By
From the * Netherlands Cancer Institute, 1066 CX Amsterdam, Netherlands; and Laboratory of
AIDS Immunology, Institute for Virus Research, Kyoto University, Kyoto, Japan
We have investigated whether in the human thymus transition of CD4+CD8+ double positive
(DP) to CD4+ or CD8+ single positive (SP) cells is sufficient for generation of functional immunocompetent T cells. Using the capacity of thymocytes to expand in vitro in response to
PHA and IL-2 as a criterion for functional maturity, we found that functional maturity of both
SP and DP thymocytes correlates with downregulation of CD1a. CD1a cells with a persistent
DP phenotype were also found in neonatal cord blood, suggesting that at least a proportion of
mature DP cells can emigrate from the thymus. The requirements for generating functional
T cells were investigated in a hybrid human/mouse fetal thymic organ culture. MHC class II-
positive, but not MHC class II-negative, mouse thymic microenvironments support differentiation of human progenitors into TCR
+CD4+ SP cells, indicating that mouse MHC class II
can positively select TCR
+CD4+ SP human cells. Strikingly, these SP are arrested in the
CD1a+ stage and could not be expanded in vitro with PHA and IL-2. CD1a+CD4+ SP thymocytes do not represent an end stage population because purified CD1a+CD4+ SP thymocytes
differentiate to expandable CD1a
cells upon cocultivation with human thymic stromal cells.
Taken together these data indicate that when CD1a+ DP TCR
low cells mature, these cells
interact with MHC, but that an additional, apparently species-specific, signal is required for
downregulation of CD1a to generate functional mature TCR
+ cells.
Tcell progenitors that develop in the thymus to mature
T cells are submitted to a series of selective events (reviewed in reference 1), the first of which takes place when
immature CD4 Here we report on the identification of downregulation
of CD1a as a hallmark for functional maturation, not only
of SP human thymocytes, but also of DP cells. To arrive at
this model, we made use of the observations that DP cells
contain in vitro clonogenic cells both in human (13, 14)
and mouse (15). These observations were intriguing because if one accepts that maturity of T cells is appropriately
reflected by their capacity to expand in vitro, some DP
cells should have been submitted to a maturation signal. The presence of both mature clonogenic DP cells and immature CD4+ SP cells (12) is difficult to reconcile with a
linear model of thymocyte differentiation from immature
CD3+CD4+CD8+ DP via immature to mature SP cells. A
possible explanation for the existence of both in vitro clonogenic mature DP thymocytes and presumably immature
SP cells could be that there are bifurcations in the pathway
of later stages of T cell development. The data presented
here are consistent with this notion, since it was found that
acquisition of functional maturity correlates perfectly with downregulation of CD1a and, most importantly, not with
downregulation of CD4 or CD8. Moreover, we show here
that MHC class II-positive, but not MHC class II-negative, mouse thymic microenvironments can support differentiation of human progenitors into CD3+CD4+ SP cells.
However, human TCR Preparation and Phenotypic Analysis of Thymocyte Subpopulations.
Thymocyte tissues were obtained from children 3 mo-10
yr of age undergoing median sternotomy and corrective cardiovascular surgery. Suspensions were made by mincing tissue and
pressing through a stainless steel mesh. Large aggregates were removed, and the cells were washed once before separating subpopulations.
CD8
CD3
cells differentiate into CD4+
CD8+ double positive (DP)1 cells. A second selection occurs when DP thymocytes differentiate into CD4+ or
CD8+ mature T cells, and is generally referred to as positive selection. It is well established that positive selection
involves sustained interactions of the TCR
heterodimer
with complexes of peptides and MHC antigens on thymic
stromal cells (reviewed in references 2). During this selection process, either CD4 or CD8 is downregulated. There
is current debate over whether downregulation of CD4 or CD8, and thus commitment to CD4+ or CD8+ T cells, is
dictated by the MHC specificity of the TCR (instructive model) (5, 6) or whether it occurs in a stochastic fashion independent of TCR/MHC interactions (selective model)
(7). In the majority of the studies addressing the issue of
positive selection, all CD3high thymocytes with a CD4 or
CD8 single positive (SP) phenotype were considered to
have completed the process of positive selection and to be
functionally mature. However, recent studies in the mouse
indicate that not all SP thymocytes that have been submitted to positive selection signals are functionally mature. It is
known that SP cells are phenotypically heterogeneous with
respect to CD24 (heat stable antigen) and CD69 (10, 11).
