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The Journal of Immunology, 2007, 178: 858-868.
Copyright © 2007 by The American Association of Immunologists, Inc.

Bone Marrow-Derived Hemopoietic Precursors Commit to the T Cell Lineage Only after Arrival in the Thymic Microenvironment1

Kornelia Heinzel*, Claudia Benz*, Vera C. Martins*, Ian D. Haidl{dagger} and Conrad C. Bleul2,*

* Department of Developmental Immunology, Max-Planck-Institute for Immunobiology, Freiburg, Germany; and {dagger} Department of Pediatrics and Department of Microbiology and Immunology, IWK Health Centre, Halifax Nova Scotia, Canada


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T lymphocytes develop in the thymus from hemopoietic precursors that commit to the T cell lineage under the influence of Notch signals. In this study, we show by single cell analyses that the most immature hemopoietic precursors in the adult mouse thymus are uncommitted and specify to the T cell lineage only after their arrival in the thymus. These precursors express high levels of surface Notch receptors and rapidly lose B cell potential upon the provision of Notch signals. Using a novel culture system with complexed, soluble Notch ligands that allows the titration of T cell lineage commitment, we find that these precursors are highly sensitive to both Delta and Jagged ligands. In contrast, their phenotypical and functional counterparts in the bone marrow are resistant to Notch signals that efficiently induce T cell lineage commitment in thymic precursors. Mechanistically, this is not due to differences in receptor expression, because early T lineage precursors, bone marrow lineage marker-negative, Sca-1-positive, c-Kit-positive and common lymphoid progenitor cells, express comparable amounts of surface Notch receptors. Our data demonstrate that the sensitivity to Notch-mediated T lineage commitment is stage-dependent and argue against the bone marrow as the site of T cell lineage commitment.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
T lymphocytes and their precursors in the adult thymus are derived from multipotent hemopoietic stem cells that reside in the bone marrow and have the capacity to self-renew (1). The adult thymus does not support self-renewing hemopoietic precursors, and hemopoietic precursors are therefore continuously imported (2). Despite intense research into thymic precursor biology, the phenotype and characteristics of the precursors that enter the adult thymus to produce mature T cells remain controversial (3, 4, 5, 6). For instance, many experimental systems have shown that thymic precursors give rise to B, NK, dendritic, and myeloid cells (7, 8, 9, 10), but it is unclear if these cells stem from precursors with multilineage potential or represent the progeny of lineage-restricted precursors that could not be separated by the markers used. Although most reports agree on the fact that the most efficient T cell progenitors in the thymus are found within the CD4low (11)/early T lineage progenitor (ETP)3 (9) population, it is still unclear if these precursors are restricted to the T cell lineage or only commit in the thymus. Commitment of hemopoietic precursors to the T lineage depends on signals mediated by Notch (12). Thus, in the absence of Notch1 signals the thymus lacks developing T cells and instead contains differentiating B cells similar to those found in the bone marrow (13). These findings have been interpreted to mean that Notch1 instructs hemopoietic precursors in the thymus to adopt a T over a B cell fate (12, 14). This view is challenged by others who propose that T cell lineage commitment occurs in the bone marrow and that precommitted progenitors colonize the thymus (15). This scenario is mainly based on data from embryonic thymus colonization and suggests that Notch merely reveals the T lineage choice by supporting further T cell differentiation steps. Thus, the nature of the hemopoietic precursor that commits to the T cell lineage and the microenvironment in which this occurs remains controversial. Indeed, staining immature thymocytes with a Notch1-specific antiserum has shown surface Notch1 on double negative (DN) 2 and DN3 but not DN1 thymocytes, which are thought to contain the potential bipotent precursors in question (16). Furthermore, the process of T cell lineage commitment has recently been shown to be a reversible and protracted process (17). If hemopoietic precursors did commit in the thymus as proposed, why are the B cells derived from thymic precursors so difficult to detect (18, 19)? We have recently identified the most immature precursors within the ETP population, which we termed the thymic multipotent precursor (TMP) population (10), and showed that three of 100 single precursors within this population have the capacity to generate mature cells of all hemopoietic lineages that develop in the thymus, namely T cells, B cells, and dendritic cells. The differentiation potential of the remaining 97 cells is an open question. If these 97 cells were T cell lineage committed, Notch could indeed simply be required to support further steps of T cell development.

The finding that cells with the same phenotype and the same T/B potential are present in the bone marrow and blood (10) immediately poses the question of why these cells do not already commit to the T cell lineage in the bone marrow. The T lineage precursor frequency among bone marrow LSK (lineage marker (Lin)–Sca-1+c-Kit+) cells is reportedly very high (20). Furthermore, the work of Zuniga-Pflücker and coworkers (21, 22) has shown that the addition of the Notch ligand Delta-like1 to a bone marrow stroma cell line is sufficient to induce T cell lineage commitment in bone marrow-derived hemopoietic precursors, indicating that thymus-specific niches are not an absolute requirement. Several reports have documented the expression of Notch ligands not only on bone marrow stroma but also on hemopoietic cells (23, 24, 25, 26), ruling out the possibility that adult thymus-settling precursors are exposed to Notch ligands only after arrival in the thymus. Consistently, several stages of B cell development in the bone marrow have been shown to express Notch target genes suggestive of Notch signaling in these cells (27).

In this study we investigated several important questions of early T cell lineage commitment. We find by single cell analyses that the majority of TMPs are uncommitted, i.e., not restricted to the T cell lineage. This finding indicates that adult thymic precursors repress non-T lineage potentials and commit to the T cell lineage only after arrival in the thymic microenvironment. We demonstrate that TMPs show surface expression of Notch receptors, express elevated levels of Notch target genes, and rapidly lose their B cell lineage potential in the presence of Notch signals. In contrast to their phenotypical and functional counterparts in bone marrow, TMPs are highly sensitive to Notch signals that induce T cell lineage commitment. This argues against the bone marrow as the site of T cell lineage commitment and may explain why T cell lineage commitment does not occur in the phenotypical and functional counterparts of TMPs in the bone marrow.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

C57BL/6, C57BL/6-Ly5.1, and CCR9-enhanced GFP (EGFP) knock-in mice that had been back-crossed to the C57BL/6 background six times were kept under specific pathogen-free conditions in the mouse facility of the Max-Planck-Institute for Immunobiology (Freiburg, Germany). To sort TMPs, 3–5 wk old mice were used. Experimentation and animal care was in accordance with the guidelines of the Max-Planck-Institute for Immunobiology.

