Physical Parameters of NK Cell Activation The Cytolytic NK Cell Immune Synapse

The surface of contact between NK cell and target cell has been termed the NK immune synapse (NKIS). Contact with a susceptible target cell has been named the cytolytic NKIS (cNKIS), and a noncytolytic interaction with a normal cell is defined as an inhibitory NKIS (iNKIS), which will be discussed later. Numerous activating receptor-ligand pairs and effector signaling molecules accumulate at the cNKIS, forming an aggregate called the central supramolec-ular activation cluster (c-SMAC) (see Fig. 1). The peripheral region (p) of the SMAC rings the c-SMAC. The p-SMAC characteristically contains NK cell adhesion molecules, including LFA-1 (CD11a) and Mac-1 (CD11b), which both tether to their ICAM-1 ligand on the target cell. CD2 is an NK cell adhesion molecule in the p-SMAC that interacts with CD58 on target cells in humans and CD48 in rodents. Accordingly, coexpression of ICAM-1 in combination with either CD48 or CD58 on target cells has been shown to initiate strong adhesion by resting NK cells [9]. The actin-binding protein talin characteristically accumulates beneath these adhesion molecules in the p-SMAC, providing a link to the cytoskeleton during cNKIS establishment [126,139,169].

During cNKIS formation, focused talin recruitment and actin polymerization in the NK cell organize the assembly of signaling molecules at the target interface. This stabilizes NK cell adhesion to the target cell and initiates subsequent actin-directed reorientation of the microtubule-organizing center (MTOC) toward the target cell [181]. Perforin-containing cytolytic granules are then shuttled to the cNKIS by microtubule-associated kinesin motors, resulting in the focused release of their contents at the target interface [90]. A ring of F-actin surrounding the site of granule release [126] is believed to localize the release of cytolytic effectors and thereby prevent killing of normal "bystander" cells. Interestingly, multiple cNKIS can simultaneously occur toward susceptible target cells on opposite poles of an NK cell, but one report suggests that c-SMAC formation can only occur at one of the multiple interfaces at a given time [169]. W├╝lfing and colleagues have provided evidence that actin polarization is a key element in a series of sequential cytoskeletal polarization events that are required to trigger NK cell cytotox-icity [181]. Furthermore, they found that cytotoxicity by NK cells was more sensitive than that of CTL to a pharmacological inhibitor of actin dynamics [181]. Separately, Orange et al. found sequential cytoskeletal reorganization requirements, in which actin function was necessary for adhesion molecule redistribution within the cNKIS, whereas tubulin assembly was not obligatory until subsequent perforin polarization toward the target [126].

Natural Killer Cell Immune Synapse

Fig. 1 The cytolytic NK immune synapse (cNKIS). A wide array of molecular interactions between receptor-ligand pairs, signaling effectors, signaling adaptors, lipid rafts, and cytoskeletal components occur at the contact interface between an NK cell (bottom) and a susceptible target cell (top). The lipid raft subdomain is designated as a speckled region of the overall plasma membrane (gray bar). The diagram is a simplified model to visualize the general physical localization of some of these molecules within the c-SMAC and p-SMAC regions of the developing cNKIS. Their locations are based on numerous immunofluorescence studies as described in the text

Fig. 1 The cytolytic NK immune synapse (cNKIS). A wide array of molecular interactions between receptor-ligand pairs, signaling effectors, signaling adaptors, lipid rafts, and cytoskeletal components occur at the contact interface between an NK cell (bottom) and a susceptible target cell (top). The lipid raft subdomain is designated as a speckled region of the overall plasma membrane (gray bar). The diagram is a simplified model to visualize the general physical localization of some of these molecules within the c-SMAC and p-SMAC regions of the developing cNKIS. Their locations are based on numerous immunofluorescence studies as described in the text

Lipid rafts are glycosphingolipid-enriched membrane microdomains that rapidly accumulate at the c-SMAC in a cytoskeleton-dependent manner [54, 104, 126, 168]. A number of important signaling markers are anchored in rafts, including the Src family PTK Lck and the linker for activation of T cells (LAT), which is a transmembrane adaptor [77, 194]. The c-SMAC becomes the focal point of intense positive signaling that is initiated by recruitment of a wide array of signaling effector proteins. The functions of these effectors will be discussed below in greater detail, but they include the membrane-active enzyme phospholipase Cy (PLCy)1, the cytosolic adaptors SLP-76, BLNK, and Wiskott-Aldrich syndrome protein (WASP), and the protein kinases Itk, Fyn, Lck, Syk, ZAP-70, and protein kinase C (PKC)-0 [127,169]. Recruitment of lipid rafts to the c-SMAC requires the Src family PTKs (Fyn and Lck), Syk family PTKs (Syk and ZAP-70), and the serine/threonine kinase, PKC-0 [13, 104]. Src homology 2 (SH2) domain-containing PTP-1 (SHP-1) is also recruited to the p-SMAC within 1 min of target cell conjugation [167, 168]. This may provide a mechanism for limiting the spread of activation beyond the c-SMAC. After 10 min of conjugation, SHP-1 enriches at the c-SMAC, possibly indicating a negative feedback role in limiting the duration of the activation response.

Many signaling events at the cNKIS lead to actin polymerization, which is crucial for cytotoxicity responses. The c-SMAC component WASP interacts with Cdc42 and the ARP2/3 complex to drive actin polymerization [128]. Patients with Wiskott-Aldrich syndrome exhibit defects in WASP and notably display impaired NK cell cytotoxicity that correlates with decreased frequency of F-actin and perforin at the cNKIS [66,127].

The above discussion has outlined the known physical parameters that occur at the cNKIS to trigger cytoskeletal polarization and the cytolytic response. The following sections will examine specific signaling pathways that are triggered downstream from activating receptors engaged with ligands at the cNKIS.

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