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Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Kyoto, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By culturing single progenitors on a monolayer of a stromal cell line that supports the development of both myeloid and B cells, it was shown that commitment to B and myeloid lineages occurred in fetal liver (FL)3 at 12 days postcoitum (dpc) 9 . However, analysis of the lineage commitment including T cell lineage had not been possible until the establishment of the multilineage progenitor (MLP) assay system 10 , in which individual progenitors are cultured with a deoxyguanosine (dGuo)-treated fetal thymus (FT) lobe in the presence of a cytokine mixture that supports the growth of B and myeloid cells. The MLP assay system is able to determine the capability of individual stem/progenitor cells to generate T, B, and myeloid cells. By using this assay, we succeeded in identifying not only multipotent progenitors (p-Multi), but also progenitors restricted to T cell lineage (p-T), B cell lineage (p-B), and myeloid lineage (p-M) in the lineage marker-negative (Lin-) c-kit+CD45+Sca-1+ (Sca-1+) population from 12-dpc FL. Bipotent progenitors generating myeloid and T cells (p-MT) and those generating myeloid and B cells (p-MB) were also detected in the Sca-1+ population.
The identification of p-T in the Sca-1+ FL cells supported the idea of prethymic commitment of the stem cells to the T cell lineage and suggested that p-T are progenitors of thymic T cells. However, the possibility has not been ruled out that the p-T represent the progenitors of extrathymically developing T cells but not those of thymic T cells. More information is necessary to determine whether the FL p-T are the progenitors of thymic T cells. In the present study, detailed characterization of progenitors in subpopulations of FL and fetal blood (FB) cells, as well as in FL of athymic nude mouse, was performed with the MLP assay system. Furthermore, T cell generating activity of p-T and p-Multi was compared in the FT organ culture system. The results of these experiments provide convincing evidence that it is p-T that migrate into the thymus to produce T cells.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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C57BL/6 (B6) mice and pregnant BALB/c-nu/+ mice time-mated with male BALB/c-nu/nu mice were purchased from Japan SLC (Shizuoka, Japan). B6Ly5.1 mice were maintained in our animal facility. B6 fetuses (15 dpc) were used in organ culture experiments as the source of FT lobes.
Abs
The following Abs were used: anti-Ly5.1 (A20-1.7; donated by
Dr. Y. Saga, Banyu Seiyaku, Tokyo, Japan), anti-Ly5.2 (AL1-4A2;
donated by Dr. I. L. Weissman, Stanford University, San Francisco,
CA), anti-Fc
RII/III (FcR) (2.4G2), anti-c-kit
(ACK-2; donated by Dr. S.-I. Nishikawa, Kyoto University),
anti-erythroid lineage cells (TER119; established by Dr. T. Kina in
our laboratory), and anti-B220 (RA-6B2; obtained from American Type
Culture Collection, Manassas, VA). Anti-Ly5.1, anti-FcR, and TER119
were labeled with FITC as described 11 . Anti-c-kit,
anti-B220, and anti-Ly5.2 were labeled with Cyanine 5 (Cy5)
(Cy5 labeling kit; Biological Detection Systems, Pittsburgh, PA), whose
fluorescence characteristics is similar to that of allophycocyanin
(APC). FITC-anti-Gr-1 and phycoerythrin (PE)-anti-Gr-1
(RA3-8C5; Caltag, San Francisco, CA), FITC-anti-B220 and
PE-anti-B220 (RA-6B2; Caltag), PE-anti-Mac-1 (M1/70;
Caltag), FITC-anti-CD8 (YTS169.4; Caltag), APC-anti-Thy-1.2
and FITC-anti-Thy1.2 (5a-8; Caltag), PE-anti-CD4 (GK1.5;
Caltag), FITC-anti-CD25 (PC61; PharMingen, San Diego, CA),
FITC-anti-CD45 and PE-anti-CD45 (30F11.1; PharMingen),
PE-anti-Sca-1 (E13-161.7; PharMingen),
FITC-anti-TCR
(GL-3; Caltag), and PE-anti-TCR
ß
(H57-597; Caltag) were also used.
