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
The pre-T cell receptor (TCR) associates with CD3-transducing subunits and triggers the
selective expansion and maturation of T cell precursors expressing a TCR-
chain. Recent experiments in pre-T
chain-deficient mice have suggested that the pre-TCR may not be required
for signaling allelic exclusion at the TCR-
locus. Using CD3-
- and CD3-
/
-deficient
mice harboring a productively rearranged TCR-
transgene, we showed that the CD3-

and CD3-
/
modules, and by inference the pre-TCR/CD3 complex, are each essential for
the establishment of allelic exclusion at the endogenous TCR-
locus. Furthermore, using
mutant mice lacking both the CD3-
and CD3-
/
genes, we established that the CD3 gene
products are dispensable for the onset of V to (D)J recombination (V, variable; D, diversity; J,
joining) at the TCR-
, TCR-
, and TCR-
loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-
locus first accessible to, and later
insulated from, the action of the V(D)J recombinase.
 |
Introduction |
Tcells can be divided into two subsets based on the
structure of their TCR. In the adult mouse, most T
cells express a TCR heterodimer consisting of
and
chains, whereas a minor population expresses an alternative
TCR isoform made of
and
chains. Each of these four
TCR chains includes a clonally variable (V)1 region encoded by genes that are assembled via somatic site-specific DNA recombination reactions. These reactions, termed
V(D)J rearrangements (D, diversity; J, joining), result in the
random recombination of V and J gene segments in TCR-
and TCR-
chain genes, and of V, D, and J gene segments
in TCR-
and TCR-
genes. V(D)J joining reactions
may result either in productive rearrangements that maintain an open reading frame throughout the gene, or in
an out-of-frame nonfunctional gene. Because T lymphocytes are diploid cells, this recombination process could, in
principle, generate T cell clones expressing two productively rearranged TCR alleles and therefore more than one
TCR-
/
or TCR-
/
chain combinations. In the mouse, the expression of a productively rearranged TCR-
chain
transgene has been shown to prevent complete V-(D)J rearrangement of endogenous TCR-
genes (1), and this has
led to the assumption that
/
T cell precursors have developed feedback inhibition mechanisms to ensure that
most mature T cell clones express one, and only one,
TCR-
/
chain combination. These mechanisms are referred to as allelic exclusion.
Intrathymic T cell development proceeds through discrete stages that can be defined on the basis of the configuration of TCR gene loci, and the expression of surface markers such as CD4 and CD8. Accordingly, the most immature
thymocytes express neither CD4 nor CD8 and are called
double negative (DN) cells. Late DN cells can mature into
CD4+CD8+ (double positive, DP) cells, a small percentage
of which develop further into CD4+CD8
or CD4
CD8+
(single positive, SP) cells. Based on the expression of CD25 and CD44, DN cells have been subdivided further and
shown to develop according to the following maturation
sequence: CD44+CD25
CD44+CD25+
CD44
/low
CD25+
CD44
/lowCD25
(2). TCR-
gene rearrangements precede rearrangements at the TCR-
locus and
proceed in two separate steps involving an initial D
J
joining event and a subsequent V
DJ rearrangement.
TCR-
gene rearrangements start at, or at the transition
to, the CD44
/lowCD25+ DN stage (2, 3), whereas the first
measurable TCR-
rearrangements occur during, or immediately after, the transition to the DP stage (4, 5). When
maturing T cells fail to rearrange their TCR genes, rearrange them nonproductively, or express TCR-
/
combinations with inappropriate specificities, they are generally
arrested at discrete developmental control points (see reviews in references 6 and 7). Molecular sensors have
evolved to couple the transition through these control
points to the attainment of certain landmark events in T
cell development. For instance, one of these sensors, known as the pre-TCR, operates at the CD44
/lowCD25+
DN stage and couples further maturation to the prior
achievement of productive TCR-
gene rearrangements.
In the pre-TCR, TCR-
is disufilde linked with a
polypeptide encoded by a nonrearranging gene and denoted as the pT
chain (8). To exert its function, the pre-TCR needs to associate with both the CD3-
/
and CD3-
dimers (9), and signal via the protein tyrosine kinases
lck and fyn (14). It has been proposed that the pre-TCR/CD3 complex triggers the selective proliferation of
TCR-
+ DN cells and concurrently drives their progression to the DP developmental stage (such transition is often
denoted as TCR-
selection). Moreover, considering that
the expression of a productively rearranged TCR-
transgene inhibits most endogenous V
to D
J
rearrangements (see above), it has been suggested that the TCR-
chain, and by extension the pre-TCR/CD3 complex, plays
a pivotal role in the enforcement of allelic exclusion at the
TCR-
locus. Therefore, disruption of the gene coding
for the pT
subunit should have prevented assembly of a
functional pre-TCR complex and affected the establishment of allelic exclusion at the TCR-
locus. However, in
pT
/
thymocytes, expression of a transgene coding for a
functional TCR-
chain was found to inhibit endogenous
V
to D
J
rearrangements to almost the same extent as in
a pT
+/+ background (18, 19). Assuming that no other
gene products can compensate for the loss of pT
(e.g., the
products of prematurely expressed TCR-
genes; reference 20), these data are inconsistent with the suggestion
that the pre-TCR/CD3 complex is involved in signaling
allelic exclusion at the TCR-
locus.
