Thymic Anlage Is Colonized by Progenitors Restricted to T, NK, and Dendritic Cell Lineages
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免疫学杂志 2005年第5期
Abstract
It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin–c-kit+IL-7R+. Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCR chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.
Introduction
T cells are mainly generated in the thymus from progenitors that originate in the extrathymic organs (1, 2). However, the issue whether T cell lineage commitment occurs prethymically or intrathymically has been a longstanding question in immunology as well as developmental biology, and still remains controversial.
In adult thymus (AT),3 the thymus-colonizing progenitors have been proposed to be common lymphoid progenitors (CLP), because the cells in the earliest thymic population showed the potential to generate T and B cells at population level analyses (3, 4, 5). Subsequently, several groups have shown the presence of progenitor populations that are more or less restricted to the lymphoid lineage (6, 7, 8, 9). These "CLP populations" have been proposed to represent the thymus-colonizing progenitor populations. Studies on Notch signaling in T cell development may also have implied the thymic colonization by CLP. Radtke et al. (10) reported that in the AT of mice carrying Notch1 gene-deficient hemopoietic cells, B cells are generated at the cost of impaired T cell development. Studies using mice with a compromised Notch signaling pathway by enforced expression of Deltex or Lunatic Fringe, or disruption of recombination signal binding protein-J, showed similar phenotypes (11, 12, 13). These results have been interpreted that CLP in the bone marrow (BM) migrate to the thymus and determine their cell fate to the T cell lineage under Notch signaling within the thymic environment. In contrast, a recent study by Allman et al. (14) reported that the earliest progenitors in the thymus are more committed to the T cell lineage than CLP in BM. So far, several studies have shown the presence of T cell lineage-restricted progenitors at "prethymic" organs such as BM or spleen (15, 16), but it remained unclarified whether these cells represent thymus-colonizing cells or progenitors of extrathymically developed T cells.
Similar studies have been made on the thymic colonization during fetal development. Progenitors restricted to the T/NK lineage have been reported to be present in 13- to 15-days postcoitus (dpc) fetal liver (FL) and blood, and these progenitors have been proposed to migrate to the fetal thymus (FT) (17). However, such progenitors can only be seen at a later stage than the first colonization period (11–13 dpc); therefore, they may not represent the initial thymic immigrants. Studies on thymic progenitors at earlier stages showed that the earliest thymic progenitors from 12-dpc fetuses were capable of generating T, B, and myeloid cells as a population (18, 19). It was further reported that the multipotent progenitors were present, albeit at a very low frequency, in the circulation during 10–12 dpc (20), and these multipotent progenitors were suggested to represent those migrating from the aorta-gonado-mesonephros (AGM) region to the FL as well as to the FT. These studies may support the idea that the earliest thymic immigrants during ontogeny are multipotent progenitors.
In contrast, by using a clonal assay system, named the multilineage progenitor assay (MLP assay), which can determine the developmental potential of individual progenitors for T, B and myeloid lineages (21), we have shown that the progenitors capable of generating T cells but not B and myeloid lineage cells (T cell progenitors) are present in prethymic organs during early ontogeny. The T cell progenitors first emerge in 10-dpc AGM region (22), and they were detected in FL from 11-dpc (23). In fetal blood (FB), the T cell progenitors emerge at 10 dpc and peak at 13 dpc (22, 24). The surface phenotype of these prethymic T cell progenitors (Lineage marker negative (Lin–) c-kit+IL-7R+) in FL and FB is indistinguishable from that of 12-dpc FT cells (19). The MLP assay also revealed that the 12-dpc FT cells contain a mixture of progenitors restricted to either the T, B, or myeloid lineage, and that the T cell progenitors represent the vast majority among them (25). These findings have led us to propose that the progenitors prethymically committed to the T cell lineage migrate to the thymus (26).
One of the major problems in these studies appeared to be a lack of the exact determination of the immediate prethymic progenitors that are just migrating into the thymus. For instance, it remains uncertain whether the cells in the earliest thymic population indeed represent the immigrants from prethymic organs, because they should have been more or less influenced by the thymic microenvironment. Also, the presence of T cell progenitors in extrathymic organs may not necessarily prove that they actually migrate into the thymus. The thymic colonization is known to start at around 11 dpc, when almost all immigrating progenitors still reside in the mesenchymal region surrounding the epithelial primordium (27, 28, 29). Harman et al. (30) recently showed that Notch signaling in thymic immigrants is activated immediately following the entrance of progenitors into the epithelial primordium. Thus, the progenitors in the mesenchymal region adjacent to the thymic epithelial primordium should represent the best source for studies on the thymic immigrants.
In the present study, we first showed that the tissue including the FT anlage and the surrounding region from 11-dpc fetus, when transplanted under the kidney capsule, gave rise to a thymus containing up to 106 thymocytes, 80% of which was donor derived. Flow cytometric and immunohistochemical analyses demonstrated that cells residing in the mesenchymal region of the 11-dpc FT anlage are exclusively Lin–c-kit+IL-7R+. By examining the developmental potential of these individual IL-7R+ cells, it was found that the vast majority of IL-7R+ cells in this region are restricted to T/NK/dendritic cell (DC) lineages. These progenitors exhibited a very high potential to proliferate before the TCR chain gene rearrangement, the hallmark of early T cell progenitors that ensures them to form a broad range of a diversified TCR repertoire. These results provide direct evidence that the first immigrants in the developing thymus are T/NK/DC progenitors.
Materials and Methods
Mice
C57BL6 (B6) mice were purchased from SLC. B6Ly5.1 mice, B6Rag-2–/– mice, and enhanced GFP transgenic (EGFP Tg) mice of B6 background (31) were maintained in our animal facility. Embryos at various stages of gestation were obtained from time-mated pregnant B6, B6Ly5.1, B6Rag-2–/–, and EGFP Tg mice. The day of finding the vaginal plug was designated as 0 dpc. Embryos with 30–35 pairs of somites were regarded as 10 dpc.
Antibodies
The following Abs were used: anti-Ly5.1 (A20), anti-Ly5.2 (104), anti-c-kit (2B8), anti-erythroid lineage cells (TER119) (32), anti-Mac-1 (M1/70), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-Thy1.2 (53-2.1), anti-CD8 (53-6.7), anti-CD4 (H129.19), anti-NK1.1 (PK136), anti-TCR (GL-3), anti-TCR (H57-597), anti-CD3 (145-2C11), anti-CD19 (1D3), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD45 (30-F11), and anti-FcRII/III (FcR; 2.4G2) were purchased from BD Pharmingen. Anti-IL-7R (A7R34) was purchased from eBioscience. TER119, anti-Gr-1, anti-B220, anti-CD19, and anti-Thy-1.2 were used as Lin markers.
Growth factors
Recombinant murine (rm) stem cell factor (SCF), rm IL-2, rm IL-3, rm Flt-3 ligand, and rm IL-7 were purchased from Genzyme-Techne.
Immunohistochemistry and immunofluorescence staining
Serial sections were prepared from snap-frozen sample as described previously (29). Sections were incubated with rabbit anti-keratin (DakoCytomation) or rabbit anti-IKAROS (33), subsequently with biotinylated anti-rabbit IgG as a secondary reagent, and developed by DAB. In immunofluorescence staining, acetone-fixed 5-µm sections were incubated with anti-IL-7R (A7R34) and rabbit anti-IKAROS, and subsequently with Alexa Fluor488 goat anti-rat IgG (H+L) conjugate (Molecular Probes) and goat anti-rabbit IgG-Texas Red conjugate (Molecular Probes) as secondary reagents. Serial sections were incubated with sheep anti-human broad spectrum cytokeratin (The Binding Site) as a primary Ab and Alexa Fluor488 donkey anti-sheep IgG (H+L) conjugate (Molecular Probes) as a secondary reagent.
Preparation of fetal cells
Fetal tissues and cells were prepared as described previously (24). Tissues containing the 11-dpc FT anlage and surrounding region were taken together, and were digested for 30 min at 37°C in the presence of collagenase (1 mg/ml) (WAKO).
Coculture with a FT lobe or with a monolayer of stromal cells
The basic procedures for the coculture with deoxyguanosin (dGuo)-treated FT lobe under high oxygen submersion conditions have been described previously (21). To investigate the frequency of progenitors that have T cell-generating potential, graded number of cells of various tissues from B6Ly5.1 fetus were cultured with a dGuo-treated FT lobe (B6Ly5.2) for 14 days, and positive and negative lobes for Ly5.1+ T cell generation were flow-cytometrically determined by staining the generated cells with Ly5.1, anti-CD4, anti-CD3, and anti-CD8. The frequency was estimated by using the Poisson distribution formula (34). Ten lobes were used for each point.
