Lack of antibody development against human FVIII in mice of subline E is caused by specific immune tolerance. Mice were treated with 8 weekly IV doses of human FVIII (A), followed by 4 weekly IV doses of human VWF (B). Blood for antibody analysis was drawn 1 week after the fourth (4xFVIII, A) and the eighth (8xFVIII, A) dose of FVIII, as well as 1 week after the fourth dose of VWF (4xVWF, B). Spleens for the analysis of ASCs were collected from 3 mice 1 week after the eighth dose of FVIII (A) and from the remaining mice 1 week after the fourth dose of VWF (B). Titers of anti–human FVIII IgG antibodies (A) and anti–human VWF IgG antibodies (B) were detected by ELISA. Antibody titers are shown for individual mice as follows: human F8 transgenic mice, male (▴) and female (●); conventional E17 F8-knockout mice: male (♢). Anti–human FVIII ASCs (A) and anti–human VWF ASCs (B) in the spleens of human F8 transgenic mice (huF8) and conventional E17 F8-knockout mice (E17F8ko) were analyzed by ELISPOT. Results obtained from a representative animal are shown. Each spot represents 1 ASC.