We have previously generated mice with a targeted mutation of the CD3-
gene (referred to as CD3-
5; reference 12). This mutation abolishes the expression of intact CD3-
polypeptides, dramatically reduces the transcription
rate of the neighboring CD3-
and CD3-
genes, and totally blocks the progression beyond the CD44
/lowCD25+
stage. The thymocytes found in CD3-
5/
5 mice contain
readily detectable levels of CD3-
, TCR-
, and pT
transcripts. However, the lack of CD3-
/
and CD3-
/
dimers is likely to prevent their pT
-TCR-
and CD3-
2
dimers from participating in the assembly of functional pre-TCR/CD3 complexes. The CD3-
5/
5 mice present several experimental advantages relative to pT
-deficient mice. First, their thymuses constitute an enriched source of
CD44
/lowCD25+ DN cells devoid of contaminating
downstream
/
T cell subsets and in which TCR-
gene
rearrangements do happen normally. Second, the CD3-
5
mutation does prevent the development of
/
T cells and
permits the analysis of early
/
T cell development in a
microenvironment insulated from the adventitious effects
resulting from the presence of
/
T cells (2, 20, 21).
Therefore, by obviating some of the experimental limitations associated with pT
/
mice, the CD3-
5/
5 mice
constitute a particularly appropriate model to determine whether the CD3-

module, and by inference the pre-TCR, is essential for the establishment of allelic exclusion
at the TCR-
locus. Here we report on experiments
showing that the CD3-

and the CD3-
/
modules of
the pre-TCR play each a pivotal role in allelic exclusion at
the TCR-
locus. In contrast, analysis of CD3-
5/
5
CD3-
/
/
double mutant mice established that the onset of V to (D)J recombination at the TCR-
, TCR-
,
and TCR-
loci can occur in the absence of CD3 subunits.
 |
Materials and Methods |
Mice.
The CD3-
5/
5 mice and CD3-
/
/
mice have
been described (12, 22). Recombination activation gene (RAG)-
1
/
mice were originally obtained from E. Spanopoulou (The
Rockefeller University, New York; 23). The P14 TCR-
transgenic mice (line 128) express a TCR-
cDNA (V
8.1-D
-J
2.4) derived from the T cell clone P14 (24). TCR-
transgenic
mice were typed for the presence of the transgene by PCR analysis of tail DNA. TCR-
transgenic mice were crossed with CD3-
5 and CD3-
/
-deficient mice to obtain CD3-
5/
5 TCR-
and CD3-
/
/
TCR-
mice. CD3-
5/
5 CD3-
/
/
double-deficient mice were derived from CD3-
5/
5 × CD3-
/
/
matings. Mice were housed in a specific pathogen-free animal facility in accordance with institutional guidelines. Mice were between 4 wk and 3 mo old when analyzed.
Antibodies and Flow Cytometry.
Biotinylated, FITC-, or PE-conjugated antibodies against CD3-
(2C11), CD4 (H129.19),
CD8 (53-6.7), CD25 (7D4), CD44 (Pgp-1), and TCR V
8 (F23.1)
were purchased from PharMingen (San Diego, CA). Biotinylated
antibodies against Mac-1 (M1/170), B220 (RA3-6B2), and Gr-1
(RA6-8c5) were from CALTAG Labs (Tebu, Le Perray en YveLines, France). Biotinylated antibodies were revealed with streptavidin tricolor (CALTAG Labs.). Cells were stained with saturating levels of
antibodies and 5-50 × 103 events (gated on forward and side
scatter) were acquired using a FACScan® flow cytometer (Becton
Dickinson, Mountain View, CA) and analyzed with Lysis II software.
Isolation of CD25+ Thymocytes.
CD25+ thymocytes were sorted
using a FACStar Plus®. Before sorting, thymus cell suspensions
were enriched for CD4
CD8
CD3
cells by one round of complement-mediated killing with a mixture of IgM anti-CD4 (clone
RL172.4), IgM anti-CD8 (clone 31M), and IgG2b anti-CD3
(clone 17A2) antibodies. Viable cells were retrieved by a density
cut using Ficoll-paque (Pharmacia, Orsay, France), stained with
propidium iodide and an FITC-conjugated anti-CD25 antibody,
and sorted for CD25high cells.
Intracellular Staining.
Expression of the transgenic TCR-
chain within the CD25+-thymocyte subset was assessed by intracellular/extracellular staining of thymocytes. Cells were first
stained for CD25 as described above. After washing in PBS supplemented with 3% FCS (PBS/FCS), cells were fixed in PBS plus
4% paraformaldehyde for 20 min at room temperature, followed
by two washing steps in PBS. Cells were then permeabilized in
PBS/FCS containing 0.1% saponin (Roth, Lauterbourg, France)
for 10 min at room temperature. Intracellular staining with a PE-conjugated anti-V
8 (MR5.2) antibody diluted in PBS/FCS plus
0.1% saponin was performed for 20 min at room temperature and
followed by three washing steps on a rocking platform using
PBS/FCS plus 0.1% saponin. Finally, cells were resuspended in
PBS and analyzed on a FACScan®.
RNA-PCR Amplification.
RNA samples were extracted from
total (CD3-
5/
5 and CD3-
5/
5 TCR-
samples) or CD25high
(sorted from the CD3-
+/+ TCR-
sample) thymocytes using
TRIzolTM (GIBCO BRL, Cergy Pontoise, France) as recommended by the manufacturer. Before conversion to cDNA, RNA
samples were treated with DNAseI-RNAse free (Pharmacia). Conversion to cDNA was done on 1 µg of total RNA using the Ready-to-GoTM T-primed first strand kit (Pharmacia). 1/15 of each reaction was used for PCR amplification. The pair of C
2 primers
used to detect transcripts incorporating the TCR C
2 exon was
as described in reference 25. They are denoted as primers 1 and 2 in Fig. 3. The sequences of the other PCR primers used in these
experiments were: CDR3 P14
: 5
-GTGATGCCGGGGGGCGGAACAC-3
; and
-globin 3
UT: 5
-GGGCATTAGCCACACCAGCCACCA-3
, and denoted in Fig. 3 as primers 3 and 4, respectively. The amplified products were analyzed on 1.5% agarose gel, transferred to nylon membrane (Gene Screen
Plus; NEN Life Science Products, LeBlanc Mesnil, France), and hybridized using a 5
-kinased oligonucleotide (C
2A; reference 26).