Growth factors
Recombinant murine (rm) IL-7 was kindly donated by Dr. Sudo (Basic Research Laboratory, Toray, Kanagawa, Japan). Commercially available rm stem cell factor (Genzyme, Cambridge, MA) and rmIL-3 (Genzyme) were also used.
High oxygen submersion (HOS) organ culture and MLP assay culture
The basic procedures for HOS culture of FT have been described previously 12 . In brief, to prepare hemopoietic cell-depleted FT lobes, thymuses obtained from 15-dpc fetuses of B6 mice were cultured on polycarbonate filters (pore size, 8 µm) (Nuclepore, Pleasanton, CA) floating on culture medium containing dGuo (1.35 mM) for 6 days in a humidified atmosphere of 5% CO2 and 95% air. The lobes were washed, and single dGuo-treated lobes were placed into wells of a 96-well V-bottom plate, to which progenitors were added. Wells along the margin of the plate were not used, but filled with water to help maintain a high humidity in the plastic bag. The plates were centrifuged at 150 x g for 5 min at room temperature, placed into a plastic bag (Ohmi Oder Air Service, Hikone, Japan), and the air inside was replaced by a gas mixture (70% O2, 25% N2, and 5% CO2). The plastic bag was incubated at 37°C. The cultures were maintained in RPMI 1640 medium (Life Technologies, Grand Island, NY) supplemented with 10% FCS (BioWhittaker, Walkersville, MD), L-glutamine (2 mM), sodium pyruvate (1 mM), sodium bicarbonate (2 mg/ml), nonessential amino acid solution (0.1 mM) (Life Technologies), 2-ME (5 x 10-5 M), streptomycin (100 µg/ml), and penicillin (100 U/ml). Medium change was performed every 5 days. After cultivation, cells were harvested, viable cells were counted by trypan blue dye exclusion, and then subjected to flow cytometric analysis.
The MLP assay system has been described in detail previously 10 . Almost all procedures are the same as in HOS culture 12 . The only differences are that always a single cell is cultured together with a dGuo-treated lobe, and that the culture medium is supplemented with rm stem cell factor (10 ng/ml), rmIL-3 (3 ng/ml), and rmIL-7 (200 U/ml) to support the growth of not only T, but also B and myeloid cells.
Flow cytometric analysis of cultured cells
Cells from both inside and outside the FT lobe were harvested from each well. One-third of each sample was stained with FITC-anti-Ly5.1 and Cy5-anti-Ly5.2 to screen for the presence of progenitor type (Ly5.1+) cells. The samples containing Ly5.1+ cells were selected for further analysis. The remaining two-thirds of cells from the selected samples were divided into two groups. One group was stained with FITC-Ly5.1, PE-anti-B220, and APC-anti-Thy1.2, and the other was stained with FITC-anti-Ly5.1, PE-anti-Mac-1, PE-anti-Gr-1, and Cy5-anti-B220. Surface phenotype was analyzed by a FACS Vantage. More detailed procedures have been described previously 10 .
RT-PCR
mRNA from 3000 cells was reverse transcribed in a 20-µl volume, and 1 µl of the cDNA generated was used in PCR reactions with the following sense and antisense primers: Aml-1, 5'-CAAGCTGAGGAGCGGCG-3', 5'-CCGACAAACCTGAGGTCGTTG-3'; Tcf-1, 5'-CCAGCTTTCTCCACTCTACG-3', 5'-TCAAGGATGGGTGGGTGAAC-3'; Mb-1, 5'-ATCATCTTGCTGTTCTGTGC-3', 5'-ACACTAACGAGGATGCTGTA-3'; c-fms, 5'-CTGGAGAAGAAATATGTGCG-3', 5'-TTCAGACCAAGCGAGAAGAT-3'; ß-actin, 5'-TCCTGTGGCATCCATGAAACT-3', 5'-GAAGCACTTGCGGTGCACGAT-3'. PCR was conducted as follows: incubation for 5 min at 94°C followed by 30 cycles of 1 min at 94°C, 1 min at 55°C (for Aml-1, at 60°C), and 2 min at 72°C. The amplified products were electrophoresed and visualized with ethidium bromide.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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FL and FB cells were obtained from 12-dpc fetuses of B6Ly5.1 mice,
and were stained with various mAbs to analyze their cell surface
phenotypes (Fig. 1
A). In the
left panels of Fig. 1A, c-kit vs Lin profiles are
shown. Mac-1 was omitted from Lin, because all progenitor cells express
Mac-1 (Ref. 13 and our unpublished data). At this gestational age,
virtually all c-kit+ cells in FL are
Lin-, and more than half of c-kit+
cells are CD45+.
