CD14⁺CD16⁻monocyte compartment is shaped by NK-cell–mediated cytotoxicity. CD14⁺CD16⁻ monocytes and preactivated NK cells were isolated from HD blood and cocultured for 18 hours (NK-Mo). After coculture, NK cells’ capacity to produce cytotoxic proteins and cytokines after PMA plus ionomycin stimuli was assessed by flow cytometry. NK cells alone were used as controls. (A) Gating strategy for the analysis of GM-CSF⁺IFN-γ⁺ and granzyme B/perforin⁺CD107a⁺ NK cells. (B) Frequency of GM-CSF⁺ IFN-γ⁺ NK cells; production (iMFI: frequency multiplied by MFI) of IFN-γ by NK cells after contact with monocytes. (C) Frequency of granzyme B/perforin⁺ within GM-CSF⁺IFN-γ⁺ NK cells; expression (MFI) of CD107a by NK cells after contact with monocytes. Cytotoxic function of NK cells was monitored during the cocultures through the assessment of target viability by flow cytometry. K-652 cells were used as positive controls for NK-cell–induced cytotoxicity. (D) Monocyte viability during coculture (5-720 minutes) with NK cells; K-562 viability during the coculture (5-720 minutes) with NK cells. (E) Overrepresentation pathway-based analysis²⁹  of NK-cell–related genes found upregulated in the transcriptome of NK-cell–primed monocytes. The input gene list (supplemental Table 1) was compared with 13-pathway databases considering a minimum overlap of 2 genes and P < .01. Each node represents a different pathway (blue), ontology category (purple), or protein-protein complex (orange); node size represents the number of genes contained in the set of a particular pathway and node color represents the P value. Nodes connected through edges share members and edges width reflect overlapping of genes between nodes. (F) Expression (MFI) of CD94 in NK cells after coculture with monocytes. (G) Expression (MFI) of HLA-E in monocytes after coculture with NK cells. (H) Gating strategy for the analysis of monocytes expressing low and high levels of HLA-E. (I) Frequency of HLA-E^low and HLA-E^high monocytes after coculture with NK cells. Monocyte viability was assessed by flow cytometry after contact with NK cells; for some groups, the CD94 receptor was blocked prior the interaction with monocytes (NK_α-CD94-Mo). (J) Frequency of CD14⁺ monocytes positive for Live/Dead dye; frequency of CD14⁺HLA-E^low monocytes. (K) Histograms and pooled expression (MFI) of HLA-ABC and B7-H6 in HLA-E^low and HLA-E^high in monocytes from both groups (Mo and NK-Mo). 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.