The Earliest Thymic T Cell Progenitors Sustain B Cell and Myeloid Lineage Potentials

The stepwise commitment from hematopoietic stem cells in the bone marrow (BM) to T lymphocyte-restricted progenitors in the thymus represents a paradigm for understanding the requirement for distinct extrinsic cues during different stages of lineage restriction from multipotent to lineage restricted progenitors. However, the commitment stage at which progenitors migrate from the BM to the thymus remains unclear. Here we provide functional and molecular evidence at the single cell level that the earliest progenitors in the neonatal thymus possessed combined granulocyte-monocyte, T and B lymphocyte, but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of thymus-seeding progenitors in the BM, which were closely related at the molecular level. These findings establish the distinct lineage-restriction stage at which the T lineage commitment transits from the BM to the remote thymus.

evidence at the single cell level that the earliest progenitors in the neonatal thymus possessed combined granulocyte-monocyte, T and B lymphocyte, but not megakaryocyte-erythroid lineage potential. These potentials were identical to those of thymus-seeding progenitors in the BM, which were closely related at the molecular level. These findings establish the distinct lineage-restriction stage at which the T lineage commitment transits from the BM to the remote thymus.
At the heart of developmental and stem cell biology, as well as regenerative medicine, is the fundamental process of lineage commitment from self-renewing multipotent stem cells to lineage-restricted progenitors. In all species and organ systems this process occurs first during embryonic development, but is recapitulated postnatally and in adult life by adult multipotent stem cells to replenish cell lineages with a limited lifespan. Hematopoiesis represents the mammalian paradigm for how multilineage diversity can be achieved through commitment of multipotent stem cells into lineage-committed progenitors and the establishment of distinct blood cell lineages 1 . However, the exact cellular commitment pathways remain unclear 1,2 .
Whereas lineage-restricted progenitors for all other blood cell lineages can be generated from self-renewing hematopoietic stem cells (HSCs) in the postnatal bone marrow (BM), the final steps of T lymphocyte lineage restriction take place in the thymus 3 . Because the thymus cannot sustain HSCs, continuous thymopoiesis can only be secured through regular replenishment by BM-resident thymus-seeding progenitors (TSPs) 4 . However, the commitment stage(s) at which these progenitors migrate from the BM to the thymus is unknown. The thymus, as the BM, harbors multiple blood cell lineages 5,6 , however the identification of multipotent progenitors that match the lineage potential of candidate TSPs in the BM has not been possible so far. The early thymic progenitors (ETPs) have been extensively studied in the adult thymus, but their exact lineage potentials and relationship to candidate TSPs in the BM have remained unclear 7 .
Recent studies that evaluated the lineage potential of ETPs at the single cell level showed that a large fraction of adult ETPs have combined T cell and myeloid (granulocytemonocyte; GM) potential 8,9 . B lineage potential was however not detected in the single, highly purified, adult ETPs, suggesting that the most primitive progenitor in the thymus might have a T-GM restricted potential 8,9 . Similar studies of the fetal thymus have supported the T-GM restricted potential of ETPs, and failed to reveal any B cell potential 10,11 . However, other studies reported even rarer adult ETPs with combined T and B (but not myeloid) cell potential 6 , and candidate TSPs identified in the BM 7 , such as common lymphoid progenitors (CLPs) 12 , lymphoid-primed multipotent progenitors (LMPPs) 13 and HSCs 1 , all possess B cell potential.