c-kit+CD45+ FL cells express Sca-1
at a broad range of levels. Sca-1-, Sca-1 low-expressing
(Sca-1low), and Sca-1 high-expressing
(Sca-1high) cell populations boxed in Fig. 1A
were used for progenitor assay. The Sca-1high population
corresponds to the
Lin-Sca-1+Mac-1+CD4-
population used in the study of Morrison et al. 13 . FB contains
virtually no Sca-1high cells, and thus the
Sca-1- and Sca-1low populations of
c-kit+CD45+ FB cells were used.
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B). Expression of Aml-1 14
is seen in all subpopulations of FL and FB cells. FL
Sca-1high cells do not at all express Tcf-1 or
Mb-1, which are specific to T or B lineage cells,
respectively 15, 16, 17 . The expression of Mb-1 seems to begin
at the Sca-1low stage of FL cells. A prominent expression
of c-fms, which is specific to myeloid lineage 18 , is
observed at Sca-1low to Sca-1- stages in FL,
although a faint band for this gene can be seen in
Sca-1high cells. Sca-1low and
Sca-1- FB cells also express c-fms at low
levels, whereas Mb-1 is virtually undetectable in these FB
cell populations. Lower levels of expression of Mb-1 and
c-fms in FB cells than in FL cells may indicate that
differentiation toward B and myeloid cells is not in progress in FB.
Expression of Tcf-1 is seen exclusively in FT cells. These
results are compatible with the previous finding in the bone marrow
that cells expressing higher levels of Sca-1 represent earlier
progenitors 19 . Clonal analysis of the progenitors in different subpopulations of FL cells
Subpopulations of FL cells expressing Sca-1 at different levels
(Fig. 1A) were isolated with a cell sorter. Single
Sca-1high, Sca-1low, and Sca-1-
cells were picked up with a micropipet, put into each well of a
V-bottom plate in which a dGuo-treated FT lobe had been placed, and
cultured in the presence of a cytokine mixture. After 10 days of
culture under HOS conditions, cells were harvested from each well,
counted, and assayed for expression of T, B, and myeloid cell markers.
In analyzing the surface phenotypes of cells generated in these
cultures, a large gating area was set in the forward-side scatter to
include all viable T, B, and myeloid cells (Fig. 2A). Fig. 2B shows
representative surface profiles of six different types of clones
observed, which are derived from p-T, p-B, p-M, p-Multi, and bipotent
progenitors p-MT or p-MB. These six types are exactly the same as those
found in our previous work 10 , and we again failed to detect any
bipotent progenitors generating T and B cells (p-TB). It should be
noted that the lineage restriction is very strict. For example, B or
myeloid cells were never generated from a p-T (top
lane of Fig. 2B), even when the recovered cells were
recultured on stromal cell monolayers (data not shown).
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summarizes the number of different
types of progenitors found in different populations of FL cells.
p-Multi are seen exclusively in the Sca-1high population,
which represents only 0.28% of 12-dpc FL cells (see Fig. 1A). Quite a large number of p-MT (17 among 115 cells
assayed) and a very small number of p-MB are also seen in the
Sca-1high population. Unipotent progenitors are mainly
found in Sca-1low and Sca-1- populations,
although p-M are abundant in the Sca-1high population. The
proportion of cells retaining progenitor activity decreases with the
reduction of Sca-1 expression levels, and only a small number of
bipotent progenitors are present in the Sca-1low
population. These results suggest that the process of restriction to T,
B, and myeloid lineages is initiated at the Sca-1high stage
and ceases at the Sca-1low stage.
