NK Cell Immunological Synapse Research Articles

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Super-Resolution Imaging of NK Cell Immunological Synapse Formation on a Supported Lipid Bilayer

Assessment of membrane lipid state at the natural killer cell immunological synapse.

The plasma membrane is a fluid structure that protects cells as one of their first barriers and actively participates in numerous biological processes in many ways including through distinct membrane sub-regions. For immunological cells, highly organized sub-compartments of plasma membranes are vital for them to sense and react to environmental changes. This includes a varying spectrum of lipid ordering in the plasma membrane which signifies or enables cellular functions. Thus, comprehensive analyses of the plasma membrane can facilitate understanding of important cell biological elements which include insights into immune cells. Here, we describe two methods that can be used to assess membrane lipid state at the natural killer cell immunological synapse via high-resolution live cell imaging techniques.

Mechanosensation and Mechanotransduction in Natural Killer Cells

Natural killer (NK) cells are a main subset of innate lymphocytes that contribute to host immune protection against viruses and tumors by mediating target cell killing and secreting a wide array of cytokines. Their functions are finely regulated by a balance between activating and inhibitory receptors and involve also adhesive interactions. Mechanotransduction is the process in which physical forces sensed by mechanosensors are translated into chemical signaling. Herein, we report findings on the involvement of this mechanism that is mainly mediated by actin cytoskeleton, in the regulation of NK cell adhesion, migration, tissue infiltration and functions. Actin represents the structural basis for NK cell immunological synapse (NKIS) and polarization of secretory apparatus. NK-target cell interaction involves the formation of both uropods and membrane nanotubes that allow target cell interaction over long distances. Actin retrograde flow (ARF) regulates NK cell signaling and controls the equilibrium between activation versus inhibition. Activating NKIS is associated with rapid lamellipodial ARF, whereas lower centripetal actin flow is present during inhibitory NKIS where β actin can associate with the tyrosine phosphatase SHP-1. Overall, a better knowledge of mechanotransduction might represent a future challenge: Realization of nanomaterials tailored for NK cells, would be important to translate in vitro studies in in vivo new immunotherapeutic approaches.

Escape of tumor cells from the NK cell cytotoxic activity

In recent years, NK cells, initially identified as potent cytotoxic effector cells, have revealed an unexpected complexity, both at phenotypic and functional levels. The discovery of different NK cell subsets, characterized by distinct gene expression and phenotypes, was combined with the characterization of the diverse functions NK cells can exert, not only as circulating cells, but also as cells localized or recruited in lymphoid organs and in multiple tissues. Besides the elimination of tumor and virus-infected cells, these functions include the production of cytokines and chemokines, the regulation of innate and adaptive immune cells, the influence on tissue homeostasis. In addition, NK cells display a remarkable functional plasticity, being able to adapt to the environment and to develop a kind of memory. Nevertheless, the powerful cytotoxic activity of NK cells remains one of their most relevant properties, particularly in the antitumor response. In this review, the process of tumor cell recognition and killing mediated by NK cells, starting from the generation of cytolytic granules and recognition of target cell, to the establishment of the NK cell immunological synapse, the release of cytotoxic molecules, and consequent tumor cell death is described. Next, the review focuses on the heterogeneous mechanisms, either intrinsic to tumors or induced by the tumor microenvironment, by which cancer cells can escape the NK cell-mediated attack.

Structural basis for RIFIN-mediated activation of LILRB1 in malaria.