The megakaryocyte-erythroid (MkE) potential of ETPs is of particular relevance for the ongoing debate as to whether the first lineage commitment step in hematopoiesis results in a strict separation into common myeloid and lymphoid lineage commitment pathways as depicted in the still prevailing text-book hierarchical model for hematopoiesis 1,14 , or whether early lymphoid progenitors sustain GM but not MkE potential 2,13,15 , as recently reported in human studies as well 16,17 . The MkE potential of purified ETPs has yet to be investigated 5,6,8,9 . The fact that no multipotent thymic progenitors with the same lineage potentials as candidate multipotent TSPs in the BM have been identified yet contributes to the considerable gap in understanding the distinct roles of the local BM and thymus environments in promoting distinct pre-thymic and thymic stages of T cell lineage commitment. Here we demonstrate at the single cell level the existence of postnatal ETPs with combined T, GM and B cell but no MkE potential, establishing the exact lineage commitment step at which the multipotent T-lymphocyte progenitors must migrate to the thymus to allow the final steps of T-lineage restriction to be completed. The data reported here provide further support of a myeloid-based model for hematopoietic lineage commitment.

Neonatal ETPs have combined T, B and GM, but not MkE potential
The current knowledge about candidate TSPs and ETPs can only be reconciled ( Supplementary Fig. 1) if either a T-GM restricted progenitor can be identified in the BM, a T-GM progenitor would be generated in the passage from the BM to the thymus and/or a thymic cell population with a combined T, GM and B cell lineage potential could be identified within or outside the ETPs. In the last scenario, the ETP could either be a lymphoid-GM restricted multipotent progenitor or a pluripotent hematopoietic stem-or progenitor cell that also possesses MkE potential. ETPs have mostly been studied in adult mice 5,6,8,9 . However, thymic involution, the physiological shrinking of the thymus with age that occurs in all vertebrates, implies that thymopoiesis, and therefore thymus seeding, is much more active in the early postnatal thymus 18 . The B cell potential of early thymocytes, at a population level, is significantly higher (although still low) in the neonatal thymus compared to the adult thymus 19 . In agreement with previous studies, Lin − CD4 − CD8α − CD25 − c-Kit hi ETPs represented only 0.01% of adult thymocytes 5 but as many as 40% of ETPs expressed cell surface Flt3, higher than previously reported 20 (Fig.  1a). Also in agreement to previous findings, a low (3.5-4.5%) but highly reproducible frequency of Flt3 expressing ETPs generated B cells 6,20 , whereas no other adult thymocyte progenitors, including Flt3 − ETPs had any detectable B cell potential ( Fig. 1b and Supplementary Fig. 2). The frequency of Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs increased more than 10-fold in newborn mice compared to adult mice (Fig. 1c), while the frequency of Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs with B cell potential was 25% ( Fig.  1d and Supplementary Fig. 3). Neither Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 − thymocytes or downstream double negative (DN) 2-4 populations in the neonatal thymus had any B cell potential (Fig. 1d). Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi newborn ETPs also produced B cells in vivo when transplanted into irradiated Rag1 −/− mice, but only very low numbers of short-lived myeloid cells ( Supplementary Fig. 4).
In addition to B cells, newborn Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs gave rise efficiently to cells of the T, NK and GM lineages, as previously demonstrated with adult ETPs (Fig. 1f,g and Supplementary Fig. 6a). In contrast, newborn ETPs were completely devoid of MkE potential (Fig. 1f). Adult ETPs lacked Mk potential as well, but, in agreement with previous studies, had GM potential ( Supplementary Fig. 6a). Quantitative gene expression analysis showed that purified Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi newborn ETPs expressed multiple GM and lymphoid, but not Mk and E affiliated genes ( Fig. 1h). Single-cell PCR experiments showed that as much as 65% of newborn Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs co-expressed GM and lymphoid genes, while lacking MkE gene expression at the single cell level (Fig. 2a).