To examine the frequency of progenitors that have B cell-generating potential, a graded number of cells was cultured on a monolayer of the stromal cell line TSt-4 for 10 days, and B cell generation was determined by examining the expression of B220 and IgM on generated cells by a flow cytometer. The frequency of myeloid-generating potential was assessed similarly, but the stromal cell line used was PA6, and Mac-1 and Gr-1 were used as myeloid cell markers. Twelve to 24 wells were examined for each point.
Cytokine-supplemented FT organ culture and flow cytometric analysis
The basic procedures for the single-cell culture with FT lobe under high oxygen submersion conditions have been described previously (21). For the analysis of T, B, and myeloid potential, SCF (10 ng/ml), IL-3 (3 ng/ml), and IL-7 (5 ng/ml) were added to the culture medium (MLP assay). After 10 days of culture, cells were harvested from each well. Details for judgment of progenitor types have been described previously (21). In the case of assessment of NK and DC potential, the cells for examination were obtained from EGFP Tg mice as has been detailed previously (35).
PCR analysis of TCR rearrangement
The basic procedures and primers for PCR have been described previously (23, 36).
Results
The earliest thymic immigrants are highly competent to produce thymocytes in vivo
The developing thymic anlage region was immunohistochemically stained with anti-keratin or anti-IKAROS Ab (Fig. 1A). IKAROS was used as a marker for hemopoietic cells (23, 37). It is seen that the first thymic immigrants appear at 11 dpc, as has been reported previously (27, 28, 29). The IKAROS+ cells in the thymic anlage area at 11 dpc reside almost exclusively in the mesenchymal region surrounding the epithelial primordium. Although some IKAROS+ cells are found very close to the outer rim of the epithelium, most of them reside apart from the epithelium, confirming our previous finding (29).
FIGURE 1. Thymic anlage of 11-dpc fetuses contains progenitors capable of forming thymic populations. A, Distribution of epithelial cells and hemopoietic cells during early thymic ontogeny. Serial sections of 10- and 11-dpc fetuses were stained with anti-keratin or anti-IKAROS Abs. B, Experimental procedure for transplantation of FT anlage. The tissue containing epithelial primordium and the surrounding mesenchyme (11 dpc), 12-dpc FT, and 13-dpc FT from B6Ly5.1 mice were transplanted under the kidney capsule of adult B6Ly5.2 mice. C, Numbers of donor-derived and recipient-derived thymocytes generated in individual grafts. Recovered cells were analyzed for the expression of Ly5.1 vs Ly5.2 to determine percentages of donor- and recipient-derived cells in the grafts. The transplanted thymus successfully developed in five of eight recipients in the 11-dpc FT group, three of four in the 12-dpc FT group, and four of five in the 13-dpc FT group. The data of three representative lobes from each group are shown. D, Representative flow cytometric profiles of cells in 11-dpc FT graft. Cells from 11-dpc FT graft (a) in C, were stained in three colors with anti-Ly5.1, anti-CD4, and anti-CD8. For comparison, a profile of neonatal thymus is shown.
We have previously shown using an in vitro system that tissues containing the thymic epithelial primordium and surrounding mesenchymal area of 11-dpc fetus (11-dpc FT anlage region) contain progenitors that can generate T cells (29). In contrast, Douagi et al. (38) showed that the progenitor activity of 12-dpc FT was too low to contribute to the first generation of thymopoiesis. Therefore, we examined in vivo the extent to which the initial thymic immigrants are able to produce thymocytes. The 11-dpc FT anlage regions were picked up from 11-dpc fetuses of C57BL6 (B6) Ly5.1 mice, and they were individually transplanted under the kidney capsule of adult B6Ly5.2 mice (Fig. 1B). For comparison, 12- and 13-dpc FT were also transplanted. After 12 days, cells were recovered from individual grafts. It was demonstrated that the progenitors that reside in the 11-dpc FT anlage region gave rise to a large number (nearly 106) of thymocytes, although the absolute number of donor-derived cells in the graft of 11-dpc FT region was smaller than those in grafts of 12-dpc FT and 13-dpc FT (Fig. 1C). CD4+CD8+ (double positive) cells as well as CD4 single-positive (SP) cells and CD8 SP cells were formed in Ly5.1+ (donor) fraction of cells in the graft of 11-dpc FT anlage region (Fig. 1D). The smaller proportion of CD4–CD8– cells in donor-derived (Ly5.1+) cells than in recipient-derived (Ly5.1–) cells may indicate that this thymus is being replaced by recipient cells. The recovered graft on the 12th day after transplantation of the 11-dpc FT anlage region can be at the same developmental stage as the thymus on day 3 after birth, which actually shows a comparable profile (Fig. 1D, far right panel). These results may indicate that the earliest thymic immigrants at 11 dpc substantially contribute to the first generation of thymocytes.
Selective migration of IL-7R-expressing cells to the thymic anlage
We next examined the surface phenotypes of cells in the 11-dpc FT anlage region (Fig. 2A). For comparison, cells from FL, FB, and other tissues, such as a portion of the head, were also examined. CD45+ cells represent all hematopoitic lineage cells except for the erythroid lineage cells (39). Virtually all Lin–CD45+ cells in all tissues examined were c-kit+ (data not shown). The absence of Lin+ cells in FT anlage region indicate that the contaminated blood cells the FT anlage region sample is quite negligible
FIGURE 2. Selective migration of Lin–c-kit+IL-7R+ cells to the 11-dpc FT anlage. A, Cells from various organs or tissues of 11-dpc fetuses were four-color stained with anti-Lin, anti-CD45, anti-FcR, and anti-IL-7R. The numbers in the panels represent the percentage of cells in each quadrant. B, A frozen section of 11-dpc fetus was stained in two colors with anti-IKAROS Ab (red) and anti-IL-7R Ab (green). A serial section was stained with anti-keratin Ab.
In 11-dpc FL, 14% of cells are Lin–CD45+, and 4 and 45% of the Lin–CD45+ cells express IL-7R and FcR, respectively (Fig. 2A). We have previously shown that Lin–c-kit+IL-7R+ (IL-7R+) population in 11- to 12-dpc FL contains mostly T cell progenitors (23). We have also shown that Lin–c-kit+FcR+ (FcR+) cells in 13-dpc FL are myeloid lineage committed (39). Thus, it can be said that the commitment toward T and myeloid lineages proceeds in the 11-dpc FL. Only 0.5% of FB cells were Lin–CD45+, and among the Lin–CD45+ FB cells, 15% were IL-7R+. The proportion IL-7R+ cells in the CD45+ population of FB is three to four times larger than that of FL cells, confirming our previous finding that T cell progenitors are preferentially released into the circulation (24). In marked contrast, a large majority (80%) of CD45+ cells in the 11-dpc FT anlage region were found to express IL-7R, while only 15% were FcR+. IL-7R–FcR– cells were very few, if any, in this region. The FcR+ cells may be myeloid precursors that distribute ubiquitously, because the cells with a similar phenotype are observed in any other tissue (Fig. 2A, and data not shown).
We then examined the IL-7R expression of cells in the 11-dpc FT anlage region by immunofluorescence staining. One section of 11-dpc fetuses was stained in two-colors with anti-IKAROS and anti-IL-7R, and another serial section was stained with anti-keratin (Fig. 2B). Note that IKAROS (red) localizes to the nucleus, while IL-7R (green) localizes to the cytoplasm. It was confirmed that virtually all IKAROS+ cells in the mesenchyme adjacent to the keratin+ epithelial primordium are IL-7R+. Taken together, these data indicate that the IL-7R+ cells selectively migrate into the thymic anlage.
The initial thymic immigrants are T cell lineage-restricted progenitors
We investigated the frequency of cells having progenitor activity for T, B, or myeloid lineages in various tissues from 11-dpc fetuses through limiting dilution analysis, and the total numbers of these progenitors per fetus were determined (Table I). Myeloid cell-generating progenitors were most abundant in FL (1100–2100 per fetus), and T cell generating progenitors and B cell generating ones were comparable to each other (300 per fetus). FB also contained many progenitors for these lineages, while the relative proportion of T cell-generating progenitors vs B cell-generating ones was much higher than that in FL. The majority of B cell-generating progenitors detected in FL and FB may represent multipotent progenitors as has been shown previously (20, 40), because most of the B cell colonies on the stromal cell layer accompanied macrophage generation (data not shown). In contrast, such multipotent progenitors may not exist in the 11-dpc FT anlage region, because B cell-generating activity was barely detected in the 11-dpc FT anlage region, indicating that this region does not contain B cell-generating progenitors such as multipotent progenitors or B cell lineage-committed progenitors.
