Natural killer cells kill extracellular Pseudomonas aeruginosa using contact-dependent release of granzymes B and H.

Pseudomonas aeruginosa is an opportunistic pathogen that often infects individuals with the genetic disease cystic fibrosis, and contributes to airway blockage and loss of lung function. Natural killer (NK) cells are cytotoxic, granular lymphocytes that are part of the innate immune system. NK cell secretory granules contain the cytolytic proteins granulysin, perforin and granzymes. In addition to their cytotoxic effects on cancer and virally infected cells, NK cells have been shown to play a role in an innate defense against microbes, including bacteria. However, it is not known if NK cells kill extracellular P. aeruginosa or how bacterial killing might occur at the molecular level. Here we show that NK cells directly kill extracellular P. aeruginosa using NK effector molecules. Live cell imaging of a co-culture of YT cells, a human NK cell line, and GFP-expressing P. aeruginosa in the presence of the viability dye propidium iodide demonstrated that YT cell killing of P. aeruginosa is contact-dependent. CRISPR knockout of granulysin or perforin in YT cells had no significant effect on YT cell killing of P. aeruginosa. Pre-treatment of YT and NK cells with the serine protease inhibitor 3,4-dichloroisocoumarin (DCI) to inhibit all granzymes, resulted in an inhibition of killing. Although singular CRISPR knockout of granzyme B or H had no effect, knockout of both in YT cells completely abrogated killing of P. aeruginosa in comparison to wild type YT cell controls. Nitrocefin assays suggest that the bacterial membrane is damaged. Inhibition of killing by antioxidants suggest that ROS are required for the bactericidal mode-of-action. Taken together, these results identify that NK cells kill P. aeruginosa through a membrane damaging, contact-dependent process that requires granzyme induced ROS production, and moreover, that granzyme B and H are redundant in this killing process.

Natural killer cells kill Burkholderia cepacia complex via a contact-dependent and cytolytic mechanism.

Burkholderia cepacia complex (Bcc), which includes B. cenocepacia and B. multivorans, pose a life-threatening risk to patients with cystic fibrosis. Eradication of Bcc is difficult due to the high level of intrinsic resistance to antibiotics, and failure of many innate immune cells to control the infection. Because of the pathogenesis of Bcc infections, we wondered if a novel mechanism of microbial host defense involving direct antibacterial activity by natural killer (NK) cells might play a role in the control of Bcc. We demonstrate that NK cells bound Burkholderia, resulting in Src family kinase activation as measured by protein tyrosine phosphorylation, granule release of effector proteins such as perforin and contact-dependent killing of the bacteria. These studies provide a means by which NK cells could play a role in host defense against Bcc infection.

ß1 Integrins Are Required To Mediate NK Cell Killing of Cryptococcus neoformans.

Cryptococcus neoformans is a fungal pathogen that causes fatal meningitis and pneumonia. During host defense to Cryptococcus, NK cells directly recognize and kill C. neoformans using cytolytic degranulation analogous to killing of tumor cells. This fungal killing requires independent activation of Src family kinase (SFK) and Rac1-mediated pathways. Recognition of C. neoformans requires the natural cytotoxicity receptor, NKp30; however, it is not known whether NKp30 activates both signal transduction pathways or whether a second receptor is involved in activation of one of the pathways. We used primary human NK cells and a human NK cell line and found that NKp30 activates SFK → PI3K but not Rac1 cytotoxic signaling, which led to a search for the receptor leading to Rac1 activation. We found that NK cells require integrin-linked kinase (ILK) to activate Rac1 for effective fungal killing. This observation led to our identification of ß1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including ß1 integrins and NKp30 and their proximal signaling pathways, are required for recognition of Cryptococcus, which activates a central cytolytic antimicrobial pathway leading to fungal killing.

Cryptococcus gattii Capsule Blocks Surface Recognition Required for Dendritic Cell Maturation Independent of Internalization and Antigen Processing.

