The CD3-gamma delta epsilon and CD3-zeta /eta Modules Are Each Essential for Allelic Exclusion at the T Cell Receptor beta  Locus but Are Both Dispensable for the Initiation of  V to (D)J Recombination at the T Cell Receptor-beta , -gamma , and -delta Loci

By Laurence Ardouin, Jamila Ismaili, Bernard Malissen, and Marie Malissen

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

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Abstract
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Materials & Methods
Results
Discussion
References

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-beta chain. Recent experiments in pre-Talpha chain-deficient mice have suggested that the pre-TCR may not be required for signaling allelic exclusion at the TCR-beta locus. Using CD3-epsilon - and CD3-zeta /eta -deficient mice harboring a productively rearranged TCR-beta transgene, we showed that the CD3-gamma delta epsilon and CD3-zeta /eta modules, and by inference the pre-TCR/CD3 complex, are each essential for the establishment of allelic exclusion at the endogenous TCR-beta locus. Furthermore, using mutant mice lacking both the CD3-epsilon and CD3-zeta /eta 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-beta , TCR-gamma , and TCR-delta loci. Thus, the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase.

    Introduction
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha  and beta  chains, whereas a minor population expresses an alternative TCR isoform made of gamma  and delta  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-alpha and TCR-gamma chain genes, and of V, D, and J gene segments in TCR-beta and TCR-delta 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-alpha /beta or TCR-gamma /delta chain combinations. In the mouse, the expression of a productively rearranged TCR-beta chain transgene has been shown to prevent complete V-(D)J rearrangement of endogenous TCR-beta genes (1), and this has led to the assumption that alpha /beta T cell precursors have developed feedback inhibition mechanisms to ensure that most mature T cell clones express one, and only one, TCR-alpha /beta 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-right-arrow CD44+CD25+right-arrow CD44-/low CD25+right-arrow CD44-/lowCD25- (2). TCR-beta gene rearrangements precede rearrangements at the TCR-alpha locus and proceed in two separate steps involving an initial Dright-arrow J joining event and a subsequent Vright-arrow DJ rearrangement. TCR-beta gene rearrangements start at, or at the transition to, the CD44-/lowCD25+ DN stage (2, 3), whereas the first measurable TCR-alpha 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-alpha /beta 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-beta gene rearrangements.

In the pre-TCR, TCR-beta is disufilde linked with a polypeptide encoded by a nonrearranging gene and denoted as the pTalpha chain (8). To exert its function, the pre-TCR needs to associate with both the CD3-gamma /epsilon and CD3-zeta 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-beta + DN cells and concurrently drives their progression to the DP developmental stage (such transition is often denoted as TCR-beta selection). Moreover, considering that the expression of a productively rearranged TCR-beta transgene inhibits most endogenous Vbeta to Dbeta Jbeta rearrangements (see above), it has been suggested that the TCR-beta chain, and by extension the pre-TCR/CD3 complex, plays a pivotal role in the enforcement of allelic exclusion at the TCR-beta locus. Therefore, disruption of the gene coding for the pTalpha subunit should have prevented assembly of a functional pre-TCR complex and affected the establishment of allelic exclusion at the TCR-beta locus. However, in pTalpha -/- thymocytes, expression of a transgene coding for a functional TCR-beta chain was found to inhibit endogenous Vbeta to Dbeta Jbeta rearrangements to almost the same extent as in a pTalpha +/+ background (18, 19). Assuming that no other gene products can compensate for the loss of pTalpha (e.g., the products of prematurely expressed TCR-alpha 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-beta locus.

