Extended Data Fig. 6: Modulation of anti-tumor immunity by engineered microbial tumor neoantigen vaccines.

a–f,h–i, BALB/c mice with established hind-flank CT26 tumors received intravenous injections of indicated therapeutic or control. a, 2 days or b–g, 8 days after treatment, tumors and TDLNs were extracted. a, Frequency of cDC2 in TDLNs (n = 9 mice per group, **P = 0.0042, **P = 0.0099, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). b–c, Lymphocytes from TDLNs were stimulated ex vivo with PMA and ionomycin in the presence of brefeldin A. b, Left: Frequency of IFN-γ⁺ Foxp3⁻CD4⁺ post-stimulation (n = 3 mice per group, *P = 0.0313, *P = 0.0246, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of TNF-α⁺ Foxp3⁻CD4⁺ T cells post-stimulation (n = 3 mice per group, *P = 0.0445, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). c, Left: Frequency of IFN-γ⁺ CD8⁺ post-stimulation (n = 3 mice per group, *P = 0.0257, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). Right: Frequency of TNF-α⁺ CD8⁺ T cells post-stimulation (n = 3 mice per group, **P = 0.0017, ***P = 0.0008, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). d, TILs were stimulated with individual 29-mer neoepitope-containing long peptides. Number IFN-γ spots (n = 8 mice per group, *P = 0.0439, *P = 0.0364, *P = 0.0281, *P = 0.0200, Kruskal-Wallis test with Dunn’s multiple comparisons test). Data are mean ± s.e.m. of background (medium control) subtracted responses. e,f, TILs were stimulated ex vivo with PMA and ionomycin in the presence of brefeldin A. e, Left: Frequency of IFN-γ⁺ Foxp3⁻CD4⁺ post-stimulation (n = 9 mice per group, *P = 0.0461, **P = 0.0014, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test). Right: frequency of IFN-γ⁺ CD8⁺ T cells post-stimulation (n = 9 mice per group, *P = 0.0486, **P = 0.0040, ****P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test). f, Frequency of IFN-γ⁺ B220⁺ B cells post-stimulation (n = 9 mice per group, *P = 0.0351, **P = 0.0010, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparisons test). g, Left: Percentage Ki-67⁺ of Foxp3⁻CD4⁺ T cells in tumors (n = 9 mice per group, *P = 0.0183, ***P = 0.0008, one-way ANOVA with Dunnett’s multiple comparisons test). Right: Percentage Ki-67⁺ of CD8⁺ T cells in tumors (n = 9 mice per group, *P = 0.0453, ns = P > 0.05, One-way ANOVA with Dunnett’s multiple comparisons test). h, Naïve, tumor-free BALB/c mice were vaccinated intravenously with the designated treatment. CT26 was engrafted on a single hind-flank after the final vaccination. Tumor growth curves (n = 8 mice per group, ****P < 0.0001, two-way ANOVA with Šídák’s multiple comparisons test). i, BALB/c mice with established CT26 tumors were treated intravenously with EcNc^{Δlon/ΔompT/LLO+} nAg¹⁹ (as in Fig. 2e). Mice that had cleared tumors, and age-matched naïve mice, were subcutaneously rechallenged with CT26 tumor cells. Tumor growth curves (n = 5 for naïve mice, 6 for nAg¹⁹). j, Left: Frequency of FoxP3⁺CD4⁺ regulatory T cells in tumors (n = 9 mice per group, *P = 0.0491, **P = 0.0072, one-way ANOVA with Holm-Šídák’s multiple comparisons test), Right: Frequency of MHCII^loF4/80⁺CD11b⁺ macrophages in tumors (n = 8 mice for PBS, 9 for other groups, *P = 0.0173, **P = 0.0057, one-way ANOVA with Dunnett’s multiple comparisons test). k, Left: Percentage PD-L1⁺ of cDC1 in TDLN (n = 5 mice per group, **P = 0.0074, ns = P > 0.05, one-way ANOVA with Dunnett’s multiple comparisons test), Right: Percentage PD-L1⁺ of cDC2 in TDLN (n = 5 mice per group, *P = 0.0103, *P = 0.0244, one-way ANOVA with Dunnett’s multiple comparisons test). a–k, Data are mean ± s.e.m.

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