Figure 3.
TLR7/8 stimulation of PBMCs induced distinct transcriptomic changes in pDCs
(A) Workflow of the scRNA-seq experiment, shown for one donor. Peripheral blood mononuclear cells (PBMCs) from time points before (TP0) and under (TP3) testosterone injections were isolated and stimulated with the TLR7/8 ligand CL097 for 2 h or left unstimulated before FACS-isolation of pDCs and subsequent scRNA-seq analysis (n = 3). Created with BioRender.com.
(B) Heatmap depicting enrichment (AUC values) of immune cell subset marker gene sets (ref.37) across all pDC clusters. Both unstimulated and stimulated pDC clusters displayed high AUC values for pDC marker genes, confirming pDC identity. Stimulated pDC clusters exhibited high AUC values for the set of ISGs, indicating effects of CL097 stimulation.
(C) Uniform manifold approximation and projection (UMAP) colored by condition. Unstimulated and stimulated pDCs from both assessed time points each form a separate group.
(D) UMAP colored by unsupervised clusters determined using Louvain method after constructing a shared nearest neighbor (SNN) graph based on k-nearest neighbors. Five clusters were associated with unstimulated pDCs and six clusters with stimulated pDCs; cluster 11 was equally distributed between both groups.
(E) Bar plots showing the percentage of cells in each pDC cluster at different stimulation conditions and time points. Cluster frequencies did not change significantly after testosterone administration (Wilcoxon signed-rank tests).