Cytokine release into culture supernatants. Purified memory B cells and purified CD4⁺ T cells were obtained from hemophilic mice treated with 4 weekly doses of FVIII. CD11c⁺ DCs were obtained from untreated hemophilic mice. Purified memory B cells were restimulated with FVIII (10 ng/mL) or FVIII (10 ng/mL) together with TLR7 agonist imiquimod (100 ng/mL) in the presence of either purified CD4⁺ T cells (A) or purified CD11c⁺ DCs (B). Cytokines released into culture supernatants were analyzed after 6 days of culture. Boxplots including median, minimum, maximum, 25% percentile, and 75% percentile of 4 independent experiments are presented. *P < .05. □ indicates FVIII only; and ▨, FVIII + TLR7 agonist imiquimod; (C) Purified CD11c⁺ DCs were incubated with FVIII (10 ng/mL) or FVIII (10 ng/mL) together with TLR7 agonist Imiquimod (100 ng/mL). INF-α released into culture supernatants was analyzed after 1, 3, and 6 days of culture. Values below detection limit (36 ng/mL) were set to zero. □ indicates FVIII only; and ■, FVIII + TLR7 agonist imiquimod.