7b). We then sought to test the antitumour efficacy of our therapeu - tics against advanced B16F10 tumours. When established orthotopic tumours were injected with microbial therapeutics intratumourally, tumours grew progressively after treatment with EcNcΔlon /ΔompT /LLO+ OVA, whereas treatment with the equal-parts combination of all seven construct-expressing strains—encompassing 42 unique B16F10 neo - antigens (nAg42)—significantly repressed growth over the same time course (Fig. 4b). Similarly, intravenous treatment with EcNcΔlon /ΔompT /LLO+ nAg42 potently restrained orthotopic tumour growth, with 72% of nAg42-treated mice alive 50 days post-tumour engraftment, whereas all control group mice succumbed to malignancy by day 24 or 30 (Fig. 4c,d and Extended Data Fig. 7c). Treatment with intravenous microbial vac - cines again induced no significant body weight change compared to PBS-treated mice (Extended Data Fig. 7d). To evaluate the tissue biodistribution of the microbial neoantigen vaccines after systemic administration in this setting, we surveyed organs after intravenous injection of EcNcΔlon /ΔompT /LLO+ nAg42. As we observed for BALB/c mice with CT26 tumours, live microbial vectors specifically colonized the B16F10 tumour at high density without detectable presence in any other organs examined (Fig. 4e). To con - firm that the microbial B16F10 neoantigen vaccine generated T cells capable of direct tumour cell killing, we treated tumour-free C57BL/6 mice intravenously with microbial therapeutics and co-incubated puri - fied splenic T cells with B16F10 tumour cells in vitro. Indeed, T cells from mice treated intravenously with EcNcΔlon /ΔompT /LLO+ nAg42 but not EcNcΔlon /ΔompT /LLO+ OVA demonstrated enhanced killing of B16F10 tumour cells (Fig. 4f). These data verify tumour-specific colonization and antigen-specific T cell induction by microbial neoantigen vaccines in ab d f gecTDLNkDa 50- 37- 25-TumourkDa MHCI/IIv 50- MHCIIa37- MHCIa 25- M1 M2 M3 M4 M5 Tumour cell Microbial treatmentAntitumour immunity A im mour cell Immune suppresion APCCD4+ T cellCD8+ T cell Tumour i.v. APCTumour: neoantigen pool NS 0123IFN /uni03B3+ of Foxp3– CD4+ (%)NS 051015IFN /uni03B3+ of CD8+ (%) CD4+ T cells CD8+ T cells NS 020406080100PD-L1+ of CD11b+F4/80+(%) 010203040PD-L1+ of CD11b+Ly6G+(%) PMNs MacrophagesEcNc/uni0394lon//uni0394ompTPBS nAg19NC LLO+ CT26 s.c. i.v. 030405060CD80+ of cDC1 (%)NS cDC1s cells0102030CD86+ of cDC1 (%)NS cDC1s cells 05060708090CD86+ of cDC2 (%) cDC2s cellsNS 05060708090CD80+ of cDC2 (%) cDC2s cellsNSTDLN TDLN h020406080Speci/f_ic lysis (%)CT26-Luc 4T1-Luc24 h4 8 h 5/1 5/1 10/1 10/1 Effector/target ratioTumour 0200400600800No. Foxp3+CD4+ per mg tumour Regulatory T cells05001,0001,500No. MHC-IIloF4/80+ per mg tumour MacrophagesTumourMHCI/IIvMHCIIaMHCIa******* ******* ************** ** ** ** *********** ** **** Fig. 3 | Microbial tumour neoantigen vaccines restructure the tumour immune microenvironment. a, Immunoblot of tumours ( n = 5) and TDLNs (n = 5). b, Left, the percentage CD80+ of cDC1s (* P = 0.0189, * P = 0.0415; NS, P > 0.05). Right, the percentage CD86+ of cDC1s (** P = 0.0061, *** P = 0.0002; NS, P > 0.05). c, Left, the percentage CD80+ of cDC2s (**** P < 0.0001; NS, P > 0.05). Right, the percentage CD86+ of cDC2s (*** P = 0.0001, **** P < 0.0001; NS, P > 0.05). d, Left, the frequency of IFNγ+Foxp3−CD4+ (n = 5 mice per group, * P = 0.0238, *P = 0.0147; NS, P > 0.05). Right, the frequency of IFNγ+CD8+ T cells ( n = 5 mice per group, ** P = 0.0015, **** P < 0.0001; NS, P > 0.05). e, CT26 TILs, specific lysis (n = 12 mice, ** P = 0.0023, ** P = 0.0027, *** P = 0.001, **** P < 