This article describes the isolation of a novel cell population (B220loc-kit+CD19) in the fetal liver that represents 70% of T-cell precursors in this organ. Interestingly, these precursors showed a bipotent T-cell and natural killer cell (NK)– restricted reconstitution potential but completely lacked B and erythromyeloid differentiation capacity both in vivo and in vitro. Moreover, not only mature T-cell receptor (TCR)αβ+ peripheral T cells but also TCRγδ+ and TCRαβ+CD8αα+ intestinal epithelial cells of extrathymic origin were generated in reconstituted mice. The presence of this population in the fetal liver of athymic embryos indicates its prethymic origin. The comparison of the phenotype and differentiation potential of B220loc-kit+CD19fetal liver cells with those of thymic T/NK progenitors indicates that this is the most immature common T/NK cell progenitor so far identified. These fetal liver progenitors may represent the immediate developmental step before thymic immigration.

Multipotent hematopoietic stem cells (HSCs) differentiate into precursors with increasingly restricted differentiation potential. This process, called lineage commitment, leads to the generation of oligopotent progenitors and finally to cells that are irreversibly engaged in a unique pathway of differentiation. The identification of progenitors at intermediate stages of differentiation is a fundamental step in understanding lineage commitment. The characterization of such intermediates within the erythromyeloid lineages has identified, in the bone marrow (BM), a granulocyte-macrophage–restricted precursor and, more recently, a common myeloid precursor and an erythrocyte-megakaryocyte precursor.1 

T-cell generation occurs in the thymus from hematopoietic precursors present in fetal liver (FL) and adult BM. Although the pathways of intrathymic T-cell differentiation are relatively well characterized, the role of the thymic microenvironment as a unique site for the induction of commitment to the T-cell lineage remains a matter of debate.2,3 Moreover, the identification of T-cell progenitors before thymic colonization remains incomplete. Cells endowed with a nonrestricted potential of differentiation into T lymphocytes have been phenotypically characterized and isolated from adult BM. Thus, HSCs as well as common lymphoid progenitors (CLPs) were purified.4 5 

The existence of committed T-cell precursors (TCPs) in the BM6 and more recently in the FL has been suggested.7,8 However, the identification of surface markers allowing the isolation of these cells has not been achieved. Populations of precursors restricted to the T-cell and natural killer cell (NK) lineages have been reported in fetal thymus, blood, and spleen.9-13 In 2 independent studies, T and NK precursors have been characterized in fetal thymus either as FcγRII/III+9 or as CD90+NK1.1+CD117+,11although the total number of precursors present at these sites was not evaluated.

We and others have previously reported that the FL, the major hematopoietic organ during embryonic life, provides TCPs with restricted potential of differentiation that continuously colonize the fetal thymus.14-16 In an attempt to identify such intermediate precursors, we isolated different fractions of FL cells and analyzed them in a quantitative manner for lymphocyte precursor activity.

Here we identify a novel population corresponding to 0.2% of FL cells that includes the majority (70%) of TCPs in this organ. These cells retained the capacity to differentiate into both T and NK progeny at the single-cell level. When transferred in vivo, they reconstituted the peripheral T and NK compartments and gave rise to intraepithelial T cells of extrathymic origin. This cell population, designated here as common T/NK cell progenitor (C-TNKP), differs both by surface marker and by gene expression analysis from previously described bipotent T/NK precursors present in fetal blood, spleen, and thymus. We propose that these cells represent the immediate developmental step before thymic immigration.

Mice

C57BL/6-Ly5.1 mice (Centre de Développement des Techniques Advances, Orleans, France), C57BL/6-Ly5.2 mice (Iffa-Credo, L'Arbresle, France), and C57BL/6-Rag2/γc−/− mice were bred in our animal facility (Institut Pasteur, Paris, France). The day of vaginal plug was considered day 0 of gestation. Timed pregnancies ofnu/+ and nu/nu embryos were obtained from CDTA by crossing female nu/+with male nu/nu mice. Homozygosity for thenu genotype was confirmed by the absence of the thymus.

Cell preparation, immunofluorescence staining, and cell sorting

All the fluorescence-labeled antibodies used in this study were from PharMingen (San Diego, CA). FL cells were depleted of erythroid precursors by magnetic bead depletion (Dynal AS, Oslo, Norway) after incubation with biotin–anti-TER119. Cells were subsequently stained with fluorescein isothiocyanate–anti-CD19 (1D3), phycoerythrin–anti-CD45R/B220 (RA3-6B2), and biotin–anti–c-kit (2B8) antibodies. Cells were separated using a FACStar Plus cell sorter (Becton Dickinson, Mountain View, CA). All sorted cell populations were found to be more than 98% pure. Intestinal intraepithelial lymphocytes (i-IELs) were isolated by density gradient as described.17 For the flow cytometric analysis, the following antibodies were used: anti–T-cell receptor (TCR)γδ (3A10), anti-TCRαβ (H57), anti-NK1.1 (PK136), anti–Gr-1 (RB6-8C5) and anti-Ly5.1 (A20), anti-CD8α (Ly2, 53-6.7), anti-CD8β (Ly3.2, 53-5.8), anti-CD4 (L3T4), and anti-CD3ε (145-2C11). Four-color analysis was performed on a FACScalibur (Becton Dickinson) and run on the Cell Quest software (version 3.2; Becton Dickinson). Dead cells were eliminated by propidium iodide exclusion.

In vitro assays

TCP potential.

FL cell suspensions were prepared from 15 to 20 embryos (B6-Ly5.1). TCP potential was determined using the fetal thymic organ culture (FTOC) assay,18 with a slight modification.15 Thymic lobes from day-14 fetuses (B6-Ly5.2) were irradiated with 3000 rad using a cesium γ irradiator. For limiting dilution analysis, cells were resuspended at 4 different concentrations in culture medium. Cells were cocultured with at least 12 individual lobes for each dilution in hanging drops. Individual lobes were examined for the presence of αβ+, γδ+, and NK1.1+ cells of donor origin (Ly5.1+cells) at day 16 of culture. No cells were observed in irradiated lobes that were not colonized. The ratio of negative lobes was scored. Frequency and absolute numbers of TCPs were calculated (based on the Poisson probability distribution) as described previously.15 

B-cell precursor potential.