In addition, CD4+ SP thymocytes with intermediate levels
of CD24 express very low levels of CD8 when analyzed
with a sensitive panning method (11). More recently, it has
been demonstrated that although the CD4+CD8low cells
had hallmarks of positive selection such as CD69 and high levels of TCR, they were not able to induce a lethal Graft
versus host disease upon transfer into irradiated allogeneic
recipients and to survive in the periphery (12). The immature CD3highCD4+CD8low cells require the thymic environment to reach the end stage of positive selection (12).
These data suggest that when functional immunocompetence of T cells is taken as the end stage of positive selection, this process is not necessarily completed when CD4 or CD8 are downregulated.
+ CD4+ SP cells selected on
mouse MHC class II continue to express CD1a and exhibit
poor clonogenic potential in vitro, suggesting that a species-specific signal is required for downregulation of CD1a and induction of functional maturity in the CD4 TCR
lineage.
cells were sorted with a FACStar plus®. Purity of
the cell populations was always >98%.
Limiting Dilution Assays.
CD1a+ and CD1a DP, CD4+ and
CD8+ SP thymocytes were plated under limiting dilution conditions in 96-well round-bottomed wells. The thymocytes were
cultured in the presence of 5.104 irradiated (3.103 rad) allogeneic
PBMC and 5.103 irradiated (5.103 rad) EBV transformed B cells
(JY) per well in 100 µl of culture medium supplemented with 0.1 µg/ml of PHA (Wellcome, Beckenham, Kent, UK) and 30 U/ml
of recombinant IL-2 (Chiron Europe, Amsterdam, Netherlands).
Culture medium consisted of Yssel's medium (17) supplemented
with 2% human serum. After 5 d of culture, 100 µl fresh culture
medium with 30 U/ml rIL-2 was added. Wells were screened
microscopically for cell growth after 2-4 wk of culture.
Hybrid Human/Mouse Fetal Thymic Organ Cultures. The in vitro development of human T and NK cells from CD34+ thymocytes was studied using the hybrid human/mouse fetal thymic organ culture (FTOC) in which human progenitor cells were cocultured with murine fetal thymuses (18). These thymuses were obtained from embryos of recombination activating gene (RAG)-1-deficient mice on days 15-16 of gestation. To investigate the role of murine MHC class II antigens in development of human cells, FTOC were set up with thymuses of MHC class II-deficient mice (19), provided by Dr. L. Glimcher (Harvard School of Public Health, Boston, MA).
The fetal thymuses were first precultured for 5 d in the presence of 1.35 mM 2-deoxyguanosine to remove endogenous thymocytes. Next, the thymic lobes were cocultured for 2 d in hanging drops in Terasaki wells with FACS® sorted human progenitor cells, transferred to nucleopore filters which were layered over gelfoam rafts in 6-well plates, and cultured for the indicated number of days. Culture medium consisted of Yssel's medium supplemented with 2% normal human serum and 5% fetal calf serum. To analyze differentiation of human cells, the mouse thymuses were dispersed into single cell suspensions and stained with mAbs specific for human cell surface antigens.Isolation of Stromal Cells. Heterogeneous cell cultures of thymic stroma were obtained for mechanic disruption of the thymic parenchyma and enzymatic treatment with collagenase and lipase and enrichment for large adherent cells (20, 21). Adherent cells were cultured in RPMI-1640 (GIBCO BRL, Gaithersburg, MD) supplemented with 10% FCS. The cultures were washed each day during the first days of culture to remove any remaining thymocytes. Stromal cells were used after two or three passages.
