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*
Servicio Común de Investigación and
Departamento de Biología Celular, Faculty of Biology, Complutense University of Madrid, Madrid, Spain
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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chain and pre-T
chain transcripts, completely lacked
NKR-P1A expressing cells, and upon limiting dilution conditions,
generated T- and T/DC-containing lobes, but no T/NK or NK ones were
found. On the other hand, the CD45+CD2- triple
negative for CD8, CD4, and CD3 Ags cell population obtained from 15-
and 16-day-old fetal rat thymus can be divided into
NKR-P1A- and NKR-P1Alow cell subpopulations
that differ in several aspects. Both cell subsets expressed pre-TCR
chain transcripts, but only the former contained fully rearranged
TCR chain transcripts. Upon limiting dilution, T cell-committed
progenitors were only found in the NKR-P1A- cell
population, whereas NK-committed progenitors were present in the
NKR-P1Alow population. More importantly, bipotential T/NK
progenitors were very rare and were found only in the
NKR-P1Alow cell population, whereas bipotential T/DC
progenitors, only previously suggested in the adult mouse thymus, were
observed frequently in the NKR-P1A-CD2- cell
subpopulation. Our results demonstrate, therefore, that a common
intrathymic T/DC intermediate represents the main T cell developmental
pathway in rat thymus. |
Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Therefore, it has not been possible to definitively conclude, from these studies, how the intrathymic development of T, NK, and DC takes place. In this report, we study the T, DC, and NK potentialities of adult and fetal rat thymic progenitors, and we conclude that clonal bipotent T/DC progenitors represent the main, if not the only, developmental intermediate in the rat thymus.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Wistar Hanover rats and C.B-17 SCID mice were used in this study. They were maintained in the animal facilities of the Facultad de Biología (Universidad Complutense de Madrid, Madrid, Spain) or Centro de Biología Molecular (Universidad Autónoma de Madrid, Madrid, Spain), respectively.
Isolation of thymic cell populations
The adult
CD45+CD2-Lin-
cell population was isolated from cell suspensions of adult thymuses
prepared in PBS containing 0.1% FCS and 5 mM EDTA, and they were
centrifuged onto a 28% BSA cushion (30 min, 400 x g,
20°C). The cell fraction at the PBS/BSA interface contained nearly
40% CD4-CD8- cells and
was further depleted of T cells, DCs, B cells, and myeloid lineages by
negative depletion with autoMACS (Miltenyi Biotec, Bergisch
Glabach, Germany) by using mouse mAb against rat CD4 (OX-38), CD8
(OX-8), CD3 (G4.18), MHC class II (OX-6), 
L chain of Igs (OX-12),
and CD11b/c (OX-42) (all hybridoma supernatants were from the European
Collection of Animal Cell Cultures), followed by rabbit anti-mouse
MACS microbeads (Miltenyi Biotec). The negative cell fraction thus
obtained, hereafter referred to as the Lineage-
(Lin-) fraction, was further enriched in
CD45+CD2- cells by
three-color FACS sorting (FACStarPlus cell
sorter; BD Biosciences, Mountain View, CA) after staining with mouse
mAb against rat CD45 (rCD45) (clone OX-1, FITC-conjugated; BD
PharMingen, San Diego, CA), CD2 (clone OX-34, PE-conjugated; BD
PharMingen), and propidium iodide, the latter to exclude nonviable
cells. Cells for further RNA/DNA extraction were sorted in serum-free
medium.
Fetal thymus (FT) cell suspensions were first depleted of CD2+ and MHC class II+ cells by negative selection in an autoMACS magnetic cell separator (Miltenyi Biotec). The CD2-MHC class II- cells thus obtained were further FACS sorted to obtain highly purified CD45+NKR-P1A- and CD45+NKR-P1Alow cell subpopulations based on rCD45 (clone OX-1, FITC-conjugated; BD PharMingen) and NKR-P1A (clone 10/78, PE-conjugated; BD PharMingen) Ag staining combined with propidium iodide.
