Staining of Treg markers in FVIII plasmid-treated HemA and HemA/Foxp3-Tg mice over time. Single-cell suspensions were prepared from spleens and peripheral blood of FVIII plasmid-treated HemA and HemA/Foxp3-Tg mice (n = 2/group) at different time points and stained for CD4, CD25, GITR, and CTLA4 expression. The cells were first gated for CD4 expression. The percentages of CD4⁺CD25⁺, CD4⁺GITR⁺, and CD4⁺CTLA4⁺ cells were determined over time; splenic cells are shown in the top panel, and peripheral blood lymphocytes in the bottom panel.