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The CD3-gammadeltaepsilon 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.

Ardouin L, Ismaili J, Malissen B, Malissen M - J. Exp. Med. (1998)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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-gammadeltaepsilon 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.

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Expression of a transgenic TCR-β chain does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε polypeptide. (A)  Relative levels of TCR-β rearrangements in CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and  total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Identical sorting windows were set up on CD25high DN cells for both the CD3-ε+/+ and  CD3-ε+/+ TCR-β samples. Considering that they contain >90% CD44−/lowCD25high DN cells (see Fig. 2), the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β  thymuses were not subjected to sorting before analysis. The extent of Dβ–Jβ and Vβ–DβJβ rearrangements were analyzed by DNA-PCR. The relative  positions of the PCR primers within the TCR-β locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving  the intronic Jβ2 3′ primer with Dβ2- (top), Vβ5- (middle) or Vβ8- (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-β transgene (Vβ8.1-Dβ-Jβ2.4) is not detectable with the pair of primers used to  reveal endogenous Vβ8–Jβ2 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-ε+/+ (WT) mice.
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Figure 4: Expression of a transgenic TCR-β chain does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε polypeptide. (A) Relative levels of TCR-β rearrangements in CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Identical sorting windows were set up on CD25high DN cells for both the CD3-ε+/+ and CD3-ε+/+ TCR-β samples. Considering that they contain >90% CD44−/lowCD25high DN cells (see Fig. 2), the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β thymuses were not subjected to sorting before analysis. The extent of Dβ–Jβ and Vβ–DβJβ rearrangements were analyzed by DNA-PCR. The relative positions of the PCR primers within the TCR-β locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving the intronic Jβ2 3′ primer with Dβ2- (top), Vβ5- (middle) or Vβ8- (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-β transgene (Vβ8.1-Dβ-Jβ2.4) is not detectable with the pair of primers used to reveal endogenous Vβ8–Jβ2 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-ε+/+ (WT) mice.

Mentions: Having established that the P14 TCR-β transgene was expressed properly at the stage at which allelic exclusion is expected to take place, we then determined its impact on TCR-β gene allelic exclusion in the presence or absence of the CD3-ε mutation. This can be assessed using a DNA-PCR assay that provides an estimation of the relative levels of Dβ→ Jβ and Vβ→ DβJβ rearrangements in various cell samples (30). As depicted in Fig. 4 (bottom diagram), primers complementary to Vβ or Dβ gene segments were used in combination with a primer positioned immediately 3′ to the Jβ2 cluster, allowing amplification of rearranged, but not germline, Vβ gene segments. The resulting PCR products were visualized by hybridization with a Jβ2-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 Dβ2 (top), Vβ5 (middle), and Vβ8 (bottom) to each of the six Jβ2 gene segments and were generated using CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Consistent with previous data indicating that β chain gene allelic exclusion acts at a point subsequent to Dβ–Jβ joining events (1), the levels of Dβ2 to Jβ2 rearrangements were almost equally high in all four samples (Fig. 4, A and B, top). Comparison of CD3-εΔ5/Δ5 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-εΔ5/Δ5). As previously documented, using total thymocytes and TCR-β transgenes unrelated to the one used herein (18, 19, 30), expression of the P14 TCR-β transgene in wild-type CD25+ DN cells (Fig. 4, lane TCRβ CD25+) resulted in a dramatic reduction of endogenous Vβ rearrangements (∼13% of control). The latter result confirms that the P14 TCR-β transgene is expressed in a functional form at the CD25+ DN stage, capable of mediating allelic exclusion at the TCR-β locus and preventing expression of a second TCR-β chain on the cell surface of transgenic SP thymocytes (Fig. 1 B). In contrast, the simultaneous presence of the CD3-εΔ5 mutation (lane TCRβ CD3-εΔ5/Δ5, Fig. 4) prevented the effects of the transgenic TCR-β chain and permitted rearrangements of endogenous TCR Vβ gene segments to occur at very substantial levels (85–91% of control). Thus, these data indicate that a functional TCR-β transgene does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε subunit.


The CD3-gammadeltaepsilon 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.

