Concentration of FVIII determines the response of FVIII-specific memory B cells and FVIII-specific T cells. CD138^– spleen cells were obtained from hemophilic mice treated with human FVIII. Cells were restimulated in vitro with human FVIII as indicated for 3 days (B) or 6 days (A,C-E). Newly formed anti-FVIII ASCs were detected by ELISPOT assay (A). ELISPOTs represent the results obtained in a typical experiment. Cell proliferation (B) and cytokine secretion into cell culture supernatants (C-E) were analyzed as described in “Materials and methods.” Presented are the means and standard deviations of triplicate cultures (B) or the medians (C-E) obtained in a typical experiment.