Table I. Numbers of progenitors per tissue of 11-dpc fetus
We next examined the commitment status of individual progenitors in FL and FT anlage region from 11-dpc fetuses by using the MLP assay. Various types of progenitors including multipotent progenitors generating T, B, and myeloid cells were detected in FcR–IL-7R– population of FL (Fig. 3A). The IL-7R+ population in 11-dpc FL contained mostly the progenitors generating only T cells (p-T) while FcR+ population exclusively contained progenitors generating only myeloid cells (p-M), confirming our previous findings (23, 39). In 11-dpc FT anlage region, it was found that IL-7R+ cells exclusively contained p-T. FcR+ population contained only p-M, although the frequency was significantly smaller than that in FL (Fig. 3B). These FcR+ p-M may correspond to the progenitors with myeloid potential detected in the thymic anlage region by the limiting dilution analysis (Table I). Very small numbers of p-T, but no other types of progenitors, were detected in the IL-7R–FcR– cells from the FT anlage region (data not shown). Together with the finding that hemopoietic cells adjacent to the epitherial primordium are mostly IL-7R+ (Fig. 2B), it was demonstrated that most of the hemopoietic progenitors migrating to the FT anlage are T cell lineage restricted.
FIGURE 3. IL-7R+ cells residing in the 11-dpc FT anlage region are T cell lineage restricted. Single cells of various populations from 11-dpc FL (A) and 11-dpc FT anlage region (B) were individually examined using the MLP assay. Cells grown in each well were recovered on day 10 and progenitor types were flow cytometrically determined. p-MTB, Progenitors generating myeloid, T, and B cells. The scales represent the numbers of progenitors detected among the 50 cells assayed. Data are representative of three independent experiments.
NK and DC potential of the earliest thymic immigrants
We have previously reported that the earliest thymic progenitors from 12-dpc FT as well as prethymic T cell progenitors in FB and FL retain the potential to generate NK cells and DC (24, 35, 41). Therefore, the tripotentiality for T, NK, and DC lineage cells can be regarded as a hallmark of early T cell progenitors. We then examined the initial thymic immigrants at a single cell level for the capability of generating T, NK, and DC lineages. A total of 40 IL-7R+ cells in 11-dpc FT region were individually cultured with a dGuo lobe in the presence of a cytokine mixture that support the generation of NK and DC. Representative FACS profile of cells of a clone containing T and NK cells (Fig. 4A) and a photograph of DC (Fig. 4B) generated from a single IL-7R+ cell are shown. The DC derived from thymic immigrants were CD11c+ and class II+ (data not shown). It was found that, of 40 cells examined, 7 cells generated T cells, and all 7 T cell progenitors also produced NK cells (Fig. 4C). Among seven T/NK bipotent progenitors, four progenitors showed DC-generating activity. The results may indicate that the earliest thymic T cell progenitors at 11 dpc retain NK and DC potentials.
FIGURE 4. Developmental potential of T cell progenitors to generate NK cells, DC, T cells, and T cells. A, Representative flow cytometric profiles of cells generated from a single T/NK progenitor. IL-7R+ cells of the 11-dpc FT anlage region from EGFP Tg mice were individually cultured with a dGuo-treated FT lobe in the presence of SCF, IL-7, IL-2, and Flt-3 ligand for 10 days. B, DC generated from a single IL-7R+ cell of the 11-dpc FT anlage region. Bar indicates 50 µm. C, A total of 40 IL-7R+ cells of the 11-dpc FT region were examined by a clonal analysis that covers T, NK, and DC lineages, and 7 showed T cell generation. The numbers of progenitors with NK and DC potential among seven T cell progenitors are shown. D, Representative flow cytometric profiles of cells generated in FT organ culture from a single cell of the 11-dpc FT anlage cell and a 14-dpc FT CD44+CD25– cell. E, Cells generated in FT organ culture from a single cell of 11-dpc FT anlage region were examined for their rearrangement status of V-J locus using PCR.
Generation of authentic thymic T cells from individual thymic immigrants
Although in Fig. 1 we have shown that the 11-dpc FT anlage region contains progenitors capable of forming a large number of thymocytes, this does not preclude the possibility that such competent progenitors represent a minority population among progenitors in the 11-dpc FT anlage region. Thus, it is important to clarify whether a majority of progenitors in FT anlage region is able to generate a broad spectrum of T cell populations, or only a limited repertoire of T cells. We addressed this issue by characterizing the T cells generated from individual progenitors of 11-dpc FT anlage region in comparison with thymic progenitors at later embryonic age (CD44+CD25– cells of 14-dpc FT). No significant difference was seen between plating efficiency (15 of 60 in the 11-dpc FT group and 17 of 60 in the 14-dpc FT group), and in both groups most of clones contained T and T cells. Flow cytometric profiles were indistinguishable regarding the proportion of double positive and CD4/8 SP populations and the ratio of T vs T cells between cells generated from a single IL-7R+ cell in the 11-dpc FT anlage region and from a single CD44+CD25– cell in 14-dpc FT (Fig. 4D). The usage of V genes in T cells was examined, and it was found that T cells generated from a single cell of 11-dpc FT anlage region cell as well as a 14-dpc FT CD44+CD25– cell exhibit rearrangement of various V genes other than the V3 gene (Fig. 4E), which is known to be preferentially rearranged at an early fetal stage (42). These results demonstrated that the majority of the earliest thymic T cell progenitors retain the potential to produce a broad range of T cell populations in both the T cell and T cell lineages.
Proliferation potential of the initial thymic immigrants before the TCR chain gene rearrangement
We have recently shown that 12-dpc FT progenitors as well as circulating prethymic T cell progenitors at 12 dpc retain a very strong capacity to proliferate before the initiation of TCR chain gene rearrangement (pre--rearrangement proliferation potential) (24, 36). Because it is expected that the more primitive progenitors have the higher pre--rearrangement proliferation potential, the assessment of this potential at a single cell level is effective in ranking each progenitor in the developmental hierarchy of T cell progenitors. We then assessed this potential by culturing individual cells in 11-dpc FT anlage region with a dGuo lobe. Cells generated from a single IL-7R+ cell of the 11-dpc FT anlage region, in comparison with those from a CD44+CD25– cell and CD44+CD25+ cell of 14-dpc FT, were analyzed by PCR for D-J rearrangement status. All clones from 11-dpc FT region exhibited almost all possible bands, indicating that these progenitors have a potential to produce highly diversified TCR chains (Fig. 5A).
FIGURE 5. Pre--rearrangement proliferation of T cell progenitors from FL, FB, and thymic anlage region of 11-dpc fetus. A, IL-7R+ FL cells and IL-7R+ cells of 11-dpc FT region, as well as CD44+CD25– and CD44+CD25+ cells of 14-dpc FT, were individually cultured with a dGuo lobe for 12 days. Genomic DNA was prepared from T cells generated in each well, and was PCR amplified using primers shown under the figure. B and C, IL-7R+ FL cells (11 FL) and IL-7R+ cells of 11-dpc FT region (11 FT), as well as CD44+CD25– and CD44+CD25+ cells of 14-dpc FT from Rag2–/– fetuses, were individually cultured with a dGuo-treated FT lobe for 10 days. B, Representative profile of cells generated from a single IL-7R+ cell in the Rag2–/– 11-dpc FT anlage region. C, Numbers of CD25+ cells in four to seven clones in each group.
We further assessed the pre--rearrangement proliferation potential by using cells from Rag2–/– mice. Rag2–/– cells from 11-dpc FT anlage region, in comparison with those from 11-dpc FL and 14-dpc FT populations, were individually cultured with a dGuo lobe. The flow cytometric profile of a representative clone generated from a IL-7R+ cell of the Rag2–/– 11-dpc FT anlage region is shown (Fig. 5B), in which the accumulation of CD44–/lowCD25+ cells, in addition to the expansion of CD44+CD25– cells, was observed. The number of generated CD25+ cells in a culture represents the pre--rearrangement proliferation potential of a seeded progenitor, because growth of Rag2–/– thymocytes is arrested at the point of TCR gene rearrangement. The numbers of CD25+ cells per clone are plotted (Fig. 5C). The difference in clonal expansion size may reflect the hierarchical relationship among progenitors. These results indicate that the initial thymic immigrants at 11 dpc locate at the highest position in the hierarchy of fetal thymic progenitors. This difference in pre--proliferation potential of progenitors between 11-dpc FT and 14-dpc FT may reflect a progenitor-progeny relationship among these progenitors, but may not be due to the difference in property of colonizing progenitors, because prethymic T cell progenitors are almost equivalent in pre--rearrangement proliferation potential during early FL ontogeny (11–15 dpc) (Ref.23 , and our unpublished observations).