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Fig. 3.
Assessment of TCR-
transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from
TCR- transgenic wild-type
thymuses (CD3- +/+ TCR- )
and total thymocytes from
TCR- transgenic CD3- 5/ 5
mice (CD3- 5/ 5 TCR- ) were
analyzed for the presence of transcripts originating from the P14
TCR- transgene using the
RNA-PCR strategy depicted in
the bottom diagram. The P14
TCR- cDNA is expressed under the control of the H-2Kb
promotor and IgH chain intronic
enhancer (EH). The 3 end of the
P14 TCR- cDNA is linked to
a genomic fragment of the human globin gene that provides
both an intron and a polyadenylation sequence (24). Owing to
the presence of this intron, primers for PCR amplification can be chosen to distinguish amplification products corresponding to transgene transcription (expected size: 0.8 kb)
from those resulting from adventitious DNA contamination (expected
size: 1.6 kb). Accordingly, an antisense primer specific for the 3 untranslated region of the human globin gene (primer 4) was used in combination with a sense primer (primer 3) straddling the sequence corresponding
to the third complementarity region of the P14 TCR- gene. RNA extracted from nontransgenic CD3- 5/ 5 thymocytes was also included as a
negative control. A second pair of primers (denoted 1 and 2) was used in
parallel to detect both endogenous and transgenic transcripts containing
the TCR C 2 exon. The products resulting from amplification with primer pairs 1 + 2 (TCR C 2) and 3 + 4 (TCR- Tg) were gel fractionated, blotted, and hybridized with a C 2-specific probe (p). The location of
specific primers are indicated by arrowheads and the transcription start
site of the TCR- transgene by an arrow. Control PCR were set up in
parallel using a pair of primers specific for the actin gene to control for
the quantity and quality of RNA in each sample, run on agarose gel, and
revealed by ethidium bromide staining (Actin). (B) The presence of the
transgenic P14 TCR- chain within the CD25+ subset present in CD3- 5 5 transgenic thymocytes was revealed by intracellular staining with
an antibody (F23.1) specific for the V 8 gene segment used by the P14
TCR chain. RAG-1 / thymocytes were also included as negative
controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3- 5/ 5 mice revealed <1% F23.1+ cells.
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Detection of Endogenous TCR Rearrangement.
Total or fractionated thymocytes were solubilized in lysis buffer (50 mM KCl, 10 mM Tris-HCl [pH 8.3], 2 mM MgCl2, 0.45% Nonidet P-40,
0.45% Tween 20, 60 µg/ml proteinase K) at a concentration of
107 cells/ml. After overnight incubation at 56°C, samples were
heated to 95°C for 30 min to inactivate proteinase K. Dilutions of
the template genomic DNA (corresponding to 105, 2 × 104, and
104 cell equivalent per 10 µl) were prepared for each sample to
demonstrate that there is a linear relationship between product
yield and the number of input target sequences. PCR amplifications were done in a final volume of 50 µl and included 10 µl of
template DNA solution, 1 µm of each primer, 200 µm of each
dNTP, 2.5 µl of PCR buffer (166 mM [NH4]2SO4, 670 mM
Tris-HCl [pH 8.8], 1 mg/ml BSA), 0.2 U Taq DNA polymerase
(GIBCO BRL) and MgCl2 at a final concentration of 2.5 mM.
Each cycle consisted of incubation at 94°C for 1 min, followed by
annealing at 63°C for 2 min and extension for 10 min at 72°C,
and was repeated 24 times. 25 µl of the reaction was fractionated
on a 1.0% agarose gel, transferred to nylon membrane (GeneScreen Plus) and hybridized with 32P-labeled oligonucleotide
probes. Hybridizing bands were quantitated using a phosphorimager (BAS 100; Fuji, Raytest France S.A.R.L., France). The oligonucleotide primers used for the analysis of TCR-
chain gene
rearrangement were as described in reference 26. PCR-based analysis of TCR-
and TCR-
chain gene rearrangements was as previously described (27). Before the analysis of the relative levels of
TCR gene rearrangements, the quality and quantity of DNA
present in each sample were checked by amplifying the nonrearranging thritorax gene (Mtrx) using primers MTRX1: 5
-AGGGTAAGCTGTGCTATGG-3
and MTRX2: 5
-AGTAGTGTTTCCTCAGTCCCC-3
.
 |
Results |
A Productively Rearranged TCR-
Transgene Is Unable to
Activate the Transition to the DP Stage in the Absence of CD3-
.
To determine whether the expression of a TCR-
chain
transgene was able to inhibit endogenous V
to D
J
rearrangements in the absence of CD3-
subunit, CD3-
5/
5
mice were crossed with transgenic mice carrying a productively rearranged V
8+ TCR-
chain derived from the
P14 T cell clone (24, 28). When expressed in a wild-type
background, this TCR-
transgene prevented endogenous
-locus gene rearrangements, as judged by the fact that
most of the SP thymocytes developing in these mice were
V
8+ (Fig. 1, compare transgenic [WT TCR-
] and nontransgenic [WT] wild-type panels). As shown in Fig. 1 A,
CD3-
5/
5 TCR-
mice had thymuses that did not develop past the DN stage and contained absolute cell numbers similar to nontransgenic CD3-
5/
5 thymuses. Thus,
expression of the P14 TCR-
transgene was unable to restore T cell development in CD3-
5/
5 mice. To specify
more precisely the effect of the TCR-
transgene on early
T cell development, we analyzed the CD44/CD25 profile of wild-type and CD3-
5/
5 DN thymocytes that developed in the absence or presence of the P14 TCR-
transgene. To this end, we gated on cells that were negative for
CD3, CD4, CD8, B cell- (B220), granulocyte- (Gr-1), and macrophage- (Mac-1) specific markers (2). As shown
in Fig. 2, comparison of DN cells from transgenic (WT
TCR-
) and nontransgenic (WT) wild-type mice indicated
that in the former there was a marked increase in the percentage of CD44
/lowCD25
thymocytes at the expense of
their immediate CD44
/lowCD25+ precursors. This finding
is in line with previous data showing that TCR-
transgenic mice exhibit CD44
/lowCD25+ cell compartments
the size of which are intermediate between those found in
nontransgenic and TCR-
/
transgenic mice (19, 20).
