By
From the * Centre d'Immunologie Institut National de la Santé et de la Recherche Médicale-Centre
National de la Recherche Scientifique de Marseille Luminy, Case 906, 13288 Marseille Cedex 9, France; Unité d'Immunologie, Centre National de la Recherche Scientifique Unité Recherche Associée
1961, Institut Pasteur, 75724 Paris, France; § Center for Cancer Research, Massachusetts Institute of
Technology, 02139 Cambridge, Massachusetts; and
INSERM U429, Hôpital Necker-Enfants
Malades, 75743 Paris, France
We have investigated the role of common chain (
c)-signaling pathways for the development
of T cell receptor for antigen (TCR)-
/
T cells. TCR-
/
-bearing cells were absent from
the adult thymus, spleen, and skin of
c-deficient (
c
) mice, whereas small numbers of thymocytes expressing low levels of TCR-
/
were detected during fetal life. Recent reports
have suggested that signaling via interleukin (IL)-7 plays a major role in facilitating TCR-
/
development through induction of V-J (variable-joining) rearrangements at the TCR-
locus.
In contrast, we detected clearly TCR-
rearrangements in fetal thymi from
c
mice (which
fail to signal in response to IL-7) and reduced TCR-
rearrangements in adult
c thymi. No
gross defects in TCR-
or TCR-
rearrangements were observed in
c
mice of any age. Introduction of productively rearranged TCR V
1 or TCR V
1/V
6 transgenes onto mice
bearing the
c mutation did not restore TCR-
/
development to normal levels suggesting that
c-dependent pathways provide additional signals to developing
/
T cells other than for
the recombination process. Bcl-2 levels in transgenic thymocytes from
c
mice were dramatically reduced compared to
c+ transgenic littermates. We favor the concept that
c-dependent receptors are required for the maintenance of TCR-
/
cells and contribute to the completion
of TCR-
rearrangements primarily by promoting survival of cells committed to the TCR-
/
lineage.
Tcells can be divided into two populations based on the
structure of their TCRs. Most T cells express TCR
heterodimers consisting of Deciphering the role played by cell-cell interactions and
soluble cytokines provided by the thymic microenvironment constitutes a central question in the development of
fetal versus adult Mice.
Mice harboring a null mutation of the and
chains, whereas a
smaller population expresses an alternative TCR made of
and
chains. These two T cell populations share a number
of features, including rearranging antigen receptor chains, the products of which associate with a set of invariant CD3
polypeptides responsible for signaling to the cell that the
TCR heterodimer has been engaged (for review see reference 1). In contrast,
/
T cells differ from
/
T cells in
their ontogeny, variable (V)1 gene repertoires, and ultimate
anatomical locations (for reviews see references 2 and 3).
/
T cells are the first TCR-expressing cells detected in
the early fetal thymus, and persist in the adult thymus in
small numbers. The first two waves of
/
T cells to appear
during a fetal development express V
5-joining
1 (J
1) and V
6-J
1 genes, both of which pair with
chains composed of V
1-D
1-J
2 segment combinations. The most
striking feature of these TCR-
/
genes is their lack of
junctional diversity (2, 3). The V
5-expressing cells migrate to the skin to become the Thy1+ dendritic epidermal
T cell (DETC) population, whereas V
6-expressing cells
migrate to mucosal surfaces lining the reproductive tract
and tongue (4). The production of V
5/V
1 and V
6/
V
1 cells slows at the end of fetal life and another wave of
TCR-
/
cells develops from this time onwards, which
uses mainly V
4 and V
1 gene segments paired with a variety of V
genes and which displays extensive junctional
diversity. In the adult, these
/
T cells constitute ~0.2%
of the thymus and following export, seed the spleen and
lymph nodes. The mechanisms that control the sequential appearance of
/
T cell subsets with distinct V gene segment usage, V(D)J junctional diversity, and unique homing
properties are unknown (5, 6). Recent evidence suggests
that the observed TCR-
and TCR-
gene rearrangements
are temporally programmed and do not rely on selection of
a particular subset of receptors among a diverse TCR-
/
combinatorial repertoire (7).
