Fig 2: pDCs and cDC1s Can Be Efficiently Generated from Human CD34+ Cord Blood Cells(A) CD34+ cord blood (CB) cells were expanded with FLT3L, SCF, TPO, and IL-7 (FST7) for 7 days. Subsequently, they were differentiated on OP9, OP9_DLL1, or OP9+OP9_DLL1 feeder layer cells, with FLT3L, TPO, and IL-7 (FT7), for 14–21 days.(B) On day 18 of differentiation, within the live-cell gate, pDCs were identified as CD206-CD14-CD123+BDCA2+ cells and cDC1s as CD206-CD14-CLEC9A+ cells positive for BDCA3 or CADM1. Plots show one experiment using CB204 donor CD34+ cells for parallel differentiation on the 3 feeder layers. Data are representative of 5 experiments each performed with cells from one or two different donors. Pie charts show mean percentage of pDCs or cDC1s within the live-cell gate from the 5 experiments.(C and D) Frequencies (C) and numbers per well (i.e., per 104 expanded CD34+ cells) (D) of cDC1s (top) and pDCs (bottom) among total live cells on day 18 after differentiation on the 3 different feeder layers. Graphs show individual results for each of the 6 donors.See also Figures S1 and S2 and Table S1.

Fig 3: pDCs and cDC1s Generated from Human CD34+ CB Cells Share Key Functional Characteristics with Their Blood CounterpartsOP9+OP9_DLL1 FT7 cultures were stimulated with the indicated agonists. cDC1s were gated as live CD14-CD206-CD123neg-to-lowBDCA2-CADM1+ cells and pDCs as CD14-CD206-CD123+BDCA2+ cells. Plots are representative of 4 donors from 2 experiments.(A) Activation marker expression.(B) Intracellular expression of IFN-a/?.(C) Intracellular expression of IL-12p40 and TNF.

Fig 4: CD1c+ DC Heterogeneity Is Evident in Human BM(A) Flow phenotyping of CD1c+ DCs from HC PB mononuclear cells (PBMCs) (representative example of n = 22), distinct from SIRPA-CD141+ cDC1s, CD123+CD303/4+ pDCs, and CD88+monocytes (Mono). CD14+CD163+BTLA- (orange), CD14-CD163+BTLA- (light orange), CD163-BTLA+CD5- (light red), and CD163-BTLA+CD5+ (red) CD1c+ DC subsets are indicated.(B) 3D representation of CD14, CD5, and BTLA expression (flow cytometry) across the CD1c+ DC population. Heatmap shows expression of CD163.(C) PCA of NanoString gene expression profiling of fluorescence-activated cell sorting (FACS)-purified DC subsets from n = 3 HC PBMCs. CD1c+ DCs were purified based on their expression of CD14, CD5, and BTLA (A).(D) Intracellular flow analysis of in vitro cytokine elaboration (percentage of positive cells) by PB monocytes (black) and CD1c+DC subsets CD14+ (orange), CD14-CD5- (gray), and CD5+ (red) from n = 9 HC donors in response to 14-h stimulation with TLR agonists (CpG, poly(I:C), CL075, and lipopolysaccharide [LPS]). p values were derived from paired two-tailed t tests; *p < 0.05; **p < 0.01; ***p < 0.005. Bars show mean ± SEM, and circles represent individual donors.(E and F) Representative examples of the flow profiling of DC subsets in human spleen (n = 3), dermis (n = 3) (E) and BM (n = 13) (F), gated as in (A). Histograms show CD163 and BTLA expression on CD14+ (orange), CD5+ (red) and CD14-CD5- (gray) CD1c+ DCs.(G) tSNE visualization of the expression of TFs and surface markers across HC PB and BM lineage(lin, CD3,19,20,56,161)-HLA-DR+ cells by CyTOF analysis. Black gates indicate the CD1c+DC population distinct from CD88+monocytes, CLEC9A+cDC1 and CD303+pDC. Red and orange gates indicate expression of lymphocyte- or monocyte-associated antigens, respectively.(H) Hierarchical clustering of single-cell transcriptomes of mature DCs from BM using all protein-coding, non-cell-cycle genes. Marker genes were identified within SC3 with parameters p < 0.01, area under the receiver operating characteristic curve (AUROC) > 0.85; cluster 1, pDCs (GZMB, JCHAIN); cluster 2, monocytes (S100A8, VCAN); cluster 3, CD14+ DC3s (HLA-DPB1); cluster 5, cDC1s (CD59). The top rows show fluorescence intensity of surface antigens (“Antigens”) from index-sorted cells, and “Phenotype” denotes their classification defined by surface markers.See also Figure S1.

