Leibson, Paul J.

Abstract The NK cell-activating receptor NKG2D plays a critical role in the destruction of malignant cells, but many of the cell-signaling mechanisms governing NKG2D-mediated cellular cytotoxicity are unknown. We have identified an NKG2D-mediated signaling pathway that governs both conjugate formation and cytotoxic granule polarization. We demonstrate that an interaction between the regulatory subunit of PI3K, p85, and the adaptor protein CrkL is required for efficient NKG2D-mediated cellular cytotoxicity. We show decreased NK cell-target cell conjugate formation in NK cells treated with PI3K inhibitors or depleted of CrkL. Independent of adhesion, we find that microtubule organization center polarization toward target cells expressing the NKG2D ligand MICA or toward anti-NKG2D-coated beads is impaired in the absence of CrkL. Ab-stimulated granule release is also impaired in NK cells depleted of CrkL. Furthermore, our data indicate that the small Ras family GTPase Rap1 is activated downstream of NKG2D engagement in a PI3K- and CrkL-dependent manner and is required for conjugate formation, MTOC (microtubule organizing center) polarization, and NKG2D-dependent cellular cytotoxicity. Taken together, our data identify an NKG2D-activated signaling pathway that collectively orchestrates NK cell adhesion, cell polarization, and granule release.

Many studies have characterized the transmembrane signaling events initiated after T-cell antigen receptor recognition of major histocompatibility complex (MHC)-bound peptides. Yet, little is known about signal transduction from a set of MHC class I recognizing receptors on natural killer (NK) cells whose ligation dramatically inhibits NK cell-mediated killing. In this study we evaluated the influence of MHC recognition on the proximal signaling events in NK cells binding tumor targets. We utilized two experimental models where NK cell-mediated cytotoxicity was fully inhibited by the recognition of specific MHC class I molecules. NK cell binding to either class I-deficient or class I-transfected target cells initiated rapid protein tyrosine kinase activation. In contrast, whereas NK cell binding to class I-deficient targets led to inositol phosphate release and increased intracellular free calcium ([Ca2+]i), NK recognition of class I-bearing targets did not induce the activation of these phospholipase C-dependent signaling events. The recognition of class I by NK cells clearly had a negative regulatory effect since blocking this interaction using anti-class I F(ab')2 fragments increased inositol 1,4,5-trisphosphate release and [Ca2+]i and increased the lysis of the targets. These results suggest that one of the mechanisms by which NK cell recognition of specific MHC class I molecules can block the development of cell-mediated cytotoxicity is by inhibiting specific critical signaling events.

Target structures important for natural killer (NK) cell recognition of virally infected cells are not well defined. Since major histocompatibility complex (MHC) class I molecules bind viral peptides during acute infection, we evaluated whether an interaction between MHC and virus might influence the susceptibility of infected cells to NK cell-mediated lysis. To control for MHC class I expression on target cells, we used either HLA class I-deficient C1R cells or C1R sublines expressing transfected HLA class I gene products. Human NK cells were unable to preferentially lyse class I-deficient C1R cells after infection with herpes simplex virus (HSV). In contrast, HLA class I transfectants were significantly more susceptible to NK cell-mediated cytotoxicity after HSV infection. This occurred for HSV-infected C1R cells expressing any of the three HLA class I gene products tested (i.e., HLA-B27, HLA-A3, or HLA-Aw68), indicating that NK cell recognition in this system does not require "self" MHC and is not unique for a single haplotype. Productive HSV infection is required for the increased killing, since inoculation with UV-inactivated virus did not lead to increased lysis. In addition, since HSV infection of the transfectants did not significantly alter the level of class I expression, the change in susceptibility appears to be due to qualitative changes in the target structures on HSV-infected, HLA class I+ targets. These results demonstrate a role for MHC class I in regulating NK cell-mediated killing of virus-infected cells.

Natural killer (NK) cells are a unique subpopulation of lymphocytes with the capability to kill malignant cells via one of two alternative mechanisms: (i) Fc receptor-dependent cytotoxicity against antibody-coated targets or (ii) direct cell-mediated cytotoxicity. However, the molecular mechanisms that trigger and subsequently regulate NK cell cytotoxicity are incompletely understood. We have therefore investigated the role of protein tyrosine phosphorylation in the transmembrane signaling initiated after Fc receptor stimulation or direct tumor cell contact in clonal CD16+/CD3- human NK cells. We report that stimulation of the Fc receptor rapidly induced the tyrosine phosphorylation of a number of NK cell proteins. These effects occurred within 2 min, were maximal at 10 min, and declined toward baseline after 60 min. In addition, Fc receptor ligation increased the in vitro protein kinase activity of NK cell phosphotyrosyl proteins. We have also demonstrated that direct contact of NK cells with K562 tumor cells induced the rapid phosphorylation of distinct NK cell phosphotyrosyl proteins. Furthermore, the protein-tyrosine kinase inhibitor herbimycin A blocked NK cell cytotoxic function in a concentration-dependent manner. Our results suggest that protein tyrosine phosphorylation is an obligatory early proximal signal in activating the cytotoxic function of NK cells.

