Tubes were inverted and allowed to dry for 6 h. Biofilms left on borosilicate tubes were stained with 0.1% (vol/vol) crystal violet for 15 min. Crystal violet stain was discarded and tubes washed three times with PBS, then inverted and allowed to dry overnight. Crystal violet-stained biofilms were dissolved with 95% ethanol and transferred to 96-well plates for measurement of absorbance at 590 nm. Phagocytosis assay Bacterial phagocytosis assays were adapted from previous work74. Culture and isolation of murine BMDMs was performed as described previously75. Bulk femoral bone marrow cells from BALB/c or C57BL/6 mice were cultured on 15 cm non-treated cell culture Petri dishes in RPMI with 20% FBS, 25 ng ml−1 M-CSF (R&D Systems) and 100 U ml−1 penicillin–streptomycin. Media was replaced with fresh media after 4 days of culture. After 7 days of culture, plates were washed with PBS and adherent macrophages were dissociated using trypsin-EDTA. Macrophages were washed in PBS, resuspended at a density of 2 × 105 cells per ml in media and 1 ml transferred to each well of 24-well plates. The 24-well plates were incubated overnight in a 37 °C incubator with humidified 5% CO2. EcN wild-type or EcNcΔlon/ΔompT, with or without a constitutive green-fluorescent protein (GFP)-expressing plasmid were cultured overnight in LB media with appropriate antibiotics. Bacterial cultures were centrifuged at 3,000g for 10 min, washed three times with sterile PBS and resuspended at a density of 4 × 108 bacteria per ml in sterile PBS. Media from wells containing adherent macrophages was aspirated, wells washed three times with PBS and 1 ml of RPMI with 5% mouse serum added to each well. Latrunculin A was added at a concen - tration of 1 μM to selected wells to inhibit phagocytosis. Next, 2 × 107 CFU of microbes were added to each well with each condition tested in triplicate. Microbial strains were incubated with BMDMs for 30 min in a 37 °C incubator at 20 rpm. After 30 min, media was aspirated and wells were washed six times with sterile ice-cold PBS. Adherent macrophages were dissociated using non-enzymatic cell dissociation buffer (Gibco), resuspended in fluorescence-activated cell sorting (FACS) buffer (PBS containing 2% FBS, 2 mM EDTA and 0.09% sodium azide) and analysed by flow cytometry. In vitro BMDM activation BMDMs were cultured as described above for phagocytosis assays. BMDMs were washed in PBS, resuspended at a density of 2 × 105 ml−1 in media and 1 ml transferred to each well of 24-well plates. The 24-well plates were incubated overnight in a 37 °C incubator with humidified 5% CO2. Wild-type EcN or EcNcΔlon/ΔompT with constitutive expression of OVA from a pUC origin plasmid were cultured overnight in LB media with appropriate antibiotics. Cultures were centrifuged at 3,000g for 10 min, washed three times with PBS and resuspended at a density of 4 × 108 bacteria ml−1 in sterile PBS. Media from wells containing macrophages was aspirated, wells were washed three times with PBS and 1 ml of RPMI with 5% mouse serum was added to each well. Next, 1 × 107 live microbes were added to each well, with each condition replicated in triplicate. Live microbial strains were incubated with BMDMs for 6 h in a 37 °C incuba - tor. After 6 h, media was aspirated and wells were washed six times with sterile ice-cold PBS. Adherent macrophages were dissociated using non-enzymatic cell dissociation buffer (Gibco), resuspended in FACS buffer and analysed by flow cytometry. DRAQ7 cell viability reagent was used to exclude dead cells (diluted 1:1,000 in FACS buffer). Extracellular antibodies for BMDM activation panel included CD80 (catalogue no. 16-10A1, Biolegend), MHC-II (catalogue no. M5/114.15.2, Biolegend), PD-L1 (catalogue no. 10F .9G2, Biolegend) and H2Kb-SIINFEKL (catalogue no. 25-D1.16, Biolegend), each used at 1:200 dilution.In vitro BMDC stimulation BMDC isolation and culture from mouse bone marrow was adapted from previous methods76. BMDCs from C57BL/6 mice were cultured on 15 cm non-treated cell culture Petri dishes in RPMI with 20% FBS, 20 ng ml−1 GM-CSF (Biolegend) and 100 U ml−1 penicillin–streptomycin. Every 1–2 days for the first 4 days, plates were gently washed and