Unlike B and T cells, NK cells do not express antigen-specific receptors, yet they can eliminate virus-infected cells and cancer cells without harming normal cells. An important component that provides specificity in target cell recognition is inhibition of NK cells by inhibitory receptors that recognize surface molecules called major histocompatibility complex (MHC) class I. MHC-specific recognition by inhibitory receptors on NK cells prevents killing of normal healthy cells. The major goal of this project is to elucidate the mechanism by which inhibitory receptors block NK cell activation. Control of natural cytotoxicity by MHC class I-specific inhibitory receptors involves recruitment of the tyrosine phosphatase SHP-1, which dephosphorylates the guanine exchange factor Vav. As Vav is essential for proper actin remodeling and synapse formation, Vav inactivation through dephosphorylation provides an efficient way to block NK cell cytotoxicity. However, we have shown recently that the inhibitory signaling pathway is more complex and involves a second component, which relies on a tyrosine phosphorylation step. The small adapter Crk is phosphorylated during inhibition by MHC class I-specific receptors. Crk phosphorylation results in its dissociation from actin cytoskeleton-associated signaling complexes. Natural killer (NK) cell inhibitory receptors recruit tyrosine phosphatases to prevent activation, induce phosphorylation and dissociation of the small adaptor Crk from cytoskeleton scaffold complexes, and maintain NK cells in a state of responsiveness to subsequent activation events. How Crk contributes to inhibition is unknown. We imaged primary NK cells over lipid bilayers carrying IgG1 Fc to stimulate CD16, and human leukocyte antigen (HLA)-E to inhibit through receptor CD94-NKG2A. HLA-Ealone induced Crk phosphorylation in NKG2A+ NK cells. At activating synapses with Fc alone, Crk was required for the movement of Fc microclusters and their ability to trigger activation signals. At inhibitory synapses, HLA-E promoted central accumulation of both Fc and phosphorylated Crk, and blocked the Fc-induced buildup of F-actin. We propose a unified model for inhibitory receptor function: Crk phosphorylation prevents essential Crk-dependent activation signals and blocks F-actin network formation, thereby reducing constraints on subsequent engagement of activation receptors.

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Sim, Malcolm J W; Malaker, Stacy A; Khan, Ayesha et al. (2017) Canonical and Cross-reactive Binding of NK Cell Inhibitory Receptors to HLA-C Allotypes Is Dictated by Peptides Bound to HLA-C. Front Immunol 8:193
Kumar, Santosh; Rajagopalan, Sumati; Sarkar, Pabak et al. (2016) Zinc-Induced Polymerization of Killer-Cell Ig-like Receptor into Filaments Promotes Its Inhibitory Function at Cytotoxic Immunological Synapses. Mol Cell 62:21-33
Sim, Malcolm J W; Stowell, Janet; Sergeant, Ruhena et al. (2016) KIR2DL3 and KIR2DL1 show similar impact on licensing of human NK cells. Eur J Immunol 46:185-91
Kumar, Santosh; Sarkar, Pabak; Sim, Malcolm J W et al. (2015) A single amino acid change in inhibitory killer cell Ig-like receptor results in constitutive receptor self-association and phosphorylation. J Immunol 194:817-26
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Burshtyn, Deborah N; Scharenberg, Andrew M; Wagtmann, Nicolai et al. (2013) Pillars article: Recruitment of tyrosine phosphatase HCP by the killer cell inhibitory receptor. Immunity. 1996. 4: 77-85. J Immunol 191:3491-9
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Kim, Hun Sik; Das, Asmita; Gross, Catharina C et al. (2010) Synergistic signals for natural cytotoxicity are required to overcome inhibition by c-Cbl ubiquitin ligase. Immunity 32:175-86
Watzl, Carsten; Long, Eric O (2010) Signal transduction during activation and inhibition of natural killer cells. Curr Protoc Immunol Chapter 11:Unit 11.9B
Das, Asmita; Long, Eric O (2010) Lytic granule polarization, rather than degranulation, is the preferred target of inhibitory receptors in NK cells. J Immunol 185:4698-704

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