MePDN inhibits NK receptor–mediated cytotoxicity and ERK phosphorylation upon NK receptor cross-linking. (A) NK cells were cultured for 5 days in the presence of IL-2 (100 U/mL) or IL-15 (20 ng/mL) with or without MePDN at the final concentration of 0.5 μg/mL. NK receptor–mediated cytotoxicity was assessed in a 4-hour ⁵¹Cr-release assay against the FcR-γ⁺ murine P815 cell line. The E/T ratio was 5:1. Data represent percentage of NK receptor–mediated cytotoxicity of MePDN-treated NK cells cultured in the presence of IL-2 (hatched bars) or IL-15 (gray bars) versus control NK cells cultured with IL-2 or IL-15 alone. Results are expressed as the mean percentage (± SEM) of cytotoxicity analyzed in 11 experiments performed with 11 different donors. Statistical significance was checked by nonparametric Mann-Whitney test. (B) After 4 days of culture, NK cells derived from the same donor analyzed in panel A and cultured with IL-2 (100 U/mL) in the presence or absence of MePDN at the final concentration of 0.5 μg/mL were starved for 4 hours at 37°C in RPMI medium with no FCS or cytokines. Cells were then incubated with the indicated mAbs and cross-linked with F(ab′)₂ GAM at 37°C for 5 minutes. Total-cell lysates were analyzed in order to detect p-ERK1/2. Cell membrane was reprobed with an antibody recognizing the native protein in order to assess comparable amounts of proteins. Similar results were obtained in the presence of IL-15. In this figure, a representative experiment of 4 is shown.