/
T cells. A variety of interleukins have
been demonstrated to affect the growth and differentiation
of TCR-
/
cells. For instance, freshly isolated
/
T cells
from fetal thymus, skin, spleen, or peritoneal cavity can
proliferate in vitro to IL-2, IL-7, or IL-15 (10), and in
utero administration of antibodies to the IL-2R
chain block
development of DETC (14). Gene ablation experiments in
vivo have confirmed some of these findings. Mice deficient in IL-2R
(shared by IL-2 and IL-15), IL-7/IL-7R
, or the
common
chain (
c; shared by IL-2, IL-4, IL-7, IL-9, and
IL-15) each have defects in
/
T cell development (15).
Still, the mechanism by which cytokine depletion affects
the differentiation program of these cells is not completely
understood. Cytokines could play a role in survival or proliferation of developing thymocytes, or alternatively might
directly influence the TCR rearrangement process. Along
these lines, experiments using mouse fetal liver cultures
supplemented with IL-7 suggested that this cytokine could specifically induce the rearrangement of TCR-
chain
genes (21), and recently IL-7R
-deficient mice were shown
to have a selective block in TCR-
gene recombination
(22). In this report, we have analyzed mice deficient in
c
to clarify the role of the
c in the development of fetal and
adult
/
T cells. Our results demonstrate that
c-containing receptor complexes play a role for TCR-
chain rearrangements in the adult, but not the fetal thymus, and more importantly, signaling through the
c provides essential survival signals for
/
T cells.
c have been
described (19),
c-deficient mice, IL-7-deficient mice (kindly
provided by R. Murray, DNAX Corp, Palo Alto, CA; reference
23), recombinase-activating gene 1-deficient mice (RAG1
/
;
ref. 24), and their littermate controls were maintained in specific pathogen-free conditions and used between 4 and 8 wk of age.
Fetal tissues were obtained from timed-pregnant mice. Day 0 of
embryonic development was considered to be the day a vaginal
plug was observed.
1 and double Tg for TCR
V
1 and TCR V
6 were constructed as follows. Genomic DNA
fragments containing the rearranged TCR V
1 and V
6 genes
were isolated from a cosmid library prepared in pWE15 using
partially digested Sau 3A I DNA from the T3.13.1 TCR-
/
hybridoma (25). The V
1-J
4 clone (45 kb) contained 10 kb of upstream sequence and extended 26 kb downstream of the C
4
exon. The V
6-D-J
1 clone (34 kb) contained 5 kb of upstream
DNA and 14 kb downstream of the C
exon. Tg constructs were
mixed and the DNA microinjected into the pronuclei of fertilized embryos. Mice carrying the Tg TCRs were identified by
PCR and backcrossed onto the C57Bl/6 background. Mice were
screened using tail DNA and primers specific for the Tg V
1
TCR: forward, 5
-CCGGCAAAAAGCAAAAAAGTT-3
; and
reverse, 5
-CCCATGATGTGCCTGACCAG-3
. PCR conditions were as follows: denaturation at 94°C for 20 s, annealing at
59°C for 25 s, and extension at 74°C for 25 s for 33 cycles.
Antibodies.
The following Abs were used as conjugates to either
FITC, PE, or biotin: anti-TCR- (H57-597), anti-TCR-
/
(GL3), anti-TCR V
5 (536), anti-CD3 (2C11), anti-CD4, anti-CD8
, anti-CD24 (J11d), anti-Mac-1 (M1/70), and anti-CD32
(2.4G2). Streptavidin Tricolor (CALTAG Labs., South San Francisco, CA) was used to detect biotinylated antibodies. The antibody specific for TCR V
1 (2.11) has been described (25). A
clonotypic antibody recognizing V
1/V
6 TCR heterodimer
(1.9) was obtained in the same fusion and its specificity verified
using a panel of
/
T cell hybridomas as described (25).
FACS® Analysis and Cell Sorting. Single cell suspensions obtained from thymus or spleen were lysed of red cells using hypotonic NH4Cl solution. DETCs were isolated from ear skin using trypsinization and mechanical disaggregation as described (26). Nonspecific binding of mAbs to FcRs was reduced by preincubation with anti-CD32 mAb for 15 min. For surface staining, cells were incubated with saturating amounts of directly conjugated mAbs for 20 min, washed twice, and incubated with streptavidin Tricolor. For Bcl-2 staining, surface antigens were stained as above, and cells were washed in PBS and fixed in PBS containing 1% paraformaldehyde/0.01% Tween-20 for 90 min on ice. Cells were subsequently incubated with hamster anti-mouse Bcl-2 (clone 3F11; PharMingen, San Diego, CA) or purified hamster Ig. Cells were washed, incubated with biotinylated goat anti- hamster Ig, and finally with streptavidin Tricolor. Cells were analyzed on a FACScan® flow cytometer using CellQuest software (Becton Dickinson, Mountain View, CA). For isolation of early thymocyte precursors, cells were stained with CD4-FITC and CD8-PE, and double negative (DN) cells sorted using a FACStar Plus® cell sorter (Becton Dickinson).
