CD39 and CD73 expression on NK cells and the effect of ADO analog on NK cell functions. Blood samples were obtained from healthy volunteers, and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll density gradient centrifugation. CD56⁺CD3⁻ NK cells were sorted out from PBMCs using FACS and cultured overnight in the presence/absence of MSCs. (A) CD39 and (B) CD73 expression was analyzed on the NK cells using flow cytometry. (C-D) NK cells were stimulated with 1000 IU/mL interleukin-2 (IL-2) in the presence/absence of 15 µM CADO and incubated overnight. Brefeldin A was added to the cultures after 1 hour of incubation. The cells were then washed, fixed, and stained intracellularly for (C) tumor necrosis factor (TNF)-α or (D) interferon (IFN)-γ. (E) NK cells were cultured with/without 15 µM CADO overnight, washed, and further incubated with K562 cells for 4 hours. CD107a surface expression was analyzed on the NK cells as a readout for NK cell degranulative capacity. (F-G) NK cells cultured with or without MSCs were cultivated in PBS with 5′AMP as a substrate for 30 minutes at 37°C. Cell-free supernatants were collected and analyzed using chromatography with tandem mass spectrometry for the levels of (F) residual 5′-AMP (as a readout of substrate utilization) or (G) ADO (as a read out of product accumulation). Paired 2-tailed Student t tests were performed using GRAPHPAD PRISM V5.00 software. Levels of significance are shown as P values (*P < .05, **P < .01). Bar graphs represent mean ± standard error of mean.