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Early T cell receptor beta gene expression is regulated by the pre-T cell receptor-CD3 complex.

Aifantis I, Feinberg J, Fehling HJ, Di Santo JP, von Boehmer H - J. Exp. Med. (1999)

Bottom Line: We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR.Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.

View Article: PubMed Central - PubMed

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

ABSTRACT
We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.

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Intracellular staining for TCR-β in CD4−8− thymocytes from C57BL/6 (WT), γc−/−, pTα−/−, CD3∈−/−, and Rag2−/− mice. (A) Total CD4−8− cells were surface stained with anti-CD25; cytoplasmic staining was performed with anti–pan TCR-β (H57) antibodies. (B) Intracellular TCR-β expression in gated small CD25+4−8− thymocytes.
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Figure 2: Intracellular staining for TCR-β in CD4−8− thymocytes from C57BL/6 (WT), γc−/−, pTα−/−, CD3∈−/−, and Rag2−/− mice. (A) Total CD4−8− cells were surface stained with anti-CD25; cytoplasmic staining was performed with anti–pan TCR-β (H57) antibodies. (B) Intracellular TCR-β expression in gated small CD25+4−8− thymocytes.

Mentions: We have analyzed thymocytes from wild-type, γc−/− 12, pTα−/− 6, CD3∈−/− 13, and Rag2−/− mice 14 in order to analyze the effect of each mutation on TCR-β gene expression in small CD25+44− cells. The subset distribution among CD4−8− cells according to CD44 and CD25 expression is shown in Fig. 1. Wild-type and γc−/− mice exhibit a similar phenotype except for an elevated proportion of CD44+25+ cells in the latter due to a partial block at this stage of development in γc−/− mice. pTα−/− mice look similar to CD3∈−/− and Rag2−/− mice, but due to their incomplete block at the CD44−25+ stage of development, contain more CD44−25− cells than the latter two strains. Of these, some 70% are γ/δ T cells 6. When intracellular TCR-β expression versus CD25 expression was analyzed in all CD4−8− cells, it became clear that wild-type and γc−/− thymocytes express TCR β chains in the majority of cells, but γc−/− thymocytes less so because of an early partial block before TCR-β rearrangement at the CD44+25+ stage 12. In these two strains, most TCR-β expression was present in CD25− cells. In contrast, in pTα−/− and CD3∈−/− mice, most TCR-β expression was found in CD25+ cells, although less completely so in pTα−/− mice because of a partial developmental block at the CD25+44− stage resulting in a population of CD25−44− cells, of which up to 70% are γ/δ T cells. Of these γ/δ T cells, up to 25% expressed cytoplasmic TCR β chains 15, which accounts for the cytoplasmic TCR-β staining in the CD25− cells in pTα−/− mice (Fig. 2 A). There is naturally no TCR-β expression in Rag2−/− mice (Fig. 2). However, this picture changed, to some extent, when the analysis was performed on smaller cells where the proportion of TCR-β+ cells among CD25+ cells was significantly decreased in wild-type and γc−/− mice but not at all or only marginally in pTα−/− and CD3∈−/− mice (Fig. 2 B). What is also apparent in Fig. 2 B is that the proportion of TCR-β1 cells among small CD25+ cells is significantly smaller in wild-type and γc−/− mice, while it is larger in pTα−/− and CD3∈−/− mice. This is due to the fact that in CD25+ cells from pTα−/− and CD3∈−/− mice, TCR-β rearrangement proceeds further than in normal mice 1617. It is also clear from Fig. 2a and Fig. b, that CD25+ cells in wild-type and γc−/− mice express on average higher TCR-β levels than CD25+ cells from pTα−/− and CD3∈−/− mice, and that with regard to this parameter there is no significant difference between CD25+ cells from pTα−/− and CD3∈−/− cells. Actually, there is a continuous spectrum of TCR-β expression rather than a discrete peak, which would be expected from a population of cells that undergoes TCR-β rearrangement and begins to express productive genes. Nevertheless, there is no doubt that the staining is specific, since there is no staining in the same population of cells in Rag2−/− mice (Fig. 2), and also because an irrelevant control antibody of the same Ig class does not stain in all different mouse strains (data not shown). Thus, all differences that exist between wild-type and CD3∈−/− mice with regard to TCR-β expression in CD25+ cells can be attributed to defective signaling by the pre-TCR rather than to an independent control of TCR-β expression by the CD3 complex alone.


Early T cell receptor beta gene expression is regulated by the pre-T cell receptor-CD3 complex.

