Inhibitory signaling blocks activating receptor clustering and induces cytoskeletal retraction in natural killer cells

Immunology Program, Memorial Sloan-Kettering Cancer Center, New York, NY 10065, USA.
The Journal of Cell Biology (Impact Factor: 9.83). 02/2011; 192(4):675-90. DOI: 10.1083/jcb.201009135
Source: PubMed


Natural killer (NK) lymphocytes use a variety of activating receptors to recognize and kill infected or tumorigenic cells during an innate immune response. To prevent targeting healthy tissue, NK cells also express numerous inhibitory receptors that signal through immunotyrosine-based inhibitory motifs (ITIMs). Precisely how signals from competing activating and inhibitory receptors are integrated and resolved is not understood. To investigate how ITIM receptor signaling impinges on activating pathways, we developed a photochemical approach for stimulating the inhibitory receptor KIR2DL2 during ongoing NK cell-activating responses in high-resolution imaging experiments. Photostimulation of KIR2DL2 induces the rapid formation of inhibitory receptor microclusters in the plasma membrane and the simultaneous suppression of microclusters containing activating receptors. This is followed by the collapse of the peripheral actin cytoskeleton and retraction of the NK cell from the source of inhibitory stimulation. These results suggest a cell biological basis for ITIM receptor signaling and establish an experimental framework for analyzing it.

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    • "60, 61, 62, 63, 64 Myosin IIA also is required for centripetal microcluster movement and this sustained retrograde flow of microclusters is required for sustained Ca2+ release and activation signaling.64, 65 Although the kinetics may be different in NK cells, microclusters of both activating and inhibitory receptors are present and appear to have similar dynamics compared with those in T cells.66, 67, 68, 69 Studies using NK target cell conjugates show that phosphorylated KIR2DL1-containing clusters originate in the periphery and coalesce in the center, thus reaffirming both the presence of microclusters and suggesting their utility in NK cells.67, "
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    ABSTRACT: Natural killer (NK) cell-mediated cytotoxicity is governed by the formation of a lytic immune synapse in discrete regulated steps, which give rise to an extensive array of cellular checkpoints in accessing NK cell-mediated cytolytic defense. Appropriate progression through these cell biological steps is critical for the directed secretion of specialized secretory lysosomes and subsequent target cell death. Here we highlight recent discoveries in the formation of the NK cell cytolytic synapse as well as the molecular steps and cell biological checkpoints required for this essential host defense process.Immunology and Cell Biology advance online publication, 21 January 2014; doi:10.1038/icb.2013.96.
    Immunology and Cell Biology 01/2014; 92(3). DOI:10.1038/icb.2013.96 · 4.15 Impact Factor
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    • "Thus, NK cell populations, which variably express different activating and inhibitory receptors, may respond differentially upon encountering a potential target cell. However, the underlying principles that control NK cell activation remain the same: activating signals emanating from their corresponding receptors (mediated by tyrosine-kinase based signal transduction pathways), are integrated with repressive signals from inhibitory receptors (mediated by protein phosphatases), culminating in either target cell killing or in unresponsiveness (27, 60). "
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    ABSTRACT: Natural killer (NK) cells are innate immune lymphocytes that function mainly as immune sentinels against viral infection and tumorigenesis. NK cell function is governed by inhibitory and activating signals arising from corresponding receptors. A prominent group of activating NK receptors is the natural cytotoxicity receptors (NCRs), which includes NKp30, NKp44, and NKp46. These receptors bind various diverse ligands of pathogenic, tumor, and even self origin. Type 1 diabetes mellitus (T1D) is a multifactorial autoimmune disease, in which insulin-producing beta (β) cells are ablated by the immune system. This killing of β cells is carried out mainly by T cells, but many other immune cells have been implicated in the pathogenesis of this disease. Importantly, NK cells were shown to be key participants in the initial autoimmune attack. It was shown that all β cells from humans and mice, healthy or sick, express an unknown ligand for the activating NKp46 receptor. In this review, we describe the role played by the NCRs in various pathologies with an emphasis on Type I diabetes.
    Frontiers in Immunology 01/2014; 5:4. DOI:10.3389/fimmu.2014.00004
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    • "Subsequently, it was shown that stimulation of NKG2A blocked the inside-out upregulation of integrin affinity in primary NK cells (10). Single cell imaging studies with NKL cells confirmed and extended these results by demonstrating that inhibitory signals antagonized IS formation, providing a reverse stop signal that encouraged migration rather than focused cytolysis (8, 9). Here, we demonstrate that engagement of ITIM-receptors disrupts IS formation in both resting and IL-2-cultured NK cells, which is largely consistent with these previous studies. "
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    ABSTRACT: Upon engagement of their cognate class I major histocompatibility complex ligands, receptors containing immunotyrosine-based inhibitory motifs (ITIMs) transduce signals that block cytolytic and inflammatory responses. In this manner, ITIM-coupled receptors play a crucial role in maintaining natural killer (NK) cell tolerance toward normal, healthy tissue. A number of studies, mostly using immortalized NK cell lines, have demonstrated that ITIM signaling functions by disrupting the cytolytic immunological synapse formed between an NK cell and its target. However, more recent imaging experiments using primary NK cells have suggested that inhibitory receptor engagement does not antagonize contact formation, casting doubt on the hypothesis that ITIM signals destabilize the synapse. To resolve this issue, we analyzed primary NK cell activation and contact formation on supported lipid bilayers containing controlled combinations of activating and inhibitory ligands. Under these conditions, we observed that ITIM signaling clearly inhibited adhesion, cell arrest, and calcium influx, three hallmarks of synapse formation. These results are consistent with previous reports showing that inhibitory receptors deliver a "reverse stop" signal, and confirm that ITIM signaling functions at least in part by destabilizing cytolytic synapse formation.
    Frontiers in Immunology 11/2013; 4:410. DOI:10.3389/fimmu.2013.00410
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