non-adherent granulocytes removed by aspirating 50% of the culture media with subsequent replacement of fresh media. On day 4, media was aspirated completely and replaced with fresh culture media with 20 ng ml−1 GM-CSF . On day 6, BMDC plates were washed with PBS and loosely adherent and non-adherent cells collected. Cells were cen - trifuged at 300g for 5 min, resuspended in fresh culture media and replated on 15 cm non-treated cell culture Petri dishes. On days 7–8, plates were washed with PBS and loosely adherent and non-adherent cells were collected. Cells were centrifuged at 300g for 5 min, resus - pended in fresh culture media at a density of 2.5 × 105 ml−1 and 200 μl was transferred to 96-well plates and incubated overnight in a 37 °C incubator. The next day, media from wells containing BMDCs was aspi - rated, and 1 ml of RPMI with 5% mouse serum was added to each well. BMDCs were pulsed with live bacteria at an multiplicity of infection of 10 for 2 h. Plates were centrifuged at 300g for 5 min, media aspirated and replaced with fresh RPMI with 10% FBS, 10 μg ml−1 gentamicin and 100 U ml−1 penicillin–streptomycin. Gentamicin concentration was increased to 40 μg ml−1 after 2–4 h. Plates were incubated for 5–48 h in a 37 °C incubator, at which time the supernatant was assessed for IL-12p70 using the Mouse IL-12p70 Quantikine ELISA Kit (R&D systems) according to the manufacturer’s instructions. OT-I and OT-II T cell stimulation and proliferation BMDCs were cultured as above, resuspended at a density of 2.5 × 105 ml−1 and 5 × 104 BMDCs transferred to 96-well plates and incubated over - night in a 37 °C incubator. The next day, media from wells containing BMDCs was aspirated, and 1 ml of RPMI with 5% mouse serum was added to each well. BMDCs were pulsed with 2 × 106 CFU of the respective bac - terial strain for 2.5 h, plates were centrifuged at 300g for 5 min, media aspirated and replaced with fresh RPMI with 10% FBS and 10 μg ml−1 gentamicin and 100 U ml−1 penicillin–streptomycin. Gentamicin con- centration was increased to 40 μg ml−1 after 2–4 h. Spleens from naive OT-I and OT-II mice were extracted, filtered through 100 μm cell strain - ers and washed in complete RPMI (RPMI-1640 supplemented with 10% (vol/vol) FBS, 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution (Gibco-11140050) and 100 U ml−1 penicillin–streptomycin). OT-I and OT-II T cells were isolated from single-cell suspensions of spleens from the respective transgenic mouse using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified OT-I and OT-II T cells were resus - pended in T cell media (complete RPMI supplemented with 50 μM β-mercaptoethanol) at a density of 5 × 105 ml−1 and 5 × 104 T cells incu- bated with 5 × 104 BMDCs pulsed with the respective microbial strains. For cytokine secretion assessment, T cells were incubated with BMDCs for 24 h, at which time supernatant was assessed for IFNγ and IL-2 using Mouse IFNgamma Quantikine ELISA Kit and Mouse IL-2 Quantikine ELISA Kit (R&D systems) according to the manufacturer’s instructions. Carboxyfluorescein succinimidyl ester (CFSE) proliferation assays were conducted as previously described77. Here, 1 × 107 OT-I or OT-II T cells were resuspended in 1 ml of room temperature PBS, and 1 μl of 5 mM CFSE (Biolegend) was added. T cells were incubated in CFSE solu - tion for 5 min at room temperature protected from light, after which time staining was quenched by adding ten times the staining volume of cell culture media. T cells were centrifuged at 300 g for 5 min, resus - pended in T cell media at a density of 5 × 105 ml−1 and incubated for an extra 10 min at room temperature. Then 5 × 104 T cells were incubated with 5 × 104 BMDCs pulsed with the respective live microbial strains. At 48 h, 50% of the media from each well was gently aspirated so as to not disturb any cells, and replaced with fresh T cell media. At 72–96 h, OT-I and OT-II T cells were collected and analysed by flow cytometry. DRAQ7 cell viability reagent was used to exclude dead cells (diluted 1:1,000 in FACS buffer). Extracellular antibody staining for CFSE assays included antimouse CD3 (catalogue no. 17A2, Biolegend), used at 1:200 dilution. Listeriolysin