TLR7 and TLR9 agonists amplify T cell–dependent and facilitate T cell–independent restimulation of FVIII-specific memory B cells. CD138⁻ (A) and CD138⁻ T⁻ (B) spleen cells were obtained from hemophilic mice treated with 4 weekly doses of 200 ng of FVIII and restimulated in vitro with FVIII in the presence of TLR7 agonist imiquimod or TLR9 agonist CpG-ODN. Newly formed FVIII-specific ASCs were detected after 6 days of culture by ELISPOT assay. The concentration of FVIII is indicated. Cells were differentiated in the presence of medium only, TLR7 agonist imiquimod (100 ng/mL) or TLR9 agonist CpG-ODN (100 ng/mL). Representative ELISPOTs are presented.