DCs and intermediate CD14⁺CD1a⁺ cells induce proliferation and RANKL expression by T cells. (A) Allostimulatory properties of monocyte-derived DCs (•), intermediate CD14⁺CD1a⁺ cells (immature DCs treated for 3 days with M-CSF and RANKL; ▴), purified monocytes (▪), and purified monocytes treated for 3 days with M-CSF and RANKL (♦), cultured with allogeneic T cells purified from 3 different donors. A total of 10³ APCs were cocultured with 10⁵ T cells. (B-E) RANKL (green) and CD3 (red) stainings of T cells, after 5 days of culture with (B) immature DCs, (C) intermediate CD14⁺CD1a⁺ cells, (D) monocytes, and (E) anti-CD3 plus anti-CD28 antibodies. Asterisks indicate immature DCs, diamonds indicate intermediate CD14⁺CD1a⁺ cells, and circles indicate monocytes. T cells were distinguished from other cells based on CD3⁺ expression and Hoechst nuclei staining (not shown).