haemolytic activity assay Sheep red blood cell (RBC) lysis by bacterial lysate was performed as described previously78. Briefly, bacteria were grown overnight in fresh LB containing appropriate antibiotics. Cultures were centrifuged at 3,000 g for 10 min, supernatants discarded and the cell pellet resus - pended to OD600 = 8 in 0.1% (w/w) bovine serum albumin (BSA) in sterile PBS titrated to pH of 5.25 with 1 M HCl. Bacteria were sonicated for 2 min. After sonication, the soluble fraction was isolated by centrifugation at 10,000 g at 4 °C for 20 min. Sheep RBCs were washed three times with PBS and resuspended at a final concentration of 6 × 108 ml−1 in 0.1% (w/w) BSA in PBS titrated to pH of 5.25. Equal parts of bacterial lysate soluble fraction and sheep RBC suspension were mixed and incubated for 15 min at 37 °C. After incubation RBC mixtures were centrifuged at 1,000g for 1 min at 4 °C and supernatant absorbance at 541 nm was then measured to quantify RBC lysis. Listeriolysin cytosolic access assay BMDMs were cultured as described above for phagocytosis assays. BMDMs were washed in PBS, resuspended at a density of 2.5 × 105 ml−1 in media, 100 ml transferred to wells of an eight-well Lab-Tek Chamber Slide system (ThermoFisher) and incubated overnight in a 37 °C incuba - tor. The next day, media from wells containing BMDMs was aspirated, and 1 ml of complete RPMI without antibiotics was added to each well. BMDMs were then pulsed with 1.25 × 106 CFU of the respective bacte- rial strain for 60 min. After the designated time media from each well was aspirated, wells were washed four times with PBS and media was replaced with fresh RPMI with 10% FBS and 40 μg ml−1 gentamicin and incubated in a stationary 37 °C incubator. After either 30 or 60 min of more incubation, media was aspirated and wells washed four times with ice-cold PBS. Then 100 ml of 100% methanol at −80 °C was then added to each well for fixation and allowed to incubate at room temperature for 10 min. Methanol was then removed, 100 ml of ice-cold PBS added to each well and slides incubated at 4 °C. Cells were permeabilized with 0.5% Triton X in PBS for 10 min. Blocking solution in 10% heat-inactivated horse serum and 3% BSA was added to each well for 30 min. After wash - ing three times, primary antibodies in 1% heat-inactivated horse serum and 1% BSA were incubated overnight at 4 °C in a humidified chamber. The next day, slides were washed with PBS three times for 10 min each and secondary antibodies were applied for 1 h at room temperature in the dark. DAPI (4′6-diamidino-2-phenylindole) was applied as part of the secondary antibody cocktail for nuclear staining. Slides were washed in PBS three times before mounting coverslips with DAKO gel and stored at 4 °C until immunofluorescence analysis. Anti-ovalbumin (catalogue no. EPR27117-90, Abcam) and anti-CD11b (catalogue no. M1/70, Abcam) primary antibodies were used for staining, both at 1:200 dilution. Animal experiments All animal experiments were approved by the Institutional Animal Care and Use Committee (Columbia University, protocol AABQ5551). The 6–7-week-old female BALB/c, C57BL/6 and B6(Cg)-Tyrc-2J/J ( Jackson Laboratories) mice were kept in accordance with all rules for animal research at Columbia University. Mice were housed in a facility with a 12 h light–dark cycle, and provided unrestricted access to both food and water. The housing facility was maintained at 21–24 °C, and kept at 40–60% humidity. Sample size was determined on the basis of our previous studies and/or pilot experiments. For subcutaneous tumour models: 5 × 106 CT26 cells in 100 μl of sterile PBS were inoculated sub - cutaneously on the hind flank of BALB/c mice, or 5 × 105 B16F10 mela- noma cells in 100 μl of sterile PBS subcutaneously on the hind flank (orthotopic) of C57BL/6 mice using a 26G needle on a 1 cc syringe. CT26 tumours were allowed to establish as indicated for each experiment, and mice were distributed between groups to equate the average start - ing tumour volume before treatment. B16F10 orthotopic tumours were allowed to establish for 9 days, and initial average tumour vol - ume equated between groups before treatment. Tumour dimensions were