Notch ligand fusion proteins

Fusion proteins were generated by cloning cDNAs encoding murine Delta-like1 Met1–His535 (GenBank accession no. X80903), Delta3 Met1–Arg468 (GenBank accession no. BC052002), Delta4 Met1-Leu525 (GenBank accession no. BC049130), Jagged1 Met1-Asp1067 (GenBank accession no. NP_038850), and Jagged2 Met1-Gly1083 (GenBank accession no. NM_010588) upstream of a splice donor into pcDNA3.1+ containing a genomic fragment encoding the human IgG1 hinge and Fc part (nt 341-1603 of GenBank accession no. AF237583, a gift by Dr. S. Krautwald and Dr. U. Kunzendorf, University of Kiel, Kiel, Germany). The Delta-like1 mFc was generated by cloning murine Delta-like1 Met1–Pro544 (GenBank accession no. X80903) and a cDNA encoding the mouse IgG2a hinge and Fc part (nt 499-1070; GenBank accession no. XM_489702) into pEF5 (Invitrogen Life Technologies). All fusion proteins were purified from supernatants of transfected HEK293 (Delta-like1, human Fc (hFc), Delta3 hFc, Delta4 hFc, Jagged1 hFc, and Jagged2 hFc) and Chinese hamster ovary (Delta-like1, mouse Fc (mFc) cells by protein A affinity chromatography according to standard methodology and quantified by Bradford protein assay (Bio-Rad).

Flow cytometric analysis and cell sorting

Cells were prepared from heterozygous CCR9-EGFP knock-in mice, purified by paramagnetic bead depletion, stained, sorted, or double-sorted for single cell experiments as described previously (10). The lineage-positive cells were excluded using the following mAbs directed against (clone name in parenthesis) anti-CD45R (RA3-6B2), anti-CD3{epsilon} (145-2C11), anti-CD8{alpha} (53-6.7), anti-CD11b (M1/70), anti-CD11c (HL3), anti-CD25 (PC61), anti-TCRbeta (H57-597), anti-TCR{gamma}{delta} (GL3), anti-NK1.1 (PK136), anti-Ter119 (Ly-76), and anti-Gr1 (RB6-8C5). LSKs were defined as Lin–CD127–Sca-1highCD117high and common lymphoid progenitors (CLPs) as Lin–CD127+Sca-1lowCD117low cells. FACS stainings using soluble Notch ligands were conducted for 40 min in PBS, 2% FCS, and 2 mM CaCl2 at room temperature followed by staining with a donkey anti-human IgG (Jackson ImmunoResearch Laboratories) or an anti-mouse IgG2a (clone R19-15; BD Pharmingen) second step. For all fusion proteins, the saturating concentration was found to be 25 nM. The pCS2+mN1FL6MT plasmid (a gift by Dr. R. Kopan, Washington University, St. Louis, MO) encoding full-length Notch1 was used to transfect HEK293 cells. Flow Jo (Tree Star) and CellQuest (BD Biosciences) were used for data analysis.

In vitro cultures of hemopoietic progenitor cells

For the culture of thymic progenitor cells layers of OP9 (provided by M. Kondo, Duke University, Durham, NC) and OP9-DL1 (provided by J. C. Zuniga-Pflücker, University of Toronto, Ontario) stromal cells as well as mixtures of OP9 and OP9-DL1 cells were plated at 2 x 103 cells per 96-well plate 48 h before progenitors were sorted. Twenty-four hours before cell sorting, the cell culture medium was replaced by RPMI 1640 containing 10% FCS, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM sodium pyruvate, and 2 x 10–5 M 2-ME. Thymic progenitors were subsequently sorted onto the different stromal cell layers and progenitors were cultured in the presence of complexed soluble Notch ligands, recombinant murine IL-7, and Flt3L, (10 ng/ml each; R&D Systems) at 37°C in a humidified chamber and 5% CO2. Notch ligand, anti-Fc mAb, and streptavidin were preclustered for 15 min at room temperature before addition to the cultures at a final concentration of 12.5 nM Notch ligand, 10 µg/ml anti-Fc mAb, and 10 µg/ml streptavidin. For OP9-DL1 to OP9 switch cultures, TMPs were cultured for 5 days and resorted as CD45+ (leukocyte common Ag) hemopoietic cells onto a fresh layer of OP9 cells. The {gamma}-secretase inhibitor N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (Calbiochem) was added to cultures at a final concentration of 1 mM. LSKs were sorted as Lin–CD127–Sca-1highCD117high and CLPs as Lin–CD127+Sca-1lowCD117low bone marrow cells, and TMPs were sorted from the thymus as Lin–CD25–CD117highCCR9EGFP high cells. For limiting dilution assays, precursors were sorted as 100, 40, 20, and 10 cells per well, and the wells containing T cells were determined 11–13 days later. At least 36 wells from at least three independent experiments were analyzed for each cell concentration. For precursor populations showing a frequency higher than one in 10, additional wells containing 4, 2, and 1 cell(s) were analyzed to cover the most informative range (28). Differentiation of TMPs toward dendritic cells was achieved by double-sorting cells onto methylcellulose (Methocult M3231; StemCell Technologies) supplemented with recombinant, murine IL-1beta (5 ng/ml), IL-3 (50 ng/ml), IL-6 (10 ng/ml), stem cell factor (SCF) (30 ng/ml), and Flt3L (30 ng/ml) (all R&D Systems). Responsive wells were scored after 4 days in culture by FACS or light microscopy as described (29).