Such observations have been generally accounted for by the fact that CD44
/lowCD25+ cells equipped with a productively rearranged TCR-
transgene progress on average
much more rapidly to the CD44
/lowCD25
stage than
their nontransgenic counterparts (29). Interestingly, the
DN cells found in the CD3-
5/
5 and CD3-
5/
5 TCR-
mice were both arrested at the same CD44
/lowCD25+
stage and lacked not only the CD44
/lowCD25
cells
proper, but also most of the CD44
/lowCD25low to
intermediates. Thus, it is likely that in the absence of CD3-

module, pT
-TCR-
P14 heterodimers were prevented
from assembling into functional pre-TCR complexes and
unable to rescue the blockade in thymic development observed in CD3-
5/
5 mice. Alternatively, the onset of expression of the P14 TCR-
transgene during T cell development may have occurred only after the CD44
/low
CD25+ stage and accounted for its failure to rescue T cell
development in CD3-
5/
5 mice.

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Fig. 1.
A transgene encoding
a productively rearranged TCR-
gene does not restore T cell development in CD3- -deficient
mice. Mice with CD3- +/+
(WT), CD3- +/+ TCR- (WT
TCR- ), CD3- 5/ 5 and CD3- 5/ 5 TCR- genotypes were
derived from a F2 intercross between the TCR- transgenic
line P14 TCR- (TCR- ) and
CD3- 5/ 5 mutant mice. (A)
Thymocytes were analyzed by
flow cytometry for the expression of CD4 versus CD8. The
percentage of cells found in each
quadrant is indicated. (B) Thymocytes were analyzed for the
expression of CD3- and V 8.
Percentage of CD3high and V 8high
cells are indicated. Considering
that the exon coding for the epitope recognized by the 2C11
anti-CD3- antibody has been
deleted in the CD3- 5 mutant
gene, and that CD3- 5/ 5 thymocytes do not express detectable levels of TCR- chain at their surface (12), the histograms obtained after staining CD3- 5/ 5 thymocytes with anti-
CD3- or anti-V 8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-
transgenic SCID mice (50) and TCR- transgenic RAG / mice (51) where transgenic TCR- chains are expressed as monomers without CD3- and
in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR- transgenic chains on the surface of CD3- 5/ 5 TCR- thymocytes
after staining with the C -specific antibody H57.597 (data not shown) and V 8-specific antibody F23.1 (compare the CD3- 5/ 5 and CD3- 5/ 5 TCR- histograms). Whether such difference resulted from the use of the P14 TCR- transgene or is rather due to the CD3- 5/ 5 background remains to be determined.
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Fig. 2.
Comparison of the
triple negative thymocyte subsets
from wild type (WT) mice and
CD3- 5/ 5 mutant mice in the
presence or absence of P14
TCR- transgene (TCR ).
Thymocytes were stained with
anti-CD3, -CD4, -CD8, -B220, -Mac-1, and Gr-1 (all biotinylated and detected with streptavidin tricolor), anti-CD44-PE, and
anti-CD25-FITC. The position
of the window (R1) used to
identify the DN T lineage cells is
shown in the top row for each
type of mouse. In the bottom
row, the DN T lineage cells
were analyzed for the expression
of CD25 and CD44. The percentage of cells found within
each quadrant is indicated.
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Considering that the P14 TCR-
transgene consists of a
TCR-
cDNA placed under the control of the H-2Kb
promotor and Ig heavy chain enhancer, its expression
within the CD25+ DN compartment should have depended solely on the activation of its transcription. To ascertain the presence of P14 TCR-
transcripts within the
CD25+ DN cell populations from CD3-
+/+ TCR-
and
CD3-
5/
5 TCR-
thymuses, we devised an RNA-PCR
assay that specifically detected the P14 TCR-
transcripts
(see legend of Fig. 3). As shown in Fig. 3 A, transcripts
originating from the P14 TCR-
transgene were readily
detectable in the CD3-
5/
5 TCR-
sample and in the
CD25+ cells sorted from CD3-
+/+ TCR-
thymuses. In
contrast, RNA extracted from CD3-
5/
5 thymocytes
contained no detectable P14 TCR-
transcripts. Note that
upon amplification with a pair of primers specific for the first exon of the TCR C
2 gene, the CD3-
5/
5 RNA
showed an hybridizing band corresponding to endogenous
(D)J-C
and V(D)J-C
transcripts (12). To exclude any
potential posttranslational regulation affecting the expression of the transgenic P14 TCR-
chains, CD3-
5/
5
TCR-
thymocytes were further analyzed by intracellular
staining with an antibody (F23.1: anti-V
8) specific for the
product of the V
gene segment used by the P14 transgenic
TCR-
chain. As shown in Fig. 3 B, most of the CD25+
cells found in CD3-
5/
5 TCR-
thymuses expressed the
intracellular transgenic TCR-
chain. Therefore, these results indicate that both the transcription and translation of
the P14 TCR-
transgene were effective at the CD44
/low
CD25+ DN stage. Consistent with the latter results, introduction of the P14 TCR-
transgene in RAG-1-deficient
mice was found to rescue the progression to the DP stage
(data not shown).
Allelic Exclusion of Endogenous TCR-
Gene Rearrangements Is
Absent in CD3-
5/
5 TCR-
Thymocytes.
