Fig 2: Distinct cDC2 Subsets Express Divergent Transcription Factors and Cell Surface Markers. Related to Figure 2(A). Enrichment of GO pathways (biological processes) in cluster 14.(B). Graph showing AUC score (x axis) for genes differentially expressed between T-bet+ and T-bet– cDC2 clusters. Earth movers distance (EMD) on the y axis. Dashed lines represent µEMD ± 3sEMD.(C). Graph showing AUC score (x axis) for genes differentially expressed between T-bet– cluster 10 and all other cDC2 clusters. EMD on the y axis. Dashed lines represent µEMD ± 3sEMD.(D). t-SNE map of splenic DCs colored by imputed expression of Clec10a and Mgl2 demonstrating co-expression of these genes by cells in cluster 10.(E). Representative flow cytometry plots showing expression of CLEC12A, CLEC10A and T-bet (RFP) in cDC2s isolated from spleen, peripheral lymph nodes (PLN), mesenteric lymph nodes (MLN), large intestine lamina propria (LI), liver and lung (left). Frequency of CLEC12A+ and CLEC10A+ cells within all cDC2s (right). Each symbol represents a single mouse. Data representative of 2 independent experiments.(E). Cytospin analysis of Wright-Giemsa stained sorted cDC2 subsets.

Fig 3: 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 4: T-bet and ROR?t Expression Delineates Distinct cDC2 Subsets(A) Heatmap reporting scaled, imputed expression for the top 10 differentially expressed genes for each cDC2 cluster identified in Figure 1E. Genes of interest are shown on the right.(B) Expression of Rorc transcript in bulk sorted T-bet– cDC2s, T-bet+ cDC2s, and cDC1s.(C) Expression of CLEC12A, Esam, and CLEC10A in cDC2s from Tbx21RFP-Cre mice. Far right: CLEC12A and CLEC10A expression within T-bet– cDC2s.(D) Summary graph for (C). Each symbol represents one mouse.(E) Enrichment of GO pathways in in T-bet+ versus T-bet– cDC2 clusters.(F) Correlation between splenic and MLN cDC2 transcriptomes.Error bars represent mean ± SEM. See also Figures S2 and S7 and Tables S1, S2, and S3.

Fig 5: Adipose eosinophil and macrophage populations are influenced by sex, diet, and RELMα.(A) t-Stochastic neighbor embedding (tSNE) analysis to identify stromal vascular fraction (SVF) leukocyte populations. (B) tSNE analyses of SVF from the different groups (male ♂, female ♀, wild-type [WT], or RELMα knockout [KO]) after 12 weeks of diet exposure (Ctr or high-fat diet [HFD]) revealed changes in eosinophil RELMα-dependent and diet-induced changes in eosinophil (red outline) and macrophage (green outline) subsets. (C, D) CD11c surface expression in CD45+MerTK+CD64+ macrophages was analyzed by tSNE, where dashed red outline shows CD11cHi cells, and quantified. (E) CD301b surface expression on SVF macrophages was examined by tSNE and quantified by mean fluorescent intensity (MFI). (F) Siglec-F surface expression on CD45+SiglecF+CD11b+ SVF eosinophils was examined by tSNE and quantified by mean fluorescent intensity (MFI). tSNE data are one representative animal per group. All other data are presented as individual points for each animal, where lines represent group means ± standard error of the mean (SEM). Statistical significance was determined by three-way analysis of variance (ANOVA) Sidak’s with multiple comparisons test (ns, no significant; *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Data are representative of 3 experiments with 4–6 mice per group.