Xenogenic fetal thymic organ culture (FTOC)
Xenogenic rat/SCID mouse FTOCs (rSCID-FTOC) were established as previously described by us (9). Briefly, 30 µl of a rat cell suspension containing variable numbers of cells was put in each well of a Terasaki plate containing one SCID mouse fetal thymic lobe (14.5- to 15-day-old fetal thymuses). After 2 days of culture in hanging drops, the lobes were transferred to FTOC conditions by placing them on the surface of 0.8-µm pore size nitrocellulose filters (Millipore, Ibérica, Spain), one per filter, which were layered over gelfoam rafts (Pharmacia-Upjohn, Madrid, Spain) in 35-mm sterile petri dishes. After 12 days of culture in these conditions, the lobes were individually dispersed into single cell-suspensions for flow cytometry (see below). Viable cells were counted by trypan blue exclusion. Cultures were maintained in a humidified incubator at 37°C and 7.5% CO2, in culture medium consisting of RPMI 1640 supplemented with 1 mM sodium pyruvate, 2 mM L-glutamine, 50 µM 2-ME, and antibiotics (all from Life Technologies, Rockville, MD) plus 10% FCS.
Flow cytometric analysis
For flow cytometric immunophenotyping, cell suspensions were
incubated with saturating concentrations of mouse mAb specific for rat
Ags. Abs used for immunofluorescence staining were either prepared in
the laboratory (purified anti-MHC class II (clone OX-6),
anti-CD53 (clone OX-44), anti-MHC class I (OX-18),
anti-CD71 (clone OX-26), biotin-labeled anti-CD45 (clone OX-1),
and anti-CD2 (clone OX-55); all from European Collection of
Animal Cell Cultures) or purchased from BD PharMingen (FITC-labeled
anti-NKR-P1A (clone 10/78), anti-CD44 (clone OX-49),
anti-CD4 (clone OX-35), anti-CD45R (His24), and anti-rat
granulocyte Ag (His48); PE-labeled anti-TCR (clone R73),
anti-TCR
(clone V65), anti-CD25 (clone OX-39),
anti-CD2 (clone OX-34), and anti-Thy1 (clone OX-7);
biotin-conjugated anti-CD54 (clone 1A29); and PerCP anti-CD8
(clone OX-8)). Either streptavidin-CyChrome (SA-CY, FL-3H; BD
PharMingen) or SA-Cy5 (FL-4H; Jackson ImmunoResearch Laboratories, West
Grove, PA) was used as a second-step reagent when a biotinylated mAb
was present. Indirect stainings were performed first and were followed
by incubation with rabbit anti-mouse Igs conjugated to FITC, PE, or
Cy5 (Jackson ImmunoResearch Laboratories). In all cases, nonspecific
binding was blocked by the addition of saturating concentrations of
purified mouse Ig (Sigma-Aldrich, Madrid, Spain) before
incubation with biotin- or fluorochrome-conjugated mAb and following
indirect stainings. Data acquisition, 20–50 x
103 events, was performed in a FACSCalibur flow
cytometer (BD Biosciences) of the Servicio Común de
Investigación (Faculty of Biology, Complutense University of
Madrid). Data were analyzed with CellQuest software (BD
Biosciences).
RNA isolation and RT-PCR analysis
RNA was extracted from cell suspensions by using TRI-Reagent solution (Molecular Research Center, Cincinnati, OH) following the manufacturer’s instructions. RT (1–1.5 µg total RNA) and PCR were performed with a RT-PCR core kit from PerkinElmer (Roche Diagnostic Systems, Mannheim, Germany).
Detection of pre-TCR chain (pT) mRNA species was conducted by
using a combination of primers that recognize highly homologous
sequences between mouse and human pT cDNA of the Ig-like
(5'-CGGCACCCCCTGGCCTTGAC-3') and transmembrane
(5'-GCTGCAGGTCAGGAGCACATC-3') domains. cDNA samples were denatured
(94°C, 1 min), annealed (60°C, 1 min), and extended (72°C, 1 min)
for 35 cycles, followed by Southern blot hybridization with a rat
pT-specific DNA probe cloned by us (9).
