Effect of anti-CD3 treatment on CD4⁺, CD8⁺ T cells, and CD4⁺Foxp3⁺ Tregs in spleen and blood. (A-C) Spleen cells were isolated from FVIII plasmid only and FVIII plasmid + anti-CD3–treated mice (n = 3/group). (A) Cells were stained and analyzed for CD4⁺, CD8⁺, and CD4⁺CD25⁺Foxp3⁺ T cells by flow cytometry 8 days after the first anti-CD3 injection and FVIII plasmid transfer. (Left panel) Representative dot plots. (Right panel) Summary of the data from 2 groups of mice. (B) Representative histograms of CD3 expression on CD4⁺Foxp3⁺ and CD4⁺Foxp3⁻ T cells obtained from FVIII plasmid only– (left panel) and FVIII plasmid + anti-CD3 (right panel)–treated mice. Dark line represents CD4⁺Foxp3⁻ cells; light line, CD4⁺Foxp3⁺ cells. (C) Detailed analysis of CD25 expression on CD4⁺Foxp3⁺ Tregs at day 8, week 4, and week 6 after treatment. (Top panel) Representative dot plots of Foxp3⁺ expression on CD4⁺ T cells (CD4⁺Foxp3⁺ cells are gated). (Bottom panel) Representative dot plots of CD25⁺ expression on CD4⁺Foxp3⁺ T cells (CD4⁺CD25^hiFoxp3⁺, CD4⁺CD25^lowFoxp3⁺, and CD4⁺CD25⁻Foxp3⁺ are each gated). Numbers are percentages of corresponding populations. Data shown are mean ± SD. **P < .01. (D-F) Blood samples were collected at serial time points from the 4 groups, including naive untreated, anti-CD3 only, FVIII plasmid only, and FVIII plasmid + anti-CD3–treated mice, stained and analyzed by flow cytometry for CD4⁺ T cells (D), CD4⁺Foxp3⁺ Tregs (E), and CD4⁺CD25⁺Foxp3⁺ Tregs (F). Data shown are mean ± SD. **P < .01 compared with FVIII plasmid only. #P < .05 compared with anti-CD3 only.