An Essential Developmental Checkpoint for Production of the T Cell Lineage

See allHide authors and affiliations

Science  02 Jul 2010:
Vol. 329, Issue 5987, pp. 93-96
DOI: 10.1126/science.1188995


In early T cell development, progenitors retaining the potential to generate myeloid and natural killer lineages are eventually determined to a specific T cell lineage. The molecular mechanisms that drive this determination step remain unclarified. We show that, when murine hematopoietic progenitors were cultured on immobilized Notch ligand DLL4 protein in the presence of a cocktail of cytokines including interleukin-7, progenitors developing toward T cells were arrested and the arrested cells entered a self-renewal cycle, maintaining non-T lineage potentials. Reduced concentrations of interleukin-7 promoted T cell lineage determination. A similar arrest and self-renewal of progenitors were observed in thymocytes of mice deficient in the transcription factor Bcl11b. Our study thus identifies the earliest checkpoint during T cell development and shows that it is Bcl11b-dependent.

Tcells are generated from multipotent hematopoietic stem cells through a series of differentiation steps. The first step in this pathway is the generation of progenitors that have lost erythroid/megakaryocyte potential but retain the capacity to generate other hemopoietic cells, including myeloid, T, and B cells (16). We and others have recently identified the next stage, in which the T cell progenitors have lost B cell potential but are still able to generate myeloid cells, dendritic cells (DCs), and natural killer (NK) cells (7, 8). Therefore, the most critical step for development of the T cell lineage is now thought to be at the point where myeloid potential is terminated.

We sought to identify the step at which progenitors become fully committed to the T cell lineage and what regulates this transition. A reliable way to substantiate that a given step is critical for the development of a lineage is to demonstrate developmental arrest at the stage before that step under particular conditions. In the case of B cell differentiation, deletion of E2a, Ebf, or Pax5 genes leads to an early developmental arrest before formation of a functional IgH chain gene; these arrested B cell progenitors undergo self-renewal and remain B lineage uncommitted, with the potential to develop along other lineages, including myeloid and T cell (911). This case illustrates that such a critical developmental checkpoint exists at the step when uncommitted B cell progenitors become determined to the B cell lineage. Unlike the B cell lineage, to date no such checkpoint has been identified for the T cell lineage before the initiation of TCR gene rearrangement.

As T cell progenitors develop, they proceed through developmental stages referred to as DN1 to DN4 (double-negative CD4CD8) that can be tracked by surface phenotype. The DN2 stage can be subdivided into two stages based on transgenic green fluorescent protein (GFP) expression controlled by the proximal lck (plck) promoter (lck is a src family kinase selectively expressed by T cells). GFP cells retain non-T lineage potential, including that for myeloid cells, DCs, and NK cells, whereas the latter stage GFP+ cells are determined to the T cell lineage (7, 12). We designate these two stages DN2mt (myeloid-T) and DN2t (T-lineage determined) and term the step between these stages the DN2-determination step. This determination step is thought to be the first critical checkpoint in T cell development (13).

We cultured lineage-negative (Lin) c-kit+ Sca-1+ (LKS) cells from 13 days post-coitum (dpc) murine fetal liver with immobilized Delta-like 4 (DLL4) protein in the presence of the cytokines SCF (stem cell factor), Flt3L (FMS-like tyrosine kinase ligand), and interleukin (IL)–7 (fig. S1). After 7 days of culture, cells remained at the DN stage (Fig. 1A, left panel), whereas in the control group, where cells were cultured with TSt-4 stromal cells expressing DLL4 (TSt-4/DLL4), generation of CD4+CD8+ double-positive (DP) cells was observed (fig. S2). Upon closer analysis on DN cells generated in the feeder-free condition, we observed that these cells resembled DN2mt cells (maintained c-kithighCD25+) (Fig. 1A, right panel) and thus named them FFDN2 cells (feeder-free-cultured DN2-like cells). By several criteria, the FFDN2 cells appeared identical to DN2mt cells: (i) they gave rise to authentic αβ T cells when transferred to a TSt-4/DLL4 stromal coculture system (fig. S3, A and B); (ii) they retained the potential to produce macrophages (Fig. 1B), NK cells, and DCs (fig. S3, C and D); (iii) intracellular T cell receptor (TCR) β chain protein was not expressed (Fig. 1C); and (iv) their gene expression profiles were similar to those of DN2mt cells (Fig. 1D and fig. S4). Furthermore, GFP expression was not observed in FFDN2 cells generated from progenitors isolated from plck-GFP mice (Fig. 1E). It is unlikely that this arrest is due to the failure of TCR gene rearrangement because enforced expression of a functional TCRβ chain gene did not prevent the developmental arrest (fig. S5). FFDN2 cells could not generate B cells (fig. S3E), indicating that dedifferentiation to more primitive progenitors did not occur in this culture system. Of note, FFDN2 cells showed an almost unlimited in vitro expansion (Fig. 1F), while essentially maintaining c-kit and CD25 expression (Fig. 1G) and a developmental potential comparable to that of freshly isolated DN2mt cells (fig. S6). Cells in the c-kit+CD25+ fraction possessed the potential to maintain long-term culture, because long-term culture could be maintained by using c-kit+CD25+ cells at the time of passage (fig. S7). Such self-renewal capacity, together with our other results, indicated that the DN2-determination step may be a critical checkpoint for T cell development.

