Fig 2: Environmental Cues Drive Distinct DC2 Differentiation Pathways within the Spleen, Related to Figure 5(A). Gating strategy for the identification of DC progenitors in the bone marrow (BM)(B). Palantir pseudo-time analysis of differentiation potential and branch probabilities from the Siglec-H+ pre-DC state to T-bet+ cDC2 and T-bet– cDC2 terminal states.(C). Plots showing Palantir differentiation potential (y axis) along Palantir pseudo-time (x axis) for Siglec-H+ DC and T-bet+ cDC2s (top) or Siglec-H+ DC and T-bet– cDC2 clusters (bottom)(D). Plots showing the top two diffusion component embeddings for Siglec-H+ DC and T-bet+ cDC2 clusters (top) or Siglec-H+ DC and Tbet– cDC2 clusters (bottom). Black arrow indicates Siglec-H+ DC cluster cells adjacent to cells from the proliferative T-bet+ cDC2 clusters 6 and 8.(E). Top panel: plots showing probability of each cell being within 20 nearest neighbors of randomly sampled shortest paths from the Siglec-H+ DC to the indicated end points. Middle panel: plots showing the proportion of cells belonging to Siglec-H+ DC, T-bet+ cDC2, or T-bet– cDC2 from 20 nearest neighbors of randomly sampled shortest paths. Bottom: plots showing diffusion distance step sizes for each step along the indicated shortest paths (bottom panel). Colors illustrate cluster membership.(F). Graph showing AUC (x axis) for genes differentially expressed between Siglec-H+ DC cluster (cluster 11) and all other cDC2 clusters. EMD on the y axis. Dashed lines represents µEMD ± 3sEMD.(G). Gating strategy for FACS-isolation of MHCII+ ILC3s: Lin = CD3, CD19, CD49b, Siglec-F.(H). Heatmap reports scaled expression of 3550 differentially expressed genes (log2FC > 1, FDR < 0.01) between ILC3s and Ror?t fm cDC2s. Selected genes listed to the right.(I). Representative flow cytometric analysis of phenotypes of splenic progeny from Tbx21RFP-cre CD45.2+Ly6C-CD64–MHCII+CD11c+Siglec-H+ pre-DCs adoptively transferred into sub-lethally irradiated CD45.1 recipient mice 7 days earlier (data from one experiment with n = 3).J. Sort purified T-bet+ or T-bet– cDC2 were cultured for 24hrs in the presence of LPS, CpG, TNF-a or IFN-?. Representative overlay histogram showing the expression of RFP(T-bet) at 24hrs. Data representative of 2 (TNF-a) or 4 (all other cytokines/TLR agonists) independent experiments, n = 2-3.

Fig 3: Single-Cell Survey Reveals Heterogeneity of cDC2s, Related to Figure 1A. Representative histogram showing expression of T-bet (RFP) in splenic cells from Tbx21RFP-cre mice.(B). Expression of T-bet in CD11b+XCR1+ DCs from the intestinal lamina propria. Data representative of > 5 independent experiments, with at least 3 mice per experiment.(C). Expression of T-bet in splenic myeloid cells. Cells were defined as: (i) Ly-6Chi monocytes (Lin –Ly6C+Ly6G–CD11b+CX3CR1+); neutrophils (Lin–Ly6C+Ly6G+); macrophages (Lin–CD64+Ly6C–). Lineages (Lin) were defined as: CD3e, CD90.2, CD19, CD49b and Siglec F. Each circle represents an individual mouse, error bars represent mean ± SEM.(D). Left: Gating strategy for single-cell sorting. DCs were defined as Lin(CD3, CD19, CD90)–Ly6C–CD64–CD11c+MHCII+. Two populations were sampled: RFP+ DCs and RFP– DCs (encompassing XCR1+ cDC1s, CD11b+RFP– and CD11b–XCR1– DCs). Right: Post-sort purity of RFP+ and RFP– cells. Contaminating population of Ly6C+ cells identifiable on post-sort purity (lower panel).(E). Similarity of splenic CD11c+MHCII+ cells to reference myeloid cells (ImmGen Consortium) Colors represent the Pearson correlation between the mean gene expression from the dendritic cell cluster in the rows and the bulk reference transcriptome in the columns.(F). Top 20 positive and negative gene loadings of PC1 for T-bet+ cDC2 clusters after cell-cycle correction (left panel). Scatterplot of PC1 and PC2 for T-bet+ cDC2 clusters after cell-cycle correction (right panel).

Fig 4: Stab2-iCreF3;R26YFP mice show reporter activity restricted to sinusoidal endothelial cells in the bone marrow.a–c Immunofluorescence (IF) staining for endothelial markers of Stab2-iCreF3;R26YFP bone marrow from 6-month-old female mice. Co-IF of YFP, DAPI and a EMCN (n = 3), b Stab2 (n = 3), c CD32b, a-SMA (n = 3), d VEGFR3, and e CAV1. Scale bars: 50 µm. f–h Hematopoietic marker staining of Stab2-iCreF3;R26YFP bone marrow from 5-month-old female mice for IF and flow cytometry analysis. f Co-IF of YFP, DAPI, CD45 (n = 3). Scale bars: 50 µm. Representative graphs of flow cytometry YFP quantification in g Lin-Sca-1+Kit+, Lin-Sca-1-Kit+ cells, T cells (CD3+), B cells (CD19+), myeloid cells (Gr1-CD11b+, Gr1+CD11b+) of g bone marrow and h spleen (n = 4). Source data for g, h are provided as a Source Data file.

Fig 5: 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.