For RT-PCR amplification of TCR V8-C mRNA species, a combination
of 5'-V8 primer (5'-GAGGCTGCAGTCAGGGAAAGC-3') with 3'-C
(5'-AGGTTTGGGTGAGTCCTCTGAC) primers was used for 35 cycles (1 min at
94°C, 1 min at 55°C, and 1 min at 72°C). Specific PCR
amplifications were detected by Southern blot hybridization with a
digoxigenin (Dig)-labeled C probe more internal than the 3'-C
primer used in the PCR.
Dig-dUTPs were obtained from Roche Diagnostic Systems. The hybridization was visualized using an alkaline-phosphatase-conjugated anti-Dig mAb (Roche Diagnostic Systems).
Primers for RT-PCR amplification of rat actin were derived from GenBank accession no v01217: forward, 5'-GATGGTGGGTATGGGTCAG-3'; reverse, 5'-GCTCATTGCCGATAGTGATG-3'.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A number of studies in mice and humans have revealed lineage
relationships between T, NK, and B cells and a subtype of DC that
presumably emerge from a common lymphoid progenitor, although later
lymphoid cell lineage developmental intermediates are still a matter of
controversy (1, 10, 11). Thus, the possible existence of a
common bipotent T/DC intrathymic progenitor has only been referred at a
population level in the adult mouse thymus (6), whereas an
immature intermediate with T/NK bipotentiality has been claimed to
occur in the mouse FT (4, 5). Interestingly, we previously
found clonal bipotent T/DC progenitors in the early rat fetal liver
(9), prompting us to investigate this subject in the rat
thymus. After whole elimination of mature intrathymic T, B, DC, and
myeloid cells (see Materials and Methods), adult thymus cell
suspensions were enriched in the
CD45+CD2- cell fraction by
FACS sorting (further referred to as CD2-
cells). These cells were 100% positive for CD44, Thy-1, MHC class I,
CD53, and the transferrin receptor, CD71 (Fig. 1
), but in sharp contrast to mouse thymic
TN cells, rat immature thymocytes did not express CD25, as previously
found by Law et al. (12), precluding a further
fractionation of the CD2- TN rat subset based on
CD44 and CD25 staining. Other tested Ags previously reported in rat
immature progenitors like the OX22 determinant of CD45 and the CD5 Ag
did not allow for a further phenotypic fractionation of the
CD2- cell population (not shown). In contrast,
because no NK cell-specific marker was included in our lineage
depletion protocol, we explored for the possible expression of NKR-P1A
by some of these cells, as reported for mouse NK1.1 in fetal thymic
progenitors (13). However, no
NKR-P1A+ cells were found in the
CD2--enriched cell fraction obtained (Fig. 1).
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transcripts (Fig. 2) and, possibly, pre-TCR-forming cells
because of the expression of pT transcripts that was also observed
(Fig. 2).
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A). Then, 12-day singly
cultured rSCID-FTOC lobes, which were reconstituted with a limiting
cell number of the CD2- cell population per
lobe, were individually analyzed by flow cytometry for the expression
of rCD45, TCR plus TCR, NKR-P1A, and MHC class II rat Ags
in four colors (Fig. 3B). In these conditions, no
rCD45+Lin- lobes were
observed, but all the rCD45+ ones contained at
least one of the lymphoid cell lineages studied. T cell-containing
lobes were present, confirming the above-described results and
suggesting the existence of T cell-committed cells already in the
CD2- adult rat thymic cell fraction. In
addition, they represented a majority of rCD45+
lobes (Fig. 3C). Along with them, some
rCD45+ lobes containing both
TCR+NKR-P1A-MHC class
II- and
TCR-NKR-P1A-MHC class
IIhigh cell populations, possibly T/DC-containing
lobes, were nicely observed, but no
rCD45+NKR-P1A+-containing
lobes developed. We further confirmed that MHC class
IIhigh-expressing cells in the lobes were true DC
by visual inspection of cytospin preparations prepared from every
cultured lobe and stained for MHC class II Ag (data not shown).
Finally, NK cell development occurred in rSCID-FTOC when a higher input
number of progenitor cells was used (by using 100 or more of these
cells), i.e., nonlimiting cell numbers (not shown), demonstrating NK
cell potentiality by these cells, but at a very low frequency to be
detected at limiting dilution. Thus, in accordance with previous
reports in mice (11) and humans (15),
immature adult rat thymic progenitors exhibit T, DC, and NK
potentialities. In addition, we demonstrate for the first time the
occurrence of clonal bipotent T/DC intrathymic progenitors in an in
vitro system that allows T, NK, and DC development.