We have previously generated mice with a targeted mutation of the CD3-epsilon gene (referred to as CD3-epsilon Delta 5; reference 12). This mutation abolishes the expression of intact CD3-epsilon polypeptides, dramatically reduces the transcription rate of the neighboring CD3-gamma and CD3-delta genes, and totally blocks the progression beyond the CD44-/lowCD25+ stage. The thymocytes found in CD3-epsilon Delta 5/Delta 5 mice contain readily detectable levels of CD3-zeta , TCR-beta , and pTalpha transcripts. However, the lack of CD3-gamma /epsilon and CD3-delta /epsilon dimers is likely to prevent their pTalpha -TCR-beta and CD3-zeta 2 dimers from participating in the assembly of functional pre-TCR/CD3 complexes. The CD3-epsilon Delta 5/Delta 5 mice present several experimental advantages relative to pTalpha -deficient mice. First, their thymuses constitute an enriched source of CD44-/lowCD25+ DN cells devoid of contaminating downstream alpha /beta T cell subsets and in which TCR-beta gene rearrangements do happen normally. Second, the CD3-epsilon Delta 5 mutation does prevent the development of gamma /delta T cells and permits the analysis of early alpha /beta T cell development in a microenvironment insulated from the adventitious effects resulting from the presence of gamma /delta T cells (2, 20, 21). Therefore, by obviating some of the experimental limitations associated with pTalpha -/- mice, the CD3-epsilon Delta 5/Delta 5 mice constitute a particularly appropriate model to determine whether the CD3-gamma delta epsilon module, and by inference the pre-TCR, is essential for the establishment of allelic exclusion at the TCR-beta locus. Here we report on experiments showing that the CD3-gamma delta epsilon and the CD3-zeta /eta modules of the pre-TCR play each a pivotal role in allelic exclusion at the TCR-beta locus. In contrast, analysis of CD3-epsilon Delta 5/Delta 5 CD3-zeta /eta -/- double mutant mice established that the onset of V to (D)J recombination at the TCR-beta , TCR-gamma , and TCR-delta loci can occur in the absence of CD3 subunits.

    Materials and Methods
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Abstract
Introduction
Materials & Methods
Results
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References

Mice. The CD3-epsilon Delta 5/Delta 5 mice and CD3-zeta /eta -/- 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-beta transgenic mice (line 128) express a TCR-beta cDNA (Vbeta 8.1-Dbeta -Jbeta 2.4) derived from the T cell clone P14 (24). TCR-beta transgenic mice were typed for the presence of the transgene by PCR analysis of tail DNA. TCR-beta transgenic mice were crossed with CD3-epsilon Delta 5 and CD3-zeta /eta -deficient mice to obtain CD3-epsilon Delta 5/Delta 5 TCR-beta and CD3-zeta /eta -/- TCR-beta mice. CD3-epsilon Delta 5/Delta 5 CD3-zeta /eta -/- double-deficient mice were derived from CD3-epsilon Delta 5/Delta 5 × CD3-zeta /eta -/- 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-epsilon (2C11), CD4 (H129.19), CD8 (53-6.7), CD25 (7D4), CD44 (Pgp-1), and TCR Vbeta 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-beta 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-Vbeta 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-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta samples) or CD25high (sorted from the CD3-epsilon +/+ TCR-beta 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 Cbeta 2 primers used to detect transcripts incorporating the TCR Cbeta 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 P14beta : 5'-GTGATGCCGGGGGGCGGAACAC-3'; and beta -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 (Cbeta 2A; reference 26).


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Fig. 3.   Assessment of TCR-beta transgene expression by RNA-PCR and intracytoplasmic staining. (A) CD25+ cells sorted from TCR-beta transgenic wild-type thymuses (CD3-epsilon +/+ TCR-beta ) and total thymocytes from TCR-beta transgenic CD3-epsilon Delta 5/Delta 5 mice (CD3-epsilon Delta 5/Delta 5 TCR-beta ) were analyzed for the presence of transcripts originating from the P14 TCR-beta transgene using the RNA-PCR strategy depicted in the bottom diagram. The P14 TCR-beta cDNA is expressed under the control of the H-2Kb promotor and IgH chain intronic enhancer (EH). The 3' end of the P14 TCR-beta cDNA is linked to a genomic fragment of the human beta  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 beta  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-beta gene. RNA extracted from nontransgenic CD3-epsilon Delta 5/Delta 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 Cbeta 2 exon. The products resulting from amplification with primer pairs 1 + 2 (TCR Cbeta 2) and 3 + 4 (TCR-beta Tg) were gel fractionated, blotted, and hybridized with a Cbeta 2-specific probe (p). The location of specific primers are indicated by arrowheads and the transcription start site of the TCR-beta 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-beta chain within the CD25+ subset present in CD3-epsilon Delta 5Delta 5 transgenic beta  thymocytes was revealed by intracellular staining with an antibody (F23.1) specific for the Vbeta 8 gene segment used by the P14 TCR beta  chain. RAG-1-/- thymocytes were also included as negative controls. Cytoplasmic staining of the CD25+ compartment from nontransgenic CD3-epsilon Delta 5/Delta 5 mice revealed <1% F23.1+ cells.

