BACKGROUND: Cannabidiol (CBD), the major non-psychoactive component of cannabis, exhibits anti-inflammatory properties, but less is known about the immunomodulatory potential of CBD on activated natural killer (NK) cells and/or their targets. Many tumor cells present heat shock protein 70 (Hsp70) on their cell surface in a tumor-specific manner and although a membrane Hsp70 (mHsp70) positive phenotype serves as a target for Hsp70-activated NK cells, a high mHsp70 expression is associated with tumor aggressiveness. This study investigated the immuno-modulatory potential of CBD on NK cells stimulated with TKD Hsp70 peptide and IL-2 (TKD+IL-2) and also on HCT116 p53wt and HCT116 p53-/- colorectal cancer cells exhibiting high and low basal levels of mHsp70 expression.
RESULTS: Apart from an increase in the density of NTB-A and a reduced expression of LAMP-1, the expression of all other activatory NK cell receptors including NKp30, NKG2D and CD69 which are significantly up-regulated after stimulation with TKD+IL-2 remained unaffected after a co-treatment with CBD. However, the release of major pro-inflammatory cytokines by NK cells such as interferon-γ (IFN-γ) and the effector molecule granzyme B (GrzB) was significantly reduced upon CBD treatment. With respect to the tumor target cells, CBD significantly reduced the elevated expression of mHsp70 but had no effect on the low basal mHsp70 expression. Expression of other NK cell ligands such as MICA and MICB remained unaffected, and the NK cell ligands ULBP and B7-H6 were not expressed on these target cells. Consistent with the reduced mHsp70 expression, treatment of both effector and target cells with CBD reduced the killing of high mHsp70 expressing tumor cells by TKD+IL-2+CBD pre-treated NK cells but had no effect on the killing of low mHsp70 expressing tumor cells. Concomitantly, CBD treatment reduced the TKD+IL-2 induced increased release of IFN-γ, IL-4, TNF-α and GrzB, but CBD had no effect on the release of IFN-α when NK cells were co-incubated with tumor target cells.
CONCLUSIONS: Cannabidiol (CBD) may potentially diminish the anti-tumor effectiveness of TKD+IL-2 activated natural killer (NK) cells.

β-(1,3/1,4)-glucan is a major component of cereal grains, such as oats and barley. In this study, we investigated the effects of cooked waxy barley, which contains β-(1,3/1,4)-glucan, on upper respiratory tract physical symptoms and mood status by performing a randomized, parallel-group, comparative trial. The primary outcome was assessed using the Wisconsin Upper Respiratory Symptom Survey-21 and Profile of Mood States second edition. Twenty-seven healthy Japanese adult participants were supplemented with 100 g of cooked waxy barley (containing 1.8 g of β-glucan) or 100 g of cooked white rice daily for 8 weeks. Participants receiving cooked waxy barley reported a reduction in cumulative days of sneezing (p < 0.05) and feeling tired (p < 0.0001) compared with the control group. After the intervention period, there were significantly less severe nasal symptoms, such as runny nose, plugged nose, and sneezing (p < 0.05), and a significantly greater reduction of the Tension-Anguish score (p < 0.05) in the barley group than in the control group. This study suggests that supplementation of cooked waxy barley containing β-(1,3/1,4)-glucan prevents or alleviates nasal upper respiratory tract symptoms and improves mood status. The findings of this study should be confirmed by double-blind trials with a larger number of participants.

OBJECTIVE: This study aimed to investigate the impact of surgical intervention on peripheral blood T lymphocyte subsets and natural killer (NK) cell activity in pediatric patients with obstructive sleep apnea hypopnea syndrome (OSAHS).
METHODS: A total of 36 OSAHS children, 32 children with tonsillar hypertrophy, and 30 healthy children were enrolled. Clinical data and polysomnography (PSG) results were collected. Peripheral blood samples were analyzed for T lymphocyte subsets, NK cells, and cytokine levels including Th1 (IFN-γ, IL-2, TNF-α), Th2 (IL-4, IL-10), and Th17 (IL-17).
RESULTS: At baseline, OSAHS children exhibited lower LSaO2 levels and higher AHI values compared to healthy children. They also showed decreased percentages of CD3 + T cells, CD4 + T cells, NK cells, and elevated CD8 + T cells and CD4+/CD8 + ratio. Levels of IFN-γ, IL-2, TNF-α, IL-4, and IL-17 were significantly lower in OSAHS children. Post-surgery improvements were observed in LSaO2, AHI, and immune markers at 3 months and 6 months. Pearson\'s correlation analysis revealed significant associations between LSaO2, AHI, and peripheral blood immune parameters at baseline and 6 months post-surgery.
CONCLUSIONS: Surgical intervention in pediatric OSAHS influences peripheral blood T lymphocyte subsets and NK cell activity. Early intervention and monitoring of immune function are crucial for the recovery and healthy development of affected children.

