F-actin dynamics stabilizes MHC class I molecules at the mature synapse of NK cells with human blood DCs. (A) Mature or immature blood DCs were stained with fluorochrome conjugated anti–HLA-ABC, -CD86, -CD83 and –HLA-DR antibodies and compared with unstained blood DCs. (B) Single cell cultures or cocultures of mature blood DCs with resting NK cells were fixed after 1 or 20 minutes of interaction and f-actin (green), nuclear DNA (blue) and MHC class I, IL-12 or IL-12R (red) were stained. Arrows point to molecule enrichment at the synapse. The graph represents the quantification of molecule staining intensity at the synapse compared with the staining at the opposite side of the same conjugated cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (C) Mature blood DCs were allowed to conjugate with resting NK cells for 20 minutes. Cocultures were fixed and stained with bodipy-conjugated phallacidin to stain f-actin (green) and anti-KIRs antibodies (red). The fluorescence intensities of KIRs and f-actin stainings were plotted along the indicated trajectory, from A to B. The synapse area in cellular conjugates is indicated by boxes. (D) Cytochalasin B was added to cocultures of mature blood DCs with resting NK cells, and conjugates were fixed after 20 minutes of interaction. F-actin (green), nuclear DNA (blue) and MHC class I (red) were stained. Arrows point to molecule enrichment at the synapse. The graph represents the quantification of molecule staining intensity at the synapse compared with the staining at the opposite side of the same conjugated cell. Values were normalized to the values of molecule distribution in unconjugated cells, assigned as 1. (E) NK cells were cultured with mature blood DCs for 6 hours and IFN-γ production by NK cells was accessed by ICS in flow cytometry. DMSO or cytochalasin B were added at 0 or 2 hours after start of the cocultures (C0 and C2, respectively). NK cells were gated as live, individual, CD3⁻CD1c⁻CD56⁺cells. Microscopy images are representative of 3 independent experiments. Original magnifications are 100× for all the microscopy images. Values on graph bars (B and D) represent medians from the analysis of at least 100 conjugates from at least 3 independent experiments. Values on graph bars in panel C represent medians from 3 independent experiments with duplicates. Error bars indicate interquartile ranges; ↑↑, fold enrichment > 2 for molecules that exist in both DC and NK cell (IL-15Rα and MHC class I); and ↑, fold enrichment > 1.5 for molecules that exist in only 1 cell (IL-12 and IL-12R); *P < .05. P values from Mann-Whitney test, nonparametric and bi-caudal. Scale bars are 10 μm.