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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) vSAG7 Transfer can be inhibited by 6E1, a vSAG7 specific monoclonal antibody. Various concentrations of the mAb 6E1 were  added in the transfer assay (6 × 104 Kmls 13.11 cells, 6 × 104 DAP DR1  cells and 5 × 104 DAP vSAG7 cells in 250 μl). After an overnight culture, supernatants were harvested and tested for IL-2 production. (b) Specific inhibition of vSAG7 transfer by an MHC class II specific mAb. Various concentrations of the mAbs XD5.117 (HLA DR) or HUT78 (human  TCR Vβ23) were added in the transfer assay. After an overnight culture,  supernatants were harvested and tested for IL-2 production.
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Figure 2: (a) vSAG7 Transfer can be inhibited by 6E1, a vSAG7 specific monoclonal antibody. Various concentrations of the mAb 6E1 were added in the transfer assay (6 × 104 Kmls 13.11 cells, 6 × 104 DAP DR1 cells and 5 × 104 DAP vSAG7 cells in 250 μl). After an overnight culture, supernatants were harvested and tested for IL-2 production. (b) Specific inhibition of vSAG7 transfer by an MHC class II specific mAb. Various concentrations of the mAbs XD5.117 (HLA DR) or HUT78 (human TCR Vβ23) were added in the transfer assay. After an overnight culture, supernatants were harvested and tested for IL-2 production.

Mentions: To further demonstrate the requirement for vSAG7 in order to obtain T cell stimulation we used the vSAG7specific mAb 6E1 to inhibit stimulation in the transfer assay. Results illustrated in Fig. 2 a clearly show that this mAb inhibits vSAG7 activity in a dose-dependent manner, as previously demonstrated for presentation of endogenously expressed vSAG7. As a control of specificity, we show that mAb 6E1 fails to inhibit SEB presentation (data not shown).


Paracrine transfer of mouse mammary tumor virus superantigen.

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

(a) vSAG7 Transfer can be inhibited by 6E1, a vSAG7 specific monoclonal antibody. Various concentrations of the mAb 6E1 were  added in the transfer assay (6 × 104 Kmls 13.11 cells, 6 × 104 DAP DR1  cells and 5 × 104 DAP vSAG7 cells in 250 μl). After an overnight culture, supernatants were harvested and tested for IL-2 production. (b) Specific inhibition of vSAG7 transfer by an MHC class II specific mAb. Various concentrations of the mAbs XD5.117 (HLA DR) or HUT78 (human  TCR Vβ23) were added in the transfer assay. After an overnight culture,  supernatants were harvested and tested for IL-2 production.
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Related In: Results  -  Collection

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Figure 2: (a) vSAG7 Transfer can be inhibited by 6E1, a vSAG7 specific monoclonal antibody. Various concentrations of the mAb 6E1 were added in the transfer assay (6 × 104 Kmls 13.11 cells, 6 × 104 DAP DR1 cells and 5 × 104 DAP vSAG7 cells in 250 μl). After an overnight culture, supernatants were harvested and tested for IL-2 production. (b) Specific inhibition of vSAG7 transfer by an MHC class II specific mAb. Various concentrations of the mAbs XD5.117 (HLA DR) or HUT78 (human TCR Vβ23) were added in the transfer assay. After an overnight culture, supernatants were harvested and tested for IL-2 production.
Mentions: To further demonstrate the requirement for vSAG7 in order to obtain T cell stimulation we used the vSAG7specific mAb 6E1 to inhibit stimulation in the transfer assay. Results illustrated in Fig. 2 a clearly show that this mAb inhibits vSAG7 activity in a dose-dependent manner, as previously demonstrated for presentation of endogenously expressed vSAG7. As a control of specificity, we show that mAb 6E1 fails to inhibit SEB presentation (data not shown).

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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