The antigen-specific restimulation of FVIII-specific memory B cells in vitro involves CD40-CD40L and B7-CD28 interactions but does not require ICOS-ICOSL interactions. Memory cell pool cells (CD138^– spleen cells) were obtained from hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII and restimulated with 10 ng/mL FVIII. Anti-FVIII ASCs were analyzed after 6 days of culture by using ELISPOT assays. To interfere with costimulatory interactions, blocking antibodies (α-CD80, α-CD86, α-ICOSL, α-CD40L as indicated) or competitor proteins (mCTLA-4/Fc, mICOS/Fc as indicated) were added to the cultures at a concentration of 10 μg/mL together with the FVIII. Appropriate isotype-matched control antibodies and human IgG1 were used at the same concentration. (A) ELISPOT data obtained in a representative experiment. (B) Mean values (n = 4) and standard deviations. All results were normalized for comparing experiments done on different days. Results obtained with FVIII only were set as 100%.