Reduction of immune responses on in vivo administration of FVIII-R2090A/K2092A/F2093A in FVIII^−/− mice. Hemophilic E17KO mice (n = 8) were injected intravenously 5 times weekly with 1 μg of FVIII WT or FVIII-R2090A/K2092A/F2093A. One week after the last injections, mice were killed, and blood samples were collected. (A-B) Anti-FVIII Ab titers from the plasma samples were evaluated by ELISA (A) and Bethesda assay (B) as described in detail in “Anti-FVIII inhibitory Ab measurements from mouse plasma by ELISA and Bethesda assay.” *P < .05 (nonparametric Mann-Whitney U test). (C) The presence of splenic ASCs producing FVIII-specific Abs was determined by ELISPOT. As a control, IgG-producing ASCs were detected. Representative wells displaying both ASCs types are shown for both groups. Spots were quantified with the AELvis reader and eli.analyze Version 6.0 software. Group injected with FVIII WT is represented by the white bars and group injected with FVIII-R2090A/K2092A/F2093A is represented by the black bars. (D-E) CD8⁻ splenocytes were assayed in a thymidine (³H) incorporation assay. Proliferation was measured after 72 hours, and thymidine was added for the last 18-20 hours. Results are shown as stimulation index (SI) from triplicate wells (mean ± SD) for both FVIII-specific (D) and nonspecific (E) proliferation. Mice injected with FVIII WT (●), group injected with FVIII-R2090A/K2092A/F2093A (○). WT indicates wild-type FVIII; and 2090/92/93, FVIII-R2090A/K2092A/F2093A.