Staining of Treg markers on endogenous CD45.1⁺T cells in Treg recipient mice. Adoptive transfer of Tregs into HemA mice, FVIII plasmid challenge, and preparation of single-cell suspensions from spleens, lymph nodes, and peripheral blood of the recipient mice were described in Figure 6. The plasmid-only–treated HemA mice without adoptive transfer were used as controls. The cells were stained with CD4, Foxp3, CD45.1, CD45.2, CD25, and CTLA4 markers. (A) Representative dot plots of stained cells. The percentages of (B) Foxp3⁺CD25⁺ cells and (C) Foxp3⁺CD25⁺CTLA4⁺ cells in CD4⁺T cells and (D) CTLA4⁺ cells in CD4⁺Foxp3⁺CD25⁺T cells isolated from Treg recipient mice were evaluated over time (spleen, ♦; lymph node, ○; peripheral blood, Δ). (E) The percentages of CD4⁺CTLA4⁺ T cells in plasmid-treated HemA control (□) and Treg recipient mice (♦) over time.