Confirmation of the critical role of Syk in IL-2 maintained LGL survival.
(A) Human fresh NK (LGL) cells, cultured in complete medium containing 100 U/mL IL-2, were incubated with 25 μM piceatannol, 25 μM LY294002, or DMSO vehicle control and were examined for cellular viability by annexin V–FITC binding and PI uptake. LGL cells were collected at 24 and 48 hours, washed in sample wash buffer, and stained with annexin V–FITC in combination with PI. Annexin V–FITC binding in piceatannol-, LY294002-, or DMSO-treated and untreated NK92 cells is shown. (B) Inhibition of IL-2–induced Akt function by inactivating Syk and the rescue of this inhibition by constitutively active PI 3-kinase. IL-2–starved LGL cells, treated with 25 μM piceatannol or DMSO, were infected with recombinant vaccinia virus encoding the constitutively active catalytic subunit of PI 3-kinase, P110*, or CD56 irrelevant control gene and then were stimulated with IL-2 (100 U/mL) for 5 minutes at 37°C. Cells were analyzed for Akt activation by Western blot analysis with Ser473-specific antibodies. (C) LGL cells were treated as described in panel B, and whole-cell lysates were analyzed by in vitro kinase assays for Akt activation with H2B as the substrate. The same membranes were stripped and reprobed with anti–pan-Akt. Results are representative of 1 of 4 independent experiments.
