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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., (©2019 Society for Leukocyte Biology.)

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., (© The Japanese Society for Immunology. 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 β1 integrin as an essential anticryptococcal receptor. These findings demonstrate that multiple receptors, including β1 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., (Copyright © 2018 by The American Association of Immunologists, Inc.)

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., (Copyright © 2018 The Authors. Published by Elsevier Inc. All rights reserved.)

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 β-1,3-glucan, a component of the fungal cell wall, binds to NKp30. We further demonstrate that β-1,3-glucan stimulates granule convergence and polarization, as shown by live cell imaging. Through Src Family Kinase signaling, β-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 β-1,3-glucan enhances fungal killing and restores defective cryptococcal killing by NK cells from HIV-positive individuals, implicating β-1,3-glucan to be both an activating ligand and a soluble PAMP that shapes NK cell host immunity.

Natural killer (NK) cells are an important contributor to innate host defense because of their role in direct microbial recognition and killing. Vitenshtein et al. make an important contribution by demonstrating that NK cells kill Candida glabrata using the NK activating receptor, NKp46, which recognizes the Epa adhesins., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

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., (Copyright © 2016 Kyei et al.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

NK cells play a central role in innate immunity, acting directly through cell-mediated cytotoxicity and by secreting cytokines. TNFα activation of TNFR2 enhances NK cell cytotoxicity, but its effects on the other essential function of NK cells - cytokine production, for which IFNγ is paramount - are poorly defined. We identify the expression of both TNFα receptors on human peripheral blood NK cells (TNFR2 > TNFR1) and show that TNFα significantly augments IFNγ production from IL-2-/IL-12-treated NK cells in vitro, an effect mimicked by a TNFR2 agonistic antibody. TNFα also enhanced murine NK cell IFNγ production via TNFR2 in vitro. In a mouse model characterized by the hepatic recruitment and activation of NK cells, TNFR2 also regulated NK cell IFNγ production in vivo. Specifically, in this model, after activation of an innate immune response, hepatic numbers of TNFR2-expressing and IFNγ-producing NK cells were both significantly increased; however, the frequency of IFNγ-producing hepatic NK cells was significantly reduced in TNFR2-deficient mice. We delineate an important role for TNFα, acting through TNFR2, in augmenting cytokine-induced NK cell IFNγ production in vivo and in vitro, an effect with significant potential implications for the regulation of innate and adaptive immune responses., (© 2016 S. Karger AG, Basel.)

Natural killer (NK) cells are a subset of immune effectors that directly bind and kill fungi via a perforin-dependent mechanism. The receptor mediating this activity and its potential role in disease remain unknown. Using an unbiased approach, we determined that NKp30 is responsible for recognition and killing of the fungal pathogens Cryptococcus and Candida. NKp30 was required for NK cell-fungal conjugate formation, phosphatidylinositol 3-kinase (PI3K) signaling, and perforin release. Because fungal infections are a leading cause of death in AIDS patients, we examined NKp30 expression in HIV-infected patients. NK cells from these patients had diminished NKp30 expression, defective perforin release, and blunted microbicidal activity. Surprisingly, interleukin-12 (IL-12) restored NKp30 expression and fungal killing. Thus, the NKp30 receptor plays a critical role in NK cell antifungal cytotoxicity, and diminished expression of NKp30 is responsible for defective antifungal activity of NK cells from HIV-infected patients, which can be corrected with IL-12., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.

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.

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment.

Malignant gliomas (MGs) are deadly brain tumors with a median survival after resection, radiotherapy and chemotherapy of only 12 months. The natural immunosuppressive state of MG patients and the locally restricted growth of MGs render this neoplasm an excellent target for immunotherapy. Consequently, several failed attempts were made to treat MGs with immune cells. Recent preclinical experimental studies, however, demonstrate that natural killer (NK) cells can kill MGs and therefore hold promise in immunotherapy of MGs. This review describes the experimental and clinical evidence that support the potential of NK cells in therapy of MGs as well as the limitations to NK therapy. Finally, we propose strategies that could circumvent mitigating factors and enhance NK cell therapy for MG patients., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.

NK cell cytotoxicity requires two positive signals for killing of tumors. Activation receptors induce polarization of the microtubule organization center and degranulation, while leukocyte function-associated antigen (LFA)-1 is required for conjugate formation and actin polymerization and under some circumstances may be sufficient for NK cell cytotoxicity. Although the receptor for direct killing of fungi is not known, CD18, the beta2 chain of LFA-1, binds components of the capsule and cell wall of the opportunistic pathogen Cryptococcus neoformans, namely the polysaccharides glucoronoxylomannan and galactoxylomannan. Herein, we also demonstrate that LFA-1 was concentrated in regions of the NK cell surface interacting with C. neoformans. Consequently, there was compelling evidence to hypothesize that NK cells would also use LFA-1 to recognize and kill C. neoformans. Using a combination of NK cell lines that did or did not express LFA-1 or by using a CD18-specific functional blocking antibody, we confirm that NK cell anti-tumor activity is critically dependent upon the expression of LFA-1. Duplicating the events of tumor cytotoxicity, NK cells form conjugates with cryptococcal targets, rearrange the cell cytoskeleton to develop an NK immunologic synapse and release perforin-containing granules; however, each of these events occurred independently of LFA-1. Furthermore, NK cell-mediated killing of C. neoformans was detectable in both NK cells pre-treated with CD18-blocking antibodies and in NK cells lacking cell surface LFA-1 expression. These results demonstrate that in the absence of LFA-1 expression, NK cells are fully capable of recognizing a target (C. neoformans) and retain all of the events required for cytotoxicity.

Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C. neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the alpha-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C. neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C. neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C. neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C. neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism.

Murine experimentation has provided many useful tools, including the ability to knockout or over-express genes and to perform experiments that are limited by ethical considerations. Over the past century, mice have imparted valuable insights into the biology of many systems, including human immunity. However, although there are many similarities between the immune response of humans and mice, there are also many differences; none is more prominent than when examining natural killer cell biology. These differences include tissue distribution, effector molecules, receptor repertoire, and cytokine responses, all of which have important implications when extrapolating the studies to the human immune responses to Cryptococcus neoformans.

Cytotoxic lymphocytes have the capacity to kill microbes directly; however, the mechanisms involved are poorly understood. Using Cryptococcus neoformans, which causes a potentially fatal fungal infection in HIV-infected patients, our previous studies showed that granulysin is necessary, while perforin is dispensable, for CD8 T lymphocyte fungal killing. By contrast, the mechanisms by which NK cells exert their antimicrobial activity are not clear, and in particular, the contribution of granulysin and perforin to NK-mediated antifungal activity is unknown. Primary human NK cells and a human NK cell line YT were found to constitutively express granulysin and perforin, and possessed anticryptococcal activity, in contrast to CD8 T lymphocytes, which required stimulation. When granulysin protein and mRNA were blocked by granulysin small interfering RNA, the NK cell-mediated antifungal effect was not affected in contrast to the abrogated activity observed in CD8 T lymphocytes. However, when perforin was inhibited by concanamycin A, and silenced using hairpin small interfering RNA, the anticryptococcal activities of NK cells were abrogated. Furthermore, when granulysin and perforin were both inhibited, the anticryptococcal activities of the NK cells were not reduced further than by silencing perforin alone. These results indicate that the antifungal activity is constitutively expressed in NK cells in contrast to CD8 T lymphocytes, in which it requires prior activation, and perforin, but not granulysin, plays the dominant role in NK cell anticryptococcal activity, in contrast to CD8 T lymphocytes, in which granulysin, but not perforin, plays the dominant role in anticryptococcal activity.