Fig 2: Mtb infection increases monocyte output from CD34+ cells in vitro.Purified CD34+ cells were exposed to live Mtb H37Rv or HK Mtb H37Rv (MOI3) for 10 days and flow cytometry was employed to determine the mature cell frequencies in the cell cultures. Representative dot plots of (a) monocytes (CD14+), (b) neutrophils (CD16+CD66b+), (c) megakaryocytes/platelets (CD41a+) and (d) classical myeloid dendritic cells (BDCA1+CD14low) in uninfected and Mtb-infected CD34+ cell cultures. Graphs show frequencies of (e) CD14+, (f) CD16+CD66b+, (g) CD41a+ and (h) BDCA1+CD14low events in uninfected (open circles), live Mtb-infected (blue diamonds) or HK Mtb-exposed (red diamonds) cell cultures at day 10. Each symbol represents one individual experiment. Results are means ± SEM of data pooled from 3 to 9 independent experiments. **p≤0.01 between Mtb vs uninfected or HK Mtb groups. (i,) Histograms show the expression of CD11b, HLA-DR, CD64 and CD16 in CD14+ events from a). Black dashed lines: Uninfected control. Blue solid lines: Mtb-infected group. Data representative of 5 independent experiments. (j,) Frequency of CD14+CD16+ events in Mtb-exposed cell cultures after 10d. Contour dot plot of CD14+CD16+ frequencies from one representative donor. Open circles: Uninfected control. Blue circles: Mtb-infected group. Each symbol represents an individual experiment. Pooled data of eight independent experiments, n = 5 different donors. p=0.076 between Mtb vs uninfected groups.10.7554/eLife.47013.014Figure 3—source data 1.Raw data from Figure 3.

Fig 3: 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 4: CD14 Expression Distinguishes between CD1c+DC Subsets Generated In Vitro(A) Gating strategy used to identify DCs and monocytes generated from HC BM CD34+ progenitors at day 21 (D21) of culture on OP9 in the presence of SCF, FL, and GM-CSF. A minimum of two antigens was used to define the following populations: CD141+CLEC9A+ cDC1s, CD123+CD303+CD304+ pDCs, CD2+CD1c+ DCs encompassing CD14+ and CD5+ populations, and CD14+CD1c-CD2- monocytes.(B) Flow analysis of the expression of population-specific markers across in vitro-generated monocytes (black), CD14+ (orange), CD5+ (red), or CD5- (pink) CD14-CD1c+ DCs as defined in (A).(C) Intracellular flow evaluation of the expression of IRF4 and IRF8 by PB and culture-derived monocytes and DCs, gated as shown in Figure 1A and (A), respectively.(D) Kinetics of DC culture output over 21 days plotted as the number of DCs or monocytes generated per CD34+ progenitor. n = 6 donors with minimum n = 3 at each time point. Dots and bars show mean and SEM.(E) Flow analysis of the expression of population-specific markers by FACS-purified PB monocytes and CD1c+ subsets at day 7 of culture.(F) Flow analysis of CD14 expression by FACS-purified PB CD1c+subsets at day 7 of culture. Histogram shows a representative example from n = 7 (CD14- DC3 and CD5- DC2) or n = 5 (CD5+ DC2) HC donors, summarized in the graph. Bars represent mean ± SEM. Circles represent individual donors. ***p < 0.005 by paired two-tailed t test.(G) PCA of NanoString gene expression of FACS-purified PB DCs (“PB”) (n = 3) and DCs derived from BM CD34+ progenitors at D21 of culture (“C”; black outline) (n = 3) after removal of a “culture signature” generated by pairwise comparison of all PB versus all culture-generated cells.(H) Heatmap of Z scores of differentially expressed signature genes (NanoString) derived from pairwise comparisons of PB CD1c+ DC subsets and monocytes, shown next to the Z scores of expression of the same genes by culture-derived CD14- and CD14+ DCs and monocytes.(I) Intracellular flow analysis of in vitro cytokine elaboration (percentage of positive cells) in response to TLR agonists, as described in Figure 1D, by CD14+CD1c- monocytes (black bars), CD14+ DC3s (orange), and CD14- DC2s (red) generated from n = 4 BM CD34+ progenitors at day 21 of culture. p values from paired two-tailed t tests; *p < 0.05; **p < 0.01; ***p < 0.005. Bars show mean ± SEM.See also Figure S2.

Fig 5: IRF8hi and IRF8lo Pathways Are Differentially Compromised in IRF8 Deficiency(A) PB flow analysis of monocytes and DCs in subjects carrying heterozygous IRF8R83C or IRF8R291Q mutation (Het), their child carrying IRF8R83C/R291Q (Bi), and a carrier of dominant-negative heterozygous mutation IRF8V426fs (Dom) compared with HC (Cont).(B) Trucount quantification of PB DCs and monocytes in subjects carrying IRF8 mutations (gating shown in Figure S6A; Hambleton et al., 2011; Bigley et al., 2018)). Cont, n = 25; Het, n = 4 (IRF8R83C, IRF8R291Q, and two subjects carrying IRF8K108E); Dom, n = 3 (IRF8V426fs); Bi, n = 2 (IRF8R83C/R291Q and IRF8K108E/K108E).(C) Flow cytometry phenotyping of CD1c+ DC subsets derived from the CD1c+CD2+ gate (gray) in (A) to identify CD14+ DC3s (orange), CD14-BTLA- DC3s (light orange), CD5-BTLA+ DC2s (light red), and CD5+BTLA+ DC2s (red).(D) Proportion of CD1c+ DC subsets (gated as in C, from the individuals represented in B). C, control; H, heterozygous parents; D, dominant-negative heterozygotes (IRF8V426fs).(E) Flow analysis of DC and monocyte precursors in PB of subjects carrying IRF8 mutations as shown, gated as in Figure 4C.(F) Proportion of DC and monocyte precursors out of all pre-DCs in PB of subjects carrying IRF8 mutations, gated as in (E). C, control; H, heterozygous; D, IRF8V426fs(G and H) Intracellular flow analysis of in vitro cytokine elaboration (percentage of positive cells) by CD14+ monocytes (black), CD14+ DC3s (orange), CD14-CD5-CD1c+ DCs (gray), and CD5+ DC2s (red) (G) and CD2+ pre-pDCs and pDCs from HC (n = 8) and subjects carrying heterozygous IRF8R83C, IRF8R291Q (mean of technical duplicates) or IRF8V426fs (IRF8, red-outlined bars) (H). See also STAR Methods and Figure 1H.Bars show mean ± SEM, and circles represent individual subjects. *p < 0.05; **p < 0.01; ***p < 0.001; ˆp = 0.053, Mann-Whitney U. See also Figure S6.