The formation of DC/NK cell conjugates is associated with Ca²⁺ influx and tyrosine phosphorylation. (A-B) mDCs form conjugates with resting NK cells. iDCs versus LPS-matured DCs (mDCs) were admixed with resting NK cells (at a 1:10 DC/NK cell ratio) and analyzed in transmission light microscopy as described in “Materials and methods.” Representative pictures of various fields are shown in panel A. More than 500 DCs were examined for their capacity to bind to resting NK cells. The percentages of DCs forming conjugates with NK cells in transmission light microscopy are shown as a mean ± SEM of 3 independent experiments (B). (C) The cell-to-cell contacts between mDCs and resting NK cells are associated with intracellular tyrosine phosphorylation. mDCs were admixed with resting NK cells. After fixation, cells were permeabilized and stained with anti-pTyr antibodies. The numbers of conjugates associated with intracellular phosphotyrosines were counted and reported as the total number of conjugates. There was an accumulation of pTyr at the DC/NK cell contact area in 85% ± 5.2% of the mDC/NK cell conjugates and in less than 10% ± 1.3% of the iDC/NK conjugates. Similar confocal images were acquired in 5 independent experiments. A representative picture of a resting NK cell interacting with an mDC (lower cell) at a contact area displaying accumulation of pTyr is shown. A more distant resting NK cell (upper cell) does not exhibit detectable pTyr. (D) Ca²⁺ influx is triggered in resting NK cells encountering mDCs. mDCs were mixed with resting autologous NK cells labeled with 4 μM Fluo4-AM and observed for 60 minutes by videomicroscopy at 37°C. One picture was acquired every 10 seconds. An increase in the intensity of Fluo-4 fluorescence of at least 50% above the level of resting NK cells was considered significant. Representative pictures of conjugate formation between mDCs and resting NK cells are shown. Intracellular Ca²⁺ raises (bottom panels) were monitored in NK cells 50 seconds after interaction with mDCs. The fluorescence intensity scale (unit of fluorescence intensity [UFI]) is shown on a gray level scale ranging from 0 to 256. The percentages of iDCs or mDCs (of 200) forming productive interactions leading to significant Ca²⁺ increases in NK cells are indicated as a mean ± SEM of 2 independent experiments (E, left graph). The mean duration of productive (with Ca²⁺) and nonproductive (without Ca²⁺) mDC/NK cell conjugates is reported in the right graph (E).