measured unblinded with a calliper every 1–3 days for calculating tumour volumes using the equation (a2 × b)/2 (a is width, b is length, where width is the smaller dimension). Group tumour sizes were com- puted as mean ± s.e.m. Body weight was measured each time tumour measurements were taken. Animals were euthanized when any of the following criteria were met: tumour burden greater than 2 cm in the largest dimension for any subcutaneous tumour, greater than 20% body weight loss, as otherwise recommended by veterinary staff or when showing clinical signs of impaired health. To examine the requirement of individual T cell populations for the efficacy of the microbial neo - antigen vaccines, mice were injected intraperitoneally with 200 μg (in 100 μl of InVivoPure pH 6.5 buffer, BioXcell) of antimouse CD4 (clone GK1.5, BioXcell), 200 μg (in 100 μl of InVivoPure pH 7.0 buffer, BioX - cell) of antimouse CD8β (clone Ly-3.2, BioXcell) or 200 μg (in 100 μl of InVivoPure pH 7.0 buffer, BioXcell) of IgG1 isotype control (clone HRPN, BioXcell) beginning 2 days before the initiation of therapeutic treatment and every 2–3 days thereafter until study endpoint. In prophylactic vaccination studies, BALB/c mice received an intrave - nous injection of either EcNcΔlon/Δ ompT /LLO+ OVA or EcNcΔlon/Δ ompT /LLO+ nAg19 every 3–5 days for a total of four injections. Four days after the final injection, 1 × 106 CT26 cells in 100 μl of sterile PBS were inoculated sub - cutaneously on the hind flank. In rechallenge studies, BALB/c mice that had cleared subcutaneous CT26 tumours on a single hind flank were engrafted with 1 × 106 CT26 cells on the opposite hind flank 100 days after tumour clearance. Age-matched naive BALB/c mice were engrafted with 1 × 106 of the same CT26 cells on a single hind flank as controls. For therapeutic studies in systemic metastases models, 5 × 105 CT26-Luc cells or 1.5 × 105 B16F10-Luc cells were injected in 100 μl of sterile PBS through the lateral tail vein with a 27G needle on 1 cc syringe. Metastases were allowed to establish for 4 days in Balb/C mice before treatment for CT26-Luc, and for 2 days in C57BL/6 albino mice (B6(Cg)-Tyrc-2J/J) for B16F10-Luc. Mice were randomly distributed between groups after metastases engraftment and before treatment. For in vivo luminescence tracking of metastases burden, mice were injected intraperitoneally with 125 μl of aqueous solution of d -Luciferin (50 mg ml−1) 6 min before imaging, and placed under isoflurane anaes - thesia for imaging using an in vivo imaging system (IVIS), with expo - sure time set to 6 min. Total flux from the lungs (CT26-Luc) or body (B16F10-Luc) was used to quantify tumour burden. For evaluation of lung metastases burden at the timepoint of treatment initiation, mice were injected intraperitoneally with 250 μl of aqueous solution of d-Luciferin (50 mg ml−1) 6 min before imaging and placed under isoflurane anaesthesia for imaging using an IVIS with exposure time set to 10 min. After in vivo IVIS analysis, mice were then re-injected with 100 μl of aqueous solution of d -luciferin (50 mg ml−1) and lungs were extracted for ex vivo IVIS imaging with exposure time set to 2 min. No formal blinding was done for in vivo experiments. For all animal experiments, intratumoural treatments were injected directly into the tumour core with care to not allow leakage of any therapeutic solution. Intravenous treatments were injected through the lateral tail vein, with care not to allow leakage of any therapeutic solution. SLP vaccination The formulation and administration of SLP vaccines was adapted from previous studies15,79,80. Each dose contained either 20 μg of each 29-mer CT26 neoantigen peptide (19 neoantigens, 380 μg of total peptide per dose) and 50 μg of poly I:C in 200 μl of 10% DMSO/90% PBS (vol/vol) in Fig. 2e and Extended Data Fig. 5e,f, or 25 μg of each 29-mer CT26 neoan - tigen peptide (19 neoantigens, 475 μg total peptide per dose) and 100 μg Articleof poly I:C in 200 μl of 10% DMSO /90% PBS (vol/vol) in Extended Data Fig. 5g. Therapeutic SLP vaccinations were administered subcutane - ously to BALB/c mice with established hind-flank CT26 tumours on the contralateral hind flank using