Immunomodulation of gene therapy with extended anti-ICOS treatment-induced activation of CD4⁺Foxp3⁺ Tregs. Lymphocytes isolated from secondary lymphoid organs of hFVIII plasmid plus anti-ICOS–treated mice were stained for various regulatory surface and intracellular markers, including CD45RB, CD62L, GITR, and CTLA4 at day 14 and 28 after plasmid transfer. For analysis, cells were gated on CD4⁺Foxp3⁺ population. (A) Expression of regulatory markers in LNs. (B) Expression of regulatory markers in spleen. The data represent delta MFI obtained by subtracting the background MFI observed in untreated naive mice.