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As mentioned above, bipotential T/NK intrathymic progenitors have been claimed as an intermediate stage in both mouse (16) and human (15) fetal thymic lymphoid development. In mice, they could occur in either CD16/CD32+ (5, 17) or NK1.1+ (13) CD44+CD25- TN fetal thymic cell populations, although their existence in adult thymus remains to be demonstrated. More importantly, a parallel examination of the possible DC potentiality of presumptive bipotent T/NK fetal thymic progenitors is still lacking in mice, whereas in humans, a high frequency of DC progenitors was reported in a fetal intrathymic T/NK bipotent population (2), although there was no clonal demonstration of those results. All these data suggest that bipotent T/NK progenitors could represent an intermediate stage in fetal thymic development that we did not detect in the adult rat thymus. Accordingly, we examined the developmental capabilities of rat fetal thymic cell populations from very early developmental days to later stages.
The first day to undoubtedly dissect the rat FT from the surrounding
connective tissue was day 15 of gestation (15 gd), which corresponds to
1–1.5 days after the first thymus seeding by
CD45+ progenitors in rats (18). At
this time point, the CD45+ cell content per
thymic lobe was 
2–5 x 103 cells,
15–20% of which have begun to express the CD2 Ag (Fig. 4). One day later (FT 16 gd), a sharp
increase in the thymus cell content took place, with a
CD45+ cell recovery of 60–80 x
103 cells per thymic lobe, which was accompanied
by an increase of both the proportion of CD2+
cells, which reached nearly 70% of the CD45+
cell fraction, and the levels of Ag expression (Fig. 4). By day 17 (FT
17 gd), the CD2+ cell population accounted for
>95% of thymic cells, as in the adult thymus (not shown). All these
results indicated that the 15 gd and 16 gd fetal thymuses of rats could
contain the most primitive thymic cells, which we further characterized
by flow cytometry.
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, both FT 15 gd and FT 16 gd cells contained a small proportion
of CD4-expressing cells that lacked CD2 or CD8 expression. In contrast,
CD8-expressing cells were undetectable until 16 gd, and they were found
to be the CD2+CD4-. From
16 gd to 17 gd, there was a sharp increase in both the proportion and
absolute numbers of
CD8+CD4- cells, and the
first CD4+CD8+ cells were
detected (Fig. 4). Upon staining for TCR, the first surface
reactive cells were detected at 17 gd (not shown).
To determine the presence of thymic DC during these early fetal stages
of rat thymus development, we first looked for MHC class II expression
on the CD45+ cells as a marker for rat thymic
DCs. Low levels of class II expression were already observed at FT 15
gd and were stronger at FT 16 gd (Fig. 5). In both cases, the
CD45+MHC class II+ cells
were CD2- cells (Fig. 5). The CD8 and CD4
expression on these cells was also examined, and although no CD8
expression was correlated with that for MHC class II, the early CD4
staining detected at fetal days 15 and 16 was nicely associated with
the CD45+class II+ cell
population, especially in the MHC class IIhigh
population of the 16-day-old FT (Fig. 5). In the adult rat, we
(19) and others (20) have reported the
occurrence of a subpopulation of CD4-expressing DC in the thymus. A
further phenotypic analysis of the class
II+CD2-CD45+
from 16-day-old FT demonstrated that they expressed higher ICAM-1,
CD53, and CD44 and lower Thy1 levels of Ag expression than those found
in the CD45+ class II-
thymic cells (Fig. 5). This phenotypic analysis suggested that these
cells corresponded to thymic DC. In addition, rat fetal thymic MHC
class IIhigh cells did not show reactivity for
the mouse mAbs His24, which recognizes the CD45R isoform in developing
rat B cells, and His48, specific for rat granulocytes, and weakly to
negative for the myeloid determinant recognized by mAb OX42,
recognizing a common epitope shared by CD11b and CD11c (not shown). In
contrast, isolated MHC class IIhigh cells from FT
16 gd ended up, after some days of culture in suspension, to a major
population of cells with long cytoplasmic processes, with an
irregularly shaped nucleus and strong MHC class II expression,
characteristic features of thymic DC (not shown).
