The frequency of CD4⁺ Foxp3⁺ T cells is significantly decreased by IL-21 and CD25 treatment in stimulation culture. MART-1–specific T-cell stimulation cultures were established using unmanipulated PBMCs (control), IL-21–treated, CD25-depleted, and CD25-depleted/IL-21–treated PBMCs. Three weeks following initial in vitro stimulation, 5 × 10⁵ cells from each experimental culture were stained with FITC-conjugated CD4, followed by intracellular staining for Foxp3 protein. Data are expressed as percentage of CD4⁺ Foxp3-positive cells among gated lymphocytes.