In situ hybridization and RT-PCR

In situ hybridization was done as previously described (30). RT-PCR analyses were conducted as described (31) using the primers for pT{alpha} (5'-ggcaccccctttccgtctc-3' and 5'-gtccaaattctgtgggtggga-3'); Deltex1 (5'-cgacttccctatggaaaacg-3' and 5'-aaagttaagatagcctcgtc-3'); Hes-1 (5'-agccagtgtcaacacgacac-3' and 5'-tgcaggttccggaggtgctt-3'), and Nrarp as described (32).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
The majority of TMPs is not restricted to the T cell lineage

Several studies have shown that adult thymic precursors have the potential to give rise to mature cells of lineages other than the T lineage such as B cells, NK cells, and myeloid cells (7, 9, 10, 33). The low frequency of these non-T lineage progenitors in a precursor population that is thought to predominantly produce T lymphocytes has frequently been interpreted to mean that the precursors are "contaminated" with rare precursors of non-T cell lineages and that most thymic precursors are T lineage committed. Consistent with this idea, a recent report (34) showed that the most immature T lineage precursors can be separated from precursor populations containing B potential by CD24 and CD117(c-kit) staining. Alternatively, non-T lineage cells in the thymus could be derived from multipotent or oligopotent precursor cells that develop mostly toward the T lineage but in rare cases give rise to non-T lineage cells. Consistent with this possibility, we have recently shown that the most immature hemopoietic precursor in the thymus, which we have termed the thymic multipotent precursor or TMP (defined by the markers Lin–CD25–CD117high CCR9EGFP high), gives rise to T, B, and dendritic cells on the single cell level (10). However, with the low frequency of bipotent T/B precursors among TMPs (~3%) the possibility exists that most TMPs are T lineage restricted. To distinguish these possibilities, we conducted the following experiments. When 570 single TMPs were double sorted and cultured under conditions permissive for T, B, and NK cell development, 85% of the responsive TMPs that generated committed T lineage cells also produced cells of a non-T lineage fate (Fig. 1A). This result indicated that the majority of TMPs are not restricted to the T cell lineage but contain the potential for the B and NK lineages. For this type of experiment it is crucial to demonstrate that the assay indeed measures clonal events. We therefore double sorted random single cells from a mixture of genetically marked TMPs onto OP9-DL1 (21) stromal layers (Fig. 1B). The double sorting of single cells from a mixture of CD45.1+ and CD45.1– TMPs consistently resulted in the generation of either CD45.1+ or CD45.1– progeny. A well that contained CD45.1+ and CD45.1– thymocytes, as would be expected to occur if more than one cell had been sorted per well, was not observed among 93 productive wells analyzed (Table I). These findings supported our conclusion that the majority of TMPs are uncommitted and not restricted to the T lineage.


Figure 1
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  		FIGURE 1. The majority of TMPs that generate T lymphocytes are not restricted to the T cell lineage. A, Single TMPs identified as Lin–CD25–CD117highCCR9EGFP high cells were double sorted from thymocytes of heterozygous CCR9-EGFP knock in mice onto mixtures of OP9:OP9-DL1 (20:1) and analyzed after 11–13 days for the presence of T, B, and NK cells. As described previously (10 ), T lineage cells were scored as CCR9EGFP+ DN3/DN4 thymocytes, B lineage cells as CD19+ cells, and NK cells as NK1.1+ cells. B, One hundred cells (top panels) or single cells (middle and lower panels) from a 1:1 mixture of CD45.1+ (isolated from F1 mice from a cross of homozygous CCR9-EGFP knock-in and C57BL/6-Ly5.1 mice) and CD45.1– (isolated from heterozygous CCR9-EGFP knock-in mice) TMPs were double-sorted on OP9-DL1 stroma cells and cultured for 11–13 days. FACS analyses of wells seeded with the indicated number of cells are shown. C, 500 TMPs and 500 bone marrow (BM) LSKs were sorted onto methylcellulose containing IL-1, IL-3, IL-6, SCF, and Flt3L and analyzed after 4 days by FACS. D, Single TMPs were cultured on methylcellulose containing IL-1, IL-3, IL-6, SCF, and Flt3L for 4 days and responsive wells were scored by light microscopy as described (29 ). Responsive wells contained clusters of 2–13 cells with numerous cytoplasmic extensions. E, Single TMPs were sorted on OP-DL1 stroma cells and cultured for 11–13 days. The wells containing T cell lineage committed cells were scored by FACS analysis as in A.

 

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  		Table I. Culture of random single cells from a 1:1 mixture of CD45.1–CD45.2+ and CD45.1+CD45.2+ TMPs on OP9-DL1

 
The cloning efficiency of single TMPs generating committed T lineage cells under conditions permissive for T, B, and NK development was only 12%, and it could therefore be argued that this assay selects for multipotent precursor cells while the T cell lineage-restricted precursors fail to develop. We therefore turned to culture conditions that efficiently support the differentiation of thymic precursors into dendritic cells (Fig. 1C). Consistent with a previous report analyzing the CD4low precursors (29), of which the TMP population represents only a small fraction, on average 67% and in some experiments up to 80% of single TMPs gave rise to small colonies of cells with the morphological features of dendritic cells (Fig. 1D). This result indicated that the vast majority of TMPs have the capacity to differentiate into dendritic cells. Specification toward the dendritic cell lineage occurs in TMPs with even higher efficiency than T cell lineage commitment, as single TMPs differentiate into T lineage-committed DN3/4s in our hands most efficiently on the stroma cell line OP9-DL1 with a cloning efficiency of ~50% (Fig. 1E).

TMPs show signs of ongoing Notch signaling

The above findings indicate that the majority of single TMPs are not T lineage restricted and that TMPs adopting a T lineage fate have to actively silence their B, NK, and dendritic cell potential. Because Notch signaling is known to play an important role in this process, we analyzed the level of Notch signaling in these precursors by semiquantitative RT-PCR for Notch target genes that have been identified in a Notch intracellular domain-transfected thymoma cell line (32, 35). All transcripts were expressed at significantly higher levels than in Lin–Sca-1+c-Kit+ LSK bone marrow cells that contain T lineage precursors at a very high frequency (20, 36) and from which the ETP population is most likely derived (10, 37) (Fig. 2A). Expression levels appeared to increase slightly from the TMP to the more mature EGFPCCR9 low ETP stage. This observation suggested that early thymic precursors receive Notch signals in the thymic microenvironment that lead to target gene expression well above levels found in the corresponding cells in the bone marrow. Increased levels of Notch target gene expression were also found in a recently described ETP subset that expresses low levels of the surface marker Flt3 (38). It was therefore important to determine the relationship between the two ETP subsets. Multiparameter flow cytometry revealed that TMPs and Flt3+ ETPs are largely overlapping populations and that TMPs but not EGFPCCR9 low ETPs express surface Flt3 (Fig. 2B). It is important to point out, however, that Flt3 levels found on TMPs are much lower than on bone marrow LSKs and that a substantial fraction of TMPs is negative for Flt3 (data not shown and Fig. 2B). Our data show that early thymic precursors express higher levels of Notch target genes than their presumptive precursors in the bone marrow, suggesting ongoing Notch signaling in these cells.


