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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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vSAG7 binds to the DR α chain. To standard conditions of  transfer assay were added various concentrations of either SEA, SEA F47,  or TSST-1. IL-2 was measured in supernatants as previously described.
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Figure 9: vSAG7 binds to the DR α chain. To standard conditions of transfer assay were added various concentrations of either SEA, SEA F47, or TSST-1. IL-2 was measured in supernatants as previously described.

Mentions: SEA binds to the α and β chains of MHC class II molecules (41a). We were thus interested to determine if the binding sites for the bacterial toxin were also involved in the binding of vSAG7. To address this question, we used a mutant of SEA, SEA F47, which has lost its ability to bind MHC class II through the α chain but is still able to bind with high affinity to the β chain of MHC class II (41a, 42). We show here (Fig. 9) that SEA F47 partially blocks the activity of vSAG7 in the transfer assay, as compared to the 20-fold inhibition induced by SEA wt. Indeed, the addition of high concentrations of SEA F47 decreases T cell stimulation by less than a twofold (∼40% of inhibition). We concluded from these studies that vSAG7 interacts mainly with MHC class II through its α chain. This result was further confirmed by the fact that high concentrations of TSST-1, a toxin known to bind exclusively to the α chain of MHC class II (43, 44), also exerted a partial (∼60%) but reproducible inhibition of vSAG7 presentation in the transfer system (Fig. 9).


Paracrine transfer of mouse mammary tumor virus superantigen.

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

vSAG7 binds to the DR α chain. To standard conditions of  transfer assay were added various concentrations of either SEA, SEA F47,  or TSST-1. IL-2 was measured in supernatants as previously described.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2196028&req=5

Figure 9: vSAG7 binds to the DR α chain. To standard conditions of transfer assay were added various concentrations of either SEA, SEA F47, or TSST-1. IL-2 was measured in supernatants as previously described.
Mentions: SEA binds to the α and β chains of MHC class II molecules (41a). We were thus interested to determine if the binding sites for the bacterial toxin were also involved in the binding of vSAG7. To address this question, we used a mutant of SEA, SEA F47, which has lost its ability to bind MHC class II through the α chain but is still able to bind with high affinity to the β chain of MHC class II (41a, 42). We show here (Fig. 9) that SEA F47 partially blocks the activity of vSAG7 in the transfer assay, as compared to the 20-fold inhibition induced by SEA wt. Indeed, the addition of high concentrations of SEA F47 decreases T cell stimulation by less than a twofold (∼40% of inhibition). We concluded from these studies that vSAG7 interacts mainly with MHC class II through its α chain. This result was further confirmed by the fact that high concentrations of TSST-1, a toxin known to bind exclusively to the α chain of MHC class II (43, 44), also exerted a partial (∼60%) but reproducible inhibition of vSAG7 presentation in the transfer system (Fig. 9).

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