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To examine whether the p-T in FL are derived from FT or whether
its generation is influenced by FT, we compared the progenitors in FL
cells from nu/nu fetuses with those from nu/+ fetuses. To clearly
discriminate nu/nu fetuses from nu/+ fetuses in littermates, 14-dpc
fetuses instead of 12-dpc fetuses were used. Because in BALB/c
background mice the expression level of Sca-1 on FL cells was too low
to be used for enrichment of progenitors 20 , a
c-kit+CD45+FcR-
population of FL cells, which had been shown to represent the most
primitive population (Ref. 21 and our unpublished data), was used.
Individual cells were cultured in the MLP assay system, and, after 10
days of culture, cells were harvested for flow cytometric analysis. The
number of different types of progenitors are shown in Table I. No difference was observed in the
frequencies of p-Multi and lineage-restricted progenitors, including
p-T, between nu/nu and nu/+ fetuses. These results indicate that the
thymus does not play any role in lineage restriction of hematopoietic
stem cells in FL.
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It is highly probable that the T cell progenitors migrate into FT
through the blood stream. We investigated the developmental capability
of individual Sca-1low and Sca-1- FB cells
(see Fig. 1A) by the MLP assay system. The results are
summarized in Fig. 4. Distribution of
different progenitors in these subpopulations of FB cells seems similar
to that in the corresponding subpopulations of FL cells (Fig. 3) in
that p-Multi do not exist and a large majority of progenitors are
lineage restricted. The only important difference is that the relative
frequency of p-T in various types of progenitors is much higher in the
FB Sca-1low population (7/16) than in the FL
Sca-1low population (3/20).
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We have previously shown that FL contains progenitors capable of generating T cells much more rapidly than those in adult bone marrow 22 . Thus, very rapid development of T cells in FT 23, 24 was attributed to the characteristics of progenitors emigrated from FL. Such a progenitor should be found among p-T, p-MT, or p-Multi. We first compared the kinetics of T cell generation from these three types of progenitors by culturing under MLP assay conditions. The results indicated that T cell generation from p-T was quicker than that from p-MT or p-Multi (data not shown).
However, MLP assay cultures may not provide optimal conditions for T
cell growth, because a cytokine mixture supporting the growth of non-T
cells is added. We employed a conventional HOS culture system 12 for
comparing the kinetics of T cell generation from different progenitors,
because we empirically know that single Sca-1high
progenitors are able to generate B and myeloid cells in addition to T
cells in the HOS culture without exogenous cytokines. Individual cells
of Sca-1low (a total of 160 cells) and
Sca-1high (a total of 80 cells) populations were cultured,
and cells generated in each well were harvested on days 6, 9, 12, and
15. As controls, groups of 5 x 102
c-kit+ FL cells and 103
c-kit+ bone marrow cells, which contain 10–15 T
cell progenitors 22 , were set up in parallel. The progenitor type,
p-T or p-Multi, was determined by the cell type generated in each well.
p-T can be easily identified, because only T cells are generated from
this type of progenitor (Fig. 2B), and p-Multi are clearly
distinguishable from p-T because T, B, and myeloid cells are generated
(see Fig. 6, lower lane). It should also be notable that the
p-T and p-Multi identified in this experiment are exclusively derived
from Sca-1low and Sca-1high populations,
respectively. It was also possible to identify p-MT-type progenitors in
HOS culture, because they generated a small number of myeloid cells in
addition to a large number of T cells (data not shown).
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A). The time course of T cell
generation from a single p-T is similar to that from 5 x
102 c-kit+ FL cells and, moreover,
similar to that of ontogenic T cell development in FT. On the other
hand, T cell generation from a single p-Multi is comparable to that
from 103 c-kit+ bone marrow cells.
p-MT type progenitors were omitted from Fig. 5A to avoid the
complexity, but the time course of T cell generation from this type of
progenitors was intermediate between those of p-T and p-Multi. These
results may indicate that the major T cell progenitors in FL emigrating
to FT are p-T. The finding that the time course of T cell generation
from c-kit+ progenitors in adult bone marrow
resembles that of p-Multi but not of p-T in FL, could indicate that no
T cell progenitors corresponding to FL p-T exist in adult bone marrow.