Discussion
The present study demonstrated that the ontogenically earliest thymic progenitors that reside in the mesenchymal region surrounding the epithelial primordium at 11 dpc of murine fetuses were T/NK/DC lineage restricted. These progenitors are considered to be authentic T cell progenitors in that they are able to generate both T and T cells, and that they retain a very high proliferation potential to form a precursor pool that would accommodate diversified TCR chains.
It has been well documented that thymic colonization by hemopoietic cells starts at 11 dpc (23, 24, 25). Several trials have been made to assess the potential of early thymic immigrants for producing thymic T cells. Owen and Ritter (43) examined the 11-dpc FT anlage in an organ culture system performed in a chamber embedded in chorioallantois of a chicken egg, and showed that it contained precursors for lymphoid cells, although the cell growth from the 11-dpc precursors was very limited. Jotereau et al. (44) have examined the progenitor potential of early FT cells by transferring the 11-dpc FT anlage or FT from 12- to 15-dpc fetuses under the kidney capsule of recipient mice, and suggested that progenitors that have migrated into the thymus before 13 dpc may mainly contribute to the fetal thymopoiesis, although in their study the absolute number and the surface phenotype of thymocytes derived from progenitors in the 11-dpc FT anlage were not shown. In contrast, Douagi et al. (38) recently showed the that the T cell producing potential of progenitors in 12-dpc FT is limited compared with that in 14-dpc FT, and speculated that the progenitors immigrating earlier than 13 dpc do not significantly contribute to the fetal T cell generation. Thus, the progenitor activity of the ontogenically earliest thymic progenitors as for the contribution to the fetal thymopoiesis has remained controversial. The present study demonstrated that the earliest thymic immigrants at 11 dpc are quite competent to generate a large number of thymocytes in vivo (Fig. 1, C and D). Of note is that the average pre--rearrangement proliferation potential of the earliest thymic immigrants at 11 dpc was even higher than that of CD44+CD25– cells in 14-dpc FT. Therefore, we propose that the ontogenically earliest thymic immigrants are not just curtain raisers, but leading actors of the first generation of thymopoiesis.
Because multipotent progenitors generating T, B, and myeloid lineages have been shown to circulate during the period of initial thymic colonization (20), it tends to be speculated that these multipotent progenitors in FB migrate into the thymus. Our present findings, together with previous studies (21, 22, 23), argue against this idea, and instead propose that progenitors that have shut off their B cell and myeloid potentials selectively migrate to the thymic analage as the initial immigrants. However, because progenitors with B cell potential, albeit at a quite low level, were found in 11-dpc FT anlage region by limiting dilution analysis (Table I), two possibilities formally remain; first, a small number of multipotent progenitors also migrate to the thymus, and second, commitment to the T cell lineage by shutting off the B cell potential is more or less incomplete.
It has previously been pointed out that the thymic immigration of progenitors during the embryonic life occurs three times in birds and twice in mice (44, 45, 46). The Lin–IL-7R+ T cell progenitors present in the marginal area of the thymic epithelium may represent those participating in the first wave of thymic immigration. Because the AGM region in addition to FL of 11-dpc fetus contains IL-7R+ T cell progenitors (22, 24), it is probable that some of the IL-7R+ T cell progenitors found in the 11-dpc FT anlage region may be direct immigrants from the AGM region. Following points still remain unclarified; whether T cell progenitors derived from the AGM region are similar to those from FL, and which sites are the major sources of thymic immigrants during the embryonic stages. In contrast, T/NK cell lineage-restricted progenitors, namely B220lowc-kit+ cells in 13- to 15-dpc FL and Thy-1+c-kitlowNK1.1+ cells in 13- to 15-dpc FB, have been shown to exist (47, 48, 49). Using a GFP-reporter system, Yokota et al. (50) have shown that a RAG1-expressing cell population that is skewed toward the T cell lineage is present in 13- to 15-dpc FL. These progenitors could represent those of the second wave of thymic immigration, because they appear in FL and FB at ontogenically later stages and they express some Lin or RAG proteins.
Our present and previous studies, using clonal analysis, showed that the thymic progenitors are tripotential for T, NK, and DC lineages. It has been shown that NK cells are produced in the FT (51). However, the thymus may not be the major site for NK cell production, because athymic mice have a normal or even higher level of NK cells. The NK potential of thymic T cell progenitors could be the vestige of phylogenic events in which T cells have evolved from primordial NK cells. It is probable that the differentiation program for NK cells is used as a base of the T cell differentiation program. In contrast, the DC program accompanying the T cell progenitors may have evolved to exploit the DC function in the thymus, because DC may play an important role in thymic negative selection (52).
Recent studies on the role of Notch1 in T cell development have proposed a model, in which the CLP are presumed to migrate to the thymus where they decide their fate under the control of Notch signals (53). However, the scheme of the CLP immigration is not the case in fetal T cell development, because the cell fate decision to the T cell lineage takes place prethymically, as has been shown in the series of our studies (26). Recently, Harman et al. (30) showed that the immigrating cells in the mesenchyme surrounding the 12-dpc epithelial pripordium did not express Hes1 nor Deltex while intrathymic cells express these molecules, indicating that the Notch1 signal is activated only after progenitors encounter the thymic epithelial cells. Our present findings, together with the finding by Harman et al., suggest that the Notch-Hes1 pathway may not be essential for T cell lineage commitment, at least in fetal T cell development. Consistent with this is the finding in Hes1-deficient mice that the expansion of intrathymic progenitors was severely impaired but the commitment status of them was not altered (54, 55). However, the possibility cannot be ruled out that a Notch signal which is too low to be monitored by Hes1 expression, or other types of Notch signaling pathways than those mediated by Notch1 and/or Hes1 molecule(s), may play a role in the T cell lineage commitment at prethymic stage.
It tends to be speculated that the thymus-colonizing progenitors in fetuses differ from those in adults, because numbers of studies have suggested the difference between fetal and adult hematopoiesis (56). Indeed, the progenitor population sharing the surface phenotype with fetal prethymic progenitors, defined as Lin–c-kithighIL-7R+ in our series of studies (23, 24), has not been reported to exist in BM or blood. However, Allman et al. (14) recently showed that the earliest progenitors in AT are not CLP but more restricted to the T cell lineage. A recent study by Porritt et al. (57) also showed that progenitors generating T cells and B cells have already been segregated in the earliest thymic population. These studies may imply that the AT is colonized by T cell lineage-committed progenitors. In contrast, Martin et al. (8) have suggested that the B220+ CLP in BM may represent progenitors bound for thymus. Very recently, a study by Schwarz et al. (58) proposed that the circulating multipotent progenitors represent the adult thymic immigrants. Further studies on the prethymic progenitors as well as the earliest thymic progenitors in both fetal and adult mice will facilitate our understanding of the molecular mechanism of T cell lineage restriction.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Wilfred T. V. Germeraad for critical reading of the manuscript.
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 work was supported by Grants 12051219, 11470086, and 10044278 from the Ministry of Education, Science, Sports and Culture, and the Special Coordination Funds of the Science and Technology Agency, Japan.
2 Address correspondence and reprint requests to Dr. Hiroshi Kawamoto, Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. E-mail address: kawamoto{at}rcai.riken.jp
3 Abbreviations used in this paper: AT, adult thymus; AGM, aorta-gonado-mesonephros; BM, bone marrow; CLP, common lymphoid progenitor; DC, dendritic cell; FB, fetal blood; FL, fetal liver; FT, fetal thymus; Lin, lineage marker; MLP assay, multilineage progenitor assay; dpc, day postcoitus; rm, recombinant murine; SCF, stem cell factor; EGFP Tg, enhanced GFP transgenic; dGuo, deoxyguanosin; SP, single positive; p-T, progenitors generating only T cells; p-M, progenitors generating only myeloid cells.
Received for publication September 1, 2004. Accepted for publication November 11, 2004.
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It remains controversial whether the thymus-colonizing progenitors are committed to the T cell lineage. A major problem that has impeded the characterization of thymic immigrants has been that the earliest intrathymic progenitors thus far identified do not necessarily represent the genuine thymic immigrants, because their developmental potential should have been influenced by contact with the thymic microenvironment. In the present study, we examined the developmental potential of the ontogenically earliest thymic progenitors of day 11 murine fetus. These cells reside in the surrounding mesenchymal region and have not encountered thymic epithelial components. Flow cytometric and immunohistochemical analyses demonstrated that these cells are exclusively Lin–c-kit+IL-7R+. Limiting dilution analyses disclosed that the progenitors with T cell potential were abundant, while those with B cell potential were virtually absent in the region of day 11 thymic anlage. Clonal analyses reveled that they are restricted to T, NK, and dendritic cell lineages. Each progenitor was capable of forming a large number of precursors that may clonally accommodate highly diverse TCR chains. These results provide direct evidence that the progenitors restricted to the T/NK/dendritic cell lineage selectively immigrate into the thymus.
