Extended Data Fig. 2: Evaluation of microbial tumor neoantigen vaccine functioning and immunologic activity in vitro.
From: Probiotic neoantigen delivery vectors for precision cancer immunotherapy
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.
