By
From the Max-Planck-Institut für Immunbiologie, D-79108 Freiburg, Germany
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Abstract |
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During thymocyte development, the clonotypic
-T cell receptor (TCR) is preceded by
sequentially expressed immature versions of the TCR-CD3 complex: the pre-TCR, containing a clonotypic TCR-
chain and invariant pre-T
, is expressed on pre-T cells before rearrangement of the TCR-
locus. Moreover, clonotype-independent CD3 complexes (CIC)
appear on pro-T cells before VDJ rearrangements of TCR-
genes. The pre-TCR is known
to mediate TCR-
selection, the prerequisite for maturation of CD4
8
double negative
(DN) thymocytes to the CD4+8+ double positive stage. A developmental function of CIC has
so far not been delineated. In mice single deficient and double deficient for CD3
/
and/or
p56lck, we observe a pronounced reduction in the proportions of CD25+ DN thymocytes that
express intracellular TCR-
chains. TCR-
transcripts are reduced in parallel with TCR-
polypeptide chains whereas no reduction in TCR-
locus rearrangements could be detected. Wild-type levels of TCR-
transcripts and of cells expressing TCR-
polypeptide chains are
induced by treatment with anti-CD3
mAb. The data suggest that the initial expression of rearranged TCR-
VDJ genes in pro-T cell to pre-T cell progression is dependent on CD3
complex signaling, and thus define a putative developmental function for CIC.
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Introduction |
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During T cell ontogeny, the complete -TCR first
appears on the cell surface during the CD4+8+ double positive (DP)1 stage of thymocyte development, after
VDJ rearrangements of both the TCR-
and the TCR-
gene loci. Its initial function is the screening of DP cells for
proper recognition of MHC-peptide complexes in the
thymic microenvironment, thus selecting the MHC-
restricted, self-tolerant repertoire of peripheral T lymphocytes (reviewed in references 1, 2). The mature
-TCR is
preceded by the pre-TCR, expressed on CD4
8
double
negative (DN) thymocytes that have rearranged TCR-
VDJ genes and before rearrangement of the TCR-
locus.
Accordingly, the pre-TCR consists of a TCR-
chain, a
surrogate TCR-
chain termed pre-T
, and components
of the CD3 complex. It serves as a screening device for
productive rearrangement of a TCR-
VDJ gene, a process termed TCR-
selection. Successful DN cells survive, proliferate, and mature to the DP stage (for review see references 3).
Expression of the pre-TCR on the thymocyte surface is
preceded by expression of CD3 complexes that lack clonotypic components, so-called clonotype-independent CD3
complexes (CIC). This has first been recognized in experiments in which immature DN thymocytes were exposed
to anti-CD3 mAb, either in thymic organ culture (7, 8) or
in vivo (9, 10). In mice genetically unable to generate a
TCR-
chain (8), treatment with anti-CD3
induced most known pre-TCR-dependent developmental responses.
In wild-type (wt) mice treatment with anti-CD3
induced
premature shutdown of TCR-
gene rearrangement,
equivalent to allelic exclusion (7). The latter experiments
suggested that CD3
is expressed at the surface of thymocytes of wt mice before TCR-
VDJ rearrangements (3, 7). The biochemical composition of CIC has first been studied by Wiest et al. (11) who reported the existence of
CD3
and CD3
dimers that are brought to the surface
of TCR-
negative immature thymocytes together with
calnexin, due to a leaky endoplasmic reticulum retention
mechanism in these cells (12). The CD3
chain is physically present in CIC (11) but not required for pre-TCR
function, as indicated by the undisturbed TCR-
selection in CD3
-deficient mutant mice (13). CD3
is functionally
associated with the pre-TCR (14) but its involvement with
CIC has not been obvious (15). CD3
cross-linking studies
have suggested a role for the src family protein tyrosine kinase p56lck (Lck) in CIC signaling (15).
