Lytic Granule Exocytosis Research Articles

LFA-1 nanoclusters integrate TCR stimulation strength to tune T-cell cytotoxic activity

T-cell cytotoxic function relies on the cooperation between the highly specific but poorly adhesive T-cell receptor (TCR) and the integrin LFA-1. How LFA-1-mediated adhesion may scale with TCR stimulation strength is ill-defined. Here, we show that LFA-1 conformation activation scales with TCR stimulation to calibrate human T-cell cytotoxicity. Super-resolution microscopy analysis reveals that >1000 LFA-1 nanoclusters provide a discretized platform at the immunological synapse to translate TCR engagement and density of the LFA-1 ligand ICAM-1 into graded adhesion. Indeed, the number of high-affinity conformation LFA-1 nanoclusters increases as a function of TCR triggering strength. Blockade of LFA-1 conformational activation impairs adhesion to target cells and killing. However, it occurs at a lower TCR stimulation threshold than lytic granule exocytosis implying that it licenses, rather than directly controls, the killing decision. We conclude that the organization of LFA-1 into nanoclusters provides a calibrated system to adjust T-cell killing to the antigen stimulation strength.

Lytic granule exocytosis at immune synapses: lessons from neuronal synapses.

Regulated exocytosis is a central mechanism of cellular communication. It is not only the basis for neurotransmission and hormone release, but also plays an important role in the immune system for the release of cytokines and cytotoxic molecules. In cytotoxic T lymphocytes (CTLs), the formation of the immunological synapse is required for the delivery of the cytotoxic substances such as granzymes and perforin, which are stored in lytic granules and released via exocytosis. The molecular mechanisms of their fusion with the plasma membrane are only partially understood. In this review, we discuss the molecular players involved in the regulated exocytosis of CTL, highlighting the parallels and differences to neuronal synaptic transmission. Additionally, we examine the strengths and weaknesses of both systems to study exocytosis.

A phenotypic screen for compounds that reverse cAMP-mediated suppression of T cell functions.

The solid tumor microenvironment (TME) suppresses immune responses. Three alterations in the TME converge on a pathway triggered by elevated cyclic AMP (cAMP) that suppresses T cell receptor (TCR) signaling. We developed a phenotypic assay to screen for small molecules that interfere with this pathway using TALL-104 human leukemic cytotoxic T lymphocytes pretreated with prostaglandin E2 to elevate cAMP. Beads coated with anti-CD3 antibodies stimulate lytic granule exocytosis, which is detected via binding of an antibody against lysosome associated membrane protein 1 (LAMP-1) measured with flow cytometry. Confirming that the assay can find compounds with desired activity, treating cells with a phorbol ester restores exocytosis. The assay behaves well in 96-well format and we screened a collection of compounds expected to have effects on epigenetic regulatory proteins. Compounds in this collection affected lytic granule exocytosis after 24-hour treatment, but none prevented cAMP from suppressing lytic granule exocytosis. We used a fully automated 384-well version of the assay to screen the Prestwick Compound Library but obtained no confirmed hits. Analyzing this assay's performance reveals two points of interest. First, cytometry offers multiple ways to quantify signals. Z' was higher using percent positive cells than mean fluorescence because the relationship between the two measures saturates, but using percent positive could make it harder to find hits in some assays. Second, variance was higher in positive controls than in negative controls in this assay, which degrades assay performance less than if variance was higher in negative controls.