Having established that the P14 TCR-
transgene was expressed properly at the stage at which allelic exclusion is expected to
take place, we then determined its impact on TCR-
gene
allelic exclusion in the presence or absence of the CD3-
mutation. This can be assessed using a DNA-PCR assay
that provides an estimation of the relative levels of D
J
and V
D
J
rearrangements in various cell samples
(30). As depicted in Fig. 4 (bottom diagram), primers complementary to V
or D
gene segments were used in combination with a primer positioned immediately 3
to the
J
2 cluster, allowing amplification of rearranged, but not
germline, V
gene segments. The resulting PCR products
were visualized by hybridization with a J
2-specific probe
after electrophoresis and blot transfer. Hybridizing bands
were quantitated using a phosphorimager and the relative
levels of rearrangements expressed as percentages of those
observed in wild-type CD25+ T cells (Fig. 4 B). The results shown in Fig. 4 corresponded to rearrangements of
D
2 (top), V
5 (middle), and V
8 (bottom) to each of the
six J
2 gene segments and were generated using CD25+
cells sorted from CD3-
+/+ (WT CD25+) and CD3-
+/+
TCR-
(TCR
CD25+) thymuses, and total thymocytes
from CD3-
5/
5 and CD3-
5/
5 TCR-
mice. Consistent with previous data indicating that
chain gene allelic
exclusion acts at a point subsequent to D
-J
joining events (1), the levels of D
2 to J
2 rearrangements were
almost equally high in all four samples (Fig. 4, A and B,
top). Comparison of CD3-
5/
5 thymocytes and wild-type
CD25+ cells indicated that the former contained V
DJ
rearrangements that were as extensive as those found in
wild-type CD25+ thymocytes (Fig. 4, lanes WT CD25+
and CD3-
5/
5). As previously documented, using total
thymocytes and TCR-
transgenes unrelated to the one
used herein (18, 19, 30), expression of the P14 TCR-
transgene in wild-type CD25+ DN cells (Fig. 4, lane
TCR
CD25+) resulted in a dramatic reduction of endogenous V
rearrangements (~13% of control). The latter result confirms that the P14 TCR-
transgene is expressed in
a functional form at the CD25+ DN stage, capable of mediating allelic exclusion at the TCR-
locus and preventing expression of a second TCR-
chain on the cell surface
of transgenic SP thymocytes (Fig. 1 B). In contrast, the simultaneous presence of the CD3-
5 mutation (lane
TCR
CD3-
5/
5, Fig. 4) prevented the effects of the
transgenic TCR-
chain and permitted rearrangements of
endogenous TCR V
gene segments to occur at very substantial levels (85-91% of control). Thus, these data indicate that a functional TCR-
transgene does not inhibit
endogenous V
to D
J
rearrangements in the absence of CD3-
subunit.

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Fig. 4.
Expression of a transgenic TCR- chain does not inhibit endogenous V to D J rearrangements in the absence of CD3- polypeptide. (A)
Relative levels of TCR- rearrangements in CD25+ cells sorted from CD3- +/+ (WT CD25+) and CD3- +/+ TCR- (TCR CD25+) thymuses, and
total thymocytes from CD3- 5/ 5 and CD3- 5/ 5 TCR- mice. Identical sorting windows were set up on CD25high DN cells for both the CD3- +/+ and
CD3- +/+ TCR- samples. Considering that they contain >90% CD44 /lowCD25high DN cells (see Fig. 2), the CD3- 5/ 5 and CD3- 5/ 5 TCR-
thymuses were not subjected to sorting before analysis. The extent of D -J and V -D J rearrangements were analyzed by DNA-PCR. The relative
positions of the PCR primers within the TCR- locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving
the intronic J 2 3 primer with D 2- (top), V 5- (middle) or V 8- (bottom) specific 5 primers were gel fractionated and detected with the intronic probe
depicted at the bottom (probe). Note that the cDNA-based P14 TCR- transgene (V 8.1-D -J 2.4) is not detectable with the pair of primers used to
reveal endogenous V 8-J 2 rearrangements. For each sample, dilutions of DNA template corresponding to 1 × 105, 2 × 104, and 1 × 104 cell equivalent were analyzed. (B) Quantification of the results shown in A. Hybridizing bands were scanned using a phosphorimager and the relative percentages of
rearrangements compared to those present in CD25+ cells from CD3- +/+ (WT) mice.
|
|
Allelic Exclusion of Endogenous TCR-
Gene Rearrangements Is
Ineffective in CD3-
/
/
TCR-
Thymocytes.
Disruption of
the CD3-
/
gene incompletely blocks the DN to DP
transition and plausibly corresponds to a leaky mutation of the pre-TCR sensor (see review in reference 31). Accordingly, CD3-
/
/
mice have small thymuses that contain
from 2-30-fold less DP cells than wild-type littermates.
These DP cells appear to have been generated via TCR-
selection since almost all of them express intracellular
TCR-
chains, a situation that contrasts with that observed
in pT
/
mice (20) and is consistent with the complete
absence of
/
T cells in CD3-
/
/
mice. However, the
DP cells found in CD3-
/
/
thymuses can be distinguished from bona fide wild-type DP cells because they
have a limited content of rearranged TCR-
gene segments (32), exhibit a reduced sensitivity to dexamethasone-induced apoptosis (15), and part of them still express CD25
(9). The split pattern of phenotypic changes elicited by the
pre-TCR in the absence of CD3-
/
subunit is likely to
reflect the fact that different cellular responses have different activation thresholds (e.g., the strength of stimulation
required for the induction of the CD4 and CD8 genes being lower than that required for triggering efficient V
J
recombination). Along that line, it was interesting to analyze whether the CD3-
/
subunit of the pre-TCR was
required for the establishment of allelic exclusion at the
TCR-
locus. To this end, CD3-
/
/
mice were crossed
with the P14 TCR-
transgenic mice and the effect of the
transgene on endogenous
locus determined with the
DNA-PCR assay described in the above paragraph. Note
that the levels of TCR-
gene rearrangement found in
CD3-
/
-deficient thymocytes are similar to those found
in wild-type littermates (12), and that the introduction of
the P14 TCR-
transgene in CD3-
/
/
mice did not
lead to any change in thymocyte cellularity and surface
phenotype (data not shown). As shown in Fig. 5, the level of D
2 to J
2 rearrangement was similar in DNA extracted from wild-type (WT), TCR-
wild-type (TCR
),
and TCR-
CD3-
/
/
mice. As previously documented for CD3-
-deficient mice (see above), allelic exclusion of the endogenous TCR-
locus was severely compromised in the absence of CD3-
/
polypeptide (Fig.
