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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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Expression of TCR-β transgenes inhibits Vγ–Jγ rearrangements. Thymocyte DNA from four types of mice (pTα+ or pTα−/− mice  with or without TCR-β transgenes [β-TG]) was amplified with primers  specific for Vγ4 and Jγ1 (nomenclature according to reference 27). Three  different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested  to ensure linearity of the signals. An aliquot of the PCR mastermix was  amplified with primers specific for the constant region of IgM to provide  an internal control for the amount of template used (Ig-C bands). Specific  bands were detected with appropriate end-labeled oligonucleotide probes.  N denotes a negative control (kidney DNA).
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Figure 4: Expression of TCR-β transgenes inhibits Vγ–Jγ rearrangements. Thymocyte DNA from four types of mice (pTα+ or pTα−/− mice with or without TCR-β transgenes [β-TG]) was amplified with primers specific for Vγ4 and Jγ1 (nomenclature according to reference 27). Three different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested to ensure linearity of the signals. An aliquot of the PCR mastermix was amplified with primers specific for the constant region of IgM to provide an internal control for the amount of template used (Ig-C bands). Specific bands were detected with appropriate end-labeled oligonucleotide probes. N denotes a negative control (kidney DNA).

Mentions: Previous experiments in normal (pTα+) mice have shown that a TCR-β transgene inhibits TCR-γ rearrangements and the generation of γδ expressing cells (32). Interestingly, the TCR-β transgene was able to suppress γ rearrangements, as measured by PCR with primers specific for Vγ4 and Jγ1, even in the absence of pTα (Fig. 4), although somewhat less efficiently than in the presence of pTα. The TCR-β transgene also mediated a strong reduction in the number of γδ cells in pTα-deficient mice (see Fig. 3 C). This effect appears particularly striking, when taking into account that the percentage and absolute number of γδ cells is strongly augmented in normal (nontransgenic) pTα−/− mice in comparison to wild-type littermates (30) (Fig. 3 C, compare the two left panels). The inhibition of TCR-γ rearrangements and the strong suppression of γδ T cell development illustrates once more that the TCR-β transgene is not innocuous despite the absence of pTα.


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)

Expression of TCR-β transgenes inhibits Vγ–Jγ rearrangements. Thymocyte DNA from four types of mice (pTα+ or pTα−/− mice  with or without TCR-β transgenes [β-TG]) was amplified with primers  specific for Vγ4 and Jγ1 (nomenclature according to reference 27). Three  different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested  to ensure linearity of the signals. An aliquot of the PCR mastermix was  amplified with primers specific for the constant region of IgM to provide  an internal control for the amount of template used (Ig-C bands). Specific  bands were detected with appropriate end-labeled oligonucleotide probes.  N denotes a negative control (kidney DNA).
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Related In: Results  -  Collection

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Figure 4: Expression of TCR-β transgenes inhibits Vγ–Jγ rearrangements. Thymocyte DNA from four types of mice (pTα+ or pTα−/− mice with or without TCR-β transgenes [β-TG]) was amplified with primers specific for Vγ4 and Jγ1 (nomenclature according to reference 27). Three different amounts of template DNA (300 ng, 200 ng, 100 ng) were tested to ensure linearity of the signals. An aliquot of the PCR mastermix was amplified with primers specific for the constant region of IgM to provide an internal control for the amount of template used (Ig-C bands). Specific bands were detected with appropriate end-labeled oligonucleotide probes. N denotes a negative control (kidney DNA).
Mentions: Previous experiments in normal (pTα+) mice have shown that a TCR-β transgene inhibits TCR-γ rearrangements and the generation of γδ expressing cells (32). Interestingly, the TCR-β transgene was able to suppress γ rearrangements, as measured by PCR with primers specific for Vγ4 and Jγ1, even in the absence of pTα (Fig. 4), although somewhat less efficiently than in the presence of pTα. The TCR-β transgene also mediated a strong reduction in the number of γδ cells in pTα-deficient mice (see Fig. 3 C). This effect appears particularly striking, when taking into account that the percentage and absolute number of γδ cells is strongly augmented in normal (nontransgenic) pTα−/− mice in comparison to wild-type littermates (30) (Fig. 3 C, compare the two left panels). The inhibition of TCR-γ rearrangements and the strong suppression of γδ T cell development illustrates once more that the TCR-β transgene is not innocuous despite the absence of pTα.

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