Fig 2: Differential IRF8 Expression Defines the Two Trajectories of DC Development(A–E) CyTOF analysis of FACS-purified CD45+lin(CD3,19,20,56,161)- PB and BM progenitors, precursors, and mature DCs and monocytes using a panel of 33 surface antigens and two intracellular stains (IRF4 and IRF8). (A) tSNE visualization of lin-HLA-DR+ cells, down-sampled to select 75,000 cells (20,000 CD11b+CD14+ monocytes, 4,000 CD11b+CD16+ monocytes, and 50,000 non-monocyte cells). PB (red) and BM (gray) cells were distinguished by differential CD45+ conjugate staining and displayed across tSNE space. (B) Heatmap of DC or monocyte-subset-specific antigens displayed on tSNE plots as in (A) (blue-yellow-red scales represent channel values). “Mature cells” plot shows the location of DC and monocyte subsets and CD34+ progenitors, identified by back-gating from bivariate plots (Figures S5B–S5D). (C) The location in tSNE space of IRF8hi (red) and IRF8lo (orange) expressing cells identified by (1) standard gating on a bivariate plot of IRF8 versus CD304 and superimposition of these gated cells on tSNE space and (2) a heatmap of IRF8 expression across all cells. (D and E) Location in tSNE space of progenitors and precursors with pDC, cDC1, or DC2 (D) and DC3 or monocyte (E) potential as defined by previous experiments, identified by back-gating from bivariate plots (Figures S5B and S5C), and heatmaps of associated antigens.(F) Diffusion map generated with 14,000 cells including GMPs, precursor and mature DCs, and monocytes. Populations were identified and color-coded according to Figures 3A (progenitors) and 4A (precursors, DCs, and monocytes), applied to CyTOF data as shown in Figures S5B and S5C. Heatmaps show the expression (log2) of IRF8 and key antigens superimposed across the diffusion map trajectories. See also Figure S5E. Diff C, diffusion component.(G) Histograms summarizing IRF8 protein expression by flow cytometry (MFI) in progenitors, precursors, and mature cells of pDC, cDC1, DC2, and DC3 lineages from BM and PB. Bars show mean ± SEM. Circles show individual donors (BM progenitors, n = 4; BM and PB precursors and mature DCs, n = 3).See also Figure S5.

Fig 3: 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 4: The treatment with lenvatinib showed cross-family compensatory mechanisms and various regulatory effects on the interaction of VEGFR2-neuropilin-1(NRP-1)-PDGFRβ. (A) HUVECs were treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF for 1 hour and then immunoprecipitated by PDGFRβ. The pulldown samples were analyzed by Western blotting for VEGFA (upper panel), phosphorylated tyrosine (p-Tyr, middle panel), and PDGFRβ (lower panel). (B) HUVECs were treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF and then immunoprecipitated by VEGFR2. The pulldown samples were analyzed by Western blotting for VEGFA (upper panel), phosphorylated tyrosine (p-Tyr, middle panel), and VEGFR2 (lower panel). (C-F) HUVECs were treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF. Then the expression of NRP-1 and VEGFR2 in indicated HUVECs was directly analyzed by flow cytometry (C, D) or after fixation and permeabilization (E, F). (G) Representative images of IF staining of NRP-1 and VEGFR2 in indicated HUVECs treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF. (H) Representative images and co-localization scatter diagrams of IF staining of NRP-1 and PDGFRβ in indicated HUVECs treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF. (I) Statistical graph of Manders’ overlap coefficient assessed by co-localization IF staining of NRP-1 and PDGFRβ in indicated HUVECs treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF. (J) HUVECs were treated with lenvatinib at different concentrations for 2 h, followed by stimulation with 60 ng/ml VEGF, and then subjected to immunoprecipitation with NRP-1 antibody. The total cell lysates were probed with anti-VEGFR2, anti-PDGFRβ, anti-NRP-1 antibodies and anti-β-actin antibodies, while the immunoprecipitates were probed with anti-VEGFR2, anti- PDGFRβ and anti-NRP-1 antibodies. The error bars represent the mean ± SD. *P < 0.05; **P < 0.01; *** P < 0.001; ****P < 0.0001; ns indicates non-significant.