T cell–dependent and T cell–independent restimulation of FVIII-specific memory B cells differ in the IgG subclass distribution of newly formed FVIII-specific ASCs. 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 or purified CD11c⁺ DCs. Newly differentiated FVIII-specific ASCs of different IgG subclasses were analyzed after 6 days of culture. Note: The control cultures of memory B cells together with CD11c⁺ DCs in the absence of TLR7 agonist did not induce any differentiation into FVIII-specific ASCs (see Figure 4A). Therefore, we did not include these data in Figure 6A. (A) Representative ELISPOTs are presented. (B) Quantification of results for T cell–dependent restimulation of memory B cells presented in (A). Results of individual ELISPOT analyses are presented. *P < .05, **P < .01, ***P < .001 for comparison of means. ○ indicates FVIII only; and ♢, FVIII + TLR7 agonist imiquimod.