Resveratrol Activates Natural Killer Cells through Akt- and mTORC2-Mediated c-Myb Upregulation

Natural killer (NK) cells are suitable targets for cancer immunotherapy owing to their potent cytotoxic activity. To maximize the therapeutic efficacy of cancer immunotherapy, adjuvants need to be identified. Resveratrol is a well-studied polyphenol with various potential health benefits, including antitumor effects. We previously found that resveratrol is an NK cell booster, suggesting that it can serve as an adjuvant for cancer immunotherapy. However, the molecular mechanism underlying the activation of NK cells by resveratrol remains unclear. The present study aimed to determine this mechanism. To this end, we investigated relevant pathways in NK cells using Western blot, real-time polymerase chain reaction, pathway inhibitor, protein/DNA array, and cytotoxicity analyses. We confirmed the synergistic effects of resveratrol and interleukin (IL)-2 on enhancing the cytolytic activity of NK cells. Resveratrol activated Akt by regulating Mammalian Target of Rapamycin (mTOR) Complex 2 (mTORC2) via phosphatase and tensin homolog (PTEN) and ribosomal protein S6 kinase beta-1 (S6K1). Moreover, resveratrol-mediated NK cell activation was more dependent on the mTOR pathway than the Akt pathway. Importantly, resveratrol increased the expression of c-Myb, a downstream transcription factor of Akt and mTORC2. Moreover, c-Myb was essential for resveratrol-induced NK cell activation in combination with IL-2. Our results demonstrate that resveratrol activates NK cells through Akt- and mTORC2-mediated c-Myb upregulation.

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p46, a Novel Natural Killer Cell–specific Surface Molecule That Mediates Cell Activation

Journal of Experimental Medicine ◽

10.1084/jem.186.7.1129 ◽

1997 ◽

Vol 186 (7) ◽

pp. 1129-1136 ◽

Cited By ~ 359

Author(s):

Simona Sivori ◽

Massimo Vitale ◽

Luigia Morelli ◽

Lorenza Sanseverino ◽

Raffaella Augugliaro ◽

...

Keyword(s):

Specific Surface ◽

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Killer Cell ◽

Cytolytic Activity ◽

Surface Molecule ◽

Limited Information ◽

Cell Clones

Limited information is available on the surface molecules that are involved in natural killer (NK) cell triggering. In this study, we selected the BAB281 monoclonal antibody (mAb) on the basis of its ability to trigger NK-mediated target cell lysis. BAB281 identified a novel NK cell–specific surface molecule of 46 kD (p46) that is expressed by all resting or activated NK cells. Importantly, unlike the NK cell antigens identified so far, the expression of p46 was strictly confined to NK cells. Upon mAb-mediated cross-linking, p46 molecules induced strong cell triggering leading to [Ca2+]i increases, lymphokine production, and cytolytic activity both in resting NK cells and NK cell clones. The p46-mediated induction of Ca2+ increases or triggering of cytolytic activity was downregulated by the simultaneous engagement of inhibitory receptors including p58, p70, and CD94/NKG2A. Both the unique cellular distribution and functional capability of p46 molecules suggest a possible role in the mechanisms of non-major histocompatibility complex–restricted cytolysis mediated by human NK cells.

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Recent Advances to Augment NK Cell Cancer Immunotherapy Using Nanoparticles

Pharmaceutics ◽

10.3390/pharmaceutics13040525 ◽

2021 ◽

Vol 13 (4) ◽

pp. 525

Author(s):

Kwang-Soo Kim ◽

Dong-Hwan Kim ◽

Dong-Hyun Kim

Keyword(s):

