By
From the * Institut Necker, Institut National de la Santé et de la Recherche Médicale, 373, F-75730
Paris, Cedex 15, France; Hôpital Necker-Enfants Malades, Institut National de la Santé et de la
Recherche Médicale, 429, F-75743 Paris, France; and § Basel Institute for Immunology, CH-4005
Basel, Switzerland
The development of pre-T cells with productive TCR- rearrangements can be mediated by
each the pre-T cell receptor (pre-TCR), the TCR-
as well as the TCR-
, albeit by distinct mechanisms. Although the TCR-
affects CD4
8
precursor cells irrespective of their
rearrangement status by TCR-
mechanisms not involving TCR-
selection, both the preTCR and the TCR-
select only cells with productive TCR-
genes for expansion and
maturation. The TCR-
appears to be much less effective than the pre-TCR because of the
paucity of TCR-
proteins in TCR-
-positive precursors since an early expressed transgenic
TCR-
can largely substitute for the pre-TCR. Thus, the TCR-
can assume a role not
only in the rescue from programmed cell death of CD4+8+ but also of CD4
8
thymocytes.
In evolution this double function of the TCR-
may have been responsible for the maturation of
T cells before the advent of the pre-TCR-
chain.
During development of Experiments in pre-TCR-deficient TCR- Mice.
The pT Antibodies and Flow Cytometry.
The following mAbs were used
for staining: anti-CD4 (H129.19, PE-conjugated; GIBCO BRL,
Gaithersburg, MD; or H129.19, FITC-conjugated; GIBCO BRL),
anti-CD8 (Ly-2, FITC-conjugated; PharMingen, San Diego, CA; or 53-6.7, biotinylated; GIBCO BRL; or 53-6.7, RED613conjugated; GIBCO BRL), anti-CD25 (3C7, PE-conjugated;
PharMingen), anti-CD44 (biotinylated KM81; American Type
Culture Collection, Rockville, MD), anti-panTCR- T cells in the thymus most
TCR genes rearrange in temporal order such that
most TCR-
rearrangement occurs before TCR-
rearrangement (1, 2). Over the years, it became clear that the
products of the rearranged genes, i.e., the TCR-
and
TCR-
chains, have an important role in controlling T cell
development: the first produced TCR-
chain covalently binds to the pre-TCR-
(pT
)1 chain (3, 4) and forms the
pre-TCR that rescues from programmed cell death CD4
8
44
25+ cells that have succeeded in TCR-
chain rearrangement. The selected cells assume the CD4
8
44
25
phenotype (5), proliferate extensively, and eventually become CD4+8+ cells that bear the TCR-
on the cell surface
while expression of the pT
is terminated (6, 7). The
CD4+8+-expressing cells are programmed to die unless the
TCR-
binds to thymic MHC molecules and cells are
rescued from cell death once more and eventually become
mature T cells that leave the thymus (8, 9). Both the preTCR and the TCR-
associate with signal-transducing
CD3 molecules and may signal through activation of src kinases like p56lck and fyn (3, 10). In fact, recent experiments
have established that p56lck- and fyn-deficient, double mutant
mice exhibited a developmental block at the CD4
8
44
25+
stage where the pre-TCR normally assumes its role (11).
Even earlier experiments in either rearrangement-deficient
RAG
/
mice (12, 13) or CD3
/
mice (14) had already
indicated that a signaling receptor that contains at least one
chain encoded by a rearranging gene was required to rescue
CD4
8
44
25+ cells from apoptotic cell death (15).
/
or pT
/
mice had shown that the pre-TCR, while having an important function in generating large numbers of CD4+8+
cells from CD4
8
precursors, was likely not to be the
only TCR able to mediate these events since both types of
mutant mice still contained significant though reduced
numbers of CD4+8+ thymocytes (6, 16). In fact, the origin
of the CD4+8+ cells in TCR-
/
mice was obscure and
the possibility was discussed that they may belong to the
lineage (16). In pT
/
mice, however, some of the CD4+8+
cells expressed TCR-
on the cell surface and could undergo positive selection to become mature T cells, i.e.,
they belonged to the
lineage. Therefore, it is important
to define alternative rescue pathways that can avoid a total
deficiency of
T cells in pT
-defective mice. Indeed, by
defining such pathways, one may gather further information on how the pre-TCR functions in immature T cells.
