Analysis of DC maturation induced by perforin-deficient NK cells. (A) Polyclonal NK-cell lines derived from either a healthy donor or an HLH patient were assessed for intracytoplasmic expression of perforin (“Materials and methods”). Open curves indicate isotypic controls, while filled curves indicate perforin expression in the HLH patient and in a healthy donor, in the lower and upper panels, respectively. (B, left) The same polyclonal NK-cell lines were analyzed for their cytolytic responses in a redirected killing assay against the FcγR⁺ target cell, P815. Specific ⁵¹Cr release was assessed either in the absence (□) or in the presence (▪) of a triggering anti-NKp30 mAb (IgG1 isotype). E/T ratio was 4:1. (Right) Cytolytic activity was also evaluated against allogeneic iDCs, either in the absence (□) or presence (▪) of anti-NKp30mAb (IgM isotype). E/T ratio was 8:1. (C) Immature DCs were cocultured with a polyclonal NK-cell line derived from a perforin-deficient HLH patient either in the absence or presence of anti-NKp30 mAb (IgM isotype). As control, iDCs were cultured with medium alone. After 2 days, DCs were harvested and analyzed for expression of CD83. Results are presented as percent of positive cells and are representative of 6 independent experiments. Horizontal bars represent markers assigned on the basis of control cells fluorescence intensity (ie, cells incubated with the second reagent alone). (D) NK cells from a healthy donor or from a perforin-deficient patient were stimulated overnight by plastic-bound anti-NKp30. Supernatants were harvested and analyzed by specific ELISA for their TNFα and IFNγ content. ▪represents TNFα release, while □ represents IFNγ release.