De novo appearance of MART1-, gp100- and/or tyrosinase-specific CD8+ T cells in 4/7 HLA-A2*0201+ melanoma patients after one cycle of DAB/IL2. Whole blood was collected from patients P7 (A), P9 (B), P14 (C) and P16 (D) throughout four cycles of DAB/IL2 administration (each cycle indicated by an arrow). The peripheral blood mononuclear cells were isolated from the whole blood by Ficoll gradient centrifugation and stained with a PE-labeled anti-CD8 monoclonal antibody and APC-labeled tetrameric HLA-A2*0201/MART1 (black) or gp100 (red) or tyrosinase (green) peptide conjugates. The peripheral blood concentration of the indicated melanoma antigen-specific CD8+ T cells was quantified by multiplying the percentage of CD8+/tetramer+ cells within the lymphocyte forward/side scatter gate by the absolute lymphocyte concentration determined using an automated hematology analyzer. Data are represented as averages ± standard error of the mean (n = 10 patients). Patient P14 did not develop detectable tyrosinase- or gp100-specific CD8+ T cells but the green line (tyrosinase) is concealing the red line (gp100) (C).