In this report we show that not only the pre-TCR but
both the TCR-
as well as the TCR-
can mediate the
differentiation of CD4
8
25+ pre-T cells albeit by distinct
mechanisms.
/
mice, TCR-
/
mice, and TCR-
/
mice have been described (6, 17, 18). TCR-
/
pT
/
mice
were bred in the animal colony of the Basel Institute for Immunology. Breeding of TCR-
/
pT
/
mice was done in the
animal facilities at the Hôpital Necker (Paris, France). C57BL/6
mice were purchased from IFFA CREDO (L'Arbresle, France).
The TCR-
transgenic mice, with a transgenic TCR specific for the male antigen (H-Y) in the context of H-2Db MHC molecules, have been described previously and were crossed on the
C57BL/6 (B6) background (19). TCR-
transgenic pT
/
mice were bred in the animal colony of the Basel Institute for Immunology. Animals were analyzed at 6-8 wk of age. Animal care was in accordance with institutional guidelines.
(H57-597,
FITC-conjugated [20]), anti-TCR-
(GL3, FITC-conjugated;
PharMingen), T3.70 (specific for the TCR-
chain of the HYreactive TCR, FITC-conjugated), and F23.1 (specific for the
TCR-
chain of HY-reactive TCR, FLUOS-conjugated [21]).
In initial experiments, it was determined whether either
the TCR- or the TCR-
could be responsible for the
production of CD4+8+ T cells in pT
/
mice by analyzing the cellular composition of thymuses from either pT
/
TCR-
/
or pT
/
TCR-
/
double mutant mice that
can only produce the
and the TCR-
, respectively.
As shown in Table 1 both types of mutant mice contained CD4+8+ T cells that were further analyzed by cytoplasmic
staining with antibodies specific for TCR-
and TCR-
chains. For this purpose cells were double stained for surface expression of CD4 and CD8 molecules as well as either for cytoplasmic TCR-
or TCR-
chains by double
fluorescence using CD4 and CD8 antibodies in one color (green) and TCR-
or TCR-
antibodies in another color
(red). In this analysis single positive CD4+8
and CD4
8+
cells show an intermediate fluorescence between that of
CD4
8
and CD4+8+ thymocytes and cells were gated accordingly into double negative, double positive (DP), and
single positive cells (Fig. 1).
Fig. 1 shows that 64% of CD48
cells in wild-type
mice expressed TCR-
chains, and that due to TCR-
selection by the pre-TCR (22) the vast majority of CD4+8+
cells contained TCR-
chains in their cytoplasm. On the
other hand, the expression of cytoplasmic TCR-
chains
was mostly restricted to CD4
8
cells. The picture was
different in pT
/
mice where, due to the diminution of
rapidly cycling TCR-
-selected CD4
8
44
25
cells (6),
only 21% of the CD4
8
cells were TCR-
positive. In
addition, only 39% of the CD4+8+ cells contained TCR-
chains in their cytoplasm indicating that in the pT
/
mice the majority of the CD4+8+ cells were generated by a
mechanism that did not involve TCR-
selection. The fact
that not all single positive cells in these mice were TCR-
+
is due to the fact that these cells are in part immature TCR-
single positive cells, on their way from CD4
8
to CD4+8+
cells. Such cells constituted a higher proportion of all cells in pT
/
mice. The TCR-
+ single positive cells had a
mature CD4+8
phenotype as confirmed by independent
three-color stainings indicating also that these cells expressed TCR-
receptors on the cell surface. These cells
were present in a higher number in pT
/
mice consistent with the notion that the pre-TCR may have a role in
regulating
rearrangement and/or expression (23 and unpublished observations).
