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The single positive T cells found in CD3-zeta/eta-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex.

Lin SY, Ardouin L, Gillet A, Malissen M, Malissen B - J. Exp. Med. (1997)

Bottom Line: CD3-zeta/eta-deficient mice have small thymuses containing cells that show a profound reduction in the surface levels of T cell receptors and terminate their differentiation at the CD4+CD8+ stage.Fusion of these peripheral T cells with a CD3-zeta-positive derivative of the BW5147 TCR-alpha-/beta- thymoma resulted in hybridomas that do express an heterogeneous set of T cell receptor alpha/beta dimers at their surface and at density comparable to those found in hybridomas derived from wild-type peripheral T cells.These results demonstrate that by increasing the density and/or output of the T cell receptors expressed in peripheral T cells, one can confer them with the capacity to respond to normal density of self-MHC molecules.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie, Institut National de la Sante et de la Recherche Medicale-Centre National de Recherche Scientifique de Marseille-Luminy, France.

ABSTRACT
CD3-zeta/eta-deficient mice have small thymuses containing cells that show a profound reduction in the surface levels of T cell receptors and terminate their differentiation at the CD4+CD8+ stage. Rather unexpectedly, CD3- or very low single positive T cells accumulate over time in the spleen and lymph nodes of CD3-zeta/eta-deficient mice after a process dependent on MHC expression. Fusion of these peripheral T cells with a CD3-zeta-positive derivative of the BW5147 TCR-alpha-/beta- thymoma resulted in hybridomas that do express an heterogeneous set of T cell receptor alpha/beta dimers at their surface and at density comparable to those found in hybridomas derived from wild-type peripheral T cells. We have investigated the specificities of these T cell receptors using spleen cells from congenic and mutant mouse strains, and showed that the majority of them readily recognized self-MHC class I or class II molecules. These results demonstrate that by increasing the density and/or output of the T cell receptors expressed in peripheral T cells, one can confer them with the capacity to respond to normal density of self-MHC molecules.

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Related in: MedlinePlus

Fusion of CD3-ζ/η−/− peripheral single positive T cells with  BW−ζ, a CD3-ζ+ derivative of the BW− thymoma, results in hybridomas  expressing TCR-α/β complexes at their surface. BW−ζ was fused to single positive T cells isolated from the spleen and lymph nodes of 3-mo-old  CD3-ζ/η−/− or -ζ/η+/+ mice. The BW−ζ fusion partner and the resulting hybridomas were analyzed by flow cytometry after staining with the  anti–CD3-ε antibody KT3, the anti–TCR-β antibody H57-597, the  anti-CD4 antibody RL172.4, or the anti-CD8 antibody 3.168.81. Considering that BW−ζ contributed trans-acting factors which inhibit the expression of the CD8-α gene in the resulting hybridomas (see text), the  histograms obtained after staining with an anti–CD8-α antibody were  used as genuine negative control histograms.
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Figure 3: Fusion of CD3-ζ/η−/− peripheral single positive T cells with BW−ζ, a CD3-ζ+ derivative of the BW− thymoma, results in hybridomas expressing TCR-α/β complexes at their surface. BW−ζ was fused to single positive T cells isolated from the spleen and lymph nodes of 3-mo-old CD3-ζ/η−/− or -ζ/η+/+ mice. The BW−ζ fusion partner and the resulting hybridomas were analyzed by flow cytometry after staining with the anti–CD3-ε antibody KT3, the anti–TCR-β antibody H57-597, the anti-CD4 antibody RL172.4, or the anti-CD8 antibody 3.168.81. Considering that BW−ζ contributed trans-acting factors which inhibit the expression of the CD8-α gene in the resulting hybridomas (see text), the histograms obtained after staining with an anti–CD8-α antibody were used as genuine negative control histograms.

