Immunomodulation of gene therapy with extended anti-ICOS treatment results in increased percentage of CD4⁺ CD25⁺ Foxp3⁺ cells. Lymphocytes of secondary lymphoid organs from 3 groups of mice treated with different regimens were stained for CD4, CD25, and intracellularly for Foxp3. (A) Representative dot plots of flow cytometry of blood samples at day 14. Time course of percentage of CD25⁺Foxp3⁺ was evaluated in CD4⁺ T cells isolated from (B) peripheral blood, (C) LNs, and (D) spleen. **P < .01, compared with groups of naive, anti-ICOS alone, and plasmid only treated mice. (E) ICOS expression on CD4⁺CD25^high, CD4⁺CD25^low, and CD4⁺CD25⁻ cell populations isolated from spleen of 3 groups of mice at day 2 to 4 after plasmid transfer. Results were shown as MFI of ICOS.