Notch ^MUT , a long neoantigen, is an endogenous complete T cell antigen of MC38. (A) The DNA sequence of WT Notch2 (left). The red arrow indicates the region deleted by the mutation in MC38. DNA sequences of mutated Notch2 in MC38 cells were obtained through WES (right). (B) The sequence of the MC38 Notch2 cDNA. (C) Tumor volumes in mice treated with Bpmel-EGFP or Bpmel-Notch2^MUT (n = 5 per group). (D) MC38 tumor volumes in naïve or Bpmel-Notch2^MUT–rejected mice (n = 5 per group). (E) Percentage of IFN-γ–releasing CD4⁺ or CD8⁺ T cells after zsGreen-HisTag or Notch2^MUT-HisTag stimulation for 16 h (n = 4 mice per group). CD3⁺ CD4⁻ T cells were identified as CD8⁺ T cells. (F) MHC II peptide-binding map of Notch2^MUT. The binding affinity of Notch2^MUT peptides spanning the Notch2^MUT sequence was predicted in silico and expressed as IC50 to I-Ab using hmMHC. The candidate peptide used for verification is marked in yellow. (G) Sorted CD4⁺ T cells from the DLN of Bpmel-Notch2^MUT tumor-bearing mice were co-cultured with DCs isolated from naïve mice and stimulated with the indicated peptides for 24 h. The left panel shows the number of IFN-γ–producing spot-forming cells (SFC) following peptide stimulation (n = 3 per group). The right panel displays three representative wells from the IFN-γ ELISPOT assay. All data are representative of three similar experiments. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison.