Combined use of IL-21 and CD25 depletion leads to significantly enhanced generation of antigen-specific cytotoxic T lymphocytes. Unmanipulated or CD25-depleted PBMCs from HLA-A2⁺ patients with melanoma were stimulated in vitro with autologous mature dendritic cells pulsed with the MART-1 M26 peptide as described in “In vitro generation of human antigen-specific CD8⁺ T cells.” Where indicated, IL-21 (30 ng/mL) was added at culture initiation. After 2 cycles of stimulation, 5 × 10⁵ cells from each experiment group were harvested and stained with 20 μg/mL peptide/MHC tetramer (PE, vertical axis) and FITC-conjugated CD4; a vital dye (DAPI) was also added to exclude dead cells. Data are expressed as percentage of tetramer-positive cells among gated lymphocytes on day 18 after second stimulation.