CD8⁺ T cells against p14 recognize TAP-deficient tumors of different histological origin. T cell clones were tested for their specificity to selectively recognize the LRPAP1_21-30 TEIPP epitope presented on TAP-impaired tumors. (A) Whisker plots of LRPAP1 gene expression in human cancers collected from the Cancer Genome Atlas database. (B) The TAP1 gene in the melanoma 518A2 was knocked out (TAP KO) using CRISPR/CAS9. In addition, the LRPAP1_21-30 epitope was knocked out in these cells (ag KO). mRNA expression of LRPAP1 was examined by qPCR. Data are shown as mean ± SD of triplicates. Significance testing: unpaired Student’s t test; n.s., not significant; ***, P < 0.001 (n = 3). (C) HLA-A*02:01 surface expression on 518A2 melanoma variants measured by flow cytometry. Plots are representative of three independent experiments. (D) GM-CSF production of T cell clone 1A8 upon 518A2 TAP KO recognition. When LRPAP1_21-30 epitope was knocked out (ag KO), recognition by the T cells was abolished. Representative data of three independent experiments are shown. Each dot represents a technical replicate. Significance testing: Student’s unpaired t test; ***, P < 0.001. (E) To control for artificial CRISPR/CAS9-mediated immunogenicity, we cocultured T cell clones specific for LRPAP1_21-30 (TAP-independent epitope) or PRAME_425-433 (TAP-dependent epitope) together with the 518A2 melanoma (WT versus TAP KO). GM-CSF cytokine response was measured. Each dot represents one independent experiment with technical triplicates. (F) HLA dependency was tested using HLA-ABC– or HLA-A*02:01–blocking antibodies. Representative data of two independent experiments are shown. Each dot represents a technical replicate. Significance testing: unpaired Student’s t test; n.s., not significant; **, P < 0.01; ***, P < 0.001. (G) TEIPP T cell specificity was determined by coculturing three different T cell clones together with TAP-proficient T1 cells and TAP-deficient T2 lymphoma cells. Cytokine responses were measured to detect T cell recognition. Representative data of three independent experiments with technical triplicates are shown. (H) HLA-A*02:01 surface expression of the TAP-impaired tumor panels as measured by flow cytometry (SKCM, melanoma; KIRC, renal carcinoma). Representative data of three independent experiments are shown. (I) Cytokine production of indicated T cell clones upon antigen recognition of four melanomas and two renal carcinomas. Each dot represents one T cell clone specific for the LRPAP1_21-30 epitope. Representative data of three independent experiments with technical triplicates are shown.