The limiting dilution culture conditions for B-cell precursor potential were described previously.19 After 10 to 14 days of culture, developing pre–B-cell colonies were scored on an inverted microscope. Individual representative colonies were further analyzed by flow cytometry. Cells from each well were further stimulated with lipopolysaccharide (LPS; Salmonella typhosa WO901; Difco) at a final concentration of 25 μg/mL, and IgM secretion was detected by enzyme-linked immunosorbent assay. Frequencies of B-cell precursors were determined according to the Poisson distribution.

NK cell precursor potential.

NK cell precursor potential in vitro was determined after culture of varying numbers (range, 20-180) of FL cells on the OP9 stromal cell line12,36 (kindly provided by Dr Kodama, Kyoto University, Yoshida, Japan), supplemented with interleukin-7 (IL-7) and c-kitligand (KL). Half of the medium was removed and changed every 4 to 6 days. IL-2 was added at day 6 of culture. Individual colonies were analyzed by flow cytometry. NK cell differentiation was also assessed when FL cells were cultured in FTOC (see above, TCP potential). IL-7, KL, and IL-2 were obtained from the supernatants of cell lines transfected with the corresponding cDNAs and titrated as described previously.20 

Erythroid and myeloid cell precursor potential.

Cells were mixed with OptiMEM, 0.8% methylcellulose (Fluka, Buchs, Switzerland), and 10% fetal calf serum (FCS), supplemented with KL, IL-3, granulocyte macrophage colony-stimulating factor (4 ng/mL), and erythropoietin (4 U/mL). Hemoglobinized clusters of fewer than 100 cells were classified as erythroid colony-forming units. Large colonies of red cells (more than 300 cells) were counted as erythroid burst-forming units, whereas colonies containing at least 2 myeloid cell types and erythroid cells were classified as mixed colony-forming units. Numbers of these colonies were counted. Individual colonies were further analyzed by May-Grünwald-Giemsa staining.

In vivo cell transfer

Sorted cells from C57BL/6 (Ly5.1) embryos were injected in the retro-orbital sinus of 400-rad irradiated C57BL/6-Rag2/γc−/− (Ly5.2) mice. After 4 to 6 weeks, cells from the peripheral blood, BM, spleen, thymus, and the small intestine (i-IELs) of the recipient mice were collected and analyzed by flow cytometry.

Purification of NK cells and cytolytic assay

Reconstituted Rag2/γc−/− mice were killed 6 weeks after transfer, and splenocytes were stained with monoclonal antibodies (mAbs) specific for CD3ε and NK1.1. CD3NK1.1+ NK cells and CD3+NK1.1 T cells were sorted and either tested for their natural cytolytic activity or expanded for 7 days in vitro (105 cells/mL) in complete RPMI medium (RPMI 1640 with 10% FCS, 10−5 M β-mercaptoethanol, 100 mg/mL streptomycin, 100 U/mL penicillin) supplemented with 1000 U recombinant hu–IL-2 (Peprotech). After 7 days, cells were harvested and a standard chromium-release assay was performed. Briefly, YAC-1 (mouse thymoma; H-2a) and P815 (mouse mastocytoma; H-2d) target cells (106) were labeled with 37.105 Bq 51Cr (ICN Pharmaceuticals, Orsay, France) for 45 minutes at 37°C. Cells were extensively washed, plated at 2 × 103 cells/well, and mixed with different numbers of effector cells. Effector cells were NK and T cells either freshly isolated from splenocytes or activated by IL-2. The radioactivity released into the cell-free supernatant was measured after 4 hours at 37°C, and the percentage specific lysis was calculated as follows: 100 × (experimental release − spontaneous release)/(maximum release − spontaneous release).

Reverse transcriptase–polymerase chain reaction

Cells were lysed in TRIzol (GIBCO-BRL, Cergy-Pontoise, France), total RNA was isolated according to the manufacturer's protocol, and cDNA was prepared as described previously.15 Gene-specific primers used for PCR have been described previously. mb-1, RAG-1, λ-5,21 TCF-1,22 Pax-5, GATA-3,7 IL-7Rα,23 universal primer for Ly49, amplifying (Ly49 C, E, F, G1-4),24 pre-TCRα (pTα),15 IL-15Rα: 5′-CCAACATGGCCTCGCCGCAGCT-3′ and 5′-TTGGGAGAGAAAGCTTCTGGCTCT-3′. The amount of cDNA in the samples was carefully standardized by real-time PCR using hypoxanthine phosphoribosyltransferase (HPRT) specific primers: 5′-CCAGCAAGCTTGCAACCTTAACAA-3′, 5′-GACTGAAAGACTTGCTCGAG-3′, and SYBR Green PCR Master Mix (Applied Biosystems, Warrington, United Kingdom). Samples were analyzed using a GeneAmp 5700 Sequence Detection System (PE Applied Biosystems). The cDNA samples were amplified for 35 cycles by the GeneAmp PCR System 9600 (Perkin Elmer, Foster City, CA). Fifteen microliters of each PCR product was subjected to electrophoresis through a 2% agarose gel and visualized after ethidium bromide staining.

Immunoscope analysis for the detection of TCRβ rearrangements

DNA was prepared according to the manufacturer's protocol (TRIzol; GIBCO-BRL). For the detection of D-Jβ rearrangements, PCR was performed on the indicated samples using a combination of 2 primers that recognize sequences 5′ of the Dβ2.1 and 3′ of the Jβ2.7.10 The PCR products were then subjected to a runoff reaction using a nested Jβ2.5 fluorescent primer.25 Runoff products were resolved on an automated 373A sequencer (Perkin-Elmer). The size and the intensity of each band were recorded and then analyzed using Immunoscope software.25 26 

Characterization of FL cells at 15 days postcoitus based on the expression of B220 and c-kit

The B220 (CD45R) marker is expressed at all stages of B-cell ontogeny. However, it is also expressed in a population of BM cells (fraction A)27,28 endowed with NK potential29and on FL lymphoid progenitors.30 CD19 thus remains the most reliable marker for B-lineage commitment.29 The expression of the receptor tyrosine kinase c-kit correlates with precursor activity and has been used to identify HSCs, CLPs, and pro–T cells.31 FL cells were isolated from 15–days postcoitus (dpc) embryos, and erythroid precursors were depleted using the TER119 mAb. TER119 FL cells were then analyzed for the expression of CD19, c-kit, and B220. The analysis identified 6 distinct populations (Figure1A). The B220+CD19+ population (a) contains pro/pre–B cells that develop in the FL.32 33 The CD19cells separate into 5 other populations (b-f) according to the level of expression of c-kit and B220. All of the above subpopulations were present at similar ratios in FL cells derived from nude (nu/nu) embryos, showing that they are not generated in the thymus (Figure 1A).

