HC/2G-1 uses the TCR α chain CDR3 for recognizing the DR4/TRAIL complex. (A) Allogeneic T cells transduced with the genes encoding wild-type HC/2G-1 TCR (WT), HC/2G-1 TCR with doubly mutated α chain and wild-type β chain (109Y-F, 112S-K), control GFP expressing retrovirus (GFP) or nothing (Untransduced) were cocultured with a panel of tumor lines. The parental HC/2G-1 T-cell clone is also shown (HC/2G-1). Red bars indicate RCC#1; orange bars, RCC#6; yellow bars, RCC#8; green bars, RCC#10; blue bars, RCC#11; indigo-blue bars, 624mel; purple bars, 938mel; and gray bars, medium alone. (B) Plate coating was done in the same way as in Figure 4B using anti-CD2 Ab, DR4-Fc, DR5-Fc, and IgG Fc in the combinations indicated. Activation of parental HC/2G-1 cells (1), allogeneic T cells transduced with GFP cDNA (2), allogeneic T cells transduced with wild-type HC/2G-1 TCR α and β chain cDNA (3), or allogeneic T cells transduced with mutated HC/2G-1 TCR α (109Y-F, 112S-K) and wild-type HC/2G-1 TCR β chain cDNA (4) were measured at 24 hours with IFN-γ ELISA. Stimulation by 624mel and RCC#6 is shown as a specificity control and by anti-CD3 Ab as a functional control. Error bars indicate SEM. Representative data from more than 3 experiments with similar results are shown.