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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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A transgene encoding  a productively rearranged TCR-β  gene does not restore T cell development in CD3-ε–deficient  mice. Mice with CD3-ε+/+  (WT), CD3-ε+/+ TCR-β (WT  TCR-β), CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β genotypes were  derived from a F2 intercross between the TCR-β transgenic  line P14 TCR-β (TCR-β) and  CD3-εΔ5/Δ5 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-ε and Vβ8.  Percentage of CD3high and Vβ8high  cells are indicated. Considering  that the exon coding for the  epitope recognized by the 2C11  anti–CD3-ε antibody has been  deleted in the CD3-εΔ5 mutant  gene, and that CD3-εΔ5/Δ5 thymocytes do not express detectable levels of TCR-β chain at their surface (12), the histograms obtained after staining CD3-εΔ5/Δ5 thymocytes with anti– CD3-ε or anti-Vβ8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-β  transgenic SCID mice (50) and TCR-β transgenic RAG−/− mice (51) where transgenic TCR-β chains are expressed as monomers without CD3-ε and  in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR-β transgenic chains on the surface of CD3-εΔ5/Δ5 TCR-β thymocytes  after staining with the Cβ-specific antibody H57.597 (data not shown) and Vβ8-specific antibody F23.1 (compare the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β histograms). Whether such difference resulted from the use of the P14 TCR-β transgene or is rather due to the CD3-εΔ5/Δ5 background remains to be determined.
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Figure 1: A transgene encoding a productively rearranged TCR-β gene does not restore T cell development in CD3-ε–deficient mice. Mice with CD3-ε+/+ (WT), CD3-ε+/+ TCR-β (WT TCR-β), CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β genotypes were derived from a F2 intercross between the TCR-β transgenic line P14 TCR-β (TCR-β) and CD3-εΔ5/Δ5 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-ε and Vβ8. Percentage of CD3high and Vβ8high cells are indicated. Considering that the exon coding for the epitope recognized by the 2C11 anti–CD3-ε antibody has been deleted in the CD3-εΔ5 mutant gene, and that CD3-εΔ5/Δ5 thymocytes do not express detectable levels of TCR-β chain at their surface (12), the histograms obtained after staining CD3-εΔ5/Δ5 thymocytes with anti– CD3-ε or anti-Vβ8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-β transgenic SCID mice (50) and TCR-β transgenic RAG−/− mice (51) where transgenic TCR-β chains are expressed as monomers without CD3-ε and in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR-β transgenic chains on the surface of CD3-εΔ5/Δ5 TCR-β thymocytes after staining with the Cβ-specific antibody H57.597 (data not shown) and Vβ8-specific antibody F23.1 (compare the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β histograms). Whether such difference resulted from the use of the P14 TCR-β transgene or is rather due to the CD3-εΔ5/Δ5 background remains to be determined.

Mentions: To determine whether the expression of a TCR-β chain transgene was able to inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε subunit, CD3-εΔ5/Δ5 mice were crossed with transgenic mice carrying a productively rearranged Vβ8+ TCR-β chain derived from the P14 T cell clone (24, 28). When expressed in a wild-type background, this TCR-β transgene prevented endogenous β-locus gene rearrangements, as judged by the fact that most of the SP thymocytes developing in these mice were Vβ8+ (Fig. 1, compare transgenic [WT TCR-β] and nontransgenic [WT] wild-type panels). As shown in Fig. 1 A, CD3-εΔ5/Δ5 TCR-β mice had thymuses that did not develop past the DN stage and contained absolute cell numbers similar to nontransgenic CD3-εΔ5/Δ5 thymuses. Thus, expression of the P14 TCR-β transgene was unable to restore T cell development in CD3-εΔ5/Δ5 mice. To specify more precisely the effect of the TCR-β transgene on early T cell development, we analyzed the CD44/CD25 profile of wild-type and CD3-εΔ5/Δ5 DN thymocytes that developed in the absence or presence of the P14 TCR-β 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-β) 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-β transgenic mice exhibit CD44−/lowCD25+ cell compartments the size of which are intermediate between those found in nontransgenic and TCR-α/β transgenic mice (19, 20). Such observations have been generally accounted for by the fact that CD44−/lowCD25+ cells equipped with a productively rearranged TCR-β 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-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β 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-γδε module, pTα–TCR-βP14 heterodimers were prevented from assembling into functional pre-TCR complexes and unable to rescue the blockade in thymic development observed in CD3-εΔ5/Δ5 mice. Alternatively, the onset of expression of the P14 TCR-β 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-εΔ5/Δ5 mice.