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B. It is indicated that p-T produces mature T cells by day
9 of culture, whereas a period of around 2 wk is required for a p-Multi
to generate mature T cells. The thymus is unable to suppress the generation of B and myeloid cells from a p-Multi
Not only p-T, but also p-B and p-M, are found in the fetal
circulation (Fig. 4). Possibility could exist that such a mixture of
progenitors produces large numbers of B and myeloid cells in the thymus
as a p-Multi does. Thus, the generation of T, B, and myeloid cells in
HOS cultures from 100 Sca-1low FB cells, which is expected
to contain all three types of unipotent progenitors but not p-Multi
(see Fig. 4A), was compared with that from a single p-Multi.
Fig. 6 (upper lane) shows an
example of the flow cytometric profile of cells generated on day 9 in a
well seeded with 100 Sca-1low FB cells. The results
indicate that a large majority of cells are T cells, although small
numbers of B and myeloid cells are also generated in this group. In
marked contrast, large numbers of B and myeloid cells in addition to T
cells are generated from a single p-Multi (lower lane
of Fig. 6). These results conform to the idea that circulating p-T are
the progenitors of thymic T cells.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Because the MLP assay is a newly devised in vitro system aiming at detecting progenitors capable of differentiating toward T, B, and myeloid lineages in a single in vitro culture, it is important to compare the detection efficiency of different progenitors determined with previously used systems. Numbers of p-B and p-M in 12-dpc FL detected by the MLP assay are comparable to previously reported values determined by a clonal assay performed on the stromal cell monolayers 9 . On the other hand, p-T have not been detected before the establishment of the MLP assay system. However, the frequency of progenitors capable of generating T cells, which probably represents the sum of p-T, p-MT, and p-Multi, has been determined by limiting dilution analysis in the intrathymus (i.t.) injection system 25 . In this report, the number of T cell progenitors per 14-dpc FL has been shown to be 1000. Because the number of p-T does not increase but slightly decreases during 12–14 dpc (H. Kawamoto et al., manuscript in preparation), and because the retention of 51Cr-labeled cells 21 h after i.t. transfer was about 1/3 26 , the number of T cell progenitors may be >3000 per 12- to 14-dpc FL. The present study showed that the total number of p-T, p-MT, and p-Multi per a 12-dpc FL is about 6000. Thus, it can be said that the MLP assay culture is comparable to the in vivo thymus in detecting T cell progenitor activity.
A more important problem could be whether the MLP assay system is
capable of detecting p-Multi as efficient as other in vitro systems.
The number of p-Multi per 12-dpc FL estimated by our MLP assay is 1100
(Fig. 3A). This value is very similar to that of bipotent
B/macrophage progenitors (1000 per 12-dpc FL) estimated by clonal assay
on the stromal cell monolayer 9 . Because only about half of such
bipotent progenitors have been estimated to retain T cell progenitor
activity 27 , the number of p-Multi type progenitors detected by
stromal cell assay may be about 500 per FL. Thus, the MLP assay is more
efficient than stromal cell assay in detecting p-Multi-type
progenitors.
In vitro findings, especially those dealing with cells retaining
differentiational potential, are always faced with the criticism
whether they exactly represents in vivo phenomena. It is difficult to
prove that the distribution of different types of progenitors detected
with the MLP assay exactly reflect the lineage commitment status in
vivo. However, as considered in the above paragraph, the detection
reliability of the MLP assay does not seem to be behind other
experimental systems. Moreover, as shown in Fig. 3, p-Multi are
exclusively found in the Sca-1high population, whereas p-T
are preferentially found in the Sca-1low population.
Further purification by surface phenotypes of different progenitors is
in progress, and recently we have succeeded in virtually completely
purifying p-T with surface markers (H. Kawamoto et al., manuscript in
preparation). Such separation and purification may highly contribute to
the reliability of experimental results obtained with the MLP assay
system.
