Lipid rafts polarized in DC and NK cells during the DC/NK cell interaction. (A-B) Confocal microscopy on mDC/NK cell cocultures stained with cholera toxin. Lipid rafts (GM1) of the mDCs (A) or of the resting NK cells (B) were stained using FITC-labeled cholera toxin. The lipid rafts of mDCs appeared as small fluorescent patches scattered on the plasma membrane in the absence of contacts (A, top left corner). In contrast, mDC lipid rafts became clustered at the mDC/NK cell interface after conjugate formation (A; DCNK-IS). A 3-dimensional reconstitution confirmed the membrane distribution of lipid rafts in the single mDC, whereas a skewed polarization was observed following DCNK-IS formation (A, right panel). The mean percentages of conjugates polarizing rafts from the DCs were 85% ± 6% in 3 independent experiments. A similar observation was made in labeled NK cells admixed with mDCs where a polarization of FITC-labeled cholera toxin was seen at the NK cell interface with mDCs. (C) Inhibitors of lipid raft mobilization prevent accumulation of phosphotyrosines at the DCNK cell interface. mDCs were pretreated with the indicated combination of MCD or cholesterol (or both) and then cocultured with NK cells (at a DC/NK cell ratio of 1:10) on slides coated with poly-L-lysine for 25 minutes, fixed, permeabilized, and stained. Results are expressed as the percentage ± SEM of conjugates associated with intracellular phosphotyrosine clustering at the DCNK-IS. One representative experiment of 3 is shown.