Introduction
T cells are mainly generated in the thymus from progenitors that originate in the extrathymic organs (1, 2). However, the issue whether T cell lineage commitment occurs prethymically or intrathymically has been a longstanding question in immunology as well as developmental biology, and still remains controversial.
In adult thymus (AT),3 the thymus-colonizing progenitors have been proposed to be common lymphoid progenitors (CLP), because the cells in the earliest thymic population showed the potential to generate T and B cells at population level analyses (3, 4, 5). Subsequently, several groups have shown the presence of progenitor populations that are more or less restricted to the lymphoid lineage (6, 7, 8, 9). These "CLP populations" have been proposed to represent the thymus-colonizing progenitor populations. Studies on Notch signaling in T cell development may also have implied the thymic colonization by CLP. Radtke et al. (10) reported that in the AT of mice carrying Notch1 gene-deficient hemopoietic cells, B cells are generated at the cost of impaired T cell development. Studies using mice with a compromised Notch signaling pathway by enforced expression of Deltex or Lunatic Fringe, or disruption of recombination signal binding protein-J, showed similar phenotypes (11, 12, 13). These results have been interpreted that CLP in the bone marrow (BM) migrate to the thymus and determine their cell fate to the T cell lineage under Notch signaling within the thymic environment. In contrast, a recent study by Allman et al. (14) reported that the earliest progenitors in the thymus are more committed to the T cell lineage than CLP in BM. So far, several studies have shown the presence of T cell lineage-restricted progenitors at "prethymic" organs such as BM or spleen (15, 16), but it remained unclarified whether these cells represent thymus-colonizing cells or progenitors of extrathymically developed T cells.
Similar studies have been made on the thymic colonization during fetal development. Progenitors restricted to the T/NK lineage have been reported to be present in 13- to 15-days postcoitus (dpc) fetal liver (FL) and blood, and these progenitors have been proposed to migrate to the fetal thymus (FT) (17). However, such progenitors can only be seen at a later stage than the first colonization period (11–13 dpc); therefore, they may not represent the initial thymic immigrants. Studies on thymic progenitors at earlier stages showed that the earliest thymic progenitors from 12-dpc fetuses were capable of generating T, B, and myeloid cells as a population (18, 19). It was further reported that the multipotent progenitors were present, albeit at a very low frequency, in the circulation during 10–12 dpc (20), and these multipotent progenitors were suggested to represent those migrating from the aorta-gonado-mesonephros (AGM) region to the FL as well as to the FT. These studies may support the idea that the earliest thymic immigrants during ontogeny are multipotent progenitors.
In contrast, by using a clonal assay system, named the multilineage progenitor assay (MLP assay), which can determine the developmental potential of individual progenitors for T, B and myeloid lineages (21), we have shown that the progenitors capable of generating T cells but not B and myeloid lineage cells (T cell progenitors) are present in prethymic organs during early ontogeny. The T cell progenitors first emerge in 10-dpc AGM region (22), and they were detected in FL from 11-dpc (23). In fetal blood (FB), the T cell progenitors emerge at 10 dpc and peak at 13 dpc (22, 24). The surface phenotype of these prethymic T cell progenitors (Lineage marker negative (Lin–) c-kit+IL-7R+) in FL and FB is indistinguishable from that of 12-dpc FT cells (19). The MLP assay also revealed that the 12-dpc FT cells contain a mixture of progenitors restricted to either the T, B, or myeloid lineage, and that the T cell progenitors represent the vast majority among them (25). These findings have led us to propose that the progenitors prethymically committed to the T cell lineage migrate to the thymus (26).
One of the major problems in these studies appeared to be a lack of the exact determination of the immediate prethymic progenitors that are just migrating into the thymus. For instance, it remains uncertain whether the cells in the earliest thymic population indeed represent the immigrants from prethymic organs, because they should have been more or less influenced by the thymic microenvironment. Also, the presence of T cell progenitors in extrathymic organs may not necessarily prove that they actually migrate into the thymus. The thymic colonization is known to start at around 11 dpc, when almost all immigrating progenitors still reside in the mesenchymal region surrounding the epithelial primordium (27, 28, 29). Harman et al. (30) recently showed that Notch signaling in thymic immigrants is activated immediately following the entrance of progenitors into the epithelial primordium. Thus, the progenitors in the mesenchymal region adjacent to the thymic epithelial primordium should represent the best source for studies on the thymic immigrants.
In the present study, we first showed that the tissue including the FT anlage and the surrounding region from 11-dpc fetus, when transplanted under the kidney capsule, gave rise to a thymus containing up to 106 thymocytes, 80% of which was donor derived. Flow cytometric and immunohistochemical analyses demonstrated that cells residing in the mesenchymal region of the 11-dpc FT anlage are exclusively Lin–c-kit+IL-7R+. By examining the developmental potential of these individual IL-7R+ cells, it was found that the vast majority of IL-7R+ cells in this region are restricted to T/NK/dendritic cell (DC) lineages. These progenitors exhibited a very high potential to proliferate before the TCR chain gene rearrangement, the hallmark of early T cell progenitors that ensures them to form a broad range of a diversified TCR repertoire. These results provide direct evidence that the first immigrants in the developing thymus are T/NK/DC progenitors.
Materials and Methods
Mice
C57BL6 (B6) mice were purchased from SLC. B6Ly5.1 mice, B6Rag-2–/– mice, and enhanced GFP transgenic (EGFP Tg) mice of B6 background (31) were maintained in our animal facility. Embryos at various stages of gestation were obtained from time-mated pregnant B6, B6Ly5.1, B6Rag-2–/–, and EGFP Tg mice. The day of finding the vaginal plug was designated as 0 dpc. Embryos with 30–35 pairs of somites were regarded as 10 dpc.
Antibodies
The following Abs were used: anti-Ly5.1 (A20), anti-Ly5.2 (104), anti-c-kit (2B8), anti-erythroid lineage cells (TER119) (32), anti-Mac-1 (M1/70), anti-Gr-1 (RB6-8C5), anti-B220 (RA3-6B2), anti-Thy1.2 (53-2.1), anti-CD8 (53-6.7), anti-CD4 (H129.19), anti-NK1.1 (PK136), anti-TCR (GL-3), anti-TCR (H57-597), anti-CD3 (145-2C11), anti-CD19 (1D3), anti-CD25 (PC61), anti-CD44 (IM7), anti-CD45 (30-F11), and anti-FcRII/III (FcR; 2.4G2) were purchased from BD Pharmingen. Anti-IL-7R (A7R34) was purchased from eBioscience. TER119, anti-Gr-1, anti-B220, anti-CD19, and anti-Thy-1.2 were used as Lin markers.
Growth factors
Recombinant murine (rm) stem cell factor (SCF), rm IL-2, rm IL-3, rm Flt-3 ligand, and rm IL-7 were purchased from Genzyme-Techne.
Immunohistochemistry and immunofluorescence staining
Serial sections were prepared from snap-frozen sample as described previously (29). Sections were incubated with rabbit anti-keratin (DakoCytomation) or rabbit anti-IKAROS (33), subsequently with biotinylated anti-rabbit IgG as a secondary reagent, and developed by DAB. In immunofluorescence staining, acetone-fixed 5-µm sections were incubated with anti-IL-7R (A7R34) and rabbit anti-IKAROS, and subsequently with Alexa Fluor488 goat anti-rat IgG (H+L) conjugate (Molecular Probes) and goat anti-rabbit IgG-Texas Red conjugate (Molecular Probes) as secondary reagents. Serial sections were incubated with sheep anti-human broad spectrum cytokeratin (The Binding Site) as a primary Ab and Alexa Fluor488 donkey anti-sheep IgG (H+L) conjugate (Molecular Probes) as a secondary reagent.
Preparation of fetal cells
Fetal tissues and cells were prepared as described previously (24). Tissues containing the 11-dpc FT anlage and surrounding region were taken together, and were digested for 30 min at 37°C in the presence of collagenase (1 mg/ml) (WAKO).
