Med. 29, 2844–2853 (2023). 55. O’Donnell, J. S., Teng, M. W. L. & Smyth, M. J. Cancer immunoediting and resistance to T cell-based immunotherapy. Nat. Rev. Clin. Oncol. 16, 151–167 (2019).Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution- NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if you modified the licensed material. You do not have permission under this licence to share adapted material derived from this article or parts of it. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creativecommons.org/licenses/by-nc-nd/4.0/. © The Author(s) 2024 ArticleMethods Cell lines The B16F10 melanoma (ATCC CRL-6475), CT26 colon carcinoma (ATCC CRL-2638) and 4T1 breast cancer (ATCC CRL-2539) authenticated cell lines were purchased directly from ATCC. CT26-Luc, B16F10-Luc and 4T1-Luc cells were lentivirally transduced with luciferase. Cells were confirmed mycoplasma free. Cells were cultured in incubators at 37 °C with atmosphere of humidified 5% CO2. B16F10 and B16F10-Luc cells were grown in DMEM supplemented with 10% (vol/vol) fetal bovine serum (FBS), 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution (Gibco-11140050) and 100 U ml−1 penicillin–streptomycin. CT26, CT26-Luc, 4T1 and 4T1-Luc cells were grown in Roswell Park Memorial Institute medium (RPMI-1640) supplemented with 10% (vol/vol) FBS, 1× GlutaMax, 1% (vol/vol) MEM non-essential amino acids solution and 100 U ml−1 penicillin–streptomycin. No commonly misi- dentified cell lines were used in this study. Exome sequencing Paired tumour and tail DNA from BALB/c mice bearing subcutane - ous CT26 tumours or C57BL/6 mice bearing subcutaneous B16F10 tumours was extracted in triplicate (n = 3 mice per tumour line) using Qiagen DNeasy Blood & Tissue Minikit following the manufacturer’s instructions. Exome capture from mouse tumour and tail DNA tripli - cates was conducted using Agilent SureSelectXT All Exon kit for target enrichment DNA library preparation56, according to the manufacturer’s instructions (Agilent). Genomic DNA was fragmented by acoustic shear - ing with a Covaris S220 instrument. Fragmented DNAs were cleaned, end-repaired and adenylated at the 3′ end. Adaptors were ligated to DNA fragments, and adaptor-ligated DNA fragments enriched with limited-cycle PCR. Adaptor-ligated DNA fragments were validated using Agilent TapeStation (Agilent) and quantified using Qubit 2.0 Fluorom - eter (ThermoFisher Scientific) and Real-Time PCR (KAPA Biosystems). Sequencing libraries were clustered onto a lane of a flow cell. After clustering, the flow cell was loaded on an Illumina HiSeq4000 Instru- ment per the manufacturer’s instructions. Samples were sequenced using 2 × 150 bp paired end configuration. Image analysis and base calling was conducted by the HiSeq Control Software. Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina bcl2fastq2.17. Sequence reads were trimmed to remove adaptor sequences and nucleotides with poor quality using Trimmomatic v.0.39 (ref. 57). Trimmed reads were aligned to the GRCm38 reference genome using the Illumina Dragen Bio-IT platform. Alignments were sorted and PCR or optical duplicates marked for generation of BAM files. Somatic single-nucleotide variants and insertion or deletion (indel) variants were called using Illumina Dra - gen58 and GATK Mutect2 (ref. 59 ). All variants from paired-normal tissue and murine variants from the dbSNP database60 were removed during the process. VCF files were left aligned and normalized, with splitting of multiallelic sites into several sites using bcftools v.1.13 (ref. 61). Only tumour-specific variants called by both algorithms were used for further analysis. RNA sequencing Tumour RNA from BALB/c mice bearing subcutaneous CT26 tumours or C57BL/6 mice bearing subcutaneous B16F10 tumours was extracted in triplicate using Qiagen RNeasy Minikit as per the manufacturer’s instructions. Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies) and RNA integrity checked using Agilent TapeStation 2400 (Agilent). RNA sequencing libraries were prepared using the NEBNext Ultra RNA library Prep Kit for Illumina as per the manufacturer’s instructions (New England Biolabs). mRNAs were enriched with Oligo(dT) beads. Enriched mRNAs were frag - mented for 15 min at 94 °C. First- and second-strand complementary DNAs (cDNAs) were synthesized subsequently. cDNA fragments were end-repaired and adenylated at 3′ ends, and universal adaptors ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. Sequencing libraries were validated on Agilent TapeStation (Agilent), and quantified using Qubit 2.0 Fluorometer (Invitrogen) and quantitative PCR (qPCR) (KAPA Biosystems). Library loading, sequencing and read trimming were done as described above. Trimmed reads were aligned to the mm10 reference using STAR aligner v.2.5.2b (ref. 62 ). Unique gene hit counts were calculated using feature counts from Subread Package v.1.5.2. Unique reads that fell within exon regions were counted. The gene hit counts table was used for expres- sion analysis using DESeq2 v.1.20.0 (ref. 63 ). Neoantigen prediction and selection Mutation-specific RNA expression and allele fraction were added to somatic VCF files using Bam-readcount64 and VAtools (http:// vatools.org). Somatic VCFs were annotated with The Ensembl Vari - ant Effect Predictor (VEP Ensembl v.104)65. Only PASS variants from VCFs were considered. Annotated VCFs were analysed using pVac - Seq for neoepitope discovery14. MHC-I affinities were predicted with NetMHCpan v.4.1 (ref. 66 ) and NetMHC v.4.0 (ref. 67 ), and MHC-II affinities were predicted with NetMHCIIpan v.4.1 (ref. 68 ) and NNa - lign v.2.0 (ref. 69 ). Exonic mutation-derived long peptides based on single-nucleotide polymorphisms (SNP) or indels predicted to generate mutant MHC-binding peptides were first filtered on the basis of the set of minimum criteria: (1) present in all tumour sample triplicates (DNA variant allele fraction ≥ 0.05) and none of the normal tissue trip - licates, (2) non-synonymous mutation resulting from either SNP or indel, (3) confirmed exonic mutation transcription (RNA variant allele fraction ≥ 0.05) and gene expression by RNA sequencing in tumour sample triplicate (transcripts per million ≥ 1), (4) at least one predicted MHC-I or MHC-II binding epitope and (5) MHC-I or MHC-II differential binding affinity17,20 (wild-type half-maximum inhibitory concentration (IC50)/mutant IC50) ≥ 1.2. Predicted neoantigens fulfilling all previous criteria were then prioritized for inclusion and selected according to the following hierarchy: (1) high predicted affinity (MHC-I or MHC-II IC50 ≤ 500 nM), (2) moderate predicted affinity (MHC-I or MHC-II IC50 500–1,000 nM) and (3) low predicted affinity (MHC-I or MHC-II IC50 1,000–5,000 nM) (Extended Data Tables 1 and 2). Strains and plasmids Plasmids were constructed using restriction-enzyme mediated and Gibson assembly cloning methods. Neoantigen construct iterations were designed and created as Geneblocks (IDT) encoding a constitu - tive promoter and 5′ untranslated region (UTR) containing selected ribosome-binding site, followed by coding region composed of mutant-residue containing long peptides connected in tandem or by various linkers as indicated. 5′ BamHI and 3′ XbaI restriction endonu- clease sites were added to constructs. Coding sequences were codon optimized for E. coli. Constructs were cloned between BamHI and XbaI restriction sites on a stabilized p246-lux CDABE plasmid where luxCDABE had been cloned out22, and flanked by 3′ λ-phage transcrip - tion terminator, with high-copy pUC origin. For protein expression assessment studies, the codon sequence for a 6×-Histidine Tag (HisTag) was added immediately before the stop codon within the neoantigen construct coding sequence by PCR amplification of full construct plas - mids with oligonucleotide containing 6×-HisTag sequence followed by kinase, ligase, DpnI enzyme mix protocol (NEB). Neoantigen con - struct plasmids were transformed into chemically competent E. coli DH5α or BL21(DE3) (New England Biolabs), or electrocompetent EcN parental strain or genetic derivatives. The parental EcN strain and all derivatives used in this study harbour an integrated luxCDABE cassette within the genome, which also contains an erythromycin resistance gene70. Plasmid encoding constitutive LLO was constructed by cloning in the hok/sok stabilization system to pCG02-p15a backbone71, PCR amplification of