To establish whether the T, B and GM potential of neonatal ETPs reflects the existence of a multipotent lympo-myeloid progenitor in the thymus, or only a mixture of lineage-restricted progenitors, we assessed the combined lineage potentials of single Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs. Single ETPs were sorted onto OP9 BM stroma to allow cells to expand for 54 hours, after which the expanded cells were split and transferred to OP9 and OP9-DL1 stroma for an additional 1 week to promote B-and combined T-and myeloid cell differentiation, respectively. Although the frequency of ETPderived clones with GM readout was reduced compared to assays in which only the GM differentiation of ETPs was promoted (Fig. 1f), we could demonstrate the existence of Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi single ETPs with combined T, B and GM lineage potentials (9.2% of clones with a lineage readout; Fig. 2b,c and Supplementary Fig. 7a). In fact, all the GM potential from wild-type ETPs was tracked to cells that not only had T cell potential as previously demonstrated 8,9 , but also possessed B lineage potential (Fig. 2c). Next, we used ETPs purified from vavP-mcl-1 transgenic mice 25 to evaluate whether enhanced cell survival could sustain short-lived myeloid cells to increase the readout for combined myeloid and lymphoid potential. Whereas the B cell potential in vavP-mcl-1 neonatal thymuses remained restricted to Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs ( Supplementary Fig. 7c), the frequency of ETPs with combined T-B-GM readout increased to 20% of all single vavP-mcl-1 ETPs (as compared to 9.2% of WT ETPs) with a lineage readout (Fig.2d,e and Supplementary Fig. 7b). These findings in single cell clonal assays establish the existence of thymic ETPs with combined T, B and GM lineage potential.

Lympho-myeloid ETPs are the most multipotent thymic progenitors
We next explored whether the Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs with combined T, B and GM lineage potentials represent the most multipotent progenitors in the neonatal thymus. The lack of detectable Mk and E potential in Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs did not rule out the presence of rare pluripotent stem-or progenitor cells in the neonatal thymus. Thus, we first used highly sensitive flow cytometry analysis to investigate the expression of three antigens, CD150 26 , CD201 27 and Mpl 28 , each highly expressed on most if not all HSCs as well as multipotent progenitors with sustained MkE potential. Neither of these antigens was expressed on Lin − CD4 − CD8α − CD25 − c-Kit hi ETPs (Fig. 3a). Similar to a subfraction of BM LMPPs, all ETPs expressed the RAG1 gene, as assessed using a green fluorescent protein (GFP) reporter under control of the Rag1 promoter 29 and most expressed the chemokine receptor CCR9 (Fig. 3b) in agreement with previous studies of adult ETPs 30,31 . No BM HSCs expressed either Rag1-GFP or CCR9 (Fig. 3b).
Whole thymocytes from neonatal mice transplanted intravenously or intrafemorally (to bypass potential changes in BM homing properties upon thymus entry) reconstituted T cells and low levels of B cells transiently (Fig. 3c,d), but failed to sustain any long-term multilineage reconstitution (Fig. 3e,f), in further support of the idea that the postnatal thymus does not harbor any HSCs. To enhance the detection of HSCs potentially present in the thymus, whole thymocytes were depleted of CD4 + and CD8 + cells and transplanted intravenously or intrafemorally (Fig. 3g,h). The absence of long-term myeloid reconstitution in all major hematopoietic organs as well as the lack of thymocyte-derived T cell progenitors in the thymus after 13 weeks in all but one transplanted mouse, further corroborated the absence of pluripotent HSCs in the thymus (Fig. 3g,h and Supplementary  Fig. 8a-d). Collectively, these results demonstrate the absence of HSCs in the postnatal thymus, compatible with ETPs with combined T, B and GM lineage potential being the most multipotent progenitors in the thymus. GFP (which also highly express interleukin 7 receptor α, IL-7Rα; Supplementary Fig. 9a) in the BM 13,32 , we next investigated the molecular relationship between ETPs and IL-7Rα + LMPPs and HSCs in the BM. We also compared ETPs to the next stages of lineage restriction in the thymus, namely Lin − CD44 + CD25 + c-Kit hi DN2 cells that sustain combined T and GM but no B lineage potential 8,9 , and Lin − CD44 − CD25 + DN3 cells, which represent the first T cell restricted progenitors in the thymus 33 . Based on global gene expression analysis 34 , ETPs clustered much closer to LMPPs in the BM than to thymic DN2s and DN3s or BM HSCs. Moreover, LMPPs clustered closer to ETPs than they did to HSCs, and DN2s closer to DN3s than ETPs (Fig. 4a). Because CLPs have been suggested to be candidate TSPs 7 and have recently been demonstrated to not only have lymphoid potential, but also sustain some myeloid potential similar to LMPPs 35 , we also compared the molecular profiles of ETPs with the two candidate TSP populations in the BM, LMPPs and Lin − CD19 − B220 − Sca-1 low c-Kit low Flt3 + IL-7Rα + Ly-6D − CLPs 36 (Fig. 4b). The newborn ETPs clustered closely with CLPs as well as LMPPs and were more distant from HSCs, DN3 and also proB cells. Moreover newborn and adult ETPs clustered closely together along with LMPPs and CLPs (Fig. 4b). These findings establish a close molecular relationship between Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs in the thymus and candidate TSPs with lympho-myeloid potential in the BM (Fig. 4a,b and Supplementary Fig.  9b,c).