In pT/
TCR-
/
mice the proportion of TCR-
+
CD4+8+ and TCR-
single positive cells was even further
reduced. When looking at the absolute numbers of various
cell subsets (Table 1 and Fig. 1) it is clear that there was a
very marked reduction in cell numbers of CD4+8+ thymocytes and more mature cells in pT
/
and pT
/
TCR-
/
mice, whereas the numbers of CD4
8
cells
were within the same range.
pT/
TCR-
/
mice also had reduced numbers of
DP cells but here the picture differed from that in pT
/
and pT
/
TCR-
/
mice in that all of the CD4+8+
cells were TCR-
positive, i.e., were exclusively generated
through a mechanism that involved TCR-
selection. The
single positive TCR-
+ cells in pT
/
TCR-
/
mice
were exported from the thymi and CD4+8
as well as
CD4
8+ cells could be detected in lymphnodes of these
mice (not shown). This excludes the possibility that these
cells belong exclusively to the NK1.1+CD4+ subset that
exhibits an unusual phenotype (24).
The above results were reproducible in the different
mice with marginal deviations in either the percentage of
cells or absolute cell numbers and are schematically presented in Fig. 2. The main message from this analysis is that
the TCR- can generate CD4+8+ cells through TCR-
selection, i.e., by intracellular or cell-autonomous signaling
only. In contrast, the TCR-
can generate CD4+8+ cells
that are either TCR-
+ or TCR-
but all TCR-
through a mechanism that may involve intercellular communication of unknown nature. If the TCR-
would
generate a significant number of DP cells by cell-autonomous signaling one might expect to find some TCR-
expression in these cells. However, the fact that the CD4+8+
cells are TCR-
negative suggests that these cells are not
selected by cell-autonomous signaling by the TCR-
even though it can not be entirely excluded that TCR-
expression is abruptly switched off in CD4+8+ cells. The
notion of intercellular communication is in line with experiments that involved transfer of
T cells into thymuses of rearrangement-deficient mice that resulted in generation
of CD4+8+ cells of host origin (25) and also with earlier
data by Shores et al. (26). Our experiments suggest that in
the latter experiments
but not
T cells promoted the
development of CD4+8+ thymocytes and make the additional point that the generation of DP cells was not due to
an artefact caused by adoptive transfer of cells.
The fact that in the absence of the pre-TCR the generation of CD4+8+ cells by the TCR- is rather inefficient,
i.e., 240 × 104 versus 2,880 × 104 in pT
/
TCR-
/
versus wild-type mice, could depend on the fact that the
TCR-
is inefficiently formed in CD4
8
cells due to the
late TCR-
rearrangement and/or the fact that TCR-
can only inefficiently replace the pre-TCR. To analyze this
question in some more detail we studied mice that express a transgenic TCR-
early in development on CD4
8
cells, i.e., TCR-
transgenic pT
/
mice. The transgenic
TCR-
could indeed overcome the cellular deficiency in
the CD4+8+ compartment as TCR-
transgenic pT
/
mice contained approximately one-half the number of thymocytes found in TCR-
transgenic pT
+ mice and
many more than the number found in nontransgenic pT
/
mice (Fig. 3). However, there was a subtle difference between TCR-
transgenic pT
+ and TCR-
transgenic
pT
/
mice in that the latter, but not the former, contained a discrete subset of CD25+ cells, indicating that in
spite of the presence of the transgenic TCR-
, the preTCR had its role in the exit from this compartment. This
could be due to the lack of expression of the transgenic TCR-
in a fraction of cells in the CD25+ compartment
of the TCR-
transgenic, pT
/
mice. This was in fact
confirmed by cytoplasmic staining: while only nine percent
of CD25+ cells in TCR-
transgenic pT
/
mice expressed the transgenic TCR-
chain the majority of these cells expressed the transgenic TCR-
chain suggesting that
expression of the two transgenes is differentially regulated
(Fig. 4). Thus, in TCR-
transgenic pT
+ mice it is the
combined action of the pre-TCR and the TCR-
(mice
that have only a TCR-
transgene still exhibit a significantly larger CD25+ compartment than TCR-
transgenic mice, not shown) that reduce the number of CD25+
cells while in TCR-
transgenic pT
/
mice this compartment is bigger in size because of the absence of the preTCR. From these data it would appear that the TCR-
can at least partially mimic the function of the pre-TCR
and that in normal mice the contribution of the TCR-
to the generation of the CD4+8+ compartment is limited
due to relatively late expression of most TCR-
chains (1, 2).
