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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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DAP vSAG7 induces Vβ6-specific expansion in vivo. 3 ×  106 DAP vSAG7 cells in 30 μl of PBS were injected in the hind footpad.  5 d later, lymph nodes were taken and CD4+ lymphocytes were analyzed  by flow cytometry for Vβ3 and Vβ6 expression. Live cells were gated by  forward and side scatter. Percentages of Vβ6-expressing cells among  CD4+ cells were respectively 11, 25, and 23%, respectively, after DAP  DR1, DAP vSAG7, and DAP DR1 vSAG7 injection. This experiment  was repeated four times and led to similar results.
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Figure 4: DAP vSAG7 induces Vβ6-specific expansion in vivo. 3 × 106 DAP vSAG7 cells in 30 μl of PBS were injected in the hind footpad. 5 d later, lymph nodes were taken and CD4+ lymphocytes were analyzed by flow cytometry for Vβ3 and Vβ6 expression. Live cells were gated by forward and side scatter. Percentages of Vβ6-expressing cells among CD4+ cells were respectively 11, 25, and 23%, respectively, after DAP DR1, DAP vSAG7, and DAP DR1 vSAG7 injection. This experiment was repeated four times and led to similar results.

Mentions: To further demonstrate that the transfer activity bears physiological relevance, experiments were set up to verify if transfer of vSAG7 from DAP vSAG7 occurs in vivo. For this purpose, mice were injected with 3 × 106 DAP vSAG7 in the hind footpad. Five days following injection, draining lymph node cells were analyzed by flow cytometry for TcR Vβ6 expression. A representative experiment is shown in Fig. 4. In this experiment, the percentage of Vβ6-expressing cells among the CD4+ cells increases from 11% (mice injected with DAP DR1 cells) in control mice to 25% in mice injected with DAP vSAG7 cells. This enhancement is comparable to the one observed after injection of DAP DR1 vSAG7 cells. As previously shown following injection of B cells or CD8+ cells expressing vSAG7, we did not observe any expansion of Vβ6+ CD8+ cells.


Paracrine transfer of mouse mammary tumor virus superantigen.

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

DAP vSAG7 induces Vβ6-specific expansion in vivo. 3 ×  106 DAP vSAG7 cells in 30 μl of PBS were injected in the hind footpad.  5 d later, lymph nodes were taken and CD4+ lymphocytes were analyzed  by flow cytometry for Vβ3 and Vβ6 expression. Live cells were gated by  forward and side scatter. Percentages of Vβ6-expressing cells among  CD4+ cells were respectively 11, 25, and 23%, respectively, after DAP  DR1, DAP vSAG7, and DAP DR1 vSAG7 injection. This experiment  was repeated four times and led to similar results.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 4: DAP vSAG7 induces Vβ6-specific expansion in vivo. 3 × 106 DAP vSAG7 cells in 30 μl of PBS were injected in the hind footpad. 5 d later, lymph nodes were taken and CD4+ lymphocytes were analyzed by flow cytometry for Vβ3 and Vβ6 expression. Live cells were gated by forward and side scatter. Percentages of Vβ6-expressing cells among CD4+ cells were respectively 11, 25, and 23%, respectively, after DAP DR1, DAP vSAG7, and DAP DR1 vSAG7 injection. This experiment was repeated four times and led to similar results.
Mentions: To further demonstrate that the transfer activity bears physiological relevance, experiments were set up to verify if transfer of vSAG7 from DAP vSAG7 occurs in vivo. For this purpose, mice were injected with 3 × 106 DAP vSAG7 in the hind footpad. Five days following injection, draining lymph node cells were analyzed by flow cytometry for TcR Vβ6 expression. A representative experiment is shown in Fig. 4. In this experiment, the percentage of Vβ6-expressing cells among the CD4+ cells increases from 11% (mice injected with DAP DR1 cells) in control mice to 25% in mice injected with DAP vSAG7 cells. This enhancement is comparable to the one observed after injection of DAP DR1 vSAG7 cells. As previously shown following injection of B cells or CD8+ cells expressing vSAG7, we did not observe any expansion of Vβ6+ CD8+ cells.

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