Natural killer cells: origin, phenotype, function

Natural killer cells (NK) are innate immune lymphocytes produced in the bone marrow. Isolation of NK cells as a separate population of lymphocytes is related to discovery of their ability to induce the death of tumor cells without prior sensitization. In this review, an attempt was made to systematize the numerous data on the biology of NK cells presented in the literature. The authors consider the stages of NK cells` differentiation from a common lymphoid progenitor (CLP) in the bone marrow, describe two functionally different populations of mature NK cells – CD56brightCDl6- and CD56dimCD16+. In addition, the role of cytokines and chemokines in the development of NK cells is discussed. The review includes data on the spectrum of molecules expressed by NK cells: adhesion molecules (LFA-1, LFA-2, LFA-3; αMβ2, αXβ2, L-selectin, VLA-4, VLA-5; PECAM-1; CEACAM-1), cytokine receptors (IL-1R, IL-2ra, IL-2Rb/IL-2Rc, IL-6Rα, IL-7Ra, IL-8R, IL-10R, IL-12Rβ1, IL-15ra, IL-18R, IL-21ra, IFNGR2, TGFBR, c-Kit, CXCR1, CXCR3, CXCR4, CCR4, CCR5, CCR6, CCR7, IChemR23, CX3CR1), as well as receptors that regulate the activity of NK cells (LILRB1, LILRB2, LILRB4; KIR2DL1-5; KIR2DS1-5; KIR3DL1-3; KIR3DS1; NKG2A, NKG2C, NKG2D; Siglec7, Siglec9; CD16; NKRP-1; TIGIT; TACTILE; NKp30, NKp44, NKp46, NKp80; LAIR-1; PD-1; TIM-3; 2B4; TLR1-9). The authors also examine the mechanisms of implementing cytotoxic activity by NK cells, including cytotoxicity, via expression of MHC-I-specific receptors, CD16 Fc receptors, receptors and ligands of apoptosis (Fas-FasL and TRAIL-TRAILR) as well as other receptors. The review describes in detail the structure of immunological synapse between the NK cell and target cell, receptor interactions, and the role of the cytoskeleton in its formation. The data are summarized on the variants of exocytosis of lytic granules by NK cells, including complete or partial fusion of vesicles with the plasma membrane, exocytosis of vesicles containing perforin and FasL, and the formation of microvesicles containing granzyme B. The review also describes data on ability of NK cells to maintain activated state for a long time, as well as to maintain contact with several targets at the same time. In addition to the functions inherent in natural killers as cells of innate immunity, the authors point out their ability to exhibit the features of cells of adaptive immunity. In general, a variety of mechanisms that regulate the activity of NK cells may complement the specific functions of lymphocytes, thus making the immune system more efficient.

Tumor Lactic Acidosis: Protecting Tumor by Inhibiting Cytotoxic Activity Through Motility Arrest and Bioenergetic Silencing

Adoptive T cell therapy (ACT) is highly effective in the treatment of hematologic malignancies, but shows limited success in solid tumors. Inactivation of T cells in the tumor milieu is a major hurdle to a wider application of ACT. Cytotoxicity is the most relevant activity for tumor eradication. Here, we document that cytotoxic T cells (CTL) in lactic acidosis exhibited strongly reduced tumor cell killing, which could be compensated partly by increasing the CTL to tumor cell ratio. Lactic acid intervened at multiple steps of the killing process. Lactic acid repressed the number of CTL that performed lytic granule exocytosis (degranulation) in tumor cell co-culture, and, additionally impaired the quality of the response, as judged by the reduced intensity of degranulation and lower secretion of cytotoxins (perforin, granzyme B, granzyme A). CTL in lactic acid switched to a low bioenergetic profile with an inability to metabolize glucose efficiently. They responded to anti-CD3 stimulation poorly with less extracellular acidification rate (ECAR). This might explain their repressed granule exocytosis activity. Using live cell imaging, we show that CTL in lactic acid have reduced motility, resulting in lower field coverage. Many CTL in lactic acidosis did not make contact with tumor cells; however, those which made contact, adhered to the tumor cell much longer than a CTL in normal medium. Reduced motility together with prolonged contact duration hinders serial killing, a defining feature of killing potency, but also locally confines cytotoxic activity, which helps to reduce the risk of collateral organ damage. These activities define lactic acid as a major signaling molecule able to orchestrate the spatial distribution of CTL inside inflamed tissue, such as cancer, as well as moderating their functional response. Lactic acid intervention and strategies to improve T cell metabolic fitness hold promise to improve the clinical efficacy of T cell–based cancer immunotherapy.

