Gene transfer of FVIII plasmid into experimental mouse strains. (A-B) FVIII levels and inhibitory antibody activity were assessed after delivery of pBS-HCRHPI-hFVIIIA into the HemA (■), Foxp3-Tg (▴), HemA/Foxp3-Tg (X), and wt mouse (♦) strains (n = 8/group). Mice were bled at regular intervals beginning on day 3, after plasmid injection. (A) Circulating FVIII activity was measured via a modified clotting assay and confirmed by a COATEST assay. (B) Inhibitory antibody titers were evaluated by Bethesda assay and expressed as Bethesda units/mL. (C-D) In vitro proliferation and suppression assay after stimulation of CD4⁺ T cells with hFVIII. (C) CD4⁺ splenic T cells isolated from wt, HemA, Foxp3-Tg, and HemA/Foxp3-Tg mice (n = 3/group) 4 weeks after FVIII plasmid treatment. Cells from untreated mice of each strain were used as controls. Triplicates of isolated CD4⁺ T cells isolated were stimulated with 4 U/mL hFVIII protein in the presence of APCs for 72 hours. Each dataset represents mean Δcpm. (D) Suppression of the proliferative response to hFVIII stimulation by Tregs in vitro. CD4⁺ T cells from spleens of FVIII plasmid-treated HemA mice were used as Teffs, and CD4⁻ cells were used as APCs. CD4⁺CD25⁺ Tregs from untreated and FVIII plasmid-treated HemA mice and CD4⁺ Tregs from untreated and FVIII plasmid-treated HemA/Foxp3-Tg mice (n = 3/group) were cocultured with the Teffs at a ratio of 1:1 Tregs to Teffs in the presence of APCs and 10 U/mL hFVIII for 3 days. [³H]Thymidine was subsequently added and cultures maintained for another 18 hours. Data are shown by percentage of suppression compared with effector cells only (without the presence of Tregs). *P < .05 between the group of effector cells only and with Tregs from Foxp3-Tg mice.