Aifantis I, Feinberg J, Fehling HJ, Di Santo JP, von Boehmer H - J. Exp. Med. (1999)

Intracellular staining for TCR-β in CD4−8− thymocytes from C57BL/6 (WT), γc−/−, pTα−/−, CD3∈−/−, and Rag2−/− mice. (A) Total CD4−8− cells were surface stained with anti-CD25; cytoplasmic staining was performed with anti–pan TCR-β (H57) antibodies. (B) Intracellular TCR-β expression in gated small CD25+4−8− thymocytes.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2195557&req=5

Figure 2: Intracellular staining for TCR-β in CD4−8− thymocytes from C57BL/6 (WT), γc−/−, pTα−/−, CD3∈−/−, and Rag2−/− mice. (A) Total CD4−8− cells were surface stained with anti-CD25; cytoplasmic staining was performed with anti–pan TCR-β (H57) antibodies. (B) Intracellular TCR-β expression in gated small CD25+4−8− thymocytes.
Mentions: We have analyzed thymocytes from wild-type, γc−/− 12, pTα−/− 6, CD3∈−/− 13, and Rag2−/− mice 14 in order to analyze the effect of each mutation on TCR-β gene expression in small CD25+44− cells. The subset distribution among CD4−8− cells according to CD44 and CD25 expression is shown in Fig. 1. Wild-type and γc−/− mice exhibit a similar phenotype except for an elevated proportion of CD44+25+ cells in the latter due to a partial block at this stage of development in γc−/− mice. pTα−/− mice look similar to CD3∈−/− and Rag2−/− mice, but due to their incomplete block at the CD44−25+ stage of development, contain more CD44−25− cells than the latter two strains. Of these, some 70% are γ/δ T cells 6. When intracellular TCR-β expression versus CD25 expression was analyzed in all CD4−8− cells, it became clear that wild-type and γc−/− thymocytes express TCR β chains in the majority of cells, but γc−/− thymocytes less so because of an early partial block before TCR-β rearrangement at the CD44+25+ stage 12. In these two strains, most TCR-β expression was present in CD25− cells. In contrast, in pTα−/− and CD3∈−/− mice, most TCR-β expression was found in CD25+ cells, although less completely so in pTα−/− mice because of a partial developmental block at the CD25+44− stage resulting in a population of CD25−44− cells, of which up to 70% are γ/δ T cells. Of these γ/δ T cells, up to 25% expressed cytoplasmic TCR β chains 15, which accounts for the cytoplasmic TCR-β staining in the CD25− cells in pTα−/− mice (Fig. 2 A). There is naturally no TCR-β expression in Rag2−/− mice (Fig. 2). However, this picture changed, to some extent, when the analysis was performed on smaller cells where the proportion of TCR-β+ cells among CD25+ cells was significantly decreased in wild-type and γc−/− mice but not at all or only marginally in pTα−/− and CD3∈−/− mice (Fig. 2 B). What is also apparent in Fig. 2 B is that the proportion of TCR-β1 cells among small CD25+ cells is significantly smaller in wild-type and γc−/− mice, while it is larger in pTα−/− and CD3∈−/− mice. This is due to the fact that in CD25+ cells from pTα−/− and CD3∈−/− mice, TCR-β rearrangement proceeds further than in normal mice 1617. It is also clear from Fig. 2a and Fig. b, that CD25+ cells in wild-type and γc−/− mice express on average higher TCR-β levels than CD25+ cells from pTα−/− and CD3∈−/− mice, and that with regard to this parameter there is no significant difference between CD25+ cells from pTα−/− and CD3∈−/− cells. Actually, there is a continuous spectrum of TCR-β expression rather than a discrete peak, which would be expected from a population of cells that undergoes TCR-β rearrangement and begins to express productive genes. Nevertheless, there is no doubt that the staining is specific, since there is no staining in the same population of cells in Rag2−/− mice (Fig. 2), and also because an irrelevant control antibody of the same Ig class does not stain in all different mouse strains (data not shown). Thus, all differences that exist between wild-type and CD3∈−/− mice with regard to TCR-β expression in CD25+ cells can be attributed to defective signaling by the pre-TCR rather than to an independent control of TCR-β expression by the CD3 complex alone.

Bottom Line: We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR.Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.

View Article: PubMed Central - PubMed

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

ABSTRACT
We have examined the question of whether there is an additional checkpoint in T cell development that regulates T cell receptor (TCR)-beta expression in CD25+44- thymocytes by mechanisms that are independent of the pre-TCR. Our analysis in various mutant mice indicates that all changes in cytoplasmic TCR-beta expression can be accounted for by pre-TCR-dependent signal mediation, putting into question the function of a putative pro-TCR.

Show MeSH