CD7-restricted binding and apoptosis induction by scFvCD7:sFasL. (A) CD7-restricted binding of scFvCD7:sFasL was assessed using CD7-positive CEM cells. Cells were incubated with scFvCD7:sFasL (solid line) or with unconditioned medium (filled area). Specific binding was demonstrated by preincubating CEM cells with mAb TH69 followed by incubation with scFvCD7:sFasL (dashed line). Binding of scFvCD7:sFasL was determined by flow cytometry using PE-conjugated anti-FasL mAb. (B) CD7-restricted induction of apoptosis by scFvCD7:sFasL was assessed using CD7-positive cells (MOLT-16, CEM, Jurkat, and HUT-78) and CD7-negative Raji cells. All cell types were treated for 16 hours with increasing concentrations of scFvCD7:sFasL, after which apoptosis was assessed by ΔΨ. Additionally, Raji cells were treated with increasing concentrations of cross-linked sFasL (cr-sFasL). Indicated values are mean + SEM of 3 independent experiments. (C) Jurkat cells were treated for 16 hours with scFvCD7:sFasL (20 ng/mL) in the presence or absence of MAb TH69 or FasL-neutralizing MAb Alf2.1. Apoptosis was assessed by staining for apoptotic DNA-fragmentation using mAb F7-26. Horizontal bars indicate the percentage of apoptosis. (D) To compare the specific apoptotic activity of scFvCD7:sFasL with that of the related TRAIL fusion protein scFvCD7: sTRAIL, Jurkat cells were treated with equimolar concentrations of scFvCD7:sFasL and scFvCD7:sTRAIL for 16h. After treatment, apoptosis was assessed by ΔΨ.