Disruption of lipid rafts inhibits FasL activity. (A) FasL-transfected 293T cells were treated with increasing concentrations of MbCD and cocultured at different effector-target ratios (E/T ratios) with Fas-sensitive target cells. After 6 hours target cell killing was assessed by the induction of DNA fragmentation. Specificity was confirmed by neutralization with anti-FasL. A typical experiment of 5 is shown. (B) FasL-transfected 293T cells were treated with increasing concentrations of filipin and cocultured with Fas-sensitive target cells at an E/T ratio of 2:1. Target cell killing was assessed by the induction of DNA fragmentation. A typical experiment of 2 is shown.(C) FasL-transfected 293T cells were treated with increasing concentrations of squalestatin and target cell killing was assessed as described (E/T ratio, 2:1). A typical experiment of 2 is shown. (D) The murine T-cell hybridoma A1.1 was treated with increasing concentrations of MbCD and then stimulated with PMA and ionomycin to induce FasL expression. Cells were then cocultured at different E/T ratios with Fas-sensitive target cells, and DNA fragmentation was measured. A typical experiment of 3 is shown. (E) Human T-cell blasts were treated as described in panel D and target cell killing was assessed. A typical experiment of 4 is shown. (F) Human T-cell blasts were treated with control or 10 mM MbCD for 20 minutes and stimulated with PMA and ionomycin overnight; IL-2 production was measured by enzyme-linked immunosorbent assay (ELISA). A typical experiment of 3 is shown.