RNA Isolation and cDNA Preparation. Total RNA was isolated from sorted cells using the guanidine thiocyanate method (22). Glycogen (20 µg; Boehringer Mannheim, Indianapolis, IN) was added to each sample to facilitate precipitation of the RNA. The cDNA was prepared with oligo(dT)15 (PharMingen, San Diego, CA) and reversed transcribed with 200 U M-MLV reverse transcriptase (GIBCO BRL). Dilutions of the cDNA in water (5 ngeq RNA/µl) were used in PCR amplification reactions.
Semi-quantitative PCR.
A semi-quantitative PCR method (23,
24) was used to compare the expression of RAG-1 in thymocyte
subpopulations. Synthetic oligonucleotides (Pharmacia LKB Biotechnology Inc., Piscataway, NJ) used as primers are as follows:
5-TATGGACAGGACTGAACGTCTTGC-3
(hypoxanthine
phosphoribosyl transferase [HPRT] sense), 5
-GACACAAACATGATTCAAATCCCTGA-3
(HPRT antisense), 5
-GAACACACTTTGCCTTCTCTTTGG-3
(RAG1 sense), 5
-CGCTTTGCCTCTTGCTTTCTCGTT-3
(RAG1 antisense). Standard curves for HPRT and RAG-1 were set up using serial dilutions of
cDNA prepared from RNA isolated from total thymus. Dilutions
of cDNA samples in water were made starting with a concentration of 0.5 ngeq RNA/µl to determine HPRT expression and 5 ngeq RNA/µl to determine RAG-1 expression. PCR was carried
out in a total volume of 50 µl consisting of 1 µM of each primer
set, 200 µM each dNTP (Pharmacia LKB Biotechnology Inc.), 2.5 mM MgCl2, 1× PCR buffer, 1 U Taq DNA polymerase
(GIBCO BRL), and 10 µl of the cDNA. Samples were covered
with 50 µl paraffin oil and heated to 94°C for 5 min and then amplified for 30 cycles of 1 min at 94°C, 1 min at 65°C, and 2 min at
72°C. After the last cycle, a final extension step at 72°C for 10 min was done. 10 µl of each PCR reaction was dot blotted on a
nylon filter (Hybond N+; Amersham Intl. Buckinghamshire,
UK). Filters were prehybridized at 55°C for at least 2 h (6× SSC,
0.5% SDS, 5× Denhardt's, and 100 mg herring sperm DNA per
liter), and hybridized overnight with an oligoprobe recognizing
specifically the HPRT or RAG-1 PCR product internal to the PCR
primers. Oligoprobes were 32P labeled according to the manufacturer's recommendations (Boehringer Mannheim). Sequences of the
probe are as follows: 5
-GTCCCCTGTTGACTGGTCATTACAAT-3
(HPRT probe), 5
-TCCTTTGAAAAGACACCTGAAGAAGC-3
(RAG1 probe). To remove any nonspecifically
bound probe, the filters were washed with excess amount of 2×
SSC; 0.1% SDS at 50°C. Quantitation of the PCR products was
done with a phosphoimager (Fujix Bas 2000; Fuji Photo Film
Co., Ltd., Tokyo, Japan) and analyzed with the supplied software.
Finally, the ratio of RAG-1/HPRT was calculated to compare
the expression of the RAG-1 mRNA in the different samples.
CD1a is a marker
that is expressed on the great majority of DP thymocytes
and part of the SP cells (25). Since this marker is not
present on mature peripheral T cells, it is generally assumed that thymic emigrants are CD1a, and that therefore,
CD1a+ thymocytes are immature. Since a proportion of the
SP cells is CD1a+, a linear model of differentiation predicts
that virtually all DP cells would be CD1a+. A nonlinear
differentiation model, however, would predict existence of
CD1a
cells among both DP and SP thymic populations.