Fig. 1.

Characterization of 15-dpc FL cells for T-cell progenitor activity: predominance of TCP in the B220lo

c-kit+CD19 fraction. FL cells from 15-dpc C57BL/6 embryos were depleted of erythroid precursors and analyzed for expression of CD19, c-kit, and B220. After flow cytometric sorting of the different subpopulations defined by these 3 markers, wild-type FL cells were tested for TCP activity in an FTOC performed under limiting dilution conditions. (A) Flow cytometric plots are shown for wild-type and nu/nu C57BL/6 FL cells. (B) Reanalysis of sorted cells from populations a and e. Sorted cells were consistently more than 98% pure. (C) The total number of TCPs in 15-dpc FL was previously determined14 and reconfirmed here. From the frequency of TCPs and the fraction that they represent in total FL cells, we calculated the total number of TCPs in each population. The bars show the percentage of the total TCP activity in each fraction. (D) Analysis of surface phenotype of sorted cells from population e after staining with indicated antibodies (solid lines). Data are representative of at least 3 independent experiments.

Fig. 1.

Characterization of 15-dpc FL cells for T-cell progenitor activity: predominance of TCP in the B220lo

c-kit+CD19 fraction. FL cells from 15-dpc C57BL/6 embryos were depleted of erythroid precursors and analyzed for expression of CD19, c-kit, and B220. After flow cytometric sorting of the different subpopulations defined by these 3 markers, wild-type FL cells were tested for TCP activity in an FTOC performed under limiting dilution conditions. (A) Flow cytometric plots are shown for wild-type and nu/nu C57BL/6 FL cells. (B) Reanalysis of sorted cells from populations a and e. Sorted cells were consistently more than 98% pure. (C) The total number of TCPs in 15-dpc FL was previously determined14 and reconfirmed here. From the frequency of TCPs and the fraction that they represent in total FL cells, we calculated the total number of TCPs in each population. The bars show the percentage of the total TCP activity in each fraction. (D) Analysis of surface phenotype of sorted cells from population e after staining with indicated antibodies (solid lines). Data are representative of at least 3 independent experiments.

Close modal

The majority of TCPs in the 15-dpc FL are present in fraction e: B220loc-kitsup+CD19

Unfractionated and sorted cells corresponding to the 6 FL subpopulations (a-f) (Figure 1A) were characterized for their TCP potential by assessing the capacity to reconstitute 14-dpc irradiated thymic lobes.18 Frequencies and total numbers of TCPs were evaluated in FTOCs performed under limiting dilution conditions. Figure1C shows the ratios of TCPs in the different subpopulations (a-f) to TCPs in total FL cells. Strikingly, population e (B220loc-kit+CD19) represents close to 70% of the TCPs in total FL cells and was thus further characterized. Figure 1D shows surface marker analysis of sorted cells from population e after staining with the indicated antibodies. These precursors were NK1.1CD90CD25CD44+.

In vitro progenitor activity of fraction e

We further evaluated the developmental potential of the cells included in fraction e. Sorted cells were independently analyzed in 3 sensitive in vitro assays supporting T, B, and erythromyeloid differentiation from uncommitted multipotent precursors.34 35 

To evaluate the origin of TCPs within fraction e, we cultured sorted cells from wild-type, nu/+, and nu/nuC57BL/6 embryos under limiting dilution conditions in FTOCs. Cells within fraction e generated T cells at a similar frequency (approximately 1 in 20 cells) (Figure2A), whereas fraction a (B220+CD19+), used as negative control, did not generate a detectable T-cell progeny (Figure 2A). This result indicates that the TCPs present in population e are of prethymic origin.

Fig. 2.

B220lo

c-kit+CD19 progenitors give rise to T and NK cells but fail to generate B and myeloid progeny in vitro. (A) B220+CD19+ (fraction a) FL cells isolated from wild-type embryos and B220loc-kit+CD19 cells (fraction e) isolated from wild-type, nu/+, andnu/nu were set under limiting dilution conditions in FTOC (upper panels) and with S17 stromal cells, KL, and IL-7 (lower panels). Plots of the percentage of negative wells per cell concentration and calculated frequencies are shown. (B) Total FL cells (■) and B220loc-kit+CD19 cells (♦) from wild-type C57BL/6 mice were cultured in methylcellulose, and erythromyeloid colonies were counted on day 7. (C) The same populations as in (B) were cultured on OP9 stromal cells in medium supplemented with IL-7, KL, and IL-2 for B, myeloid, and NK cell development. Cells were stained and analyzed by flow cytometry after 10 days of culture.

Fig. 2.

B220lo

c-kit+CD19 progenitors give rise to T and NK cells but fail to generate B and myeloid progeny in vitro. (A) B220+CD19+ (fraction a) FL cells isolated from wild-type embryos and B220loc-kit+CD19 cells (fraction e) isolated from wild-type, nu/+, andnu/nu were set under limiting dilution conditions in FTOC (upper panels) and with S17 stromal cells, KL, and IL-7 (lower panels). Plots of the percentage of negative wells per cell concentration and calculated frequencies are shown. (B) Total FL cells (■) and B220loc-kit+CD19 cells (♦) from wild-type C57BL/6 mice were cultured in methylcellulose, and erythromyeloid colonies were counted on day 7. (C) The same populations as in (B) were cultured on OP9 stromal cells in medium supplemented with IL-7, KL, and IL-2 for B, myeloid, and NK cell development. Cells were stained and analyzed by flow cytometry after 10 days of culture.

