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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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In vitro transfer of  vSAG7 from class II− cells to class  II+ cells. (a and b) Expression levels of vSAG7 and actin mRNA in  different clones of DAP cells  transfected with vSAG7. RNA  was extracted from cloned DAP  vSAG7 transfectants. 10 μg of  RNA were run on a 1.2% agarose-formaldehyde gel and transferred on a nylon membrane.  Membranes were then hybridized  with either actin (a) or vSAG7 (b)  radiolabeled probes and exposed  for 24 h. Each track represents an  individual clone. (c) Murine fibroblasts transfected with vSAG7 can  stimulate T cell hybridomas in the  presence of DAP DR1 cells. 6 ×  104 DAP DR1 cells and 6 × 104  Kmls 13.11 cells were incubated  overnight with either 5 × 104  DAP cells or various amounts of  DAP vSAG7 cells. Supernatants  were then harvested and tested for  IL-2 activity. (d and e) Levels of  vSAG7 expression by DAP vSAG7  clone 18 and DAP DR1 vSAG7  are comparable. RNA was extracted from DAP vSAG7 clone  18 and DAP DR1 vSAG7 clone  3B2, run on a 1.2% agarose-formaldehyde gel and hybridized with  actin (d) or vSAG7 (  f ) radiolabeled probes. (  f ) Levels of stimulation in the transfer assay (DAP  vSAG7 + DAP DR1) and in direct presentation (DAP DR1  vSAG7) are comparable. 6 × 104  Kmls 13.11 cells were cocultured  in 250 μl either with 6 × 104  DAP DR1 cells and 5 × 104 DAP  vSAG7 clone 18 cells, or with 6 ×  104 DAP DR1 vSAG7 cells. Supernatants were then harvested  and tested for IL-2 production.
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Figure 1: In vitro transfer of vSAG7 from class II− cells to class II+ cells. (a and b) Expression levels of vSAG7 and actin mRNA in different clones of DAP cells transfected with vSAG7. RNA was extracted from cloned DAP vSAG7 transfectants. 10 μg of RNA were run on a 1.2% agarose-formaldehyde gel and transferred on a nylon membrane. Membranes were then hybridized with either actin (a) or vSAG7 (b) radiolabeled probes and exposed for 24 h. Each track represents an individual clone. (c) Murine fibroblasts transfected with vSAG7 can stimulate T cell hybridomas in the presence of DAP DR1 cells. 6 × 104 DAP DR1 cells and 6 × 104 Kmls 13.11 cells were incubated overnight with either 5 × 104 DAP cells or various amounts of DAP vSAG7 cells. Supernatants were then harvested and tested for IL-2 activity. (d and e) Levels of vSAG7 expression by DAP vSAG7 clone 18 and DAP DR1 vSAG7 are comparable. RNA was extracted from DAP vSAG7 clone 18 and DAP DR1 vSAG7 clone 3B2, run on a 1.2% agarose-formaldehyde gel and hybridized with actin (d) or vSAG7 (  f ) radiolabeled probes. (  f ) Levels of stimulation in the transfer assay (DAP vSAG7 + DAP DR1) and in direct presentation (DAP DR1 vSAG7) are comparable. 6 × 104 Kmls 13.11 cells were cocultured in 250 μl either with 6 × 104 DAP DR1 cells and 5 × 104 DAP vSAG7 clone 18 cells, or with 6 × 104 DAP DR1 vSAG7 cells. Supernatants were then harvested and tested for IL-2 production.

Mentions: The vSAG7 gene was transfected into the MHC class II− DAP3 murine fibroblastic line. Seven clones of G418-resistant cells were analyzed by Northern Blot. Equal loading of all wells was confirmed by comparable levels of actin mRNA in all clones tested (Fig. 1 a). When compared to other clones, clones 17 and 18 showed significantly higher levels of vSAG7 mRNA (Fig. 1 b). Clone 2 also expressed vSAG7 mRNA, although at lower levels. To verify the capacity of MHC class II− vSAG7+ cells to transfer vSAGs, we developed a transfer assay in which equal numbers (6 × 104) of DAP DR1 cells and the Vβ6+ vSAG7-responsive Kmls 13.11 hybridomas were cocultured for 16 to 20 h together with various amounts of the DAP vSAG7 cells. T cell stimulation was assessed by measuring IL-2 present in the supernatants. The three vSAG7+ clones (clones 2, 17, and 18) were able to induce a dose dependent T cell activation in several independent experiments. A representative experiment is shown in Fig. 1 c where increases in the production of IL-2 ranged from 10- to 20-fold as compared to cocultures of DAP vSAG7 cells and untransfected DAP cells. The latter indicated the absolute prerequisite for MHC class II molecules in order to obtain vSAG7 presentation. Levels of IL-2 production were directly correlated with the number of cells expressing vSAG7 or with the levels of vSAG7 expressed by the class II− fibroblasts. Indeed, little or no stimulation was observed with less than 2 × 103 DAP vSAG7 cells, whereas maximal response was obtained with 5 × 104 DAP vSAG7 cells per well.


Paracrine transfer of mouse mammary tumor virus superantigen.

