Depletion of CD4⁺ICOS⁺ T cells in mice with extended 4-week anti-ICOS treatment. Lymphocytes isolated from secondary lymphoid organs of 4 groups of mice, namely, untreated control, anti-ICOS alone, hFVIII plasmid only, and hFVIII plasmid plus anti-ICOS–treated mice, were stained for ICOS and CD4 markers. (A) Left panel, representative dot plots of flow cytometry of blood and spleen samples at day 2 after indicated treatment. Right panel, statistical analysis of percentage of ICOS⁺CD4⁺ cells in 4 groups of mice (n = 3). (B-D) Time course of percentage of CD4⁺ T cells in peripheral blood (B), LNs (C), and spleen (D) against isolated total mononuclear cells. *P < .05, **P < .01, compared with groups of naive, anti-ICOS only, and plasmid-only treated mice. N and P indicate untreated naive mice and mice treated with hFVIII plasmid only, respectively.