Fig 1: DC3s Are a Discrete Subset of CD88-CD1c+CD163+ Cells in Human Peripheral Blood(A) Gating strategy used to define mononuclear phagocytes expressing CD14 and/or CD1c. Cells expressing CD14 and/or CD1c were sorted by flow cytometry from 3 healthy donors and pooled before scRNA-seq analysis. To improve the resolution of CD1c+ subsets, the cellular input was enriched in CD1high cells (Figure S1A). Single cells were isolated using a droplet-based approach and sequenced. Dimensionality reduction of scRNA-seq data was performed using dimensionality reduction (t-distributed stochastic neighbor embedding [tSNE]). Clusters A, B, C, and D were identified using the shared nearest neighbor (SNN) clustering algorithm. Each dot represents an individual cell (n = 1,622).(B) Hierarchal clustering of groups A, B, C, and D based on average gene expression (14,933 genes).(C) Absolute number of differentially expressed genes (DEGs) for pairwise comparisons between groups A, B, and D.(D) Heatmaps displaying relative expression of up to 20 DEGs defining each cluster.(E) Violin plots illustrating expression probability distributions across clusters of representative DEGs (226 total DEGs). Feature plots display the average expression of groups of genes (identified in violin plots) in each cell of the tSNE plot defined in (A).(F) Expression distribution across clusters A, B, C, and D of gene signatures identified by Villani et al. (2017) and Yin et al. (2017). (**p < 0.01, ****p < 0.0001, one-way ANOVA test)(G) Identification of 4 subsets within CD14lo to hi CD1clo to hi cells by unsupervised clustering of flow cytometry data using the FlowSOM algorithm. tSNE and unsupervised clustering were performed using the following markers: CD88, CD1c, FceRI, CD14, CD163, BTLA, CD123, and CD5. tSNE plots (right) display the relative expression of each marker among the subsets. Dot plots (below) show the expression of specific markers in clusters 1, 2, and 3 when combined in 2-dimensional analysis.(H) Improved gating strategy for identification of cDC2s, DC3s, and CD14+ monocytes in circulating PBMCs and histograms showing expression of S100A8/9, FceRI, CD5, CD14, CD116, and CD206.(I) Principal-component analysis (PCA) for bulk-sequenced mononuclear phagocyte populations as defined in (H).(J) Cluster dendrogram of the different cell types using the 2,000 most variable genes.(K) Heatmaps comparing the relative expression of markers discriminating clusters in scRNA-seq analysis (A, B, C, and D, left) and in bulk RNA-seq analysis on sorted subsets based on the gating strategy defined in (H) (right).See also Figure S1 and Table S1.