Cryptococcus gattii is an emerging fungal pathogen on the west coast of Canada and the United States that causes a potentially fatal infection in otherwise healthy individuals. In previous investigations of the mechanisms by which C. gattii might subvert cell-mediated immunity, we found that C. gattii failed to induce dendritic cell (DC) maturation, leading to defective T cell responses. However, the virulence factor and the mechanisms of evasion of DC maturation remain unknown. The cryptococcal polysaccharide capsule is a leading candidate because of its antiphagocytic properties. Consequently, we asked if the capsule of C. gattii was involved in evasion of DC maturation. We constructed an acapsular strain of C. gattii through CAP59 gene deletion by homologous integration. Encapsulated C. gattii failed to induce human monocyte-derived DC maturation and T cell proliferation, whereas the acapsular mutant induced both processes. Surprisingly, encapsulation impaired DC maturation independent of its effect on phagocytosis. Indeed, DC maturation required extracellular receptor signaling that was dependent on TNF-α and p38 MAPK, but not ERK activation, and the cryptococcal capsule blocked this extracellular recognition. Although the capsule impaired phagocytosis that led to pH-dependent serine-, threonine-, and cysteine-sensitive protease-dependent Ag processing, it was insufficient to impair T cell responses. In summary, C. gattii affects two independent processes, leading to DC maturation and Ag processing. The polysaccharide capsule masked extracellular detection and reduced phagocytosis that was required for DC maturation and Ag processing, respectively. However, the T cell response was fully restored by inducing DC maturation.

Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans.

The activity of Rac in leukocytes is essential for immunity. However, its role in NK cell-mediated anti-microbial signaling remains unclear. In this study, we investigated the role of Rac in NK cell mediated anti-cryptococcal killing. We found thatCryptococcus neoformansindependently activates both Rac and SFK pathways in NK cells, and unlike in tumor killing,Cryptococcusinitiated a novel Rac → PI3K → Erk cytotoxicity cascade. Remarkably, Rac was not required for conjugate formation, despite its essential role in NK cytotoxicity againstC. neoformans Taken together, our data show that, unlike observations with tumor cells, NK cells use a novel Rac cytotoxicity pathway in conjunction with SFK, to killC. neoformans.

Glycyrrhizin inhibits highly pathogenic H5N1 influenza A virus-induced pro-inflammatory cytokine and chemokine expression in human macrophages.

Hypercytokinaemia is thought to contribute to highly pathogenic H5N1 influenza A virus disease. Glycyrrhizin is known to exert immunomodulatory and anti-inflammatory effects and therefore a candidate drug for the control of H5N1-induced pro-inflammatory gene expression. Here, the effects of an approved parenteral glycyrrhizin preparation were investigated on H5N1 virus replication, H5N1-induced pro-inflammatory responses, and H5N1-induced apoptosis in human monocyte-derived macrophages. Glycyrrhizin 100 µg/ml, a therapeutically achievable concentration, impaired H5N1-induced production of CXCL10, interleukin 6, and CCL5 and inhibited H5N1-induced apoptosis but did not interfere with H5N1 replication. Global inhibition of immune responses may result in the loss of control of virus replication by cytotoxic immune cells including natural killer cells and cytotoxic CD8(+) T-lymphocytes. Notably, glycyrrhizin concentrations that inhibited H5N1-induced pro-inflammatory gene expression did not affect cytolytic activity of natural killer cells. Since H5N1-induced hypercytokinaemia is considered to play an important role within H5N1 pathogenesis, glycyrrhizin may complement the arsenal of potential drugs for the treatment of H5N1 disease.

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells.

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (NS1 IAV). The cells were used to investigate the influence of NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with DeltaNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with DeltaNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells.

Primary Esophageal Tuberculosis Without Dysphagia or Odynophagia in a Patient Without HIV.

We report a case of primary esophageal tuberculosis in a 35-year-old woman without HIV who presented with a month's history of epigastric and chest pain without dysphagia or odynophagia and was found to have histologic evidence of multiple caseating granulomata on esophageal biopsy, which was confirmed positive for Mycobacterium tuberculosis complex DNA and cultures.

Phagosomal F-Actin Retention by Cryptococcus gattii Induces Dendritic Cell Immunoparalysis.

