KpOmpA and flagellin trigger α-defensin synthesis and release by NK cells. Intracellular α-defensin expression was analyzed using (A) FACS in freshly and highly purified human NK cells and CD56⁺CD3⁺ T cells and using (B) confocal microscopy in NK cells (magnification: × 300 and × 1000 in left and right panels, respectively). (B, inset) Cells labeled with isotype control mAb (magnification: × 600). (C) α-defensin mRNA expression in NK cells either unstimulated or stimulated for 2 hours with KpOmpA in the absence or in the presence of IL-2 was analyzed using RT-PCR. RNA integrity and cDNA synthesis were verified by amplifying GAPDH cDNA. (D-E,G) Human CD56⁺CD3-NK cells were unstimulated or stimulated with 1 μM KpOmpA, flagellin, or TT in the absence or in the presence of 200 U/mL IL-2 or of 0.1% contaminating neutrophils, when indicated. After 2 hours, cells were stained for intracellular α-defensin expression analysis by cytometry. (F) α-Defensin released by NK cells and CD56⁺CD3⁺ cells activated by KpOmpA or flagellin in the absence or presence of IL-2 was quantified in cell culture supernatants using ELISA. Data are expressed in MFI (A,D,E,G) and in ng/mL, mean ± SD, n = 3 (F).