A flow-cytometry-based assay to assess the cytolytic activity against tumor cells by combination of mouse MAIT cells and natural killer cells
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ABSTRACT:
SUBMITTER: Sugimoto C
PROVIDER: S-EPMC10628808 | biostudies-literature | 2023 Oct
REPOSITORIES: biostudies-literature
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Project description:Background: Measurement of natural killer (NK) cell function has important clinical utility in several diseases. Although the flow cytometry (FC)-based 4-h NK cytotoxicity assay using peripheral blood mononuclear cells (PBMCs) in the clinical laboratory has been used for this purpose, this assay requires large amounts of blood and a rapid PBMC isolation step. Here, we developed an FC-based overnight NK cytotoxicity assay using whole blood (WB), and applied it to patients with liver diseases. Methods: Peripheral blood of healthy volunteers (n = 28) and patients with liver diseases, including hepatocellular carcinoma (n = 19) and liver cirrhosis (n = 7), was analyzed for complete blood count, absolute NK cell count, and NK cell activity (NKA). NKA was evaluated in three assay types: an FC-based overnight WB NK cytotoxicity assay using carboxyfluorescein diacetate succinimidyl ester-labeled K562 cells in the presence of various cytokine combinations [including interleukin (IL)-2, IL-18, and IL-21], an FC-based 4-h PBMC NK cytotoxicity assay, and an FC-based CD107a degranulation assay using WB and PBMCs. Results: Optimal cytokine combinations for NK cell activation in WB were determined (IL-2/IL-18, IL-2/IL-21, and IL-2/IL-18/IL-21). A good correlation was observed between WB and PBMC NK cytotoxicity assays; absolute NK cell counts were better correlated with the WB NK cytotoxicity assay than with the PBMC NK cytotoxicity assay. This WB NK cytotoxicity assay showed that patients with liver diseases had significantly lower NK cytotoxicity than healthy volunteers, under stimulation with various cytokines (p < 0.001). Conclusion: The proposed FC-based overnight WB NK cytotoxicity assay correlates well with the conventional 4-h PBMC NK cytotoxicity assay, demonstrating future potential as a supportive assay for clinical laboratory research and observational studies.
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Project description:Identifying differential protein expression is routinely used to delineate natural killer (NK) cells from various sample cohorts. This protocol describes key steps for NK cell analysis: identifying human NK cells using flow gating, data export from FlowJo, data loading in R, dimensionality reduction and visualization with Uniform Manifold Approximation and Projection, and generalized linear modeling with CyotGLMM. These analyses can help generate potential biomarkers of interest to identify NK cells across aging, treatment groups, and others. For complete details on the use and execution of this protocol, please refer to Kroll et al. (2022).1.
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Project description:Artemisinin, a chemical compound used for the treatment of malaria, has been known to show anti-cancer activity. However, the effect of this chemical on natural killer (NK) cells, which are involved in tumor killing, remains unknown. Here, we demonstrate that artemisinin exerts a potent anti-cancer effect by activating NK cells. NK-92MI cells pre-treated with artemisinin were subjected to a cytotoxicity assay using K562 cells. The results showed that artemisinin significantly enhances the cytolytic activity of NK cells in a dose-dependent manner. Additionally, the artemisinin-enhanced cytotoxic effect of NK-92MI cells on tumor cells was accompanied by the stimulation of granule exocytosis, as evidenced by the detection of CD107a expression in NK cells. Moreover, this enhancement of cytotoxicity by artemisinin was also observed in human primary NK cells from peripheral blood. Our results suggest that artemisinin enhances human NK cell cytotoxicity and degranulation. This is the first evidence that artemisinin exerts antitumor activity by enhancing NK cytotoxicity. Therefore, these results provide a deeper understanding of the action of artemisinin and will contribute to the development and application of this class of compounds in cancer treatment strategies.
