Id2 is induced by TGF-β1 costimulation in LC progenitors, but ectopic Id2 is not sufficient to substitute for TGF-β1. (A) Relative Id2 mRNA levels in CD34⁺ progenitors stimulated with SCF, Flt3L, GM-CSF, and TNFα ± TGF-β1 for the indicated time points determined by real-time PCR. Values were normalized to GAPDH expression and compared with Id2 mRNA levels in unstimulated cells (0 h). One representative of 3 independent experiments is shown. (B) CD34⁺ cord-blood cells were transduced with Id2-IRES-GFP or empty control vector and then stimulated in serum-free medium supplemented with the cytokine combination SCF/Flt3L/GM-CSF/TNFα with or without TGF-β1 as indicated. Day-7-generated Id2-transduced (center diagram, without TGF-β1) or control-transduced (left: without TGF-β1, right: with TGF-β1) cultures are compared. GFP⁺ cells were gated in a separate FACS diagram (not shown) and were further analyzed for the expression of Langerin versus CD1a. One representative of 5 independent experiments is shown. Numbers in each quadrant indicate the percentage of cells in that quadrant.