Inhibition of memory B-cell restimulation by high-dose FVIII involves caspase activation and Fas-dependent mechanisms. CD138^– spleen cells were obtained from hemophilic mice immunized with human FVIII and restimulated with either 0.01 or 20 μg/mL as indicated. Newly formed ASCs were analyzed after 6 days of culture by ELISPOT assays. (A) Some of the cultures were preincubated with a broad-spectrum caspase inhibitor (Z-VAD-FMK) for 4 hours before FVIII was added. As a positive control for apoptosis, part of the cultures was incubated with 0.01 μg/mL FVIII in the presence of an agonistic anti–murine Fas antibody (Jo2). (B) Some of the cultures were incubated in the presence of Fas-Fc. As a negative control for Fas-Fc, part of the cultures was incubated in the presence of a human monoclonal IgG1. Presented is a typical experiment. Spleens of 5 mice were pooled for cell preparation. All experiments (n = 3) gave similar results.