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-beta chain gene rearrangement were as described in reference 26. PCR-based analysis of TCR-gamma and TCR-delta 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
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Materials & Methods
Results
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References
A Productively Rearranged TCR-beta Transgene Is Unable to Activate the Transition to the DP Stage in the Absence of CD3-epsilon .

To determine whether the expression of a TCR-beta chain transgene was able to inhibit endogenous Vbeta to Dbeta Jbeta rearrangements in the absence of CD3-epsilon subunit, CD3-epsilon Delta 5/Delta 5 mice were crossed with transgenic mice carrying a productively rearranged Vbeta 8+ TCR-beta chain derived from the P14 T cell clone (24, 28). When expressed in a wild-type background, this TCR-beta transgene prevented endogenous beta -locus gene rearrangements, as judged by the fact that most of the SP thymocytes developing in these mice were Vbeta 8+ (Fig. 1, compare transgenic [WT TCR-beta ] and nontransgenic [WT] wild-type panels). As shown in Fig. 1 A, CD3-epsilon Delta 5/Delta 5 TCR-beta mice had thymuses that did not develop past the DN stage and contained absolute cell numbers similar to nontransgenic CD3-epsilon Delta 5/Delta 5 thymuses. Thus, expression of the P14 TCR-beta transgene was unable to restore T cell development in CD3-epsilon Delta 5/Delta 5 mice. To specify more precisely the effect of the TCR-beta transgene on early T cell development, we analyzed the CD44/CD25 profile of wild-type and CD3-epsilon Delta 5/Delta 5 DN thymocytes that developed in the absence or presence of the P14 TCR-beta 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-beta ) 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-beta transgenic mice exhibit CD44-/lowCD25+ cell compartments the size of which are intermediate between those found in nontransgenic and TCR-alpha /beta transgenic mice (19, 20). Such observations have been generally accounted for by the fact that CD44-/lowCD25+ cells equipped with a productively rearranged TCR-beta 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-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta 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-gamma delta epsilon module, pTalpha -TCR-beta P14 heterodimers were prevented from assembling into functional pre-TCR complexes and unable to rescue the blockade in thymic development observed in CD3-epsilon Delta 5/Delta 5 mice. Alternatively, the onset of expression of the P14 TCR-beta 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-epsilon Delta 5/Delta 5 mice.


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Fig. 1.   A transgene encoding a productively rearranged TCR-beta gene does not restore T cell development in CD3-epsilon -deficient mice. Mice with CD3-epsilon +/+ (WT), CD3-epsilon +/+ TCR-beta (WT TCR-beta ), CD3-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta genotypes were derived from a F2 intercross between the TCR-beta transgenic line P14 TCR-beta (TCR-beta ) and CD3-epsilon Delta 5/Delta 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-epsilon and Vbeta 8. Percentage of CD3high and Vbeta 8high cells are indicated. Considering that the exon coding for the epitope recognized by the 2C11 anti-CD3-epsilon antibody has been deleted in the CD3-epsilon Delta 5 mutant gene, and that CD3-epsilon Delta 5/Delta 5 thymocytes do not express detectable levels of TCR-beta chain at their surface (12), the histograms obtained after staining CD3-epsilon Delta 5/Delta 5 thymocytes with anti- CD3-epsilon or anti-Vbeta 8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-beta transgenic SCID mice (50) and TCR-beta transgenic RAG-/- mice (51) where transgenic TCR-beta chains are expressed as monomers without CD3-epsilon and in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR-beta transgenic chains on the surface of CD3-epsilon Delta 5/Delta 5 TCR-beta thymocytes after staining with the Cbeta -specific antibody H57.597 (data not shown) and Vbeta 8-specific antibody F23.1 (compare the CD3-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta histograms). Whether such difference resulted from the use of the P14 TCR-beta transgene or is rather due to the CD3-epsilon Delta 5/Delta 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-epsilon Delta 5/Delta 5 mutant mice in the presence or absence of P14 TCR-beta transgene (TCRbeta ). 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.

