Generation of RCC-reactive CD8⁺ T cells from HLA-class Ia-matched healthy donors. In allogeneic MLTC-1 and MLTC-2, CD8⁺ T cells isolated from PBMCs of healthy donors 860 and 880 with a purity of more than 98% were stimulated weekly with HLA Ia–matched RCC lines MZ1257-RCC and MZ1851-RCC, respectively. HLA class Ia genotypes of donors and RCC lines are shown in Table 1. Both RCC lines were negative for HLA class II in flow cytometry. Specificity of MLTC responders was analyzed on day 25 of culture in 20-hour IFN-γ ELISPOT assays. (A) Targets were the RCC stimulator cell lines, the hematopoietic counterparts (ie, EBV-LCL or PHA-activated PBMC) derived from the same RCC patients, and NK target K562. PHA-activated PBMCs of patient MZ1257 and EBV-LCL of patient MZ1851 were not available. (B) HLA restriction of anti-RCC reactivity was determined by adding mAbs specific for total HLA class I, HLA-A2, HLA-A3, HLA-B/-C alleles, or HLA-DR. Each bar represents the mean spot number of triplicates with 10 000 MLTC responders seeded per well.