Natural killer (NK) cells provide a unique opportunity to study integrin signaling in the absence of inside-out signals. In contrast to T cells where inside-out signaling is required to activate beta-2 integrin LFA-1, LFA-1 on primary NK cells binds directly to ICAM-1, which is sufficient to induce polarization of granules (aka secretory lysosomes) to the site of contact with target cells, but not degranulation. Conversely, binding of Fc receptor CD16 to IgG1 results in unpolarized degranulation. We took a proteomics approach to examine signaling by LFA-1. Primary human NK cells were stimulated on plates coated with ICAM-1. Tyrosine-phosphorylated proteins and their associated proteins were pulled down with mAb 4G10, eluted with phenyl-phosphate, and analyzed by mass spectrometry. Unique proteins enriched after stimulation by LFA-1, but not by CD16 were validated by immunoblotting. Through siRNA-mediated silencing, we showed that integrin-linked kinase (ILK), gamma-parvin, RhoGEF7, and Pyk2, all known to control cell polarity during migration, and that leupaxin were critical for LFA-1-dependent granule polarization. Proximity ligation assays to image protein complexes in cells revealed an enhanced association of ILK with LFA-1 upon ICAM-1 binding. Polarity in migrating cells is controlled also by a Cdc42-dependent pathway, which involves Par6, APC, and phosphorylation of kinase GSK3beta. Binding to ICAM-1 induced ILK-dependent GSK3beta phosphorylation in NK cells. Silencing of Cdc42, Par6 and APC blocked granule polarization to the NKtarget cell contact site. In addition, Pyk2, leupaxin, and CLIP-170 were required not only for MTOC polarization, but also for granule convergence to the MTOC. Our work has revealed that granule polarization induced by LFA-1 in NK cells uses a signaling pathway similar to that used to establish polarity during cell migration. Therefore, granule polarization induced by LFA-1 in NK cells uses a signaling pathway similar to that used to establish polarity during migration of adherent cells. Binding of natural killer (NK) cell inhibitory receptors to MHC class I molecules (MHC-I) confers increased responsiveness to NK cells by a process known as NK cell licensing/education. The number of IR engaged with MHC and the strength of MHC binding to IR calibrate the potential responsiveness of each NK cell for cytotoxicity and cytokine secretion. Reduced MHC-I expression or a lack of inhibitory receptors for MHC-I results in diminished NK cell responsiveness. It is still not clear how licensing affects different steps in NK cell cytotoxicity, such as the contribution of the β2 integrin LFA-1, which is essential for tight conjugation with target cells and for lytic granule polarization. Binding of cytotoxic lymphocytes to the adhesion ligand ICAM-1 requires an open conformation of LFA-1, which is regulated by inside-out signals from other receptors. In this study, we evaluated whether unlicensed NK cells have a defect in inside-out signaling to LFA-1 for conjugate formation or in outside-in signaling by LFA-1 for lytic granule polarization. Unlicensed NK cells did not form as many stable conjugates with target cells. The reduction of NK cell conjugation to target cells was not attributed to altered β2 integrin LFA-1 properties but was instead due to reduced inside-out signaling to LFA-1 by activating receptors. For those unlicensed NK cells that did form conjugates, LFA-1-dependent granule polarization was similar to that in licensed NK cells. Thus, licensing controls signals as proximal as inside-out signaling by activating receptors but not integrin outside-in signaling for granule polarization.

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