Phospholipase C-γ2 is essential for NK cell cytotoxicity and innate immunity to malignant and virally infected cells

Abstract

Phospholipase C-␥2 (PLC-␥2) is a key component of signal transduction in leukocytes. In natural killer (NK) cells, PLC-␥2 is pivotal for cellular cytotoxicity; however, it is not known which steps of the cytolytic machinery it regulates. We found that PLC-␥2-deficient NK cells formed conjugates with target cells and polarized the microtubule-organizing center, but failed to secrete cytotoxic granules, due to defective calcium mobilization. Consequently, cytotoxicity was completely abrogated in PLC-␥2-deficient cells, regardless of whether targets expressed NKG2D ligands, missed self major histocompatibility complex (MHC) class I, or whether NK cells were stimulated with IL-2 and antibodies specific for NKR-P1C, CD16, CD244, Ly49D, and Ly49H. Defective secretion was specific to cytotoxic granules because release of IFN-␥ on stimulation with IL-12 was normal. Plcg2 ؊/؊ mice could not reject MHC class I-deficient lymphoma cells nor could they control CMV infection, but they effectively contained Listeria monocytogenes infection.

Line of attack: NK cell specificity and integration of signals

“…As a result of this multiplicity, only a few molecules have been shown to be required for natural cytotoxicity in mouse knockout models. Phospholipase C (PLC)-γ2 is an important mediator of degranulation, cytotoxicity, and cytokine production [38][39][40]. Pharmacological inhibition of PLC-γ abrogates degranulation, cytotoxicity and cytokine production by human NK cells, in addition to early signals for intracellular calcium mobilization [41].…”

PI5KI-dependent signals are critical regulators of the cytolytic secretory pathway

“…5,6,8,31 Increasing evidence highlights that distinct signals promote granule polarization and secretion. 40 In human NK cells, PI3K plays a pivotal role in ADCC and natural cytotoxicity against certain tumor cells 5,41 ; such kinase leads to the formation of the critical membrane-bound second messenger, PIP3, which acts as a docking site for several PH domain-containing proteins, such as Vav family proteins, in turn responsible for the activation of Rho family small G proteins.…”

“…2,3 Several structurally distinct receptors have been implicated in the activation of NK-cell cytolytic machinery: when cross-linked by the corresponding ligands on target cell, they trigger multiple and intersecting signaling pathways responsible for functional activation. 4 A vast array of activating NK receptors belonging to different families are coupled to the lipid modifying enzymes phosphatidylinositol3-kinase (PI3K) and phospholipase C␥ (PLC␥), which provide signals critically required for the activation of the cytolytic machinery [5][6][7][8] ; notably, both enzymes use the membrane phospholipid phosphatidylinositol-4,5-bisphosphate (PIP2) as common substrate. 9 Besides its role as signaling intermediate, PIP2 also acts as critical regulator of various cellular processes, including actin remodeling, membrane and vesicle trafficking, adhesion, and ion transport.…”

“…Recently, in both human and mouse, the secretory step, but not the polarization of lytic granules, has been shown to depend on Ca 2ϩ flux. 8,47 Accordingly, in fresh resting NK cells, the failure of a given receptor, such as NKG2D, to induce granule exocytosis correlates with the lack of Ca 2ϩ response on receptor stimulation. 4 We show here that both PI5KI␣ and PI5KI␥ are required for the efficient PLC␥ activity: in silenced cells, we observed almost complete inhibition of 2B4-induced IP3 generation; the decreased response is not associated with impaired PLC␥ tyrosine phosphorylation, indicating that both ␣-and ␥-dependent PIP2 pools are not redundantly required for PLC␥ activity.…”

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3β

“…The ligation or antibody dependent cross linking of the ITAM-dependent receptors initiates NK cell signal transduction cascade by phosphorylation of tyrosine residues located within conserved ITAM sequences through Src protein tyrosine kinase (PTK) family [20]. These in turn allows the consequent recruitment and activation of Syk and/or ZAP70 PTK and the transmembrane adaptor proteins, linker for activation of T cells, LAT and LAT2 [20,21], further leading to the recruitment and activation of downstream effector molecules such as PI3K [5,22,23], the phospholipases PLCγ1 and PLCγ2 [24][25][26][27], the guanosine triphosphate-guanosine diphosphate exchange factors Vav2 and Vav3, [28], the extracellular signal-regulated kinase (ERK) [29], and finally triggers both cytotoxicity and cytokine production [20]. On the other hand, ligation of DAP10 dependent receptor (NKG2D) directly recruits PI3K and Grb2-Vav1 complex [15][16][17][18][19], triggering cytotoxicity but not cytokine production [30,31].…”