To more specifically look at T cell and GM lineage affiliated gene expression, T and GM gene lists were derived from the literature and from previously published data sets 37,38 (Supplementary Methods). When T and GM lineage affiliated programs were compared, adult and neonatal ETPs clustered closely for both, and closer to LMPPs and CLPs than to DN2 and DN3 in the thymus and HSCs in the BM (Fig. 5a,b and Supplementary Fig. 9d,e).
Of particular interest, many T cell affiliated genes that eventually become dramatically upregulated in DN2s and further upon T cell commitment in DN3s, have already been upregulated in LMPPs (as compared to HSCs) and CLPs, with less change from LMPPs and CLPs to ETPs (Fig. 5a). Using quantitative RT-PCR, some of the myeloid, lymphoid, T cell and Notch related genes were investigated in greater detail (Fig. 6). In addition to confirming the combined expression of GM (Mpo, Csf1r, Csf2rb,) and lymphoid (sterile IgH, Il7r, Rag1) lineage affiliated genes, these data also showed that characteristic early T cell specific genes (Ptcra, Cd3e, Cd3g) were not significantly upregulated in neither multipotent LMPPs nor ETPs. In contrast, Notch1 was upregulated in LMPPs and further in ETPs, and in agreement with this, the Notch target genes Il2ra (Cd25), Gata3 and in particular Hes1 39 were upregulated in the transition from LMPPs to ETPs. Finally, whereas HSCs lack expression of Ccr7 and Ccr9 30,31 , encoding critical chemokine receptors for migration to the thymus, these genes were drastically upregulated in LMPPs, in further support of the idea that LMPPs are TSPs. Collectively, these results demonstrate that ETPs and candidate TSPs such as LMPPs and CLPs, have closely related gene expression profiles, reinforcing that ETPs more likely are derived from lympho-myeloid restricted progenitors than HSCs in the BM.

DISCUSSION
Here we identify ETPs in the neonatal thymus with combined T, B and GM, but no MkE lineage potential, and we demonstrate a close functional and molecular linkage between ETPs and candidate TSPs in the BM.
The observation that ETPs lack MkE potential is of key importance towards reconciling the ongoing debate regarding the roadmap for hematopoietic lineage commitment, as the classical model for hematopoietic lineage commitment predicts that the first lineage commitment step of HSCs results in a strict separation of myelo-erythroid and lymphoid commitment pathways 1,14 . According to this model, any cell with combined lymphoid and GM potential should also possess MkE potential. However, the current study demonstrates that Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs with combined T, B and GM lineage potential were devoid of Mk and E lineage potentials. These cells co-express, at the single cell level, lymphoid and GM, but not MkE related genes, similar to LMPPs with identical lineage potentials in the BM 13,34 . Thus, this study provides support for a myeloid-based lineage commitment model 2,13,15,16,17 , by demonstrating the existence of T-B-GM restricted progenitors in the postnatal thymus. Such cells have been previously identified in the BM and the fetal liver 13,34 .
