Fig. 4: Vitd3 and dexamethasone alters metabolic and signaling networks in immune-suppressive phenotypes of tol-DC.

A Schematic diagram of tolerogenic DC treatment conditions. Control (black), vitd3+dexa (purple) and vitd3 (orange) cells sampled at iDC, actDC, and mDC were subjected to dimensionality reduction using UMAP. Single-cell heatmaps were overlayed on concatenated (iDC, actDC, and mDC) samples to depict immune marker expression changes between maturation stages in control and tolerogenic cell clusters. B Boxplots represent changes in SCENITH puromycin protein synthesis (gMFI puromycin) levels across DC stages and treatment conditions (N = 3 donors). C Overview of kinetic changes and differences in percentual (left panel) and protein synthesis-adjusted (right panel) SCENITH metabolic parameters between control, vitd3+dexa (purple) and vitd3-treated (orange) DC across differentiation timeline. Connecting lines visualize average pathway changes (precursor stages represent 3 independent donors, iDC, actDC, and mDC represent SIX independent donors). Statistical significance of pairwise comparisons between control and vitd3-dexa-tol (purple asterisk) and control and vitd3-tol samples (orange asterisk) analyses are depicted. D Integrated clustering heatmap of DC activation stages based on median arcsinh transformed expression values for scMEP metabolic regulators (N = 3). Bottom heatmap annotations include donor labels, treatment conditions and DC differentiation stages. Fluorescent quantitation of mitochondrial size (Mitotracker Deep Red) along with protein translation/ATP levels are annotated in the form of a heatmap. Point annotations representing lactate and glucose supernatant measurements were determined in iDC, actDC, and mDC. Heatmap annotation for DC immune signatures are located at the top of the clustering matrix. scMEP markers are colored according to their metabolic pathway activity. E Kinetic profiles for calculated median scMEP pathways scores for control (black), vitd3+dexa (purple) and vitd3 (orange)-treated DC across DC maturation timeline. Connecting lines visualize mean pathway changes (N = 3). Statistical significance of pairwise comparisons between control and vitd3-dexa-tol (purple asterisk) and control and vitd3-tol samples (orange asterisk) analyses are depicted. F Glucose and lactate measurements in control and tolerogenic DC culture supernatants are shown. Of note glucose level measurement increase in the media between d3 and iDC stage is due to media change at day 3. Three technical replicates from three donors are presented with error bars indicating standard deviation. Multiple comparisons statistical significance in B, C, E, F was calculated via one-way ANOVA with Tukey’s post-hoc test. For all panels, P-values are represented as *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. p-values < 0.05 were considered statistically significant (ns). Box plots indicate second and third quantile (box), median (horizontal line) and 1.5× the interquartile range (whiskers). Source data are provided as a Source Data file.