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)

A transgene encoding  a productively rearranged TCR-β  gene does not restore T cell development in CD3-ε–deficient  mice. Mice with CD3-ε+/+  (WT), CD3-ε+/+ TCR-β (WT  TCR-β), CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β genotypes were  derived from a F2 intercross between the TCR-β transgenic  line P14 TCR-β (TCR-β) and  CD3-εΔ5/Δ5 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-ε and Vβ8.  Percentage of CD3high and Vβ8high  cells are indicated. Considering  that the exon coding for the  epitope recognized by the 2C11  anti–CD3-ε antibody has been  deleted in the CD3-εΔ5 mutant  gene, and that CD3-εΔ5/Δ5 thymocytes do not express detectable levels of TCR-β chain at their surface (12), the histograms obtained after staining CD3-εΔ5/Δ5 thymocytes with anti– CD3-ε or anti-Vβ8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-β  transgenic SCID mice (50) and TCR-β transgenic RAG−/− mice (51) where transgenic TCR-β chains are expressed as monomers without CD3-ε and  in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR-β transgenic chains on the surface of CD3-εΔ5/Δ5 TCR-β thymocytes  after staining with the Cβ-specific antibody H57.597 (data not shown) and Vβ8-specific antibody F23.1 (compare the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β histograms). Whether such difference resulted from the use of the P14 TCR-β transgene or is rather due to the CD3-εΔ5/Δ5 background remains to be determined.
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Related In: Results  -  Collection

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Figure 1: A transgene encoding a productively rearranged TCR-β gene does not restore T cell development in CD3-ε–deficient mice. Mice with CD3-ε+/+ (WT), CD3-ε+/+ TCR-β (WT TCR-β), CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β genotypes were derived from a F2 intercross between the TCR-β transgenic line P14 TCR-β (TCR-β) and CD3-εΔ5/Δ5 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-ε and Vβ8. Percentage of CD3high and Vβ8high cells are indicated. Considering that the exon coding for the epitope recognized by the 2C11 anti–CD3-ε antibody has been deleted in the CD3-εΔ5 mutant gene, and that CD3-εΔ5/Δ5 thymocytes do not express detectable levels of TCR-β chain at their surface (12), the histograms obtained after staining CD3-εΔ5/Δ5 thymocytes with anti– CD3-ε or anti-Vβ8 antibodies were used as genuine negative control histograms. Note that in contrast to the situation previously observed in TCR-β transgenic SCID mice (50) and TCR-β transgenic RAG−/− mice (51) where transgenic TCR-β chains are expressed as monomers without CD3-ε and in a phosphatidyl inositol-linked form, we have not been able to detect P14 TCR-β transgenic chains on the surface of CD3-εΔ5/Δ5 TCR-β thymocytes after staining with the Cβ-specific antibody H57.597 (data not shown) and Vβ8-specific antibody F23.1 (compare the CD3-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β histograms). Whether such difference resulted from the use of the P14 TCR-β transgene or is rather due to the CD3-εΔ5/Δ5 background remains to be determined.
Mentions: To determine whether the expression of a TCR-β chain transgene was able to inhibit endogenous Vβ to DβJβ rearrangements in the absence of CD3-ε subunit, CD3-εΔ5/Δ5 mice were crossed with transgenic mice carrying a productively rearranged Vβ8+ TCR-β chain derived from the P14 T cell clone (24, 28). When expressed in a wild-type background, this TCR-β transgene prevented endogenous β-locus gene rearrangements, as judged by the fact that most of the SP thymocytes developing in these mice were Vβ8+ (Fig. 1, compare transgenic [WT TCR-β] and nontransgenic [WT] wild-type panels). As shown in Fig. 1 A, CD3-εΔ5/Δ5 TCR-β mice had thymuses that did not develop past the DN stage and contained absolute cell numbers similar to nontransgenic CD3-εΔ5/Δ5 thymuses. Thus, expression of the P14 TCR-β transgene was unable to restore T cell development in CD3-εΔ5/Δ5 mice. To specify more precisely the effect of the TCR-β transgene on early T cell development, we analyzed the CD44/CD25 profile of wild-type and CD3-εΔ5/Δ5 DN thymocytes that developed in the absence or presence of the P14 TCR-β 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-β) 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-β transgenic mice exhibit CD44−/lowCD25+ cell compartments the size of which are intermediate between those found in nontransgenic and TCR-α/β transgenic mice (19, 20). Such observations have been generally accounted for by the fact that CD44−/lowCD25+ cells equipped with a productively rearranged TCR-β 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-εΔ5/Δ5 and CD3-εΔ5/Δ5 TCR-β 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-γδε module, pTα–TCR-βP14 heterodimers were prevented from assembling into functional pre-TCR complexes and unable to rescue the blockade in thymic development observed in CD3-εΔ5/Δ5 mice. Alternatively, the onset of expression of the P14 TCR-β 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-εΔ5/Δ5 mice.

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