Fig 1: Phenotypic profiling of dexamethasone-mediated tolerogenic dendritic cells. (A) Schematic representation of the experimental approach, comparing dendritic cell (DC) with tolerogenic dendritic cell (tolDC) differentiation. (B) DC and tolDC were cocultured with CD8+ cells for 5 days. The final CFSE signal of CD8 + cells is shown (left panel). CD8+ with only CD3/CD28 T-activator beads (C+) or alone (C–) are also shown. In the right panel, the average proliferation is shown (mean ± standard error of the mean (SEM)) (n = 4). (C) IL-10, TNFa, IL-12p70 and IL1-ß production of DC and tolDC, after 5 days of differentiation and 24 h of LPS (10 ng/µl) and IFNg (20 ng/µl) stimuli. P-values of paired t-tests are shown (n = 4) (ns P > 0.05, * P < 0.05). (D) Box-plots of CD80, CD83, CD86 and HLA-DR surface expression (Median Fluorescence Intensity) in DCs and tolDCs in steady-state or stimulated with LPS (10 ng/µl) and IFNg (20 ng/µl) (n = 6) (ns P > 0.05, ** P < 0.01, *** P = 0.001). (E) Gene expression heatmap of differentially expressed genes comparing tolDCs with DCs and also displaying the gene expression values of the precursor cell type (MO) (logFC > 0.5, FDR < 0.05). Scaled fluorescence values of expression arrays are shown, ranging from -2 (lower gene expression, green) to +2 (higher gene expression, orange) (n = 3). (F) Gene ontology (GO) over-representation of GO Biological Process categories. Fold change of tolDC induced genes over background and -log10(FDR) of Fisher's exact tests are shown. (G) Discriminant regulon expression analysis (DoRothEA) of tolDC compared with DC. Only transcription factors with FDR <0.05 are shown. NES and logFC of transcription factor expression are depicted. (H) T-distributed stochastic neighbor embedding (t-SNE) plot of the aggregated and batch-corrected gene expression data from our study (MO, DC and tolDC) and two additional public datasets (GSE40484 (moMAC, moDC, cDC2, CM (classical MOs) and NCM (non-classical MOs) and GSE99056 (M-MAC (M2 macrophages) and GM-MAC (M1 macrophages)). The four different groups obtained using k-means clustering are represented with grey ellipses of multivariate t-distributions.