To examine this issue, we performed three parameter flow
cytometric analysis of CD1a, CD4, and CD8, which confirmed earlier data that the vast majority of DP cells, and
around 40% of the SP cells, express CD1a (Fig. 1). A very
small percentage of the DP cells, however, is clearly negative for CD1a. Interestingly, the levels of CD4 and CD8
on the CD1a
DP cells are lower than on total DP thymocytes (Fig. 1), suggesting that downregulation of either
one of these coreceptors had already been initiated before
completion of CD1a downregulation. To address whether
the CD1a
cells are functionally mature, we performed a
limiting dilution of CD1a
and CD1a+ cells. Table 1
shows that the cloning efficiencies of CD1a
DP, CD4+,
and CD8+ SP thymocytes in one representative experiment were 24, 41, and 34%, respectively. In sharp contrast,
virtually none of the CD1a+ DP or CD1a+ SP cells could
be cloned. The lack of clonogenic potential of CD1a+ subsets was not due to an inhibiting effect of the anti-CD1a antibody, since cloning efficiencies of unseparated SP cells
plated in presence or absence of anti-CD1a mAb were virtually identical (results not shown). The in vitro expandable
DP thymocytes could be cloned, and the majority of the
DP clones maintained their DP phenotype for a prolonged
period of time (results not shown) which is consistent with
data published previously (13, 14). These data conclusively
demonstrate that the clonogenic potential of SP and DP
thymocytes resides exclusively in the CD1a
subset, and
that functional maturation, as defined by the ability to
clonally expand, can already be manifested at the DP stage of thymocyte maturation.
|
The
results of the limiting dilution experiments indicate that
CD1a+CD3high SP thymocytes are functionally immature.
The observation that the great majority of the CD3high cells
in the thymus express the activation marker CD69, which is induced after positive selection (26) suggests, however, that positive selection signals have been delivered to a
significant proportion of the CD1a+ SP cells. To further
substantiate whether the CD1a+ SP cells have been submitted to selection signals, we investigated not only expression
of CD69, but also Bcl-2 which is associated with positive
selection as well (29, 30). In addition, expression of CD27
was analyzed. CD27 is expressed on most CD3high human
thymocytes, and may also be associated with positive selection (31). Three parameter analysis of CD1a+ SP cells confirms that the majority of these cells express CD69, Bcl-2,
and CD27 (Table 2). These data are consistent with expression profiles of CD69 (25), Bcl-2 (32), and CD27 (31)
on total human CD3high thymocytes published previously.
Besides upregulation of CD69, positive selection also results in downregulation of RAG-1 (28, 33). A semi-quantitative reverse transcriptase-PCR of the CD1a+ and CD1a
SP populations revealed that CD8+CD1a+ cells still express
levels of RAG-1 which are comparable to that of total thymocytes (Fig. 2 A). The levels of RAG mRNA in CD1a+
CD4+ SP cells, however, are much lower than that of total
cells (Fig. 2 B). Taken together, these data can be interpreted to indicate that CD1a+ SP cells express some, but
not all, features of cells that have received a TCR-mediated
positive selection signal.
Development of Human CD4+ SP Cells in a Mouse Fetal Thymus Requires Mouse MHC Class II Antigens, but the Mouse Thymus Is Inefficient at Inducing Maturation of Human CD4+ SP T Cells.
Recently it was demonstrated that
human progenitor cells can develop in mouse thymic organs in a FTOC (18, 34). Human progenitor cells developed into SP cells, but human stromal cells were not detectable in such cultures (36). Human CD4 can replace mouse CD4 in development of mouse MHC class II-restricted
T cells (38). To address the question of whether interaction
of human CD4 with mouse MHC class II can select human
CD4+ T cells, FTOC were performed with thymi from
MHC class II-positive and MHC class II-deficient mice.
The mouse thymi were reconstituted with CD34+CD1a
postnatal thymocytes that include the most primitive thymic T cell precursors (39, 40). After incubation in a MHC
class II-positive murine thymic microenvironment, 6.5% of
the harvested cells were TCR
+ CD4+ SP (Fig. 3 A). By
contrast, the number of TCR
+CD4+ SP T cells recovered from thymi of MHC class II-deficient mice was reduced considerably to 0.46% in experiment 1 (Fig. 3 B)
and 0.05% in experiment 2 (Fig. 3 C), compared to that recovered from thymi of MHC class II-positive mice (6-10%
in four independent experiments). A significant portion of
the TCR
+ cells that developed in a class II MHC-positive thymic environment expressed CD69 (Fig. 3 D), indicating that some cells were activated, presumably as a consequence of selection via the TCR. These findings indicate
that mouse MHC class II antigens can positively select human CD4+ cells. It is noteworthy that very few human
CD8+TCR
+ SP cells could be recovered from the FTOC
with human CD34+CD1a
cells and the mouse thymi.
There were more CD3+CD8+ SP cells present and >90%
of those cells express TCR
(results not shown). One
possible reason for this is that mouse MHC class I does not
efficiently select human CD8+ T cells, despite the fact that
human CD8 is able to functionally interact with the
3 domain of mouse H2Kb (41). Another explanation is that in
addition to class I MHC, other signals are required for selection of CD8+ T cells.
Having established that MHC class II antigens can support development of human CD4+ SP T cells, we next investigated whether the mouse thymic environment can induce downregulation of CD1a and functional maturation.
Early thymic CD34+CD1a progenitors were isolated and
cultured in FTOC for 4 wk. Analysis of the cells harvested
from the FTOC revealed the presence of TCR
and
TCR
high cells. Almost all TCR
high cells expressed
CD1a, while most TCR
high cells lacked CD1a (Fig. 4).
Immature TCR
dim cells mostly expressed CD1a (Fig. 4).
Stimulation of the cells harvested from the FTOC with a
feeder cell mixture, PHA, and IL-2 resulted in expansion
mostly of TCR
+ cells and few TCR
+ cells (Fig. 5).
Most of those TCR
+ cells that were expanded expressed CD4 (Fig. 5). These data demonstrate that although
the mouse MHC class II-positive mouse thymic environment can support development of CD4+ SP thymocytes, it
is very inefficient in induction of functional maturation of
these cells. By contrast, the mouse thymic microenvironment efficiently induces maturation of TCR
+ cells. Thus,
failure of the mouse MHC class II-positive environment to
induce functional maturation of TCR
+ cells is not due
to a intrinsic incapability to support maturation of human
T cells.
Differentiation of CD1a+ to CD1a
The
presence of CD1a+ and CD1a SP thymocytes raises the
question whether CD1a+ SP cells are the direct precursors
of CD1a
SP cells. An alternative possibility would be that the
CD1a
SP cells are derived from the CD1a
DP cells and
that CD1a+ SP cells represent a dead-end lineage. To investigate this, we cocultured purified CD1a+CD4+ SP cells
with short term cultured human thymic stromal cells. This
coculture resulted in a gradual downregulation of CD1a
which was completed on day 12 (Fig. 6 A). Phenotypic
analysis of these cells reveals that they express high levels of
TCR
and CD4. Unexpectedly, many of these cells also
express CD8
(Fig. 6 B). These differentiated CD1a
cells
could be expanded in vitro and the phenotype did not alter upon expansion (Fig. 6 B). These data indicate that CD1a+
CD4+ SP cells can differentiate to expandable CD1a
CD4+
SP cells and also to expandable CD1a
CD4+CD8
+ cells.
Presence of DP Cells in Neonatal Cord Blood.
As indicated
in Fig. 1, the thymus contains expandable CD1a DP cells.
Although not shown here, we were able to clone DP cells and these clones maintained a persistent DP phenotype upon
long-term culture in accord with data published (13, 14).
The presence of DP cells, expressing several characteristics
of maturity, raises the question whether these cells are able
to migrate from the thymus. DP cells could be observed in
T cell samples from neonatal cord blood (Fig. 7) in percentages ranging from 0.5 to 3% of the total number of
CD3+ T cells (n = 4). All DP cord blood cells express
CD3 and CD27, and they lack CD1a or the activation antigen CD69 (Fig. 7). Further analysis of these cells indicate
that the majority of these cells express CD45RA, and are
negative for CD45RO and Fas (Fig. 7) suggesting that
these DP cells represent naive, not memory, cells. Moreover, like in the thymus (42), both CD8+
CD4+ and
CD4+CD8
+
+ cells could be observed. These observations are compatible with the notion that DP cells can migrate out of the thymus.
