F-actin remodeling is essential for the maintenance of MHC class I/KIRs inhibitory signaling at the mature synapse between mature DCs and resting NK cells. (A) NK cells were cultured alone or with mature DCs for 6h, in the presence of BFA, and IFN-γ production by NK cells was assessed by ICS, followed by flow cytometry. DMSO and cytochalasin B were added at 0 or 2 hours after start of the culture (C0 and C2, respectively). NK cells were gated as live, individual, CD3⁻CD56⁺cells. (B) Mature DCs and resting NK cells were prestained with vital dyes and cocultured for 6 hours, in the presence of BFA. IFN-γ production by DC-conjugated NK cells was assessed by ICS in flow cytometry. DMSO or cytochalasin B were added at 0 or 2 hours after start of the coculture (C0 and C2, respectively). IFN-γ⁺ NK cell-conjugated DCs (in red) were gated as indicated, and could be detected despite the auto-fluorescence of unconjugated DCs (blue). (C) Cytochalasin B was added to cocultures of mature DCs and resting NK cells, and conjugates were fixed after 20 minutes of interaction. F-actin (green), nuclear DNA (blue) and CD94, KIRs, IL-15Rα, or 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. (D) Resting NK cells or mature DCs were preincubated with anti-KIRs or anti-MHC class I blocking antibodies (HLA class I specific IgG2a in left and IgM in right panel), respectively, or with the isotype matched antibody controls. DMSO or cytochalasin B were added to cultures as in panel A. Plots are representative of at least 3 independent experiments. Values on graph bars represent medians from at least 3, or 2 for panel B, independent experiments with duplicates. Error bars indicate interquartile ranges. ↑↑ Fold enrichment > 2; *P < .05, **P < .001 and ***P < .0001. P values from Mann-Whitney test, nonparametric, and bi-caudal. Scale bars are 10 μm.