A function for CIC in normal thymocyte differentiation
has so far not been recognized. On the contrary, the nature
of the block in thymic development in CD3-deficient
mice (16) has argued against a functional role of CIC: the
DN stage of thymocyte development can be divided by the
marker antigens CD44 and CD25 into four consecutive
subsets: CD44+CD25
, CD44+CD25+, CD44
CD25+,
and CD44
CD25
(17). TCR-
locus VDJ rearrangement begins during the CD44+CD25+ stage and peaks at
the CD44
CD25+ stage (18, 19). TCR-
polypeptide
chains are first detected in CD44
CD25+ DN cells (20,
21). TCR-
positive cells proceed to the CD44
CD25
stage and subsequently become DP cells (20). The
block in thymocyte development in CD3
-deficient mice
lies between the CD44
CD25+ and the CD44
CD25
stages and has thus been indistinguishable from that in mice deficient for RAG1/2 or for several other components required for a functional pre-TCR (reviewed in reference
24). This has been taken as evidence against a functional
role of CD3 complexes before the expression of the TCR-
chain and the formation of the pre-TCR.
In this paper, we report experiments on several strains of
mutant mice with graded defects in CD3 complex signaling, suggesting a possible functional role for CIC. In mice
deficient for Lck (25), or for CD3/
(26), the generation of up to 15% of the wt number of DP thymocytes suggests residual pre-TCR/CD3 signaling activities. A drastic
further reduction in DP cells in mice double deficient for
CD3
/
and Lck suggests a more pronounced impairment in pre-TCR function with both defects combined. In addition, mice single deficient or double deficient (sd or dd, respectively) for CD3
/
and/or Lck have moderately or severely reduced numbers, respectively, of DN thymocytes
with intracellular TCR-
polypeptide chains. TCR-
VDJ
transcripts are reduced in parallel with TCR-
polypeptide
chains whereas no reduction in TCR-
locus rearrangements could be detected. Stimulation with anti-CD3
mAb
restores TCR-
expression in thymocytes of mice double
deficient for CD3
/
and Lck to the wt level. The results
suggest that expression of rearranged TCR-
VDJ genes in
pro-T cells requires signals from a functional CD3 complex, possibly assigning a developmental function to CIC.
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Materials and Methods |
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Mice.
BALB/c mice, mice deficient of p56lck (25), and mice deficient of CD3mAbs and Flow Cytometry.
The following mAbs, unlabeled or labeled with either FITC, phycoerythrin or biotin, were purchased from PharMingen (San Diego, CA): anti-CD4 (H129.19), anti-CD8 (53-6.7), anti-TCR-Treatment with Anti-CD3 mAb.
Reaggregate Thymic Organ Culture.
Reaggregate thymic organ culture (RTOC) were set up essentially as previously described (31, 32). To test the effect of anti-CD3Analysis of TCR- Gene Rearrangements by Semiquantitative
PCR.
Estimation of TCR- VDJC mRNAs by Semiquantitative Reverse
Transcription PCR.
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Results |
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Previous results have shown that mice single deficient for CD3 /
(
-sd) or Lck (Lck-sd) generate ~5-15% of the wt number of DP cells (25). As shown by the data in Fig. 1 (top),
the development of DP thymocytes is virtually completely
blocked in mice double deficient of CD3
/
and Lck (
/
Lck-dd). As previously shown, CD25 is maintained on
some of the DP cells in the single deficient mice (24, 25). A
detailed characterization of the block in pre-TCR-dependent thymic development in
/Lck-dd mice has recently
been reported (Würch, A., J. Biro, I. Falk, and K. Eichmann, manuscript submitted for publication).