Close modal

To evaluate the B-cell differentiation potential, we cultured the same populations under clonal conditions with the stromal cell line S17 in the presence of IL-7 and KL. Sorted cells from fraction a gave rise to B-cell colonies at a frequency of approximately 1 in 4 cells (Figure2A). In contrast, cells from fraction e failed to generate a B-lineage progeny. The absence of B cells in these cultures was confirmed by the absence of IgM-secreting cells after LPS stimulation (data not shown).

The myeloid and erythroid potential of cells from fraction e was addressed by colony formation in methylcellulose. Unfractionated FL cells gave rise to colonies at a frequency of approximately 1 colony-forming unit per 500 plated cells (Figure 2B). Both homogeneous and mixed colonies were identified after May-Grünwald-Giemsa staining of cytospin preparations. Developing colonies included granulocytes and macrophages, and a few colonies also included erythroid cells (data not shown). In contrast, progenitors in fraction e failed to generate colonies in this assay even when up to 15 000 cells were plated (Figure 2B).

An additional characterization of the lymphoid and myeloid differentiation potential was performed in a culture system using the stromal cell line OP912,36 and exogenous interleukins. These conditions permit the development of NK, B, and myeloid cells. Figure 2C shows that both total FL cells and sorted progenitors from fraction e were capable of giving rise to NK cells. Moreover, whereas unfractionated FL cells generated CD19+ B cells and myeloid cells, as indicated by the expression of Gr-1 and Mac-1, precursors in fraction e failed to generate these lineages (Figure 2C), ruling out that this fraction contained the previously identified uncommitted precursors present in FL.7 8 

Evidence for a bipotent T/NK progenitor in FL by single-cell analysis

The limiting dilution analysis allowed us to conclude that cells within fraction e contained T and NK precursors, either as a bipotent T/NK or as a mixture of NK and T committed progenitors. To address these possibilities, we investigated the differentiation potential of individual cells in fraction e. Cells were micromanipulated under direct microscopic inspection and allowed to colonize single irradiated fetal thymic lobes. A representative analysis of the cells generated in independent lobes is shown in Figure 3. Expression of T and NK cell markers was examined by gating on Ly5.1+ donor-derived cells. T cells expressing TCRαβ or TCRγδ as well as NK+ cells expressing only NK1.1 were present. NK1.1+TCRαβ cells were also generated.

Fig. 3.

In vitro generation of T and NK cells from single B220lo

c-kit+CD19 progenitors.Sorted cells were seeded at one cell per well in Terasaki plates. Wells were individually checked under a microscope, and wells containing a single cell were identified. Single cells were then placed in a hanging drop with one irradiated thymic lobe from Ly5 congenic embryos. Flow cytometric analysis of individual lobes was done at day 16 of FTOC.

Fig. 3.

In vitro generation of T and NK cells from single B220lo

c-kit+CD19 progenitors.Sorted cells were seeded at one cell per well in Terasaki plates. Wells were individually checked under a microscope, and wells containing a single cell were identified. Single cells were then placed in a hanging drop with one irradiated thymic lobe from Ly5 congenic embryos. Flow cytometric analysis of individual lobes was done at day 16 of FTOC.

Close modal

Under these conditions, the T-cell readout frequency of sorted CD44+CD25+ fetal thymocytes was 1 in 5 cells.15 Using cells from fraction e, we observed thymic reconstitution in 5% (3 of 60) of the individual lobes. Although this efficiency remains low, lobes in which only T or NK cells developed were not observed. Under limiting dilution conditions, where more than one cell from fraction e was used in FTOC, independent NK or T-cell reconstitution was never observed. However, lobes containing only NK progeny were observed when cells from fraction f were seeded in FTOC (data not shown). Together these data indicate that both T and NK potentials in cells from fraction e were present in the same precursor, which we will define as C-TNKP (common T/NK cell progenitor), demonstrating for the first time the existence of such a bipotent cell in FL.

TCRβ chain gene rearrangement and gene expression analysis

We next investigated whether TCRβ rearrangements have been initiated in these progenitors. DNA was isolated from cells of fraction e before and after FTOC, and rearrangements of the TCRβ genes were examined by PCR. As shown in Figure 4A, D-Jβ rearrangements were not observed in cells from fraction e. In contrast, a diverse pattern of rearrangements was observed when these progenitors were cultured in irradiated fetal thymic lobes. These data suggest that rearrangements in the TCRβ locus occur after thymic seeding and commitment to the T lineage. Recent findings,37 showing that the NK potential is maintained in thymic progenitors until TCRβ gene rearrangements occur, support our conclusion.

Fig. 4.

B220lo

c-kit+CD19 cells retain their TCRβ locus in a germline configuration but express T-lineage–specific genes. (A) DNA was prepared from 5 × 103 cells from fraction e before and after FTOC. Rearrangement of the TCRβ genes was examined by a sensitive 2-step PCR using pairs of primers (1 and 2) followed by a runoff using the fluorescent primer 3, as shown in the higher panel. TCRβ gene rearrangement was not observed in cells from fraction e. In contrast, diverse Dβ-Jβ rearrangements (indicated by arrows) were observed after FTOC. (B) RT-PCR analysis of cells from population e (line 2) for the indicated genes. Controls (line 3) included 15-dpc fetal thymocytes, CD19+B220+ cells, and NK1.1+CD3 mature NK cells. S17 cells were used as a negative control (line 1). The amount of cDNA was carefully standardized according to HPRT transcripts.

Fig. 4.

B220lo

c-kit+CD19 cells retain their TCRβ locus in a germline configuration but express T-lineage–specific genes. (A) DNA was prepared from 5 × 103 cells from fraction e before and after FTOC. Rearrangement of the TCRβ genes was examined by a sensitive 2-step PCR using pairs of primers (1 and 2) followed by a runoff using the fluorescent primer 3, as shown in the higher panel. TCRβ gene rearrangement was not observed in cells from fraction e. In contrast, diverse Dβ-Jβ rearrangements (indicated by arrows) were observed after FTOC. (B) RT-PCR analysis of cells from population e (line 2) for the indicated genes. Controls (line 3) included 15-dpc fetal thymocytes, CD19+B220+ cells, and NK1.1+CD3 mature NK cells. S17 cells were used as a negative control (line 1). The amount of cDNA was carefully standardized according to HPRT transcripts.

