Fig 2: Human DC Heterogeneity, Related to Figure 7(A). Violin plots showing expression distribution of mouse DC subset marker genes across human peripheral blood DC and monocyte clusters identified in Villani et al. (2017).(B). Representative flow cytometric analysis of mouse peripheral blood cDC2s showing absence of T-bet (RFP)+ cDC2s.(C). Gating strategy for FACS-isolation of human spleen DCs for scRNA-seq. DCs were defined as live, LIN(CD3,CD56,CD19)-CD14–CD11C+HLA-DR+.(D). Representative flow cytometry analysis of human spleen cDC2s gated as Lin(CD3,CD56,CD19)–CD14–CD11c+HLA-DR+CD123–XCR1–CLEC4A+ cells. Left panel: cell surface expression of CD1c and CLEC10A by cDC2s. Right panel: overlay of CLEC10A+ and CLEC10A– cDC2s distinguished by differential expression of CLEC4A and FceR1a. Summary bar graphs show frequency of CD1C+CLEC10A+ and CD1C+CLEC10A– cDC2s as a percentage of cDC2s (n = 4 individuals).(E). t-SNE embedding of 9,315 FACS-isolated CD45+ immune cells from two melanoma tumors. Colors indicate unsupervised clustering by Phenograph (left panel) or classification based on expression of canonical markers and correlations with bulk RNA-seq data (right panel). Each dot represents an individual cell.(F). Pearson correlations between cluster centroids in (F) and bulk RNA-seq data from purified immune populations (Jeffrey et al., 2006, Novershtern et al., 2011)(G). t-SNE map of 2,122 myeloid cells identified in (F). Colors indicate patient sample (left) or unsupervised clustering by Phenograph (right panel). Each dot represents an individual cell.(H). Heatmap of normalized, log transformed and MAGIC imputed expression of top 20 differentially expressed genes, defined by the highest earth mover’s distance (EMD), per Phenograph cluster in E. The colored bar at the top of the heatmap shows assignment of cells to clusters labeled in F, right panel.(I). t-SNE map of human melanoma myeloid cells (H) colored by imputed expression of labeled genes.

Fig 3: Conservation of DC Subsets across Species(A) t-SNE map of human peripheral blood DCs from Villani et al. (2017), colored by cell type or log-transformed expression of labeled genes (middle and right panel).(B) Expression of CD1c and CLEC10A within peripheral blood cDC2s. Bar graph shows summary frequencies for four individual donors.(C) Distribution of expression changes between T-bet+ and T-bet– cDC2s. Genes up- or downregulated in human “DC2” versus “DC3” cDC2s (Villani et al., 2017) are shown.(D) t-SNE map of 4,465 human splenic DCs. Colors indicate unsupervised clustering by Phenograph (left) or classification based on expression of canonical markers (right). Each dot represents an individual cell.(E) Expression of canonical myeloid genes across the transcriptionally defined DC clusters from (D).(F) Heatmap reporting scaled, imputed expression for the top differentially expressed genes for each cluster identified in (D).(G) Pearson correlation between human and mouse spleen scRNA-seq DC clusters. Mouse spleen DC clusters from Figure 1E.(H) Heatmap reporting scaled, imputed expression for the 103 top varying TF genes across cDC2s in each species. Rows are ordered by the TF gene cluster determined using Phenograph and columns are ordered by the annotated cDC2 cluster.(I) Heatmap reporting overlap of top varying TF genes for mouse and human TF gene clusters identified in (H). Each colored count indicates the number of TFs that belong to both the mouse (rows) and human TF gene cluster (columns) indicated. TF gene clusters are further annotated with the cDC2 subset concordant with their expression profile in (H).(J) t-SNE map of human splenic DCs colored by imputed expression of canonical human DC genes (top) or cDC2 lineage-defining TF and marker genes (middle and bottom) identified in Figures 2A and 3D.(K) Diffusion component analysis of human spleen DC clusters identified in (D) illustrating gradients from “AS” DCs to cDC2A and cDC2B clusters.See also Figure S6 and Tables S5, S6, and S7.