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Allelic exclusion in pTalpha-deficient mice: no evidence for cell surface expression of two T cell receptor (TCR)-beta chains, but less efficient inhibition of endogeneous Vbeta--> (D)Jbeta rearrangements in the presence of a functional TCR-beta transgene.

Krotkova A, von Boehmer H, Fehling HJ - J. Exp. Med. (1997)

Bottom Line: Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion.Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta6 or Vbeta8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression.Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha, it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha-/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-beta chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-beta rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-beta rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-alpha (pTalpha) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta6 or Vbeta8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTalpha-deficient mice expressing a functionally rearranged TCR-beta transgene. Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha, it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha-/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.

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Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD3−4−8−) thymocytes. (A) Gates used to isolate  CD25+CD44−/low triple-negative thymocytes. Thymocytes from three  mice of the same genotype were pooled and stained as described in Fig. 3  B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and  CD8-expressing cells) were eliminated by electronic gating (data not  shown). The panels representing the B6 and the TCR-β-transgenic,  pTα+ mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pTα−/− animals)  had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show  the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details,  see legend to Fig. 6. In this particular experiment, Vβ11 rearrangements  involving all Jβ2 elements, except Jβ2.6, were not appropriately amplified  at the highest template concentration (lane 4). This phenomenon is due  to excess template DNA and is not related to the mouse genotype, because a failure to amplify Vβ→ Jβ rearrangements that correspond to  larger PCR bands than Vβ→ Jβ2.6 rearrangements has also been observed  in a few other experiments at the highest DNA concentration when the  template DNA was derived from wild-type or nontransgenic pTα−/−  mice.
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Figure 7: Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD3−4−8−) thymocytes. (A) Gates used to isolate CD25+CD44−/low triple-negative thymocytes. Thymocytes from three mice of the same genotype were pooled and stained as described in Fig. 3 B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and CD8-expressing cells) were eliminated by electronic gating (data not shown). The panels representing the B6 and the TCR-β-transgenic, pTα+ mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pTα−/− animals) had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details, see legend to Fig. 6. In this particular experiment, Vβ11 rearrangements involving all Jβ2 elements, except Jβ2.6, were not appropriately amplified at the highest template concentration (lane 4). This phenomenon is due to excess template DNA and is not related to the mouse genotype, because a failure to amplify Vβ→ Jβ rearrangements that correspond to larger PCR bands than Vβ→ Jβ2.6 rearrangements has also been observed in a few other experiments at the highest DNA concentration when the template DNA was derived from wild-type or nontransgenic pTα−/− mice.

Mentions: Because the proportion of various thymocyte subpopulations is markedly altered in pTα-deficient mice, owing to the severe block in αβ T cell development (30), we were concerned that our results might have been influenced inappropriately by differences in the cellular composition of thymi from TCR-β–transgenic pTα+ and pTα−/− mice, respectively, although it was difficult to envisage specifically how this could have mimicked enhanced endogeneous rearrangements in the absence of pTα. Nevertheless, to exclude such a possibility completely and to allow a comparison of equivalent thymic subpopulations, we verified our PCR analysis of endogeneous Vβ→ (D)Jβ rearrangements with DNA from sorted CD25+CD44−/low DN thymocytes, which represent the developmental stage at which TCR-β rearrangements predominantly occur (12, 14). Fig. 7 shows that the result of this analysis was exactly the same as with unfractionated thymocytes; although the transgenic TCR-β chain could inhibit endogeneous Vβ→ (D)Jβ rearrangements in the absence of pTα (Fig. 7, compare lanes 4, 8, 12 with lanes 3, 7, 11), the degree of inhibition was less pronounced than in CD25+ thymocytes expressing pTα. Taken together, our data clearly indicate that the pTα chain contributes to the inhibition of endogeneous Vβ rearrangements by productive TCR-β transgenes.


Allelic exclusion in pTalpha-deficient mice: no evidence for cell surface expression of two T cell receptor (TCR)-beta chains, but less efficient inhibition of endogeneous Vbeta--> (D)Jbeta rearrangements in the presence of a functional TCR-beta transgene.