Close modal

We analyzed by reverse transcriptase–polymerase chain reaction (RT-PCR) the expression of genes that play a role in early B, T, or NK cell differentiation. As shown in Figure 4B, sorted cells from fraction e failed to express B-lineage–specific genes (λ-5, mb-1, and Pax-5). They expressed IL-7Rα and, at barely detectable levels, RAG-1 transcripts. Interestingly, these progenitors expressed the T-lineage–specific transcription factor GATA-3 and low levels of TCF-1. Expression of the pTα gene was not detected under the same PCR conditions. We also did not detect expression of genes associated with NK lineage differentiation, such as IL-15Rα and Ly49. Expression of both T- and NK-related genes has been observed in the NK1.1+CD90+ pT/NK progenitors derived from the fetal thymus, blood, and spleen, suggesting that these may represent a more mature stage of differentiation.12 38 

B220loc-kit+CD19FL progenitors reconstitute both the T and NK, but neither the B-cell nor the myeloid, compartments of Rag2/γc−/−mice

To assess the hematopoietic reconstitution potential in vivo of cells in fraction e, we intravenously injected sorted B220loc-kit+CD19 cells into sublethally irradiated mice carrying mutations in both the Rag2 and the common cytokine receptor γc genes.39 The additional absence of NK cells in the Rag2/γc−/− mice40 (Figure5A) compared with Rag2−/− mice makes them ideal hosts to analyze the potential to generate B, T, and NK cells. Rag2/γc−/−mice were intravenously injected with 7 × 104TER119 FL cells or 104 cells from fraction e, both numbers corresponding to 500 TCPs in the respective populations.

Fig. 5.

In vivo progenitor activity of B220lo

c-kit+CD19 FL cells.Total 15-dpc FL cells and sorted B220loc-kit+CD19 cells (fraction e) were injected into irradiated C57BL/6-Rag2/γc−/− mice at 7 × 104 and 104 cells per mouse, respectively, corresponding to an equivalent number of 500 TCPs. Four weeks after cell transfer, the blood (A), thymus (B), BM (C), and i-IELs (D) were analyzed by flow cytometry. The presence of donor-derived cells was assessed by gating on Ly5.1+ cells. Numbers indicate the relative percentage within the indicated gates. The results are representative of 4 experiments.

Fig. 5.

In vivo progenitor activity of B220lo

c-kit+CD19 FL cells.Total 15-dpc FL cells and sorted B220loc-kit+CD19 cells (fraction e) were injected into irradiated C57BL/6-Rag2/γc−/− mice at 7 × 104 and 104 cells per mouse, respectively, corresponding to an equivalent number of 500 TCPs. Four weeks after cell transfer, the blood (A), thymus (B), BM (C), and i-IELs (D) were analyzed by flow cytometry. The presence of donor-derived cells was assessed by gating on Ly5.1+ cells. Numbers indicate the relative percentage within the indicated gates. The results are representative of 4 experiments.

Close modal

The analysis of blood samples from mice injected with total FL cells revealed the presence of TCRαβ+ mature T cells, NK1.1+IL2Rβ+ NK cells, and also B220+IgD+ mature B cells 4 to 6 weeks after cell transfer. In contrast, sorted cells from fraction e, although capable of generating T and NK cells, lacked any detectable B-cell reconstitution potential (Figure 5A). The main hematopoietic sites (BM and thymus) of the recipient mice were analyzed at 4 weeks after transfer for the presence of donor-derived myeloid, B, T, and NK cells. Mice having transplantation with either unfractionated FL cells or sorted cells from fraction e contained T cells in the thymus (Figure5B). At this time point, most progeny derived from fraction e had already progressed to the mature CD4+ or CD8+simple positive stage, and consistently 70% of these thymocytes expressed high levels of TCRαβ. The majority (76%) of the progeny of unfractionated FL cells were at the double positive stage, and only 30% were TCRαβ+. Mice injected with total FL cells showed sustained T-cell lymphopoiesis 8 weeks after injection. In contrast, in recipients of cells from fraction e, T-cell generation was no longer observed (data not shown). These data suggest that progenitors included in fraction e have a limited self-renewal potential and are able to generate only a transient wave of T-cell reconstitution.

The analysis of BM cells showed a striking difference in the B and myeloid repopulation capacity of the 2 sets of reconstituted mice (Figure 5C). In support of the in vitro data, fraction e was devoid of B and myeloid cell precursor activity, as shown by the total absence of B220+IgD+ and Gr-1+ cells, which were generated only from unfractionated 15-dpc FL (Figure 5C). It is interesting to note that a minor B220lo population was detected in mice reconstituted with fraction e; those donor-derived cells were shown to be B220loIgDNK1.1+.

To exclude the possibility that an early transient wave of B-cell repopulation was generated in the mice receiving B220loc-kit+CD19transplants, we analyzed the peripheral blood within 2 weeks after cell transfer. Mice reconstituted with unfractionated FL cells contained significant levels of donor-derived B220+IgD+cells in the peripheral blood. In contrast, mice having transplantation with fraction e showed no repopulation in the peripheral blood at this time point (data not shown).

Evidence for the existence of T cells of extrathymic origin within the intestinal epithelium41 led us to ascertain whether the progenitors in fraction e could contain precursors for this T-cell subset. Rag2/γc−/− mice reconstituted with progenitors from fraction e contained αβ and γδ T cells in the i-IEL compartment (Figure 5D). TCRαβ+ cells expressing the CD8αα homodimer, characteristic of extrathymic i-IELs, were also present in these mice.

Population e generates cytolytic NK cells in vivo

The spleens of mice reconstituted with population e contained a subset of cells phenotypically indistinguishable from mature NK cells that we tested for their cytolytic activity. Splenic NK1.1+CD3 but not CD3+NK1.1 cells generated in vivo from population e exhibited low but detectable levels of cytolysis against YAC-1 cells (Figure 6A). IL-2–activated CD3NK1.1+ cells showed strong cytolytic activity against NK-sensitive YAC-1 thymoma cells (Figure 6A) but not against NK-resistant P815 mastocytoma cells (Figure 6B). The cytolytic activity of NK cells generated in vivo from population e was comparable to that displayed by NK cells isolated from control B6 mice (Figure6B). These data demonstrate that the B220loc-kit+CD19subpopulation can give rise to functional NK cells in vivo.

