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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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Specificity of hybridomas generated from single  positive peripheral T cells isolated from wild-type mice of  H-2b or H-2k haplotypes (A),  CD3-ζ/η−/− mutant mice of  H-2b (B), or H-2k (C) haplotypes.  IL-2 production of the various hybridomas was measured after incubation without any stimulus (Medium), with irradiated C57BL/6  (H-2b) or C57BL/6-H-2k (H-2k)  splenocytes (106 cells/well), or in  wells coated with the anti– CD3-ε mAb 2C11 (10 μg/ml).
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Figure 4: Specificity of hybridomas generated from single positive peripheral T cells isolated from wild-type mice of H-2b or H-2k haplotypes (A), CD3-ζ/η−/− mutant mice of H-2b (B), or H-2k (C) haplotypes. IL-2 production of the various hybridomas was measured after incubation without any stimulus (Medium), with irradiated C57BL/6 (H-2b) or C57BL/6-H-2k (H-2k) splenocytes (106 cells/well), or in wells coated with the anti– CD3-ε mAb 2C11 (10 μg/ml).

Mentions: To examine the specificities of the heterogeneous TCR-α/β dimers expressed on the Z−B and Z−K sets of hybridomas, we tested them for IL-2 production in response to spleen cells from C57BL/6 or C57BL/6-H-2k mice. All the hybridomas were capable of IL-2 production in response to immobilized anti–CD3-ε mAb. Hybridomas from CD3-ζ/η−/−, H-2b animals showed three distinct patterns of response. As shown in Fig. 4 B, one hybridoma (Z−B17.20) failed to react with H-2b or H-2k splenocytes, while another one (Z−B21.17) reacted with both H-2k and H-2b splenocytes and produced IL-2 when grown in medium alone. However, the most remarkable pattern of reactivity was observed with the eight remaining Z−B hybridomas in that they reacted with C57BL/6 syngeneic spleen cells but failed to respond with spleen cells prepared from congenic C57BL-6-H-2k mice. (This major reactivity pattern is illustrated by hybridoma Z−B5.16 in Fig. 4 B). On the other hand, six of the eight hybridomas derived from CD3-ζ/η−/−, H-2k animals showed a reciprocal pattern of reactivity. For example, as illustrated in Fig. 4 C for hybridoma Z−K8.36, they did recognize C57BL/6-H-2k cells, but not C57BL/6 cells. The two remaining hybridomas of the Z−K set reacted with both spleen cell populations but, in contrast to hybridoma Z−B21.17, did not produce any detectable IL-2 when cultured in medium alone (compare hybridomas Z−K37.4 and Z−B21.17 in Fig. 4). Despite the expression of TCR associated with the same complement of CD3 polypeptides and in amounts comparable to or even greater than those found at the surface of the Z−B and Z−K sets of hybridomas, none of the T cell hybridomas derived from CD3-ζ/η+/+ animals had autoreactivity (sets Z+B and Z+K in Fig. 4 A). The consistency of the latter result with previous reports obtained with the BW− fusion partner (28, 29), clearly indicates that fusion with the BW−ζ derivative does not in itself confer some artifactual reactivities to the resulting hybridomas. As summarized in Fig. 5, these results showed that the majority of the TCRα/β+ hybridomas established from CD3-ζ/η−/− animals specifically reacted with syngeneic spleen cells.


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)

Specificity of hybridomas generated from single  positive peripheral T cells isolated from wild-type mice of  H-2b or H-2k haplotypes (A),  CD3-ζ/η−/− mutant mice of  H-2b (B), or H-2k (C) haplotypes.  IL-2 production of the various hybridomas was measured after incubation without any stimulus (Medium), with irradiated C57BL/6  (H-2b) or C57BL/6-H-2k (H-2k)  splenocytes (106 cells/well), or in  wells coated with the anti– CD3-ε mAb 2C11 (10 μg/ml).
© Copyright Policy
Related In: Results  -  Collection

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Figure 4: Specificity of hybridomas generated from single positive peripheral T cells isolated from wild-type mice of H-2b or H-2k haplotypes (A), CD3-ζ/η−/− mutant mice of H-2b (B), or H-2k (C) haplotypes. IL-2 production of the various hybridomas was measured after incubation without any stimulus (Medium), with irradiated C57BL/6 (H-2b) or C57BL/6-H-2k (H-2k) splenocytes (106 cells/well), or in wells coated with the anti– CD3-ε mAb 2C11 (10 μg/ml).
Mentions: To examine the specificities of the heterogeneous TCR-α/β dimers expressed on the Z−B and Z−K sets of hybridomas, we tested them for IL-2 production in response to spleen cells from C57BL/6 or C57BL/6-H-2k mice. All the hybridomas were capable of IL-2 production in response to immobilized anti–CD3-ε mAb. Hybridomas from CD3-ζ/η−/−, H-2b animals showed three distinct patterns of response. As shown in Fig. 4 B, one hybridoma (Z−B17.20) failed to react with H-2b or H-2k splenocytes, while another one (Z−B21.17) reacted with both H-2k and H-2b splenocytes and produced IL-2 when grown in medium alone. However, the most remarkable pattern of reactivity was observed with the eight remaining Z−B hybridomas in that they reacted with C57BL/6 syngeneic spleen cells but failed to respond with spleen cells prepared from congenic C57BL-6-H-2k mice. (This major reactivity pattern is illustrated by hybridoma Z−B5.16 in Fig. 4 B). On the other hand, six of the eight hybridomas derived from CD3-ζ/η−/−, H-2k animals showed a reciprocal pattern of reactivity. For example, as illustrated in Fig. 4 C for hybridoma Z−K8.36, they did recognize C57BL/6-H-2k cells, but not C57BL/6 cells. The two remaining hybridomas of the Z−K set reacted with both spleen cell populations but, in contrast to hybridoma Z−B21.17, did not produce any detectable IL-2 when cultured in medium alone (compare hybridomas Z−K37.4 and Z−B21.17 in Fig. 4). Despite the expression of TCR associated with the same complement of CD3 polypeptides and in amounts comparable to or even greater than those found at the surface of the Z−B and Z−K sets of hybridomas, none of the T cell hybridomas derived from CD3-ζ/η+/+ animals had autoreactivity (sets Z+B and Z+K in Fig. 4 A). The consistency of the latter result with previous reports obtained with the BW− fusion partner (28, 29), clearly indicates that fusion with the BW−ζ derivative does not in itself confer some artifactual reactivities to the resulting hybridomas. As summarized in Fig. 5, these results showed that the majority of the TCRα/β+ hybridomas established from CD3-ζ/η−/− animals specifically reacted with syngeneic spleen cells.

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