CD56 regulates human NK cell cytotoxicity through Pyk2.

Human natural killer (NK) cells are defined as CD56+CD3-. Despite its ubiquitous expression on human NK cells the role of CD56 (NCAM) in human NK cell cytotoxic function has not been defined. In non-immune cells, NCAM can induce signaling, mediate adhesion, and promote exocytosis through interactions with focal adhesion kinase (FAK). Here we demonstrate that deletion of CD56 on the NK92 cell line leads to impaired cytotoxic function. CD56-knockout (KO) cells fail to polarize during immunological synapse (IS) formation and have severely impaired exocytosis of lytic granules. Phosphorylation of the FAK family member Pyk2 at tyrosine 402 is decreased in NK92 CD56-KO cells, demonstrating a functional link between CD56 and signaling in human NK cells. Cytotoxicity, lytic granule exocytosis, and the phosphorylation of Pyk2 are rescued by the reintroduction of CD56. These data highlight a novel functional role for CD56 in stimulating exocytosis and promoting cytotoxicity in human NK cells.

Molecular regulation of the plasma membrane-proximal cellular steps involved in NK cell cytolytic function.

Natural killer (NK) cells, cytolytic lymphocytes of the innate immune system, play a crucial role in the immune response against infection and cancer. NK cells kill target cells through exocytosis of lytic granules that contain cytotoxic proteins, such as perforin and granzymes. Formation of a functional immune synapse, i.e. the interface between the NK cell and its target cell enhances lysis through accumulation of polymerized F-actin at the NK cell synapse, leading to convergence of lytic granules to the microtubule organizing center (MTOC) and its subsequent polarization along microtubules to deliver the lytic granules to the synapse. In this review, we focus on the molecular mechanisms regulating the cellular processes that occur after the lytic granules are delivered to the cytotoxic synapse. We outline how - once near the synapse - the granules traverse the clearings created by F-actin remodeling to dock, tether and fuse with the plasma membrane in order to secrete their lytic content into the synaptic cleft through exocytosis. Further emphasis is given to the role of Ca2+ mobilization during degranulation and, whenever applicable, we compare these mechanisms in NK cells and cytotoxic T lymphocytes (CTLs) as adaptive immune system effectors.

PHENOTYPIC AND FUNCTIONAL CHARACTERISTICS OF MICROVESICLES PRODUCED BY NATURAL KILLER CELLS

Natural killer (NK) cells are of special interest among a multitude of microvesicle (MV) source cells. NK cells are a lymphocyte subpopulation performing contact cytolysis of virus-infected cells and tumor cells. Each of the NK cell populations has a unique receptor repertoire on its surface and, thus, unique functions. During their contact with a target cell, the most common mechanism of cytolysis is an exocytosis of lytic granules. However, some indirect evidence suggests that MV with CD56 phenotype and leukocyte-derived MV with various phenotypes are present in the peripheral blood plasma.This research is aimed to study the phenotype, composition and cytotoxic activity of microvesicles produced by NK cells. The analysis of receptor expression showed that MV, as well as source cells of the NK-92 cell line, had a similar CD56 molecule expression profile. The expression profile in MV differs from the same in source cells by higher CD119 and CD11b expression and by lower CD18 expression. Culturing of NK-92 cells in the presence of PMA, IL-1β, TNFα, IFNγ resulted in alterations of cell phenotypes and MV. Immunoblots revealed a change of perforin and granzyme B (GrB) in MV. The analysis of the cytotoxic activity of NK-92 cells in a natural killer in vitro assay employing K562 target cells demonstrated that MV obtained from TNFα-activated cells of the NK-92 cell line increased the cytotoxicity of the same TNFα-activated NK-92 cells regarding cytotoxicity levels. This coincides with the previously revealed increased content of GrB in MV obtained from TNFα-activated cells of the NK-92 cell line. To sum up depending on the cytokine NK-92 cells produce MV that differ in their phenotype, composition and activity. Any changes in MV composition can result in changes in their functional activity: in particular, changes can increase the cytotoxic activity of NK cells of the NK-92 cell line. Thus, besides a well-known and proved way for GrB delivery to a target cell, we can suggest an additional way – the transportation of GrB within MV.

