hnCD16-iPSC–derived NK cells are functionally mature and do not downregulate CD16 expression upon activation. (A) Unmodified iNK cells, hnCD16-iNK cells, and adult PB-NK cells were stained and analyzed by flow cytometry for CD56 and CD16 and the indicated NK cell surface receptors. In each panel, red line: isotype control; blue line: stained sample. Data were repeated independently in 3 separate experiments. (B) hnCD16-iNK cells, unmodified iNK cells, or PB-NK cells were stimulated as indicated for 4 hours, and CD16 expression was determined by flow cytometry (n = 4-6 per group). (C) Representative flow cytometric analysis of CD16 and TNF-α expression on unmodified iNK cells, hnCD16-iNK cells, and PB-NK cells that were left unstimulated or stimulated with K562 cells or PMA/ionomycin. (D) Representative flow cytometric analysis of intracellular TNF-α and IFN-γ production after a 4-hour incubation with culture media only (unstimulated), with P815 cells, or with P815 cells + anti-CD16 antibody. Data in panels C-D were repeated in 3 separate experiments.