The Plasmodium species that cause malaria are obligate intracellular parasites, and disease symptoms occur as they replicate within human blood. Despite risking immune detection, the parasite delivers proteins that bind host receptors to infected erythrocyte surfaces. In the causative agent of the most deadly human malaria, Plasmodium falciparum, RIFINs form the largest erythrocyte surface protein family1. Some RIFINs can bind inhibitory immune receptors, acting as targets for unusual antibodies containing a LAIR1 ectodomain2-4, or as ligands for LILRB15. RIFINs stimulate LILRB1 activation and signalling5, thereby potentially dampening human immune responses. To understand this process, we determined a structure of a RIFIN bound to LILRB1. We show that the RIFIN mimics the natural activating ligand of LILRB1, MHC class I, in its LILRB1-binding mode. A single RIFIN mutation disrupts the complex, blocks LILRB1 binding by all tested RIFINs and abolishes signalling in a reporter assay. In a supported lipid bilayer system, which mimics NK cell activation by antibody- dependent cell-mediated cytotoxicity, both RIFIN and MHC are recruited to the NK cell immunological synapse and reduce cell activation, as measured by perforin mobilisation. Therefore, LILRB1-binding RIFINs mimic the binding mode of the natural ligand of LILRB1 and suppress NK cell function.

PD-1 blocks lytic granule polarization with concomitant impairment of integrin outside-in signaling in the natural killer cell immunological synapse

The inhibitory receptor programmed cell death protein 1 (PD-1) is upregulated on a variety of immune cells, including natural killer (NK) cells, during chronic viral infection and tumorigenesis. Blockade of PD-1 or its ligands produces durable clinical responses with tolerable side effects in patients with a broad spectrum of cancers. However, the underlying molecular mechanisms of how PD-1 regulates NK cell function remain poorly characterized. We sought to determine the effect of PD-1 signaling on NK cells. PD-1 was overexpressed in CD16-KHYG-1 (a human NK cell line with both antibody-dependent cellular cytotoxicity through CD16 and natural cytotoxicity through NKG2D) cells and stimulated by exposing the cells to NK-sensitive target cells expressing programmed death ligand 1 (PD-L1). PD-1 engagement by PD-L1 specifically blocked NK cell-mediated cytotoxicity without interfering with the conjugation between NK cells and target cells. Further examination showed that PD-1 signaling blocked lytic granule polarization in NK cells, which was accompanied by failure of integrin-linked kinase, a key molecule in the integrin outside-in signaling pathway, to accumulate in the immunological synapse after NK-target cell conjugation. Our results suggest that NK cell cytotoxicity is inhibited by PD-1 engagement, which blocks lytic granule polarization to the NK cell immunological synapse with concomitant impairment of integrin outside-in signaling. This study provides novel mechanistic insights into how PD-1 inhibition disrupts NK cell function.

Immunology: Is Actin at the Lytic Synapse a Friend or a Foe?

Cytotoxic Tcells and natural killer cells defend us against disease by secreting lytic granules. Whether actin facilitates or thwarts lytic granule secretion has been an open question. Recent results now indicate that the answer depends on the maturation stage of the immune cell-target cell contact.

Termination of the Activating NK Cell Immunological Synapse Is an Active and Regulated Process.

Cellular cytotoxicity is essential for the elimination of virus-infected and cancerous cells by NK cells. It requires a direct cellular contact through the establishment of an immunological synapse (IS) between the NK cell and the target cell. In this article, we show that not only the establishment of the IS, but also its maintenance is a highly regulated process. Ongoing receptor-proximal signaling events from activating NK cell receptors and actin dynamics were necessary to maintain a stable contact in an energy-dependent fashion, even after the IS was formed successfully. More importantly, the initiation of a contact to a new susceptible target cell resulted in accelerated detachment from an old target cell. We propose that the maintenance of an existing IS is a dynamic and regulated process to allow for effective serial killing of NK cells.