Signaling pathways involved in the T-cell-mediated immunity against Epstein-Barr virus: Lessons from genetic diseases.

Primary immunodeficiencies (PIDs) provide researchers with unique models to understand in vivo immune responses in general and immunity to infections in particular. In humans, impaired immune control of Epstein-Barr virus (EBV) infection is associated with the occurrence of several different immunopathologic conditions; these include non-malignant and malignant B-cell lymphoproliferative disorders, hemophagocytic lymphohistiocytosis (HLH), a severe inflammatory condition, and a chronic acute EBV infection of T cells. Studies of PIDs associated with a predisposition to develop severe, chronic EBV infections have led to the identification of key components of immunity to EBV - notably the central role of T-cell expansion and its regulation in the pathophysiology of EBV-associated diseases. On one hand, the defective expansion of EBV-specific CD8 T cells results from mutations in genes involved in T-cell activation (such as RASGRP1, MAGT1, and ITK), DNA metabolism (CTPS1) or co-stimulatory pathways (CD70, CD27, and TNFSFR9 (also known as CD137/4-1BB)) leads to impaired elimination of proliferating EBV-infected B cells and the occurrence of lymphoma. On the other hand, protracted T-cell expansion and activation after the defective killing of EBV-infected B cells is caused by genetic defects in the components of the lytic granule exocytosis pathway or in the small adapter protein SH2D1A (also known as SAP), a key activator of T- and NK cell-cytotoxicity. In this setting, the persistence of EBV-infected cells results in HLH, a condition characterized by unleashed T-cell and macrophage activation. Moreover, genetic defects causing selective vulnerability to EBV infection have highlighted the role of co-receptor molecules (CD27, CD137, and SLAM-R) selectively involved in immune responses against infected B cellsvia specific T-B cell interactions.

A Multiplexed Assay That Monitors Effects of Multiple Compound Treatment Times Reveals Candidate Immune-Enhancing Compounds

We previously developed a flow cytometry assay that monitored lytic granule exocytosis in cytotoxic T lymphocytes stimulated by contacting beads coated with activating anti-CD3 antibodies. That assay was multiplexed in that responses of cells that did or did not receive the activating stimulus were distinguished via changes in light scatter accompanying binding of cells to beads, allowing us to discriminate compounds that activate responses on their own from compounds that enhance responses in cells that received the activating stimulus, all within a single sample. Here we add a second dimension of multiplexing by developing means to assess in a single sample the effects of treating cells with test compounds for different times. Bar-coding cells before adding them to test wells lets us determine compound treatment time while also monitoring activation status and response amplitude at the point of interrogation. This multiplexed assay is suitable for screening 96-well plates. We used it to screen compounds from the National Cancer Institute, identifying several compounds that enhance anti-LAMP1 responses. Multiple-treatment-time (MTT) screening enabled by bar-coding and read via high-throughput flow cytometry may be a generally useful method for facilitating the discovery of compounds of interest.

C2 Domains of Munc13-4 Are Crucial for Ca2+-Dependent Degranulation and Cytotoxicity in NK Cells.

