NK cells converge lytic granules to promote cytotoxicity and prevent bystander killing.
ABSTRACT: Natural killer (NK) cell activation triggers sequential cellular events leading to destruction of diseased cells. We previously identified lytic granule convergence, a dynein- and integrin signal-dependent movement of lysosome-related organelles to the microtubule-organizing center, as an early step in the cell biological process underlying NK cell cytotoxicity. Why lytic granules converge during NK cell cytotoxicity, however, remains unclear. We experimentally controlled the availability of human ligands to regulate NK cell signaling and promote granule convergence with either directed or nondirected degranulation. By the use of acoustic trap microscopy, we generated specific effector-target cell arrangements to define the impact of the two modes of degranulation. NK cells with converged granules had greater targeted and less nonspecific "bystander" killing. Additionally, NK cells in which dynein was inhibited or integrin blocked under physiological conditions demonstrated increased nondirected degranulation and bystander killing. Thus, NK cells converge lytic granules and thereby improve the efficiency of targeted killing and prevent collateral damage to neighboring healthy cells.
Project description:Natural killer (NK) cells play a central role during innate immune responses by eliminating pathogen-infected or tumorigenic cells. In the microenvironment, NK cells encounter not only target cells but also other cell types including non-target bystander cells. The impact of bystander cells on NK killing efficiency is, however, still elusive. In this study we show that the presence of bystander cells, such as P815, monocytes or HUVEC, enhances NK killing efficiency. With bystander cells present, the velocity and persistence of NK cells were increased, whereas the degranulation of lytic granules remained unchanged. Bystander cell-derived H<sub>2</sub>O<sub>2</sub> was found to mediate the acceleration of NK cell migration. Using mathematical diffusion models, we confirm that local acceleration of NK cells in the vicinity of bystander cells reduces their search time to locate target cells. In addition, we found that integrin ? chains (?1, ?2 and ?7) on NK cells are required for bystander-enhanced NK migration persistence. In conclusion, we show that acceleration of NK cell migration in the vicinity of H<sub>2</sub>O<sub>2</sub>-producing bystander cells reduces target cell search time and enhances NK killing efficiency.
Project description:NK cells are cytotoxic lymphocytes important in defense against viral infection and cancer. NK cells mediate cytotoxicity predominantly through directed secretion of lytic granules, which are specialized lysosome-related organelles, containing effector molecules such as perforin and granzymes. Although many requirements for lytic granule transport to, and secretion at, the NK cell lytic synapse are known, the minimum number of degranulation events required by an NK cell to kill its target is unknown. We performed high-resolution four-dimensional confocal microscopy of human NK-target cell conjugates to quantify NK cell degranulation (using a degranulation indicator, LAMP-1-pHluorin) as well as target cell death. Despite containing almost 200 granules, we found that an individual NK cell needed only two to four degranulation events, on average, to mediate target cell death. Although NK cells released approximately one-tenth of their total lytic granule reserve upon a single target, they required just over one-hundredth of their total lytic granules to kill a target cell. Importantly, the kinetics of NK cell killing correlated to the size of and the amount of effector molecules contained within lytic granules, as well as the temporal, but not spatial, organization of degranulation events. Thus, our study answers a fundamental question as to how many degranulation events it takes for a human NK cell to kill its target.
Project description:NK cells provide host defense by killing viral-infected and cancerous cells through the secretion of preformed lytic granules. Polarization of the lytic granules toward the target cell is dependent on an intact microtubule (MT) network as well as MT motors. We have recently shown that DOCK8, a gene mutated in a primary immunodeficiency syndrome, is involved in NK cell killing in part through its effects on MT organizing center (MTOC) polarization. In this study, we identified Hook-related protein 3 (HkRP3) as a novel DOCK8- and MT-binding protein. We further show that HkRP3 is present in lytic granule fractions and interacts with the dynein motor complex and MTs. Significantly, depletion of HkPR3 impaired NK cell cytotoxicity, which could be attributed to a defect in not only MTOC polarity, but also impaired clustering of lytic granules around the MTOC. Our results demonstrate an important role for HkRP3 in regulating the clustering of lytic granules and MTOC repositioning during the development of NK cell-mediated killing.
Project description:Natural killer (NK) cells kill a target cell by secreting perforin into the lytic immunological synapse, a specialized interface formed between the NK cell and its target. Perforin creates pores in target cell membranes allowing delivery of proapoptotic enzymes. Despite the fact that secreted perforin is in close range to both the NK and target cell membranes, the NK cell typically survives while the target cell does not. How NK cells preferentially avoid death during the secretion of perforin via the degranulation of their perforin-containing organelles (lytic granules) is perplexing. Here, we demonstrate that NK cells are protected from perforin-mediated autolysis by densely packed and highly ordered presynaptic lipid membranes, which increase packing upon synapse formation. When treated with 7-ketocholesterol, lipid packing is reduced in NK cells making them susceptible to perforin-mediated lysis after degranulation. Using high-resolution imaging and lipidomics, we identified lytic granules themselves as having endogenously densely packed lipid membranes. During degranulation, lytic granule-cell membrane fusion thereby further augments presynaptic membrane packing, enhancing membrane protection at the specific sites where NK cells would face maximum concentrations of secreted perforin. Additionally, we found that an aggressive breast cancer cell line is perforin resistant and evades NK cell-mediated killing owing to a densely packed postsynaptic membrane. By disrupting membrane packing, these cells were switched to an NK-susceptible state, which could suggest strategies for improving cytotoxic cell-based cancer therapies. Thus, lipid membranes serve an unexpected role in NK cell functionality protecting them from autolysis, while degranulation allows for the inherent lytic granule membrane properties to create local ordered lipid "shields" against self-destruction.