Figure 2
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  		FIGURE 2. TMPs express elevated levels of Notch target genes. A, Semiquantitative RT-PCR was conducted on 5-fold dilutions of cDNA generated from sorted bone marrow (BM) LSKs, TMPs, and a slightly more differentiated population among ETPs expressing low levels of CCR9EGFP (EGFPlow (EGFPlo) ETPs) (10 ) for which the FACS analysis is shown in B. HPRT, hypoxanthine phosphoribosyltransferase. B, TMPs, but not a slightly more differentiated population among ETPs expressing low levels of CCR9EGFP (EGFPlow ETPs), show Flt3 surface expression. Top left and top middle plots are gated on Lin– cells and the top right plot on thymic Lin–Sca-1+c-Kit+ LSK cells as shown in the top middle plot. The bottom histogram plots are gated on EGFPlow ETPs and TMPs and the gray curve shows staining with an isotype control. Plots are shown in a biexponential format that avoids displaying the cell populations squished against an axis.

 
TMPs express Notch receptors on the surface

If TMPs actively repressed their non-T lineage potentials and committed to the T lineage in the thymic microenvironment, they would be expected to express Notch receptor on the cell surface. This point was crucial, because a previous study (16) failed to demonstrate Notch1 expression on DN1 thymocytes despite its well-documented importance in T cell lineage specification. On the mRNA level, we found expression of all four Notch receptors in TMPs by RT-PCR (our unpublished data). To detect protein expression on the surface of TMPs, we generated fusion proteins of the murine Notch ligands Delta-like1, Delta3, Delta4, Jagged1, and Jagged2 and either the hFc or the mouse Fc (mFc) portion of Ig including the hinge region. Such fusion proteins have previously been shown to physically interact with soluble and membrane-bound Notch receptors (39, 40). Fusion proteins purified by affinity chromatography showed saturable, concentration-dependent staining on Ba/F3 cells (Fig. 3A) that required the presence of calcium (Fig. 3B). For reasons that were not further investigated, the Delta3 hFc fusion protein showed no specific binding above levels that were seen with the human Fc portion alone. Transfection of HEK293 cells with full-length Notch1 significantly increased Notch ligand staining above endogenous Notch receptor levels present in HEK293 cells (41, 42), indicating that the staining was Notch receptor specific (our unpublished data). To identify the Notch ligand fusion that worked best in FACS analysis, we stained full-length Notch1-transfected HEK293 cells with saturating concentrations of the fusion proteins (Fig. 3C). The highest levels of staining in these experiments were consistently found with the D4hFc fusion protein, and we therefore analyzed the expression of Notch receptors on thymocytes with this reagent. Consistent with a previous report detecting surface expression of Notch1 (16), the highest levels of Notch receptors were found on CD4–CD8–CD3{epsilon}– triple negative (TN) cells with only low levels of receptor expression on double positive and single positive thymocytes (Fig. 3D). Fig. 3D shows that the majority of TN1 thymocytes are either negative for Notch receptors or express them at levels well below those of TN2 and TN3 thymocytes. In contrast, TMPs, which represent only a minute subset within the TN1 population (10), stained positive for D4hFc binding and showed Notch receptor expression at levels identical with TN2/TN3 thymocytes. Further analyses excluding cells expressing lineage markers showed that the entire ETP population (corresponding to fraction DN1ab), which includes TMPs but not the noncanonical precursors DN1c, DN1d, and DN1e (34), expressed significant levels of surface Notch receptor (Fig. 3E). Although this approach cannot distinguish between the different Notch receptors, the data indicate that TMPs, but not the majority of DN1s outside of the ETP gate, express high levels of Notch receptors.


Figure 3
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  		FIGURE 3. TMPs and ETPs but not other thymocytes present in the DN1 population show surface expression of Notch receptors. A, Staining of soluble Notch ligand fusion proteins is concentration dependent. Ba/F3 cells were incubated with the indicated amounts of Delta-like1 mFc (D1mFc) (given as µg/ml), and staining was visualized by incubation with an anti-mouse Fc mAb. B, Staining with soluble Notch ligand fusion proteins is calcium dependent. Ba/F3 cells were stained with 2 µg/ml D1mFc either in the absence or the presence of 2 mM EGTA. A sample without a fusion protein was used as negative control. C, HEK293 cells transfected with full-length Notch1 were stained using purified Notch ligand fusion proteins, each at a saturating concentration of 25 nM. Fusion proteins consisted of the extracellular domains of Delta-like1 (D1), Delta4 (D4), Jagged1 (J1) and Jagged2 (J2) fused to either murine IgG2a (mFc) or human IgG1 (hFc) hinge and Fc part. A purified protein lacking the Notch ligand part (hFc) was used as control. D, CD4–CD8–CD3{epsilon}– (TN) thymocytes, coreceptor double-positive (DP), coreceptor single-positive thymocytes (SP), and TMPs were stained with D4hFc (dark line) and Fc protein lacking a Notch ligand (hFc) (gray area). E, ETPs (corresponding to fraction DN1ab) and noncanonical precursors DN1c, DN1d, and DN1e were stained with D4hFc (dark line) and Fc protein lacking a Notch ligand (hFc) (gray area). The dot plot is gated on Lin–/low (Lin–/lo)CD25–CD44+ thymocytes. Plots are shown in a biexponential format that avoids displaying the cell populations squished against an axis.

 
Intriguingly, none of the other fusion proteins, namely Delta-like1 hFc, Delta-like1 mFc, Jagged1 hFc, and Jagged2 hFc, showed staining on thymocytes above background levels (our unpublished data). This finding is in contrast to the staining seen on Ba/F3 cells (Fig. 3, A and B) and Notch1-transfected HEK293 cells (Fig. 3C) and suggests that the avidity of the Notch receptor-Ig-fusion protein interactions differ for Notch receptors on thymocytes and cell lines. From their biologic activity it is clear that all fusion proteins do interact with Notch receptors on thymocytes (see below). The fact that most of these interactions cannot be detected by flow cytometry indicates low avidities of these interactions. Because soluble Ig fusion proteins are not the natural ligand and amino acids very close to the transmembrane domain omitted from the construct could be crucial for binding, conclusions concerning in vivo receptor-ligand avidities cannot be drawn easily using this approach.