0.0001; NS, P > 0.05, Kruskal–Wallis test with Dunn’s multiple comparisons test). f, Left, the percentage PD-L1+ of Ly6G+CD11b+ (n = 8 mice for PBS, n = 9 for other groups, **P = 0.0037, ** P = 0.0059). Right, the percentage PD-L1+ of CD11b+F4/80+ (n = 8 mice for PBS, n = 9 for all other groups, ** P = 0.0010; NS, P > 0.05). PMNs, polymorphonuclear cells. g, Left, the number of Foxp3+CD4+ T cells mg−1 (n = 9 mice per group * P = 0.0131, * P = 0.0241). Right, the number of MHC-IIlo+F4/80+ CD11b+ macrophages per mg ( n = 8 mice for PBS, n = 9 for other groups, *P = 0.0385, * P = 0.0407). h, Immunologic mechanism. b–d,f,g, One-way ANOVA with a–d, Tukey’s, f, Dunnett’s or g, left, Holm–Šídák’s or right, Dunnett’s multiple comparisons test. b,c, n = 9 mice per group. b–g, Data are mean ± s.e.m. Gel source data are in Supplementary Fig. 2. 458 | Nature | Vol 635 | 14 November 2024 ArticleB16F10 melanoma. To assess the dependency of antitumour efficacy on CD4+ and CD8+ T cells in vivo, we depleted either CD4+ or CD8+ T cells from mice treated intravenously with EcNcΔlon/Δ ompT /LLO+ nAg42. We found that depletion of either CD4+ or CD8+ T cells ablated therapeutic effi- cacy, indicating that both conventional CD4+ and cytotoxic CD8+ T cells are required for productive antitumour immunity in vivo (Fig. 4g and Extended Data Fig. 7e). Immunity and metastases in melanoma To characterize the immunologic changes associated with antitumour efficacy in this tumour model, we performed immunophenotyping of orthotopic B16F10 tumours 8 days post-intravenous microbial treatment. Tumours treated intravenously with EcNcΔlon/Δ ompT /LLO+ nAg42 had significantly higher numbers and frequencies of cDC1s and cDC2s, conventional CD4+ and cytotoxic CD8+ T cells, natural killer (NK) cells and inflammatory monocytes (Fig. 4h,i and Extended Data Fig. 7f–j). Analyses of the intratumoural lymphoid compartment showed enhanced expression of CD69 on Foxp3−CD4+ and CD8+ TILs, and significantly increased frequencies of IFNγ secreting conventional Foxp3−CD4+ and cytotoxic CD8+ TILs after restimulation with PMA and ionomycin in EcNcΔlon/Δ ompT /LLO+ nAg42-treated tumours, indicating enhanced T cell activation and effector cytokine production within the TME (Fig. 5a and Extended Data Fig. 8a). Tumour-infiltrating Foxp3−CD4+ and CD8+ T cells and NK cells also expressed significantly higher levels of Granzyme-B after EcNcΔlon /ΔompT /LLO+ nAg42 treatment, suggestive of amplified cytolytic function (Fig. 5b,c ). Consistent with enduring activity of antitumour immunity, we also observed higher levels of proliferating tumour-infiltrating CD4+ and CD8+ T cells and NK cells as assessed by Ki-67 staining (Extended Data Fig. 8b). In addition to the enhanced activation of tumour-infiltrating T and NK cells, treatment with EcNcΔlon/Δ ompT /LLO+ nAg42 significantly reduced TIM-1 expression by tumour-infiltrating CD19+ B cells and thus the fre - quency of regulatory TIM-1+ B cells—an important immunosuppressive cell population in the B16F10 model44—and increased B cell prolifera- tion (Fig. 5d and Extended Data Fig. 8c). Moreover, EcNcΔlon/Δ ompT /LLO+ nAg42 vaccination reduced the frequency of immunosuppressive Foxp3+ regulatory T cells, myeloid-derived suppressor cells (MDSCs) and MHC-IIlo macrophages within tumours (Fig. 5e and Extended Data Fig. 8d). Infiltrating monocytes and dendritic cells in EcNcΔlon/Δ ompT /LLO+ nAg42-treated tumours showed increased expression of MHC-II (Extended Data Fig. 8e), suggestive of enhanced antigen presentation capacity. Overall, these data demonstrate that intravenous microbial tumour neoantigen vaccination mediates