In the immune system, degranulation/exocytosis from lymphocytes is crucial for life through facilitating eradication of infected and malignant cells. Dysfunction of the NK cell exocytosis process has been implicated with devastating immune diseases, such as familial hemophagocytic lymphohistiocytosis, yet the underlying molecular mechanisms of such processes have remained elusive. In particular, although the lytic granule exocytosis from NK cells is strictly Ca2+-dependent, the molecular identity of the Ca2+ sensor has yet to be identified. In this article, we show multiple lines of evidence in which point mutations in aspartic acid residues in both C2 domains of human Munc13-4, whose mutation underlies familial hemophagocytic lymphohistiocytosis type 3, diminished exocytosis with dramatically altered Ca2+ sensitivity in both mouse primary NK cells as well as rat mast cell lines. Furthermore, these mutations within the C2 domains severely impaired NK cell cytotoxicity against malignant cells. Total internal reflection fluorescence microscopy analysis revealed that the mutations strikingly altered Ca2+ dependence of fusion pore opening of each single granule and frequency of fusion events. Our results demonstrate that both C2 domains of Munc13-4 play critical roles in Ca2+-dependent exocytosis and cytotoxicity by regulating single-granule membrane fusion dynamics in immune cells.

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.

Nanoscale Dynamism of Actin Enables Secretory Function in Cytolytic Cells

SummaryNatural killer (NK) cells are innate immune effectors that lyse virally infected and tumorigenic cells through the formation of an immunological synapse. Actin remodeling at the lytic immunological synapse is a critical requirement for multiple facets of cytotoxic function. Activating receptor and integrin signaling leads to the regulated turnover and remodeling of actin, which is required for adhesion, sustained receptor signaling, and ultimately exocytosis. NK cells undergo lytic granule exocytosis in hypodense regions of a pervasive actin network. Although these requirements have been well demonstrated, neither the dynamic regulation of synaptic actin nor its specific function, however, has been determined at a nanoscale level. Here, live-cell super-resolution microscopy demonstrates nanoscale filamentous actin dynamism in NK cell lytic granule secretion. Following cell spreading, the overall content of the branched actin network at an immune synapse is stable over time and contains branched actin fibers and discrete actin foci. Similar actin architecture is generated in cytolytic T cells, although the timescale differs from that of NK cells. Individual filament displacement leads to stochastic clearance formation and disappearance, which are independent of lytic granule positioning. Actin dynamism is dependent upon branched network formation mediated by Arp2/3 and contractility generated by myosin IIA. Importantly, the use of small-molecule inhibitors demonstrates that actin dynamism is ultimately needed for granule secretion. Thus, we describe a requirement for nanoscale actin fiber rearrangement in generating the complex actin architecture that enables lytic granule secretion.

A Case of Type 2 Hypersensitivity to Rasburicase Diagnosed with a Natural Killer Cell Activation Assay

Drug hypersensitivity reactions can lead to different clinical pictures depending on the underlying immunological mechanism. Diagnosis tests are already available to assess the most frequent drugs hypersensitivity reactions, which are mediated by specific IgE or T cells. However, it remains challenging to diagnose type 2 hypersensitivity reactions (T2HR), which can lead to severe cytopenia and liver failure. Here, we describe a case of T2HR to rasburicase, an uricolytic agent used to prevent tumor lysis syndrome. In this patient, sensitization was associated with the production of specific IgG able to bind to leukocytes. We found that patient NK cells were specifically activated in the presence of rasburicase and autologous serum, which led to exocytosis of lytic granules. This antibody-dependent cell cytotoxicity mechanism may lead to cytopenia observed in the patient. Moreover, this NK cell activation assay could be used to improve the diagnosis of a T2HR to rasburicase and, by extent, to other drugs. These data also suggest that NK cells could play an important role in the pathophysiological mechanism of T2HR.

