High concentrations of ATG induce apoptosis of PBL. (A-B) PBLs were incubated with rabbit (▪) or horse (•) ATGs, F(ab′)₂ fragments of ATG (□), or nonmitogenic ATG number 5 (○) over a range of concentrations. In panel A, [³H]TdR uptake was measured after 72 hours of culture. In panel B, apoptosis was measured by surface binding of annexin V after 24 hours of culture. (C) Kinetics of ATG-induced apoptosis. PBLs were incubated with NRS alone (○), rabbit ATGs (250 μg/mL; ▪), or horse ATGs (500 μg/mL; •), and apoptosis was measured by surface binding of annexin V. (A-C) Results are expressed as the mean ± SEM from triplicate measurements of one experiment representative of several independent experiments. (D) PBLs were incubated for 24 hours in the presence of NRS (control) or ATG (250 μg/mL). Anti-CD95 (7C11; 1 μg/mL) was added to 3-day-activated lymphoblasts during 24 hours. DNA fragmentation was analyzed by flow cytometry staining using the F7-26 mAb. The percentage of cells with fragmented DNA is indicated for each histogram. Results shown are representative of 4 independent experiments. (E) Morphologic features of cells after treatment with NRS or ATG were observed using transmission electron microscopy. Original magnification, × 10 000.