The antigen-specific restimulation of FVIII-specific memory B cells in vivo shows the same dependence on costimulatory interactions as the restimulation in vitro. Memory cell pool cells (CD138^– spleen cells) were obtained from hemophilic mice treated with 4 intravenous doses of 200 ng (80 U/kg) FVIII and transferred into naive hemophilic mice. (A) Twenty-four hours after transfer, mice were treated with a single dose of either 200 ng FVIII (⋄) or formulation buffer (○). As a negative control, naive hemophilic mice that did not receive any cell transfer were treated with a single intravenous dose of 200 ng FVIII (▵). (B-D) Twenty-two hours after transfer, mice were treated with intravenous doses of either 100 μg α-CD80 and 100 μg α-CD86 (B; ▪), 200 μg α-CD40L antibody (C; ▪), 200 μg α-ICOSL antibody (D; ▪), or 200 μg appropriate isotype control antibodies (B-D; ♦). Two hours after the application of the antibodies, mice received a single intravenous dose of FVIII. Blood samples were obtained at 3, 7, 14, 21, and 49 days after treatment with FVIII and analyzed for anti-FVIII antibody titers by ELISA. Data shown represent mean values (n=5) and SD.