Article Extended Data Fig. 2 | Evaluation of microbial tumor neoantigen vaccine functioning and immunologic activity in vitro. a, Plasmid copy number in wildtype EcN or cryptic plasmid cured EcNc ( n = 3 per group). b , Upper: relative immunoblot chemiluminescent intensity of synthetic neoantigen construct MHCI/IIv expression in wildtype EcN vs. derivative strains ( n = 3 biological replicates), Lower: representative immunoblot of construct MHCI/IIv expressed in wildtype EcN and derivative strains. c , Upper: relative immunoblot chemiluminescent intensity of synthetic neoantigen construct MHCIa expression in wildtype EcN vs. derivative strains ( n = 3 biological replicates), Lower: representative immunoblot of construct MHCIa expressed in wildtype EcN and derivative strains. d , Biofilm formation quantified for wildtype EcN, and derivative strains by crystal violet stain assay. (**** P < 0.0001, ns = P > 0.05, One-way ANOVA with Tukey’s multiple comparison test, n = 9 biological replicates for EcN, 10 for EcNc WT, 12 for other groups). e ,f, MFI of e , Left: H2kb-SIINFEKL complex and Right: MHC-II, or f , Left: CD80 and Right: PD-L1 for BMDM incubated with the indicated live microbial strain or media for 6 h (*P = 0.0231, * P = 0.0414, *** P = 0.0002, *** P = 0.0005, **** P < 0.0001, ns = P > 0.05, one-way ANOVA with Tukey’s multiple comparisons test, n = 4 biological replicates per group). g , Left: sheep red blood cells (RBCs) were incubated with lysate from EcNcΔlon /ΔompT with (LLO+) or without (LLO−) cytosolic LLO expression. Absorbance at 541 nm ( n = 3 biological replicates per group). Right: percentage of live BMDM after incubation with indicated live microbial strain or control for 6 h ( n = 4 biological replicates per group). h, Immunoblot depicting expression of neoantigen constructs MHCIa, MHCIIa, and MHCI/IIv in EcNcΔlon /ΔompT with (LLO+) or without (LLO−) co-expression of cytosolic LLO. i, Immunofluorescence microscopy analysis of BMDM co-incubated with Left: EcNcΔlon /ΔompT OVA or, Right: EcNcΔlon /ΔompT /LLO+ OVA. j , Naïve OT-II T cells were incubated with BMDC’s pulsed with the indicated condition. Left: IFN-γ quantification in supernatant of OT-II cultures (**** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test, n = 3 biological replicates per group), Middle: IL-2 quantification in supernatant of OT-II culture (**** P < 0.0001, one-way ANOVA with Tukey’s multiple comparisons test, n = 3 biological replicates per group). Right: representative histogram depicting CFSE dilution of stimulated OT-II T cells. a –g,j, Data are mean ± s.e.m. Gel source data in Supplementary Figs. 1 and 2.