Mentions: To assess whether the single positive T cells accumulating at a low rate in the periphery of CD3-ζ/η−/− mice synthesized TCR-αβ heterodimers, we intended to fuse these cells with the BW− thymoma and convert them into T cell hybridomas. BW− is a variant of the BW5147 thymoma that lacks functional TCR-α and -β genes and has been extensively used to analyze the specificity of TCR expressed on heterogeneous populations of nontransformed T cells (e.g., references 28 and 29). Considering that BW− cells do not transcribe the CD3-ζ/η gene (13) and are, as such, unsuitable for experiments involving CD3-ζ/η–deficient T cells, we first transfected them with a plasmid directing the expression of CD3-ζ polypeptides. Because the CD3-η polypeptide does not appear endowed with unique signaling capacities relative to CD3-ζ (30, 31), we limited our effort to the construction of a derivative of BW−, denoted BW−ζ, and expressing only the CD3-ζ products (see Materials and Methods). As expected, transfection into BW− of the CD3-ζ gene alone did not lead to the appearance of surface molecules reactive with either an anti–CD3-ε or anti–TCR-β mAb (Fig. 3, top). Cells from spleen, inguinal, and mesenteric lymph nodes from 3-mo-old CD3-ζ/η−/− mice of H-2b (Z−B) or H-2k (Z−K) haplotypes were isolated and separately fused with BW−ζ. Sets of control hybridomas were prepared similarly from spleen and lymph node cells from 3-mo-old wild-type littermates of H-2b (Z+B) or H-2k (Z+K) haplotypes. Once produced, T cell hybridomas from individual wells were expanded briefly and analyzed for expression of CD3 at their surface. 20% of the hybridomas from CD3-ζ/η+/+ mice expressed CD3 at their surface, the remaining CD3-negative hybridomas being likely TCR loss variants. Of the hybridomas derived from CD3-ζ/η−/− mice, 5% did express CD3 at their surface and in amounts no lower than on hybridomas derived from CD3-ζ/η+/+ mice (compare hybridomas Z+K 2.3, Z−B17.20, and Z−K3.13 in Fig. 3). The lack of detectable CD3 expression on a larger proportion of the hybridomas derived from CD3-ζ/η−/− mice probably resulted from the fact that some of them had additionally lost the transfected CD3-ζ gene during their establishment. All the CD3+ hybridomas (10 from CD3-ζ/η−/−, H-2b animals, 8 from CD3-ζ/η−/−, H-2k animals, and 10 from CD3-ζ/η+/+ animals of H-2b or H-2k haplotypes) were cloned and further analyzed using antibodies specific for the TCR-α/β, TCR-γ/δ, CD4, or CD8 molecules. All the CD3+ hybridomas were readily stained with an antibody against the constant region of the TCR-β chain (Fig. 3 and data not shown). When further analyzed with a panel of anti-Vα and anti-Vβ mAb, they were found to express the products of gene segments belonging to the Vα2, Vα3.2, Vα8, Vβ2, Vβ6, Vβ8, Vβ12, or Vβ14 subfamilies. These results suggest that the CD3+ hybridomas derived from CD3-ζ/η−/− and CD3-ζ/η+/+ animals are not grossly skewed toward a peculiar Vα or Vβ usage. Approximately half of the CD3+ hybridomas displayed a CD4+CD8− phenotype (Fig. 3). Considering that BW5147 and its derivatives have been shown to contribute trans-acting factors capable of silencing the expression of the CD8-α gene (32), it is possible that the CD4−CD8− phenotype of some of the remaining hybridomas resulted from their derivation from T cells belonging to the CD8+ lineage.


The single positive T cells found in CD3-zeta/eta-/- mice overtly react with self-major histocompatibility complex molecules upon restoration of normal surface density of T cell receptor-CD3 complex.

Lin SY, Ardouin L, Gillet A, Malissen M, Malissen B - J. Exp. Med. (1997)

Fusion of CD3-ζ/η−/− peripheral single positive T cells with  BW−ζ, a CD3-ζ+ derivative of the BW− thymoma, results in hybridomas  expressing TCR-α/β complexes at their surface. BW−ζ was fused to single positive T cells isolated from the spleen and lymph nodes of 3-mo-old  CD3-ζ/η−/− or -ζ/η+/+ mice. The BW−ζ fusion partner and the resulting hybridomas were analyzed by flow cytometry after staining with the  anti–CD3-ε antibody KT3, the anti–TCR-β antibody H57-597, the  anti-CD4 antibody RL172.4, or the anti-CD8 antibody 3.168.81. Considering that BW−ζ contributed trans-acting factors which inhibit the expression of the CD8-α gene in the resulting hybridomas (see text), the  histograms obtained after staining with an anti–CD8-α antibody were  used as genuine negative control histograms.
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Related In: Results  -  Collection

