hFVIII-alum increases the tolerogenicity of anti-CD4 mAb. (A) C57Bl/6 and BALB/c 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 or 110 following the last administration of anti-CD4. (B-C) Serum concentration of hFVIII-specific IgG1 in C57Bl/6 mice (B) (n = 6; **P < .01) and in BALB/c mice (C) (n = 6; *P < .05; **P < .01). (D) hFVIII-alum and anti-CD4 were administered through different routes (as indicated in the figure label) according to the schedule represented in panel A. One group of mice was treated with an isotype control. All mice were then exposed to hFVIII intravenously and serum anti-hFVIII IgG1 was quantified. Animals that received hFVIII-alum together with anti-CD4 remained tolerant regardless of the administration route (n = 6; ***P < .001). (E) Persistent serum levels of hFVIII did not prevent the generation of hFVIII-specific IgG1 in the presence of anti-CD4. C57Bl/6 mice were treated with anti-CD4 mAbs in the days indicated above. Some mice were treated with 1 U hFVIII every other day for 2 weeks. We found that animals exposed to multiple administrations of hFVIII still produced higher titers of anti-hFVIII IgG1 than animals treated with hFVIII-alum (n = 6; ***P < .001). (F) Administration of the nondepleting anti-CD4 mAb (YTS177.9) does not lead to CD4 T-cell depletion. DO11.10.Rag^−/− mice were treated with 2 × 1 mg YTS177.9, and splenocytes were examined by flow cytometry for evidence of T-cell depletion at day 7 (n = 5; P = nonsignificant). i.m., intramuscular; i.p., intraperitoneal; i.v., intravenous; n.s., nonsignificant; s.c., subcutaneous.