Figure 6.

The RNF10 uPeptide generates an immunostimulatory epitope recognized by human autologous T cells

(A) CD8⁺ T cells were primed by autologous DCs loaded with uPeptide antigens. The CTLs were restimulated with peptides overnight. The uPeptide-specific T cell response was evaluated using an IFN-γ ELISpot. Representative ELISpot images are shown; spot count per 5 × 10⁴ T cells is presented. (B) The primed T cells were restimulated with uPeptide antigens overnight and evaluated by flow cytometry; representative flow cytometry plots of intracellular cytokine staining are shown. Percentages indicate proportions of CD8⁺ T cells producing different cytokines (mean ± SD, n = 3). (C) CD8⁺ T cells were primed by autologous DCs loaded with uPeptide antigens. The primed T cells were then stained with the uRNF10 RLFGQQQRA-HLA-A∗02:01 tetramer. Numbers in plots denote percentages of the tetramer⁺CD8⁺ T cells (mean ± SD, n = 3). (D) The primed T cells were co-cultured with MIA PaCa-2 cells for 18 h and their cytotoxicity against MIA PaCa-2 cells was assessed by lactate dehydrogenase (LDH) assay (mean ± SD, n = 3). (E) The T cells were co-cultured with CellTrace CMFDA-labeled MIA PaCa-2 cells for 18 h. Tumor cell death was examined by PI uptake via flow cytometry. Percentages of dead tumor cells are shown (mean ± SD, n = 3). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, compared with untreated T cells (−). p-values were determined by two-tailed one-way ANOVA with Dunnett’s multiple-comparisons test.