Engraftment of CD45.2⁺ Tregs isolated from FVIII plasmid-treated HemA/Foxp3-Tg mice in CD45.1⁺HemA mice. A total of 3 × 10⁶ T cells was isolated from CD45.2⁺HemA/Foxp3-Tg mice (n = 22 with 2 mice per time point) 2 weeks after FVIII plasmid treatment, and adoptively transferred into CD45.1⁺HemA mice at day 0. The recipient mice were subsequently challenged with 100 μg of FVIII plasmid by hydrodynamic injection at day 1. Single-cell suspensions were prepared from spleens, lymph nodes, and peripheral blood of recipient mice after adoptive transfer at different time points. The cells were stained with CD4, CD45.1, CD45.2, CD25, Foxp3, and CTLA4 markers. The percentages of engrafted CD4⁺Foxp3⁺CD45.2⁺T cells were evaluated over 120 days. Insert shows the time curve over 14 days (spleen, ♦; lymph node, ○; peripheral blood, Δ).