CTA regulates antitumor immunity through phenotypic changes in T cells. (A) Predicted affinity of Notch2MUT-derived potent epitopes for H2-Kb and H2-Db (left) and for I-Ab (right). The NetMHCpan algorithm and the HMM-based binding predictor hmMHC were used for analysis. (B) The experimental procedure for ex vivo antigen stimulation. 13 days after Bpmel-Notch2MUT injection, DLN cells were stimulated with zsGreen-HisTag or Notch2MUT-HisTag protein that was purified from MC38 cells overexpressing zsGreen-HisTag or Notch2MUT-HisTag. (C) All frameshift mutations in Bpmel cells were identified using WES. Sequencing results are listed in Table S5. (D) DLNs were isolated from Bpmel tumor–bearing mice on day 13. Cells were stimulated for 6 h with the two predicted MHC I epitopes of USP49, and IFN-γ release was detected (right panels). (E) Translated non-self peptide after frameshift 1 knock-in or frameshift 2 knock-in on MagD1 gene locus. (F) SIY-specific CD8⁺ T cells were detected using the SIY tetramer 1 wk after electrotransfection; the β-galactosidase tetramer was used as a negative control. (G and H) FCM analysis of CX3CR1 and CD27 expression in CD8⁺ or CD4⁺ T cells in DLNs of MC38 tumor-bearing mice 15 days after tumor inoculation (n = 5 per group). (I) Tumor volumes in MC38-bearing mice electrotransfected with Bpmel-β-actin-OVAII, pCAGGS-SIY, pCAGGS-OVAII, or a mixture of pCAGGS-SIY and pCAGGS-OVAII, or pCAGGS-SIY-OVAII. Statistical analysis was performed using one-way ANOVA followed by Dunnett’s multiple comparison. All data, except for WES data, are representative of triplicate experiments.