Restimulation of FVIII-specific memory B cells in vivo. 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. 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 (▵). Blood samples were obtained 1, 3, 7, 14, 42, and 84 days after treatment and analyzed for anti-FVIII antibody titers by ELISA. Each point represents a single mouse. The same mice were analyzed for each time point.