CD14⁺CD16⁻monocytes are instructed by the polarization of IFN-γ at the NKp30-mediated junction with NK cells. Preactivated NK cells and CD14⁺CD16⁻ prestained monocytes (violet), isolated from HD blood, were cocultured for 6 hours in the presence of Live/Dead viability dye (blue). After coculture, cells were fixed and stained for IFN-γ (green) and phalloidin (red). In all experiments and groups, remaining platelets accompanied the monocyte fractions. For some cocultures, the NKp30 receptor was blocked prior the interaction with monocytes (NK_α-NKp30-Mo). (A) IFN-γ dynamics during the interaction of NK cells with monocytes. (B) Distribution of IFN-γ in nonagglomerated NK cells (arrow). (C) Distribution of IFN-γ during the interaction of NK cells with dying monocytes (arrow). (D) Presence of IFN-γ in nanotube-like structures (arrow). (E) Frequency and expression (MFI) of CD27, CD244 and NKp30 on NK cells cultured alone (NK) or after 18 hours of coculture with CD14⁺CD16⁻ monocytes (NK + Mo). (F) Distribution of IFN-γ during the interaction of NKp30-blocked NK cells with monocytes. (G) IFN-γ dynamics during the interaction of NKp30-blocked NK cells with monocytes. (H) Total fluorescence intensity of IFN-γ determined at the junction formed between NK cells and monocytes. Prestained NK cells (green) and monocytes (violet) were cocultured and live-cell imaging was recorded for up to 12 hours. (I) Captures of the interaction between NK cells and monocytes. (J) Interaction time of monocytes with NK cells vs monocytes with NKp30-blocked NK cells. Error bars represent standard deviation. Data are representative of at least 2 or more independent experiments combined; n = 2-3 HDs per experiment. Statistical comparisons were performed with the unpaired Student t test with Welch correction. *P < .05, **P < .01, ***P < .001. See “Confocal microscopy” for stains used; original magnifications ×20 (A-B,F,I) and ×40 (C-D,G).