PCR Analysis. DNA samples were extracted from total or fractionated populations of fetal or adult thymocytes using the salting-out technique (27). PCR reactions were done in a final volume of 20 µl and included a maximum of 100 ng of template DNA, 1 mM of each primer, 200 µM of each deoxynucleotide triphosphate, and 0.2 U of Taq DNA polymerase. Whole reaction mixtures were run on a 1.5% agarose gel, blotted to nylon membrane (Gene Screen Plus, New England Nuclear, Boston, MA), and hybridized with 32P-labeled oligonucleotide probes.
The oligonucleotides and PCR conditions used for the analysis of TCR-In vitro data
have suggested an important role for IL-2, IL-7, and IL-15
in the survival, proliferation, and differentiation of cells belonging to the /
T cell lineage (10). Because the
c
receptor plays an integral role in the function of these cytokine receptors, we anticipated that
c
mice would exhibit a defect in
/
T cell development. As most adult
/
T cells fail to express the CD4 and CD8 coreceptors and
are found in the DN subset of T cells, we examined wild-type (wt) and
c
DN thymocytes for the presence of cells
bearing TCR-
/
receptors. Compared to control mice,
adult thymi from
c
mice showed a complete absence of
TCR-
/
+ cells (Fig. 1). Further analysis of the peripheral
lymphoid organs, skin, and small intestine of adult
c-deficient mice failed to demonstrate any TCR-
/
+ cells in
the animal (Fig. 1 and data not shown). These results confirm and extend previous observations demonstrating the
strict dependence on
c-containing receptors for the development of all types of
/
T cells present in adult mice
(15).
Considering that /
T cells constitute a minor cell population in the adult thymus, we next analyzed fetal thymi
from
c
mice, since they contain a higher frequency of
/
T cells (9). Total thymocyte cell numbers were clearly reduced in
c
mice relative to control mice at all stages of
fetal development examined (Table 1) although CD4 and
CD8 expression was unaltered (data not shown). An ~10-fold reduction in cell number is apparent in
c
fetal thymi,
yet with age, thymocyte cell numbers increase in parallel
with
c+ mice. This suggests that alternative
c-independent signaling pathways (including that of the receptor tyrosine kinase c-kit; reference 30) support continuous thymic
seeding and permit progressive thymocyte accumulation.
Analysis of fetal (days 16-18)
c-deficient thymocytes revealed a reduced percentage of cells expressing TCR-
/
heterodimers (Fig. 2 and data not shown) and suggested
that
/
T cells might have some capacity to develop in
that context, but might subsequently be lost, perhaps due
to poor survival or failure to mature. Consistent with this
hypothesis, the few fetal TCR-
/
thymocytes found in
c
mice have an immature phenotype characterized by
low levels of TCR-
/
heterodimers and high levels of
heat-stable antigen (HSAhi; Fig. 2). Similar observations
have been made in IL-7-deficient mice (16), pointing to
IL-7 as the
c-dependent cytokine responsible for the defect observed in
c
thymi.
|
Taken together, these observations confirm the major
role played by the IL-7/c-signaling pathway in
/
T cell
development (16), but also suggest that factors independent of
c and IL-7 are present in the fetal, but not the
adult, thymus and can support the appearance of the most
immature
/
T cells. Recently, a novel cytokine, thymic
stromal cell-derived lymphopoeitin (TSLP; reference 31),
has been identified which shares biological activities with
IL-7 and uses the IL-7R
chain for signaling (32). We
would hypothesize that TSLP could partially replace IL-7
during fetal development in
c
mice. Two additional observations support this view. First, thymocytes from
c
mice respond to TSLP (33) and second, mice with a deletion of the IL-7R
chain have neither fetal nor adult
TCR-
/
cells detectable by FACS® analysis (17). Therefore, the spectrum of cytokine receptors expressed by fetal
and adult immature thymocytes may be identical, but fetal
versus adult thymic stroma may differ in their abilities to
produce cytokines, like TSLP. Along this line, it will be interesting to determine the relative expression of TSLP in
fetal and adult thymi.