Fig 5: Two Trajectories of DC Development Connect the Progenitor Compartment with Mature DCs(A) Flow gating strategy used to identify DCs and their precursors in BM, including CD141+ cDC1s; CD1c+ DCs; AXL+CD5+ cells composed of CD123hiCD11c- (light pink) and CD123intCD11c+ (dark pink) fractions; CD2+ (light blue) and CD2- (dark blue) pDCs; CD123+CD303/4lo cells (turquoise); SIRPA/B-CD123intCD141- (lightest purple) and CD141lo (light purple) populations; and CD123-SIRPA/B CD34int (brown), CD34-CD2+ (dark orange), and CD34-CD2- (gray) precursors.(B) The output of in vitro culture of CD34int DC precursors FACS-purified from BM using the gating strategy described in (A). Population-specific output is expressed as a proportion (%) of the total cells captured by all DC and monocyte gates. CD123hi303/4lo (six experiments from four donors; 6;4); CD2+ pre-pDCs (5;3); CD123hi5+ (4;3); CD123int5+ (4;3); CD34intCD123int (4;4); CD34intSIRPA+ (5;5); SIRPA+2+ (4;4); SIRPA+2- (4;4). Bars represent mean + SEM, and circles represent individual experiments. Significant differences in the proportional output of DC2s versus DC3s (red) or DC3s versus monocyte (black) are indicated: *p < 0.05; **p < 0.01 (paired, two-tailed t test).(C) Flow gating strategy from (A) applied to lin-HLA-DR+ cells from HC BM fractionated by high, intermediate, and low CD34 expression, next to blood (columns) for comparison of antigen expression levels among progenitor, precursor, and mature populations. Individual DC lineages are ordered in rows.(D) Proliferative potential of FACS-purified DC and DC precursors estimated by CFSE dilution (see STAR Methods). CD34+progenitors and CD14+monocytes were included as positive and negative controls, respectively. The CFSE dilution histograms for each precursor are grouped and ordered according to their proposed position in the developmental trajectory for each DC lineage. Plots shown are representative of n = 3 experiments (summarized in Figure S4H).(E and F) tSNE visualization of the first 20 principal components (explaining 35% total variance) of the transcriptomes of 244 single cells adaptively sampled from lin-HLA-DR+ CD34neg-int precursor and mature DC populations of BM. tSNE plots are annotated by the gate of origin from index-linked flow (E) or by 15 clusters generated from hierarchical clustering of all protein-coding non-cell-cycle genes (F), independently of surface phenotype (Figure S4K).(G) Heatmaps showing the expression of key surface antigens (SAs) (index-linked flow) or log2 gene expression of TFs and FLT3 (scRNA-seq) across the tSNE plot in (E) and (F). Black circles represent regions of high or low IRF8 expression, marked Al or Bl, respectively. The differential expression patterns of these regions correspond to the patterns of regions “A” (IRF8hiCD123intGMP) and “B” (IRF8loGMP33+) in Figures 3E–3G.(H and I) Diffusion map generated with all protein-coding, non-cell-cycle genes to infer pseudo-temporal ordering of cells and reconstruct lineage branching. (H) Cells are colored according to the hierarchical clusters generated in Figure S4K. (I) IRF8 expression (log2). Diff C, diffusion component.(J) Violin plot of differential IRF8 expression (log2) in clusters 10 (SIRPA+34int) and 12 (early pre-DC2). **p < 0.001 by Mann-Whitney U.(K and L) MFI of intracellular IRF8 by flow analysis across gates identifying BM 34intSIRPA+ pre-DC3s and pre-mono and CD123hiCD5+ early pre-DC2s (K) and CD5+ DC2s and CD5- DC3s (L) (n = 4). *p = 0.028 by Mann-Whitney U.See also Figure S4.