Expression of wild-type and gene-optimized human melanoma–specific TCRs. Activated PBMCs were transduced with wild-type or codon-optimized Gp100-specific R6C12 and Mart-1–specific DMF4 TCRs. TCR expression was determined 4 days after transduction by flow cytometric analysis using (A) anti-CD8 and anti-Vβ12 (top panels) or anti-Vβ8 (bottom panels), or (B) anti-CD8 and A2.1-Mart-1_{(26-35, 27 A>L)} tetramer (top panels) or A2.1-Gp100_{(209-217, 210 T>M)} tetramer (bottom panels). The numbers indicate the percentage of Vβ⁺ or tetramer⁺ CD8⁺ cells. (C,D) Retrovirus encoding wt (□) or opt (■) DMF4 (C) or R6C12 TCR (D) was titrated on Jurkat/MA cells by adding the indicated amount of viral supernatant to a total volume of 1 mL. Four days after transduction, cells were analyzed by flow cytometry for TCR expression by anti-TCRαβ antibody staining.