We considered a
number of nonexclusive hypotheses to explain the pronounced negative effect of c deficiency on
/
T cell development: (a)
c may be required for committment to the
TCR-
/
lineage (defect in
/
-
/
lineage branching),
(b)
c may be required for initiation or completion of the
site-specific DNA recombination process affecting the
TCR-
and/or TCR-
loci (defect in TCR rearrangements), or (c) signals via
c may be essential for the survival
of cells during the process of
/
T cell differentiation.
To explore these different possibilities, we analyzed the
status of TCR gene rearrangements in fetal and adult thymocytes from wt and c-deficient mice using a polymerase
chain reaction technique that can specifically assess the
presence of rearranged DNA from the various TCR gene
loci (9, 28). The results shown in Fig. 3 were generated using fetal (day 17) thymocytes and PCR primer pairs specific
for the V
5-J
1, V
1-J
4, V
1-J
2, and V
4-J
1 rearrangements. Although V
4-J
1 and V
1-J
4 rearrangements were nearly unaffected in
c
fetal thymi, the canonical V
1-J
2 and V
5-J
1 rearrangements found in
thymocytes destined to seed the skin epithelium (4) were
clearly reduced (Fig. 3). As expected from the fact that the
c mutation does not block the development of
/
T cells (19, 20), DNA samples from
c thymi contained D
-J
and V
-D
-J
rearrangements that were as diverse as
those found in wt samples (data not shown).
Similar studies were performed on adult c
thymi. As
shown in Fig. 4, the V
1-J
4 rearrangements detected in
4-wk-old
c
thymi represent 23% of those observed in wt
thymi, respectively. Only limited V
4-J
1 rearrangements
are observed in adult
c
thymi, representing ~1% of the
levels observed in adult wt thymi. In contrast, the extent of
TCR-
rearrangements found in adult thymi from
c-mutant
mice resembled those present in wt adult thymi (Fig. 4 B,
top). Thus, these results suggest that the
c mutation has little effect on adult TCR-
rearrangements, but appears to
selectively reduce adult TCR-
rearrangements. Parallel studies using IL-7-deficient mice demonstrated a similar
defect in TCR-
rearrangements (Table 2).
|
Most developing /
T cells contain TCR-
gene segments that have rearranged during the DN stages of development (34, 35). As a result, TCR-
/
+ thymocytes retain TCR-
locus sequences (36), which could account for the split phenotype observed for the TCR-
and TCR-
loci in unfractionated adult
c
thymocytes (Fig. 4). We
therefore examined TCR-
rearrangements in CD4
CD8
precursors from
c mutant mice. As summarized in Table
3, the extent of TCR-
rearrangement present in
c
DN
thymocytes was found to be similar to that observed in total
c
thymocytes and in CD3
5/
5 thymocytes, which
are an enriched source of
c+ DN cells with normal levels
of TCR-
gene rearrangements (34). Therefore, these
findings demonstrate that TCR-
genes do rearrange in the
CD4
CD8
precursors isolated from adult
c
mice.
TCR-
rearrangements in
c
DN thymocytes were also
reduced (data not shown).
|
In conclusion, our analysis of TCR- and TCR-
rearrangements in fetal and adult
c
thymi distinguishes
TCR-
/
cell development during these two stages. Although noncanonical TCR-
and TCR-
rearrangements
were both present during the fetal period, TCR-
, but not
TCR-
, rearrangements were severely reduced in the adult
c
thymus. The implications of these observations are the
following. First, during fetal life, the absence of
c signaling
pathways does not impair the ability to rearrange the
TCR-
or TCR-
loci. This suggests that
c-independent
factors may compensate for the lack of IL-7/
c-mediated
signals. Second, the fact that fetal and adult thymi from
c
mice do contain TCR-
and TCR-
rearrangements, but
fail to give rise to mature
/
T cells strongly suggests that
other defects (e.g., survival of already committed or successfully rearranged
/
T cells) likely accounts for the defective
/
T cell development observed in
c thymi.
To further address potential defects in /
-
/
lineage branching and/
or
/
T cell survival, we crossed
c-deficient mice with
mice Tg for a functionally rearranged TCR V
1 gene or
with double Tg mice harboring the same TCR V
1 and a
productively rearranged TCR V
6 gene. The T3.13.3 hybridoma from which these rearranged TCR chains were
isolated corresponds to the subset of adult TCR-
/
cells
and the TCR V
1/V
6 heterodimer demonstrates extensive junctional diversity (25). In addition, the V
1 Tg construct contains the necessary flanking DNA sequences to
ensure proper expression in TCR-
/
precursors, as well
as the silencer element required to prevent its adventitious
expression in TCR-
/
lineage cells (39). Founder mice
expressing the V
1 or V
1/V
6 constructs were identified and crossed onto the
c-deficient background.
