CTL clone B5 recognizes an HLA-A3–associated antigen expressed by renal and pancreatic carcinoma cell lines and their LCL counterparts. CTL B5 was cloned from allogeneic MLTC-1 that sensitized CD8⁺ T lymphocytes of healthy donor 860 against the HLA class Ia–matched RCC line MZ1257-RCC. (A) Lytic activity of CTL B5 was tested against MZ1257-RCC (▪), MZ1257-LCL (▴), and K562 (○) in a 4-hour ⁵¹Cr-release assay. Results shown are percentage of cytolysis at indicated effector-to-target (E/T) ratios. (B) Effect of different anti–HLA class I mAbs on recognition of MZ1257-RCC (▪) and MZ1257-LCL (□) by CTL B5 in 20-hour IFN-γ ELISPOT assay. (C) Full-length HLA-A*03011 cDNA cloned from MZ1257-RCC and inserted into pcDNA3.1(+) expression vector was transiently transfected in 293T cells. Transfectants were tested for recognition by CTL B5 in a 20-hour IFN-γ ELISPOT assay. In a control experiment, MZ1257-RCC–derived HLA-A*03011 cDNA was able to present a known HLA-A3–restricted peptide antigen derived from GPNMB_V2^mut cDNA to antimelanoma CTL line MLTC18 (GPNMB_V2^mut cDNA and MLTC18 provided by V.L. and T.W.). (D) Cross-reactivity of CTL B5 tested in 20-hour IFN-γ ELISPOT analyses against HLA-A3⁺ RCC, non-RCC tumor, and LCL cell lines. LCL indicates Epstein-Barr virus (EBV)–transformed B-lymphoblastoid cell line; MEL, melanoma cell line; LC, lung cancer cell line; COC, colon cancer cell line; PC, pancreatic-carcinoma cell line; and GBC, gallbladder-carcinoma cell line. Results shown are a representative part of 25 HLA-A3⁺ targets totally tested. All spot assays were performed with 5000 CTLs seeded per well. Each bar represents the mean spot number of triplicates.