Considering that the P14 TCR-beta transgene consists of a TCR-beta 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-beta transcripts within the CD25+ DN cell populations from CD3-epsilon +/+ TCR-beta and CD3-epsilon Delta 5/Delta 5 TCR-beta thymuses, we devised an RNA-PCR assay that specifically detected the P14 TCR-beta transcripts (see legend of Fig. 3). As shown in Fig. 3 A, transcripts originating from the P14 TCR-beta transgene were readily detectable in the CD3-epsilon Delta 5/Delta 5 TCR-beta sample and in the CD25+ cells sorted from CD3-epsilon +/+ TCR-beta thymuses. In contrast, RNA extracted from CD3-epsilon Delta 5/Delta 5 thymocytes contained no detectable P14 TCR-beta transcripts. Note that upon amplification with a pair of primers specific for the first exon of the TCR Cbeta 2 gene, the CD3-epsilon Delta 5/Delta 5 RNA showed an hybridizing band corresponding to endogenous (D)J-Cbeta and V(D)J-Cbeta transcripts (12). To exclude any potential posttranslational regulation affecting the expression of the transgenic P14 TCR-beta chains, CD3-epsilon Delta 5/Delta 5 TCR-beta thymocytes were further analyzed by intracellular staining with an antibody (F23.1: anti-Vbeta 8) specific for the product of the Vbeta gene segment used by the P14 transgenic TCR-beta chain. As shown in Fig. 3 B, most of the CD25+ cells found in CD3-epsilon Delta 5/Delta 5 TCR-beta thymuses expressed the intracellular transgenic TCR-beta chain. Therefore, these results indicate that both the transcription and translation of the P14 TCR-beta transgene were effective at the CD44-/low CD25+ DN stage. Consistent with the latter results, introduction of the P14 TCR-beta transgene in RAG-1-deficient mice was found to rescue the progression to the DP stage (data not shown).

Allelic Exclusion of Endogenous TCR-beta Gene Rearrangements Is Absent in CD3-epsilon Delta 5/Delta 5 TCR-beta Thymocytes.

Having established that the P14 TCR-beta transgene was expressed properly at the stage at which allelic exclusion is expected to take place, we then determined its impact on TCR-beta gene allelic exclusion in the presence or absence of the CD3-epsilon mutation. This can be assessed using a DNA-PCR assay that provides an estimation of the relative levels of Dbeta right-arrow Jbeta and Vbeta right-arrow Dbeta Jbeta rearrangements in various cell samples (30). As depicted in Fig. 4 (bottom diagram), primers complementary to Vbeta or Dbeta gene segments were used in combination with a primer positioned immediately 3' to the Jbeta 2 cluster, allowing amplification of rearranged, but not germline, Vbeta gene segments. The resulting PCR products were visualized by hybridization with a Jbeta 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 Dbeta 2 (top), Vbeta 5 (middle), and Vbeta 8 (bottom) to each of the six Jbeta 2 gene segments and were generated using CD25+ cells sorted from CD3-epsilon +/+ (WT CD25+) and CD3-epsilon +/+ TCR-beta (TCRbeta CD25+) thymuses, and total thymocytes from CD3-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta mice. Consistent with previous data indicating that beta  chain gene allelic exclusion acts at a point subsequent to Dbeta -Jbeta joining events (1), the levels of Dbeta 2 to Jbeta 2 rearrangements were almost equally high in all four samples (Fig. 4, A and B, top). Comparison of CD3-epsilon Delta 5/Delta 5 thymocytes and wild-type CD25+ cells indicated that the former contained Vright-arrow DJ rearrangements that were as extensive as those found in wild-type CD25+ thymocytes (Fig. 4, lanes WT CD25+ and CD3-epsilon Delta 5/Delta 5). As previously documented, using total thymocytes and TCR-beta transgenes unrelated to the one used herein (18, 19, 30), expression of the P14 TCR-beta transgene in wild-type CD25+ DN cells (Fig. 4, lane TCRbeta CD25+) resulted in a dramatic reduction of endogenous Vbeta rearrangements (~13% of control). The latter result confirms that the P14 TCR-beta transgene is expressed in a functional form at the CD25+ DN stage, capable of mediating allelic exclusion at the TCR-beta locus and preventing expression of a second TCR-beta chain on the cell surface of transgenic SP thymocytes (Fig. 1 B). In contrast, the simultaneous presence of the CD3-epsilon Delta 5 mutation (lane TCRbeta CD3-epsilon Delta 5/Delta 5, Fig. 4) prevented the effects of the transgenic TCR-beta chain and permitted rearrangements of endogenous TCR Vbeta gene segments to occur at very substantial levels (85-91% of control). Thus, these data indicate that a functional TCR-beta transgene does not inhibit endogenous Vbeta to Dbeta Jbeta rearrangements in the absence of CD3-epsilon subunit.


