Fig 2: Flow cytometry analysis from approximately day 31 of co-cultureCells were harvested after 3 weeks of MS5 co-culture and stained for surface markers.(A) Cells were gated for singlets and scatter and then for viability (7AAD) and CD45 expression as indicated in the figure (top panel). Expression of CD19 and CD34 is shown within the 7AAD-CD45+ fraction (second panel, left). The CD19 positive fraction is further gated for expression of CD10 and CD20 (right). Counting beads were used in this experiment and can be seen in the scatter plot in the top middle panel.(B) Cells were gated for singlets and scatter (as in Figure 8A, plots not shown). Contour plot of viability (7AAD) and CD45 expression as shown in the figure (left). Further gating on 7AAD-CD45+ cells for B lymphoid (CD19) and, within the CD19- gate, myeloid (CD11b and CD14) markers are shown to the right.

Fig 3: Polarization of low-grade inflammatory monocytes through the reduction of IRAK-M.(a–e) BMMs from C57 BL/6 mice were cultured with M-CSF (10 ng ml-1) in the presence LPS for 5 days and SP600125 was added to some cultures. Fresh LPS and SP600125 was added every 2 days. (a) JNK phosphorylation within CD11b+Ly6C++ monocytes treated with super-low-dose LPS (0.1 ng ml-1) was determined by flow cytometry. (b) Fluorescent RNA probe for miR-24-3p was added to the cell cultures 16 h before harvesting. The expression levels of miR-24-3p within CD11b+Ly6C++ monocytes were examined by flow cytometry. (c) The expression levels of SR-B1 within CD11b+Ly6C++ monocytes were examined by flow cytometry. (d,e) Western blot analyses of SMAD4 and IRAK-M expression in the cell cultures. (f,g) Peripheral blood mononuclear cells collected from healthy individuals were cultured with M-CSF (100 ng ml-1) in the presence super-low-dose LPS (5 pg ml-1) for 2 days. The frequency of inflammatory monocytes (f) and SMAD4 expression within CD14+CD16+ population (g) was determined by flow cytometry. Quantified data are shown from cells with indicated treatment (n=3) and results are representative of three experiments. Error bars show means±s.e.m.; NS, not significant; *P<0.05, **P<0.01 and ***, P<0.001; (a,f,g) Student's t-test; (b) one-way analysis of variance.

Fig 4: Gating Strategy. Overlapping events were ruled out from the FSC-H/FSC-A analysis. Subsequently, the populations were selected by size and complexity. From the lymphocyte region, T lymphocytes were identified as CD3 + and CD4 + cells, and CD19+ for B lymphocytes. Monocytes were identified as CD14+ cells. Once the three populations had been selected, the positive cells for CD74, CD44, CXCR2, CXCR4 and CXCR7 receptors were identified from the pre-established FMOs (CD44 are represented in CD4+ T lymphocytes, CD74 in B lymphocytes, CD44 in monocytes and CXCR2 in granulocytes). FSC-H/FSC-A; FSC-Height (FSC-H) by FSC-Area (FSC-A). FMO; Fluoresence minus one.

Fig 5: 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.