backbone with SLC cloned out, and Gibson assembly of Geneblock encoding LLO under constitutive promoter and 5′ UTR containing selected ribosome-binding site. Constitutive LLO plasmids were transformed into electrocompetent EcN parental and genetic derivative strains. Strains were cultured in Luria-Bertani (LB) medium with antibiotics for plasmid retention (pUC:kanamycin 50 μg ml−1, p15a:spectinomycin 50 μg ml−1) in a 37 °C orbital incubator. Construction of cryptic plasmid-cured EcN EcN cryptic plasmids were cured with Cas9-mediated double-strand break, as described previously27. Briefly, EcN was transformed with pFREE or pCryptDel4.8 to cure the cryptic plasmids pMUT1 or pMUT2, respectively. The transformants were grown overnight and diluted 1:1,000 the next day into fresh LB containing 0.2% rhamnose and 0.43 μM anhydrotetracycline. After 24 h of incubation, the culture was streaked onto LB plates without antibiotics and incubated overnight in a 30 °C incubator. Colonies were screened with colony PCR to verify the loss of cryptic plasmids. Construction of genetic knockout strains Genetic knockouts were performed using the lambda red recombina- tion system72. In brief, EcNc was transformed with pKD46. Transfor - mants were grown at 30 °C in LB with ampicillin and l-arabinose, then made electrocompetent. The chloramphenicol resistance cassette with corresponding overhangs for each target gene for deletion was prepared by PCR amplification of pKD3. Electroporation was performed using 100 μl of competent cells and 50–300 ng amplified DNA. After 2 h of recovery, cells were plated on LB agar containing chloramphenicol and incubated at 37 °C overnight. Target gene deletion was verified by colony PCR. For excision of the antibiotic resistance marker, pCP20 was transformed, and the transformants were plated on fresh LB plates containing ampicillin and incubated at 30 °C overnight. Selected colo - nies were then inoculated onto fresh LB plates without antibiotics and cultured at 43 °C overnight for induction of flippase and plasmid curing. Clones were subsequently screened for loss of antibiotic resistance. qPCR for PCN Copy number variant plasmids were constructed from a high-copy pUC-GFP22 plasmid. The plasmid backbone excluding the pUC origin was PCR-amplified and Gibson assembled with sc101*, p15A or ColE1 origin of replication insert. The respective inserts were prepared from PCR amplification of template plasmid pCG02_sc101*, pCG02_p15A or pTH05_ColE1. Plasmid copy number (PCN) was determined as reported previously22, in which the relative abundance of plasmid DNA compared to genomic DNA is measured by qPCR. Briefly, strains with the plasmid of interest were grown at 37 °C overnight in fresh LB with appropriate antibiotics. Cells were collected by centrifugation at 3,000g at 4 °C for 10 min, the supernatant removed and the cell pellet resuspended in distilled water for optical density measurement at 600 nm (OD600) equal to 1. Resuspended cells were fivefold serially diluted. Samples were denatured at 95 °C for 10 min and 2 μl of each sample dilution was added into 18 μl of NEB Luna Universal qPCR Master Mix in each well of a 96-well plate. Then 25-fold diluted samples were used for the measure- ment of crossing point values: the cycle number when amplified sample fluorescence exceeds the background. Fivefold diluted samples were used for generation of the standard curve for PCR efficiency (E). E was defined from the slope (S ) of each standard curve with the equation E = 5(−1/S ) and PCN was determined with the equation PCN = (EGCTG)/ (EPCTP), where respective values for genomic DNA are denoted by a subscript G and plasmid DNA by subscript P. Immunoblot and ELISA For immunoblot and ELISA, a C-terminal 6×-HisTag was attached to each neoantigen construct. Strains expressing neoantigen construct with C-terminal 6×-HisTag were grown overnight in LB media with appropriate antibiotics. Equalization of OD600 measurement to match colony-forming units (CFU) per ml (CFU ml−1) between all cultures was done before all sample processing. CFU-matched cultures were cen - trifuged at 3,000g at 4 °C for 10 min. For immunoblot, samples were resuspended in B-PER lysis reagent (ThermoFisher Scientific) con - taining 250 U ml−1 benzonase nuclease (Millipore Sigma) and 1 U ml−1 rLysozyme (Millipore Sigma) and placed on an orbital shaker for 15 min at room temperature. Samples were centrifuged at 10,000g for 20 min at 4 °C to separate soluble and insoluble fractions or total lysate used directly. Processed samples were mixed with SDS-loading buffer with 5 mM dithiothreitol, boiled and subject to immunoblot analysis. For relative quantification of immunoblot chemiluminescent intensity, target protein bands on the same blot were normalized to the loading control band DnaK for the same sample. DnaK loading controls were always run on the same gel as target proteins. Normalized values were divided to provide relative intensity values. Mouse anti-6×His (αTHE) was purchased from GenScript, mouse anti-DnaK was purchased from Abcam (8E2/2). αTHE and 8E2/2 antibodies were used at 1:5,000 dilution. For HisTag ELISA, samples were resuspended in ice-cold PBS con - taining HAL T protease inhibitor cocktail (ThermoFisher Scientific). Samples were sonicated on ice for 2 min total time. Sonicated samples were centrifuged at 10,000g for 20 min at 4 °C. Soluble sample fractions were analysed using GenScript HisTag ELISA Detection Kit as per the manufacturer’s instructions. For ex vivo immunoblot analysis, BALB/c mice bearing estab - lished hind-flank CT26 tumours were injected intravenously with the EcNcΔlon/ΔompT /LLO+ nAg19-His strain cocktail that contains all three neoanti - gen constructs, in which each construct (MHCIa, MHCIIa and MHCI/IIv) contained a C-terminal 6×-HisTag. Then 48 h after treatment, tumours and TDLNs were extracted from mice and placed in B-PER lysis reagent (ThermoFisher Scientific) with 250 U ml−1 benzonase nuclease (Milli - pore Sigma), and homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes). Tissue homogenate was sonicated on ice for 3 min. Sonicated samples were centrifuged at 10,000g for 20 min at 4 °C to separate soluble and insoluble fractions, and fractions subsequently resuspended and diluted in lysis buffer. Sample fractions were mixed with SDS-loading buffer with 5 mM dithiothreitol, boiled and subject to immunoblot analysis. For ex vivo IL-12p70 ELISA analysis, BALB/c mice bearing established hind-flank CT26 tumour were injected intratumourally with PBS, EcNcΔlon/ΔompT or EcNcΔlon/ΔompT /LLO+. Then 4–24 h after treatment, tumours were extracted and placed in ice-cold PBS containing HAL T protease inhibitor cocktail without dimethylsulfoxide (DMSO) (ThermoFisher Scientific). Tumours were homogenized using a gentleMACS tissue dissociator (Miltenyi Biotec, C-tubes), and centrifuged at 3,000g for 10 min at 4 °C. The supernatant was then collected and centrifuged at 10,000 g for 20 min at 4 °C to separate soluble and insoluble fractions. IL-12p70 in soluble sample fractions was analysed using the Mouse IL-12p70 Quantikine ELISA Kit (R&D systems) according to the manu- facturer’s instructions. Blood bactericidal assay EcN wild-type or EcNcΔlon/Δ ompT were cultured overnight in LB media without antibiotics. Cultures were centrifuged at 3,000g for 10 min, resuspended in 1 ml of ice-cold sterile PBS and normalized to OD600 = 1. Then 50 μl of OD600 = 1 microbe suspension was added to 1 ml of single donor human whole blood (Innovative Research) in triplicate and incu - bated in a 37 °C stationary incubator. After 2 h of incubation, a sample was taken from each blood–microbe mixture and serial dilution was prepared in PBS. Dilutions were plated on LB agar with erythromycin (25 μg ml−1). After incubation overnight at 37 °C, colonies were quanti - fied by spot-forming assay and CFU ml−1 blood was calculated. Biofilm assay Biofilm formation assays were conducted as described previously73. Briefly, EcN wild-type, cryptic plasmid-cured (EcNc), Lon knockout Article(EcNcΔlo n), OmpT knockout (EcNcΔompT) or double protease knockout (EcNcΔlon/ΔompT) were cultured for 48 h in LB media with 25 μg ml−1 eryth - romycin in borosilicate glass tubes in a 30 °C stationary incubator, with tube caps wrapped with parafilm to prevent evaporation. At 48 h, cultures were discarded and borosilicate tubes were washed three times with PBS.