Coculture with a FT lobe or with a monolayer of stromal cells
The basic procedures for the coculture with deoxyguanosin (dGuo)-treated FT lobe under high oxygen submersion conditions have been described previously (21). To investigate the frequency of progenitors that have T cell-generating potential, graded number of cells of various tissues from B6Ly5.1 fetus were cultured with a dGuo-treated FT lobe (B6Ly5.2) for 14 days, and positive and negative lobes for Ly5.1+ T cell generation were flow-cytometrically determined by staining the generated cells with Ly5.1, anti-CD4, anti-CD3, and anti-CD8. The frequency was estimated by using the Poisson distribution formula (34). Ten lobes were used for each point.
To examine the frequency of progenitors that have B cell-generating potential, a graded number of cells was cultured on a monolayer of the stromal cell line TSt-4 for 10 days, and B cell generation was determined by examining the expression of B220 and IgM on generated cells by a flow cytometer. The frequency of myeloid-generating potential was assessed similarly, but the stromal cell line used was PA6, and Mac-1 and Gr-1 were used as myeloid cell markers. Twelve to 24 wells were examined for each point.
Cytokine-supplemented FT organ culture and flow cytometric analysis
The basic procedures for the single-cell culture with FT lobe under high oxygen submersion conditions have been described previously (21). For the analysis of T, B, and myeloid potential, SCF (10 ng/ml), IL-3 (3 ng/ml), and IL-7 (5 ng/ml) were added to the culture medium (MLP assay). After 10 days of culture, cells were harvested from each well. Details for judgment of progenitor types have been described previously (21). In the case of assessment of NK and DC potential, the cells for examination were obtained from EGFP Tg mice as has been detailed previously (35).
PCR analysis of TCR rearrangement
The basic procedures and primers for PCR have been described previously (23, 36).
Results
The earliest thymic immigrants are highly competent to produce thymocytes in vivo
The developing thymic anlage region was immunohistochemically stained with anti-keratin or anti-IKAROS Ab (Fig. 1A). IKAROS was used as a marker for hemopoietic cells (23, 37). It is seen that the first thymic immigrants appear at 11 dpc, as has been reported previously (27, 28, 29). The IKAROS+ cells in the thymic anlage area at 11 dpc reside almost exclusively in the mesenchymal region surrounding the epithelial primordium. Although some IKAROS+ cells are found very close to the outer rim of the epithelium, most of them reside apart from the epithelium, confirming our previous finding (29).
FIGURE 1. Thymic anlage of 11-dpc fetuses contains progenitors capable of forming thymic populations. A, Distribution of epithelial cells and hemopoietic cells during early thymic ontogeny. Serial sections of 10- and 11-dpc fetuses were stained with anti-keratin or anti-IKAROS Abs. B, Experimental procedure for transplantation of FT anlage. The tissue containing epithelial primordium and the surrounding mesenchyme (11 dpc), 12-dpc FT, and 13-dpc FT from B6Ly5.1 mice were transplanted under the kidney capsule of adult B6Ly5.2 mice. C, Numbers of donor-derived and recipient-derived thymocytes generated in individual grafts. Recovered cells were analyzed for the expression of Ly5.1 vs Ly5.2 to determine percentages of donor- and recipient-derived cells in the grafts. The transplanted thymus successfully developed in five of eight recipients in the 11-dpc FT group, three of four in the 12-dpc FT group, and four of five in the 13-dpc FT group. The data of three representative lobes from each group are shown. D, Representative flow cytometric profiles of cells in 11-dpc FT graft. Cells from 11-dpc FT graft (a) in C, were stained in three colors with anti-Ly5.1, anti-CD4, and anti-CD8. For comparison, a profile of neonatal thymus is shown.
We have previously shown using an in vitro system that tissues containing the thymic epithelial primordium and surrounding mesenchymal area of 11-dpc fetus (11-dpc FT anlage region) contain progenitors that can generate T cells (29). In contrast, Douagi et al. (38) showed that the progenitor activity of 12-dpc FT was too low to contribute to the first generation of thymopoiesis. Therefore, we examined in vivo the extent to which the initial thymic immigrants are able to produce thymocytes. The 11-dpc FT anlage regions were picked up from 11-dpc fetuses of C57BL6 (B6) Ly5.1 mice, and they were individually transplanted under the kidney capsule of adult B6Ly5.2 mice (Fig. 1B). For comparison, 12- and 13-dpc FT were also transplanted. After 12 days, cells were recovered from individual grafts. It was demonstrated that the progenitors that reside in the 11-dpc FT anlage region gave rise to a large number (nearly 106) of thymocytes, although the absolute number of donor-derived cells in the graft of 11-dpc FT region was smaller than those in grafts of 12-dpc FT and 13-dpc FT (Fig. 1C). CD4+CD8+ (double positive) cells as well as CD4 single-positive (SP) cells and CD8 SP cells were formed in Ly5.1+ (donor) fraction of cells in the graft of 11-dpc FT anlage region (Fig. 1D). The smaller proportion of CD4–CD8– cells in donor-derived (Ly5.1+) cells than in recipient-derived (Ly5.1–) cells may indicate that this thymus is being replaced by recipient cells. The recovered graft on the 12th day after transplantation of the 11-dpc FT anlage region can be at the same developmental stage as the thymus on day 3 after birth, which actually shows a comparable profile (Fig. 1D, far right panel). These results may indicate that the earliest thymic immigrants at 11 dpc substantially contribute to the first generation of thymocytes.
Selective migration of IL-7R-expressing cells to the thymic anlage
We next examined the surface phenotypes of cells in the 11-dpc FT anlage region (Fig. 2A). For comparison, cells from FL, FB, and other tissues, such as a portion of the head, were also examined. CD45+ cells represent all hematopoitic lineage cells except for the erythroid lineage cells (39). Virtually all Lin–CD45+ cells in all tissues examined were c-kit+ (data not shown). The absence of Lin+ cells in FT anlage region indicate that the contaminated blood cells the FT anlage region sample is quite negligible
FIGURE 2. Selective migration of Lin–c-kit+IL-7R+ cells to the 11-dpc FT anlage. A, Cells from various organs or tissues of 11-dpc fetuses were four-color stained with anti-Lin, anti-CD45, anti-FcR, and anti-IL-7R. The numbers in the panels represent the percentage of cells in each quadrant. B, A frozen section of 11-dpc fetus was stained in two colors with anti-IKAROS Ab (red) and anti-IL-7R Ab (green). A serial section was stained with anti-keratin Ab.
In 11-dpc FL, 14% of cells are Lin–CD45+, and 4 and 45% of the Lin–CD45+ cells express IL-7R and FcR, respectively (Fig. 2A). We have previously shown that Lin–c-kit+IL-7R+ (IL-7R+) population in 11- to 12-dpc FL contains mostly T cell progenitors (23). We have also shown that Lin–c-kit+FcR+ (FcR+) cells in 13-dpc FL are myeloid lineage committed (39). Thus, it can be said that the commitment toward T and myeloid lineages proceeds in the 11-dpc FL. Only 0.5% of FB cells were Lin–CD45+, and among the Lin–CD45+ FB cells, 15% were IL-7R+. The proportion IL-7R+ cells in the CD45+ population of FB is three to four times larger than that of FL cells, confirming our previous finding that T cell progenitors are preferentially released into the circulation (24). In marked contrast, a large majority (80%) of CD45+ cells in the 11-dpc FT anlage region were found to express IL-7R, while only 15% were FcR+. IL-7R–FcR– cells were very few, if any, in this region. The FcR+ cells may be myeloid precursors that distribute ubiquitously, because the cells with a similar phenotype are observed in any other tissue (Fig. 2A, and data not shown).
We then examined the IL-7R expression of cells in the 11-dpc FT anlage region by immunofluorescence staining. One section of 11-dpc fetuses was stained in two-colors with anti-IKAROS and anti-IL-7R, and another serial section was stained with anti-keratin (Fig. 2B). Note that IKAROS (red) localizes to the nucleus, while IL-7R (green) localizes to the cytoplasm. It was confirmed that virtually all IKAROS+ cells in the mesenchyme adjacent to the keratin+ epithelial primordium are IL-7R+. Taken together, these data indicate that the IL-7R+ cells selectively migrate into the thymic anlage.
The initial thymic immigrants are T cell lineage-restricted progenitors
We investigated the frequency of cells having progenitor activity for T, B, or myeloid lineages in various tissues from 11-dpc fetuses through limiting dilution analysis, and the total numbers of these progenitors per fetus were determined (Table I). Myeloid cell-generating progenitors were most abundant in FL (1100–2100 per fetus), and T cell generating progenitors and B cell generating ones were comparable to each other (300 per fetus). FB also contained many progenitors for these lineages, while the relative proportion of T cell-generating progenitors vs B cell-generating ones was much higher than that in FL. The majority of B cell-generating progenitors detected in FL and FB may represent multipotent progenitors as has been shown previously (20, 40), because most of the B cell colonies on the stromal cell layer accompanied macrophage generation (data not shown). In contrast, such multipotent progenitors may not exist in the 11-dpc FT anlage region, because B cell-generating activity was barely detected in the 11-dpc FT anlage region, indicating that this region does not contain B cell-generating progenitors such as multipotent progenitors or B cell lineage-committed progenitors.
