Recognition of different HLA-A2–positive target cells by allo-HLA–reactive T-cell clones and confirmation of their single peptide specificity by transduction of shRNA specific for their respective recognized Ags. (A) The allo-HLA–reactive T-cell clones were stimulated with HLA-A2–negative EBV-LCLs (A2neg LCL), HLA-A2–positive EBV-LCLs (A2pos LCL), HLA-A2⁺ fibroblasts derived from 2 different individuals (FIB1 and FIB2), K562 cells transduced with HLA-A2 (K562+A2), HLA-A2–positive melanoma cell line 1.14 (Mel 1.14), and HLA-A2–positive renal cell carcinoma cell line 1774 (RCC 1774). Reactivity of one representative clone, clone HSS12, is shown. (B) Allo-HLA–reactive T-cell clones HSS12, HSS23, HSS41, and HSS47 were tested against fibroblasts transduced with lentiviral vectors encoding shRNAs specific for the genes of USP11, FDPS, VPS13B, or ATXN10 in combination with the puromycin resistance gene. shRNA-transduced cells were cultured for 14 days with puromycin (4 μg/mL) and used as stimulator cells for the corresponding allo-reactive T-cell clone and a control allo-reactive T-cell clone. Nontransduced cells (nTd) were used as control stimulator cells. By quantitative real-time PCR, the down-regulation of mRNA of the different genes compared with nontransduced cells was analyzed, the fold decrease for USP11 = 5, FDPS = 31, VPS13B = 5, ATXN10 = 10. (C) HSS11 and HSS12 were tested against DRA^+/+ (CBA) and DRA^−/− (EBA) fibroblasts transduced with lentiviral vectors encoding shRNAs specific for the genes of DRA or THRAP4, selected for 14 days with puromycin and cultured for 72 hours with 100 IU of IFNγ/mL. Down-regulation of mRNA of DRA and THRAP4 was analyzed by qRT-PCR, the decrease for THRAP4 in CBA and EBA was 10-fold, the decrease for DRA in CBA was 4-fold. No expression of DRA was measured in EBA.