Expression of the V1 Tg alone in mice with or without
the
c mutation did not alter absolute thymocyte cell numbers or the expression of mature CD4 or CD8 single positive thymocytes (Table 4; Fig. 5). Total thymocyte preparations from nontransgenic littermates contained only a
very small percentage of cells marked with the anti-TCR
V
1 antibody, whereas
c+ V
1 Tg animals demonstrate
an increase in both the frequency (Fig. 5) and absolute
numbers of V
1
/
T cells (Table 4). Importantly, the
V
1+ cells were negative for TCR-
chains, demonstrating that the Tg was correctly expressed in TCR-
/
cells
and that the flanking silencer element was operative in the
/
T cells found in these Tg mice (Fig. 5).
c
TCR-
Tg animals showed a population of V
1+ cells in total thymus preparations; these Tg+ thymocytes were more clearly
demonstrated in the DN compartment (Fig. 5). Despite expression of the Tg V
1 chain,
/
T cells in
c
mice were
still severely reduced. Compared with
c+ Tg animals, there
was a 120-fold reduction in absolute numbers of V
1+ cells
(Table 4). Considering that the
c chain plays a role in the
generation of the earliest noncommitted thymic precursors (CD44+CD25
cells; reference 40) which are 15-fold reduced in
c
mice (data not shown), part of the dramatic
reduction in V
1+ thymocytes in
c
Tg mice stems from
the limited number of thymocyte precursors available to
express the Tg V
1 receptor. Taking this into account,
/
T cells are still eightfold reduced (120-fold/15-fold) in
c
Tg mice relative to
c+ Tg controls, suggesting that additional mechanisms are responsible for the defective
/
T
cell development. Similar results were obtained using mice
expressing the same TCR V
1 chain and a rearranged TCR
V
6 (Fig. 6 and data not shown). We conclude that a rearranged TCR-
/
Tg does not restore normal
/
T cell
development in the absence of
c. Moreover, since
c
mice
can express the TCR-
/
transgenes, a defect in TCR-
/
-
/
lineage branching can be effectively ruled out.
|
Considering that thymically derived /
T cells seed the
spleen and lymph nodes of postnatal mice, we further examined the peripheral lymphoid compartments for transgenic
/
T cells. As shown in Fig. 6, a large population of cells
expressing the transgenic TCR-
/
receptor are present in
the spleen of
c+ Tg animals; these cells coexpress CD3,
but are TCR-
negative (data not shown) and accumulate
to levels which are approximately six-fold higher in absolute numbers than
c+ nontransgenic animals (Table 4). In
contrast, the periphery of
c
Tgs contain only a few cells
expressing the Tg TCR-
/
receptor, and these cells fail to
accumulate in the spleen. In terms of absolute numbers,
c
Tg
/
cells are reduced 142-fold compared with
c+
Tg littermates (Table 4). Taken together, our results suggest a major role for
c-dependent signals in the survival of
/
T cells.
To investigate whether c
Tg
/
T cells had a defect
in survival, we examined Tg thymocytes for the expression
of the antiapoptotic factor, Bcl-2. Engagement of
c-dependent receptors has been shown to maintain high levels of
Bcl-2, which appear to protect lymphoid cells from cell death
(41). Although V
1+ thymocytes from
c+ mice expressed Bcl-2, Tg+
/
T cells from
c
mice were essentially negative for Bcl-2 (Fig. 7). These results are consistent with a defect in
/
T cell survival in the absence of
c, although the relative contribution of Bcl-2 in supporting
/
T cell development remains to be determined.
Concluding Remarks.
Most /
T cells start their development within the thymus where they rearrange their
TCR-
and TCR-
genes via site-specific DNA recombination reactions triggered by the specialized stromal microenvironment found in the thymus. IL-2, IL-7, and IL-15
bind to specific receptors that share the
c and these cytokines have been postulated to play an important role in the
survival, growth, and differentiation of
/
cells (10).