Fig. 1

Immobilized DLL4 with a combination of cytokines induces self-renewing expansion of immature thymocytes. (A) LKS progenitor cells (200 cells) from 13 dpc murine fetal liver were cultured with immobilized Fc-DLL4 supplemented with 10 ng/ml of SCF, IL-7, and Flt3L for 7 days. Generated cells were harvested and stained with the indicated antibodies and analyzed by a flow cytometry. Data are representative of four independent experiments. (B) A photomicrograph of macrophages generated from FFDN2 cells. FFDN2 cells induced from green mouse progenitors in a similar manner as (A) were sorted and cultured with TSt-4 stromal cells for 14 days in the presence of 10 ng/ml M-CSF (macrophage colony-stimulating factor). Macrophages are seen as large GFP+ cells. Scale bar, 100 μm. (C) Expression of intracellular (ic) TCRβ in FFDN2 cells, in negative control cells generated in feeder-free culture using only the Fc portion (Control), and in positive control CD4+CD8+ DP cells from an adult thymus. (D) mRNA expression of lineage-specific genes in cells derived from DN1, DN2mt, DN3, DP, and FFDN2 cells determined by quantitative reverse transcription polymerase chain reaction (RT-PCR). Expression was normalized to acidic ribosomal protein (ARP) mRNA expression, and the mean + SD of triplicate samples is shown. Data are representative of three independent experiments. (E) Flow cytometric analysis of GFP expression in FFDN2 cells generated from progenitors of plck-GFP mice in comparison with cells generated under TSt-4/DLL4 conditions (Control). Data are representative of three independent experiments. (F) A growth curve of FFDN2 cells. Viable cells were enumerated at the indicated time points. (G) c-kit versus CD25 expression by FFDN2 cells after long-term culture. Fetal liver LKS cells were cultured under feeder (–) conditions for 30 days and then analyzed by flow cytometry. Data are representative of four independent experiments.

To investigate the molecular mechanisms of T cell lineage determination, we searched for an environmental cue that could drive the arrested cells through the DN2-determination step. After testing various cytokines and Notch ligand conditions in the feeder-free culture system, we found that FFDN2 cells initiate differentiation when the concentration of IL-7 is reduced on day 7 of culture (10 ng/ml to 1 ng/ml). In this induction system, GFP+ DN3 cells appear on day 3 after IL-7 reduction (Fig. 2A). These cells did not express myeloid-lineage transcription factors PU.1 (Sfpi1) and C/EBPα, whereas T cell lineage–associated genes such as lck, Tcf1, pTα, and Bcl11b were markedly up-regulated (Fig. 2B). Notably, cells in these cultures developed up to the αβTCR-expressing CD4+CD8+ DP stage (Fig. 2C and figs. S8 and S9). Although the kinetics of DP cell growth was delayed compared with that in the TSt-4/DLL4 feeder cell culture system, the final yield of DP cells was nearly identical (fig. S10). The DP cells generated by reducing the concentration of IL-7 appeared to be authentic DP cells, because they give rise to CD4 and CD8 single-positive (SP) cells when transferred to a fetal thymus organ culture system (fig. S11). These results demonstrated that αβTCR+ cells can be generated from prethymic progenitors in a “feeder-free” culture system and that the TCRβ-selection, which is thought to serve as the critical checkpoint for preTCR formation in progenitors, does not require additional environmental factors in this feeder-free culture system.