5, compare V to DJ rearrangements in lanes TCR
and
TCR
CD3-
/
/
). Thus, these data suggest that the signals conveyed by the partial pre-TCR/CD3 complexes
found in CD3-
/
-deficient mice are unable to trigger
TCR-
allelic exclusion.

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Fig. 5.
Expression of a transgenic TCR- chain does not inhibit endogenous V to D J
rearrangements in the absence of
CD3- / polypeptide. The relative levels of TCR- rearrangements found in CD3- / +/+
(WT), CD3- / +/+ TCR-
(TCR ), and CD3- / /
TCR- thymocytes were determined as described in the legend
of Fig. 4.
|
|
The Onset of V to DJ Recombination at the TCR-
, -
, and
-
Loci Can Occur in the Absence of Both CD3-
and CD3-
/
Polypeptides.
The molecular mechanisms regulating the
development of B cells and
/
T cells display striking
similarities (see review in references 33 and 34). For instance, pre-B cells express a B cell analogue of the pre-T
cell receptor called the pre-B cell receptor. The pre-B cell
receptor associates with Ig
/Ig
transducing subunits and
triggers both the selective amplification/maturation of
IgH+ pre-B cells and establishement of allelic exclusion at
the IgH locus (34). Ig
-deficient mice show a complete
block in B cell development at a stage corresponding to the
CD44
/lowCD25+ stage of T cell development (35). Interestingly, VH to DHJH rearrangements were found to be
severely reduced in Ig
-deficient mice, whereas DH to JH
rearrangements proceeded normally. This indicated that Ig-
may play an important regulatory role in the onset of VH
to DHJH recombination. When bred separately, the CD3-
5
and CD3-
/
mutations had no discernible effect on the
occurrence and extent of V
to D
J
recombination (Figs.
4 and 5). Therefore, the V to DJ recombination events affecting TCR-
and IgH loci may be subjected to distinct
regulatory signals. It is also possible, however, that the
CD3-
and CD3-
/
chains play redundant regulatory roles in the onset of V
to D
J
recombination. To address this question, mice lacking both proteins were derived from a F2 intercross between CD3-
5/
5 and CD3-
/
/
mice. As shown in Fig. 6, mice lacking both CD3-
and CD3-
/
chains had thymuses the size and surface
phenotype of which closely resemble those found in parental CD3-
5/
5 mice (Fig. 7, compare the CD4/CD8 and
CD44/CD25 profiles of panels 
5/
5/
/
+/+ and 
5/
5/
/
/
). Interestingly,
+/+
/
/
and
+/
5
/
/
thymuses displayed markedly different CD4/CD8 phenotypes,
the latter closely resembling in size and composition those
developing in 
5/
5
/
/
double-mutant mice (Fig. 6).
Thus, in the absence of CD3-
/
chains, the CD3-
5
mutation manifests a clear gene-dosage effect, suggesting
that in a CD3-
/
-less context, it is the CD3-
subunits
that limit the number of pre-TCR subcomplexes available
for driving the transition to the DP stage.

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|
Fig. 6.
T cell development
in CD3- 5/ 5CD3- / /
double mutant mice. Mice with
 5/ 5 / +/+, +/+ / / , +/ 5
/ / , and  5/ 5 / / genotypes were derived from an F2 intercross between CD3- 5/ 5 and
CD3- / / mutant mice. Total thymocytes were analyzed by
flow cytometry for the expression of CD4 versus CD8 (A) and
CD25 versus CD44 (B). The
percentages of cells found in
each quadrant is indicated.
|
|

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Fig. 7.
V to D J rearrangements are not affected in CD3- 5/ 5
CD3- / / double mutant mice. The relative levels of TCR- rearrangements found in  5/ 5 / +/+, +/+ / / , and  5/ 5 / / thymocytes were determined as described in the legend of Fig. 4.
|
|
Considering that thymocytes that lack both CD3-
and
CD3-
/
genes are still capable of reaching the CD44
/low
CD25+ DN stage during which V
to D
J
recombination normally happens (Fig. 6 B), we analyzed the status of
their TCR-
loci using the DNA-PCR assay previously
described in the legend of Fig. 4. As shown in Fig. 7, CD3-
5/
5CD3-
/
/
double mutant mice contained D
J
and V
D
J
rearrangements, the extent of which was
similar to those found in the parental single mutant thymocytes. Finally, we examined the effects of the lack of
both CD3-
and CD3-
/
on the rearrangement of TCR-
and -
genes using a DNA-PCR approach (27). As shown
in Fig. 8, the absence of both CD3-
and CD3-
/
had little effect on the extent and timing of TCR-
and -
gene
rearrangements.

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Fig. 8.
Relative levels of
TCR- and TCR- gene rearrangements in RAG-1 / ,
CD3- 5/ 5, CD3- / / ,
CD3- 5/ 5, CD3- / / , and
wild-type thymocytes. DNA extracted from thymocytes of fetuses at day 17 of gestation (wt
E17) and of 4-6-wk-old wild-type (WT) and mutant mice was
amplified with PCR primer pairs
specific for the V 1-J 2, V 4-
J 1, V 5-J 1, V 5-J 1, V 4-J 1,
and V 1-J 2 rearrangements.