An actin cytoskeletal barrier inhibits lytic granule release from natural killer cells in patients with Chediak-Higashi syndrome

BackgroundChediak-Higashi syndrome (CHS) is a rare disorder caused by biallelic mutations in the lysosomal trafficking regulator gene (LYST), resulting in formation of giant lysosomes or lysosome-related organelles in several cell types. The disease is characterized by immunodeficiency and a fatal hemophagocytic lymphohistiocytosis caused by impaired function of cytotoxic lymphocytes, including natural killer (NK) cells.ObjectiveWe sought to determine the underlying biochemical cause of the impaired cytotoxicity of NK cells in patients with CHS.MethodsWe generated a human cell model of CHS using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology. We used a combination of classical techniques to evaluate lysosomal function and cell activity in the model system and super-resolution microscopy to visualize F-actin and lytic granules in normal and LYST-deficient NK cells.ResultsLoss of LYST function in a human NK cell line, NK92mi, resulted in inhibition of NK cell cytotoxicity and reproduced other aspects of the CHS cellular phenotype, including the presence of significantly enlarged lytic granules with defective exocytosis and impaired integrity of endolysosomal compartments. The large granules had an acidic pH and normal activity of lysosomal enzymes and were positive for the proteins essential for lytic granule exocytosis. Visualization of the actin meshwork openings at the immunologic synapse revealed that the cortical actin acts as a barrier for secretion of such large granules at the cell-cell contact site. Decreasing the cortical actin density at the immunologic synapse or decreasing the lytic granule size restored the ability of LYST-deficient NK cells to degranulate and kill target cells.ConclusionThe cortical actin and granule size play significant roles in NK cell cytotoxic function. We present evidence that the periodicity of subsynaptic actin is an important factor limiting the release of large lytic granules from NK cells from patients with CHS and could be a novel target for pharmaceutical intervention.

Syntaxin 4 mediates endosome recycling for lytic granule exocytosis in cytotoxic T-lymphocytes.

Adaptive and innate immunity utilize the perforin-killing pathway to eliminate virus-infected or cancer cells. Cytotoxic T-lymphocytes (CTLs) and natural killer cells mediate this process by releasing toxic proteins at the contact area with target cells known as immunological synapse (IS). Formation of a stable IS and exocytosis of toxic proteins requires persistent fusion of Rab11a recycling endosomes with the plasma membrane (PM) that may assure the delivery of key effector proteins. Despite the importance of the recycling endosomal compartment, the membrane fusion proteins that control this process at the IS remain elusive. Here, by performing knockdown experiments we found that syntaxin 4 (STX4) is necessary for cytotoxic activity and CD107a degranulation against target cells in a similar fashion to syntaxin 11, which is involved in lytic granule (LG) exocytosis and immunodeficiency when it is mutated. Using total internal reflection fluorescent microscopy we identified that STX4 mediates fusion of EGFP-Rab11a vesicles at the IS. Immunoprecipitation experiments in lysates of activated CTLs indicate that endogenous STX4 may drive this fusion step by interacting with cognate proteins: Munc18-3/SNAP23/VAMP7 and/or VAMP8. These results reveal the role of STX4 in mediating fusion of Rab11a endosomes upstream of lytic granules (LGs) exocytosis and further demonstrate the importance of this pathway in controlling CTL-mediated cytotoxicity.

Development of an Enhanced Phenotypic Screen of Cytotoxic T-Lymphocyte Lytic Granule Exocytosis Suitable for Use with Synthetic Compound and Natural Product Collections

We previously developed an assay of cytotoxic T-lymphocyte lytic granule exocytosis based on externalization of LAMP-1/CD107A using nonphysiological stimuli to generate maximal levels of exocytosis. Here, we used polystyrene beads coated with anti-CD3 antibodies to stimulate cells. Light scatter let us distinguish cells that contacted beads from cells that had not, allowing comparison of signaling events and exocytosis from stimulated and unstimulated cells in one sample. Bead stimulation resulted in submaximal exocytosis, making it possible to detect compounds that either augment or inhibit lytic granule exocytosis. Coupled with the assay’s ability to distinguish responses in cells that have and have not contacted a stimulatory bead, it is possible to detect three kinds of compounds: inhibitors, stimulators, which cause exocytosis, and augmenters, which enhance receptor-stimulated exocytosis. To validate the assay, we screened a set of synthetic compounds identified using our previous assay and a library of 320 extracts prepared from tunicate-associated bacteria. One of the extracts augmented exocytosis threefold. Activity-guided fractionation and structure elucidation revealed that this compound is the known PKC activator teleocidin A-1. We conclude that our modified assay is suitable for screening synthetic compound plates and natural product collections, and will be useful for identifying immunologically active small molecules.