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This paper is concerned with events before the formation of the pre-TCR, i.e., with factors that control the initial expression of TCR- VDJ genes. The experiments
were stimulated by the unexpected observation that total
DN thymocytes of single deficient and in particular of
/Lck-dd mice contain reduced proportions of cells with intracellular TCR-
chains (Fig. 1, middle). As previously shown,
thymocytes harboring intracellular TCR-
polypeptide
chains first appear among CD44
CD25+ DN cells (20,
21). Whereas typically 40-50% of CD44
CD25+ cells are
TCR-
+, this proportion increases to >90% in the
CD44
CD25
subset, due to TCR-
selection. Accordingly, with considerable individual variation,
50% of
the DN thymocytes of adult wt mice may consist of
CD44
CD25
cells. In contrast, as the formation of this
subset is pre-TCR-dependent, it is drastically reduced in
single deficient mice and in
/Lck-dd mice (Fig. 1, top,
and Würch, A., J. Biro, I. Falk, and K. Eichmann, manuscript submitted for publication). Therefore, we compared TCR-
expression of wt and mutant mice by excluding
CD44
CD25
DN cells from the analysis, i.e., by gating
on DN cells positive for CD44 and/or CD25 (Fig. 1, bottom). For the sake of convenience, cells gated in this way
will be referred to as CD25+ DN cells, accounting for
>85% of this subset in wt and mutant mice. In both kinds
of single deficient mice, the proportions of TCR-
+ cells
among CD25+ DN cells are <20% compared with >40%
in wt mice. A more pronounced reduction of TCR-
+
cells is seen in the CD25+ DN cells of
/Lck-dd mice,
ranging from 4-10% in individual mice. In addition, the
intensity of intracellular TCR-
staining is decreased, particularly in thymocytes of
/Lck-dd mice. These results
suggest that mice with compromised CD3 signaling possess reduced numbers of TCR-
+ CD25+ DN thymocytes.
The extent of the reduction appears to be proportional to
the severity of the defect in CD3 signaling.
The reduced number of TCR-+
CD25+ DN cells in
/Lck-dd mice may in principle be
due to a reduced generation rate, a reduced proliferation
rate, or an increased death rate of cells in this subset, or any
combination of these factors. A reduced rate of generation
of TCR-
+ thymocytes in mice deficient in CD3 complex
signaling may be due to compromised TCR-
locus VDJ
rearrangement. Semiquantitative analyses of DJ and VDJ
rearrangements in purified CD25+ DN thymocytes of wt,
-sd, Lck-sd, and
/Lck-dd mice are shown in Fig. 2. If reduced TCR-
rearrangements were responsible for the
low numbers of TCR-
+ CD25+ DN cells in single deficient and
/Lck-dd mice, this should be reflected in weaker
PCR signals. In particular, signal intensities of
/Lck-dd thymocytes should be 5-10-fold weaker than that of wt
CD25+ DN thymocytes, i.e., shifted by at least one dilution step. The data do not support this possibility, as they
fail to reveal significant quantitative differences between
the four strains of mice. There are minor quantitative variations, but these vary between experiments and may reflect
individual and/or technical variability. Within the limits of
this semiquantitative method, TCR-
DJ and VDJ rearrangements appear to proceed normally in thymocytes of
mice deficient in CD3 complex signaling.
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If the paucity of TCR-+ DN
cells in single deficient and
/Lck-dd mice was in any way
related to the deficiency in CD3 signaling it should be possible to increase the number of TCR-
+ DN cells by stimulating the thymocytes with anti-CD3
mAb. Previously we and others have shown that treatment of thymocytes of
various pre-TCR mutant mice with anti-CD3
mAb leads
to thymocyte proliferation and differentiation to the DP
stage (8, 15, 38). Although in these previous experiments no attempts were made to separate proliferation
from differentiation, it seemed desirable for the present experiments to distinguish the putative anti-CD3
induced differentiation to TCR-
+ cells from the expansion of
the preexisting TCR-
+ population through anti-CD3
induced proliferation. If an increase in the size of the
TCR-
+ subset depended on proliferation, the paucity of
TCR-
+ cells would most likely be due to insufficient expansion. An increase in the size of the TCR-
+ subset independent of proliferation would support the possibility that CD3-mediated signals were involved in the control of
TCR-
expression.