immunologic restructuring within the melanoma TME, recruiting APCs and activating NK cells, and CD4+ and CD8+ T cells while diminishing immunosuppressive cell populations. Given the robust antitumour efficacy induced by intravenous vaccina - tion in orthotopic B16F10, we investigated the efficacy of EcNcΔlon /ΔompT /LLO+ nAg42 in established, systemic B16F10-Luc metastases (Extended Data Fig. 8f). Whereas systemic metastases rapidly progressed in PBS or EcNcΔlon/Δ ompT /LLO+ OVA treated mice, EcNcΔlon/Δ ompT /LLO+ nAg42 strongly inhibited metastatic growth (Fig. 5f,g and Extended Data Fig. 8g,h). 75-ab dc 25-kDaIa1 - -Ia2Ia3I/IIv1 DnaK EcNc/uni0394lon//uni0394ompT/LLO +B16F10 Neoantigen selection LonOmpT +LLOMicrobial neoantigen vaccine ef LODEcNc/uni0394lon//uni0394ompT/LLO+ nAg42 TumourTDLNBrainHeartLungsLiverSpleenKidneys Bladder102103104105106107108CFU g–1 B16F10 s.c. i.v. B16F10 s.c. i.v. nAg42EcNc/uni0394lon//uni0394ompT LLO+ OVAPBS 05 10 1505001,0001,5002,000 Time (days post-treatment)Tumour volume (mm3) EcNc/uni0394lon//uni0394ompT LLO+ OVA nAg42PBS *** 01 02 03 04 05 0020406080 Time (days post-engraftment)Survival (%) B16F10 s.c.i.t. 05 10 1505001,0001,5002,000 Time (days post-treatment)nAg42EcNc/uni0394lon//uni0394ompT LLO+ OVATumour volume (mm3) gh i 050100150200No. CD8+ CTLs (per mg tumour)NS CD4+ T cells CD8+ T cellsNo. Foxp3–CD4+ T cells (per mg tumour)NS 020406080100 cDC1s cells05101520No. CD301b+ cDC2 (per mg tumour)NS 0204060No. CD103+XCR1+ cDC1 (per mg tumour)NS cDC2s cellsnAg42EcNc/uni0394lon//uni0394ompT/LLO+ OVA PBSEffector/target ratio 05 10 1505001,0001,5002,000 Time (days post-treatment)nAg42 (iso)LLO+ OVA nAg42 (/uni03B1CD4) nAg42 (/uni03B1CD8) B16F10 s.c. i.v.EcNc/uni0394lon//uni0394ompTTumour volume (mm3) NSNS10/1 20/1 40/1nAg42EcNc/uni0394lon//uni0394ompT LLO+ OVAPBS 020406080Speci/f_ic lysis (%)NS NS NSNSMHC 100 nAg42EcNc/uni0394lon//uni0394ompT/LLO+ OVA PBS **** ******** **** *** * ** **** ** **** ************* Fig. 4 | Microbial antitumour vaccine efficacy in orthotopic melanoma. a, Melanoma therapeutic design. Left, a Circos plot, mutanome B16F10. Right, an immunoblot with neoantigen constructs. b –e,g–i, C57BL/6 mice with orthotopic B16F10 tumours treated starting 9 days post-engraftment. b, Intratumoural treatment every 3–5 days. Tumour growth curves ( n = 7 mice per group, **** P < 0.0001; NS, P > 0.05). c, Intravenous treatment every 3–5 days. Tumour growth curves (**** P < 0.0001; NS, P > 0.05). d, Kaplan– Meier survival curves, mice from c (*** P = 0.0001, log-rank Mantel–Cox test). e, Intravenous treatment, EcNcΔlon /ΔompT /LLO+ nAg42 (n = 6 mice). Microbial CFU g−1, LOD = 1 × 103 CFU g−1. f, Purified splenic T cells, specific lysis B16F10-Luc ( n = 5 mice per group, *** P = 0.001, **** P < 0.0001; NS, P > 0.05). g, Intravenous treatment every 3–5 days. Tumour growth curves ( n = 6 mice for OVA, n = 7 for isotype, n = 8 for other groups, ** P = 0.0082, **** P < 0.0001; NS, P > 0.05). h,i, Intravenous treatment on days 9 and 12. h , Left, the number of CD103+XCR1+ cDC1 per mg (* P = 0.0103, ** P = 0.0030; NS, P > 0.05). Right, the number of CD301b+ cDC2 per mg (** P = 0.0038, ** P = 0.0064; NS, P > 0.05). i, Left, the number of Foxp3−CD4+ T cells per mg (** P = 0.0015, *** P = 0.0008; NS, P > 0.05). Right, the number of CD8+ T cells mg−1 (** P = 0.0022, ** P = 0.0047; NS, P > 0.05). b,c,g, two-way ANOVA, or f ,h,i, one-way ANOVA, with b , Šídák’s or c ,f–i, Tukey’s multiple comparisons test. h ,i, n = 8 mice for nAg42, n = 7 for other groups. c,d, n = 5 mice for PBS, n = 7 for other groups. b,c,e–i, Data are mean ± s.e.m. Gel source data are in Supplementary Fig.