Fig. 5.

Spectral validation and immunogenicity analyses of uORF-derived tumor antigens. a–c Validation of the experimentally eluted peptides a uP_{ATF5_A*02}, b uP_{MAPK1_A*03}, and c uP_{TMEM203_B*07/C*16}. Comparison of fragment spectra (m/z on x-axis) of HLA uLigands eluted from primary samples (identification) to their corresponding isotope-labeled synthetic peptides (validation, mirrored on the x-axis) with the calculated spectral correlation coefficient (R²). Identified b- and y-ions are marked in red and blue, respectively. Ions containing isotope-labeled amino acids are marked with asterisks. d–f Naïve CD8⁺ T cells were primed in vitro using HLA uLigand-loaded aAPCs with the HLA-A*02-, -A*03-, and -B*07-restricted peptides d uP_{ATF5_A*02}, e uP_{MAPK1_A*03}, and f uP_{TMEM203_B*07/C*16}, respectively. Graphs show single, viable cells stained for CD8 and PE-conjugated multimers of indicated specificity. The left panels show HLA uLigand tetramer staining, the right panels (negative control) depict tetramer staining of T cells from the same donor primed with an HLA-matched control peptide. g–i Functional characterization of HLA uLigand-specific CD8⁺ T cells by intracellular cytokine staining. Representative examples of IFN-γ and TNF production (upper panels) as well as CD107a expression (lower panels) after stimulation of g uP_{ATF5_A*02}-, h uP_{MAPK1_A*03}-, and i uP_{TMEM203_B*07/C*16}-specific CD8⁺ T cells with the HLA-A*02-, -A*03-, and -B*07-restricted peptides uP_{ATF5_A*02}, uP_{MAPK1_A*03}, and uP_{TMEM203_B*07/C*16}, respectively (left panels) compared to a negative HLA-matched control peptide (right panels)