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Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein.

Aifantis I, Pivniouk VI, Gärtner F, Feinberg J, Swat W, Alt FW, von Boehmer H, Geha RS - J. Exp. Med. (1999)

Bottom Line: Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion.The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage.Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et Recherche Medicale (INSERM) U373, Hôpital Necker Enfants-Malades, Paris cedex 15, France.

ABSTRACT
Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

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Thymocytes from TCR-α/β–transgenic SLP-76−/− mice fail to suppress V to DJ rearrangements in the endogenous TCR-β locus. 20 ng of genomic DNA isolated from DN thymocytes of SLP-76+/−, SLP-76+/−-tg, SLP-76−/−, and SLP-76−/−-tg was amplified with primers that specifically detect rearrangements among Vβ5, Vβ8, Vβ10, and DJβ2. Equivalent loading was confirmed by amplification of a fragment of the Cμ gene (IgM). The PCR products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled oligonucleotide probes specific for Jβ2.7 and Cμ. For each Vβ, there are six bands that correspond to rearrangements to the DJβ2 segments DJβ2.1 through DJβ2.7 (Jβ2.6 is a pseudogene and is not rearranged). Similar results were obtained in two other experiments.
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Figure 5: Thymocytes from TCR-α/β–transgenic SLP-76−/− mice fail to suppress V to DJ rearrangements in the endogenous TCR-β locus. 20 ng of genomic DNA isolated from DN thymocytes of SLP-76+/−, SLP-76+/−-tg, SLP-76−/−, and SLP-76−/−-tg was amplified with primers that specifically detect rearrangements among Vβ5, Vβ8, Vβ10, and DJβ2. Equivalent loading was confirmed by amplification of a fragment of the Cμ gene (IgM). The PCR products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled oligonucleotide probes specific for Jβ2.7 and Cμ. For each Vβ, there are six bands that correspond to rearrangements to the DJβ2 segments DJβ2.1 through DJβ2.7 (Jβ2.6 is a pseudogene and is not rearranged). Similar results were obtained in two other experiments.

Mentions: In normal thymocytes, introduction of functionally rearranged TCR-β transgenes leads to inhibition of rearrangements in the endogenous TCR-β locus at the V to DJ step 35. Furthermore, introduction of a TCR-β transgene in pTα−/− and p56lck−/− also leads to inhibition of endogenous TCR-β rearrangements 1336. To examine if introduction of the transgenic TCR into SLP-76–deficient thymocytes could also inhibit endogenous Vβ to DJβ rearrangements, we used a semiquantitative PCR assay as previously described 1637. In these experiments, Vβ to DJβ2 rearrangements involving representative Vβ segments (Vβ5, Vβ8, and Vβ10) were amplified from genomic DNA samples isolated from DN thymocytes of TCR-transgenic or nontransgenic SLP-76+/− or SLP-76−/− mice. The identity of the resulting DNA fragments corresponding to rearrangements between a particular Vβ and one of the Jβ segments was confirmed by Southern blot analysis with a Jβ2.7-specific probe. In agreement with our previously published observations 17, Vβ to DJβ rearrangements were readily detectable in both SLP-76+/− and SLP-76−/− thymocytes (Fig. 5, lanes 1, 3, 5, 7, 9, and 11). As expected, introduction of the TCR-α/β transgene into SLP-76+/− thymocytes almost completely blocked Vβ to DJβ rearrangements of the endogenous TCR-β genes (Fig. 5, lanes 1, 2, 5, 6, 9, and 10). In contrast, rearrangements of the endogenous TCR-β genes occurred at comparable levels in both TCR-transgenic and nontransgenic SLP-76−/− thymocytes (Fig. 5, lanes 3, 4, 7, 8, 11, and 12). These results suggest that SLP-76 is essential for transgene-mediated inhibition of TCR-β locus rearrangement.


Allelic exclusion of the T cell receptor beta locus requires the SH2 domain-containing leukocyte protein (SLP)-76 adaptor protein.

