Dose-dependent inhibition of memory B-cell restimulation by FVIII. CD138^– spleen cells were obtained from hemophilic mice immunized with human FVIII (A,D), murine FVIII (B), or OVA (C) as described in “Materials and methods.” Newly formed anti–human FVIII (A,D), anti–murine FVIII (B), or anti-OVA (C) ASCs were detected by ELISPOT analysis after culture of CD138^– spleen cells in the presence of human or murine FVIII at the concentrations indicated. (C) CD138^– spleen cells were stimulated with a fixed concentration of OVA and human FVIII at the concentration indicated. (D) CD138^– spleen cells were cultured with either human FVIII or thrombin-cleaved human FVIII. ELISPOTs represent the results obtained in a typical experiment. The graphs represent mean values (n = 7) and standard errors of the mean. Results were normalized for comparing experiments done on different days by setting responses obtained with 0.01 μg/mL FVIII (A) or 0.01 μg/mL OVA (C) at 100%. Spleens from 5 mice were pooled for cell preparation in each experiment. **P < .01; ***P < .001.