Pemphigus vulgaris (PV) is a rare autoimmune disorder where autoantibodies target the desmosomal proteins resulting in blistering of oral mucosa and skin. While the pathogenesis of PV is mainly mediated by the adaptive immune system, key players of innate immunity are also emerging. This study outlines the phenotypic as well as functional attributes of NK cells in PV. Through in-depth analysis using flow cytometry we identified an increase in the frequency of CD56+ CD3- NK cells and their subtypes in periphery. Along with this there is an increased frequency of IFNγ+ CD56bright CD16dim NK cells. mRNA expression of sorted NK cells for differentially expressed genes, particularly key transcription factors such as T-bet and EOMES, as well as surface receptors like NKG2D and KIR2D, and the cytokine IFNγ, displayed significant upregulation. A significant activation of NK cells was seen in the disease state. The levels of perforin and IFNγ were significantly elevated in the culture supernatants of patients. Additionally, a significantly higher cytotoxicity of NK cells in PV was observed. In lesioned tissues of PV, NK related markers were significantly increased. Lastly, we observed NK cells using confocal microscopy in the tissue biopsies of patients which showed significant infiltration of CD56+ CD3- NK cells at the lesional sites. This study aimed to shed light on the pivotal role of NK cells in the immunopathology of PV, offering a thorough understanding of their behaviour and changes in expression which might help in contributing to the development of novel therapeutics.

Natural killer cell-derived extracellular vesicles (NK-EVs) are candidate biotherapeutics against various cancers. However, standardised potency assays are necessary for a reliable assessment of NK-EVs\' cytotoxicity. This study aims to thoroughly evaluate a highly sensitive resazurin phenoxazine-based cell viability potency assay (measurement of the cellular redox metabolism) for quantifying the cytotoxicity of NK-EVs against leukaemia K562 cells (suspension model) and breast cancer MDA-MB-231 cells (adherent model) in vitro. The assay was evaluated based on common analytical parameters setforth by regulatory guidelines, including specificity, selectivity,accuracy, precision, linearity, range and stability. Our results revealed that this resazurin-based cell viability potency assay reliably and reproducibly measured a dose-response of NK-EVs\' cytotoxic activity against both cancer models. The assay showed precision with 5% and 20% variation for intra-run and inter-run variability. The assay signal showed specificity and selectivity of NK-EVs against cancer target cells, as evidenced by the diminished viability of cancer cells following a 5-hour treatment with NK-EVs, without any detectable interference or background. The linearity analysis of target cancer cells revealed strong linearity for densities of 5000 K562 and 1000 MDA-MB-231 cells per test with a consistent range. Importantly, NK-EVs\' dose-response for cytotoxicity showed a strong correlation (|ρ| ∼ 0.8) with the levels of known cytotoxic factors associated with the NK-EVs\' corona (FasL, GNLY, GzmB, PFN and IFN-γ), thereby validating the accuracy of the assay. The assay also distinguished cytotoxicity changes in degraded NK-EVs, indicating the ability of the assay to detect the potential loss of sample integrity. Compared to other commonly reported bioassays (i.e., flow cytometry, cell counting, lactate dehydrogenase release assay, DNA-binding reporter assay and confluence assay), our results support this highly sensitive resazurin-based viability potency assay as a high-throughput and quantitative method for assessing NK-EVs\' cytotoxicity against both suspension and adherent cancer models for evaluating NK-EVs\' biotherapeutics.