TMPs lose their B cell potential rapidly and irreversibly

Next, we investigated the kinetics with which TMPs lose their capacity to give rise to B cells and commit to the T cell lineage. This was an important issue, because Notch-mediated T cell lineage specification in fetal liver precursors has recently been shown to be an asynchronous and protracted process that maintains non-T cell lineage potentials, e.g., B cell potential, in progenitor cells over extended periods of time (17). If TMPs committed to the T cell lineage in the same way, it would be very unlikely that this could take place in the thymus because B cell potential in early thymic precursors has been notoriously difficult to detect (10, 18, 19, 38). We have shown previously that within the ETP population only TMPs retain the capacity to give rise to B cells, whereas more mature stages have lost this capacity (10). Consistently, 10 of 16 wells seeded with 100 TMPs onto the bone marrow stroma cell line OP9 (43) contained CD19+ B cells after 12 days of culture (Fig. 4A). Wells containing developing B cells were almost entirely lost when TMPs were cultured for 5 days on OP9-DL1 stromal layers and resorted as CD45+ (leukocyte common Ag) hemopoietic cells onto a fresh layer of OP9 cells (Fig. 4A). Thus, in contrast to fetal liver precursors, TMPs rapidly lose their ability to give rise to B cells when placed on OP9-DL1 cells. To rule out the possibility that OP9-DL1 cells were inadvertently transferred during the resort, all experiments were conducted using OP9 cells without a GFP marker, which allowed us to distinguish between OP9 and OP9-DL1 cells. None of the switch cultures was found to contain significant amounts of GFP+, i.e., OP-DL1 stroma cells (Fig. 4B). This result indicated that TMPs cultured in the presence of membrane-bound Notch ligands rapidly lose their B cell lineage potential. Similarly, TMPs cultured on OP9 cells rapidly lose their capacity for T lineage specification (Fig. 5). When TMPs cultured on OP9 stroma layers were switched to OP9-DL1 stroma after 2, 3, 4, 5, or 6 days, wells containing T lineage cells rapidly declined the longer TMPs had been cultured on OP9. Conversely, the number of wells that contained B lineage cells that proliferated even in the presence of Delta-like1 ligand increased. The rapid loss of T lineage cells in this experiment is consistent with the finding that T cell development requires continuous or recurrent Notch signaling (19, 44). Collectively, our data demonstrate that TMPs rapidly lose their B and T cell lineage potential when cultured on OP9-DL1 and OP9 stroma layers, respectively, and that TMPs commit to either lineage irreversibly and as a homogeneous population.


Figure 4
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  		FIGURE 4. TMPs rapidly lose their B cell lineage potential on OP9-DL1 stroma cells. A, TMPs were sorted as 100 cells per well either on OP9 stroma cells and analyzed after 11–12 days (upper panels) or first on OP9-DL1 and resorted after 5 days onto OP9 stroma cells and analyzed after 12 or 16 days as shown in the diagram (lower panels). FACS plots to the right are gated on NK1.1–CD19– cells, i.e., the lower left quadrant of FACS plots to the left. The number of wells seeded with 100 TMPs that gave rise to B cells is indicated. B, If OP9-DL1 stroma cells had inadvertently been transferred to the OP9 culture when TMPs were cultured as shown in the scheme in A, they would appear as large, complex cells in forward scatter/side scatter and express high levels of GFP (see analysis of OP9-DL1 cells in the panels on the right). Panels to the left show a representative analysis of a switch culture 11 days after resorting showing only few cells in the OP9-DL1 gate. Our previous analyses have shown that B cell development from TMPs proceeds efficiently even in the presence of 5% OP9-DL1 cells (10 ). Therefore, even if switch cultures indeed contained low amounts of contaminating OP9-DL1 cells, these would not be sufficient to abrogate B cell development.

 

Figure 5
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  		FIGURE 5. TMPs lose their T cell lineage potential on OP9 stroma cells. TMPs sorted as 100 cells per well onto OP9 stroma cells were transferred after 2, 3, 4, 5, or 6 days onto OP9-DL1 stroma cells. Wells were analyzed 12 days after the switch as shown in the diagram. The frequency of wells containing T and B cells is plotted against the day the culture was switched from the OP9 to the OP9-DL1 stroma and the number of analyzed wells is indicated.