Fig. 2

Reduction of IL-7 concentration induces the generation of DP cells in feeder-free culture. (A) LKS cells (200 cells) from 13 dpc fetal liver were cultured with immobilized Fc-DLL4 in the presence of SCF, IL-7, and Flt3L (10 ng/ml). After 7 days, the concentration of IL-7 was maintained or reduced to 1 ng/ml, and the cells were cultured for an additional 3 days. Cells were analyzed by flow cytometry. Data are representative of three independent experiments. (B) mRNA expression of lineage-associated genes in cells cultured in the presence of 10 ng/ml or 1 ng/ml of IL-7 in the same manner as (A). Expression was normalized to ARP mRNA expression, and the mean + SD of triplicate samples is shown. Data are representative of three independent experiments. (C) Flow cytometric analysis of cells generated 7 days after switching to cultures with either high or low IL-7 concentration. Cells were analyzed for the expression of CD4 versus CD8 and TCRβ versus TCRγδ. Data are representative of five independent experiments.

Often transcription factors regulate cell lineage determination steps. Among genes up-regulated by our induction system, we focused on Bcl11b, a T cell lineage–specific transcription factor originally identified as a tumor suppressor in murine T cell lymphoma (14). Bcl11b-deficient mice exhibit impaired thymocyte development around the DN3 to immature SP stage because of an inability to rearrange the Vβ to Dβ gene segments (15). We carefully reexamined the phenotype of fetal thymus cells from Bcl11b-deficient mice and found that, at 18 dpc, there was a developmental arrest at the DN2 stage (Fig. 3A). Despite this, the absolute number of DN2 cells was not increased (Fig. 3B), indicating that self-renewing expansion is not so prominent in vivo, a difference that could be due to the limited niche space in the thymus for early progenitors. We cultured these DN2 cells under TSt-4/DLL4 conditions, which can support T cell differentiation up to the DP stage. In such cultures, Bcl11b−/− cells continued to proliferate even after 4 weeks, maintaining their DN2 surface phenotype (Fig. 3C). Similar to FFDN2 cells, Bcl11b−/− DN2 cells exhibited features of DN2mt cells, including the potential to develop into macrophages and NK cells (Fig. 3D), and loss of B cell potential (fig. S12).

Fig. 3

Bcl11b is essential for T cell lineage determination. (A) Flow cytometric analysis of fetal thymocytes from Bcl11b−/− mice. Profiles are shown for CD4 versus CD8 of 18 dpc fetal thymocytes, and c-kit versus CD25 of cells gated in upper panels, from the indicated mice. For each group, more than five mice were individually analyzed, and representative profiles are shown. (B) Absolute numbers of total thymocytes and DN2 cells in 18 dpc fetuses of the indicated Bcl11b genotypes. More than five mice were individually analyzed for each group, and the mean + SD is shown. (C) Flow cytometric analysis of c-kit versus CD25 of fetal liver LKS cells from Bcl11b−/− mice cultured on TSt-4/DLL4 stromal cells for 30 days. Data are representative of three independent experiments. (D) Generation of macrophages and NK cells from cultured Bcl11b−/− fetal liver cells. The c-kit+ CD25+ cells shown in (C) were cultured (200 cells per well) for 7 days with TSt-4 cells in the presence of M-CSF (left panel) or IL-15 (right panel) and analyzed for macrophage and NK cell markers by flow cytometry. Data are representative of three independent experiments. (E) Fetal liver cells from Bcl11b+/+ or Bcl11b−/− mice (Ly5.2) were transferred into lethally irradiated mice (Ly5.1). Flow cytometric profiles of reconstituted thymocytes of recipient mice 8 weeks after transfer are shown. In right panels, profiles of cells gated on CD3CD4CD8 [triple negative (TN)] fraction are shown. For each group, more than five mice were individually analyzed, and representative data are shown.

Bcl11b deficiency is lethal around the neonatal period (15). To investigate whether the developmental arrest of Bcl11b−/− progenitors is seen in the adult thymus, where T cells are continuously generated, we produced chimeric mice by transferring Bcl11b−/− fetal liver cells into irradiated B6Ly5.1 congenic mice. At 8 weeks after transfer, we observed nearly complete developmental arrest at the DN2 stage, with only a few DP cells (Fig. 3E). Similar to ex vivo fetal thymocytes of Bcl11b−/− mice and cultured Bcl11b−/− DN2 cells, the arrested DN2 cells were equivalent to DN2mt cells. There was no increase in thymic B cells in the recipients of the Bcl11b−/− fetal liver cells (fig. S13), indicating that the Bcl11b−/− DN2 cells that developed in the thymus did not dedifferentiate into more primitive progenitors in vivo.

