Fig. 2: Stimulation of human MoDCs in vitro with (R848+TDB) induced antigen cross-presentation of internalized proteins.
Phosphoproteomic analysis of human MoDCs identified the phosphorylation of 17 proteins belonging to the MHC class I processing and presentation pathway (Reactome 19282). a STRING analysis illustrates the order of interaction between these identified proteins. Proteins significantly phosphorylated upon treatment are indicated in bold. b, c The phosphorylation of nine of these proteins was significantly increased upon stimulation, shown as Box-whisker plots, center:mean, boxes:75th and 25th percentile, whiskers: minimum and maximum values). b The treatment-specific phosphorylation of four proteins was confirmed by western blotting after separation of phosphorylated and unphosphorylated species of each protein using phos-tag gel electrophoresis(representative images of three biological replicates). Cells from adult study participants were used for this. c Comparative intensity of the five remaining analytes with significant differences in phosphorylation N = 9 newborns, 4 adults, error bars indicate mean+SEM. Statistical comparisons in (b, c) are indicated by connecting lines and employed two-tailed Wilcoxon rank-sum test (*p < 0.05, **p < 0.01, ***P < 0.001). d Cross-presentation was confirmed in a MoDC:CD8 co-culture assay. MoDCs were pulsed with 1 µg/ml soluble protein antigen as indicated, in the presence or absence of adjuvants as indicated on the axes, and subsequently cultured with autologous CD4+ or CD8+ T cells for 4 days before evaluation of antigen-specific T cell activation (n = 5 adults, 2-way ANOVA with Tukey post-hoc test, error bars indicate mean+SEM. *p < 0.05, **p < 0.01, ***P < 0.001). Source data are provided as a Source Data file.