To distinguish these possibilities, we titrated the amounts
of anti-CD3 injected per
/Lck-dd mouse (data not
shown) and identified a low dose that induces differentiation without increasing the spontaneous proliferation of
thymocytes. The absence of additional proliferation is documented in Fig. 3, which compares the BrdU incorporation into thymocytes of
/Lck-dd mice during 3 d, untreated or after injection of 15 µg anti-CD3
. We observe
three distinct populations in untreated mice: cells with
maximal BrdU labeling have passed two or more S phases,
cells with submaximal labeling have passed one S phase,
and cells without label have not divided. In mice injected
with anti-CD3
the vast majority of cells show homogeneous submaximal label. The data suggest that thymocytes of untreated mice divide heterogeneously and asynchronously with a mean division rate of approximately once per
3 d. Injection of 15 µg anti-CD3
synchronizes cell division such that almost all of the cells divide once during the
3 d. Accordingly, total thymocyte numbers of injected
mice remain within the range observed for untreated mice.
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Fig. 4 shows experiments in which /Lck-dd mice were
injected with 15 µg anti-CD3
on day 0 and their thymocytes analyzed for CD4 and CD8 and for intracellular
TCR-
on each of three subsequent days. The proportion
of TCR-
+ DN cells increases slightly but not significantly
above the range observed in untreated mice. However, beginning with day 2 we observe the appearance of DP cells
containing increasing proportions of TCR-
+ cells, reaching >80% on day 3. Since DP thymocytes on day 3 account for ~50%, >40% of all thymocytes are now TCR-
+. As virtually all cells have divided once during the 3 d,
this increased proportion of TCR-
+ cells cannot have
arisen by proliferation of the preexisting TCR-
+ population. These data therefore suggest that cross-linking of CD3
induces de novo expression of intracellular TCR-
polypeptide chains in
/Lck-dd thymocytes.
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From the data in Fig. 4 it appears that induction of
TCR- expression by anti-CD3
in
/Lck-dd thymocytes
occurs mostly after the cells acquire the DP phenotype.
Since this would indicate a reversal of the physiological order of events, it was of interest to test TCR-
induction by
anti-CD3
in subpopulations of DN thymocytes. As shown
in Fig. 5, untreated
/Lck-dd mice generate very few CD44
CD25
cells. However, TCR-
+ cells are enriched
in this population suggesting rudimentary TCR-
selection (Würch, A., J. Biro, I. Falk, K. Eichmann, manuscript submitted for publication). The CD44
CD25
population
increases rapidly on days 1 and 2 after anti-CD3
, i.e., before the appearance of significant numbers of DP cells (compare to Fig. 4, data on the same individual mice).
These CD44
CD25
DN cells contain enhanced proportions of TCR-
+ cells, suggesting an induction of TCR-
expression before acquisition of the DP stage, at least in a
proportion of cells. Nevertheless, little TCR-
induction is
seen in CD25+ DN cells, suggesting that stimulation with
anti-CD3
induces TCR-
expression and subsequent differentiation in rapid succession.
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The experiment in Fig. 5 was complemented by DNA
staining in order to assess anti-CD3 induced alterations in
cell cycle in DN subpopulations, as well as putative apoptotic effects. Thymocytes with subdiploid DNA content
are not detected in either untreated or treated mice, arguing against the possibility that the observed effects are influenced by massive cell death. The CD25+ DN population
contains a small proportion of cells in S/G2/M that does
not vary after injection of anti-CD3
. The few CD44
CD25
cells in untreated
/Lck-dd mice contain more
than twice as many cells in S/G2/M, consistent with the
maintenance of limited TCR-
selection. Whereas the
number of CD44
CD25
cells increases after injection of
anti-CD3
, a transient decrease in the proportion of CD44
CD25
cells in S/G2/M is seen on day 1. This may indicate a transient cell cycle arrest, possibly the basis for the
anti-CD3
induced synchronization of proliferation suggested by the data in Fig. 3. These data exclude the possibility that injection of 15 µg anti-CD3
selectively stimulates proliferation in the CD44
CD25
population and
further support our conclusion that induction of TCR-
expression in
/Lck-dd thymocytes is independent of proliferation.
