In Vitro and In Vivo Evidence That the PP2A Inhibitor SET Regulates IFN-γ Production in Monokine-Stimulated Natural Killer Cells.

Monokines (i.e. IL-12, IL-18 and IL-15) induce natural killer (NK) cells to produce interferon-γ (IFN-γ), which is critical for monocyte clearance of infectious pathogens and tumor surveillance. To identify new regulators of IFN-γ production we performed oligonucleotide array analysis of unstimulated and IL-12- and IL-18-stimulated NK92 cells. Among the subset of mRNAs differentially regulated in monokine-stimulated cells, we found SET, a potent inhibitor of the protein phosphatase type 2A (PP2A). SET mRNA and/or protein levels were upregulated in IL-12/IL-18- and IL-12/IL-15-stimulated primary human NK cells. Interestingly, the SET protein is also selectively increased in the resting CD56^bright NK subset, which is a potent producer of IFN-γ relative to the CD56^dim NK subset. To determine whether SET positively regulates IFN-γ production by inhibiting PP2A activity, we employed RNAi and interfered with SET expression in NK92 cells. SET downregulation inhibited IFN-γ secretion by IL-12/IL-18, IL-12/IL-15- or IL-15/IL-18-stimulated NK92 cells. By contrast, ectopic SET expression increased IFN-γ production in monokine-stimulated NK92 and primary human NK cells. Because downregulation of SET augmented PP2A activity in NK92 cells, we sought to investigate whether pharmacologic activation of PP2A inhibits the ability of NK cells to produce IFN-γ. Indeed, suppression of IFN-γ expression and secretion was also observed upon treatment of NK92 and primary NK cells with 1,9-dideoxy-forskolin, a known inducer of PP2A activity. Accordingly, NK cells from mice treated with 1.9-dideoxy-forskolin produced less IFN-γ in response to in vivo monokine stimulation than did NK cells from vehicle-treated mice. Mechanistically, activation of PP2A by SET knock-down or 1,9-dideoxy-forskolin treatment leads to inhibition of ERK1/2, p65RelA and STAT5 activity in monokine-stimulated NK cells. Because these signaling molecules are important for IFN-γ production by monokine-stimulated NK cells, our results strongly suggest that monokine induction of SET expression in NK cells is essential for limiting PP2A activity that, otherwise, would negatively impact the ability of NK cells to produce and release optimal levels of IFN-γ.