PCR products were gel fractionated and the corresponding Southern blots hybridized with
labeled oligonucleotide probes.
|
|
 |
Discussion |
We showed that CD3-

and CD3-
/
modules are
each essential for the establishment of allelic exclusion at
the TCR-
locus. Their mandatory contribution to the activation of this negative feedback loop probably relates to
the role they play in the assembly and function of the pre-TCR. In contrast, analysis of TCR-
transgenic, pT
/
mice showed that TCR-
chains can trigger allelic exclusion without being associated with a pT
chain (18, 19).
However, in the two experimental systems used to assess
the role of pT
in the establishment of allelic exclusion at
the TCR-
locus, significant variations were observed in
the levels of inhibition of endogeneous TCR-
gene rearrangements and accounted for by the presence of distinct
TCR-
transgene copy numbers and/or insertion sites (18, 19). Regardless of these variations, the discrepancy that exists between the pT
- and CD3-deficient mice with regard
to the establishment of allelic exclusion at the TCR-
locus can be explained by the presence within the CD25+
DN cells of low constitutive levels of V
J
recombination that occur before signaling through the pre-TCR. In
pT
/
mice, and only in pT
/
mice, the resulting
TCR-
chains are likely to contribute to the premature assembly of TCR-
/
complexes capable of signaling maturation as well as allelic exclusion via their associated CD3 subunits (20). However, if V
-J
rearrangements do occur
in CD25+ DN cells, it is at a frequency at least 100-fold
lower than that observed in DP cells (32). Thus, premature
TCR-
chain expression can only account for part of the
effects observed with the transgenic TCR-
chain in the absence of pT
. As suggested by Krotkova et al. (19), the capacity of the transgenic TCR-
to signal allelic exclusion
independently of pT
may also relate to its capacity to be
expressed in a phosphatidyl inositol-linked form at the surface of CD25+ cells. (As discussed in the legend of Fig. 1,
we have not been able to detect P14 TCR-
chains on the
surface of CD3-
5/
5 TCR-
thymocytes.) Therefore, the
occurrence of TCR-
allelic exclusion in the absence of
pT
chain is likely to result from the combination of inappropriate expression of the transgenic TCR-
chains and
premature TCR-
chain expression. Irrespective of these
considerations, our data clearly exclude a model in which TCR-
chains can signal TCR-
allelic exclusion in the
mere absence of any of the CD3 components thought to be
part of the pre-TCR/CD3 sensor.
Our data also bear on the causal relationships between
pre-TCR-induced cell proliferation and the establishment
of TCR-
allelic exclusion. It has been suggested that
preTCR-induced cell cycle progression is essential for the
establishment of allelic exclusion at the TCR-
locus (36-39;
see also references 40 and 41 in the case of B cell development). As outlined in Fig. 9, one or more rounds of DNA
replication are speculated to enable the reprogrammation of
the chromatin structure of the TCR-
loci and make them
inaccessible to the V(D)J recombinase. According to that
model, the lack of TCR-
allelic exclusion observed in the
CD3-
5/
5 thymuses would be fully accounted for by the
fact that their TCR-
pT
heterodimers are prevented
from inducing cell cycle entry. Mice carrying a mutation in
the lck gene display a pronounced thymic atrophy associated with a dramatic reduction in the number of DP cells
(42). In these mutant mice, TCR-
gene allelic exclusion is not severely compromised as the presence of a productively rearranged TCR-
transgene resulted in an almost
complete inhibition of endogenous TCR-
gene rearrangements (43). Considering that TCR-
transgenic,
CD3-
/
/
thymuses display the same composition and
cellularity as TCR-
transgenic, lck
/
thymuses (compare
our data with those of Wallace et al., reference 43), it came
as a surprise to find that there was in the former a clear dissociation between the transition to the DP stage and the establishment of TCR-
gene allelic exclusion. Thus, in the absence of CD3-
/
subunit, TCR-
selection may have
led to differentiation rather than proliferation and, consistent with the above model, resulted in the lack of TCR-
gene allelic exclusion. However, the frequency of dividing
early DP cells is only slightly smaller in CD3-
/
/
mice
than in wild-type littermates, indicating that CD3-
/
-less pre-TCR complexes are still capable of triggering cell cycle
entry (9). Collectively, these observations suggest that burst
of cell divisions induced by the pre-TCR may be enabling
rather than inductive for the establishment of TCR-
gene
allelic exclusion, and that the pre-TCR is likely to contribute additional signals to effect TCR-
gene allelic exclusion. According to that view and under physiological conditions, the signals emanating from both the lck- and
CD3-
/
-less pre-TCR complexes suffice to trigger cell
cycle entry and CD4/CD8 expression, whereas only those
emanating from the former can reach the higher threshold
plausibly required to activate the regulatory loop required
for mediating allelic exclusion (denoted as 5 in Fig. 9).
However, it should be noted that upon massive and artefactual cross-linking, even the partial pre-TCR complexes
expressed at the surface of CD3-
/
/
DN thymocytes
are capable of inducing both maturation to the DP stage
and TCR-
gene allelic exclusion (as suggested by the finding that most of the CD3-
/
/
DP cells that develop
after injection of anti-CD3-
antibodies do not contain intracellular TCR-
chains; reference 44). Therefore, our
results are reminiscent of those obtained with the TCR
complexes expressed on mature T cells (e.g., reference 45)
in that they suggest that different pre-TCR-mediated responses display distinct activation thresholds.

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|
Fig. 9.