 
TMPs are highly sensitive to Notch signals for T cell lineage commitment

The presented data support the notion that multipotent precursors travel from the bone marrow to the thymus and we wondered why these precursors would not commit to the T lineage already in the bone marrow. T lineage precursor frequency in bone marrow lymphoid-primed multipotent progenitors and CLPs has been shown to be very high (20, 45), and both Notch ligands and receptors are widely expressed in the bone marrow (12). The finding that T cells develop on the bone marrow stroma cell line OP9, which overexpresses Delta-like1, suggests similar requirements for early B and T cell development (21). However, the sensitivity of bone marrow in comparison with thymic T lineage precursors to Notch signals has, to date, not been investigated. To address this question experimentally we developed a culture system that allowed us to titrate the Notch signal strength by using complexed, soluble Notch ligands to drive T lineage commitment in precursor populations cultured on OP9 stroma cells. Soluble Notch ligands have been shown to induce Notch signaling when clustered into higher molecular complexes (46, 47). The Notch signaling strength that can be achieved with complexed soluble ligand has been shown to be much lower than that achieved with membrane-bound ligand (47). Because hemopoietic precursors do not develop into T lineage cells on OP9 cells in the absence of added Notch signals, this system was found to be ideal for determining the requirements for T cell lineage commitment. Hemopoietic precursors were sorted from heterozygous CCR9-EGFP knock-in mice, and the readout for this assay was the generation of DN3/4-committed T lineage cells that can be identified by high level CCR9EGFP expression (10). The requirement for Notch signals at the subsequent DN3/4 to double-positive beta-selection step (48, 49) was not investigated in this study. The generation of DN3/4 committed T lineage cells from TMPs cultured on OP9 cells required the addition of higher molecular complexes of cross-linked soluble Delta-like1 (Fig. 6, A and B). Fig. 6A shows that only the combination of the Delta-like1 Fc fusion protein, an Fc-specific biotinylated mAb, and streptavidin resulted in significant numbers of positive cultures. All three components were preclustered before addition to the cultures, and omitting any one of them abrogated T lineage commitment. The addition of complexed Fc-protein lacking the Notch ligand domain was equally ineffective. Whether a mouse or a human Fc portion together with the appropriate cross-linking Ab was used was irrelevant for the outcome of the experiment. Typical cultures contained not only NK1.1–CD19–CD90(Thy-1)+ lymphocytes that expressed high levels of EGFPCCR9 indicative of committed DN3/4 stage thymocytes but also NK cells (Fig. 6B). This finding indicated that the soluble Notch ligand, in contrast to the membrane-bound ligand on OP9-DL1 stroma, does not entirely suppress non-T cell lineages, suggesting that the Notch signal strength exerted by soluble Notch ligand in this assay was lower than that on OP9-DL1. Soluble ligand-induced Notch signals were mediated by a {gamma}-secretase-dependent mechanism (Fig. 6C), and T lineage commitment of TMPs cultured with a soluble Notch ligand required the same 12 days observed in cultures on a membrane-bound ligand (10). Similarly, mature B cells and NK cells developed with the same kinetics found on mixtures of OP9/OP9-DL1. This observation indicated that the mode of Notch ligand presentation, membrane bound or soluble, did not affect the developmental kinetics. Using a complexed, soluble Notch ligand we could now titrate the amount of Notch signal strength required to induce T lineage commitment in TMPs (Fig. 6D). Increasing concentrations of ligand suppressed B lineage commitment and produced increasing numbers of wells containing T lineage-committed cells up to a concentration of 12.5 nM Delta-like1 when almost all wells, each seeded with 100 TMPs, contained T lineage cells. This observation was consistent with the idea that there is a threshold level of Notch signaling that is required for a precursor to commit to the T lineage. Fig. 6D shows that for TMPs 12.5 nM Delta-like1 is the lowest effective concentration to efficiently induce T lineage commitment. To compare the threshold levels of productive Notch signals between bone marrow and thymic precursors, we determined the frequency of T lineage precursors capable of responding to 12.5 nM Delta-like1 by limiting dilution assays (Table II). Although bone marrow LSKs (defined as Lin–CD127–Sca-1highCD117high bone marrow precursors), CLPs (Lin–CD127+Sca-1lowCD117low bone marrow precursors), and TMPs (Lin–CD25–CD117highCCR9EGFP high thymocytes) contain T cell lineage precursors at a frequency between 1 in 3 to 1 in 50 when assayed on OP9-DL1 stroma, only TMPs contain T lineage precursors at a detectable frequency that respond to complexed, soluble ligand. This difference was particularly striking for CLPs, which showed a high frequency of T lineage precursors on OP9-DL1. These results indicate that the Notch signal strength required to commit multipotent precursors in the bone marrow to the T lineage is considerably higher than for precursors in the thymus. Differential responsiveness of T lineage precursors could be explained if thymus but not bone marrow precursors received Notch signals in their respective niches. Thymic but not bone marrow precursors would then be "preactivated" by ongoing Notch signaling if, for example, bone marrow precursors lacked Notch receptors or if Notch ligands were absent from the bone marrow precursor niche. But unresponsiveness to soluble ligand cannot be explained by the lack of Notch receptors on bone marrow precursors, at least for CLPs and for CCR9EGFP+ LSKs, as these subpopulations express high levels of Notch receptors on the surface (Fig. 6E). Furthermore, it is very unlikely that thymic but not bone marrow precursors are exposed to Notch ligands in their respective niche, as LSKs have been shown to express Jagged2 (50), a Notch ligand that is capable of driving T cell lineage commitment (see below). It could then be argued that TMPs are more sensitive to Notch ligand-mediated T lineage commitment because they have been exposed longer to Notch signals than their bone marrow counterparts and therefore require only little additional Notch signal for T lineage commitment. This would be consistent with their increased expression of Notch target genes in comparison to LSK cells (Fig. 2A). RT-PCR analyses revealed that CLPs also express elevated levels of Notch target genes (compare Figs. 6F with 2A) despite their unresponsiveness to 12.5 nM Delta-like1 (Table II). In conclusion, we find that the differential responsiveness of bone marrow vs thymic precursors to low level Notch signals cannot be easily explained by their differential exposure to Notch signals in their respective niche. We therefore favor the idea that T lineage commitment is actively repressed in bone marrow precursors.


Figure 6
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  		FIGURE 6. Complexed, soluble Notch ligands induce T cell lineage commitment in TMPs. A, One hundred TMPs were cultured in the presence of complexed, soluble Notch ligands. Purified Notch ligand-Fc fusion proteins, cross-linking biotinylated Abs, and streptavidin were preclustered and added to OP9 cultures of TMPs on the day of initiation as indicated. Wells were analyzed 11–13 days later for T lineage committed CCR9EGFP+ DN3/4 thymocytes as shown in B. The number of wells that contained T lineage cells among analyzed wells is indicated. D1, Delta-like1. B, The culture of TMPs in the presence of Delta-like1 mFc, biotinylated anti-mFc mAb, and streptavidin results in the generation of CCR9EGFP+ DN3/4 thymocytes. The left panel shows all lymphoid cells, the middle panel is gated on NK1.1–CD19– cells, and the right panel is gated on CD4–CD8–CD3{epsilon}– (TN) cells. C, Complexed, soluble ligands mediate T cell lineage commitment through a {gamma}-secretase-dependent mechanism. TMPs were cultured in the presence of 1 mM N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester {gamma}-secretase inhibitor or carrier alone and wells were scored 11–13 days after initiation of the culture. D, 100 TMPs were cultured in the presence of different concentrations of preclustered soluble Delta-like1 mFc (D1mFc) and wells were scored for T and B cells 11–13 days after initiation of the culture. E, CLPs and bone marrow LSKs express high levels of Notch receptors. Notch receptor expression was measured by Delta4 hFc (D4hFc) staining on bone marrow cells electronically gated for CLPs, CCR9EGFP– and CCR9EGFP+ LSKs. F, Semiquantitative RT-PCR was conducted on 5-fold dilutions of cDNA generated from sorted bone marrow (BM) LSKs and CLPs. HPRT, hypoxanthine phosphoribosyltransferase.