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.

Chediak-Higashi syndrome: Lysosomal trafficking regulator domains regulate exocytosis of lytic granules but not cytokine secretion by natural killer cells

Mutations in lysosomal trafficking regulator (LYST) cause Chediak-Higashi syndrome (CHS), a rare immunodeficiency with impaired cytotoxic lymphocyte function, mainly that of natural killer (NK) cells. Our understanding of NK cell function deficiency in patients with CHS and how LYST regulates lytic granule exocytosis is very limited. We sought to delineate cellular defects associated with LYST mutations responsible for the impaired NK cell function seen in patients with CHS. We analyzed NK cells from patients with CHS with missense mutations in the LYST ARM/HEAT (armadillo/huntingtin, elongation factor 3, protein phosphatase 2A, and the yeast kinase TOR1) or BEACH (beige and Chediak-Higashi) domains. NK cells from patients with CHS displayed severely reduced cytotoxicity. Mutations in the ARM/HEAT domain led to a reduced number of perforin-containing granules, which were significantly increased in size but able to polarize to the immunologic synapse; however, they were unable to properly fuse with the plasma membrane. Mutations in the BEACH domain resulted in formation of normal or slightly enlarged granules that had markedly impaired polarization to the IS but could be exocytosed on reaching the immunologic synapse. Perforin-containing granules in NK cells from patients with CHS did not acquire certain lysosomal markers (lysosome-associated membrane protein 1/2) but were positive for markers of transport vesicles (cation-independent mannose 6-phosphate receptor), late endosomes (Ras-associated binding protein 27a), and, to some extent, early endosomes (early endosome antigen 1), indicating a lack of integrity in the endolysosomal compartments. NK cells from patients with CHS had normal cytokine compartments and cytokine secretion. LYST is involved in regulation of multiple aspects of NK cell lytic activity, ranging from governance of lytic granule size to control of their polarization and exocytosis, as well as regulation of endolysosomal compartment identity. LYST functions in the regulated exocytosis but not in the constitutive secretion pathway.

MID2 can substitute for MID1 and control exocytosis of lytic granules in cytotoxic T cells.

We have recently shown that the E3 ubiquitin ligase midline 1 (MID1) is upregulated in murine cytotoxic lymphocytes (CTL), where it controls exocytosis of lytic granules and the killing capacity. Accordingly, CTL from MID1 knock-out (MID1(-/-)) mice have a 25-30% reduction in exocytosis of lytic granules and cytotoxicity compared to CTL from wild-type (WT) mice. We wondered why the MID1 gene knock-out did not affect exocytosis and cytotoxicity more severely and speculated whether MID2, a close homologue of MID1, might partially compensate for the loss of MID1 in MID1(-/-) CTL. Here, we showed that MID2, like MID1, is upregulated in activated murine T cells. Furthermore, MID1(-/-) CTL upregulated MID2 two-twenty-fold stronger than CTL from WT mice, suggesting that MID2 might compensate for MID1. In agreement, transfection of MID2 into MID1(-/-) CTL completely rescued exocytosis of lytic granules in MID1(-/-) CTL, and vice versa, knock-down of MID2 inhibited exocytosis of lytic granules in both WT and MID1(-/-) CTL, demonstrating that both MID1 and MID2 play a central role in the regulation of granule exocytosis and that functional redundancy exists between MID1 and MID2 in CTL.