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The mechanisms of mAb-induced ADCC have been well established. However, the ADCC bioassays used to quantify mAb-induced ADCC require continued development/refinement to properly assess and compare the potency of newly developed therapeutic mAbs and biosimilars to meet regulatory requirements. We used trastuzumab and a lactate dehydrogenase (LDH)-based ADCC bioassay as a model to define critical parameters of the ADCC bioassay, describing how several bioassay parameters, including preparation of effector cells, E/T ratio, target cell selection, bioassay media components, and treatment time can influence the data quality of the ADCC activity. We confirm that a 4 to 24 h recovery cultivation is required to restore peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cell activity toward ADCC when using cryopreserved PBMCs. Furthermore, we delineated the cellular mechanisms underlying the restored ADCC activity following the recovery cultivation. We observed that CD69, an early marker of NK cell activation, was upregulated and a new subset CD56dim/CD16dim population was dramatically increased in the recovered NK cells, which led to an increase in expression and secretion of perforin, granzyme B, and cytokine production. This study provides comprehensive technical insights into ADCC bioassay optimization to inform trastuzumab biosimilar development. The knowledge gained from this study can also be leveraged to guide bioassay development for therapeutic mAbs with ADCC as the primary mechanism of action.

Chimeric antigen receptor- (CAR-)modified T cells have been successfully used to treat blood cancer. With the improved research on anti-tumour adoptive cell therapy, researchers have focused on immune cells other than T lymphocytes. Natural killer (NK) cells have received widespread attention as barriers to natural immunity. Compared to T lymphocyte-related adoptive cell therapy, the use of NK cells to treat tumours does not cause graft-versus-host disease, significantly improving immunity. Moreover, NK cells have more sources than T cells, and the related modified cells are less expensive. NK cells function through several pathways in anti-tumour mechanisms. Currently, many anti-tumour clinical trials have used NK cell-related adoptive cell therapies. In this review, we have summarized the recent progress in NK cell-related adoptive cellular immunotherapy for tumour treatment and propose the current challenges faced by CAR-NK cell therapy.

Glioblastoma (GBM) poses a significant challenge in oncology and stands as the most aggressive form of brain cancer. A primary contributor to its relentless nature is the stem-like cancer cells, called glioblastoma stem cells (GSCs). GSCs have the capacity for self-renewal and tumorigenesis, leading to frequent GBM recurrences and complicating treatment modalities. While natural killer (NK) cells exhibit potential in targeting and eliminating stem-like cancer cells, their efficacy within the GBM microenvironment is limited due to constrained infiltration and function. To address this limitation, novel investigations focusing on boosting NK cell activity against GSCs are imperative. This study presents two streamlined image-based assays assessing NK cell migration and cytotoxicity towards GSCs. It details protocols and explores the strengths and limitations of these methods. These assays could aid in identifying novel targets to enhance NK cell activity towards GSCs, facilitating the development of NK cell-based immunotherapy for improved GBM treatment.

Chimeric antigen receptor (CAR) cell therapies utilize CARs to redirect immune cells towards cancer cells expressing specific antigens like human epidermal growth factor receptor 2 (HER2). Despite their potential, CAR T cell therapies exhibit variable response rates and adverse effects in some patients. Non-invasive molecular imaging can aid in predicting patient outcomes by tracking infused cells post-administration. CAR-T cells are typically autologous, increasing manufacturing complexity and costs. An alternative approach involves developing CAR natural killer (CAR-NK) cells as an off-the-shelf allogeneic product. In this study, we engineered HER2-targeted CAR-NK cells co-expressing the positron emission tomography (PET) reporter gene human sodium-iodide symporter (NIS) and assessed their therapeutic efficacy and PET imaging capability in a HER2 ovarian cancer mouse model.NK-92 cells were genetically modified to express a HER2-targeted CAR, the bioluminescence imaging reporter Antares, and NIS. HER2-expressing ovarian cancer cells were engineered to express the bioluminescence reporter Firefly luciferase (Fluc). Co-culture experiments demonstrated significantly enhanced cytotoxicity of CAR-NK cells compared to naive NK cells. In vivo studies involving mice with Fluc-expressing tumors revealed that those treated with CAR-NK cells exhibited reduced tumor burden and prolonged survival compared to controls. Longitudinal bioluminescence imaging demonstrated stable signals from CAR-NK cells over time. PET imaging using the NIS-targeted tracer 18F-tetrafluoroborate ([18F]TFB) showed significantly higher PET signals in mice treated with NIS-expressing CAR-NK cells.Overall, our study showcases the therapeutic potential of HER2-targeted CAR-NK cells in an aggressive ovarian cancer model and underscores the feasibility of using human-derived PET reporter gene imaging to monitor these cells non-invasively in patients.