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Figure 3: Fusion of CD3-ζ/η−/− peripheral single positive T cells with BW−ζ, a CD3-ζ+ derivative of the BW− thymoma, results in hybridomas expressing TCR-α/β complexes at their surface. BW−ζ was fused to single positive T cells isolated from the spleen and lymph nodes of 3-mo-old CD3-ζ/η−/− or -ζ/η+/+ mice. The BW−ζ fusion partner and the resulting hybridomas were analyzed by flow cytometry after staining with the anti–CD3-ε antibody KT3, the anti–TCR-β antibody H57-597, the anti-CD4 antibody RL172.4, or the anti-CD8 antibody 3.168.81. Considering that BW−ζ contributed trans-acting factors which inhibit the expression of the CD8-α gene in the resulting hybridomas (see text), the histograms obtained after staining with an anti–CD8-α antibody were used as genuine negative control histograms.
Mentions: To assess whether the single positive T cells accumulating at a low rate in the periphery of CD3-ζ/η−/− mice synthesized TCR-αβ heterodimers, we intended to fuse these cells with the BW− thymoma and convert them into T cell hybridomas. BW− is a variant of the BW5147 thymoma that lacks functional TCR-α and -β genes and has been extensively used to analyze the specificity of TCR expressed on heterogeneous populations of nontransformed T cells (e.g., references 28 and 29). Considering that BW− cells do not transcribe the CD3-ζ/η gene (13) and are, as such, unsuitable for experiments involving CD3-ζ/η–deficient T cells, we first transfected them with a plasmid directing the expression of CD3-ζ polypeptides. Because the CD3-η polypeptide does not appear endowed with unique signaling capacities relative to CD3-ζ (30, 31), we limited our effort to the construction of a derivative of BW−, denoted BW−ζ, and expressing only the CD3-ζ products (see Materials and Methods). As expected, transfection into BW− of the CD3-ζ gene alone did not lead to the appearance of surface molecules reactive with either an anti–CD3-ε or anti–TCR-β mAb (Fig. 3, top). Cells from spleen, inguinal, and mesenteric lymph nodes from 3-mo-old CD3-ζ/η−/− mice of H-2b (Z−B) or H-2k (Z−K) haplotypes were isolated and separately fused with BW−ζ. Sets of control hybridomas were prepared similarly from spleen and lymph node cells from 3-mo-old wild-type littermates of H-2b (Z+B) or H-2k (Z+K) haplotypes. Once produced, T cell hybridomas from individual wells were expanded briefly and analyzed for expression of CD3 at their surface. 20% of the hybridomas from CD3-ζ/η+/+ mice expressed CD3 at their surface, the remaining CD3-negative hybridomas being likely TCR loss variants. Of the hybridomas derived from CD3-ζ/η−/− mice, 5% did express CD3 at their surface and in amounts no lower than on hybridomas derived from CD3-ζ/η+/+ mice (compare hybridomas Z+K 2.3, Z−B17.20, and Z−K3.13 in Fig. 3). The lack of detectable CD3 expression on a larger proportion of the hybridomas derived from CD3-ζ/η−/− mice probably resulted from the fact that some of them had additionally lost the transfected CD3-ζ gene during their establishment. All the CD3+ hybridomas (10 from CD3-ζ/η−/−, H-2b animals, 8 from CD3-ζ/η−/−, H-2k animals, and 10 from CD3-ζ/η+/+ animals of H-2b or H-2k haplotypes) were cloned and further analyzed using antibodies specific for the TCR-α/β, TCR-γ/δ, CD4, or CD8 molecules. All the CD3+ hybridomas were readily stained with an antibody against the constant region of the TCR-β chain (Fig. 3 and data not shown). When further analyzed with a panel of anti-Vα and anti-Vβ mAb, they were found to express the products of gene segments belonging to the Vα2, Vα3.2, Vα8, Vβ2, Vβ6, Vβ8, Vβ12, or Vβ14 subfamilies. These results suggest that the CD3+ hybridomas derived from CD3-ζ/η−/− and CD3-ζ/η+/+ animals are not grossly skewed toward a peculiar Vα or Vβ usage. Approximately half of the CD3+ hybridomas displayed a CD4+CD8− phenotype (Fig. 3). Considering that BW5147 and its derivatives have been shown to contribute trans-acting factors capable of silencing the expression of the CD8-α gene (32), it is possible that the CD4−CD8− phenotype of some of the remaining hybridomas resulted from their derivation from T cells belonging to the CD8+ lineage.

Bottom Line: CD3-zeta/eta-deficient mice have small thymuses containing cells that show a profound reduction in the surface levels of T cell receptors and terminate their differentiation at the CD4+CD8+ stage.Fusion of these peripheral T cells with a CD3-zeta-positive derivative of the BW5147 TCR-alpha-/beta- thymoma resulted in hybridomas that do express an heterogeneous set of T cell receptor alpha/beta dimers at their surface and at density comparable to those found in hybridomas derived from wild-type peripheral T cells.These results demonstrate that by increasing the density and/or output of the T cell receptors expressed in peripheral T cells, one can confer them with the capacity to respond to normal density of self-MHC molecules.

View Article: PubMed Central - PubMed

Affiliation: Centre d'Immunologie, Institut National de la Sante et de la Recherche Medicale-Centre National de Recherche Scientifique de Marseille-Luminy, France.

ABSTRACT
CD3-zeta/eta-deficient mice have small thymuses containing cells that show a profound reduction in the surface levels of T cell receptors and terminate their differentiation at the CD4+CD8+ stage. Rather unexpectedly, CD3- or very low single positive T cells accumulate over time in the spleen and lymph nodes of CD3-zeta/eta-deficient mice after a process dependent on MHC expression. Fusion of these peripheral T cells with a CD3-zeta-positive derivative of the BW5147 TCR-alpha-/beta- thymoma resulted in hybridomas that do express an heterogeneous set of T cell receptor alpha/beta dimers at their surface and at density comparable to those found in hybridomas derived from wild-type peripheral T cells. We have investigated the specificities of these T cell receptors using spleen cells from congenic and mutant mouse strains, and showed that the majority of them readily recognized self-MHC class I or class II molecules. These results demonstrate that by increasing the density and/or output of the T cell receptors expressed in peripheral T cells, one can confer them with the capacity to respond to normal density of self-MHC molecules.

Show MeSH
Related in: MedlinePlus