Even though /Lck-dd mice generate minimal numbers
of peripheral CD3 positive cells (data not shown), it was
possible that in vivo injection of anti-CD3
mAb induces
such cells to release cytokines that influence thymocytes
(39), possibly including the induction of TCR-
in the
thymus. To exclude this possibility, RTOC were set up
with
/Lck-dd thymocytes preincubated with anti-CD3
or HIg for 2 h in suspension, then extensively washed, and
thereafter reaggregated with thymic stroma cells. As shown
in Fig. 6, after 3 d of RTOC such anti-CD3
-treated
/Lck-dd thymocytes contain increased proportions of TCR-
+
cells, some of which have acquired CD4/CD8 expression.
Induction is less efficient than in vivo, most likely owing to
the brief exposure to the antibody. After RTOC, HIg-treated (and untreated, not shown) thymocytes also have a
slightly enhanced TCR-
expression compared with ex
vivo cells, perhaps due to experimental handling. The data
suggest that TCR-
induction occurs by direct ligation of
CD3
on the surface of
/Lck-dd thymocytes.
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The levels of TCR- VDJC mRNAs
were estimated by semiquantitative RT-PCR. Purified
DN and DP thymocytes of wt mice, DN cells of untreated
/Lck-dd mice, and DN and DP thymocytes of
/Lck-dd mice 3 d after injection of 15 µg anti-CD3
mAb, were
analyzed. The results in Fig. 7 reveal ~10-fold lower V
8
transcript levels in DN cells of
/Lck-dd mice compared
with wt DN cells. Slightly less pronounced differences are
seen for V
5 transcripts. Injection of anti-CD3
induces
V
8 and V
5 mRNAs in DN cells nearly to wt levels, and
the DP cells induced in anti-CD3
treated
/Lck-dd mice
display wt levels of V
8 and V
5 mRNAs as well. The
data exclude the possibility that TCR-
expression in
/Lck-dd mice is limited by some form of translational or posttranslational regulation. The good correlations between
levels of TCR-
VDJC mRNAs and the proportions of
cells expressing TCR-
polypeptide chains suggest that
TCR-
expression in the thymus of
/Lck-dd mice is impaired at the mRNA level.
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Discussion |
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The work presented in this paper was stimulated by the
phenotype of /Lck-dd thymocytes which features a drastic
reduction in the numbers of TCR-
+ CD25+ DN cells
and reduced TCR-
mRNA levels in this population.
This was unexpected as a pre-TCR-associated block in
thymocyte development should leave maturation up to the
TCR-
+ CD25+ DN stage unperturbed. Experiments
were performed to link this phenotype to the defect in
CD3 signaling in
/Lck-dd mice, and to distinguish among
three nonmutually exclusive mechanisms that could account for this phenotype: a block in the generation, a block
in the proliferation, or a shorter lifetime of TCR-
+ cells.
A causal relationship between defective CD3 complex
signaling and the paucity of TCR-+ cells in the mutant
mice is suggested by two lines of evidence. First, the extent
in reduction of TCR-
+ cells appears to reflect the severity
in the malfunction of the CD3 complex. Whereas mice
single-deficient for CD3
/
or for Lck possess ~50% of
the wt number of TCR-
+ CD25+ DN cells, the number
further drops to ~10% in
/Lck-dd mice. It is not excluded that Lck and perhaps also the CD3
/
module may
have roles in other signaling pathways in immature thymocytes, but the aggravation associated with the combined
deficiency strongly suggests the involvement of the CD3
complex. Second, the reduction in TCR-
+ cells including
TCR-
mRNA levels is corrected by treating
/Lck-dd mice with anti-CD3
mAb. We excluded that the induction is mediated by long range in vivo effects, suggesting a
direct induction via CD3
on the thymocyte surface.
A defect in the generation of TCR-+ cells in
/Lck-dd
mice would suggest a role for CD3 complexes before the
appearance of TCR-
chains, i.e., clonotype-independent
CD3 components. In contrast, a block in proliferation or a
shortened survival time of TCR-
+ cells would indicate
regulation by the pre-TCR-associated CD3 complex.
