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Paracrine transfer of mouse mammary tumor virus superantigen.

Delcourt M, Thibodeau J, Denis F, Sekaly RP - J. Exp. Med. (1997)

Bottom Line: We show that this transfer activity is paracrine but does not require cell-to-cell contact.Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7.Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie Institut de Recherches Cliniques de Montréal, Canada.

ABSTRACT
Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive V beta s. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V beta s led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II+ cells in mice led to expansion of V beta 6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

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(a) A panel of vSAG7-expressing hybridomas expressing Vβ6  or Vβ8.1 respond to the transferred vSAG7. 6 × 104 T cell hybridomas  cells Kmls 13.11 (Vβ6), Kmls 12.6 (Vβ6), RG17 (Vβ6), and KR3 (Vβ8.1)  were tested in direct presentation assays (overnight coculture with 6 ×  104 DAP DR1 vSAG7 cells) or in transfer assays (overnight coculture  with 6 × 104 DAP DR1 cells and 6 × 104 DAP vSAG7 cells) for IL-2  production. (b) Presentation of the transferred vSAG7 is not restricted to  DAP DR1 cells. 6 × 104 DAP DR1 cells or 6 × 104 CH12 B lymphoma  cells were cocultured overnight with 5 × 104 DAP vSAG7 cells and 6 ×  104 Kmls 13.11 cells. Supernatants were then harvested and tested for IL-2  activity.
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Figure 3: (a) A panel of vSAG7-expressing hybridomas expressing Vβ6 or Vβ8.1 respond to the transferred vSAG7. 6 × 104 T cell hybridomas cells Kmls 13.11 (Vβ6), Kmls 12.6 (Vβ6), RG17 (Vβ6), and KR3 (Vβ8.1) were tested in direct presentation assays (overnight coculture with 6 × 104 DAP DR1 vSAG7 cells) or in transfer assays (overnight coculture with 6 × 104 DAP DR1 cells and 6 × 104 DAP vSAG7 cells) for IL-2 production. (b) Presentation of the transferred vSAG7 is not restricted to DAP DR1 cells. 6 × 104 DAP DR1 cells or 6 × 104 CH12 B lymphoma cells were cocultured overnight with 5 × 104 DAP vSAG7 cells and 6 × 104 Kmls 13.11 cells. Supernatants were then harvested and tested for IL-2 activity.

Mentions: vSAG7 not only reacts with Vβ6-expressing T cells, but also Vβ8.1expressing T cells. A panel of Vβ6- (Kmls 13.11, Kmls 12.6 and RG17)- or Vβ8.1 (KR3)-expressing hybridomas was used to demonstrate that transfer of vSAG7 was capable of stimulating T cells expressing different vSAG7responding TCRs. All these hybridomas were stimulated at comparable levels by vSAG7 both in the transfer assay and in direct presentation by DAP DR1 vSAG7 cells (Fig. 3 a).


Paracrine transfer of mouse mammary tumor virus superantigen.

Delcourt M, Thibodeau J, Denis F, Sekaly RP - J. Exp. Med. (1997)

(a) A panel of vSAG7-expressing hybridomas expressing Vβ6  or Vβ8.1 respond to the transferred vSAG7. 6 × 104 T cell hybridomas  cells Kmls 13.11 (Vβ6), Kmls 12.6 (Vβ6), RG17 (Vβ6), and KR3 (Vβ8.1)  were tested in direct presentation assays (overnight coculture with 6 ×  104 DAP DR1 vSAG7 cells) or in transfer assays (overnight coculture  with 6 × 104 DAP DR1 cells and 6 × 104 DAP vSAG7 cells) for IL-2  production. (b) Presentation of the transferred vSAG7 is not restricted to  DAP DR1 cells. 6 × 104 DAP DR1 cells or 6 × 104 CH12 B lymphoma  cells were cocultured overnight with 5 × 104 DAP vSAG7 cells and 6 ×  104 Kmls 13.11 cells. Supernatants were then harvested and tested for IL-2  activity.
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Related In: Results  -  Collection

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Figure 3: (a) A panel of vSAG7-expressing hybridomas expressing Vβ6 or Vβ8.1 respond to the transferred vSAG7. 6 × 104 T cell hybridomas cells Kmls 13.11 (Vβ6), Kmls 12.6 (Vβ6), RG17 (Vβ6), and KR3 (Vβ8.1) were tested in direct presentation assays (overnight coculture with 6 × 104 DAP DR1 vSAG7 cells) or in transfer assays (overnight coculture with 6 × 104 DAP DR1 cells and 6 × 104 DAP vSAG7 cells) for IL-2 production. (b) Presentation of the transferred vSAG7 is not restricted to DAP DR1 cells. 6 × 104 DAP DR1 cells or 6 × 104 CH12 B lymphoma cells were cocultured overnight with 5 × 104 DAP vSAG7 cells and 6 × 104 Kmls 13.11 cells. Supernatants were then harvested and tested for IL-2 activity.
Mentions: vSAG7 not only reacts with Vβ6-expressing T cells, but also Vβ8.1expressing T cells. A panel of Vβ6- (Kmls 13.11, Kmls 12.6 and RG17)- or Vβ8.1 (KR3)-expressing hybridomas was used to demonstrate that transfer of vSAG7 was capable of stimulating T cells expressing different vSAG7responding TCRs. All these hybridomas were stimulated at comparable levels by vSAG7 both in the transfer assay and in direct presentation by DAP DR1 vSAG7 cells (Fig. 3 a).

Bottom Line: We show that this transfer activity is paracrine but does not require cell-to-cell contact.Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7.Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

View Article: PubMed Central - PubMed

Affiliation: Laboratoire d'Immunologie Institut de Recherches Cliniques de Montréal, Canada.

ABSTRACT
Transfer of vSAG7, the endogenous superantigen encoded in the Mtv7 locus, from MHC class II to MHC class II+ cells has been suggested to occur both in vivo and in vitro. This transfer usually leads to the activation and deletion of T cells expressing responsive V beta s. However, there is no direct molecular evidence for such a transfer. We have developed an in vitro system which confirms this property of vSAGs. vSAG7 was transfected into a class II murine fibroblastic line. Coculture of these cells with class II+ cells and murine T cell hybridomas expressing the specific V beta s led to high levels of IL-2 production which was specifically inhibited by vSAG7- and MHC class II-specific mAbs. Moreover, injection of vSAG7+ class II+ cells in mice led to expansion of V beta 6+ CD4+ cells. We show that this transfer activity is paracrine but does not require cell-to-cell contact. Indeed, vSAG7 was transferred across semi-permeable membranes. Transfer can occur both from class II+ and class II+ cells, indicating that MHC class II does not sequester vSAG7. Finally, competition experiments using bacterial toxins with well defined binding sites showed that the transferred vSAG7 fragment binds to the alpha 1 domain of HLA-DR.

Show MeSH
Related in: MedlinePlus