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Monokines (i.e., interleukin [IL]-12, -18, and -15) induce natural killer (NK) cells to produce interferon-gamma (IFN-gamma), which is a critical factor for immune surveillance of cancer and monocyte clearance of infection. We show that SET, which is a potent inhibitor of protein phosphatase type 2A (PP2A) activity, is highly expressed in human CD56bright NK cells, which produce more IFN-gamma than CD56dim NK cells. SET was up-regulated upon monokine stimulation of primary human NK cells. Furthermore, ectopic overexpression of SET significantly enhanced IFN-gamma gene expression in monokine-stimulated NK cells. In contrast, RNAi-mediated suppression of SET expression renders NK cells inefficient in producing high levels of IFN-gamma in response to monokine costimulation. Mechanistically, suppression of PP2A activity by SET is important for IFN-gamma gene expression in NK cells. In fact, treatment of primary human NK cells with the PP2A activator 1,9-dideoxy-forskolin, as well as administration of the drug to C57BL/6 mice, significantly reduced NK-dependent IFN-gamma production in response to monokine treatment. Further, SET knockdown or pharmacologic activation of PP2A diminished extracellular signal-regulated kinase 1/2, p65RelA, signal transducer and activator of transduction 4 (STAT4), and STAT5 activity in monokine-stimulated NK cells, potentially contributing to the reduction in IFN-gamma gene expression. Thus, SET expression is essential for suppressing PP2A phosphatase activity that would otherwise limit NK cell antitumoral and/or antiinflammatory functions by impairing NK cell production of IFN-gamma.

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 CD56bright NK subset, which is a potent producer of IFN-γ relative to the CD56dim 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.