The similar stage of arrest in the DLL4/IL-7 cultures and in the Bcl11b−/− mice suggested that the arrest in the cultures may be due to a failure to up-regulate Bcl11b. To examine this possibility, we retrovirally transduced Bcl11b cDNA into fetal liver LKS cells and cultured these transduced cells under DLL4/IL-7 conditions. The Bcl11b-transduced cells could give rise to DN3 cells even in the presence of a high concentration of IL-7 (Fig. 4A), and TCRβ gene rearrangement was enhanced (Fig. 4B), whereas myeloid-lineage–associated genes were suppressed (Fig. 4C), demonstrating that Bcl11b expression eliminated the DN2 arrest that occurred in the DLL4/IL-7 cultures.

Fig. 4

Enforced expression of Bcl11b abrogated the DN2 arrest in the DLL4/IL-7 cultures. (A to C) LKS cells from 13 dpc B6 fetal liver were transduced with murine stem cell virus (MSCV)–Bcl11b or MSCV-control vector. Two days later, GFP+ cells were sorted and cultured with immobilized Fc-DLL4 in the presence of 10 ng/ml of SCF, IL-7, and Flt3L for 7 days. Flow cytometric profiles of CD4 versus CD8, c-kit versus CD25, and CD44 versus CD25 expression (A), icTCRβ expression (B), and mRNA expression (C) in generated cells are shown. Expression of mRNA was normalized to ARP mRNA expression, and the mean + SD of triplicate samples is shown. Data are representative of three independent experiments.

As has been reported (16), the absence of Bcl11b had a severe impact on the generation of thymic αβT cells, whereas there was little effect on the generation of γδ T cells (fig. S14A). The same is true for cells generated in the DLL4/IL-7 cultures (fig. S14B). These results suggested that the segregation to the γδT cell lineage occurs before the DN2mt stage, although the possibility still remains that the γδ T cells that had been generated from “leaky” DN3 cells underwent compensatory proliferation.

The developmental steps just after the formation of preTCR (DN3 stage) and αβ TCR (DP stage) serve as critical checkpoints (16, 17), and cells that fail to pass these points succumb to apoptotic cell death. In contrast, the arrested progenitors at the DN2-determination step enter a self-renewal cycle. The appearance of self-renewing progenitors among Bcl11b−/− thymocytes may explain the previous findings that loss-of-function mutations in the Bcl11b gene are frequently observed in murine T cell lymphomas induced by γ irradiation (14) and that chromosomal aberration disrupting Bcl11b gene was identified in human T cell acute lymphoblastic leukemia cases (18), because the acquisition of self-renewal capacity is regarded as the first step in leukemia development. In this context, a similar outcome was recently observed when Lmo2, a known oncogene, was overexpressed in thymocytes and caused the cells to enter a self-renewal cycle in vivo (19).

The present study thus defines a Bcl11b-driven checkpoint at which T cell progenitors terminate non-T-lineage potential in order to become determined to the T cell lineage (fig. S15). Our finding that Bcl11b up-regulation can be triggered by an extrinsic cue, diminished IL-7, suggests that progression through the DN2-determination step is instructed by environmental signals in the thymus. It is quite likely that the reduction in IL-7 signaling is a physiological mediator of this step, because the IL-7R is dramatically down-regulated at the transition from the DN2 to the DN3 stage (20). Considering that Bcll1b is thought to be a transcriptional repressor, we speculate that Bcl11b directly suppresses myeloid-lineage–associated genes, such as PU.1 or C/EBPα, and that such suppression is critical for differentiation toward the T cell fate.

A recent study demonstrated that Bcl11b is expressed in the T cell–like lymphoid cells of lamprey (21). Because a Bcl11b homolog has not been found in animals other than vertebrates (fig. S16), we propose that Bcl11b arose in phylogeny to construct a new lineage distinct from the preexisting innate type killer cells.

Supporting Online Material

Materials and Methods

Figs. S1 to s16


References and Notes

  1. The authors are grateful to C. Murre, T. Kadesch, Y. Agata, and S. Yamasaki for providing us with reagents and protocols, and to P. Burrows for critical reading of the manuscript. This work was partially supported by Grant-in-Aid for Young Scientists (A) from the Ministry of Education, Science, Sports, and Culture, Japan.

Stay Connected to Science

Navigate This Article