The present study showed that the frequency of p-T in nu/nu mice is the
same as that in nu/+ mice (Table I). This finding not only indicated
that the p-T are not derived from FT, but also that the emergence of
p-T is not influenced by FT. The present study further provided
evidence supporting that p-T are the progenitors of thymic T cells.
First, p-T produces only T cells, whereas p-Multi produces a large
number of B and myeloid cells in the thymic lobe as well, even without
exogenous cytokines (Fig. 6). Secondly, the time course of T cell
generation from a p-T, but not from a p-Multi, conforms to that of
ontogenic T cell generation in FT (Fig. 5). Thirdly, p-T are abundant
but p-Multi are scarcely detectable in FB (Fig. 4). These results, in
combination with our recent findings that FT does not contain any
p-Multi or early progenitors 28 , may indicate that it is p-T but not
the hematopoietic stem cells that migrate into the thymus to produce T
cells.
p-Multi were exclusively found in the Sca-1high population
in which lineage-specific genes are barely expressed (Fig. 1B). Detection of a large number of p-MT in this population
(17 among 115 cells assayed) may indicate that restriction to p-M and
p-T begins at this very early stage. However, the number of p-M was
much larger than that of p-T in this population. This could be due to
the preferential generation of p-M from a p-MT or p-Multi, or that p-M
retains self-renewal capacity. We failed to detect any p-B in 115
Sca-1high cells investigated in this experiment, and the
number of p-B was the lowest among three types of lineage-restricted
progenitors in 12-dpc fetuses (Figs. 3 and 4). However, the frequency
of p-B depends on the strain of mouse and also on the gestational age
(H. Kawamoto et al., manuscript in preparation). For example, it is
indicated in Table I that the frequency of p-B in BALB/c fetuses (14
dpc) is higher than that of p-T.
The relative frequency of p-T in FB was much higher than that in FL
(Figs. 3 and 4), suggesting that they are actively emigrating from FL.
The abundance of p-T in circulation is consistent with the previous
findings that the FT at this gestational age (11–12 dpc) is able to
accept extrathymic progenitors 29 . It should also be pointed out that
the p-T in FL and FB we determined are
c-kit+Thy-1-, the phenotype being
distinct from c-kitlowThy-1+ T cell
progenitors previously reported to be present in FB of 15.5-dpc fetuses
30 .
Previous investigation on the developmental potential of progenitors
performed by culturing single cells on a stromal cell monolayer has
revealed the presence of bipotent progenitors generating myeloid and B
cells in the FB of 10- to 12-dpc fetuses, and such progenitors were
shown to be multipotent 27 . On the other hand, we were unable to
detect any p-Multi among a total of 160 c-kit+
FB cells from 12-dpc fetuses (Fig. 4), indicating that the frequency of
p-Multi in 12-dpc FB, should they exist, is very low (<200 per whole
blood). However, our failure of detecting p-Multi in FB may not be
inconsistent with the findings of Delassus and Cumano 27 , because the
number of circulating "multipotent" progenitors estimated by this
group is very small (30 per a 12-dpc fetus), and, moreover, the number
drastically declines between 12 and 13 dpc.
Although the presence of common lymphoid progenitors has generally been presumed, no p-TB-type progenitors were detected in either FL or FB. This is confirmed in a large number of experiments in which we have investigated more than 2000 c-kit+ cells from FL, FB, and FT (data not shown). The failure to detect any p-TB-type progenitors is in contrast with the fact that other types of bipotent progenitors, p-MT and p-MB, are constantly detected. The presence of common lymphoid progenitors has been suggested by analyzing the developmental potential of a purified population from adult thymus 31, 32 or bone marrow 33 . However, these findings are not based on clonal assay systems necessary for elucidating the potential of individual progenitors to give rise to different lineage cells. Another finding popularly thought to be compatible with the idea of a common lymphoid progenitor could be provided by Ikaros gene knockout mice 34 , because in these mice the development of T, B, and NK cells is severely impaired. However, because granulocyte generation is also abolished in these mice, the results may indicate that Ikaros plays a crucial role in differentiation of both myeloid and lymphoid lineages. An alternative interpretation could be that the common progenitor exists that is capable of generating T cells, B cells, NK cells, and granulocytes.