 

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  		Table II. T cell lineage precursor frequency among bone marrow and thymic hemopoietic precursors

 
Both Delta and Jagged ligands induce T cell lineage commitment in TMPs

The data to date indicate that uncommitted precursors generated in the bone marrow adopt a T lineage fate in the thymus through Notch signals and we therefore wanted to identify the functionally relevant Notch ligands. The earliest immigrants to the adult thymus have been shown to enter at the corticomedullary junction (51) and migrate toward the subcapsular zone by a CCR9-dependent mechanism (52), and relevant Notch ligands would have to be expressed accordingly. Intriguingly, in situ hybridization studies on adult thymus sections revealed detectable expression only for Delta4 and Jagged2 (Fig. 7A). Riboprobes for Delta-like1, Delta3, and Jagged1 showed specific staining in the developing brain (D1 and D3) and the developing vasculature (J1) as expected (our unpublished data), demonstrating that the probes were functional. RT-PCR analyses of adult thymic stroma detected transcripts for all five ligands (our unpublished data), but expression levels are apparently below the sensitivity of in situ hybridization. The data indicate that Delta4 and Jagged2 but not the other Notch ligands are expressed at clearly detectable levels in the relevant thymic niche, i.e., the corticomedullary junction and the cortex. Concerning the functional role of these ligands, two reports have shown that Delta but not Jagged ligands are capable of inducing T cell lineage commitment, as neither Jagged1 transfectants of S17 cells (53) nor OP9 cells (54) generated T lineage cells from multipotent hemopoietic precursors. This had been interpreted to mean that Delta and Jagged ligands transmit qualitatively distinct signals. The data presented here indicate that the exact quantification of Notch ligand is crucial, but the amount of surface Delta or Jagged had not been determined in those studies. To compare the quality of Notch signals generated by different Notch ligands side by side, we added preclustered ligands to TMPs cultured on OP9. We found that not only Delta4 but also Jagged1 and Jagged2 produced DN3/4-committed T lineage cells in these cultures (Fig. 7B). All three ligands were found to efficiently induce T cell lineage commitment in >85% of the wells seeded with 100 TMPs in the presence 12.5 nM soluble ligand (Fig. 7C), the same molar concentration that we found to be effective for Delta-like1 (Fig. 6D). No difference was observed in the kinetics of T lineage commitment and the kinetics of the development of non-T lineage cells compared with cultures on OP9/OP9-DL1 mixtures or in the presence of D1hFc. As expected, T lineage cells generated by treating TMPs with complexed, soluble Jagged ligands expressed transcripts for Deltex1, GATA-3, CD4, CD8, and CD3 (Fig. 7D). We conclude that not only Delta but also Jagged ligands induce T cell lineage specification in uncommitted hemopoietic precursors isolated from the thymus, suggesting functional redundancy among these Notch ligands in the thymus.


Figure 7
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  		FIGURE 7. Delta and Jagged ligands induce T cell lineage commitment in TMPs. A, In situ hybridization on adult thymic sections with probes specific for all known Notch ligands. A white line marks the corticomedullary junction. M, Medulla; scale bar, 100 µm. B, The culture of 100 TMPs in the presence of complexed, soluble Delta4-Fc, Jagged1-Fc, and Jagged2-Fc results in the generation of CCR9EGFP+ DN3/4 thymocytes. FACS plots to the left are gated on NK1.1–CD19– cells and FACS plots to the right are gated on CD4–CD8–CD3{epsilon}– (TN) cells. C, The frequency of wells containing mature cells of the T, NK, or B lineage or no cells (NG, No growth) 11–13 days after culture of 100 TMPs in the presence of complexed, soluble Notch ligands (D1, Delta-like 1; D4, Delta4; J1, Jagged1; J2, Jagged2) is shown. All ligands were added as purified proteins at 12.5 nM. The data is based on at least 16 different wells from at least three different experiments. D, RT-PCR analysis of productive wells after 11–13 days of culture of TMPs in the presence of complexed, soluble Delta-like1 (D1), Delta4 (D4), Jagged1 (J1), and Jagged2 (J2).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
T cells are derived from multipotent hemopoietic precursors. The hemopoietic precursor that commits to the T cell lineage by repressing alternative fates and the microenvironment in which T cell lineage commitment occurs is unclear. There is good evidence for both scenarios, i.e. T lineage commitment in precursors in the thymus (12) and in precursors in the bone marrow (15). In this study we present several lines of evidence for a model in which multipotent or oligopotent hemopoietic precursor cells travel from the bone marrow to the thymus where, under the influence of Notch signals, they give rise to mainly T lymphocytes and small amounts of B cells and dendritic cells. First, we show on the single cell level that the majority of the earliest thymic precursors are uncommitted and retain the capacity to produce B, NK, and dendritic cells. This finding indicates that repression of alternative fates and commitment to the T cell lineage occurs in precursors in the thymus and not the bone marrow. Second, we find that thymic precursors supplied with a Notch signal rapidly and irreversibly lose non-T lineage potentials in vitro, which is consistent with the small number of thymocytes with non-T lineage potential found in vivo. Third, our data supply evidence that thymic precursors are far more sensitive to T lineage commitment mediating Notch signals than are bone marrow precursors.

