IgG subclass distribution of anti-FVIII antibodies and anti-FVIII ASCs. (A) The IgG subclass distribution of anti-FVIII antibodies in blood plasma was compared with the IgG subclass distribution of anti-FVIII ASCs in spleen and bone marrow of hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII. Titers of anti-FVIII antibodies were determined by ELISA (each point represents an individual mouse), and anti-FVIII ASCs were analyzed by ELISPOT assays (spleens and bone marrow of 3 mice were pooled). Blood and tissues were obtained from the same mice. (B) IgG subclass distribution of anti-FVIII ASCs that were differentiated in vitro from CD138^– spleen cells. Spleen cells were obtained from hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII and depleted of CD138⁺ ASCs. CD138^– cells were subsequently restimulated with 10 ng/mL FVIII. Anti-FVIII ASCs were analyzed after 6 days of culture using ELISPOT assays.