Fig. 6.

Cytolytic activity of NK cells generated in vivo.

CD3NK1.1+ NK cells (circles) and CD3+NK1.1 T cells (triangles) were purified from splenocytes of mice reconstituted with population e. (A) Freshly sorted cells were tested for cytolytic activity versus YAC-1 thymoma targets. (B) Sorted NK cells (circles) and T cells (triangles) from mice reconstituted with population e (empty symbols) and control B6 mice (filled symbols) were cultured in vitro for 7 days in IL-2 and thereafter tested for cytolytic activity versus sensitive YAC-1 and resistant P815 target cells.

Fig. 6.

Cytolytic activity of NK cells generated in vivo.

CD3NK1.1+ NK cells (circles) and CD3+NK1.1 T cells (triangles) were purified from splenocytes of mice reconstituted with population e. (A) Freshly sorted cells were tested for cytolytic activity versus YAC-1 thymoma targets. (B) Sorted NK cells (circles) and T cells (triangles) from mice reconstituted with population e (empty symbols) and control B6 mice (filled symbols) were cultured in vitro for 7 days in IL-2 and thereafter tested for cytolytic activity versus sensitive YAC-1 and resistant P815 target cells.

Close modal

We have quantitatively characterized TCPs in different subsets of FL cells. This analysis allowed us to isolate a novel population of hematopoietic progenitors that represent the majority (70%) of TCPs in 15-dpc FL. Cells within this population lack in vitro and in vivo potential for B and erythromyeloid differentiation. These precursors were shown to differentiate into T and NK cells by single-cell fate analysis. Moreover, precursors uniquely committed to either the T or NK lineage were not detected in single-cell or limiting dilution assays, arguing for the absence of more restricted progenitors among this subset. This conclusion is supported by recent studies indicating that T-lineage restriction from a bipotent p-T/NK occurs in the thymus.11 37 Consistent with this notion, the cell population described here is of prethymic origin. We designate this cell population as common T/NK progenitor (C-TNKP).

We show that the frequency of T-cell generation in vitro in FL-derived C-TNKP cells is approximately 1 in 20 cells. It should be noted that a pure population of TCPs (ie, CD44+CD25+prothymocytes) has a plating efficiency of 1 in 5 cells in this assay. This result indicates that not all cells in FTOC conditions colonize the thymic lobes, and furthermore not all cells will efficiently interact with the thymic stroma to initiate the T-cell differentiation program. Thus, the true frequency of TCPs in the population we describe could be much higher. It should also be pointed out that the frequency of T-cell generation obtained here is comparable to that obtained for CLPs from BM (1:21).5 The cloning efficiency for B-cell precursor detection in vitro is comparable to that found for T cells in FTOCs. Thus, 1 in 4 CD19+ FL cells can generate B-cell colonies in the presence of stromal cells, KL, and IL-7. The fact that we are comparing potentials of differentiation using tests with similar plating efficiencies reinforces our conclusion that B-cell differentiation potential is not retained in B220loc-kit+CD19FL precursors.

When injected into Rag2/γc−/− mice, FL-derived B220loc-kit+CD19 cells were shown to be effective in the repopulation of the T and NK cell compartment of Rag2/γc-deficient mice, further confirming the restricted differentiation potential of these progenitors assessed in vitro. The limited self-renewal potential of these precursors is shown by a skewed ratio of CD4/CD8 single- to double-positive cells 4 weeks after transfer and by the virtual absence of thymocytes 4 weeks later. The transient reconstitution observed contrasts with a sustained thymopoiesis obtained with total FL cells known to harbor HSCs. Thus, the self-renewing progenitors in the BM that ensure constant generation of TCPs are most likely the stem cells because the common lymphoid precursors (CLPs), the immediate precursors of the C-TNKP, were also shown to have transient reconstitution capacity.5Although the BM of mice reconstituted with fraction e from FL showed no signs of differentiation of B or myeloid cells, a minor population of B220loNK1.1+ donor-derived cells was detected. They most likely correspond to mature NK cells known to express low levels of B220 that differentiated in situ.42 

The analysis of i-IELs of the reconstituted mice showed the presence of donor-derived cells that reconstituted this particular environment with ratios of γδ- and αβ-expressing T cells comparable to those found in normal mice. Moreover, TCRαβ and γδ cells expressing the homodimer CD8αα were also generated. Both populations have been shown to undergo extrathymic differentiation.41 We conclude that FL-derived C-TNKPs are able to reconstitute not only the thymic but also the extrathymic T-cell compartments. Precursors for i-IELs have been described in a specialized structure named cryptopatches.43 In addition, putative IEL precursors have been reported to express low levels of B220.44 45 The similarity between this phenotype and that expressed in the population described here raises the possibility of a lineage relationship between these subsets. However, further investigations are required to formally assess this possibility.

Although sharing the common capacity to differentiate into both T and NK cells, the population described here expressed a different set of surface markers compared with the p-T/NK previously identified in the fetal thymus, blood, and spleen but absent from FL, the major hematopoietic organ during embryonic life.12 These were shown to be NK1.1+, CD117lo, and CD90+; in contrast, the FL C-TNKPs described here are NK1.1, CD117high, and CD90.

Gene expression analysis revealed additional differences between these populations. NK1.1+CD90+CD117loprecursors expressed the T-cell–specific transcript pTα and genes associated with NK lineage differentiation such as IL-15Rα and NKR-P1 family members. In contrast, the population described here has undetectable levels of pTα and IL-15Rα transcripts. However, 2 transcription factors associated with T-cell development (GATA-3 and TCF-1) were expressed in both populations. All together, this suggests that C-TNKP in FL represents a more immature population than NK1.1+CD90+CD117lo precursors. On the other hand, GATA-3 is expressed at low levels in common lymphoid progenitors (CLPs) while it is up-regulated in thymic pro-T cells. In conclusion, the C-TNKP can be placed in an intermediate developmental stage between the CLP and the p-T/NK identified in fetal blood and thymus (Figure 7).

Fig. 7.

Model for prethymic T and NK lineage commitment.