Although diverse signaling events are initiated by stimulation of multichain immune recognition receptors on lymphocytes, it remains unclear as to which specific signal transduction pathways are functionally linked to granule exocytosis and cellular cytotoxicity. In the case of natural killer (NK) cells, it has been presumed that the rapid activation of protein kinase C (PKC) enables them to mediate antibody-dependent cellular cytotoxicity (ADCC) and "natural" cytotoxicity toward tumor cells. However, using cloned human NK cells, we determined here that Fc receptor stimulation triggers granule release and ADCC through a PKC-independent pathway. Specifically, pretreatment of NK cells with the selective PKC inhibitor, GF109203X (using concentrations that fully blocked phorbol myristate acetate/ionomycin-induced secretion) had no effect on FcR-initiated granule release or ADCC. In contrast, FcR ligation led to the rapid activation of phosphatidylinositol 3-kinase (PI 3-kinase), and inhibition of this enzyme with the selective inhibitor, wortmannin, blocked FcR-induced granule release and ADCC. Additional experiments showed that, whereas FcR-initiated killing was wortmannin sensitive and GF109203X insensitive, natural cytotoxic activity toward the tumor cell line K562 was wortmannin insensitive and GF109203X sensitive. Taken together, these results suggest that: (a) PI 3-kinase activation induced by FcR ligation is functionally coupled to granule exocytosis and ADCC; and (b) the signaling pathways involved in ADCC vs natural cytotoxicity are distinct.

Crosslinking of the low affinity immunoglobulin G (IgG) Fc receptor (Fc gamma R type III) on natural killer (NK) cells initiates antibody-dependent cellular cytotoxicity. During this process, Fc gamma R stimulation results in the rapid activation of phospholipase C (PLC), which hydrolyzes membrane phosphoinositides, generating inositol-1,4,5-trisphosphate and sn-1,2-diacylglycerol as second messengers. We have recently reported that PLC activation after Fc gamma R stimulation can be inhibited by a protein tyrosine kinase (PTK) inhibitor. Based on the paradigm provided by the receptor tyrosine kinases, we investigated whether PLC-gamma 1 and/or PLC-gamma 2 are expressed in NK cells, and whether the PLC-gamma isoforms are tyrosine phosphorylated in response to Fc gamma R stimulation. Immunoblotting analyses with PLC-gamma 1- and PLC-gamma 2-specific antisera demonstrate that both isoforms are expressed in human NK cells. Furthermore, Fc gamma R crosslinking triggers the tyrosine phosphorylation of both PLC-gamma 1 and PLC-gamma 2 in these cells. Phosphorylation of both isoforms is detectable within 1 min, and returns to basal level within 30 min. Pretreatment with herbimycin A, a PTK inhibitor, blocked the Fc gamma R-induced tyrosine phosphorylation of PLC-gamma 1 and PLC-gamma 2, and the subsequent release of inositol phosphates. These results suggest that Fc gamma R-initiated phosphoinositide turnover in human NK cells is regulated by the tyrosine phosphorylation of PLC-gamma. More broadly, these observations demonstrate that nonreceptor PTK(s) activated by crosslinkage of a multisubunit receptor can phosphorylate both PLC-gamma isoforms.

AbstractNatural killer (NK) cells are a subpopulation of lymphocytes that can mediate cytotoxicity of certain tumor cells, virus‐infected cells, and normal cells. In addition to their cytotoxic potential, NK cells secrete a variety of cytokines and chemokines that can modulate the function, growth, and differentiation of other immune cells. These different responses are initiated by the interaction of specific NK surface receptors with defined soluble or cell‐associated ligands. There are several different types of receptors on the NK cell surface including “triggering” receptors, adhesion molecules, cytokine receptors, and MHC‐recognizing killer‐cell inhibitory receptors. The functional response of an NK cell is the result of the integration of signals transduced by these different types of receptors. Some of these signaling pathways are similar to other lymphoid cells, but there are also unique features employed by NK cells. This overview focuses on receptor‐initiated signaling pathways that modulate NK functions.