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Fig. 4.   Expression of a transgenic TCR-beta chain does not inhibit endogenous Vbeta to Dbeta Jbeta rearrangements in the absence of CD3-epsilon polypeptide. (A) Relative levels of TCR-beta rearrangements in CD25+ cells sorted from CD3-epsilon +/+ (WT CD25+) and CD3-epsilon +/+ TCR-beta (TCRbeta CD25+) thymuses, and total thymocytes from CD3-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta mice. Identical sorting windows were set up on CD25high DN cells for both the CD3-epsilon +/+ and CD3-epsilon +/+ TCR-beta samples. Considering that they contain >90% CD44-/lowCD25high DN cells (see Fig. 2), the CD3-epsilon Delta 5/Delta 5 and CD3-epsilon Delta 5/Delta 5 TCR-beta thymuses were not subjected to sorting before analysis. The extent of Dbeta -Jbeta and Vbeta -Dbeta Jbeta rearrangements were analyzed by DNA-PCR. The relative positions of the PCR primers within the TCR-beta locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving the intronic Jbeta 2 3' primer with Dbeta 2- (top), Vbeta 5- (middle) or Vbeta 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-beta transgene (Vbeta 8.1-Dbeta -Jbeta 2.4) is not detectable with the pair of primers used to reveal endogenous Vbeta 8-Jbeta 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-epsilon +/+ (WT) mice.

Allelic Exclusion of Endogenous TCR-beta Gene Rearrangements Is Ineffective in CD3-zeta /eta -/- TCR-beta Thymocytes.

Disruption of the CD3-zeta /eta 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-zeta /eta -/- 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-beta selection since almost all of them express intracellular TCR-beta chains, a situation that contrasts with that observed in pTalpha -/- mice (20) and is consistent with the complete absence of gamma /delta T cells in CD3-zeta /eta -/- mice. However, the DP cells found in CD3-zeta /eta -/- thymuses can be distinguished from bona fide wild-type DP cells because they have a limited content of rearranged TCR-alpha 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-zeta /eta 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 Valpha right-arrow Jalpha recombination). Along that line, it was interesting to analyze whether the CD3-zeta /eta subunit of the pre-TCR was required for the establishment of allelic exclusion at the TCR-beta locus. To this end, CD3-zeta /eta -/- mice were crossed with the P14 TCR-beta transgenic mice and the effect of the beta  transgene on endogenous beta  locus determined with the DNA-PCR assay described in the above paragraph. Note that the levels of TCR-beta gene rearrangement found in CD3-zeta /eta -deficient thymocytes are similar to those found in wild-type littermates (12), and that the introduction of the P14 TCR-beta transgene in CD3-zeta /eta -/- mice did not lead to any change in thymocyte cellularity and surface phenotype (data not shown). As shown in Fig. 5, the level of Dbeta 2 to Jbeta 2 rearrangement was similar in DNA extracted from wild-type (WT), TCR-beta wild-type (TCRbeta ), and TCR-beta CD3-zeta /eta -/- mice. As previously documented for CD3-epsilon -deficient mice (see above), allelic exclusion of the endogenous TCR-beta locus was severely compromised in the absence of CD3-zeta /eta polypeptide (Fig. 5, compare V to DJ rearrangements in lanes TCRbeta and TCRbeta CD3-zeta /eta -/-). Thus, these data suggest that the signals conveyed by the partial pre-TCR/CD3 complexes found in CD3-zeta /eta -deficient mice are unable to trigger TCR-beta allelic exclusion.


