Highly purified memory B cells require CD4⁺ T-cell help for differentiation into FVIII-specific ASCs. (A) Purified memory B cells and CD4⁺ T cells were obtained from hemophilic mice treated with 4 weekly doses of FVIII. Cells were cultured in the presence of FVIII only, FVIII and TLR7 agonist imiquimod (100 ng/mL), or FVIII and a mixture of TLR7 agonist imiquimod (100 ng/mL) and TLR9 agonist CpG-ODN (100 ng/mL). The concentration of FVIII is indicated. Newly differentiated FVIII-specific ASCs were analyzed after 6 days of culture. Representative ELISPOTs are presented. (B) Purified naive B cells were obtained from untreated hemophilic mice, and CD4⁺ T cells were obtained from hemophilic mice treated with 4 weekly doses of FVIII. Naive B cells together with CD4⁺ T cells were cultured in the presence of FVIII only, FVIII and TLR7 agonist imiquimod (100 ng/mL), or FVIII and a mixture of TLR7 agonist Imiquimod (100 ng/mL) and TLR9 agonist CpG-ODN (100 ng/mL). FVIII was used at a concentration of 10 ng/mL. Newly differentiated FVIII-specific IgG ASCs and IgM ASCs were analyzed after 6 days of culture. Representative ELISPOTs are presented. (C) Purified memory B cells were obtained from hemophilic mice treated with 4 weekly doses of FVIII. Cells were cultured in the presence of FVIII and TLR7 agonist imiquimod or FVIII and a mixture of TLR7 agonist imiquimod (100 ng/mL) and TLR9 agonist CpG-ODN (100 ng/mL). The concentration of FVIII is indicated. Newly differentiated FVIII-specific ASCs were analyzed after 6 days of culture. Representative ELISPOTs are presented.