Expansion of PBMCs pretreated with denileukin diftitox by CMV and MART-1 peptides. PBMCs from a CMV⁺ donor were treated in vitro with denileukin diftitox or media for 18 hours. (A) Cells were replated in 10 U/mL IL-2 for 3 days and then cultured with CMVpp65 peptide and IL-2. Cells were analyzed by peptide–MHC tetramer staining on days 7 and 10 of culture. Data are presented as the percentage of CD8⁺CMV⁺ cells for untreated and denileukin diftitox–pretreated PBMCs. (B) Cells were replated in 10 U/mL IL-2 for 3 days and were then stimulated with MART-1 (26-35) peptide and IL-2. Peptide–MHC tetramer staining was performed on day 8 of culture. These cells were then restimulated with autologous PBMCs pretreated with denileukin diftitox (or media) as described, pulsed with MART-1 peptide, and irradiated. Cells were again analyzed by MART-1–specific tetramer 8 days after this second stimulation. We present the percentage of CD8⁺MART-1⁺ cells for untreated and denileukin diftitox–pretreated cells for both the first and second stimulations. Boxes on the graphs represent cells gated for analysis of percentage positive CD8 and tetramer.