Immunomodulatory activity of CpG-ODN in vitro when added with a time delay. CD138⁻ spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and in vitro restimulated with either an inhibiting concentration (20 μg/mL) of FVIII (A and B) or a stimulating concentration (10 ng/mL) of FVIII (C and D). Then, 100 ng/mL CpG-ODN or medium were added either together with FVIII on day 0 or at different times (days 1-5) after FVIII. Newly differentiated anti-FVIII ASCs were analyzed by ELISPOT assay 6 days after the addition of CpG-ODN. All results were normalized in relation to results obtained with 10 ng/mL FVIII without CpG-ODN (0/6), which was set to 100%. Arithmetic means of a representative experiment are presented. (A) Twenty μg/mL FVIII without CpG-ODN; (B) 20 μg/mL FVIII + 100 ng/mL CpG-ODN; (C) 10 ng/mL FVIII without CpG-ODN; and (D) 10 ng/mL FVIII + 100 ng/mL CpG-ODN.