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). In addition,
NKR-P1Ahigh cells appeared for the first time in
the FT 16 gd, associated with low levels of CD2 and Thy-1 Ags,
suggesting their presumptive relationship to rat NK cells. In contrast,
as NKR-P1A expression has been also reported in adult rat DCs
(21), we confronted NKR-P1A and MHC class II Ag expression
with that of CD45 and CD2. As it can be seen in Fig. 6, NKR-P1A and MHC
class II Ag expression defined two mutually excluding cell populations.
Accordingly, three
CD45+CD2- cell
subpopulations can be defined in the early rat FT:
NKR-P1A- class II+,
NKR-P1Alow class II-, and
NKR-P1A- class II-.
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and pT transcripts. As shown in Fig. 7, fully rearranged TCR V-C
transcripts were only detected in the NKR-P1A-
cell population, but to the contrary, pT transcripts were detected
in both the NKR-P1Alow and the
NKR-P1A-CD2- cells.
Accordingly, NKR-P1A- cells could contain
T-committed cells, whereas the NKR-P1Alow
could contain the above-mentioned reported T/NK bipotent
progenitors.
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+ and TCR+
thymocytes (not shown), as the only cell lineage growing in the lobes
(Fig. 8). Under the same limiting cell
number conditions, we also found a lower but reproducible number of
lobes containing both T and DC cell lineages, whereas the lowest
frequency of rCD45+ corresponded to those that
contained all of the three cell lineages studied (Fig. 8). When
limiting numbers of the NKR-P1Alow cell
subpopulation were used in the reconstitution of SCID-FTOC, we
exclusively found NK-containing lobes, whereas no T- or DC-containing
lobes grew (Fig. 8). However, under nonlimiting conditions, 100 or more
cells of the NKR-P1Alow cell population gave rise
to 100% reconstituted lobes, all of which contained NK cells. Some of
these also developed T cells, but DC-containing lobes remained rare
(not shown), indicating that T/NK bipotent progenitors could exist in
this fetal thymic population, but at a very low frequency to be
detected in limiting dilution conditions.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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We had already observed bipotent T/DC progenitors in the early rat fetal liver (9) where, in contrast, T/NK cells were rarely detected. It is still not known whether these results have implications for the commitment status of cell progenitors colonizing the thymus. In this regard, we have demonstrated DC- and, more importantly, T cell-committed progenitors in the early rat fetal liver, which could represent prethymic progenitors for these lymphoid cell lineages or an extrathymic pathway of their development (9).
In contrast, the existence of a common T/DC progenitor has remained largely unexplored because previous published articles have mainly focused on the common T/NK progenitor (2, 4, 5, 13). It is important, however, to remark that these studies were conducted with fetal but not adult thymic cells and did not examine the situation of DC. In contrast, most of those studies were based on cytokine-supplemented cultures, which could give rise to unpredictable results. Thus, Ikawa et al. (5) improved the proportion of NK cells to the detriment of T cell production in in vitro assays supplemented with IL-2. In these experiments the authors, however, did not show the effect of IL-2 addition on the reported frequency of clonal progenitors, although, as previously shown by others (3), it is presumable that thymic progenitors are selectively influenced toward NK differentiation upon IL-2 addition. More importantly, Márquez et al. (22) have shown that the addition of IL-2 to suspension cultures of early intrathymic human progenitors results in the appearance of NK cells with a reduction of DC cell recovery. Recently, the unpredictable influence of cytokines in cell lineage decisions of uncommitted progenitors has also been pointed out by Kondo et al. (23), who redirected common lymphoid progenitor toward myeloid differentiation. Finally, in agreement with our results, adult intrathymic CD44+CD25+ TN cells rarely form NK cells (5, 8), but still retain a robust capacity to develop T cells and DC upon both in vivo i.v. transfer (6) or in in vitro assays (7).
As stated above, the existence of a lymphoid DC lineage is widely supported by a large body of data in human and mouse (1, 11, 15, 24), and although there are no available specific markers for rat lymphoid DC, our current results make this situation now applicable to this species. With respect to this, experiments are in progress for further phenotypic characterization of rat DC derived from T/DC single colonized lobes.
