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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.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.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.

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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Vβ8.1/8.2 versus Vβ-pool staining of lymph node cells from  TCR-β-transgenic mice expressing (pTα+) or lacking (pTα−/−) a functional pTα gene (four-color analysis). The transgene-encoded receptor  contains the Vβ8.2 element. Staining and gating was done as described in  Fig. 1.
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Figure 2: Vβ8.1/8.2 versus Vβ-pool staining of lymph node cells from TCR-β-transgenic mice expressing (pTα+) or lacking (pTα−/−) a functional pTα gene (four-color analysis). The transgene-encoded receptor contains the Vβ8.2 element. Staining and gating was done as described in Fig. 1.

Mentions: For flow cytometry, single cell suspensions from thymi and lymph nodes (axial, mesenteric, inguinal) were prepared in PBS containing 2% calf serum (CS). The number of viable cells was determined using a Coulter counter. Thymocytes were stained at 5 × 106 cells per ml in PBS, 2% CS containing the relevant antibodies at saturating concentrations. When pools of anti-Vβ reagents were used, it proved necessary to dialyze the antibody mix containing all first-step antibodies immediately before the staining, to eliminate the cytotoxicity of the concentrated reagents. Dialysis was performed at 4°C in ultra thimbles (Schleicher & Schuell UH 100/10; cutoff ∼10,000 MM) against two changes of PBS, 2% CS for 2 × 30 min. Phenotypes and proportions of thymocyte subsets were analyzed by three-color flow cytometry using a FACScan® (Beckton Dickinson, Mountain View, CA) and the Lysis II program. The data depicted in Figs. 2 and 3 were analyzed with the program Cellquest (Becton Dickinson). Dead cells were excluded from the analysis by forward- and side-scatter gating, when analyzing numerically small subpopulations also by addition of propidium iodine (PI) and gating on PI− cells. Four-color analyses and cell sorting were performed on a FACStar Plus® (Beckton Dickinson) equipped with a pulse processor for forward-scatter width (FSC-W) in order to gate out cell doublets.


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)

Vβ8.1/8.2 versus Vβ-pool staining of lymph node cells from  TCR-β-transgenic mice expressing (pTα+) or lacking (pTα−/−) a functional pTα gene (four-color analysis). The transgene-encoded receptor  contains the Vβ8.2 element. Staining and gating was done as described in  Fig. 1.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199014&req=5

Figure 2: Vβ8.1/8.2 versus Vβ-pool staining of lymph node cells from TCR-β-transgenic mice expressing (pTα+) or lacking (pTα−/−) a functional pTα gene (four-color analysis). The transgene-encoded receptor contains the Vβ8.2 element. Staining and gating was done as described in Fig. 1.
Mentions: For flow cytometry, single cell suspensions from thymi and lymph nodes (axial, mesenteric, inguinal) were prepared in PBS containing 2% calf serum (CS). The number of viable cells was determined using a Coulter counter. Thymocytes were stained at 5 × 106 cells per ml in PBS, 2% CS containing the relevant antibodies at saturating concentrations. When pools of anti-Vβ reagents were used, it proved necessary to dialyze the antibody mix containing all first-step antibodies immediately before the staining, to eliminate the cytotoxicity of the concentrated reagents. Dialysis was performed at 4°C in ultra thimbles (Schleicher & Schuell UH 100/10; cutoff ∼10,000 MM) against two changes of PBS, 2% CS for 2 × 30 min. Phenotypes and proportions of thymocyte subsets were analyzed by three-color flow cytometry using a FACScan® (Beckton Dickinson, Mountain View, CA) and the Lysis II program. The data depicted in Figs. 2 and 3 were analyzed with the program Cellquest (Becton Dickinson). Dead cells were excluded from the analysis by forward- and side-scatter gating, when analyzing numerically small subpopulations also by addition of propidium iodine (PI) and gating on PI− cells. Four-color analyses and cell sorting were performed on a FACStar Plus® (Beckton Dickinson) equipped with a pulse processor for forward-scatter width (FSC-W) in order to gate out cell doublets.

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.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.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.

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