Mice were vaccinated twice with adjuvant alone (Control), with HA515, or with the 3 immunogenic neoepitopes (DHX58, CAND1, and ADGRF5-II; Neo-vax). (A–C) One week after the last vaccination, mice were injected i.v. with CFSE-labeled target cells: CFSE^hi cells were pulsed with indicated peptides, whereas CFSE^lo cells were unpulsed. Lymph nodes and spleen were harvested 24 hours later, and pulsed and unpulsed target cells were quantified by flow cytometry after gating on viable CD19⁺ CFSE⁺ cells. (A) Percentage of cytotoxicity toward HA515-paulsed target cells (n = 3 mice per group), calculated as indicated in Methods. *P < 0.05, with unpaired 2-tailed Welch’s t test. (B) Representative flow cytometry plots showing the killing of target cells in lymph nodes. (C) Percentage of cytotoxicity toward neoepitope-pulsed target cells determined in the lymph nodes and spleen (n = 3–7 mice per group). *P < 0.05, **P < 0.01, with Kruskal-Wallis and Dunn’s multiple-comparison tests. Data are pooled from 2 independent experiments. (D) Vaccine-draining lymph node cells were co-cultured with CFSE^hi target cells pulsed with indicated neoepitopes, whereas CFSE^lo cells were pulsed with WT peptides. For HA515 peptide, CFSE^lo cells were unpulsed. MHC-I–blocking antibody was added as indicated. After incubation for 16–18 hours, cells were harvested for flow cytometry quantification of viable CD19⁺CFSE⁺ target cells, and the percentage of cytotoxicity toward mutated versus WT peptide-loaded targets was calculated. *P < 0.05, with unpaired 2-tailed Welch’s t test. Data are representative of 2 independent experiments. (E–G) In vitro killing of 4T1 target cells was tested as described in the schema (E). (F) Representative flow plots after gating on viable CFSE⁺ cells. (G) Percentage of cytotoxicity toward irradiated cells versus untreated cells, calculated as described in Methods. **P < 0.01, with unpaired 2-tailed Welch’s t test. All data are expressed as mean ± SEM.