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

In vitro transfer of  vSAG7 from class II− cells to class  II+ cells. (a and b) Expression levels of vSAG7 and actin mRNA in  different clones of DAP cells  transfected with vSAG7. RNA  was extracted from cloned DAP  vSAG7 transfectants. 10 μg of  RNA were run on a 1.2% agarose-formaldehyde gel and transferred on a nylon membrane.  Membranes were then hybridized  with either actin (a) or vSAG7 (b)  radiolabeled probes and exposed  for 24 h. Each track represents an  individual clone. (c) Murine fibroblasts transfected with vSAG7 can  stimulate T cell hybridomas in the  presence of DAP DR1 cells. 6 ×  104 DAP DR1 cells and 6 × 104  Kmls 13.11 cells were incubated  overnight with either 5 × 104  DAP cells or various amounts of  DAP vSAG7 cells. Supernatants  were then harvested and tested for  IL-2 activity. (d and e) Levels of  vSAG7 expression by DAP vSAG7  clone 18 and DAP DR1 vSAG7  are comparable. RNA was extracted from DAP vSAG7 clone  18 and DAP DR1 vSAG7 clone  3B2, run on a 1.2% agarose-formaldehyde gel and hybridized with  actin (d) or vSAG7 (  f ) radiolabeled probes. (  f ) Levels of stimulation in the transfer assay (DAP  vSAG7 + DAP DR1) and in direct presentation (DAP DR1  vSAG7) are comparable. 6 × 104  Kmls 13.11 cells were cocultured  in 250 μl either with 6 × 104  DAP DR1 cells and 5 × 104 DAP  vSAG7 clone 18 cells, or with 6 ×  104 DAP DR1 vSAG7 cells. Supernatants were then harvested  and tested for IL-2 production.
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Related In: Results  -  Collection

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Figure 1: In vitro transfer of vSAG7 from class II− cells to class II+ cells. (a and b) Expression levels of vSAG7 and actin mRNA in different clones of DAP cells transfected with vSAG7. RNA was extracted from cloned DAP vSAG7 transfectants. 10 μg of RNA were run on a 1.2% agarose-formaldehyde gel and transferred on a nylon membrane. Membranes were then hybridized with either actin (a) or vSAG7 (b) radiolabeled probes and exposed for 24 h. Each track represents an individual clone. (c) Murine fibroblasts transfected with vSAG7 can stimulate T cell hybridomas in the presence of DAP DR1 cells. 6 × 104 DAP DR1 cells and 6 × 104 Kmls 13.11 cells were incubated overnight with either 5 × 104 DAP cells or various amounts of DAP vSAG7 cells. Supernatants were then harvested and tested for IL-2 activity. (d and e) Levels of vSAG7 expression by DAP vSAG7 clone 18 and DAP DR1 vSAG7 are comparable. RNA was extracted from DAP vSAG7 clone 18 and DAP DR1 vSAG7 clone 3B2, run on a 1.2% agarose-formaldehyde gel and hybridized with actin (d) or vSAG7 (  f ) radiolabeled probes. (  f ) Levels of stimulation in the transfer assay (DAP vSAG7 + DAP DR1) and in direct presentation (DAP DR1 vSAG7) are comparable. 6 × 104 Kmls 13.11 cells were cocultured in 250 μl either with 6 × 104 DAP DR1 cells and 5 × 104 DAP vSAG7 clone 18 cells, or with 6 × 104 DAP DR1 vSAG7 cells. Supernatants were then harvested and tested for IL-2 production.
Mentions: The vSAG7 gene was transfected into the MHC class II− DAP3 murine fibroblastic line. Seven clones of G418-resistant cells were analyzed by Northern Blot. Equal loading of all wells was confirmed by comparable levels of actin mRNA in all clones tested (Fig. 1 a). When compared to other clones, clones 17 and 18 showed significantly higher levels of vSAG7 mRNA (Fig. 1 b). Clone 2 also expressed vSAG7 mRNA, although at lower levels. To verify the capacity of MHC class II− vSAG7+ cells to transfer vSAGs, we developed a transfer assay in which equal numbers (6 × 104) of DAP DR1 cells and the Vβ6+ vSAG7-responsive Kmls 13.11 hybridomas were cocultured for 16 to 20 h together with various amounts of the DAP vSAG7 cells. T cell stimulation was assessed by measuring IL-2 present in the supernatants. The three vSAG7+ clones (clones 2, 17, and 18) were able to induce a dose dependent T cell activation in several independent experiments. A representative experiment is shown in Fig. 1 c where increases in the production of IL-2 ranged from 10- to 20-fold as compared to cocultures of DAP vSAG7 cells and untransfected DAP cells. The latter indicated the absolute prerequisite for MHC class II molecules in order to obtain vSAG7 presentation. Levels of IL-2 production were directly correlated with the number of cells expressing vSAG7 or with the levels of vSAG7 expressed by the class II− fibroblasts. Indeed, little or no stimulation was observed with less than 2 × 103 DAP vSAG7 cells, whereas maximal response was obtained with 5 × 104 DAP vSAG7 cells per well.

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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