FVIII activity levels and anti-FVIII inhibitor titers in plasma of FVIII transgenic following FVIII immunization in the presence of IFA. (A) Immunization schemes for T2F8 and 2bF8 transgenic (left) and WT (control; right) mice. Preimmune blood was collected for all strains for FVIII:C assay. For T2F8 and 2bF8 transgenic mice that express hBDDFVIII, animals were immunized with rhF8 at 600 U/kg in the presence of IFA. Blood samples were collected 2 weeks after immunization. Because WT have full-length endogenous murine FVIII, mice received rhfF8 at 600 U/kg plus IFA and further blood collection was made the following week (week 3 overall). (B) Functional FVIII activity (FVIII:C) levels in T2F8 transgenic mice before and after immunization. FVIII:C levels were determined by chromogenic assay. Two lines of the endothelial-specific hBDDFVIII-expressing T2F8 transgenic mice with differing numbers of gene insertions (T2F8^Tg[ES#5] and T2F8^Tg[ES#65]) were used. WT mice were used as a control. (C) Comparison of inhibitor titers in T2F8 (EC-specific FVIII expression) and 2bF8 (platelet-specific FVIII expression) transgenic mice after a single dose of rhF8 immunization (week 2). The unpaired 2-tailed Student t test was used to analyze data.