(A) Protocol of the in vivo experiment. Male E16 mice were primed with two immunogenic doses of rFVIII (1μg in 100μl PBS) intravenously on day 0 and 7. After the confirmation of detectible serum anti-FVIII antibody levels on day 21, the mice were randomly divided into three groups (n = 4 per group): OVA-BAR hTregs (squares), FVIII-BAR hTregs (circles), and PBS (diamonds). Each group of mice received a booster dose of rFVIII i.v. on day 28, followed by the i.v. injection of 2×106 FVIII-BAR hTregs, control OVA-BAR hTregs, or PBS, respectively. The mice were bled weekly after day 35, and the serum anti-FVIII antibody levels were measured by an ELISA assay as above. (B) Antibody titers before and after infusion of BAR Tregs. The antibody levels were measured by an ELISA assay. The data were expressed as mean ± SEM. *p < 0.05 between the FVIII-BAR hTregs group and the control OVA-BAR hTregs or PBS group. The non-normal distribution data was analyzed by Mann Whitney U test. Representative result from two independent experiments is shown.