Project description:The formation of a dynamic, actin-rich immunological synapse (IS) and the polarization of cytolytic granules toward target cells are essential to the cytotoxic function of NK cells. Following polarization, lytic granules navigate through the pervasive actin network at the IS to degranulate and secrete their toxic contents onto target cells. We examined lytic granule motility and persistence at the cell cortex of activated human NK cells, using high-resolution total internal reflection microscopy and highly quantitative analysis techniques. We illustrate that lytic granules are dynamic and observe substantial motility at the plane of the cell cortex prior to, but not after, degranulation. We also show that there is no significant change in granule motility in the presence of Latrunculin A (which induces actin depolymerization), when added after granule polarization, but that there is a significant decrease in lytic granule persistence subsequent to degranulation. Thus, we show that lytic granules are highly dynamic at the cytolytic human NK cell IS prior to degranulation and that the persistence of granules at the cortex following exocytosis requires the integrity of the synaptic actin network.
Project description:Natural killer (NK) cells are critically involved in the early immune response against various intracellular pathogens, including <i>Coxiella burnetii</i> and <i>Chlamydia psittaci</i> <i>Chlamydia</i>-infected NK cells functionally mature, induce cellular immunity, and protect themselves by killing the bacteria in secreted granules. Here, we report that infected NK cells do not allow intracellular multiday growth of <i>Coxiella</i>, as is usually observed in other host cell types. <i>C. burnetii</i>-infected NK cells display maturation and gamma interferon (IFN-γ) secretion, as well as the release of <i>Coxiella</i>-containing lytic granules. Thus, NK cells possess a potent program to restrain and expel different types of invading bacteria via degranulation. Strikingly, though, in contrast to <i>Chlamydia</i>, expulsed <i>Coxiella</i> organisms largely retain their infectivity and, hence, escape the cell-autonomous self-defense mechanism in NK cells.
Project description:Natural killer (NK) cells play important roles in host immunity by killing virus-infected and tumor cells. Killing of the target cell is achieved by formation of an immune synapse and localized secretion of lytic granules containing perforin and granzymes. Here, we demonstrate that Wiskott-Aldrich syndrome protein (WASp)-interacting protein (WIP), important in generation of a large complex of proteins involved in actin cytoskeleton rearrangements, is indispensable for NK cell cytotoxicity. WIP knockdown completely inhibited cytotoxicity, whereas overexpression of WIP enhanced NK cell cytolytic ability. WIP was found to colocalize with lytic granules. WIP segregated to the lysosomal fraction, where granzyme B activity was also found, and the interaction between WIP and granules was independent of WASp. Importantly, WIP knockdown inhibited polarization of lytic granules to the immune synapse, but not conjugate formation. These results indicate that WIP is involved in lytic granule transport and is essential for regulation of NK cell cytotoxic function.
Project description:NK cells eliminate viral-infected and malignant cells through a highly orchestrated series of cytoskeletal rearrangements, resulting in the release of cytolytic granule contents toward the target cell. Central to this process is the convergence of cytolytic granules to a common point, the microtubule-organizing center (MTOC), before delivery to the synapse. In this study, we show that vasodialator-stimulated phosphoprotein (VASP), an actin regulatory protein, localizes to the cytolytic synapse, but surprisingly, shows no impact on conjugate formation or synaptic actin accumulation despite being required for human NK cell-mediated killing. Interestingly, we also find that a pool of VASP copurifies with lytic granules and localizes with lytic granules at the MTOC. Significantly, depletion of VASP decreased lytic granule convergence without impacting MTOC polarization. Using the KHYG-1 cell line in which lytic granules are in a constitutively converged state, we find that either VASP depletion or F-actin destabilization promoted spreading of formerly converged granules. Our results demonstrate a novel requirement for VASP and actin polymerization in maintaining lytic granule convergence during NK cell-mediated killing.
Project description:Secretory lysosomes of natural killer (NK) cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Lysosome-associated membrane protein (LAMP) 1/CD107a is used as a marker for NK-cell degranulation, but its role in NK-cell biology is unknown. We show that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNA interference (RNAi) cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi network-derived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity.
Project description:Natural killer (NK) cell-mediated killing involves the membrane fusion of preformed lytic granules. While the roles of actin and microtubules are well accepted during this process, the function of septins, another cytoskeletal component that associates with actin and microtubules, has not been investigated. Here we show that genetic depletion or pharmacologic stabilization of the septin cytoskeleton significantly inhibited NK cell cytotoxicity. Although the stabilization of septin filaments impaired conjugate formation, depletion of septin proteins had no impact on conjugate formation, lytic granule convergence, or MTOC polarization to the cytotoxic synapse (CS). Interestingly, septins copurify and accumulate near the polarized lytic granules at the CS, where they regulate lytic granule release. Mechanistically, we find that septin 7 interacts with the SNARE protein syntaxin 11 and facilitates its interaction with syntaxin binding protein 2 to promote lytic granule fusion. Altogether, our data identify a critical role for septins in regulating the release of lytic granule contents during NK cell-mediated killing.