Clinical Trials ◽

Nk Cells ◽

Cancer Immunotherapy ◽

Tumor Cells ◽

Therapeutic Efficacy ◽

Nk Cell ◽

Cell Cancer ◽

Immune Surveillance ◽

Cytolytic Activity ◽

Contact Frequency

Among various immunotherapies, natural killer (NK) cell cancer immunotherapy using adoptive transfer of NK cells takes a unique position by targeting tumor cells that evade the host immune surveillance. As the first-line innate effector cell, it has been revealed that NK cells have distinct mechanisms to both eliminate cancer cells directly and amplify the anticancer immune system. Over the last 40 years, NK cell cancer immunotherapy has shown encouraging reports in pre-clinic and clinic settings. In total, 288 clinical trials are investigating various NK cell immunotherapies to treat hematologic and solid malignancies in 2021. However, the clinical outcomes are unsatisfying, with remained challenges. The major limitation is attributed to the immune-suppressive tumor microenvironment (TME), low activity of NK cells, inadequate homing of NK cells, and limited contact frequency of NK cells with tumor cells. Innovative strategies to promote the cytolytic activity, durable persistence, activation, and tumor-infiltration of NK cells are required to advance NK cell cancer immunotherapy. As maturing nanotechnology and nanomedicine for clinical applications, there is a greater opportunity to augment NK cell therapeutic efficacy for the treatment of cancers. Active molecules/cytokine delivery, imaging, and physicochemical properties of nanoparticles are well equipped to overcome the challenges of NK cell cancer immunotherapy. Here, we discuss recent clinical trials of NK cell cancer immunotherapy, NK cell cancer immunotherapy challenges, and advances of nanoparticle-mediated NK cell therapeutic efficacy augmentation.

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miR-1224 contributes to ischemic stroke-mediated natural killer cell dysfunction by targeting Sp1 signaling

Journal of Neuroinflammation ◽

10.1186/s12974-021-02181-4 ◽

2021 ◽

Vol 18 (1) ◽

Author(s):

Yan Feng ◽

Yan Li ◽

Ying Zhang ◽

Bo-Hao Zhang ◽

Hui Zhao ◽

...

Keyword(s):

Ischemic Stroke ◽

Nk Cells ◽

Natural Killer ◽

Brain Ischemia ◽

Cell Activation ◽

Nk Cell ◽

Cell Activity ◽

Passive Transfer ◽

Immune Defense ◽

Nk Cell Activity

Abstract Background Brain ischemia compromises natural killer (NK) cell-mediated immune defenses by acting on neurogenic and intracellular pathways. Less is known about the posttranscriptional mechanisms that regulate NK cell activation and cytotoxicity after ischemic stroke. Methods Using a NanoString nCounter® miRNA array panel, we explored the microRNA (miRNA) profile of splenic NK cells in mice subjected to middle cerebral artery occlusion. Differential gene expression and function/pathway analysis were applied to investigate the main functions of predicted miRNA target genes. miR-1224 inhibitor/mimics transfection and passive transfer of NK cells were performed to confirm the impact of miR-1224 in NK cells after brain ischemia. Results We observed striking dysregulation of several miRNAs in response to ischemia. Among those miRNAs, miR-1224 markedly increased 3 days after ischemic stroke. Transfection of miR-1224 mimics into NK cells resulted in suppression of NK cell activity, while an miR-1224 inhibitor enhanced NK cell activity and cytotoxicity, especially in the periphery. Passive transfer of NK cells treated with an miR-1224 inhibitor prevented the accumulation of a bacterial burden in the lungs after ischemic stroke, suggesting an enhanced immune defense of NK cells. The transcription factor Sp1, which controls cytokine/chemokine release by NK cells at the transcriptional level, is a predicted target of miR-1224. The inhibitory effect of miR-1224 on NK cell activity was blocked in Sp1 knockout mice. Conclusions These findings indicate that miR-1224 may serve as a negative regulator of NK cell activation in an Sp1-dependent manner; this mechanism may be a novel target to prevent poststroke infection specifically in the periphery and preserve immune defense in the brain.

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789 Generation of a Bicycle NK-TICA™, a novel NK cell engaging molecule to enhance targeted tumor cytotoxicity

Journal for ImmunoTherapy of Cancer ◽

10.1136/jitc-2021-sitc2021.789 ◽

2021 ◽

Vol 9 (Suppl 3) ◽

pp. A824-A824

Author(s):

Fay Dufort ◽

Christopher Leitheiser ◽

Gemma Mudd ◽

Julia Kristensson ◽

Alexandra Rezvaya ◽

...

