Comparison of the gene-optimized 1D3 TCR and wild-type DMF4 TCR. Activated PBMCs were transduced with titrated viral supernatants of pMX vectors encoding the DMF4 wild-type or gene-optimized receptor or the 1D3 gene-optimized receptor. (A) Four days after transduction, expression in PBMCs was determined by staining with anti-CD8 and A2.1-Mart-1_{(26-35, 27 A>L)} tetramer. The numbers in the top-right and bottom-right corners indicate the percentage of tetramer⁺ CD8⁺ and tetramer⁺ CD8⁻ cells, respectively. (B) Transduced cells were incubated with T2 cells loaded with the indicated Mart-1_(26-35) peptide concentrations. After 5 hours of incubation, cells were stained with anti-CD8, and intracellular cytokine production was determined by anti-IFN-γ staining. The percentage of IFN-γ⁺ CD8⁺ cells is shown for wild-type DMF4 (▵), gene-optimized DMF4 (▴), gene-optimized 1D3 (■), or nontransduced lymphocytes (□). Error bars represent standard deviations (n = 2). (C) Tetramer⁺ CD8⁺ cells were sorted and 1 week later incubated with T2 cells loaded with the indicated Mart-1_(26-35) peptide concentrations. After 5 hours of incubation, cells were stained with anti-CD8, and intracellular cytokine production was determined by anti-IFN-γ staining. The percentage of IFN-γ⁺ CD8⁺ cells is shown for sorted wild-type DMF4 (▵), gene-optimized DMF4 (▴), and gene-optimized 1D3 (■) transduced PBMCs.