Ardouin L, Ismaili J, Malissen B, Malissen M - J. Exp. Med. (1998)

Expression of a transgenic TCR-β chain does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε polypeptide. (A)  Relative levels of TCR-β rearrangements in CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and  total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Identical sorting windows were set up on CD25high DN cells for both the CD3-ε+/+ and  CD3-ε+/+ TCR-β samples. Considering that they contain >90% CD44−/lowCD25high DN cells (see Fig. 2), the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β  thymuses were not subjected to sorting before analysis. The extent of Dβ–Jβ and Vβ–DβJβ rearrangements were analyzed by DNA-PCR. The relative  positions of the PCR primers within the TCR-β locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving  the intronic Jβ2 3′ primer with Dβ2- (top), Vβ5- (middle) or Vβ8- (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-β transgene (Vβ8.1-Dβ-Jβ2.4) is not detectable with the pair of primers used to  reveal endogenous Vβ8–Jβ2 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-ε+/+ (WT) mice.
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Figure 4: Expression of a transgenic TCR-β chain does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε polypeptide. (A) Relative levels of TCR-β rearrangements in CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Identical sorting windows were set up on CD25high DN cells for both the CD3-ε+/+ and CD3-ε+/+ TCR-β samples. Considering that they contain >90% CD44−/lowCD25high DN cells (see Fig. 2), the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β thymuses were not subjected to sorting before analysis. The extent of Dβ–Jβ and Vβ–DβJβ rearrangements were analyzed by DNA-PCR. The relative positions of the PCR primers within the TCR-β locus are depicted by arrows in the bottom diagram. Products derived from PCR reactions involving the intronic Jβ2 3′ primer with Dβ2- (top), Vβ5- (middle) or Vβ8- (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-β transgene (Vβ8.1-Dβ-Jβ2.4) is not detectable with the pair of primers used to reveal endogenous Vβ8–Jβ2 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-ε+/+ (WT) mice.
Mentions: Having established that the P14 TCR-β transgene was expressed properly at the stage at which allelic exclusion is expected to take place, we then determined its impact on TCR-β gene allelic exclusion in the presence or absence of the CD3-ε mutation. This can be assessed using a DNA-PCR assay that provides an estimation of the relative levels of Dβ→ Jβ and Vβ→ DβJβ rearrangements in various cell samples (30). As depicted in Fig. 4 (bottom diagram), primers complementary to Vβ or Dβ gene segments were used in combination with a primer positioned immediately 3′ to the Jβ2 cluster, allowing amplification of rearranged, but not germline, Vβ gene segments. The resulting PCR products were visualized by hybridization with a Jβ2-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 Dβ2 (top), Vβ5 (middle), and Vβ8 (bottom) to each of the six Jβ2 gene segments and were generated using CD25+ cells sorted from CD3-ε+/+ (WT CD25+) and CD3-ε+/+ TCR-β (TCRβ CD25+) thymuses, and total thymocytes from CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β mice. Consistent with previous data indicating that β chain gene allelic exclusion acts at a point subsequent to Dβ–Jβ joining events (1), the levels of Dβ2 to Jβ2 rearrangements were almost equally high in all four samples (Fig. 4, A and B, top). Comparison of CD3-εΔ5/Δ5 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-εΔ5/Δ5). As previously documented, using total thymocytes and TCR-β transgenes unrelated to the one used herein (18, 19, 30), expression of the P14 TCR-β transgene in wild-type CD25+ DN cells (Fig. 4, lane TCRβ CD25+) resulted in a dramatic reduction of endogenous Vβ rearrangements (∼13% of control). The latter result confirms that the P14 TCR-β transgene is expressed in a functional form at the CD25+ DN stage, capable of mediating allelic exclusion at the TCR-β locus and preventing expression of a second TCR-β chain on the cell surface of transgenic SP thymocytes (Fig. 1 B). In contrast, the simultaneous presence of the CD3-εΔ5 mutation (lane TCRβ CD3-εΔ5/Δ5, Fig. 4) prevented the effects of the transgenic TCR-β chain and permitted rearrangements of endogenous TCR Vβ gene segments to occur at very substantial levels (85–91% of control). Thus, these data indicate that a functional TCR-β transgene does not inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε subunit.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: 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.

ABSTRACT
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-gammadeltaepsilon 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.

Show MeSH
Related in: MedlinePlus