In this paper we have investigated acquisition of functional maturity by human thymocytes. The hypothesis that forms the basis of this study is that maturity of T cells is appropriately and faithfully reflected by their capacity to expand in vitro. We think that this is the case because in vitro expandability is a general property of mature peripheral T cells. Moreover, T cell clones derived from mature thymocytes can mediate cytotoxic activities and produce cytokines upon stimulation in vitro (data not shown). Accepting our premise, the data argue that some DP are mature, while a considerable proportion of the SP cells in the human thymus are functionally immature. Most importantly, acquisition of functional maturity correlates with downregulation of CD1a. The cognizance that CD1a marks immature cells within thymocyte subpopulations allowed a meaningful and detailed analysis of these cells and a comparison with functionally mature thymocytes.
Our findings that CD1a+ SP cells are not clonogenic in vitro confirm and extend findings of Vanhecke et al. who investigated the clonogenic potential of CD4+ SP human thymocyte subsets (42). The CD1a+CD4+ SP thymocytes acquire the capacity to expand in vitro after cocultivation with short term cultured thymic stromal cells. This acquisition paralleled downregulation of CD1a. The observations identifying functionally immature CD1a+CD4+ SP cells are compatible with recent findings in the mouse by Dyall et al. (12) who demonstrated that a proportion of CD4+ SP murine thymocytes are functionally immature by several criteria. In many respects, the immature CD1a+CD3+ CD4+ SP thymocytes in the human thymus are similar to the functionally immature CD3+CD4+ SP in the mouse thymus (12). The immature mouse CD4+ SP thymocytes can be distinguished from mature cells by virtue of their expression of CD24 and high levels of CD69 (12). CD8 was not detectable by fluorimetric analysis, but the fact that the immature CD4+ SP mouse cells can be retained on antiCD8 immobilized on plastic indicates that low levels of CD8 are present on the immature CD4+ SP cells (12). Similar to the immature CD3+CD4+ SP population in the mouse, the human CD1a+CD4+ cells express CD69 and Bcl-2, indicative for cells that have been submitted to a positive selection signal (26). In addition, most CD1a+CD4+ SP cells express CD27, which may be another marker that is induced by positive selection (31, 42, 43). Human CD1a+CD3+ CD4+ SP cells expressed RAG-1, though in lower levels than total thymocytes. However, our inability to analyse RAG expression in individual cells precludes consideration of the possibility that a proportion of CD4+ CD1a+ SP cells are negative for RAG-1. Also, the CD8+ SP population in the human thymus contains functionally immature CD1a+ cells. The observation that heat stable antigen+CD8+ SP cells are present in the mouse thymus (10) suggested that there are immature cells also within the CD8+ SP thymic population, but the functional activity of those cells was not tested. The immature human CD1a+ CD8+ SP cells are similar to the CD1a+CD4+ SP cells in that the majority expresses CD69, Bcl-2, and CD27, but differ in expression levels of RAG-1, which are much higher than on CD1a+ CD4+ SP cells.
Although the expression of CD27, CD69, Bcl-2, and
high levels of CD3 on part of the immature CD1a+ DP
and almost all CD1a+ SP thymocytes indicates that these
CD1a+ cells have been submitted to a positive selection
signal, it is clear that a transition to CD1a cells is required
to confer functional maturity to these thymocytes. Two
possible mechanisms for the discrepancy between upregulation of CD27, CD69, and Bcl-2, and the CD1a+ to
CD1a
switch can be put forward. One is that downregulation of CD1a and acquisition of functional competence
requires a much greater sustained MHC/TCR interaction
than induction of CD69. This idea would take into consideration data from mouse studies indicating that consecutive, or perhaps even continual, TCR/MHC interactions are required to complete positive selection (44). A second possibility is that MHC/TCR interactions are sufficient for upregulation of CD69, but that an additional signal is required for downregulation of CD1a and acquisition
of functional maturity. Our experiments with the hybrid
human/mouse FTOC system provides support for the notion that two signals are required for induction of the functional program in immature thymocytes. We observed that
although interactions of mouse MHC class II antigens with
human CD4 and TCR drive generation of CD4+ SP cells,
the mouse thymic microenvironment is very inefficient in
downregulating CD1a and inducing functional maturation of CD4+ TCR
T cells. The inefficiency of mouse
thymic microenvironment to induce functional maturity
in CD4+ TCR
+ T cells is not due to, for example, tissue culture conditions since TCR
cells mature efficiently in the mouse FTOC. Moreover, we observed that
cocultivation of CD1a+ CD4+ SP cells with human thymic
stromal cells results in differentiation to CD1a
cells.