Therefore, it is important to discriminate between these alternatives. Thymocytes of untreated
/Lck-dd mice proliferate asynchronously such that about one-fourth divides
more than once, one-half divides just once, and one-fourth
does not divide during 3 d. The mean rate of division thus
appears to be once in 3 d, i.e., 3 d appears to be the mean
turnover period for thymocytes of untreated
/Lck-dd
mice. By titration of anti-CD3
we identified a low dose at
which proliferation and turnover appear to be synchronized but not quantitatively altered. The subset of TCR-
+ cells increases selectively ~10-fold in
/Lck-dd mice
treated with this dose of anti-CD3
, whereas the total thymic cellularity remains within the range of untreated mice.
Synchronization extends to the CD44
CD25
cells, excluding selective proliferation in the induced cell population. These data prove that the numerical increase of the TCR-
+ subset after injection of anti-CD3
is independent of proliferation. As a second possibility, the increased
proportion of the TCR-
+ subset after anti-CD3
may be
caused by a selective increase in longevity of the TCR-
+
cells in
/Lck-dd mice. As outlined above, cells turnover
once during 3 d so that full survival of all TCR-
+ cells
may result in an increase of merely twofold within 3 d after
injection of anti-CD3
. Thus, in order to account for the observed proportion of >40% TCR-
+ cells after 3 d, the
numbers of TCR-
negative cells would have to be reduced at least fourfold, which would require substantial cell
death and result in a significant decrease in thymic cellularity. Such a scenario is not consistent with our data which
therefore argues against the possibility that anti-CD3
increases the number of TCR-
+ cells by increasing their
longevity. Assuming that the anti-CD3
treatment corrects
the physiological defects associated with CD3 malfunction,
these results argue against the possibility that the paucity of
TCR-
+ cells in
/Lck-dd mice is mainly caused by impaired proliferation or a reduced survival time. Hence, the
paucity of TCR-
+ cells in
/Lck-dd mice appears to be
mainly a consequence of the impaired generation of cells
that express intracellular TCR-
polypeptide chains.
Among the biosynthetic levels at which the generation
of TCR-+ cells could be compromised we considered the
rearrangement of TCR-
VDJ genes and their expression
at the mRNA levels. An astounding fact in lymphopoiesis
has been the striking parallelism in the early phases of T and
B cell development, including the molecular design and the
selection processes mediated by the pre-TCR and the pre-BCR (40). Recent data by Gong and Nussenzweig (41) have shown that the Ig
signaling component of the BCR
is required for rearrangement of the Igµ chain genes in immature B cells. In contrast, we could not detect evidence
for impaired TCR-
rearrangements in the CD3 signaling-deficient mice studied in this report. It should be pointed
out that we used a semiquantitative method to estimate the
frequencies of TCR-
rearrangement, so that moderate quantitative differences might have been missed. Nevertheless, using similar semiquantitative technology, we see
striking quantitative differences between TCR-
VDJC
mRNA levels of wt and
/Lck-dd mice. Indeed, the
mRNA levels fully account for the differences in the proportions of TCR-
+ cells in wt and
/Lck-dd mice.
Therefore, we suggest that the paucity of TCR-
+ cells in
/Lck-dd mice is predominantly caused by a block in the
expression of rearranged TCR-
VDJ genes at the mRNA
level. So far we do not know whether regulation takes
place at a transcriptional or posttranscriptional level. Other
examples for a control of mRNA levels by CD3 signaling
in immature thymocytes include the RAG genes (42) and
germline transcripts of TCR-
(43) and TCR-
genes
(42).
