Cooperation of PU.1 and TGF-β1 in LC induction from progenitor cells. (A-D) CD34⁺ cord-blood cells were transduced with PU.1-IRES-GFP or empty control vector and then stimulated in serum-free medium supplemented with the cytokine combinations SCF/Flt3L/GM-CSF or SCF/Flt3L/GM-CSF plus TGF-β1 as indicated. (A) Schematic representation of the retroviral vectors used in the experiments (left). Representative day-5-generated gene-transduced cultures were FACS sorted into GFP⁺ and GFP^- cell fractions and were subsequently analyzed for PU.1 or actin using Western blot analysis (right). (B-D) Day 7-generated PU.1- or control-transduced cultures are compared. GFP⁺ cells were gated on a separate FACS diagram (not shown) and were further analyzed for the expression of informative marker molecules (B: Langerin or CD40 vs CD1a; C: CD80, CD86, or CD83 vs CD1a). (C) Culture morphology of PU.1- or control-transduced cultures with or without TGF-β1 as indicated. Data in panels B, C, and D are representative of 9, 6, and 3 independent experiments, respectively. Data in panel D are representative of 3 independent experiments. (E) CD34⁺ cord-blood cells were transduced with PU.1-IRES-GFP (PU.1) or empty control vector (CTRL) and were then stimulated in culture medium supplemented with the cytokine combinations SCF/Flt3L/GM-CSF and 10% FCS in the absence or presence of 10 μg/mL neutralizing antibody specific for TGF-β1. Gated GFP⁺ cells were analyzed for Langerin versus CD1a expression. The data shown represent 1 of 3 experiments with comparable results. Numbers in quadrants of diagrams indicate the percentage of cells in that quadrant.