Cryptococcus gattii is a major cause of life-threatening mycosis in immunocompetent individuals and responsible for the ongoing epidemic outbreak of cryptococcosis in the Pacific Northwest of North America. This deadly fungus is known to evade important host immune responses, including dendritic cell (DC) maturation and concomitant T cell immunity, via immune evasion mechanisms that remain unclear. Here, we demonstrate that primary human DCs phagocytose C. gattii but the maturation of phagosomes to phagolysosomes was blocked as a result of sustained filamentous actin (F-actin) that entrapped and concealed the phagosomes from recognition. Superresolution structured illumination microscopy (SR-SIM) revealed that the persistent phagosomal F-actin formed a cage-like structure that sterically hindered and functionally blocked the fusion of lysosomes. Blocking lysosome fusion was sufficient to inhibit phagosomal acidification and subsequent intracellular fungal killing by DCs. Retention of phagosomal F-actin by C. gattii also caused DC immunoparalysis. Disrupting the retained F-actin cage with cytochalasin D not only restored DC phagosomal maturation but also promoted DC costimulatory maturation and robust T cell activation and proliferation. Collectively, these results reveal a unique mechanism of DC immune evasion that enhances intracellular fungal pathogenicity and may explain suppressed cell-mediated immunity.IMPORTANCECryptococcus yeast species typically display characteristics of opportunistic pathogens, with the exception of C. gattii, which can cause life-threatening respiratory and disseminated brain infections in otherwise healthy people. The pathogenesis of C. gattii is not well understood, but an important characteristic is that C. gattii is capable of evading host cell-mediated immune defenses initiated by DCs. Here, we report that when virulent C. gattii becomes ingested by a DC, the intracellular compartment containing the fungi is covered by a persistent protein cage structure consisting of F-actin. This F-actin cage acts as a barrier to prevent interaction with other intracellular compartments, and as a result, the DC fails to kill the fungi and activate important cell-mediated immune responses. We propose that this unique immune evasion mechanism permits C. gattii to remain unchallenged within host cells, leading to persistent infection.

Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method.

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity.

Microbial killing by NK cells.

It is now evident that NK cells kill bacteria, fungi, and parasites in addition to tumor and virus-infected cells. In addition to a number of recent publications that have identified the receptors and ligands, and mechanisms of cytotoxicity, new insights are reflected in the reports from researchers all over the world at the 17th Meeting of the Society for Natural Immunity held in San Antonio, TX, USA from May 28 through June 1, 2018. We will provide an overview of the field and discuss how the presentations at the meeting might shape our knowledge and future directions in the field.

Granule-Dependent NK Cell Killing of Cryptococcus Requires Kinesin to Reposition the Cytolytic Machinery for Directed Cytotoxicity.

Cryptococcus is the most important cause of fungal meningitis in immunocompromised individuals. Host defense against Cryptococcus involves direct killing by NK cells. That NK cells from HIV-infected patients fail to polarize perforin to the microbial synapse and kill C. neoformans led us to explore the mechanisms used to reposition and polarize the cytolytic granules to the synapse. Using live-cell imaging, we observed microtubule and granule movements in response to Cryptococcus that revealed a kinesin-dependent event. Eg5-kinesin bound to perforin-containing granules and was required for association with the microtubules. Inhibition of Eg5-kinesin abrogated dynein-dependent granule convergence to the MTOC and granule and MTOC polarization to the synapse and suppressed NK cell killing of Cryptococcus. In contrast, Eg5-kinesin was dispensable for tumor killing. This reveals an alternative mechanism of MTOC repositioning and granule polarization, not used in tumor cytotoxicity, in which Eg5-kinesin is required to initiate granule movement, leading to microbial killing.

Identification of the fungal ligand triggering cytotoxic PRR-mediated NK cell killing of Cryptococcus and Candida.

Natural killer (NK) cells use the activating receptor NKp30 as a microbial pattern-recognition receptor to recognize, activate cytolytic pathways, and directly kill the fungi Cryptococcus neoformans and Candida albicans. However, the fungal pathogen-associated molecular pattern (PAMP) that triggers NKp30-mediated killing remains to be identified. Here we show that ß-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that ß-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, ß-1,3-glucan increases expression and clustering of NKp30 at the microbial and NK cell synapse to induce perforin release for fungal cytotoxicity. Rather than blocking the interaction between fungi and NK cells, soluble ß-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating ß-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

Histone deacetylase inhibitors suppress natural killer cell cytolytic activity.

