Identification of neoantigens of Bpmel. (A) Distribution of exome mutations at the chromosomal level in Bpmel. Colors indicate the number of mutations. This WES analysis identified 14,035 exomic mutations. (B) Workflow for the identification of Bpmel neoantigens. The numbers of peptide candidates screened at each step are shown. (C) Mutation types of Bpmel, as identified through WES analysis. At the RNA level, 328 missense variants were identified in 4,934 genes. (D) The 328 epitopes generated by missense mutation were screened based on their predicted affinities for H2-Kb and H2-Db. The top 1% of the affinity ranking was selected as high-affinity peptides (left and center). Candidates were further selected based on the ratio of affinity before and after mutation and on fragments per kb of transcript per million mapped reads (FPKM) (right). This screening identified 44 candidates (detailed sequences are listed in Table S3). (E) The 44 selected peptides were synthesized for the in vitro T cell screening assay. CD8⁺ T cells were isolated from the DLNs of Bpmel-bearing mice and stimulated with the indicated peptides (5 µg/ml) for 8 h. The percentage of IFN-γ–releasing CD8⁺ T cells was evaluated using IFN-γ–catching assays. Four WT and mutant peptide sequences that induced IFN-γ secretion are shown (center). (F) DLN single cells were stimulated with the indicated peptides (5 µg/ml) for 8 h. IFN-γ secretion was detected using the ELISpot assay. OVAI was used as a negative control. Dot numbers are shown. All four candidates except AZIN1^MUT promoted CD8⁺ T cell release of IFN-γ. (G) Control tetramers (Tet-ctrl), Scap^MUT, and ARAF^MUT tetramers (the AIFM^MUT MHC tetramer could not be constructed) were used to stain CD8⁺ TILs. Here, 0.17–3.03% of ARAF^MUT-specific CD8⁺ T cells were detected in the CD8⁺ TILs of all Bpmel-bearing mice (n = 5 per group). Representative images are shown.