Thus, all of the three known TCRs can have a role in
promoting the development of pre-T cells: the TCR-
most likely by intercellular communication that furthers the
development of CD4+8+ cells irrespective of whether or not
they have succeeded in TCR-
rearrangement, the TCR-
that depends strictly on intracellular, cell-autonomous signals generated by the TCR-
chains and the pre-TCR
that operates by a similar mechanism as the TCR-
but is
much more efficient because of the early and abundant expression of the pT
gene during the phase of TCR-
rearrangement. Therefore, only mice that cannot produce any
of these receptors will exhibit complete arrest at the CD4
8
stage of development as evident in RAG
/
mice or mice that
are deficient in both TCR-
and TCR-
chains and therefore, can make neither TCR-
, pre-TCR, nor TCR-
(14). In normal mice, the contribution of the TCR-
in
development of cells of the
lineage appears to be limited based on the fact that the vast majority of CD4+8+ cells
are TCR-
+ and thus are TCR-
selected. Likewise, in
normal mice, the contribution of the TCR-
to the transition of DN to DP cells may be limited because of the small
number of DP cells in pT
/
TCR-
/
mice. However,
in the absence of pT
these receptors avoid a severe immunodeficiency by enabling the formation of a significant number of mature
T cells. It would appear that both the preTCR and the TCR-
do not only mediate maturation
but also proliferation since in wild-type mice and pT
/
TCR-
/
mice the proportion of large CD4+8+ blasts
that are derived from dividing CD4
8
precursors (15) is
very similar (Table 2). There are only slightly fewer blasts
in pT
/
TCR-
/
mice indicating that also the TCR-
generates dividing CD4+8+ cells.
|
With regard to the role of the src kinases in early development, our data is consistent with the notion that signaling through the pre-TCR involves both lck and fyn kinases
but is equally consistent with the idea that the fyn kinase is
involved only in signaling through the TCR- or -
,
and thereby responsible for the incomplete developmental
arrest observed in lck
/
mice. The fact that the TCR-
promotes development much in the same way as the preTCR, i.e., by cell-autonomous signaling and thereby TCR-
selection, suggests that T cell development may have proceeded in this way before the advent of the pre-TCR-
chain in evolution and that the pre-TCR had simply the
advantage of making the pairing of a single TCR-
chain
with different TCR-
chains more effective.
Address correspondence to Harald von Boehmer, Institut Necker, INSERM 373, 156, rue de Vaugirad, F-75730 Paris, Cedex 15, France.
Received for publication 20 December 1996 and in revised form 5 March 1997.
1 Abbreviations used in this paper: DP, double positive; pTWe thank Diane Mathis for critical review of the manuscript.
This work was supported in part by the Institut National de la Santé et Recherche Médicale, (Paris), and by the Faculté Necker Enfants Malades, Déscartes Université (Paris). J. Buer is supported by a grant from the Deutsche Forschungsgemeinschaft. I. Aifantis is a recipient of a Biotechnology grant from the European Commission. J.P. DiSanto is supported by a grant from the Association Pour La Recherche Contre Le Cancer. H. von Boehmer is supported by the Institut Universitaire de France. The Basel Institute for Immunology is supported by Hoffman-La Roche (Basel).
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