(A) Effect of immunosuppressive agents on ATG-mediated apoptosis. PBL were cultured for 3 days with PHA (5 μg/mL) and CsA (250 ng/mL), FK506 (10 nmol/L) or RPM (60 nmol/L) were added at the onset of the culture. Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342, 20 hours after treatment with ATG no. 95-07 or their F(ab′)2 fragments at 10 μg/mL. (B) Effect of addition of exogenous IL-2 or IFN-γ. PBL were cultured for 3 days with PHA (5 μg/mL); medium alone (gray bars) or CsA (250 ng/mL) (black bars) were added at the onset of the culture. Recombinant IL-2 (25 U/mL) or rIFN-γ (500 U/mL) was added during the last 24 hours of activation. Apoptosis was determined by fluorescence microscopy after staining with Hoechst 33342, 20 hours after treatment with ATG no. 95-07, their F(ab′)2 fragments at 10 μg/mL or the CH11 (1 μg/mL) MoAb. (Results are expressed as mean ± SEM of three different experiments).
