Fig. 5. Microbial neoantigen vectors restructure the tumour immune microenvironment and suppress established metastatic melanoma.

a–e, Mice with orthotopic B16F10 received an intravenous treatment on day 9 and 12 post-engraftment. a, Left, experimental schematic. Middle, the frequency of IFNγ⁺Foxp3⁻CD4⁺ post-stimulation (*P = 0.0335, **P = 0.0040; NS, P > 0.05). Right, the frequency of IFNγ⁺CD8⁺ T cells (****P < 0.0001; NS, P > 0.05). b, Left, the frequency of Granzyme-B⁺Foxp3⁻CD4⁺ (**P = 0.0024, **P = 0.0041; NS, P > 0.05). Right, the frequency of Granzyme-B⁺ CD8⁺ T cells (*P = 0.0495, **P = 0.0014; NS, P > 0.05). c, Frequency of Granzyme-B⁺NK1.1⁺ NK cells (****P < 0.0001; NS, P > 0.05). d, Left, the median fluorescence intensity (MFI) of TIM-1 on CD19⁺ B cells (*P = 0.0457, **P = 0.0029, ****P < 0.0001). Right, the frequency of TIM-1⁺CD19⁺ B cells of CD45⁺ cells (*P = 0.0442; NS, P > 0.05). e, Left, the frequency of Foxp3⁺CD4⁺ T cells of CD4⁺ cells (**P = 0.0035, **P = 0.0038). Right, the frequency of MHCII⁻Ly6c⁺ MDSCs of CD45⁺ cells (*P = 0.0440; NS, P > 0.05). f–h, C57BL/6 mice (n = 5 mice per group) with 2-day established B16F10-Luc systemic metastases. Intravenous treatment every 3–5 days. f, Representative systemic metastases luminescence. g, Mean total flux from systemic metastases (***P > 0.0003; NS, P > 0.05, two-way ANOVA with Dunnett’s multiple comparisons test). h, Kaplan–Meier survival curve for mice with B16F10-Luc systemic metastases (**P = 0.0015, log-rank Mantel–Cox test). a–e, One-way ANOVA with a–c, Tukey’s, d, left, Tukey’s or right, Dunnett’s and e, Dunnett’s multiple comparisons test. a–e, n = 8 mice for nAg⁴², n = 7 for other groups. a–e,g, Data are mean ± s.e.m.

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