Fig. 4: CD11c⁺Siglec-H⁺Ly6D^hiZbtb46^–CCR9^loB220^lo precursors contribute to cDCs via a Zbtb46⁺Ly6D⁺ intermediary stage.

a Siglec-H^– pre-cDC, Siglec-H⁺ pre-cDC, Siglec-H⁺Zbtb46⁺Ly6D⁺ cells as well as lo-lo, lo-hi precursors and pDCs (gated Siglec-H⁺Ly6D^hiZbtb46^– as shown in Fig. 3a) were sorted from Lineage-depleted BM cells of heterozygous Zbtb46^gfp mice and cultured for 3 days with Flt3L on EL08-1D2 stromal cells. The phenotype of the progeny was analyzed by flow cytometry. Percentages of pDCs (circles) or cDCs (triangles) within CD11c⁺ progeny are shown (mean ± SEM, n = 5); pDC vs cDC output was compared using paired, two-sided t-tests with Holm-Šídák correction for multiple testing. Adjusted p-values: <0.05(*), <0.005(**), <0.0.001(***). b Phenotype of cells derived from the indicated populations after 3 days of culture (representative results, n = 5). A potential transition from Ly6D⁺Zbtb46^– to Zbtb46⁺Ly6D^– is indicated by the dotted arrows. c The indicated populations were sorted and cultured for 3 days with Flt3L without stromal cells, then analyzed by flow cytometry. UMAP analysis of concatenated CD11c⁺ cells from all samples. cDCs generated from each precursor subset were gated as CD11c⁺Zbtb46^highMHCII^high and projected onto the UMAP, together with pDCs generated from lo-hi precursors for comparison. d Heatmap of hierarchically clustered log₂ normalized relative gene expression (scaled per gene) in the progeny of the indicated DC precursors before or after 1, 2 and 3 days of culture with Flt3L measured by qRT-PCR (grey: not detectable). Gene expression in freshly isolated BM and spleen pDC and cDC served as a reference (mean values of 2 independent experiments are shown). e CellTrace Blue proliferation dye signal in the CD11c⁺ fraction of the indicated input cells after 3d of culture (representative results, n = 5). f Expression of the indicated markers in the undivided fraction of CD11c⁺ cells generated from lo-lo precursors after 3 days. g Zbtb46 and Ly6D expression in lo-lo precursor cells directly after sorting and after 20 h of culture with Flt3L. The precentage of Zbtb46⁺Ly6D⁺ cells is shown in the gates. h Percentage of Zbtb46⁺Ly6D⁺ cells within CD11c⁺ cells derived from lo-lo precursors after 20 h of culture (mean ± SEM, n = 3).