Taken together, these observations raise the possibility that
species-specific activating or costimulatory molecules are required for efficient maturation of human CD4+ T cells.
In this paper, we confirm and extend earlier findings
(13, 14) that the human thymus contains in vitro expandable DP cells. In vitro expandable DP thymocytes have also
been found in the mouse (15). In vitro expandability of human DP thymocytes correlates perfectly with completion
of downregulation of CD1a, as was also the case for SP
cells. Our observations that DP cells are present in the periphery of neonates suggest that some mature CD1a DP
cells may migrate out of the thymus. Most of these peripheral DP cells have characteristics of naive cells in that they
express CD45RA and are negative for CD45RO and Fas,
which are selectively expressed on memory cells. As was
also found within the mature DP thymocytes, cord blood
DP T cells lack CD1a and a proportion lacks CD8
as
well. The characteristics of these DP cord blood cells make
it unlikely that they are derived from peripheral SP cells
that have upregulated CD4 or CD8 due to activation. It
seems, therefore, that the CD4CD8 phenotype becomes
stable once the DP cells have been submitted to a maturation signal. The fact that cloned lines of DP thymocytes
with sustained CD4CD8 phenotype can be established is
consistent with this notion. It is relevant to note that cloned
lines of DP T cells have been established from peripheral T
cells (47). It is possible that those clones originated from
cells that emigrated from the thymus as DP cells.
The presence of both mature clonogenic DP cells and
immature SP cells is difficult to reconcile with a generalized
linear model of thymocyte differentiation from immature
TCR+CD4+CD8+ DP cells via immature to mature SP
cells. It is possible that for some thymocytes, completion of
positive selection and acquisition of functional competence
can occur at the DP stage, while for others this could happen at the SP stage. However, at least part of the CD1a
DP cells could be derived not from CD1a+ DP cells but
from CD1a+CD4+ SP cells. This is suggested by the experiments in which CD1a+CD4+ SP thymocytes were cocultured with short term cultures of postnatal stromal cells.
The cells harvested from such cocultures lacked CD1a and
expressed CD3 and CD4, but a large proportion of these cells coexpress CD8
. This phenotype persisted after expansion of these cells. It is also possible that some of the
CD1a
DP cells are derived from CD1a
SP cells as suggested by the experiments depicted in Fig. 6. Finally, we
have considered the possibility that the CD1a
DP cells are
derived from cells that never expressed CD1. This cannot
be excluded; however, we consider this unlikely since virtually all CD3
CD4+ immature SP cells considered to be
the precursors of the DP cells, express CD1a (39). Further
experiments are needed to elucidate the differentiation patterns of thymocytes after being submitted to positive selection mediated by the MHC/TCR interaction.
Address correspondence to Hergen Spits, The Netherlands Cancer Institute, Plesmanlaan 121, 1066 CX Amsterdam, The Netherlands.
Received for publication 30 April 1996
This work was supported by a grant from the Dutch Cancer Foundation (No. NKI 95-960).We thank F. Couwenberg for technical assistance, Drs. L. Lanier, J.H. Phillips, E. Reinherz, R. van Lier, and G. Aversa for their gifts of mAbs and Drs. L. Glimcher and A. Kruisbeek for making available the MHC class II KO mice. We confer our thanks to Dr. A. Kruisbeek for a critical review of the manuscript.
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