Differentiation of DC_NK depends on direct contact between NK cells and monocytes and is mediated by GM-CSF and CD40L expressed by NK cells. (A) CD14⁺ monocytes were cultured in medium alone (filled gray histogram), in direct contact with NK cells (- - -), or with NK cells on opposite sides of a transwell membrane (–). Cells were stained on day 6 using mAb against CD14, CD86, DC-SIGN and CD40. A combined gate was set on monocytes/DCs based on the FSC:SSC profiles and NK cells were excluded from this gate. Results are representative of 3 separate experiments. (B) Isolated CD56^bright (•) and CD56^dim NK cells (■) derived from PB were cultured at 10⁵ cells/well for 72 hours in medium alone, or in the presence of various concentrations of IL-15 as indicated. GM-CSF in the cell-free culture supernatant was measured by ELISA. The data show mean (± SD) of 3 separate experiments. (C) CD14⁺ monocytes were cocultured with autologous NK cells in medium alone, or medium containing mouse IgG1 isotype control Ab, or in the presence of neutralizing mAb against either GM-CSF or CD154 as indicated on the top of each contour graph. Cells were stained on day 6 using mAb against DC-SIGN, CD40, CD80, and CD14. A combined gate was set on monocytes/DCs based on their CD56⁻ and FSC:SSC profiles, and quadrants are set according to the isotype control staining. Numbers indicate percentage of cells within each quadrant. Results are representative of 2 separate experiments.