Krotkova A, von Boehmer H, Fehling HJ - J. Exp. Med. (1997)

Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD3−4−8−) thymocytes. (A) Gates used to isolate  CD25+CD44−/low triple-negative thymocytes. Thymocytes from three  mice of the same genotype were pooled and stained as described in Fig. 3  B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and  CD8-expressing cells) were eliminated by electronic gating (data not  shown). The panels representing the B6 and the TCR-β-transgenic,  pTα+ mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pTα−/− animals)  had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show  the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details,  see legend to Fig. 6. In this particular experiment, Vβ11 rearrangements  involving all Jβ2 elements, except Jβ2.6, were not appropriately amplified  at the highest template concentration (lane 4). This phenomenon is due  to excess template DNA and is not related to the mouse genotype, because a failure to amplify Vβ→ Jβ rearrangements that correspond to  larger PCR bands than Vβ→ Jβ2.6 rearrangements has also been observed  in a few other experiments at the highest DNA concentration when the  template DNA was derived from wild-type or nontransgenic pTα−/−  mice.
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Figure 7: Analysis of genomic DNA isolated from sorted CD25+ triple-negative (CD3−4−8−) thymocytes. (A) Gates used to isolate CD25+CD44−/low triple-negative thymocytes. Thymocytes from three mice of the same genotype were pooled and stained as described in Fig. 3 B. FITC-positive cells (macrophages, granulocytes, B cells, CD4-, and CD8-expressing cells) were eliminated by electronic gating (data not shown). The panels representing the B6 and the TCR-β-transgenic, pTα+ mice have a much higher density of dots, because CD4- and CD8-expressing thymocytes from these mice (but not from pTα−/− animals) had been depleted with complement before staining, so that triple-negative thymocytes were already strongly enriched. The lower panels show the purity of the populations after sorting. (B) PCR-based analysis of genomic DNA from sorted CD25+ triple-negative thymocytes. For details, see legend to Fig. 6. In this particular experiment, Vβ11 rearrangements involving all Jβ2 elements, except Jβ2.6, were not appropriately amplified at the highest template concentration (lane 4). This phenomenon is due to excess template DNA and is not related to the mouse genotype, because a failure to amplify Vβ→ Jβ rearrangements that correspond to larger PCR bands than Vβ→ Jβ2.6 rearrangements has also been observed in a few other experiments at the highest DNA concentration when the template DNA was derived from wild-type or nontransgenic pTα−/− mice.
Mentions: Because the proportion of various thymocyte subpopulations is markedly altered in pTα-deficient mice, owing to the severe block in αβ T cell development (30), we were concerned that our results might have been influenced inappropriately by differences in the cellular composition of thymi from TCR-β–transgenic pTα+ and pTα−/− mice, respectively, although it was difficult to envisage specifically how this could have mimicked enhanced endogeneous rearrangements in the absence of pTα. Nevertheless, to exclude such a possibility completely and to allow a comparison of equivalent thymic subpopulations, we verified our PCR analysis of endogeneous Vβ→ (D)Jβ rearrangements with DNA from sorted CD25+CD44−/low DN thymocytes, which represent the developmental stage at which TCR-β rearrangements predominantly occur (12, 14). Fig. 7 shows that the result of this analysis was exactly the same as with unfractionated thymocytes; although the transgenic TCR-β chain could inhibit endogeneous Vβ→ (D)Jβ rearrangements in the absence of pTα (Fig. 7, compare lanes 4, 8, 12 with lanes 3, 7, 11), the degree of inhibition was less pronounced than in CD25+ thymocytes expressing pTα. Taken together, our data clearly indicate that the pTα chain contributes to the inhibition of endogeneous Vβ rearrangements by productive TCR-β transgenes.

Bottom Line: Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion.Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta6 or Vbeta8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression.Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha, it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha-/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.

View Article: PubMed Central - PubMed

Affiliation: Basel Institute for Immunology, CH-4005 Basel, Switzerland.

ABSTRACT
Although individual T lymphocytes have the potential to generate two distinct T cell receptor (TCR)-beta chains, they usually express only one allele, a phenomenon termed allelic exclusion. Expression of a functional TCR-beta chain during early T cell development leads to the formation of a pre-T cell receptor (pre-TCR) complex and, at the same developmental stage, arrest of further TCR-beta rearrangements, suggesting a role of the pre-TCR in mediating allelic exclusion. To investigate the potential link between pre-TCR formation and inhibition of further TCR-beta rearrangements, we have studied the efficiency of allelic exclusion in mice lacking the pre-TCR-alpha (pTalpha) chain, a core component of the pre-TCR. Staining of CD3+ thymocytes and lymph node cells with antibodies specific for Vbeta6 or Vbeta8 and a pool of antibodies specific for most other Vbeta elements, did not reveal any violation of allelic exclusion at the level of cell surface expression. This was also true for pTalpha-deficient mice expressing a functionally rearranged TCR-beta transgene. Interestingly, although the transgenic TCR-beta chain significantly influenced thymocyte development even in the absence of pTalpha, it was not able to inhibit fully endogeneous TCR-beta rearrangements either in total thymocytes or in sorted CD25+ pre-T cells of pTalpha-/- mice, clearly indicating an involvement of the pre-TCR in allelic exclusion.

Show MeSH
Related in: MedlinePlus