Fig. 4: Validation of MHC-I binding and immunogenic potential of neoantigen peptides from mutated FABPs.

a, Overlay histograms showing MHC-I binding for H-2K^b (left graph) and H-2D^b (right graph)-specific neoantigen peptides. Numbers next to histograms represent GeoMean fluorescence intensity. b, Ex vivo re-stimulation assay to assess the immunogenicity of neoantigen peptides. Scatter plot with bar showing normalized IFN-γ production of splenocytes from mice (n = 3) immunized with neoantigen peptide-pulsed Flt3L BM-DC following restimulation with the corresponding peptide relative to cultures without peptide. The horizontal line represents a fold increase of 1; that is, induction of a peptide-specific CD8 T cell response was undetectable. Data shown are mean ± standard deviation (s.d.) of three biological replicates. c, Immunogenic peptides from b were retested in a separate experiment. Graphs show IFN-γ production of splenocytes from immunized mice after re-stimulation with neoantigen peptides derived from mutated spectrin β2 (left), mutated myosin light chain 2 (88D4a, middle) or mutated myosin 7b (88D12a, right). Data in scatter plots represent five biological replicates. Also shown are the mean ± s.d. P values were analyzed using a ratio paired t test: mut spectrin (P = 0.0001), 88D4a (P = 0.003) and 88D12a (P = 0.0047). d, Tumor growth profile of either MCA205 mutant spectrin-β2 (R₉₁₃→L)^high versus MCA205 spectrin-β2 WT^high implanted into sGSN^KO (n = 9 and n = 10 mice, respectively) and WT (n = 11 and n = 10 mice, respectively) (left) or MCA205 mutant spectrin-β2 (R₉₁₃→L)^low versus MCA205 spectrin-β2 WT^low implanted into sGSN^KO (n = 10 and n = 10 mice, respectively) and WT (n = 11 and n = 12 mice, respectively) (right). Data are from one experiment. e, Tumor growth profile of MCA205 mutant spectrin-β2 (R₉₁₃→L)^low implanted into sGSN^KO (n = 16), sGSN^KO DNGR-1^KO (n = 19) mice, and WT (n = 17) mice. Data are representative of two independent experiments. Mean tumor volumes ± s.e.m. in d,e were compared using the Bonferroni-corrected two-way ANOVA. Where indicated, ****P < 0.0001, **P = 0.0055, ns, not significant.

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