Functional analysis of melanoma-specific TCRs. (A,B) Human PBMCs were transduced with titrated aliquots of virus as detailed in Figure 3. Five days after transduction, cell cultures transduced with the Mart-1–specific TCRs DMF4 (▴), 1D3 (●), or 2C2 (■) were incubated with T2 cells loaded with the indicated concentrations of Mart-1_(26-35) peptide. As a control, transduced cells were incubated with T2 cells loaded with the highest concentration of Gp100_(209-217) peptide (open symbols). (C,D) Five days after transduction, cell cultures transduced with Gp100 R6C12 TCR (♦) were incubated with T2 cells loaded with the indicated concentrations of Gp100_(209-217) peptide. As a control, transduced cells were also incubated with T2 cells loaded with the highest concentration of Mart-1_(26-35) peptide (open symbols). After 5 hours of incubation, cells were stained with FITC anti-CD8 and PE anti-CD4, and intracellular cytokine production was determined using APC anti-IFN-γ. The percentage of IFN-γ–positive CD8 cells (A,C) and CD4 cells (B,D) is shown. Error bars in panels A-D represent standard deviations (n = 3). (E,F) Lysis of melanoma cell lines in a ⁵¹Cr-release assay. Twelve days after transduction, 1D3 TCR–transduced (E) or R6C12 TCR–transduced (F) cells were cocultured with different HLA-A2.1⁺, Mart⁺, and Gp100⁺ cell lines: AKR (●), GDO (■), and 526 (♦). The HLA-A2.1⁻, Mart⁺, and Gp100⁺ cell line 938 (▴) was used as a control. Coculture of nontransduced cells with melanoma cell lines is indicated by open symbols: AKR (○), GDO (□), 526 (◇), and 938 (▵). Cells were incubated at the indicated effector-target ratios for 4 hours, after which the percentage of lysis was determined. Error bars represent standard deviations (n = 3).