Anti-RCC CTLs with HLA- and non–HLA-restricted cytotoxicity or with restriction by a nonclassical HLA-Ib molecule. CTL clones K34 and J23 were isolated from allo-MLTC-1 on CD8⁺ T lymphocytes of healthy donor 860 and the HLA class Ia–matched RCC line MZ1257-RCC. (A,D) Lysis on MZ1257-RCC (▪), MZ1257-LCL (▴), and K562 (○) by CTLs was determined in 4-hour ⁵¹Cr-release assays. Results are percentage of cytolysis at indicated E/T ratios. (B,E) IFN-γ ELISPOT assays were performed on MZ1257-RCC in the presence of anti–HLA class I mAbs to identify CTL restriction elements. (C) Reactivity of CTL K34 was tested against allogeneic HLA-Cw7⁺ target cell lines in IFN-γ spot assays. (F) IFN-γ spot production of CTL J23 was assayed against target cell lines matched or mismatched for single HLA-Ia alleles. ELISPOT results shown are representative of 50 different targets completely tested. They were obtained in 20-hour assays on 5000 CTLs/well in triplicates.