RCC recognition by HC/2G-1 is blocked by anti-TRAIL Ab and expression of DR4 (TRAIL-R1) and CD58 on target cells confers HC/2G-1 recognition. (A) RCC and EBV-B lines from 10 patients were cocultured with HC/2G-1 cells, and IFN-γ in the culture supernatant was detected by ELISA after 20 hours of coculture. Cell line numbers from the same patient are matched between EBV-B and RCC. HC/2G-1 was derived from the patient 1. Error bars indicate SEM. (B) HEK-293 and its mock-, DR4-, or DR5-transduced variants were cocultured with HC/2G-1 T cells, and T-cell activation was measured by IFN-γ ELISA. Error bars indicate SEM. (C) HC-2G-1 cells were preincubated with either an isotype-matched control Ab (gray bars) or an anti-TRAIL Ab (black bars), and cocultured with various RCC cell lines (left panel) or enzyme-digested fresh RCC cells (middle panel). In the right panel, MART1_27-35-reactive control CD8⁺ tumor-infiltrating lymphocytes (JKF6) were similarly preincubated with Abs and cocultured with 624mel, a human melanoma cell line. After a 20-hour coculture, IFN-γ in the supernatant was measured by ELISA. Error bars indicate SEM. (D-F) cDNA library expression screening using CHO cells stably transduced with DR4 as the transfection target identified CD58 as the second crucial element. The effect of DR4 and CD58 expression on recognition was specific to HC/2G-1 cells. CHO and CHO/hDR4 cells were transiently transfected with GFP, CD58, CD54, CD102, or CD106 cDNA expression plasmids. After 24 hours, HC/2G-1 T cells (D), MART1_27-35-reactive negative control CD8 T cells (E), or HLA-DRβ1*0401–restricted gp100 44-59-reactive negative control CD4 T cells (F) were added and IFN-γ in the supernatant was measured by ELISA at 20 hours of coculture. Error bars indicate SEM. Representative results from at least 3 similar experiments are presented.