Keyword(s):

Phage Display ◽

Nk Cells ◽

Natural Killer ◽

Tumor Antigen ◽

Cell Activation ◽

Immune Cell ◽

Nk Cell ◽

High Affinity ◽

Tumor Cytotoxicity ◽

Immune Oncology

BackgroundNatural killer (NK) cells are immune cells that can detect and eliminate tumor cells and bridge innate to adaptive immune responses. Tumor specific activation of NK cells is thus an area of active investigation in immune oncology, but to date has relied on complex biologic modalities (e.g., antibodies, fusion proteins, or cell therapies), each of which has inherent disadvantages in this application. Thus, alternative approaches are warranted. Bicycle® are small (ca. 1.5 kDa), chemically synthetic, structurally constrained peptides discovered via phage display and optimized using structure-driven design and medicinal chemistry approaches. We have now applied this technology to identify Bicycles that bind specifically to the key activating receptors, NKp46 and CD16a. When chemically coupled to tumor antigen binding Bicycles this results in highly potent, antigen-dependent receptor activation and NK cell activation. We term this new class of fully synthetic molecules Bicycle® natural killer- tumor-targeted immune cell agonists (NK-TICAs™) and we will describe their discovery and evaluation in this presentation.MethodsUsing our unique phage display screening platform, we have identified high affinity, selective binders to NKp46 and CD16a. By conjugating the Bicycle® NK cell-engaging binders to a model tumor antigen EphA2-binding Bicycle®, we have developed a bifunctional Bicycle NK-TICA™ molecule. In in vitro functional assays, we evaluated the ability of the Bicycle NK-TICAs™ to induce NK cell activation as well as cell-mediated cytotoxicity and cytokine production in NK-tumor co-culture assays.ResultsWe have developed a novel modular compound with high affinity and selectivity to NK cell receptors with specific tumor targeting capability. We demonstrate potent, selective binding of our Bicycles to receptor-expressing cells and the capability of the bifunctional molecule to induce NK cell function. With Bicycle's novel NK-TICA™ compound, we demonstrate engagement of NK cells, specific activation and function of NK cells, and enhanced EphA2-expressing tumor cytotoxicity, in a dose dependent manner.ConclusionsBicycle NK-TICAs™ are novel therapeutic agents capable of enhancing the landscape of immune oncology. We hypothesize that utilization of Bicycle NK-TICA™ as a multifunctional immune cell engager will promote modulation of NK cells, and infiltration and anti-tumor activity of NK cells in solid tumors. The data presented here provide initial proof of concept for application of the Bicycle technology to drive NK cell-mediated tumor immunity.

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Natural Killer Cells: Potential Biomarkers and Therapeutic Target in Autoimmune Diseases?

Frontiers in Immunology ◽

10.3389/fimmu.2021.616853 ◽

2021 ◽

Vol 12 ◽

Author(s):

Elena Gianchecchi ◽

Domenico V. Delfino ◽

Alessandra Fierabracci

Keyword(s):

Systemic Sclerosis ◽

Autoimmune Diseases ◽

Cytotoxic Activity ◽

Nk Cells ◽

Natural Killer ◽

T Cell Activation ◽

Cell Activation ◽

Nk Cell ◽

Killer Cell

Autoimmune diseases recognize a multifactorial pathogenesis, although the exact mechanism responsible for their onset remains to be fully elucidated. Over the past few years, the role of natural killer (NK) cells in shaping immune responses has been highlighted even though their involvement is profoundly linked to the subpopulation involved and to the site where such interaction takes place. The aberrant number and functionality of NK cells have been reported in several different autoimmune disorders. In the present review, we report the most recent findings regarding the involvement of NK cells in both systemic and organ-specific autoimmune diseases, including type 1 diabetes (T1D), primary biliary cholangitis (PBC), systemic sclerosis, systemic lupus erythematosus (SLE), primary Sjögren syndrome, rheumatoid arthritis, and multiple sclerosis. In T1D, innate inflammation induces NK cell activation, disrupting the Treg function. In addition, certain genetic variants identified as risk factors for T1D influenced the activation of NK cells promoting their cytotoxic activity. The role of NK cells has also been demonstrated in the pathogenesis of PBC mediating direct or indirect biliary epithelial cell destruction. NK cell frequency and number were enhanced in both the peripheral blood and the liver of patients and associated with increased NK cell cytotoxic activity and perforin expression levels. NK cells were also involved in the perpetuation of disease through autoreactive CD4 T cell activation in the presence of antigen-presenting cells. In systemic sclerosis (SSc), in addition to phenotypic abnormalities, patients presented a reduction in CD56hi NK-cells. Moreover, NK cells presented a deficient killing activity. The influence of the activating and inhibitory killer cell immunoglobulin-like receptors (KIRs) has been investigated in SSc and SLE susceptibility. Furthermore, autoantibodies to KIRs have been identified in different systemic autoimmune conditions. Because of its role in modulating the immune-mediated pathology, NK subpopulation could represent a potential marker for disease activity and target for therapeutic intervention.

