Fig. 1: Definition of the precise neoepitope of Ccdc85c^MUT that mediates tumor rejection.

a The sequences of the 18-mer wild type and mutant peptides derived from Ccdc85c gene as well as their corresponding allelic fractions (the number of mutant/normal reads divided by the total number of reads (coverage) at a specific genomic position) are shown. b The top panel shows a schematic diagram of immunization and tumor challenge in BALB/cJ mice. The bottom panel (left) shows tumor growth in BALB/cJ mice immunized with Ccdc85c^MUT or Ccdc85c^WT and challenged with Meth A as described in Methods. Each line represents tumor growth in a single mouse (n = 5 mice per group). AUC for each group is plotted in the panel on the right. Data are presented as mean ± SD. P values were calculated using 1-way ANOVA test adjusted for multiple comparisons (Tukey’s multiple comparison test). c Several truncated versions of the 18-mer Ccdc85c^MUT peptide were tested in tumor rejection assay. BALB/cJ mice were immunized and tumor challenged. Each line represents tumor growth in a single mouse. Although mice were immunized with individual peptides, the data for multiple peptides are grouped into one with the composition of the peptides shown on the right. The tumor rejection data for individual peptides are shown in Supplementary Fig. 1. Tumor rejection score (TRS) for each neoepitope is shown in the yellow box, where five represents a complete tumor protection and zero means no tumor rejection. d On the left panel, total Area Under the Curve (AUC) scores for each group in B are plotted. Each bar shows the average total AUC score for the indicated group (TRS = 5; n = 35, TRS = 4–4.5; n = 40, TRS = 3–3.3; n = 25, TRS = 2; n = 30, TRS = 0.1–1.5; n = 60). Error bars represent standard deviation (SD). The P values corresponding to the comparison of TRS = 0 with TRSs 5.0, 4.0–4.5 and 3.0–3.3 were respectively <0.0001, <0.0001 and 0.0002. P values were calculated using 1-way ANOVA test adjusted for multiple comparisons. On the right, peptides with the highest and the lowest TRS are shown. e Targeted MS-based detection of TYIRPFETKVK and YIRPFETKVK among MHC I peptides eluted from BMDCs pulsed with the 18-mer Ccdc85c^MUT. Heavy labeled synthetic peptides were spiked into the peptide samples; the labeled amino acid is marked with a bold character and the mutation is in red. Matched peak lists for the “heavy” and “light” ions were extracted and monitored, while only single charge y ions were plotted. See Methods for details. f Predicted (by NetMHC4.0) and measured IC50 values of the binding of candidate precise neoepitopes of Ccdc85c^MUT to K^d are shown. The candidate neoepitopes include those defined by tumor rejection and MS as in panels c and e. The other three candidate neoepitopes were predicted by NetMHC4.0 alone and were not active in tumor rejection. The affinities of the MS/TRS predicted neoepitopes were also measured for D^d and L^d; measured affinities were below the level of detection.