The molecular characterization and the analysis of the potential of differentiation of FL-derived B220loc-kit+CD19progenitors place this population (C-TNKP) in an intermediate developmental stage between the CLP and the p-T/NK identified in fetal blood and thymus (see “Discussion” for further details).

Fig. 7.

Model for prethymic T and NK lineage commitment.

The molecular characterization and the analysis of the potential of differentiation of FL-derived B220loc-kit+CD19progenitors place this population (C-TNKP) in an intermediate developmental stage between the CLP and the p-T/NK identified in fetal blood and thymus (see “Discussion” for further details).

Close modal

A continuous flow of immigrants seems necessary to ensure a constant T-cell generation in the thymus.46 It is conceivable that multiple cell types such as HSCs, CLPs, and p-T/NK are involved in this process. Our own previous results showed that the fetal thymus is seeded by increasing numbers of TCPs.15 The quantitative data presented here indicate the following: (1) During mid-gestation, the major population of FL cells endowed with T-cell differentiation potential are committed to the T/NK lineage; (2) this population can reconstitute both the conventional thymic and the extrathymic T-cell subsets; and (3) both by surface marker and gene expression pattern, they differ from previously described thymic and blood-derived pT/NK cells. We propose that if constant thymic immigration occurs, the population described here likely constitutes a major component of this process.

The isolation of a homogeneous population of prethymic bipotent T/NK precursors will allow the identification of genes involved in early stages of T-cell commitment and differentiation and eventually will allow us to understand the molecular basis for thymic immigration. Moreover, the capacity of a human counterpart of this cell population to reconstitute efficiently the T-cell compartment could be used to prevent lymphopenia following stem cell transplantation and could therefore be of high therapeutic interest.

We thank Mathias Haury, Anne Louise for cell sorting, Pablo Pereira for advice in the i-IEL preparations, and Laurent Boucontet for help with real-time PCR analysis.

The Unité du Développement des Lymphocytes is supported by grants from the ANRS and “Ligue Nationale Contre le Cancer” as a registered laboratory. I.D. is supported by a fellowship from the Ligue Nationale Contre le Cancer.

The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked “advertisement” in accordance with 18 U.S.C. section 1734.