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NT5E/CD73 as Correlative Factor of Patient Survival and Natural Killer Cell Infiltration in Glioblastoma

Journal of Clinical Medicine ◽

10.3390/jcm8101526 ◽

2019 ◽

Vol 8 (10) ◽

pp. 1526 ◽

Cited By ~ 7

Author(s):

Jiao Wang ◽

Sandro Matosevic

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Patient Survival ◽

Nk Cell ◽

Killer Cell ◽

Surface Protein ◽

The Cancer Genome Atlas ◽

Negative Prognostic Factor ◽

A Cell

CD73, a cell-surface protein encoded by the gene NT5E, is overexpressed in glioblastoma (GBM), where it contributes to the tumor’s pathophysiology via the generation of immunosuppressive adenosine. Adenosinergic signaling, in turn, drives immunosuppression of natural killer (NK) cells through metabolic and functional reprogramming. The correlation of CD73 with patient survival in relation to GBM pathology and the intratumoral infiltration of NK cells has not been comprehensively studied before. Here, we present an analysis of the prognostic relevance of CD73 in GBM based on transcriptional gene expression from patient data from The Cancer Genome Atlas (TCGA) database. Utilizing bioinformatics data mining tools, we explore the relationship between GBM prognosis, NT5E expression, and intratumoral presence of NK cells. Our analysis demonstrates that CD73 is a negative prognostic factor for GBM and that presence of NK cells may associate with improved prognosis. Moreover, the interplay between expression of NT5E and specific NK genes hints to potential functional effects of CD73 on NK cell activation.

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Enhancing network activation in natural killer cells: predictions from in silico modeling

Integrative Biology ◽

10.1093/intbio/zyaa008 ◽

2020 ◽

Vol 12 (5) ◽

pp. 109-121

Author(s):

Sahak Z Makaryan ◽

Stacey D Finley

Keyword(s):

Nk Cells ◽

Natural Killer ◽

In Silico ◽

Intracellular Signaling ◽

Cell Activation ◽

Nk Cell ◽

Mechanistic Model ◽

Cell Killing ◽

Published Data ◽

Network Activation

Abstract Natural killer (NK) cells are part of the innate immune system and are capable of killing diseased cells. As a result, NK cells are being used for adoptive cell therapies for cancer patients. The activation of NK cell stimulatory receptors leads to a cascade of intracellular phosphorylation reactions, which activates key signaling species that facilitate the secretion of cytolytic molecules required for cell killing. Strategies that maximize the activation of such intracellular species can increase the likelihood of NK cell killing upon contact with a cancer cell and thereby improve efficacy of NK cell-based therapies. However, due to the complexity of intracellular signaling, it is difficult to deduce a priori which strategies can enhance species activation. Therefore, we constructed a mechanistic model of the CD16, 2B4 and NKG2D signaling pathways in NK cells to simulate strategies that enhance signaling. The model predictions were fit to published data and validated with a separate dataset. Model simulations demonstrate strong network activation when the CD16 pathway is stimulated. The magnitude of species activation is most sensitive to the receptor’s initial concentration and the rate at which the receptor is activated. Co-stimulation of CD16 and NKG2D in silico required fewer ligands to achieve half-maximal activation than other combinations, suggesting co-stimulating these pathways is most effective in activating the species. We applied the model to predict the effects of perturbing the signaling network and found two strategies that can potently enhance network activation. When the availability of ligands is low, it is more influential to engineer NK cell receptors that are resistant to proteolytic cleavage. In contrast, for high ligand concentrations, inhibiting phosphatase activity leads to sustained species activation. The work presented here establishes a framework for understanding the complex, nonlinear aspects of NK cell signaling and provides detailed strategies for enhancing NK cell activation.

