E2/ERα signaling promotes development of DCs that harbor increased amounts of IRF4 protein. (A) Lin⁻ BM cells were incubated with 1nM E2 or vehicle in hormone-deficient medium. On day 7, IRF4 protein expression in CD11c⁻ and CD11c⁺ cells was determined. Shown are: binding of anti-IRF4 Ab to cells from vehicle-treated cultures (dotted line); binding of anti-IRF4 Ab to cells from E2-treated cultures (solid line); binding of isotype control IgG on cells from the E2-treated cultures (shaded gray). MFI values for anti-IRF4 binding with or without E2 are indicated (with value for isotype control IgG subtracted). Data are representative of 3 experiments. (B-E) MPs were incubated in standard medium with ICI 182,780 (100nM) or vehicle. On day 7, the amount of IRF4 protein expression in CD11c⁻ and CD11c⁺ cells was determined. (B) CD11c expression on all cells; the percentages of CD11c⁻ and CD11c⁺ cells are indicated. (C) Anti-IRF4 binding on CD11c⁻ and CD11c⁺ cells in the presence of ICI 182,780 (dotted line) or vehicle (solid line). The binding of isotype control IgG on cells in vehicle-treated cultures is indicated (shaded gray). MFI values for anti-IRF4 binding with or without ICI 182,780 are indicated (with value for isotype control IgG subtracted). (D) Within CD11c⁺ cells, CD11b^int DCs express higher levels of IRF4 than CD11b^hi DCs. The percentage of DCs within each gate is indicated. (E) MFI values for anti-IRF4 binding with or without ICI 182,780 are indicated for each DC subset. Data are representative of 2 experiments.