1
Akashi
K
Traver
D
Miyamoto
T
Weissman
IL
A clonogenic common myeloid progenitor that gives rise to all myeloid lineages.
Nature.
404
2000
193
197
2
Shortman
K
Wu
L
Early T lymphocyte progenitors.
Annu Rev Immunol.
14
1996
29
47
3
Rodewald
HR
Fehling
HJ
Molecular and cellular events in early thymocyte development.
Adv Immunol.
69
1998
1
112
4
Spangrude
GJ
Heimfeld
S
Weissman
IL
Purification and characterization of mouse hematopoietic stem cells [published erratum appears in Science. 1989;244:1030].
Science.
241
1988
58
62
5
Kondo
M
Weissman
IL
Akashi
K
Identification of clonogenic common lymphoid progenitors in mouse bone marrow.
Cell.
91
1997
661
672
6
Abramson
S
Miller
RG
Phillips
RA
The identification in adult bone marrow of pluripotent and restricted stem cells of the myeloid and lymphoid systems.
J Exp Med.
145
1977
1567
1579
7
Kawamoto
H
Ikawa
T
Ohmura
K
Fujimoto
S
Katsura
Y
T cell progenitors emerge earlier than B cell progenitors in the murine fetal liver.
Immunity.
12
2000
441
450
8
Kawamoto
H
Ohmura
K
Fujimoto
S
Katsura
Y
Emergence of T cell progenitors without B cell or myeloid differentiation potential at the earliest stage of hematopoiesis in the murine fetal liver.
J Immunol.
162
1999
2725
2731
9
Rodewald
HR
Moingeon
P
Lucich
JL
Dosiou
C
Lopez
P
Reinherz
EL
A population of early fetal thymocytes expressing Fc gamma RII/III contains precursors of T lymphocytes and natural killer cells.
Cell.
69
1992
139
150
10
Rodewald
H-R
Kretzschmar
K
Takeda
S
Hohl
C
Dessing
M
Identification of pro-thymocytes in murine fetal blood: T lineage commitment can precede thymus colonization.
EMBO J.
13
1994
4229
4240
11
Carlyle
JR
Michie
AM
Furlonger
C
et al
Identification of a novel developmental stage marking lineage commitment of progenitor thymocytes.
J Exp Med.
186
1997
173
182
12
Carlyle
JR
Zuniga-Pflucker
JC
Requirement for the thymus in alphabeta T lymphocyte lineage commitment.
Immunity.
9
1998
187
197
13
Michie
AM
Carlyle
JR
Schmitt
TM
et al
Clonal characterization of a bipotent T cell and NK cell progenitor in the mouse fetal thymus.
J Immunol.
164
2000
1730
1733
14
Ema
H
Douagi
I
Cumano
A
Kourilsky
P
Development of T cell precursor activity in the murine fetal liver.
Eur J Immunol.
28
1998
1563
1569
15
Douagi
I
Andre
I
Ferraz
JC
Cumano
A
Characterization of T cell precursor activity in the murine fetal thymus: evidence for an input of T cell precursors between days 12 and 14 of gestation.
Eur J Immunol.
30
2000
2201
2210
16
Kawamoto
H
Ohmura
K
Katsura
Y
Presence of progenitors restricted to T, B, or myeloid lineage, but absence of multipotent stem cells, in the murine fetal thymus.
J Immunol.
161
1998
3799
3802
17
Ishikawa
H
Li
Y
Abeliovich
A
Yamamoto
S
Kaufmann
SH
Tonegawa
S
Cytotoxic and interferon gamma-producing activities of gamma delta T cells in the mouse intestinal epithelium are strain dependent.
Proc Natl Acad Sci U S A.
90
1993
8204
8208
18
Jenkinson
EJ
Franchi
LL
Kingston
R
Owen
JJT
Effect of deoxyguanosine on lymphopoiesis in the developing thymus rudiment in vitro: application in the production of chimeric thymus rudiments.
Eur J Immunol.
12
1982
583
587
19
Cumano
A
Paige
CJ
Enrichment and characterization of uncommitted B-cell precursors from fetal liver at day 12 of gestation.
EMBO J.
11
1992
593
601
20
Godin
I
Garcia-Porrero
JA
Dieterlen-Lievre
F
Cumano
A
Stem cell emergence and hemopoietic activity are incompatible in mouse intraembryonic sites.
J Exp Med.
190
1999
43
52
21
Li
YS
Hayakawa
K
Hardy
RR
The regulated expression of B lineage associated genes during B cell differentiation in bone marrow and fetal liver.
J Exp Med.
178
1993
951
960
22
Hattori
N
Kawamoto
H
Fujimoto
S
Kuno
K
Katsura
Y
Involvement of transcription factors TCF-1 and GATA-3 in the initiation of the earliest step of T cell development in the thymus.
J Exp Med.
184
1996
1137
1147
23
Yokota
Y
Mansouri
A
Mori
S
et al
Development of peripheral lymphoid organs and natural killer cells depends on the helix-loop-helix inhibitor Id2.
Nature.
397
1999
702
706
24
Toomey
JA
Shrestha
S
de la Rue
SA
et al
MHC class I expression protects target cells from lysis by Ly49-deficient fetal NK cells.
Eur J Immunol.
28
1998
47
56
25
Pannetier
C
Cochet
M
Darche
S
Casrouge
A
Zoller
M
Kourilsky
P
The sizes of the CDR3 hypervariable regions of the murine T-cell receptor beta chains vary as a function of the recombined germ-line segments.
Proc Natl Acad Sci U S A.
90
1993
4319
4323
26
Bousso
P
Wahn
V
Douagi
I
et al
Diversity, functionality, and stability of the T cell repertoire derived in vivo from a single human T cell precursor.
Proc Natl Acad Sci U S A.
97
2000
274
278
27
Allman
D
Li
J
Hardy
RR
Commitment to the B lymphoid lineage occurs before DH-JH recombination.
J Exp Med.
189
1999
735
740
28
Hardy
RR
Carmack
CE
Shinton
SA
Kemp
JD
Hayakawa
K
Resolution and characterization of pro-B and pre-pro-B cell stages in normal mouse bone marrow.
J Exp Med.
173
1991
1213
1225
29
Rolink
A
ten Boekel
E
Melchers
F
Fearon
DT
Krop
I
Andersson
J
A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors.
J Exp Med.
183
1996
187
194
30
Sagara
S
Sugaya
K
Tokoro
Y
et al
B220 expression by T lymphoid progenitor cells in mouse fetal liver.
J Immunol.
158
1997
666
676
31
Akashi
K
Kondo
M
Weissman
IL
Role of interleukin-7 in T-cell development from hematopoietic stem cells.
Immunol Rev.
165
1998
13
28
32
Strasser
A
Rolink
A
Melchers
F
One synchronous wave of B cell development in mouse fetal liver changes at day 16 of gestation from dependence to independence of a stromal cell environment.
J Exp Med.
170
1989
1973
1986
33
Rolink
A
Melchers
F
Molecular and cellular origins of B lymphocyte diversity.
Cell.
66
1991
1081
1094
34
Cumano
A
Paige
CJ
Iscove
NN
Brady
G
Bipotential precursors of B cells and macrophages in murine fetal liver.
Nature.
356
1992
612
615
35
Godin
I
Dieterlen-Lièvre
F
Cumano
A
Emergence of multipotent hemopoietic cells in the yolk sac and paraaortic splanchnopleura in mouse embryos, beginning at 8.5 days postcoitus.
Proc Natl Acad Sci U S A.
92
1995
773
777
36
Nakano
T
Kodama
H
Honjo
T
Generation of lymphohematopoietic cells from embryonic stem cells in culture.
Science.
265
1994
1098
1101
37
Ikawa
T
Kawamoto
H
Fujimoto
S
Katsura
Y
Commitment of common T/natural killer (NK) progenitors to unipotent T and NK progenitors in the murine fetal thymus revealed by a single progenitor assay.
J Exp Med.
190
1999
1617
1625
38
Bruno
L
Rocha
B
Rolink
A
von Boehmer
H
Rodewald
HR
Intra- and extra-thymic expression of the pre-T cell receptor alpha gene.
Eur J Immunol.
25
1995
1877
1882
39
DiSanto
JP
Muller
W
Guy-Grand
D
Fischer
A
Rajewsky
K
Lymphoid development in mice with a targeted deletion of the interleukin 2 receptor gamma chain.
Proc Natl Acad Sci U S A.
92
1995
377
381
40
Colucci
F
Soudais
C
Rosmaraki
E
Vanes
L
Tybulewicz
VL
Di Santo
JP
Dissecting NK cell development using a novel alymphoid mouse model: investigating the role of the c-abl proto-oncogene in murine NK cell differentiation.
J Immunol.
162
1999
2761
2765
41
Rocha
B
Vassalli
P
Guy-Grand
D
The V beta repertoire of mouse gut homodimeric alpha CD8+ intraepithelial T cell receptor alpha/beta + lymphocytes reveals a major extrathymic pathway of T cell differentiation.
J Exp Med.
173
1991
483
486
42
Puzanov
IJ
Bennett
M
Kumar
V
IL-15 can substitute for the marrow microenvironment in the differentiation of natural killer cells.
J Immunol.
157
1996
4282
4285
43
Saito
H
Kanamori
Y
Takemori
T
et al
Generation of intestinal T cells from progenitors residing in gut cryptopatches [see comments].
Science.
280
1998
275
278
44
Page
ST
Bogatzki
LY
Hamerman
JA
et al
Intestinal intraepithelial lymphocytes include precursors committed to the T cell receptor alpha beta lineage.
Proc Natl Acad Sci U S A.
95
1998
9459
9464
45
Page
ST
van Oers
NS
Perlmutter
RM
Weiss
A
Pullen
AM
Differential contribution of Lck and Fyn protein tyrosine kinases to intraepithelial lymphocyte development.
Eur J Immunol.
27
1997
554
562
46
Le Douarin
NM
Cell migrations in embryos.
Cell.
38
1984
353
360

Author notes

Iyadh Douagi, Unité du Développement des Lymphocytes, Département d'Immunologie, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris, Cedex 15, France; e-mail: idouagi@pasteur.fr.

Sign in via your Institution