As in mice (25), the intrathymic development of rat NK
cells takes place very early in rat ontogeny, preceding the appearance
of mature T cells. In agreement, the first
NKR-P1AhighCD2+ cells
detected in the rat thymus during ontogeny were found at 16 gd when
TCR-expressing cells are still lacking. Interestingly, mature NK
cells at 16 gd are preceded by a population of
NKR-P1AlowCD2- cells at 15
gd, which is mainly committed to the NK cell lineage. This situation is
similar to that reported in mouse fetal ontogeny for the NK1.1 molecule
and NK-committed clonal progenitors (25). Despite these
results, the significance of intrathymic NK cell development still
remains intriguing, as nude animals contain extrathymically derived NK
cells (26). This intrathymic NK cell development could
reflect a special situation during ontogeny, as in the adult thymus,
this developmental pathway should be extremely rare. Recently, Id-2 and
Id-3 transcriptional inhibitors of bHLH factors have been shown to
block the development of both human T and DC from a common progenitor,
while promoting the NK cell fate (27). Thus, it could be
that unknown microenvironmental signals, which promote NK cell fate
through the regulation of Id proteins, block the T and DC development
from a common T/NK/DC progenitor to a similar extent, further
precluding a common T/NK intermediate as an obligatory T cell
developmental pathway over the T/DC demonstrated in this study. In
contrast, a parallel development of thymic T and DC, as demonstrated in
mice (11), could be significantly relevant for eliminating
potentially auto-reactive T cell clones during T cell development,
while the significance of an intrathymic NK cell development still
remains to be understood.
Expression of NKR-P1 by a subset of mature T cells has been correlated with a special T cell subset (28) and, in some cases, with an activated state of normal T cells (29). Remarkably, the rarely developed T cells in rSCID-FTOC colonized by the NKR-P1AlowCD2- 16 FT population did not show a preferential expression of the NKR-P1 Ag. However, some of these NK-T cells developed in rSCID-FTOC from fetal liver progenitors (14) and from adult and fetal thymic progenitors (results not shown), further precluding these fetal thymic NKR-P1Alow progenitors as an alternative route for the development of NK-T cells.
In summary, the present results conclusively demonstrate the occurrence of a common T/DC rather than a T/NK bipotential intermediate as the main, if not the only, pathway of rat T cell development in both adult and FT. Its existence in human and mouse thymus remains to be further demonstrated.
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Acknowledgments |
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Footnotes |
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2 Address correspondence and reprint requests to Dr. Agustín G. Zapata, Departamento de Biología Celular, Facultad de Biología, Universidad Complutense de Madrid, 28040 Madrid, Spain. E-mail address: sci{at}eucmax.sim.ucm.es
3 Abbreviations used in this paper: DC, dendritic cell; FTOC, fetal thymic organ culture; rSCID-FTOC, xenogenic rat/SCID mouse FTOCs; pT, pre-TCR chain; TN, triple negative for CD8, CD4 and CD3 Ags; rCD45, rat CD45; FT, fetal thymus; gd, gestational day; Lin-, Lineage-; Dig, digoxigenin.
Received for publication May 9, 2001. Accepted for publication July 18, 2001.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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RII/III contains precursors of T lymphocytes and natural killer cells. Cell 69:139.[Medline]
T cell differentiation in mouse fetal thymic ontogeny. J. Immunol. 160:744.This article has been cited by other articles:
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  H. Q. Shen, M. Lu, T. Ikawa, K. Masuda, K. Ohmura, N. Minato, Y. Katsura, and H. Kawamoto T/NK Bipotent Progenitors in the Thymus Retain the Potential to Generate Dendritic Cells J. Immunol., October 1, 2003; 171(7): 3401 - 3406. [Abstract] [Full Text] [PDF]
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E. Donskoy and I. Goldschneider Two Developmentally Distinct Populations of Dendritic Cells Inhabit the Adult Mouse Thymus: Demonstration by Differential Importation of Hematogenous Precursors Under Steady State Conditions J. Immunol., April 1, 2003; 170(7): 3514 - 3521. [Abstract] [Full Text] [PDF]
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