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Fig. 5.   Expression of a transgenic TCR-beta chain does not inhibit endogenous Vbeta to Dbeta Jbeta rearrangements in the absence of CD3-zeta /eta polypeptide. The relative levels of TCR-beta rearrangements found in CD3-zeta /eta +/+ (WT), CD3-zeta /eta +/+ TCR-beta (TCRbeta ), and CD3-zeta /eta -/- TCR-beta thymocytes were determined as described in the legend of Fig. 4.

The Onset of V to DJ Recombination at the TCR-beta , -gamma , and -delta Loci Can Occur in the Absence of Both CD3-epsilon and CD3-zeta /eta Polypeptides.

The molecular mechanisms regulating the development of B cells and alpha /beta 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 Igalpha /Igbeta transducing subunits and triggers both the selective amplification/maturation of IgH+ pre-B cells and establishement of allelic exclusion at the IgH locus (34). Igbeta -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 Igbeta -deficient mice, whereas DH to JH rearrangements proceeded normally. This indicated that Ig-beta may play an important regulatory role in the onset of VH to DHJH recombination. When bred separately, the CD3-epsilon Delta 5 and CD3-zeta /eta mutations had no discernible effect on the occurrence and extent of Vbeta to Dbeta Jbeta recombination (Figs. 4 and 5). Therefore, the V to DJ recombination events affecting TCR-beta and IgH loci may be subjected to distinct regulatory signals. It is also possible, however, that the CD3-epsilon and CD3-zeta /eta chains play redundant regulatory roles in the onset of Vbeta to Dbeta Jbeta recombination. To address this question, mice lacking both proteins were derived from a F2 intercross between CD3-epsilon Delta 5/Delta 5 and CD3-zeta /eta -/- mice. As shown in Fig. 6, mice lacking both CD3-epsilon and CD3-zeta /eta chains had thymuses the size and surface phenotype of which closely resemble those found in parental CD3-epsilon Delta 5/Delta 5 mice (Fig. 7, compare the CD4/CD8 and CD44/CD25 profiles of panels epsilon Delta 5/Delta 5/zeta /eta +/+ and epsilon Delta 5/Delta 5/ zeta /eta -/-). Interestingly, epsilon +/+ zeta /eta -/- and epsilon +/Delta 5 zeta /eta -/- thymuses displayed markedly different CD4/CD8 phenotypes, the latter closely resembling in size and composition those developing in epsilon Delta 5/Delta 5 zeta /eta -/- double-mutant mice (Fig. 6). Thus, in the absence of CD3-zeta /eta chains, the CD3-epsilon Delta 5 mutation manifests a clear gene-dosage effect, suggesting that in a CD3-zeta /eta -less context, it is the CD3-epsilon 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-epsilon Delta 5/Delta 5CD3-zeta /eta -/- double mutant mice. Mice with epsilon Delta 5/Delta 5zeta /eta +/+, epsilon +/+zeta /eta -/-, epsilon +/Delta 5 zeta /eta -/-, and epsilon Delta 5/Delta 5zeta /eta -/- genotypes were derived from an F2 intercross between CD3-epsilon Delta 5/Delta 5 and CD3-zeta /eta -/- 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.   Vbeta to Dbeta Jbeta rearrangements are not affected in CD3-epsilon Delta 5/Delta 5 CD3-zeta /eta -/- double mutant mice. The relative levels of TCR-beta rearrangements found in epsilon Delta 5/Delta 5zeta /eta +/+, epsilon +/+zeta /eta -/-, and epsilon Delta 5/Delta 5zeta /eta -/- thymocytes were determined as described in the legend of Fig. 4.