It is very likely that the real frequency of neonatal Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs with T-B-GM potential is higher than what we were able to demonstrate. Analysis of clones grown from single newborn ETPs demonstrated that most ETPs with T cell potential simultaneously possessed B cell potential, but less than 20% of these also revealed GM potential in WT mice, although under optimized GM conditions more than 50% of ETPs demonstrated GM potential. We speculated that the reduced GM readout in the multilineage clonal assay reflected the short life-span of vulnerable myeloid cells, and in agreement with this MCL-1 enhanced the detection of neonatal ETPs with combined T, B and GM potential, most likely through increased survival of myeloid cells.
Our findings also suggest that the identified T-B-GM restricted progenitor is the most multipotent progenitor in the neonatal thymus, as we failed to detect any MkE lineage potential or MkE-specific gene expression within highly purified ETPs. Furthermore, we also demonstrate that there are no phenotypic or in vivo reconstituting HSCs or multipotent progenitors in the neonatal thymus.
Previous studies have suggested that the earliest fetal thymic progenitors in the embryo have combined T cell and myeloid, but no B cell lineage potential 10,11 , raising the possibility that the progenitors seeding the embryonic thymus might be distinct and more committed than in the postnatal thymus. In contrast with the neonatal thymus which we concentrated on in this study, it remains unclear if the adult thymus is also seeded with ETPs with combined T, GM and B lineage potentials. As thymopoiesis is much decreased in the adult compared to the newborn thymus, it can be predicted that the most multipotent ETPs are much more infrequent in adult than in the neonatal thymus. While the low frequency of B lineage potential of adult ETPs 6,20 does not allow a clear test of the combined lineage potential of these cells using the current clonal lineage potential assay, it is notable that rare Lin − CD4 − CD8α − CD25 − c-Kit hi Flt3 hi ETPs were the only adult thymocytes with B cell potential 6,20 . In addition, the current study showed that adult ETPs, like neonatal ETPs, have GM, but not Mk potential, while global gene expression analysis pinpointed a very close molecular relationship between neonatal and adult ETPs. Collectively these datasets suggest that adult thymuses, as the neonatal thymuses, might also seeded by rare T-B-GM progenitors, which we could unequivocally identify in the neonatal thymus. Nevertheless, there are distinct differences between fetal, neonatal and adult hematopoietic stem and progenitor cells. The regulated migration of TSPs to the thymus might also differ in the newborn and the adult, so it remains possible that the lineage potentials of newborn and adult TSPs might be different.
While these studies establish the extent of ETP multipotentiality (T-B-GM) and the close phenotypic and molecular relationship between ETPs and LMPPs and CLPs with the same lineage potentials in the BM 13,32,34,35 , they do not exclude the possibility that other candidate progenitors in the BM might seed the thymus 40 . A recent study suggested that T-GM-restricted progenitors might exist in the BM 41 , although such progenitors remain to be purified and characterized in further detail. The GM potential of ETPs is limited, and recent studies have suggested that it has little if any functional significance with regard to these progenitors acting as myeloid progenitors in the thymus 35,42 . Likewise, it seems unlikely that ETPs have any important physiological role as B cell progenitors. Rather, the significance of these sustained lineage potentials of ETPs is to provide a better understanding of the required lineage restriction steps for lineage commitment from pluripotent HSCs in the BM to a T cell restricted progenitor in the thymus. Specifically, progenitors with combined T-B-GM potential, such as LMPPs and CLPs, are derived in the BM from HSCs that have shut down the MkE transcriptional programs and lineage potentials. Unlike HSCs, LMPPs and CLPs upregulate CCR9 30,31 , which enables their transfer to the thymus 30,31 . Migration to the thymus