Table I. Numbers of progenitors per tissue of 11-dpc fetus
We next examined the commitment status of individual progenitors in FL and FT anlage region from 11-dpc fetuses by using the MLP assay. Various types of progenitors including multipotent progenitors generating T, B, and myeloid cells were detected in FcR–IL-7R– population of FL (Fig. 3A). The IL-7R+ population in 11-dpc FL contained mostly the progenitors generating only T cells (p-T) while FcR+ population exclusively contained progenitors generating only myeloid cells (p-M), confirming our previous findings (23, 39). In 11-dpc FT anlage region, it was found that IL-7R+ cells exclusively contained p-T. FcR+ population contained only p-M, although the frequency was significantly smaller than that in FL (Fig. 3B). These FcR+ p-M may correspond to the progenitors with myeloid potential detected in the thymic anlage region by the limiting dilution analysis (Table I). Very small numbers of p-T, but no other types of progenitors, were detected in the IL-7R–FcR– cells from the FT anlage region (data not shown). Together with the finding that hemopoietic cells adjacent to the epitherial primordium are mostly IL-7R+ (Fig. 2B), it was demonstrated that most of the hemopoietic progenitors migrating to the FT anlage are T cell lineage restricted.
FIGURE 3. IL-7R+ cells residing in the 11-dpc FT anlage region are T cell lineage restricted. Single cells of various populations from 11-dpc FL (A) and 11-dpc FT anlage region (B) were individually examined using the MLP assay. Cells grown in each well were recovered on day 10 and progenitor types were flow cytometrically determined. p-MTB, Progenitors generating myeloid, T, and B cells. The scales represent the numbers of progenitors detected among the 50 cells assayed. Data are representative of three independent experiments.
NK and DC potential of the earliest thymic immigrants
We have previously reported that the earliest thymic progenitors from 12-dpc FT as well as prethymic T cell progenitors in FB and FL retain the potential to generate NK cells and DC (24, 35, 41). Therefore, the tripotentiality for T, NK, and DC lineage cells can be regarded as a hallmark of early T cell progenitors. We then examined the initial thymic immigrants at a single cell level for the capability of generating T, NK, and DC lineages. A total of 40 IL-7R+ cells in 11-dpc FT region were individually cultured with a dGuo lobe in the presence of a cytokine mixture that support the generation of NK and DC. Representative FACS profile of cells of a clone containing T and NK cells (Fig. 4A) and a photograph of DC (Fig. 4B) generated from a single IL-7R+ cell are shown. The DC derived from thymic immigrants were CD11c+ and class II+ (data not shown). It was found that, of 40 cells examined, 7 cells generated T cells, and all 7 T cell progenitors also produced NK cells (Fig. 4C). Among seven T/NK bipotent progenitors, four progenitors showed DC-generating activity. The results may indicate that the earliest thymic T cell progenitors at 11 dpc retain NK and DC potentials.
FIGURE 4. Developmental potential of T cell progenitors to generate NK cells, DC, T cells, and T cells. A, Representative flow cytometric profiles of cells generated from a single T/NK progenitor. IL-7R+ cells of the 11-dpc FT anlage region from EGFP Tg mice were individually cultured with a dGuo-treated FT lobe in the presence of SCF, IL-7, IL-2, and Flt-3 ligand for 10 days. B, DC generated from a single IL-7R+ cell of the 11-dpc FT anlage region. Bar indicates 50 µm. C, A total of 40 IL-7R+ cells of the 11-dpc FT region were examined by a clonal analysis that covers T, NK, and DC lineages, and 7 showed T cell generation. The numbers of progenitors with NK and DC potential among seven T cell progenitors are shown. D, Representative flow cytometric profiles of cells generated in FT organ culture from a single cell of the 11-dpc FT anlage cell and a 14-dpc FT CD44+CD25– cell. E, Cells generated in FT organ culture from a single cell of 11-dpc FT anlage region were examined for their rearrangement status of V-J locus using PCR.
Generation of authentic thymic T cells from individual thymic immigrants
Although in Fig. 1 we have shown that the 11-dpc FT anlage region contains progenitors capable of forming a large number of thymocytes, this does not preclude the possibility that such competent progenitors represent a minority population among progenitors in the 11-dpc FT anlage region. Thus, it is important to clarify whether a majority of progenitors in FT anlage region is able to generate a broad spectrum of T cell populations, or only a limited repertoire of T cells. We addressed this issue by characterizing the T cells generated from individual progenitors of 11-dpc FT anlage region in comparison with thymic progenitors at later embryonic age (CD44+CD25– cells of 14-dpc FT). No significant difference was seen between plating efficiency (15 of 60 in the 11-dpc FT group and 17 of 60 in the 14-dpc FT group), and in both groups most of clones contained T and T cells. Flow cytometric profiles were indistinguishable regarding the proportion of double positive and CD4/8 SP populations and the ratio of T vs T cells between cells generated from a single IL-7R+ cell in the 11-dpc FT anlage region and from a single CD44+CD25– cell in 14-dpc FT (Fig. 4D). The usage of V genes in T cells was examined, and it was found that T cells generated from a single cell of 11-dpc FT anlage region cell as well as a 14-dpc FT CD44+CD25– cell exhibit rearrangement of various V genes other than the V3 gene (Fig. 4E), which is known to be preferentially rearranged at an early fetal stage (42). These results demonstrated that the majority of the earliest thymic T cell progenitors retain the potential to produce a broad range of T cell populations in both the T cell and T cell lineages.
Proliferation potential of the initial thymic immigrants before the TCR chain gene rearrangement
We have recently shown that 12-dpc FT progenitors as well as circulating prethymic T cell progenitors at 12 dpc retain a very strong capacity to proliferate before the initiation of TCR chain gene rearrangement (pre--rearrangement proliferation potential) (24, 36). Because it is expected that the more primitive progenitors have the higher pre--rearrangement proliferation potential, the assessment of this potential at a single cell level is effective in ranking each progenitor in the developmental hierarchy of T cell progenitors. We then assessed this potential by culturing individual cells in 11-dpc FT anlage region with a dGuo lobe. Cells generated from a single IL-7R+ cell of the 11-dpc FT anlage region, in comparison with those from a CD44+CD25– cell and CD44+CD25+ cell of 14-dpc FT, were analyzed by PCR for D-J rearrangement status. All clones from 11-dpc FT region exhibited almost all possible bands, indicating that these progenitors have a potential to produce highly diversified TCR chains (Fig. 5A).
FIGURE 5. Pre--rearrangement proliferation of T cell progenitors from FL, FB, and thymic anlage region of 11-dpc fetus. A, IL-7R+ FL cells and IL-7R+ cells of 11-dpc FT region, as well as CD44+CD25– and CD44+CD25+ cells of 14-dpc FT, were individually cultured with a dGuo lobe for 12 days. Genomic DNA was prepared from T cells generated in each well, and was PCR amplified using primers shown under the figure. B and C, IL-7R+ FL cells (11 FL) and IL-7R+ cells of 11-dpc FT region (11 FT), as well as CD44+CD25– and CD44+CD25+ cells of 14-dpc FT from Rag2–/– fetuses, were individually cultured with a dGuo-treated FT lobe for 10 days. B, Representative profile of cells generated from a single IL-7R+ cell in the Rag2–/– 11-dpc FT anlage region. C, Numbers of CD25+ cells in four to seven clones in each group.
We further assessed the pre--rearrangement proliferation potential by using cells from Rag2–/– mice. Rag2–/– cells from 11-dpc FT anlage region, in comparison with those from 11-dpc FL and 14-dpc FT populations, were individually cultured with a dGuo lobe. The flow cytometric profile of a representative clone generated from a IL-7R+ cell of the Rag2–/– 11-dpc FT anlage region is shown (Fig. 5B), in which the accumulation of CD44–/lowCD25+ cells, in addition to the expansion of CD44+CD25– cells, was observed. The number of generated CD25+ cells in a culture represents the pre--rearrangement proliferation potential of a seeded progenitor, because growth of Rag2–/– thymocytes is arrested at the point of TCR gene rearrangement. The numbers of CD25+ cells per clone are plotted (Fig. 5C). The difference in clonal expansion size may reflect the hierarchical relationship among progenitors. These results indicate that the initial thymic immigrants at 11 dpc locate at the highest position in the hierarchy of fetal thymic progenitors. This difference in pre--proliferation potential of progenitors between 11-dpc FT and 14-dpc FT may reflect a progenitor-progeny relationship among these progenitors, but may not be due to the difference in property of colonizing progenitors, because prethymic T cell progenitors are almost equivalent in pre--rearrangement proliferation potential during early FL ontogeny (11–15 dpc) (Ref.23 , and our unpublished observations).
