Fig 1: DL1-NOTCH2 Optimizes the Differentiation of cDC1s from the BMTotal murine BM cells were cultured in the presence of FLT3L alone (FL), FLT3L with control OP9 cells (FL+OP9) or FLT3L with OP9 cells expressing Notch ligand DL1 (FL+Notch). OP9 cells were added on day 3, and BM cells were analyzed on day 7 of differentiation.(A) Representative staining plots of differentiated BM cells. The top row shows total live cells with B220+ CD11clo pDCs and B220- CD11chi cDCs highlighted; the other rows show gated cDCs with CD11bhi cDC2s and/or CD11b- cDC1s highlighted.(B) The expression of CD8a on gated cDCs in BM cultures differentiated as in (A).(C) The subset composition of differentiated BM cells. Shown are fractions of pDCs (of total live cells) and cDC subsets (of gated B220- CD11c+ MHC class II+ cDCs) and the absolute number of these subsets per 105 initial BM cells. cDC1s were defined either as CD24+ or CD8a+. Data points represent values from BM cultures of individual mice pooled from 2 experiments; bars represent mean.(D) Representative expression of the indicated surface markers on gated CD24+ cDC1s from FL or FL+Notch cultures. The expression of cDC1 markers on cDC2s is included as a control.(E) Representative expression of CD11c and MHC class II on DC subsets from FL or FL+Notch cultures. Subsets were gated, omitting the marker that is shown in each case.(F) Representative expression of Esam on cDC2s from FL or FL+Notch cultures. The expression on cDC1s is included as a control; the dotted line represents negative staining.(G) The effect of NOTCH2 blockade on the expression of CD8a on cDC1s and of Esam on cDC2s. BM cells were differentiated in FL or FL+Notch cultures in the presence of control immunoglobulin G (IgG) or anti-N2; the fluorescence intensity of marker expression in the indicated subsets is shown. Data represent mean ± SD of 5 parallel cultures for anti-N2 and IgG and 9 cultures pooled from 2 experiments for controls.(H) The effect of transcription factor deletion on DC differentiation. BM from control wild-type (WT) mice or mice with DC-specific deletion of Irf8 (Irf8 conditional knockout, cKO) or with germline deletion of Irf8 or Batf3 were cultured in FL or FL-Notch cultures. Shown is the fraction of the indicated DC subsets among total live cells; data points represent values from individual mice pooled from three (for Irf8) or two (for Batf3) experiments; bars represent mean.Statistical significance: ***p < 0.001, **p < 0.01, *p < 0.05.