Ig isotypes and IgG subclasses of anti-FVIII ASC in vitro differentiated in the presence of FVIII and 100 μg/mL CpG-ODN. CD138⁻ spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and restimulated with stimulating (10 ng/mL) and inhibiting (1 and 20 μg/mL) concentrations of FVIII in the presence of medium (control) or 100 ng/mL CpG-ODN (CpG). Newly formed anti-FVIII ASC were detected by ELISPOT assay after 6 days of culture. Arithmetic means of a representative experiment are presented. Medium control without FVIII (○); 10 ng/mL FVIII (●); 1 μg/mL FVIII (▴); and 20 μg/mL FVIII (▵).