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation.

Inhibition of apoptosis prevents West Nile virus induced cell death.

BACKGROUND: West Nile virus (WNV) infection can cause severe meningitis and encephalitis in humans. Apoptosis was recently shown to contribute to the pathogenesis of WNV encephalitis. Here, we used WNV-infected glioma cells to study WNV-replication and WNV-induced apoptosis in human brain-derived cells. RESULTS: T98G cells are highly permissive for lytic WNV-infection as demonstrated by the production of infectious virus titre and the development of a characteristic cytopathic effect. WNV replication decreased cell viability and induced apoptosis as indicated by the activation of the effector caspase-3, the initiator caspases-8 and -9, poly(ADP-ribose)polymerase (PARP) cleavage and the release of cytochrome c from the mitochondria. Truncation of BID indicated cross-talk between the extrinsic and intrinsic apoptotic pathways. Inhibition of the caspases-8 or -9 inhibited PARP cleavage, demonstrating that both caspases are involved in WNV-induced apoptosis. Pan-caspase inhibition prevented WNV-induced apoptosis without affecting virus replication. CONCLUSION: We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death.

Granule-Dependent Natural Killer Cell Cytotoxicity to Fungal Pathogens.

Natural killer (NK) cells kill or inhibit the growth of a number of fungi including Cryptococcus, Candida, Aspergillus, Rhizopus, and Paracoccidioides. Although many fungi are not dangerous, invasive fungal pathogens, such as Cryptococcus neoformans, cause life-threatening disease in individuals with impaired cell-mediated immunity. While there are similarities to cell-mediated killing of tumor cells, there are also important differences. Similar to tumor killing, NK cells directly kill fungi in a receptor-mediated and cytotoxic granule-dependent manner. Unlike tumor cell killing where multiple NK cell-activating receptors cooperate and signal events that mediate cytotoxicity, only the NKp30 receptor has been described to mediate signaling events that trigger the NK cell to mobilize its cytolytic payload to the site of interaction with C. neoformans and Candida albicans, subsequently leading to granule exocytosis and fungal killing. More recently, the NKp46 receptor was reported to bind Candida glabrata adhesins Epa1, 6, and 7 and directly mediate fungal clearance. A number of unanswered questions remain. For example, is only one NK cell-activating receptor sufficient for signaling leading to fungal killing? Are the signaling pathways activated by fungi similar to those activated by tumor cells during NK cell killing? How do the cytolytic granules traffic to the site of interaction with fungi, and how does this process compare with tumor killing? Recent insights into receptor use, intracellular signaling and cytolytic granule trafficking during NK cell-mediated fungal killing will be compared to tumor killing, and the implications for therapeutic approaches will be discussed.

Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients.

UNLABELLED: Cryptococcus neoformans is a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and kill C. neoformans via a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans Moreover, those NK cells that bound to C. neoformans failed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response to C. neoformans Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12. IMPORTANCE: The mechanisms by which NK cells bind directly to pathogens and deploy their deadly cytolytic machinery during microbial host defense are only beginning to be elucidated. With the goal of understanding this process, we used NK cells from HIV-infected patients, which were known to have a defect in killing of Cryptococcus neoformans Taking advantage of previous studies that had shown that IL-12 restored killing, we used the cytokine as a gain-of-function approach to define the relevance of multiple steps in the recognition and cytolytic pathway. We demonstrated that NK cells from HIV-infected patients failed to kill Cryptococcus due to defects in perforin expression, granule polarization, and release of perforin. Additionally, IL-12 restored recognition of C. neoformans through binding of the NK-activating receptor NKp30. These observations identify important mechanisms used by NK cells to kill microbes and determine that defects in NK cells from HIV-infected patients are reversible.

Valproic acid modulates NCAM polysialylation and polysialyltransferase mRNA expression in human tumor cells.