appears critical for the next T-lineage restriction steps, first to a T-GM 8,9 and finally to a fully T cell restricted progenitor. In addition to providing novel insights into the normal stepwise process of T lineage commitment in the BM and thymus, the present studies are also of considerable relevance for a recently identified clinically, phenotypically and molecularly distinct group of mixed T-GM acute lymphoblastic leukemias (ALLs) which are predominantly observed in children but also seen in adults, and termed ETP leukemias 43 . Furthermore, the sustained B cell potential of ETPs might explain why the MLL-AF4 fusion oncogene, highly specific for human B cell malignancies, can give rise to B cell malignancies even if targeted to thymic progenitors 44

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Stem-and progenitor cell purification
All cell sorting experiments were performed on a BD FACSAriaIIu cell sorter (BD Biosciences) with a total mean cell sorting purity of 99% ± 2% for Flt3 hi ETPs, based on reanalysis for all gating steps and therefore all antigens defining Flt3 hi ETPs (Lin − CD4 − CD8α − CD25 + c-Kit hi Flt3 hi ) ( Supplementary Fig. 3). Single cells were seeded by an automated cell deposition unit (ACDU) providing single cells in >99% of the wells, and no wells with more than 1 cell. FACS analyses were performed on a BD LSRII (BD Biosciences). See Supplementary Table 1 for instrument configurations. Subsequent data analyses were performed with the FlowJo analysis software (TreeStar Inc). Cells used in cell sorting experiments were either unenriched or enriched for CD117 with MACS cell separation (Miltenyi Biotec) followed by Fc-block incubation and staining with anti-mouse antibodies (see Supplementary Table 2 for antibody information). Fluorescence-minus-one controls as well as negative populations were used as gate-setting controls.

Single cell and quantitative PCR
Multiplex single-cell RT-PCR was performed as previously described 34 Table 4 for TaqMan assays used.

In vitro cultures
GM and Mk lineage potentials were analyzed as previously described 32 (see Supplementary  Table 5). Mk potential was additionally evaluated using the Megacult collagen-based assay (StemCell Technologies). 200 ETPs or 100,000 unfractionated control BM cells were plated in Megacult-collagen media supplemented with growth factors (see Supplementary Table 5). Mk colonies were evaluated after 7 days with Acetylthiocholiniodide (Sigma) staining according to manufacturer's instructions.
OP9 and OP9-DL1 stromal cells were kindly provided by Dr A. Cumano. To evaluate the individual GM, B, T and NK cell lineage potentials single ETPs were FACS sorted onto monolayers of OP9 or OP9-DL1 stromal cells as previously described (see Supplementary  Table 5). Myeloid lineages formed on OP9 stroma co-cultures were evaluated after 6 days by morphology analysis of May-Grünwald (Sigma) and Giemsa (Fluka) stained slides.
To analyze combined lineage potentials, single cell deposition was performed (ensured as specified above), onto OP9 cells supplemented with hFLT3L, mScf and hIL-7.
Approximately 54 hours later, the co-culture was split to a secondary layer of OP9 with the same condition to promote B cells and a layer of OP9-DL1 with cytokines (hFLT3L, mSCF, hIL-7, hIL-6, mIL-3, hG-CSF, mGM-CSF, hCSF-1) to promote T and myeloid cell differentiation. The timing of the first culture period was optimized to give the highest frequency possible for all lineages in the combined lineage potential assay. One week after transfer to the second culture, clones were evaluated for the presence of CD19 + B cells, CD4 + CD8α + NK1.1 − and/or Thy1.2 + CD25 + NK1.1 − T cells and Mac-1 + F4/80 + myeloid cells. Clones expressing Mac-1 and/or F4/80 were additionally evaluated by morphology. The timing of the readout was optimized to the earliest time-point where T cells could be detected, and at which time myeloid progeny would still be possible to detect. However, as the differentiation time is longer for lymphoid cells, the production of myeloid progeny is most likely underestimated.
All frequencies of single cell assays are expressed as total frequencies, that is relative to the total number of cells plated unless otherwise stated.