Four-dimensional correlation between frequency of degranulation and Natural Killer (NK) cell cytotoxicity

Abstract NK cells are cytotoxic lymphocytes required for early control of viral infections and tumor immunosurveillance. NK cytotoxicity occurs through directed secretion of lytic granules (LG) containing effector molecules such as perforin and granzymes. Cytoskeletal reorganization during the formation of an NK-cell immunological synapse (IS) enables delivery of LG to the IS. Fusion of LG with the NK cell membrane results in exocytosis of its contents into the IS leading to target cell death. While the requirements for LG transport and exocytosis are largely known, the minimum number of degranulation events sufficient for an NK cell to kill its target is unknown. In this study we performed four-dimensional quantification of degranulation events using a degranulation indicator (LAMP1-pHluorin) in concert with a target cell death sensor. Confocal microscopy of live single NK-target cell conjugates allowed visualization and measurement of the entire NK-cell kill. Degranulation events were quantified in LAMP1-pHluorin transduced human NK cell lines against their known targets. Killing exhibited two distinct patterns, fast and slow. In one NK cell line the minimal effective degranulation events for fast killing were almost half of that for slow killing and were similar to the minimal effective degranulation events in the other NK cell line. LD50 for vital dye extinction was calculated and was mediated by a surprisingly small number of degranulation events. Importantly, a strong temporal correlation existed between the appearance of degranulation events and target cell killing. With this study we have quantified NK cell degranulation at a single cell level and defined parameters of degranulation that correlate with NK-cell cytotoxicity.

Measurement of Lytic Granule Convergence After Formation of an NK Cell Immunological Synapse.

Natural killer (NK) cells contain specialized lysosome-related organelles termed lytic granules allowing them to mediate cytotoxicity against tumorigenic or virally infected target cells. NK cells polarize their lytic granules toward a target cell via the microtubule-organizing center (MTOC). Prior to that, however, lytic granules converge to the MTOC along microtubules utilizing minus-end-directed microtubule motors. Herein we describe how to visualize and quantify lytic granule convergence using confocal microscopy to gain quantitative insight into NK cell cytotoxicity and its regulation.

High- and Super-Resolution Microscopy Imaging of the NK Cell Immunological Synapse.

Recent advances in imaging technology have enabled significant advances in the study of NK cell cytotoxic effector function through quantitative analysis of the NK cell immunological synapse. This can include the use of high- and super-resolution microscopy to quantify dynamics of cytoskeletal elements and the role they play in the regulation and execution of NK cell directed secretion. Here we describe a protocol for the recapitulation of the NK cell lytic synapse on glass, the acquisition of microscopy images, and suggested approaches for the processing and analysis of microscopy data.

UNC-45A Is a Nonmuscle Myosin IIA Chaperone Required for NK Cell Cytotoxicity via Control of Lytic Granule Secretion.

NK cell's killing is a tightly regulated process under the control of specific cytoskeletal proteins. This includes Wiskott-Aldrich syndrome protein, Wiskott-Aldrich syndrome protein-interacting protein, cofilin, Munc13-4, and nonmuscle myosin IIA (NMIIA). These proteins play a key role in controlling NK-mediated cytotoxicity either via regulating the attachment of lytic granules to the actin-based cytoskeleton or via promoting the cytoskeletal reorganization that is requisite for lytic granule release. UNC-45A is a highly conserved member of the UNC-45/CRO1/She4p family of proteins that act as chaperones for both conventional and nonconventional myosin. Although we and others have shown that in lower organisms and in mammalian cells NMIIA-associated functions, such as cytokinesis, cell motility, and organelle trafficking, are dependent upon the presence of UNC-45A, its role in NK-mediated functions is largely unknown. In this article, we describe UNC-45A as a key regulator of NK-mediated cell toxicity. Specifically we show that, in human NK cells, UNC-45A localize at the NK cell immunological synapse of activated NK cells and is part of the multiprotein complex formed during NK cell activation. Furthermore, we show that UNC-45A is disposable for NK cell immunological synapse formation and lytic granules reorientation but crucial for lytic granule exocytosis. Lastly, loss of UNC-45A leads to reduced NMIIA binding to actin, suggesting that UNC-45A is a crucial component in regulating human NK cell cytoskeletal dynamics via promoting the formation of actomyosin complexes.