Polysialic acid (PSA) is a dynamically regulated carbohydrate modification of the neural cell adhesion molecule NCAM, which has been linked to cancer development and dissemination. Two enzymes, the polysialyltransferases ST8SiaIV and ST8SiaII, are known to be involved in the polysialylation of NCAM. The antiepileptic drug valproic acid (VPA) is associated with anti-cancer activity. In this study, VPA blocked the adhesion of several neuroectodermal tumor cell lines to human umbilical vein endothelial cells. Furthermore, VPA induced intracellular PSA accumulation and enhanced expression of PSA-NCAM on the cell surface. Using a semiquantitative RT-PCR strategy, VPA was shown to up-regulate ST8SiaIV mRNA, whereas ST8SiaII mRNA was down-regulated by this compound. Our data indicate that increased expression of ST8SiaIV enables accelerated polysialylation of NCAM, which might be coupled to a loss of adhesive functions of tumor cells.

Mycophenolate mofetil increases adhesion capacity of tumor cells in vitro.

BACKGROUND: The immunosuppressive drug mycophenolate mofetil (MMF) reduces expression of the heterophilic binding elements intercellular adhesion molecule-1 and vascular cell adhesion molecule-1 and thereby prevents attachment of alloactivated leukocytes to donor endothelium. The authors speculated that MMF might further diminish receptors of the immunoglobulin superfamily which, however, act as homophilic binding elements. Because decrease of homophilic adhesion receptors correlates with tumor dissemination and metastasis, MMF could trigger development or recurrence of neoplastic tumors. METHODS: The authors analyzed the influence of MMF on homotypic adhesion receptors and its consequence for tumor cell attachment to an endothelial cell monolayer. Neuroblastoma (NB) cells, which self-aggregate by means of the homophilic-binding element neural cell adhesion molecule (NCAM), were used. Effects of MMF on the 140- and 180-kDa NCAM isoforms were investigated quantitatively by flow cytometry, Western blot, and reverse-transcriptase (RT) polymerase chain reaction (PCR). The relevance of NCAM for tumor cell binding was proven by treating NB with NCAM antisense oligonucleotides. RESULTS: MMF profoundly increased the number of adherent NB cells, with a maximum effect at 0.1 microM, compared with controls. Decrease of NCAM on the cell surface was detected by flow cytometry. Western blot and RT-PCR demonstrated reduced protein and RNA levels of the 140- and 180-kDa isoforms. Treatment of NB cells with NCAM antisense oligonucleotides showed that reduced NCAM expression leads to enhanced tumor cell adhesion. CONCLUSIONS: MMF decreases NCAM receptors, which is associated with enhanced tumor cell invasiveness. The authors conclude that an MMF-based immunosuppressive regimen might increase the risk of tumor metastasis if this process is predominantly conveyed by means of homophilic adhesion proteins.

Myxoma virus infection promotes NK lysis of malignant gliomas in vitro and in vivo.

Myxoma virus (MYXV) is a well-established oncolytic agent against different types of tumors. MYXV is also known for its immunomodulatory properties in down-regulating major histocompatibility complex (MHC) I surface expression (via the M153R gene product, a viral E3-ubiquitin ligase) and suppressing T cell killing of infected target cells. MHC I down-regulation, however, favors NK cell activation. Brain tumors including gliomas are characterized by high MHC I expression with impaired NK activity. We thus hypothesized that MYXV infection of glioma cells will promote NK cell-mediated recognition and killing of gliomas. We infected human gliomas with MYXV and evaluated their susceptibility to NK cell-mediated cytotoxicity. MYXV enhanced NK cell-mediated killing of glioma cells (U87 cells, MYXV vs. Mock: 51.73% vs. 28.63%, P = .0001, t test; U251 cells, MYXV vs. Mock: 40.4% vs. 20.03%, P .0007, t test). Using MYXV M153R targeted knockout (designated vMyx-M153KO) to infect gliomas, we demonstrate that M153R was responsible for reduced expression of MHC I on gliomas and enhanced NK cell-mediated antiglioma activity (U87 cells, MYXV vs. vMyx-M153KO: 51.73% vs. 25.17%, P = .0002, t test; U251 cells, MYXV vs. vMyx-M153KO: 40.4% vs. 19.27, P = .0013, t test). Consequently, NK cell-mediated lysis of established human glioma tumors in CB-17 SCID mice was accelerated with improved mouse survival (log-rank P = .0072). These results demonstrate the potential for combining MYXV with NK cells to effectively kill malignant gliomas.