It cannot be excluded that TCR- chains are expressed
in most
/Lck-dd thymocytes at a level below detection by
intracellular staining. If this were the case, a role for the
pre-TCR in TCR-
expression could be rescued by assuming that a low basal level of TCR-
chains is synthetized CD3-independently, giving rise to the formation of a
few pre-TCR complexes on the cell surface; subsequent
full TCR-
expression requires positive feedback by signaling through the pre-TCR. In the absence of experimental support for such a scenario, we think that a more plausible interpretation of the present experiments is that the
CD3-dependent signals inducing TCR-
expression in
immature thymocytes are generated by CIC. CIC may thus
represent developmentally relevant CD3 complexes. As
CIC are expressed on thymocytes at the pro-T cell stage
we suggest the term pro-TCR, in analogy to the pre-TCR. It remains to be elucidated in which way the pro-TCR generates the signals required for TCR-
expression.
Speculations with regard to a ligand for the pre-TCR have
been discussed at length (40, 44) and the arguments, which
apply to the pro-TCR as well, shall not be repeated here.
At present the evidence appears to favor the possibility that
the immature forms of the TCR may generate signals
without a requirement for ligand recognition (45). Additional open questions include whether the pro-TCR contains other components, in addition to those of the CD3
complex. An obvious candidate is pre-T
(46), which has
been shown to be present at the mRNA level in pro-T cells (47). A surrogate TCR-
chain may also be considered, as well as other proteins that have been found associated with the pre-TCR (48, 49).
In /Lck-dd mice, a small residual CD3 signaling activity
is suggested by a small amount of pre-TCR-dependent
TCR-
selection spontaneously taking place. Recent work
on the characterization of mice double deficient for the src-family protein tyrosine kinases Lck and Fyn has revealed
that both are involved in pre-TCR signaling (50, 51). Although Lck seems to be part of the major pathway, Fyn can
partially replace Lck in its absence. Therefore, it is reasonable to assume that the small residual CD3 signaling activity, as well as the activation of
/Lck-dd thymocytes by
anti-CD3
, is mediated by a cooperation between CD3
and Fyn. Although this activity is insufficient for the generation of significant numbers of DP cells, it suffices to mediate ~10% of wt TCR-
expression. This finding indicates
a relatively low signaling requirement of TCR-
expression, and is consistent with a hypothesis in which successive
CD3-dependent steps in thymocyte development require stepwise increasing signal intensities (52). It would be of interest to study TCR-
expression in the thymus of mice in
which CD3 signaling is totally abolished.
What could be the biological purpose of a mechanism
that places TCR- expression under the control of CD3
signaling? Optimal CD3 signaling requires expression of
multiple gene products including those of the CD3 complex proper, downstream elements such as protein tyrosine
kinases of the src and syk families, as well as other known
and unknown factors. It is possible that the correct timing in the activation of the large array of involved genes is not free of errors. Placing TCR-
expression under the control
of CD3 signaling would provide an elegant mechanism for
a selective recruitment of signal-competent cells into the
developmental process, and for excluding signal-incompetent cells from this process. Future studies will have to test
the hypothesis that ordered and complete expression of the
CD3-associated signaling system may be error prone. If so,
TCR-
expression would define a novel CD3-dependent checkpoint in early thymocyte development, preceding
TCR-
selection.
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Footnotes |
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Address correspondence to Klaus Eichmann, Max-Planck-Institut für Immunbiologie, Stübeweg 51, D-79108 Freiburg, Germany. Phone: 49-761-5108-541; Fax: 49-761-5108-545; E-mail: eichmann{at}immunbio.mpg.de
Received for publication 31 July 1998.
This paper was written while K. Eichmann was a Scholar-in-Residence at the Fogarty International Center for Advanced Study in the Health Sciences, National Institutes of Health, Bethesda, Maryland.We thank Drs. B. and M. Malissen for mice; Drs. A. Singer, P. Love, and I. Haidl for critical reading of the manuscript; and Mr. H. Kohler and Ms. P. Wehrstedt for able technical assistance.
Abbreviations used in this paper BrdU, 5-bromodeoxyuridine; CIC, clonotype-independent CD3 complex; dd, double deficient; DN, double negative; DP, double positive; HPRT, hypoxanthine phosphoribosyltransferase; Lck, p56lck; RT, reverse transcription; RTOC, reaggregate thymic organ culture; sd, single deficient; wt, wild-type.
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References |
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