Considering that thymocytes that lack both CD3-epsilon and CD3-zeta /eta genes are still capable of reaching the CD44-/low CD25+ DN stage during which Vbeta to Dbeta Jbeta recombination normally happens (Fig. 6 B), we analyzed the status of their TCR-beta loci using the DNA-PCR assay previously described in the legend of Fig. 4. As shown in Fig. 7, CD3-epsilon Delta 5/Delta 5CD3-zeta /eta -/- double mutant mice contained Dbeta right-arrow Jbeta and Vbeta right-arrow Dbeta Jbeta 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-epsilon and CD3-zeta /eta on the rearrangement of TCR-gamma and -delta genes using a DNA-PCR approach (27). As shown in Fig. 8, the absence of both CD3-epsilon and CD3-zeta /eta had little effect on the extent and timing of TCR-gamma and -delta gene rearrangements.


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Fig. 8.   Relative levels of TCR-gamma and TCR-delta gene rearrangements in RAG-1-/-, CD3-epsilon Delta 5/Delta 5, CD3-zeta /eta -/-, CD3-epsilon Delta 5/Delta 5, CD3-zeta /eta -/-, 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 Vdelta 1-Jdelta 2, Vdelta 4- Jdelta 1, Vdelta 5-Jdelta 1, Vgamma 5-Jgamma 1, Vgamma 4-Jgamma 1, and Vgamma 1-Jgamma 2 rearrangements. PCR products were gel fractionated and the corresponding Southern blots hybridized with labeled oligonucleotide probes.

    Discussion
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

We showed that CD3-gamma delta epsilon and CD3-zeta /eta modules are each essential for the establishment of allelic exclusion at the TCR-beta 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-beta transgenic, pTalpha -/- mice showed that TCR-beta chains can trigger allelic exclusion without being associated with a pTalpha chain (18, 19). However, in the two experimental systems used to assess the role of pTalpha in the establishment of allelic exclusion at the TCR-beta locus, significant variations were observed in the levels of inhibition of endogeneous TCR-beta gene rearrangements and accounted for by the presence of distinct TCR-beta transgene copy numbers and/or insertion sites (18, 19). Regardless of these variations, the discrepancy that exists between the pTalpha - and CD3-deficient mice with regard to the establishment of allelic exclusion at the TCR-beta locus can be explained by the presence within the CD25+ DN cells of low constitutive levels of Valpha right-arrow Jalpha recombination that occur before signaling through the pre-TCR. In pTalpha -/- mice, and only in pTalpha -/- mice, the resulting TCR-alpha chains are likely to contribute to the premature assembly of TCR-alpha /beta complexes capable of signaling maturation as well as allelic exclusion via their associated CD3 subunits (20). However, if Valpha -Jalpha 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-alpha chain expression can only account for part of the effects observed with the transgenic TCR-beta chain in the absence of pTalpha . As suggested by Krotkova et al. (19), the capacity of the transgenic TCR-beta to signal allelic exclusion independently of pTalpha 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-beta chains on the surface of CD3-epsilon Delta 5/Delta 5 TCR-beta thymocytes.) Therefore, the occurrence of TCR-beta allelic exclusion in the absence of pTalpha chain is likely to result from the combination of inappropriate expression of the transgenic TCR-beta chains and premature TCR-alpha chain expression. Irrespective of these considerations, our data clearly exclude a model in which TCR-beta chains can signal TCR-beta 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-beta allelic exclusion. It has been suggested that preTCR-induced cell cycle progression is essential for the establishment of allelic exclusion at the TCR-beta 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-beta loci and make them inaccessible to the V(D)J recombinase. According to that model, the lack of TCR-beta allelic exclusion observed in the CD3-epsilon Delta 5/Delta 5 thymuses would be fully accounted for by the fact that their TCR-beta pTalpha 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-beta gene allelic exclusion is not severely compromised as the presence of a productively rearranged TCR-beta transgene resulted in an almost complete inhibition of endogenous TCR-beta gene rearrangements (43). Considering that TCR-beta transgenic, CD3-zeta /eta -/- thymuses display the same composition and cellularity as TCR-beta 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-beta gene allelic exclusion. Thus, in the absence of CD3-zeta /eta subunit, TCR-beta selection may have led to differentiation rather than proliferation and, consistent with the above model, resulted in the lack of TCR-beta gene allelic exclusion. However, the frequency of dividing early DP cells is only slightly smaller in CD3-zeta /eta -/- mice than in wild-type littermates, indicating that CD3-zeta /eta -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-beta gene allelic exclusion, and that the pre-TCR is likely to contribute additional signals to effect TCR-beta gene allelic exclusion. According to that view and under physiological conditions, the signals emanating from both the lck- and CD3-zeta /eta -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-zeta /eta -/- DN thymocytes are capable of inducing both maturation to the DP stage and TCR-beta gene allelic exclusion (as suggested by the finding that most of the CD3-zeta /eta -/- DP cells that develop after injection of anti-CD3-epsilon antibodies do not contain intracellular TCR-beta 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-beta locus. Upon entering the CD44-/low CD25+ compartment, the TCR Vbeta gene segment cluster [(V)n] become accessible to the V(D)J recombinase (dashed lines sandwiching the TCR-beta alleles a and b). At that stage of development, the pTalpha and CD3 components of the pre-TCR are already available and it is the TCR-beta 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 Vbeta gene (VDJ+) as a result of their first attempt of rearrangement (step 1, see reference 29). The resulting TCR-beta 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-beta alleles), thereby restricting such a T cell to the expression of only a single TCR-beta 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 Vbeta to Dbeta Jbeta 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 Vbeta to Dbeta Jbeta rearrangements can be attempted quasisimultaneously on both beta  alleles, may explain the presence of rare cells with two productively rearranged TCR-beta alleles (52, 53). Based on the comparison of TCR-beta transgenic, p56lck-/-, and TCR-beta transgenic, CD3-zeta /eta -/- mice (see Discussion), it is tempting to speculate that TCR-beta 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-beta locus, and thereby permits factor(s) induced by the second pathway (step 5) to act and render the TCR-beta 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-beta allelic exclusion (39).

