Tolerance induction requires IL-10 and is independent of Foxp3⁺ Treg cells. (A) Mice were treated with hFVIII-alum and anti-CD4 as described in Figure 1A. Different groups were also treated with the indicated mAbs. All mice were exposed to 1 U hFVIII on days 30 and 60. Serum concentrations of hFVIII-specific IgG1 were determined on days 30, 60, and 90 (n = 5; **P < .01; ***P < .001). (B-C) Representative dot-plots (B) and quantification of splenic CD4⁺CD25⁺Foxp3⁺ T cells (C) from animals treated with anti-CD4 mAb or an isotype control on day 90 (n = 5; P = nonsignificant). (D) T/B monoclonal mice (T-Bmc) and T-Bmc Foxp3-deficient mice (T-Bmc FoxP3sf) were immunized on days 1 and 14 with OVA-HA-alum in the presence of nondepleting anti-CD4 or an isotype control. Serum HA-specific IgG1 was determined on day 24 (n = 5; ***P < .001). (E) IL-10^−/− and IL-10^+/+ mice were treated with hFVIII-alum and anti-CD4 mAbs as described above. The serum concentration of hFVIII-specific IgG1 was quantified on day 90 (n = 6; ***P < .001). (A-E) Data are representative of 2 independent experiments.