Molecular mechanisms of functional natural killer deficiency in patients with partial DiGeorge syndrome

DiGeorge syndrome affects more than 3.5 million persons worldwide. Partial DiGeorge syndrome (pDGS), which is characterized by a number of gene deletions in chromosome 22, including the chicken tumor virus number 10 regulator of kinase (Crk)-like (CrkL) gene, is one of the most common genetic disorders in human subjects. To date, the role of natural killer (NK) cells in patients with pDGS remains unclear. We sought to define the effect of pDGS-related Crk haploinsufficiency on NK cell activation and cytotoxic immunological synapse (IS) structure and function. Inducible CrkL-silenced NK cells were used to recapitulate the pDGS, CrkL-haploinsufficient phenotype. Findings were validated by using NK cells from patients with actual pDGS. Ultimately, deficits in the function of NK cells from patients with pDGS were restored by lentiviral transduction of CrkL. Silencing of CrkL expression inhibits NK cell function. Specifically, pDGS haploinsufficiency of CrkL inhibits accumulation of activating receptors, polarization of cytolytic machinery and key signaling molecules, and activation of β2-integrin at the IS. Reintroduction of CrkL protein restores NK cell cytotoxicity. CrkL haploinsufficiency causes functional NK deficits in patients with pDGS by disrupting both β2-integrin activation and activating receptor accumulation at the IS. Our results suggest that NK cell IS quality can directly affect immune status, providing a potential target for diagnosis and therapeutic manipulation in patients with pDGS and in other patients with functional NK cell deficiencies.

Super-resolution imaging of the natural killer cell immunological synapse on a glass-supported planar lipid bilayer.

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

Super-resolution Imaging of the Natural Killer Cell Immunological Synapse on a Glass-supported Planar Lipid Bilayer

The glass-supported planar lipid bilayer system has been utilized in a variety of disciplines. One of the most useful applications of this technique has been in the study of immunological synapse formation, due to the ability of the glass-supported planar lipid bilayers to mimic the surface of a target cell while forming a horizontal interface. The recent advances in super-resolution imaging have further allowed scientists to better view the fine details of synapse structure. In this study, one of these advanced techniques, stimulated emission depletion (STED), is utilized to study the structure of natural killer (NK) cell synapses on the supported lipid bilayer. Provided herein is an easy-to-follow protocol detailing: how to prepare raw synthetic phospholipids for use in synthesizing glass-supported bilayers; how to determine how densely protein of a given concentration occupies the bilayer's attachment sites; how to construct a supported lipid bilayer containing antibodies against NK cell activating receptor CD16; and finally, how to image human NK cells on this bilayer using STED super-resolution microscopy, with a focus on distribution of perforin positive lytic granules and filamentous actin at NK synapses. Thus, combining the glass-supported planar lipid bilayer system with STED technique, we demonstrate the feasibility and application of this combined technique, as well as intracellular structures at NK immunological synapse with super-resolution.

Analysis of NKp46 Receptor Function at the NK Cell Immunological Synapse.

Analysis of NKp46 Receptor Function at the NK Cell Immunological Synapse.