A model accounting
for the role of the pre-TCR/
CD3 complex in the establishment of allelic exclusion at the
TCR- locus. Upon entering
the CD44 /low CD25+ compartment, the TCR V gene segment cluster [(V)n] become
accessible to the V(D)J recombinase (dashed lines sandwiching the
TCR- alleles a and b). At that
stage of development, the pT
and CD3 components of the
pre-TCR are already available
and it is the TCR- polypeptides that constitute the rate limiting factor in the assembly of the pre-TCR/CD3 complex. From a
cohort of nine CD44 /low CD25+
triple negative thymocytes, three
are expected to produce a functional V gene (VDJ+) as a result
of their first attempt of rearrangement (step 1, see reference 29).
The resulting TCR- polypeptide participates in the assembly
of a pre-TCR complex (step 2). As soon as assembled, this complex triggers (step 3) the transition beyond the CD44 /low CD25+ stage and activates a
negative feedback loop that will close the accessibility of the second, partially rearranged, allele (allele b) to the V(D)J recombinase (continuous lines sandwiching the TCR- alleles), thereby restricting such a T cell to the expression of only a single TCR- chain allele. The p56lck kinase (lck) constitutes one
of the effector operating downstream of the pre-TCR/CD3 complex since the overexpression of a catalytically active form of p56lck inhibits endogenous
V to D J rearrangements while inducing coincidently the transition to the DP stage (30). As proposed previously (29), time delay along this negative
feedback loop, and/or the existence of a few cells in which V to D J rearrangements can be attempted quasisimultaneously on both alleles, may explain the presence of rare cells with two productively rearranged TCR- alleles (52, 53). Based on the comparison of TCR- transgenic, p56lck / , and
TCR- transgenic, CD3- / / mice (see Discussion), it is tempting to speculate that TCR- gene allelic exclusion is brought about via two contingent pathways. One of which (step 4b), by inducing cell proliferation and DNA replication, enables the reprogrammation of the chromatin structure at
the TCR- locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR- locus inaccessible to further V(D)J
recombination. Note that the degradation of RAG-2, which results from cyclin-dependent kinase phosphorylation (loop 4a; reference 38) and occurs
during the burst of divisions associated with the transition from the DN to the DP stage, appears to constitute a fail-safe mechanism not essential for the
execution of TCR- allelic exclusion (39).
|
|
Complexes consisting of calnexin and of CD3-
/
or
CD3-
/
pairs can be expressed at low levels at the surface
of DN thymocytes (46). Upon cross-linking with anti-
CD3-
antibodies, they can induce the progression to the
DP stage even in the absence of TCR-
and pT
chains
(10, 11, 47). It is unlikely, however, that such CD3-calnexin complexes have a normal signaling function before
pre-TCR expression as CD3-
5/
5 mice produce T cells
that can reach the CD44
/lowCD25+ stage and faithfully
initiate V
to D
J
rearrangements (12). Our analysis of
thymocytes lacking both the CD3-
and CD3-
/
chains
emphasizes that the CD3 subunits start to function only immediately before the CD44
/lowCD25high to CD44
/low
CD25
transition (i.e., at a time when the pre-TCR is expected to operate). Based on the above results, the observation that overexpression of various CD3-
transgenes
blocks thymocyte development before the CD25+ DN
stage (44), can be plausibly accounted for by the fact that when overexpressed the CD3-
polypeptides can sequester
effector or adaptor molecules belonging to signaling cassettes involved in the progression to the CD25+ DN stage
(e.g., those operated by c-kit and the common cytokine receptor
chain; reference 48). Collectively, our findings strongly suggest that the CD3 components are differentially
involved in the sequential events that make the TCR-
locus first accessible to, and later insulated from, the action of
the V(D)J recombinase. In contrast, during B cell development, the Ig
transducing subunit appears to play a unique
role in the initiation of VH to DHJH recombination, independent of, and prior to, its function as a component of the
pre-B cell receptor (34, 35). Additionally, we have found
that none of the CD3 components are required for the
completion of TCR-
and TCR-
chain gene rearrangements. These results suggest that TCR-
and TCR-
gene
rearrangements are probably not subjected to stepwise epigenetic controls analogous to those that affect TCR-
and
TCR-
gene rearrangements and rely on the sequential
expression of CD3-associated pre-TCR and TCR sensors. Finally, in the case of the
/
T cell lineage, it should be
emphasized that the raison d'être of the pre-TCR may be
that
/
T cells undergo a second step of selection known
as TCR-
/
selection, and that there is a limited number
of stromal cell niches capable of supporting such a selection
event (49). Thereby, by triggering the selective expansion
and maturation of only those T cell precursors expressing a
TCR-
chain, the pre-TCR is likely to allow this limited
number of cell niches not to be swamped with nonselectable (i.e., TCR-
) DP cells, and maximize the efficacy of
TCR-
/
selection.
Address correspondence to Dr. Bernard Malissen, Centre d'Immunologie INSERM-CNRS de Marseille-Luminy, Case 906, 13288 Marseille Cedex 9, France. Phone: 33-4-91-26-94-18; FAX: 33-4-91-26-94-30;
E-mail: bernardm{at}ciml.univ-mrs.fr
We thank Yujiro Tanaka for stimulating discussions, Pierre Golstein for comments on the manuscript, Jorg
Fehling and Harald von Boehmer for sharing unpublished data with us, Hanspeter Pircher for providing the
TCR-
transgenic mice, Nicole Brun and Marc Barad for expert assistance with cell sorting, Corinne
Béziers-La-Fosse for graphic art, and Véronique Préau and Noëlle Guglietta for typing the manuscript.
This work was supported by institutional grants from Centre National de la Recherche Scientifique and Institut National de la Santé et de la Recherche Médicale, and by specific grants from Association pour la Recherche sur le Cancer, Ligue Nationale contre le Cancer (Comité des Bouches-du-Rhône) and the Commission of the European Communities. J. Ismaili was supported by a postdoctoral fellowship from
Commission of the European Communities (CHRXCT94-0584) and L. Ardouin by a predoctoral fellowship from Ministère de l'Education Nationale, de la Recherche et de la Technologie.
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