Figure 1.  Reciprocal stimulation of resting NK cells and autologous iDCs. Cultured iDCs and resting, purified NK cells were incubated alone or in combination for 18 h (E–G) or 48 h (A–D). After culture the cells were analyzed by flow cytometry for the expression of CD86 (DCs; A–C), CD69 (NK; D) or CD69 and CD25 (NK; E–G). Specific analysis of NK cell or DC subsets was achieved by a combination of gating on forward versus side scatter differences and staining with antibodies for CD56 (NK) or CD86 (DC). (A) iDCs were incubated alone (▪) or with NK cells (□) at a ratio of 5:1 (NK/DC) in the presence of the indicated doses of LPS. The percentage of CD86bright DC was determined by specific gating. (B) The representative experiment shown was performed in the presence of 6.25 ng/ml of LPS and the iDCs were incubated alone (gray bar); with NK cells at a 5:1 (NK/DC) ratio (black bar); with NK cells (5:1) plus 10 μg/ml of a neutralizing antibody for TNF-α (white bar); or with NK cells (5:1) but separated in trans-wells (striped bar). Changes in DC maturation were determined by measuring CD86 mean fluorescence intensity (MFI). (C) iDCs were incubated alone (DCs) or with NK cells at the indicated ratios (NK/DC) in the presence (▴) or absence (▵) of 6.25 ng/ml of LPS. DC maturation was determined as in A. (D) NK cells were incubated for 48 h either alone (NK) or with iDCs at the indicated ratios (NK/DC) in the presence (▴) or absence (▵) of 10 ng/ml of LPS. NK cell activation was determined by the percentage of cells expressing CD69. (E–G) Flow cytometry dot plots from a representative experiment measuring NK cell activation marker expression (CD69 and CD25). NK cells were incubated for 18 h: alone (E), with iDCs at an NK/DC ratio of 1:5 (F), or with iDCs (NK/DC, 1:5), but separated in trans-wells (G).