus. These discrepancies suggest that potent in vitro assays can impose myeloid potential onto lymphoidrestricted progenitors such as thymic pro T cells, and BM CLPs. Under particular conditions, it seems possible to override the normal developmental potential of progenitors. Thus, more sensitive assays for hematopoiesis do not necessarily produce physiological results. The cells that migrate from the BM to the thymus require further investigation to understand their identity, migration characteristics, turnover, release and homing mechanisms into thymic entry niches. Further challenging questions will address how pathways from HSCs to the thymus are disturbed under pathological conditions such as infection, myeloablation followed by BM transplantation, and under myelotoxic and thymotoxic conditions such as steroid treatment, ionizing radiation, or chronic diseases associated with catabolic stress. Acknowledgements We thank Hans Jo¨rg Fehling for collaborations on underlying fate mapping studies, and Carmen Blum, Hans Jo¨rg Fehling, Thorsten Feyerabend and Vera Martins for critical reading of the manuscript. SMS is supported by a fellowship from the DFG (Schl-1897/1-1). HRR is supported by the DFG through SFB 497-B5 and KFO 142-P8, and receives funding from the European Research Council under the European Community’s Seventh Framework Programme (FP7/20072013) / ERC grant agreement no. 233074.
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