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SARS-CoV-2 Spike 1 Protein Controls Natural Killer Cell Activation via the HLA-E/NKG2A Pathway

Cells ◽

10.3390/cells9091975 ◽

2020 ◽

Vol 9 (9) ◽

pp. 1975 ◽

Cited By ~ 2

Author(s):

Daria Bortolotti ◽

Valentina Gentili ◽

Sabrina Rizzo ◽

Antonella Rotola ◽

Roberta Rizzo

Keyword(s):

Epithelial Cells ◽

Nk Cells ◽

Natural Killer ◽

Cell Activation ◽

Nk Cell ◽

Killer Cell ◽

Lung Epithelial Cells ◽

Lung Epithelial ◽

Spike Proteins

Natural killer cells are important in the control of viral infections. However, the role of NK cells during severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection has previously not been identified. Peripheral blood NK cells from SARS-CoV and SARS-CoV-2 naïve subjects were evaluated for their activation, degranulation, and interferon-gamma expression in the presence of SARS-CoV and SARS-CoV-2 spike proteins. K562 and lung epithelial cells were transfected with spike proteins and co-cultured with NK cells. The analysis was performed by flow cytometry and immune fluorescence. SARS-CoV and SARS-CoV-2 spike proteins did not alter NK cell activation in a K562 in vitro model. On the contrary, SARS-CoV-2 spike 1 protein (SP1) intracellular expression by lung epithelial cells resulted in NK cell-reduced degranulation. Further experiments revealed a concomitant induction of HLA-E expression on the surface of lung epithelial cells and the recognition of an SP1-derived HLA-E-binding peptide. Simultaneously, there was increased modulation of the inhibitory receptor NKG2A/CD94 on NK cells when SP1 was expressed in lung epithelial cells. We ruled out the GATA3 transcription factor as being responsible for HLA-E increased levels and HLA-E/NKG2A interaction as implicated in NK cell exhaustion. We show for the first time that NK cells are affected by SP1 expression in lung epithelial cells via HLA-E/NKG2A interaction. The resulting NK cells’ exhaustion might contribute to immunopathogenesis in SARS-CoV-2 infection.

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Aberrant anti-viral response of natural killer cells in severe asthma

European Respiratory Journal ◽

10.1183/13993003.02422-2018 ◽

2020 ◽

Vol 55 (5) ◽

pp. 1802422

Author(s):

Justine Devulder ◽

Cécile Chenivesse ◽

Valérie Ledroit ◽

Stéphanie Fry ◽

Pierre-Emmanuel Lobert ◽

...

Keyword(s):

Nk Cells ◽

Natural Killer ◽

Severe Asthma ◽

Cell Activation ◽

Mononuclear Cells ◽

Nk Cell ◽

Peripheral Blood Mononuclear ◽

Healthy Donors ◽

Asthma Patients ◽

Ifn Γ

Rhinovirus infections are the main cause of asthma exacerbations. As natural killer (NK) cells are important actors of the antiviral innate response, we aimed at evaluating the functions of NK cells from severe asthma patients in response to rhinovirus-like molecules or rhinoviruses.Peripheral blood mononuclear cells from patients with severe asthma and healthy donors were stimulated with pathogen-like molecules or with the rhinoviruses (RV)-A9 and RV-2. NK cell activation, degranulation and interferon (IFN)-γ expression were analysed.NK cells from severe asthma patients were less cytotoxic than those from healthy donors in response to toll-like receptor (TLR)3, TLR7/8 or RV-A9 but not in response to RV-2 stimulation. Furthermore, when cultured with interleukin (IL)-12+IL-15, cytokines which are produced during viral infections, NK cells from patients with severe asthma were less cytotoxic and expressed less IFN-γ than NK cells from healthy donors. NK cells from severe asthmatics exhibited an exhausted phenotype, with an increased expression of the checkpoint molecule Tim-3.Together, our findings indicate that the activation of NK cells from patients with severe asthma may be insufficient during some but not all respiratory infections. The exhausted phenotype may participate in NK cell impairment and aggravation of viral-induced asthma exacerbation in these patients.

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