Inhibition of memory B-cell restimulation by high-dose FVIII is irreversible. CD138^– spleen cells were obtained from hemophilic mice immunized with human FVIII and restimulated with either 20 or 0.01 μg/mL FVIII. After 3 days of culture, cells were washed, split into 2 aliquots, and cultured for another 6 days as indicated. Newly formed ASCs were analyzed by ELISPOT. Presented is a typical experiment. Spleens of 5 mice were pooled for cell preparation. All experiments (n = 3) gave similar results.