Complexes consisting of calnexin and of CD3-gamma /epsilon or CD3-delta /epsilon pairs can be expressed at low levels at the surface of DN thymocytes (46). Upon cross-linking with anti- CD3-epsilon antibodies, they can induce the progression to the DP stage even in the absence of TCR-beta and pTalpha chains (10, 11, 47). It is unlikely, however, that such CD3-calnexin complexes have a normal signaling function before pre-TCR expression as CD3-epsilon Delta 5/Delta 5 mice produce T cells that can reach the CD44-/lowCD25+ stage and faithfully initiate Vbeta to Dbeta Jbeta rearrangements (12). Our analysis of thymocytes lacking both the CD3-epsilon and CD3-zeta /eta 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-epsilon transgenes blocks thymocyte development before the CD25+ DN stage (44), can be plausibly accounted for by the fact that when overexpressed the CD3-epsilon 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 gamma  chain; reference 48). Collectively, our findings strongly suggest that the CD3 components are differentially involved in the sequential events that make the TCR-beta locus first accessible to, and later insulated from, the action of the V(D)J recombinase. In contrast, during B cell development, the Igbeta 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-gamma and TCR-delta chain gene rearrangements. These results suggest that TCR-gamma and TCR-delta gene rearrangements are probably not subjected to stepwise epigenetic controls analogous to those that affect TCR-alpha and TCR-beta gene rearrangements and rely on the sequential expression of CD3-associated pre-TCR and TCR sensors. Finally, in the case of the alpha /beta T cell lineage, it should be emphasized that the raison d'être of the pre-TCR may be that alpha /beta T cells undergo a second step of selection known as TCR-alpha /beta 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-beta chain, the pre-TCR is likely to allow this limited number of cell niches not to be swamped with nonselectable (i.e., TCR-beta -) DP cells, and maximize the efficacy of TCR-alpha /beta selection.

    Footnotes

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

Received for publication 30 September 1997.

1   Abbreviations used in this paper: D, diversity; DN, double negative; DP, double positive; J, joining; PBS/FCS, PBS supplemented with 3% FCS; RAG, recombination activation gene; SP, single positive; V, variable.

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-beta 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.

    References
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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