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Project description:BackgroundNatural killer (NK) cells have antiviral and antitumor activity that could be harnessed for the treatment of infections and malignancies. To maintain cell viability and enhance antiviral and antitumor effects, NK cells are frequently treated with cytokines. Here they performed an extensive assessment of the effects of cytokines on the phenotype and function of human NK cells.MethodsThey used cytometry by time-of-flight (CyTOF) to evaluate NK cell repertoire changes after stimulation with interleukin (IL)-2, IL-15 or a combination of IL-12/IL-15/IL-18. To analyze the high dimensional CyTOF data, they used several statistical and visualization tools, including viSNE (Visualization of t-Distributed Stochastic Neighbor Embedding), Citrus (Cluster identification, characterization, and regression), correspondence analysis, and the Friedman-Rafsky test.ResultsAll three treatments (IL-2, IL-15, and IL-12/IL-15/IL-18) increase expression of CD56 and CD69. The effects of treatment with IL-2 and IL-15 are nearly indistinguishable and characterized principally by increased expression of surface markers including CD56, NKp30, NKp44, and increased expression of functional markers, such as perforin, granzyme B, and MIP-1β. The combination of IL-12/IL-15/IL-18 induces a profound shift in the repertoire structure, decreasing expression of CD16, CD57, CD8, NKp30, NKp46, and NKG2D, and dramatically increasing expression of IFN-γ.ConclusionsCyTOF provides insights into the effects of cytokines on the phenotype and function of NK cells, which could inform future research efforts and approaches to NK cell immunotherapy. There are several analytical approaches to CyTOF data, and the appropriate method should be carefully selected based on which aspect of the dataset is being explored. © 2016 International Clinical Cytometry Society.
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Project description:Natural killer (NK) cells are known to have effector and cytolytic properties to kill virus infected or tumor cells spontaneously. Due to these properties, NK cells have been used as an adoptive cellular therapy to control tumor growth in various clinical trials but have shown limited clinical benefits. This indicates that our knowledge about phenotypic and functional differences in NK cells within the tumor microenvironment and secondary lymphoid tissues is incomplete. In this work, we report that B16F10 cell-induced melanoma recruits the CD11b+CD27+ subset of NK cells at a very early stage during tumor progression. These intratumoral NK cells showed increased expression of CD69, reduced inhibitory receptor KLRG1, and decreased proliferative ability. As compared to splenic NK cells, intratumoral NK cells showed decreased expression of activating receptors NKG2D, Ly49D and Ly49H; increased inhibitory receptors, NKG2A and Ly49A; decreased cytokines IFN? and GM-CSF; decreased cytokine receptors IL-21R, IL-6R?, and CD122 expression. Depletion of NK cells led to decrease peripheral as well as intratumoral effector CD4+T-bet+ cells (Th1), and increased tumor growth. Furthermore, purified NK cells showed increased differentiation of Th1 cells in an IFN?-dependent manner. Anti-NKG2D in the culture promoted differentiation of effector Th1 cells. Collectively, these observations suggest that intratumoral NK cells possess several inhibitory functions that can be partly reversed by signaling through the NKG2D receptor or by cytokine stimulation, which then leads to increased differentiation of effector Th1 cells.
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Project description:Mitochondrial autophagy, also known as mitophagy, is an autophagosome-based mitochondrial degradation process that eliminates unwanted or damaged mitochondria after cell stress. Most studies dealing with mitophagy rely on the analysis by fluorescence microscopy of mitochondrial-autophagosome colocalization. However, given the fundamental role of mitophagy in the physiology and pathology of organisms, there is an urgent need for novel quantitative methods with which to study this process. Here, we describe a flow cytometry-based approach to determine mitophagy by using MitoTracker Deep Red, a widely used mitochondria-selective probe. Used in combination with selective inhibitors it may allow for the determination of mitophagy flux. Here, we test the validity of the use of this method in cell lines and in primary cell and tissue cultures.
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Project description:BackgroundRecently, allogeneic natural killer (NK) cells have gained considerable attention as promising immunotherapeutic tools due to their unique biological functions and characteristics. Although many NK expansion strategies have been reported previously, a deeper understanding of cryopreserved allogeneic NK cells is needed for specific therapeutic approaches.MethodsWe isolated CD3-CD56+ primary natural killer (pNK) cells from healthy donors and expanded them ex vivo using a GMP-compliant method without any feeder to generate large volumes of therapeutic pNK cells and cryopreserved stocks. After validation for high purity and activating phenotypes, we performed RNA sequencing of the expanded and cryopreserved pNK cells. The pNK cells were used against various cancer cell lines in 7-AAD/CFSE cytotoxicity assay. For in vivo efficacy study, NSG mice bearing subcutaneous cisplatin-resistant A2780cis xenografts were treated with our pNK cells or cisplatin. Antitumor efficacy was assessed by measuring tumor volume and weight.ResultsCompared to the pNK cells before expansion, pNK cells after expansion showed 2855 upregulated genes, including genes related to NK cell activation, cytotoxicity, chemokines, anti-apoptosis, and proliferation. Additionally, the pNK cells showed potent cytolytic activity against various cancer cell lines. Interestingly, our activated pNK cells showed a marked increase in NKp44 (1064-fold), CD40L (12,018-fold), and CCR5 (49-fold), and did not express the programmed cell death protein 1(PD-1). We also demonstrated the in vitro and in vivo efficacies of pNK cells against cisplatin-resistant A2780cis ovarian cancer cells having a high programmed death-ligand 1(PD-L1) and low HLA-C expression.ConclusionsTaken together, our study provides the first comprehensive genome wide analysis of ex vivo-expanded cryopreserved pNK cells. It also indicates the potential use of expanded and cryopreserved pNK cells as a highly promising immunotherapy for anti-cancer drug resistant patients.