It has been a long standing question in early T cell development if the B cells, NK cells, and dendritic cells that have been derived from thymic precursors in various experimental settings stem from precursors with multilineage potential or represent the progeny of lineage-restricted precursors that could not be separated by the markers used (8). This question can only be answered by an assay that measures clonal events and allows the simultaneous differentiation of T and non-T lineage cells. Using such an assay we show that the majority of TMPs are not restricted to the T cell lineage and we therefore favor the idea that hemopoietic cells of non-T lineage stem from precursors with oligolineage or multilineage potential. Clonal analyses of fetal thymic precursors have been conducted for many years by the Kawamoto/Katsura laboratory in organ cultures (55) and more recently by the Zuniga-Pflücker laboratory on OP9-DL1 stroma (44). But, comparison to the adult situation is difficult: Most reports do not find B precursor activity in fetal thymic precursors (56, 57), and fetal liver progenitors with B potential have been shown to emerge later in ontogeny than progenitors with T potential (58). The latter observation is consistent with the finding that phenotypically and functionally distinct precursors enter the fetal thymus over time (59, 60). This led to the notion that in the embryo T and B cell development proceeds either in separate pathways or that B cell lineage potential is lost prethymically. In the adult, in contrast, most reports do find B potential in thymic precursors (7, 9, 33, 38), and our demonstration of a bipotent T/B precursor among TMPs (10) suggests that in the absence of Notch signals these cells will generate B cells. Thus, there is little evidence for a multipotent precursor that enters the embryonic thymus. Using the same clonal assay the Kawamoto/Katsura laboratory (61) has recently shown that most ETPs retain the capacity to produce NK cells and dendritic cells, which is in agreement with our data, but B potential was not detected in these studies. The reason for this difference may be the low frequency of B precursors in TMPs (~1:30) analyzed here that represent only 20% of ETPs and contain all of the B potential in this subset (10).

To analyze the requirements for T cell lineage commitment in TMPs, we have developed a novel culture system that uses complexed, soluble Notch ligands on TMPs cultured on OP9 stroma. For studying the effects of Notch signaling on hemopoietic cells, the use of soluble Notch ligands has several advantages over currently available assay systems. By varying the amount of soluble ligand, it is possible to titrate the Notch signal strength that is required to commit multipotent precursors to the T cell lineage. Using this approach we find that the requirements of T lineage precursors in bone marrow and thymus to commit to the T cell lineage differ vastly. Although both CLPs and TMPs respond to Notch signals supplied by OP9-DL1 stroma with similar efficiencies, CLPs cannot be committed to the T cell lineage with a TMP-effective dose of the soluble Notch ligand. We conclude that the sensitivity to Notch-mediated T lineage commitment is stage dependent and that T lineage commitment is harder to achieve at the bone marrow precursor stage. Elucidating the molecular basis of this difference will require further work. The simplest explanation for this finding would be that the thymic but not the bone marrow precursors are primed by Notch signals in their respective niche and therefore require less additional Notch signal to commit. But both Notch ligands and Notch receptors are present also in the bone marrow, and both CLPs and TMPs express elevated levels of Notch target genes. A recent report indicates that the retroviral re-expression of E47 in E2A-deficient hemopoietic progenitors induces Hes-1 expression (62), suggesting that Hes-1 expression in CLPs may in part be Notch independent. Ongoing work therefore aims to elucidate how much Notch signaling occurs in CLPs, what the transcriptional program is that weak (soluble Notch ligands) vs strong (OP9-DL1) Notch signals initiate in these cells, and how these parameters differ in TMPs. In this respect, it is important to keep in mind that the bone marrow precursor that travels to the thymus is most likely an extremely rare cell (63). The low frequency of responsive cells in LSKs and CLPs may therefore reflect the fact that the majority of these cells develop independently of the T cell lineage. Further work is being undertaken to identify bone marrow precursors with high Notch ligand sensitivity.

Previous reports have used {gamma}-secretase inhibitors to determine the Notch signaling requirements of fetal thymocytes (44, 64) and found that different levels of Notch signaling inhibition affect distinct steps of T lineage specification. Our own attempts to culture adult TMPs in fetal thymic organ culture in the presence of several different {gamma}-secretase inhibitors have failed to reliably produce B cell development, which may suggest that these molecules interfere with the intramembranous cleavage of other essential molecules in adult but not embryonic hemopoietic precursors. The Bernstein group (65) has recently shown that lower densities of plate-bound Delta-like1 expand early B lineage cells from bone marrow LSKs, whereas higher densities expand early T lineage cells. Although our data clearly agree with the effects on T lineage cells, we have not found effects of low levels of complexed, soluble Notch ligand on the cloning efficiency or the extent of expansion of B lineage cells (compare Fig. 6D with Fig. 4A (the number of B cell wells found in the absence of soluble ligand is typically 40–60% of wells); also, our unpublished data).

Another advantage of the soluble Notch ligand-OP9 culture system is that the effects of different Notch ligands can be analyzed side by side using conditions that differ only in the extracellular Notch ligand domain. Previous reports that analyzed the functionality of Jagged1 by retroviral transduction of S17 (53) and OP9 (54) cells indicated that Delta and Jagged ligands transmit signals of different quality to hemopoietic cells. Because the amounts of functional Notch ligands on such stroma cells are difficult to enumerate, the mRNA expression levels of Delta and Jagged ligands never match, and the overexpression of one Notch ligand appears to affect the expression level of another Notch ligand (54), we would argue that soluble ligands are better suited to allow side by side comparisons of Notch ligands. Concentrating on the T cell lineage commitment step of T cell development in this study, we have not addressed the Notch-dependent DN3/4 to DP beta-selection step, and it will be interesting to see how beta-selection is influenced by different Notch levels of different ligands. Our results indicate that Delta-like1, Delta4, Jagged1, and Jagged2 are equally capable of instructing TMPs to adopt a T cell lineage fate and predict that all of these ligands may be able to complement the lack of another in vivo. As the absence of Delta-like1 does not interfere with T cell development in vivo (66), this may be true not only for single ligands but also for combinations thereof.


   Acknowledgments
 
We are indebted to C. Sainz and M. Konrath for excellent technical support and to A. Würch and J. Wersing for cell sorting. We thank T. Boehm for his support and discussions on the manuscript.


   Disclosures
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This project was supported by Deutsche Forschungsgemeinschaft Grant SFB620/A7. V.C.M. was supported by a grant from the Fundação para a Ciência e a Tecnologia. Back

2 Address correspondence and reprint requests to Dr. Conrad C. Bleul, Max-Planck-Institute for Immunobiology; Department of Developmental Immunology, Stübeweg 51, 79108 Freiburg, Germany. E-mail address: bleul{at}immunbio.mpg.de Back

3 Abbreviations used in this paper: ETP, early T lineage progenitor; CLP, common lymphoid progenitor; DN, double negative; EGFP, enhanced GFP; hFc, human Fc; Lin, lineage marker; mFc, mouse Fc; SCF, stem cell factor; TMP, thymic multipotent precursor; TN, triple negative. Back

Received for publication August 25, 2006. Accepted for publication October 23, 2006.


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 Introduction
 Materials and Methods
 Results
 Discussion
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