One million pltLys-iTreg or purTGFβ-iTreg cells were adoptively transferred by IV injection into F8null/B6 mice followed by rhF8 (Xyntha; Pfizer Inc., New York, NY) immunization weekly at 50 U/kg per week IV for a total of 4 weeks. One week after the last immunization, blood samples were collected. The titers of anti-FVIII inhibitory antibodies (inhibitors) and total anti-FVIII antibodies were determined by Bethesda assay and enzyme-linked immunosorbent assay (ELISA), respectively, as described in our previous report.16 (link),19 (link) Six days after the iTreg cell transfer, CD4+ T cells from peripheral blood were analyzed by flow cytometry for enhanced green fluorescent protein (EGFP) expression to assess the recovery of the infused iTregs. CD45.1 and CD45.2 were used as congenic markers.