In a recent study using mice deficient in IL-7R
chains,
TCR-
gene rearrangements were found to be selectively
abolished, and as a consequence, these mice lacked both fetal
and adult TCR-
/
cells (22). The authors concluded that
ligands binding to the IL-7R
chain (IL-7 and TSLP) are
likely to be mandatory for the process of TCR-
rearrangements within intrathymic TCR-
/
cell precursors
(22). Although we do not refute this conclusion, our data
reveal an additional function of
c-containing receptors
(likely due to IL-7), that is independent of the TCR-
rearrangement process. Thymocytes from adult
c-deficient mice do not contain detectable TCR-
/
cells and showed
only low levels of TCR-
rearrangements, thereby limiting
the potential synthesis of TCR-
polypeptides. Complementation of such
c-deficient mice with TCR-
and
TCR-
/
transgenes only partially rescued thymic
/
T
cell development and did not permit accumulation of peripheral
/
T cells. Therefore, the developmental blockade affecting the adult
/
T cell lineage in
c-deficient
mice results not only from the limited amounts of rearranged TCR-
genes but also from the fact that IL-7 promotes the survival of
/
T cell precursors containing
TCR-
and TCR-
/
polypeptides. The survival role
played by IL-7 in
/
T cell development is supported by our observation that fetal thymocytes from
c-deficient
mice do contain TCR-
and TCR-
rearrangements, but
fail to generate appreciable numbers of
/
T cells intrathymically or to export them to the periphery. Further
evidence is provided by the fact that
c
/
T cells contain dramatically reduced levels of the antiapoptotic factor
Bcl-2. Previous reports have shown that engagement of
c-containing receptors maintains cellular Bcl-2 protein levels
(41), thereby promoting lymphoid cell survival. Whether
the near-absent Bcl-2 levels are directly responsible for the
survival defect in
c
/
T cells or simply an epiphenomenon related to decreased cell survival remains to be determined.
Thus, we would like to propose that for /
T cells, the
primary function of
c-containing receptors is to promote
survival. The ability of the
c
fetal thymus to support the
survival of
/
T cells (possibly via TSLP) would explain
the presence of TCR-
rearrangements in IL-7-deficient
mice and
c-deficient mice, and their mere absence in IL-7R
-deficient mice. After successful expression of a functional TCR-
/
complex,
/
T cells would still require
signals via
c to survive, mature, and seed the periphery.
This idea gains support from the analysis of IL-7-deficient
mice, where V
5loHSAhi fetal thymocytes are readily detectable, but fail to mature into V
5hiHSAlo cells (16). As a result, skin DETCs are not detectable in IL-7-deficient mice
(data not shown).
Based on these results, one is led to ask why development of /
T cells is less severely impaired in
c-deficient
mice than that of
/
T cells. In the
/
lineage, it has
been recently documented that TCR-
rearrangements are
accompanied by a selective process allowing only those
cells displaying a productively rearranged V
gene to reach
the next stage of differentiation (
selection). At a later
time point, a second phase of selection, denoted TCR-
/
selection, occurs to ensure MHC restriction and self tolerance. For
/
T cells, TCR-
and TCR-
rearrangements
are probably achieved concurrently and not subjected to
pre-TCR-based epigenetic control mechanisms operating
during
/
T cell development (34). In this model of
/
T development,
/
T cell precursors might not receive
any pre-TCR or TCR signals, and engagement of
c-containing cytokine receptors may constitute the only means
to support the survival of these cells. In marked contrast, in
/
T cell precursors, the
c-dependent survival signals
and the pre-TCR dependent survival signals may partially
overlap, explaining how, in the absence of survival signals
dependent on
c-containing receptors, signals emanating at
a latter time point from the pre-TCR may rescue the development of a few
/
T cell precursors.
Address correspondence to James P. DiSanto, INSERM U429, Pavillon Kirmisson, Hôpital Necker-Enfants Malades, 149 rue de Sèvres, Paris F-75743 France. Phone: 33-1-44-49-50-51; FAX: 33-1-45-75-13-02; E-mail: disanto{at}necker.fr
Received for publication 22 April 1997 and in revised form 4 August 1997.
1 Abbreviations used in this paper:This work was supported by grants from the Centre National de la Recherche Scientifique, the Institut National de la Sante et de la Recherche Medicale, the Association pour le Recherche sur le Cancer, Ligue Nationale Contre le Cancer, and the Commission of the European Communities.
We thank R. Murray (DNAX Corp.) for kindly providing IL-7-deficient mice, S.Y. Huang for help in generating the TCR Tg mice, and A. Wilson, D. Guy-Grand, and F. Rieux-Laucat for helpful discussions.
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