a 29G needle. SLP vaccine groups were treated on the same days as microbial therapeutic groups. Ex vivo lung histology Explanted lungs from BALB/C mice bearing CT26-Luc metastases or C57BL/6 albino mice (B6(Cg)-Tyrc-2J/J) bearing B16F10-Luc metastases were washed three times in PBS and placed in 10% formalin. After at least 24 h of fixation, lungs were transferred to 70% ethanol and subse - quently embedded in paraffin. Then 50 μm consecutive sections were stained with haematoxylin and eosin. Lung sections were analysed for the presence of tumour foci. Microbial administration for in vivo experiments For therapeutic administration, bacterial strains were grown overnight in fresh LB media containing the appropriate antibiotics. Overnight cultures were centrifuged at 3,000g at 4 °C for 10 min and washed three times with ice-cold sterile PBS. Microbes were delivered intra - tumourally at a concentration of 5 × 108 CFU ml−1 in sterile PBS, with 20 μl of injected using a 1 cc syringe with a 29G needle. For intravenous treatment, 100 μl of microbes were delivered at a concentration of 1 × 108 CFU ml−1 in sterile PBS, through the lateral tail vein using a 1 cc syringe with a 29G needle. Biodistribution and in vivo bacterial dynamics For biodistribution experiments, BALB/c mice bearing established hind-flank CT26 or lung metastatic CT26-Luc tumours were injected intravenously with 100 μl of 1 × 108 CFU ml−1 EcNcΔlon/ΔompT /LLO+. Then 96–120 h after a single i.v. injection for hind-flank tumours or at end - point for lung metastases, tumours or tumour-bearing lungs and other organs were extracted from mice, weighed and homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes). Homogen- ates were serially diluted in sterile PBS and plated on LB agar plates at 37 °C overnight. Colonies were quantified per organ and computed as CFU per gram of tissue (CFU g−1). For tracking bacterial colonization of subcutaneous tumours by microbial luminescence, tumour-bearing mice treated intratumourally or intravenously with wild-type EcN parental strain or genetic derivates were imaged using IVIS at various time points. For abscopal experiments, treated and untreated tumours were harvested 14 days after a single intratumoural bacterial injection. Ex vivo T cell killing assay For B16F10-Luc specific killing, naive tumour-free C57BL/6 mice were injected intravenously every 4 days with PBS, EcNcΔlon/Δ ompT /LLO+ OVA or nAg42 for a total of four doses. Five days after the final dose spleens from treated mice were extracted, filtered through 100 μm cell strainers and washed in complete RPMI. T cells were isolated from single-cell suspensions of spleens from the respective mouse using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in T cell media for use in the specific lysis assay. The luciferase-based killing assay was adapted from previous meth - ods81. B16F10-Luc target cells were grown for 48 h in the presence of 100 U ml−1 murine IFNγ. Target cells were gathered and plated at 1 × 104 cells per well in a 96-well plate. After 12 h, T cells were added to each well to achieve designated effector-to-target ratios (10:1, 20:1 or 40:1). After 42 h of co-incubation, 50 U ml−1 IL-2 was added to all wells. For CT26-Luc versus 4T1-Luc luciferase-based specific killing assay: BALB/c mice with established hind-flank CT26 tumours were treated intravenously with EcNcΔlon /ΔompT /LLO+ nAg19 on days 0 and 3. On day 8, tumours were extracted and mechanically homogenized, followed by digestion with collagenase A (1 mg ml−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l-glutamine, 1% penicillin–streptomycin and 10 mM HEPES) with gentamicin (40 μg ml−1) for 1 h at 37 °C on a shaker platform at 150 rpm. Tumour homogenates were filtered through 100 μm cell strainers and washed in T cell media. Tumour-infiltrating T cells were isolated from single-cell suspensions of tumours from mice using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in T cell media. CT26-Luc or 4T1-Luc target cells were grown for 12 h in the presence of 100 U ml−1 murine IFNγ. Target cells were gathered and plated at 1 × 104 cells per well in a 96-well plate. After 12 h, T cells were added to each well