Fig. S3 . The correlation coefficient r is shown for each Listeria -synthetic peptide pair (see methods). E , subcellular localization prediction of the 12 identified Listeria antigens ( top ) and the entire Listeria proteome ( bottom ) by PSORTdb 4.0 . " title="... antigens ( top ) and the entire Listeria proteome ( bottom ) by PSORTdb 4.0 . " property="contentUrl" width="100%" height="100%"/>
Figure Legend Snippet: Detection of high confident Listeria immunopeptides. A , principal component analysis (PCA) of peptide intensities coherently cluster the Listeria -infected ( red ) and uninfected samples ( blue ). B , volcano plots indicating the higher abundance of the Listeria peptides ( red ) in infected samples. C , heatmap of z-scored Listeria peptide intensities in the TMT and label-free samples. D , all 12 high-confident Listeria immunopeptide sequences were synthesized to compare their synthetic and experimental fragmentation spectra, confirming the bona fide identification of the four peptides shown here and in Fig. S3 . The correlation coefficient r is shown for each Listeria -synthetic peptide pair (see methods). E , subcellular localization prediction of the 12 identified Listeria antigens ( top ) and the entire Listeria proteome ( bottom ) by PSORTdb 4.0 .

Techniques Used: Infection, Synthesized

Figure Legend Snippet: CD8 T cell responses of MHC class I–associated peptides after DC + peptide prime and Δ actA- Δ inlB-L. monocytogenes booster immunization. A , C57Bl/6 mice were infected i.v. with Δ actA- Δ inlB-L. monocytogenes . Seven days later, splenocytes were harvested and stimulated with the indicated peptides (1 μM final concentration) for 5 h in the presence of brefeldin A prior to the detection of CD8 T cell–derived IFN-ɣ and TNF. Data presented are from two mice (m1 and m2) from one representative experiment of two with similar results. N = 4 mice total analyzed for all peptides. B , experimental design. C57Bl/6 mice were immunized i.v. with dendritic cells (DC) coated with the identified peptides derived from LMON_0576 or LMON_2148 antigens. Seven days later, immunized mice were boosted with Δ actA- Δ inlB-L. monocytogenes (LM). Fourteen days after boosting, splenocytes were harvested and stimulated with the indicated peptides (1 μM final concentration) for 5 h in the presence of brefeldin A prior to the detection of CD8 T cell–derived IFN-ɣ and TNF. C , representative flow plots of IFN-ɣ and TNF in gated CD8 T cells from DC-2148 peptide primed, LM boosted mice. Left panel, no peptide stimulation; right panel, LMON_2148 peptide stimulation. D , representative flow plots of IFN-ɣ and TNF in gated CD8 T cells from DC-0576 peptide primed, LM boosted mice. Left panel, no peptide stimulation; right panel, LMON_0576 peptide stimulation. Numbers represent % of cells in each quadrant. E cumulative data from three mice in each group in one experiment that is representative of two similar experiments.

Techniques Used: Infection, Concentration Assay, Derivative Assay

Figure Legend Snippet: Assessment of protective immunity after DC-0576 priming and boosting with mRNA-LNP vaccine expressing the LMON_0576 antigen. A , experimental design. A group of C57Bl/6 mice were DC + 0576 peptide primed. Sixteen days later, some of these mice were boosted with an mRNA vaccine expressing the LMON_0576 antigen or Δ actA- Δ inlB-L. monocytogenes . B , representative flow plots of LMON_0576 peptide-stimulated IFN-ɣ production by gated CD8 T cells from the blood in the indicated group at 24 days after initial DC + 0576 prime. C , cumulative data showing % IFN-ɣ production from five mice per group at 24 days after DC + 0576 prime. D , immunized mice and naïve controls were challenged i.v. with virulent LM. Colony-forming units per gram of liver ( left ) and spleen ( right ) at 3 days post challenge. Each dot is an individual mouse. Data analyzed by one-way ANOVA.

Techniques Used: Expressing