Nondepleting anti-CD4 induces long-term protection from inhibitor formation in HemA mice. (A) HemA mice were treated with 1 U hFVIII or HFVIII-alum and 2 × 1 mg nondepleting anti-CD4 (or isotype control) on the indicated days, followed by subsequent administrations of 1 U hFVIII as represented. The presence of serum hFVIII-specific immunoglobulin was investigated at days 30, 60, and 90 following last administration of anti-CD4. (B) Quantification of serum hFVIII-specific IgG1 from C57Bl/6 HemA mice at days 30 and 90, following multiple exposures to hFVIII (n = 8; ***P < .001). (C) Quantification of FVIII inhibitors from the serum of C57Bl/6 mice at day 90 (n = 8; **P < .01). (D) Serum hFVIII-specific IgG1 from BALB/c HemA mice (n = 4; **P < .01). (E) Quantification of hFVIII inhibitors from BALB/c HemA mice (n = 4; *P < .05). (F) C57Bl/6 HemA mice were treated on days 1 and 14 with hFVIII-alum (intraperitoneally or subcutaneously) and anti-CD4 (intraperitoneally or intravenously). A control group remained untreated. Subsequent challenges were done by intravenous administration of hFVIII, in a total of 4 injections, given twice weekly starting on day 21. Quantification of the anti-hFVIII IgG1 levels and BU, measured 1 week after the final exposure to hFVIII (n = 6; *P < .05). (G) Quantification of IL-10 concentration in supernatants of splenocytes from C57Bl/6 HemA mice, collected at day 7 following initial treatment with HFVIII-alum plus anti-CD4. The splenocytes were stimulated with anti-CD3 for 3 days (n = 3; *P < .05). Data are representative of 2 independent experiments. ip, intraperitoneal; iv, intravenous; sc, subcutaneous.