The failure of detecting p-TB-type progenitors may lead to the idea that p-T and p-B are either directly generated from p-Multi or through the p-MT and p-MB stages, respectively, rather than through a common lymphoid progenitor. This is consistent with the previous findings of Lemischka and colleagues 6 that, in the experiment of retrovirus-mediated gene introduction into hematopoietic progenitors in bone marrow, common gene integration sites were found between T and myeloid cells and between B and myeloid cells, but not between T and B cells. In this context, it is worth noting that many cases of human leukemia have been reported that are thought to be derived from a p-MT 35, 36 or a p-MB 37, 38 . A close relation between B and myeloid lineages have also been suggested by the presence of cells displaying characteristics of B cells and macrophages 39 . It should be pointed out that T and B cells use a variety of very similar molecules in Ag recognition as well as in signaling and gene activation, which are encoded by genes that have evolved from common ancestral genes. Expression of these genes may be initiated at the progenitor stage. It seems vital for p-T to avoid any differentiational errors that could be caused by expressing a similar set of genes related to B cell development. Bypassing a common lymphoid progenitor stage could be an important strategy for preventing such an occurrence. Recently, it was reported that progenitors that can generate both T and B cells are present in bone marrow of adult mice 40 . The possibility may exist that the process of lineage commitment in adult bone marrow is ruled differently from that in FL. However, because their experimental system is not effective in detecting other types of progenitors, it is still unclear whether the stage of the common lymphoid progenitor they detected is on the major pathway of generating T and B cells. A more exact understanding of lineage commitment in the adult bone marrow will be reached after the application of the MLP assay system.
The establishment of the MLP assay system, which is effective in discriminating the developmental potential of individual stem/progenitor cells, enabled us to identify different types of progenitors in subpopulations of FL and FB cells. The present study mainly focused on T cell development in the fetus and provided evidence supporting that p-T produced in FL are the progenitors migrating into the thymus to produce T cells. In this article, the p-T is defined as a unipotent progenitor capable of generating T cells but not B or myeloid cells. However, very recently we found that a large proportion of p-T in 12-dpc FT retains the capability to generate NK cells in an environment where IL-2 was added to the dGuo-treated lobe (our unpublished results). Therefore, it is highly probable that p-T in FL are able to generate NK cells. This is consistent with a recent finding by Carlyle and Zúñiga-Pflücker 41 that Thy-1+c-kitlowNK1.1+ population in FB is capable of generating T and NK cells, thus suggesting the presence of T/NK progenitors. However, it is still unclear whether the NK progenitor is exclusively related with p-T or whether all FL p-T are able to generate NK cells. It has also been shown that thymic T cell progenitors retain a potential to generate dendritic cells 42 . More detailed investigation is required to clarify the relationship between p-T and progenitors of NK and dendritic cells.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Yoshimoto Katsura, Department of Immunology, Institute for Frontier Medical Sciences, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan. E-mail address:
3 Abbreviations used in this paper: FL, fetal liver; dGuo, deoxyguanosine; dpc, days postcoitum; FB, fetal blood; FT, fetal thymus; HOS, high oxygen submersion; Lin, lineage markers; MLP, multilineage progenitor; p-B, progenitors capable of generating B cells but not T or myeloid cells; p-M, progenitors capable of generating B cells but not T or myeloid cells; p-MB, bipotent progenitors capable of generating myeloid and B cells but not T cells; p-MT, bipotent progenitors capable of generating myeloid and B cells but not T cells; p-Multi, multipotent progenitors; p-T, progenitors capable of generating T cells but not B or myeloid cells; FcR, FcRII/III; Cy5, Cyanine 5; APC, allophycocyanin; PE, phycoerythrin; rm, recombinant murine.
Received for publication August 13, 1998. Accepted for publication November 30, 1998.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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