Cell:cell interactions in the immune system

Video podcast available Go to www.immunologicalreviews.com to watch an interview with Guest Editor Facundo Batista. The cells of the immune system are continually on the move! Naive T cells in a human collectively crawl over 10 000 miles a day in search of antigens (1). Each dendritic cell (DC) touches 5000 T cells an hour to scan repertoires over a million deep (2, 3). The adaptive immune system functions through a vast network of provisional cell–cell interactions that are continuously changing. When this frenetic scanning finds a fit between T-cell antigen receptor (TCR) and DC antigen, the interaction can be prolonged with a pronounced deceleration of the T cell and finally a long-lived interaction that has been described as an immunological synapse (2, 4). In vitro studies on the immunological synapse suggest a profound supramolecular organization that serves focused trans-synaptic and juxtracrine information transfer between cells (5–8). Immunological synapses are not restricted to T cells but are also formed by natural killer (NK) cells, B cells, granulocytes, and phagocytes using related immunotyrosine-based activation motif-containing receptors (9, 10). These interactions are profoundly influenced by the three-dimensional (3D) tissue environment (11). This volume of Immunological Reviews takes a broader look at the cell–cell interactions in the immune system from the molecular organization of antigen receptors during surveillance to the organization of stromal cell networks in lymph nodes, bone marrow, and the lung (Fig. 1). The organization of the plasma membrane is a fundamental issue in biology that sets the stage for cell–cell interaction and communication. The classical fluid mosaic model suggested membrane proteins diffusing in a lipid sea with potential for interactions with cytoplasmic and extra-cytoplasmic binding partners that account for regulation and processes like adhesion and receptor capping. Observations on lipid phases in model systems with disordered, liquidordered, and gel phases in order of increasing viscosity suggested that such phases could also exist in biological membranes on some scale (12). Biophysical observations of liquid-ordered domains enriched in various lipid-modified proteins have led to models in which lipids have a role in providing a higher level of organization (13). Most recently, evidence for protein-driven domains, referred to as Immunological Reviews 2013 Vol. 251: 7–12 Printed in Singapore. All rights reserved

Important Role for NKp30 in Synapse Formation and Activation of NK Cells

Natural killer (NK) cells play an important role in host defense against viral infections and tumor cells. NK cells require contact with target cells and must subsequently form an NK cell immunological synapse (NKIS). The NKIS represents the interface formed between NK cells and their target cells via receptor recognition and adhesion molecule binding, and it is essential for NK cell cytotoxicity, which is mediated through degranulation and release of cytolytic enzymes. In this study, NKp30-mediated signaling and the role of NKp30 in the formation of the NKIS were investigated. The results from confocal microscopy studies showed that the activating receptor, NKp30, and the adhesion molecule, LFA-1 (CD11a), accumulated at the interface between NK92 cells, which are a human NK cell line, and HeLa cells, which are a human tumor cell line (NK92-HeLa). Blockade of NKp30 inhibited NK cell degranulation, cytotoxicity and cytokine secretion but did not affect the conjugation between NK and target cells. Additionally, ligation with HeLa cells activated the Erk1/2 signaling pathway in NK92 cells, and blockade of NKp30 decreased the phosphorylation of Erk1/2. Therefore, NKp30 is a triggering receptor downstream of adhesion and plays an important role in NK cell activation, degranulation and cytotoxicity.

A Targeted siRNA Screen Identifies Regulators of Cdc42 Activity at the Natural Killer Cell Immunological Synapse

Natural killer (NK) cells kill tumor cells and virally infected cells, and an effective NK cell response requires processes, such as motility, recognition, and directional secretion, that rely on cytoskeletal rearrangement. The Rho guanosine triphosphatase (GTPase) Cdc42 coordinates cytoskeletal reorganization downstream of many receptors. The Rho-related GTPase from plants 1 (ROP1) exhibits oscillatory activation behavior at the apical plasma membrane of growing pollen tubes; however, a similar oscillation in Rho GTPase activity has so far not been demonstrated in mammalian cells. We hypothesized that oscillations in Cdc42 activity might occur within NK cells as they interact with target cells. Through fluorescence lifetime imaging of a Cdc42 biosensor, we observed that in live NK cells forming immunological synapses with target cells, Cdc42 activity oscillated after exhibiting an initial increase. We used protein-protein interaction networks and structural databases to identify candidate proteins that controlled Cdc42 activity, leading to the design of a targeted short interfering RNA screen. The guanine nucleotide exchange factors RhoGEF6 and RhoGEF7 were necessary for Cdc42 activation within the NK cell immunological synapse. In addition, the kinase Akt and the p85α subunit of phosphoinositide 3-kinase (PI3K) were required for Cdc42 activation, the periodicity of the oscillation in Cdc42 activity, and the subsequent polarization of cytotoxic vesicles toward target cells. Given that PI3Ks are targets of tumor therapies, our findings suggest the need to monitor innate immune function during the course of targeted therapy against these enzymes.