Fig 2: In vitro induction of DCs from HSPCs. (A) Workflow for the induction of HSPC-DCs. (B) Flow cytometry of pDCs (CD66b-CD14-CD1c-CD123+CD303-) and CD1c+cDCs (CD66b-CD14-CD1c+) populations after 14 days of induction from CD34+HSPCs. On day 14, (C) After stimulated with the TLR4 and TLR7 agonists for 2 hours, mRNA of FACS sorter purified CD1c+cDCs was extracted to measure gene expression using real time RT-PCR. (D) CD123+pDCs and CD303+pDCs were highly purified using FACs sorter. Then both populations were stimulated with the TLR7 and TLR9 for 2 hours, mRNA was collected to measure gene expression using real time RT-PCR. Error bars indicate mean ± SD. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 3: Sepsis patients exhibit increased levels of late apoptosis on DC subsets rather than on monocytes. (A) Flow detection of apoptosis of DC subsets. (B) Percentage of early apoptotic pDC and late apoptotic/dead pDC from healthy donors(n = 10), sepsis 1-2d (n = 16) and sepsis 3-7d (n = 8). (C) Percentage of early apoptotic CD1c+cDC and late apoptotic/dead CD1c+cDC from healthy donors (n = 10), sepsis 1-2d (n = 21) and sepsis 3-7d (n = 14). (D) Percentage of early apoptotic monocytes and late apoptotic/dead monocytes from healthy donors (n = 30), sepsis 1-2d (n = 22) and sepsis 3-7d (n = 12). Error bars indicate mean ± SD. *P < 0.05; **P < 0.01; ****P < 0.0001.

Fig 4: Immune cells exist in preovulatory follicles. (A) UMAP showing immune cell types in preovulatory follicles. (B) The proportion of immune cells in each preovulatory follicle sample. (C) Flow cytometry analysis identifying CD45+ immune cells, macrophages (sorted with CD45 and CD68), DCs (sorted with CD1C and HLA-DR), T cells (sorted with CD45 and CD3) and neutrophils (sorted with CD11B and CD15) in a single preovulatory follicle.

Fig 5: CD1c+ DCs in LPS-BAL are likely to be tissue-recruited blood CD1c+ DCs. a Expression of surface antigens by flow cytometry on DC2/3 from HC blood (green), SS-BAL (blue) and LPS-BAL (red) relative to isotype control (gray). Antigens predicted to discriminate between blood and tissue CD1c+ DCs were tested. Representative plots from more than three experiments are shown. b Dendrogram showing hierarchical clustering of monocyte/macrophage and DC gene expression by indicated subsets isolated from HC blood and SS/LPS BAL. In vitro monocyte-derived DCs were also included. Clustering used Pearson correlation metric. Monocyte/macrophage and DC identifying genes were taken from McGovern et al.28. The 25 genes available on the NanoString Immunology v2 panel were used. c Proliferation of allogeneic peripheral blood T cells measured by CSFE dilution during 7-day co-culture with or without MP subsets isolated from LPS BAL. Flow cytometry plots are gated on CD3+ T cells from a representative experiment. Summary graph shows 2–3 replicates per subset. Bars show mean ± SEM. Means were compared by one-way ANOVA with Dunnett’s multiple comparison test of DC2/3 against other subsets. *p < 0.05. d Analysis of differentially expressed genes (DEGs) by DC2/3 from SS/LPS-BAL and HC blood. Comparisons were made by unpaired t-test with p < 0.05 and >3-fold difference in mean expression. Venn diagram shows DEGs in DC2/3 from SS-BAL, LPS-BAL, and HC blood. Circles represent DEGs between SS BAL and blood (cyan; 154 genes, 33%) and between LPS-BAL and blood (yellow; 124 genes, 39%). The overlapping circles represents DEGs shared between comparisons (100 genes, 21%). The top 10 upregulated genes (standard type) and top 5 downregulated genes (italic type) in BAL relative to blood are listed. Heatmap shows DEGs common to DC2/3 in both SS-BAL and LPS-BAL relative to HC blood. Genes with >10-fold difference in mean expression are shown. Genes discussed in the text are colored burgundy. e Heatmap showing DEGs between DC2/3 from SS-BAL and LPS-BAL. Genes with >10-fold difference in mean expression are shown. Genes discussed in the text are colored burgundy