Aifantis I, Pivniouk VI, Gärtner F, Feinberg J, Swat W, Alt FW, von Boehmer H, Geha RS - J. Exp. Med. (1999)

Thymocytes from TCR-α/β–transgenic SLP-76−/− mice fail to suppress V to DJ rearrangements in the endogenous TCR-β locus. 20 ng of genomic DNA isolated from DN thymocytes of SLP-76+/−, SLP-76+/−-tg, SLP-76−/−, and SLP-76−/−-tg was amplified with primers that specifically detect rearrangements among Vβ5, Vβ8, Vβ10, and DJβ2. Equivalent loading was confirmed by amplification of a fragment of the Cμ gene (IgM). The PCR products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled oligonucleotide probes specific for Jβ2.7 and Cμ. For each Vβ, there are six bands that correspond to rearrangements to the DJβ2 segments DJβ2.1 through DJβ2.7 (Jβ2.6 is a pseudogene and is not rearranged). Similar results were obtained in two other experiments.
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Related In: Results  -  Collection

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Figure 5: Thymocytes from TCR-α/β–transgenic SLP-76−/− mice fail to suppress V to DJ rearrangements in the endogenous TCR-β locus. 20 ng of genomic DNA isolated from DN thymocytes of SLP-76+/−, SLP-76+/−-tg, SLP-76−/−, and SLP-76−/−-tg was amplified with primers that specifically detect rearrangements among Vβ5, Vβ8, Vβ10, and DJβ2. Equivalent loading was confirmed by amplification of a fragment of the Cμ gene (IgM). The PCR products were separated on a 1.5% agarose gel, transferred to a nylon membrane, and hybridized with radiolabeled oligonucleotide probes specific for Jβ2.7 and Cμ. For each Vβ, there are six bands that correspond to rearrangements to the DJβ2 segments DJβ2.1 through DJβ2.7 (Jβ2.6 is a pseudogene and is not rearranged). Similar results were obtained in two other experiments.
Mentions: In normal thymocytes, introduction of functionally rearranged TCR-β transgenes leads to inhibition of rearrangements in the endogenous TCR-β locus at the V to DJ step 35. Furthermore, introduction of a TCR-β transgene in pTα−/− and p56lck−/− also leads to inhibition of endogenous TCR-β rearrangements 1336. To examine if introduction of the transgenic TCR into SLP-76–deficient thymocytes could also inhibit endogenous Vβ to DJβ rearrangements, we used a semiquantitative PCR assay as previously described 1637. In these experiments, Vβ to DJβ2 rearrangements involving representative Vβ segments (Vβ5, Vβ8, and Vβ10) were amplified from genomic DNA samples isolated from DN thymocytes of TCR-transgenic or nontransgenic SLP-76+/− or SLP-76−/− mice. The identity of the resulting DNA fragments corresponding to rearrangements between a particular Vβ and one of the Jβ segments was confirmed by Southern blot analysis with a Jβ2.7-specific probe. In agreement with our previously published observations 17, Vβ to DJβ rearrangements were readily detectable in both SLP-76+/− and SLP-76−/− thymocytes (Fig. 5, lanes 1, 3, 5, 7, 9, and 11). As expected, introduction of the TCR-α/β transgene into SLP-76+/− thymocytes almost completely blocked Vβ to DJβ rearrangements of the endogenous TCR-β genes (Fig. 5, lanes 1, 2, 5, 6, 9, and 10). In contrast, rearrangements of the endogenous TCR-β genes occurred at comparable levels in both TCR-transgenic and nontransgenic SLP-76−/− thymocytes (Fig. 5, lanes 3, 4, 7, 8, 11, and 12). These results suggest that SLP-76 is essential for transgene-mediated inhibition of TCR-β locus rearrangement.

Bottom Line: Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion.The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage.Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Institut National de la Santé et Recherche Medicale (INSERM) U373, Hôpital Necker Enfants-Malades, Paris cedex 15, France.

ABSTRACT
Signaling via the pre-T cell receptor (TCR) is required for the proliferative expansion and maturation of CD4(-)CD8(-) double-negative (DN) thymocytes into CD4(+)CD8(+) double-positive (DP) cells and for TCR-beta allelic exclusion. The adaptor protein SH2 domain-containing leukocyte protein (SLP)-76 has been shown to play a crucial role in thymic development, because thymocytes of SLP-76(-/-) mice are arrested at the CD25(+)CD44(-) DN stage. Here we show that SLP-76(-/-) DN thymocytes express the pre-TCR on their surfaces and that introduction of a TCR-alpha/beta transgene into the SLP-76(-/-) background fails to cause expansion of DN thymocytes or developmental progression to the DP stage. Moreover, analysis of TCR-beta rearrangement in SLP-76(-/-) TCR-transgenic mice or in single CD25(+)CD44(-) DN cells from SLP-76(-/-) mice indicates an essential role of SLP-76 in TCR-beta allelic exclusion.

Show MeSH
Related in: MedlinePlus