to achieve designated effector-to-target ratios (5:1 or 10:1), with 50 U ml−1 IL-2 added to all wells. Luminescence from each well was quantified after addition of One-Glo Luciferase Assay System (Promega), as per the manufacturer’s instructions, after 24–96 h of coculture. Minimum lysis wells contained only the respective luciferase-expressing tumour target cells. In maxi - mum lysis wells, 20 μl of media was replaced with 20 μl of 3% Triton X-100 60 min before luminescence readout. Specific lysis (%) was cal- culated using the luminescence values of the respective conditions with the following formula: 100 − (100 × ((sample − maximum lysis)/ (minimum lysis − maximum lysis))). IFNγ ELISpot BALB/c mice with established hind-flank CT26 tumours were treated intravenously with EcNcΔlon/Δ ompT /LLO+ nAg19 on day 0 and 3. On day 8, tumours were extracted and mechanically homogenized, followed by digestion with collagenase A (1 mg ml−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l -glutamine, 1% penicillin–streptomy - cin and 10 mM HEPES) with gentamicin (40 μg ml−1) for 1 h at 37 °C on a shaker platform at 150 rpm. Tumour homogenates were filtered through 100 μm cell strainers and washed in RPMI containing CTL-Wash Supplement (Immunospot) and 1% l -glutamine. Splenocytes from naive BALB/c mice were isolated in the same way, without digestion. T cells were isolated from single-cell suspensions of tumours from mice using the EasySep Mouse T Cell Isolation Kit (StemCell Technologies) according to the manufacturer’s instructions. Purified T cells were resuspended in CTL-Test Medium supplemented with 1% l-glutamine for use in the enzyme-linked immunosorbent spot (ELISpot) assay. Mouse IFNγ Single-Color ELISpot plates and kits were purchased from Immunospot. ELISpot plates were prepared as per manufacturer’s instructions. Here, 5 × 105 naive splenocytes were plated with 2 × 104 TILs per well in 200 μl CTL-Test Medium supplemented with 1% l -glutamine and gentamicin (30 μg ml−1). Then 29-mer synthetic neoantigen or negative control (OVA) peptides were added to each well at a final con- centration of 5 μg ml−1. Cells were stimulated overnight in a stationary 37 °C incubator with atmosphere of humidified 5% CO2. After incuba- tion, plates were developed as per the manufacturer’s protocol and spots quantified using a CTL Immunospot S6 Universal machine and CTL ImmunoSpot software v.7.0.24.0. Flow cytometry immunophenotyping For CT26 flow-cytometric immunophenotyping, BALB/c mice with hind-flank CT26 tumours received intravenous treatment with the indicated microbial therapeutic or PBS on day 0. Two or 8 days after treatment, TDLNs and/or tumours were extracted. For B16F10 flow-cytometric immunophenotyping, C57BL/6 mice with hind-flank, orthotopic B16F10 tumours received intravenous treatment with the indicated microbial therapeutic or PBS on day 0 and 3. Eight days after treatment, tumours were extracted. Lymphoid and myeloid immune subsets were isolated from tumour tissue by mechanical homogenization of tumour or TDLN tissue, followed by digestion with collagenase A (1 mg ml−1, Roche) and DNase I (0.5 μg ml−1, Roche) in isolation buffer (RPMI-1640 with 5% FBS, 1% l-glutamine, 1% penicillin– streptomycin and 10 mM HEPES) for 1 h at 37 °C for tumours or 30 min at 37 °C for TDLNs, on a shaker platform at 150 rpm. For ex vivo lymphocyte stimulation with PMA and ionomycin, TDLNs were not digested beforehand. Tumour and TDLN homogenates were filtered through 100 μm cell strainers and washed in isolation buffer. To measure overall cytokine production by T cells, cells were stimulated for 3 h with PMA (50 ng ml−1, Sigma-Aldrich) and ionomycin (1 nM, Calbiochem) in the presence of brefeldin A (1 μg ml−1). To measure neoantigen-specific cytokine production by T cells, cells were stimu- lated for 5 h with pools of peptides (2 μg ml−1) representing the neo - antigens encoded in therapeutic strains in the presence of brefeldin A (1 μg ml−1). Cells were stained in FACS buffer. Ghost Dye cell viability reagent was used to exclude dead cells (diluted 1:1,000 in PBS). Extra - cellular antibodies for lymphoid immunophenotyping included: CD4 (RM4-5, Biolegend), NKp46 (29A1.4, BD Biosciences), NK1.1 (PK136, Biolegend), CD45 (30-F11, BD Biosciences), B220 (RA3-6B2, BD Bio - sciences), CD19 (6D5, Biolegend), CD8a (53-6.7, Biolegend), TIM-1 (RMT1-4, BD Biosciences) and CD69 (H1.2F3, BD Biosciences). After extracellular staining, cells were washed with FACS buffer, and fixed using the FOXP3/transcription factor staining buffer set (Tonbo), as per the manufacturer’s instructions. Intracellular antibodies for lym- phoid immunophenotyping included: Foxp3 (FJK-16s, Thermo), CD3ε (145-2C11, Biolegend), TCRβ (H57-507, BD Biosciences), Ki-67 (SolA15, Thermo), Granzyme-B (QA16A02, Biolegend), TNF (MP6-XT22, Bioleg - end) and IFNγ (XMG1.2, Biolegend). For myeloid immunophenotyping, extracellular antibodies included: Ly6C (HK1.4, Biolegend), I-A/I-E (M5/114.15.2, BD Biosciences), XCR1 (ZET, Biolegend), CD11b (M1/70, Biolegend), CD103 (2E7, Biolegend), CD45 (30-F11, BD Biosciences), F4/80 (BM8, Biolegend), CD11c (HL3, BD Biosciences), CD172a/SIRPα (P84, Biolegend), Ly6G (1A8, Biolegend and BD Biosciences), PD-L1 (10 F .9G2, Biolegend), CD301b (URA-1, Biolegend), CD3 (145-2C11, Biole - gend), CD19 (1D3, Biolegend), NK1.1 (PK136, Biolegend), NKp46 (29A1.4, Biolegend) CD64 (X54-5/7.1, Biolegend), CD80 (16-10A1, Biolegend) and CD86 (GL-1, BD Biosciences). All antibodies for flow cytometry were used at a 1:200 dilution. After staining, cells were washed and resuspended with FACS buffer for flow cytometry analysis using a BD LSRFortessa or Cytek Aurora cell analyser. FACS Diva or SpectroFlo software was used for data acquisition. Collected flow cytometry data were analysed using FlowJo. Synthetic peptides Synthetic peptides representing neoantigens for lymphocyte restimu - lation assays and vaccination were synthesized by and purchased from Peptide v.2.0. All peptides were above or equal to 95% purity. Statistics and reproducibility Statistical analyses and P value calculations were performed using GraphPad Prism v.9 and v.10. For each experiment, the particular sta- tistical analysis is detailed in the respective figure legend. A two-tailed unpaired Student’s t-test, one-way analysis of variance (ANOVA) or two-way ANOVA with appropriate post hoc test was used for data that were roughly normally distributed. For analysis of Kaplan–Meier sur- vival experiments, the log-rank (Mantel–Cox) test was used. All analyses were two-tailed. For all statistical analyses, NS denotes not significant, which is P > 0.05. For Fig. 4a and Extended Data Fig. 7b, immunoblot data are repre - sentative of four independent experiments. In Extended Data Figs. 1b and 2h, immunoblot data are representative of three independent experiments. Immunofluorescence data in Extended Data Fig. 2i are representative of three independent experiments. Histology data in Extended Data Figs. 5h and 8f are representative of three independent experiments. All other results in the paper were replicated at least two to three times in independent experiments. Biological materials availability Reasonable requests for biological materials used in this study will be promptly reviewed by Columbia Technology Ventures to verify whether the request is subject to any intellectual property or confidentiality obligations. Any materials that can be shared will be released through a material transfer agreement. Reporting summary Further information on research design is available in the Nature Port - folio Reporting Summary linked to this article. Data availability Whole-exome sequencing and RNA sequencing data generated for this study are deposited under umbrella BioProject accession number PRJNA1025007 . Whole-exome data are available on the Short Read Archive with BioProject ID PRJNA1024050 . RNA sequencing data are available on National Center for Biotechnology Information Gene Expression Omnibus with accession number GSE244808 . All other data are available within the article or its Supplementary Informa - tion. Datasets used for analyses in this study were: Ensembl release 102 M. musculus GRCm38 gene annotations (GRCm38, https://useast. ensembl.org/Mus_musculus/Info/Index, accession GCA_000001635.8) and dbSNP build 142 (ftp.ncbi.nih.gov/snp ). Source data are provided with this paper.