Signaling mechanisms modulating synergistic activation of natural killer cells

Abstract

Natural Killer (NK) cells are discriminated among innate lymphoid cells (ILCs) with their cytolytic activity that has a key role in the control of viral infection and tumorigenesis. NK cells not only kill virus-infected or transformed cells including cancer stem cells by releasing cytotoxic granules, but also contribute to modulate diverse immune responses by secretion of cytokines (e.g., IFN-γ and TNF-α) and chemokines (e.g., MIP-1α and MIP-1β). Therefore, the malfunction of NK cells is linked various diseases, and the manipulation of NK cells function have hold promise in therapeutic applications. However, the limited information for the mechanisms regulating activation of NK cells has hampered the progression toward therapeutic strategies. Accordingly, understanding accurate activating and inhibitory receptors signaling mechanisms controlling NK cells function can augment successful NK cell-based therapies. The triggering of NK cell effector functions relies on the engagement of activating or inhibitory receptors for ligands on their target cells. The balanced signals from activating and inhibitory receptors are known to dictate NK cells activity. However, even in the case of inhibitory signal is absent, activating signal from single coactivation receptor is insufficient to induce enough activation of resting NK cells. Signals from multiple activating receptors, not a single receptor, are required for NK cell functions. And only specific combinations among them can induce additive or synergistic enhancement of NK cells activity. For this reason, the study of signaling mechanisms modulating synergistic activation of NK cells is critical for harnessing the arm of NK cells for clinical purposes. In this study, two critical signaling molecules for immune cell functions, glycogen synthase kinase (GSK)-3β and NF-κB are examined for regulation of synergistic activation of NK cells, and their interaction mechanisms with other signaling pathways are investigated. In a synergistic activation model of NK cells that combines NKG2D and 2B4 activating receptors, representative non-ITAM-associated activating receptors, GSK-3β negatively regulated the synergistic activation of both cytotoxicity and cytokine secretion. The individual or combinational stimulation of NKG2D and 2B4 induced inhibitory phosphorylation of GSK-3, and the extent of phosphorylation in accordance with the level of NK cell activity. The inhibition of GSK-3β by siRNA knockdown or pharmacological inhibition increased NK cell function, but GSK-3α knockdown had no effect. This negative role of GSK-3β was dependent on its kinase activity. The regulation of NK cell function by GSK-3β was a common mechanism for both NKL cell line and primary NK cells. And NK cell activations by either ITAM-associated or non-ITAM activating receptors were regulated by GSK-3β. NF-κB is a well-known transcription factor that is critical for diverse immune responses. Nevertheless, its role in NK cells activated by multiple activating receptors during target cell recognition is not defined. Synergistic combination of NKG2D, 2B4, or DNAM-1 induced synergistic and sufficient activation of NF-κB pathway that is linked to synergistic NK cell activity, whereas single receptor stimulation was insufficient. Receptors cooperation for NF-κB activation was regulated at the level of Vav1 phosphorylation, the 1st checkpoint, and this Vav1-dependent synergistic signal also cooperates with separate PI3K-Akt signal for synergistic p65 phosphorylation, the 2nd checkpoint, in a stepwise manner. As confirmed with a X-linked lymphoproliferative disease (XLP) NK cells model that is defective for 2B4 receptor signaling because of mutations of SAP adaptor, stepwise checkpoint signaling mechanism for NF-κB activation was suggested. These newly uncovered signaling mechanisms during multiple receptor stimulation provides new insights of receptor cooperation to determine specificity and magnitude of NK cell activation and bases for establishing NK cell-based therapeutic strategies.