Long-term protective effect of CD4⁺Foxp3⁺ Tregs in FVIII plasmid-treated HemA mice. CD4⁺ T cells were isolated from HemA/Foxp3-Tg mice 1, 2, 6, and 9 weeks after FVIII plasmid treatment (n = 3/group) and adoptively transferred to naive HemA mice. Recipient mice, a control group of naive HemA mice (n = 3), and a control group receiving CD4⁺ T cells derived from untreated HemA/Foxp3-Tg mice were challenged with FVIII plasmid injection 1 day after adoptive transfer. All treated mice were then subjected to a second challenge with FVIII plasmid injection 16 weeks after the first injection. (A) FVIII activity and (B) anti-FVIII antibody titers were evaluated over time, as described in Figure 2.