Apoptosis of T lymphocytes at high concentrations of ATG is not the consequence of an AICD process. (A) PBLs were activated by rabbit ATG at the indicated concentration in the presence or absence of ZB4 (2.5 μg/mL) and TNF-R-Ig (20 μg/mL), and apoptosis was measured at 24 hours. (B) Activated lymphoblasts were treated for 24 hours with either medium, CD178 (100 ng/mL) in the presence or absence of ZB4 (2.5 μg/mL), or TNF-α (50 ng/mL) in the presence or absence of TNF-R-Ig (20 μg/mL). (A-B) Apoptosis was measured by surface binding of annexin V. (C) Analysis of caspase-8 processing in the presence of rabbit ATG. PBLs were treated with ATG for 24 hours. As positive control, activated lymphoblasts were treated with anti-CD95 (7C11; 1 μg/mL) for 6 hours. (D) Kinetics of FLIPs expression in PBLs treated with rabbit ATG (50 or 250 μg/mL). (C-D) Cells were lysed after a wash with PBS, and proteins were loaded and separated on 12% SDS-PAGE followed by Western blotting with the anti-caspase-8 mAb (C) or the anti-FLIP mAb (D). Amounts of loaded proteins have been controlled for homogeneity by probing membranes with an anti-β-actin mAb. Results from one experiment representative of 2 independent experiments showing similar results. (E) PBLs were incubated with NRS or ATG (250 μg/mL) for 24 hours in the presence of CsA (1 μg/mL), RPM (500 nM), or dexamethasone (1 μM). Apoptosis was measured by surface binding of annexin V, and results shown are representative of 2 independent experiments.