Discussion
The present study demonstrated that the ontogenically earliest thymic progenitors that reside in the mesenchymal region surrounding the epithelial primordium at 11 dpc of murine fetuses were T/NK/DC lineage restricted. These progenitors are considered to be authentic T cell progenitors in that they are able to generate both T and T cells, and that they retain a very high proliferation potential to form a precursor pool that would accommodate diversified TCR chains.
It has been well documented that thymic colonization by hemopoietic cells starts at 11 dpc (23, 24, 25). Several trials have been made to assess the potential of early thymic immigrants for producing thymic T cells. Owen and Ritter (43) examined the 11-dpc FT anlage in an organ culture system performed in a chamber embedded in chorioallantois of a chicken egg, and showed that it contained precursors for lymphoid cells, although the cell growth from the 11-dpc precursors was very limited. Jotereau et al. (44) have examined the progenitor potential of early FT cells by transferring the 11-dpc FT anlage or FT from 12- to 15-dpc fetuses under the kidney capsule of recipient mice, and suggested that progenitors that have migrated into the thymus before 13 dpc may mainly contribute to the fetal thymopoiesis, although in their study the absolute number and the surface phenotype of thymocytes derived from progenitors in the 11-dpc FT anlage were not shown. In contrast, Douagi et al. (38) recently showed the that the T cell producing potential of progenitors in 12-dpc FT is limited compared with that in 14-dpc FT, and speculated that the progenitors immigrating earlier than 13 dpc do not significantly contribute to the fetal T cell generation. Thus, the progenitor activity of the ontogenically earliest thymic progenitors as for the contribution to the fetal thymopoiesis has remained controversial. The present study demonstrated that the earliest thymic immigrants at 11 dpc are quite competent to generate a large number of thymocytes in vivo (Fig. 1, C and D). Of note is that the average pre--rearrangement proliferation potential of the earliest thymic immigrants at 11 dpc was even higher than that of CD44+CD25– cells in 14-dpc FT. Therefore, we propose that the ontogenically earliest thymic immigrants are not just curtain raisers, but leading actors of the first generation of thymopoiesis.
Because multipotent progenitors generating T, B, and myeloid lineages have been shown to circulate during the period of initial thymic colonization (20), it tends to be speculated that these multipotent progenitors in FB migrate into the thymus. Our present findings, together with previous studies (21, 22, 23), argue against this idea, and instead propose that progenitors that have shut off their B cell and myeloid potentials selectively migrate to the thymic analage as the initial immigrants. However, because progenitors with B cell potential, albeit at a quite low level, were found in 11-dpc FT anlage region by limiting dilution analysis (Table I), two possibilities formally remain; first, a small number of multipotent progenitors also migrate to the thymus, and second, commitment to the T cell lineage by shutting off the B cell potential is more or less incomplete.
It has previously been pointed out that the thymic immigration of progenitors during the embryonic life occurs three times in birds and twice in mice (44, 45, 46). The Lin–IL-7R+ T cell progenitors present in the marginal area of the thymic epithelium may represent those participating in the first wave of thymic immigration. Because the AGM region in addition to FL of 11-dpc fetus contains IL-7R+ T cell progenitors (22, 24), it is probable that some of the IL-7R+ T cell progenitors found in the 11-dpc FT anlage region may be direct immigrants from the AGM region. Following points still remain unclarified; whether T cell progenitors derived from the AGM region are similar to those from FL, and which sites are the major sources of thymic immigrants during the embryonic stages. In contrast, T/NK cell lineage-restricted progenitors, namely B220lowc-kit+ cells in 13- to 15-dpc FL and Thy-1+c-kitlowNK1.1+ cells in 13- to 15-dpc FB, have been shown to exist (47, 48, 49). Using a GFP-reporter system, Yokota et al. (50) have shown that a RAG1-expressing cell population that is skewed toward the T cell lineage is present in 13- to 15-dpc FL. These progenitors could represent those of the second wave of thymic immigration, because they appear in FL and FB at ontogenically later stages and they express some Lin or RAG proteins.
Our present and previous studies, using clonal analysis, showed that the thymic progenitors are tripotential for T, NK, and DC lineages. It has been shown that NK cells are produced in the FT (51). However, the thymus may not be the major site for NK cell production, because athymic mice have a normal or even higher level of NK cells. The NK potential of thymic T cell progenitors could be the vestige of phylogenic events in which T cells have evolved from primordial NK cells. It is probable that the differentiation program for NK cells is used as a base of the T cell differentiation program. In contrast, the DC program accompanying the T cell progenitors may have evolved to exploit the DC function in the thymus, because DC may play an important role in thymic negative selection (52).
Recent studies on the role of Notch1 in T cell development have proposed a model, in which the CLP are presumed to migrate to the thymus where they decide their fate under the control of Notch signals (53). However, the scheme of the CLP immigration is not the case in fetal T cell development, because the cell fate decision to the T cell lineage takes place prethymically, as has been shown in the series of our studies (26). Recently, Harman et al. (30) showed that the immigrating cells in the mesenchyme surrounding the 12-dpc epithelial pripordium did not express Hes1 nor Deltex while intrathymic cells express these molecules, indicating that the Notch1 signal is activated only after progenitors encounter the thymic epithelial cells. Our present findings, together with the finding by Harman et al., suggest that the Notch-Hes1 pathway may not be essential for T cell lineage commitment, at least in fetal T cell development. Consistent with this is the finding in Hes1-deficient mice that the expansion of intrathymic progenitors was severely impaired but the commitment status of them was not altered (54, 55). However, the possibility cannot be ruled out that a Notch signal which is too low to be monitored by Hes1 expression, or other types of Notch signaling pathways than those mediated by Notch1 and/or Hes1 molecule(s), may play a role in the T cell lineage commitment at prethymic stage.
It tends to be speculated that the thymus-colonizing progenitors in fetuses differ from those in adults, because numbers of studies have suggested the difference between fetal and adult hematopoiesis (56). Indeed, the progenitor population sharing the surface phenotype with fetal prethymic progenitors, defined as Lin–c-kithighIL-7R+ in our series of studies (23, 24), has not been reported to exist in BM or blood. However, Allman et al. (14) recently showed that the earliest progenitors in AT are not CLP but more restricted to the T cell lineage. A recent study by Porritt et al. (57) also showed that progenitors generating T cells and B cells have already been segregated in the earliest thymic population. These studies may imply that the AT is colonized by T cell lineage-committed progenitors. In contrast, Martin et al. (8) have suggested that the B220+ CLP in BM may represent progenitors bound for thymus. Very recently, a study by Schwarz et al. (58) proposed that the circulating multipotent progenitors represent the adult thymic immigrants. Further studies on the prethymic progenitors as well as the earliest thymic progenitors in both fetal and adult mice will facilitate our understanding of the molecular mechanism of T cell lineage restriction.
Disclosures
The authors have no financial conflict of interest.
Acknowledgments
We thank Dr. Wilfred T. V. Germeraad for critical reading of the manuscript.
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 work was supported by Grants 12051219, 11470086, and 10044278 from the Ministry of Education, Science, Sports and Culture, and the Special Coordination Funds of the Science and Technology Agency, Japan.
2 Address correspondence and reprint requests to Dr. Hiroshi Kawamoto, Laboratory for Lymphocyte Development, RIKEN Research Center for Allergy and Immunology, 1-7-22, Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan. E-mail address: kawamoto{at}rcai.riken.jp
3 Abbreviations used in this paper: AT, adult thymus; AGM, aorta-gonado-mesonephros; BM, bone marrow; CLP, common lymphoid progenitor; DC, dendritic cell; FB, fetal blood; FL, fetal liver; FT, fetal thymus; Lin, lineage marker; MLP assay, multilineage progenitor assay; dpc, day postcoitus; rm, recombinant murine; SCF, stem cell factor; EGFP Tg, enhanced GFP transgenic; dGuo, deoxyguanosin; SP, single positive; p-T, progenitors generating only T cells; p-M, progenitors generating only myeloid cells.
Received for publication September 1, 2004. Accepted for publication November 11, 2004.
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