Production of anti-FVIII antibodies by ASCs in vitro does not depend on any of the costimulatory interactions tested. Spleen cells and bone marrow cells were obtained from hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII and analyzed for ASCs by ELISPOT assays. All results were normalized for comparing experiments done on different days. The ELISPOTs above each graph represent the results obtained in a typical experiment. To interfere with B7-CD28, CD40-CD40L, and ICOS-ICOSL 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 at a concentration of 10 μg/mL at the beginning of the cultures. Appropriate isotype-matched control antibodies and human IgG1 were used as controls at the same concentration. Mean values (n = 3) and SDs are presented.