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Project description:Expression levels of cellular proteins can be affected by various perturbations, such as genetic knockout of interactors, drug treatments or cell stress. To specifically measure the effects on protein levels post-synthesis under different experimental conditions, it is important to compensate for transcriptional and other upstream changes. Here, we provide a protocol for a dual-fluorescence flowcytometry-based assay to determine protein levels. The protein of interest is genetically linked to enhanced GFP (eGFP) followed by a viral 2A self-cleaving peptide sequence and mCherry. As a result, translation of the reporter construct leads to two fluorescent protein products from the same mRNA template, which enables unambiguous protein expression analysis with normalization across samples.
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Project description:BackgroundNatural Killer (NK) cells are effector lymphocytes of the innate immune system and are subclassed into CD56BrightCD16Dim/- and CD56DimCD16+ NK cells. Intracellular calcium (Ca2+) is fundamental to regulate a number of intracellular signalling pathways and functions in NK cells, which are essential in mediating their natural cytotoxic function. Transient receptor potential melastatin 2 (TRPM2) is a Ca2+-permeable non-selective cation channel that possesses a critical role in calcium-dependent cell signalling to maintain cellular homeostasis. TRPM2 and CD38 protein surface expression has yet to be determined on NK cells using flow cytometry. Characterisation of TRPM2 has been previously identified by in vivo models, primarily using methods such as genetic remodification, immunohistochemistry and whole cell electrophysiology. The aim of this study was to develop an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cell subsets using an antibody that has not been previously applied using flow cytometry.ResultsAt 2 h/1 h, TRPM2 (Fig. 2 A, B, p < 0.05) and TRPM2/CD38 (Fig. 3A, B, p < 0.05) surface expression significantly increased between 1:300 and 1:50 at 2 h/1 h. TRPM2/CD38 surface expression furthermore increased between 1:100 and 1:50 at 2 h/1 h (Fig. 3A, p < 0.05). Interestingly, TRPM2/CD38 surface expression significantly decreased from 1:50 to 1:5 on CD56BrightCD16Dim/- NK cells. These consistent findings highlight that 1:50 is the optimal antibody dilution and threshold to measure TRPM2 and TRPM2/CD38 surface expression on NK subsets. 2 h/1 h was determined as the optimal incubation period to ensure a sufficient timeframe for maximal antibody binding and surface expression.ConclusionFor the first time, we describe an in vitro methodology to characterise TRPM2 and CD38 surface expression on NK cells in healthy participants. Finally, using an antibody that has not been previously applied in flow cytometry, we determined an antibody concentration and incubation time that is robust, rapid and sensitive for the application of flow cytometry.
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Project description:Progress in our understanding of MR1-restricted mucosa-associated invariant T (MAIT) cells has raised interest in harnessing these cells for immunotherapy. The innate-like response characteristics, abundance in the blood, donor-unrestricted nature, and tropism for tissues make MAIT cells suitable candidates for adoptive cell transfer therapies. However, reliable methods and tools to utilize MAIT cells in such approaches are lacking. Here, we established methodology for efficient expansion of human MAIT cells in culture with high purity and yield, while preserving their functional response toward their natural ligand and increasing their cytotoxic potential. The cultured MAIT cells retained their effector memory characteristics without signs of terminal differentiation and expressed a more diverse set of chemokine receptors, potentially widening their already broad tissue tropism. To investigate the potential of MAIT cells in a context outside their main role in controlling bacterial infection, we engineered cultured MAIT cells with a new TCR specificity to mediate effective antiviral HLA class I-restricted effector function. In summary, we developed robust and effective methodology for the expansion of human MAIT cells with enhanced cytolytic capacity and for their engineering with a new specificity. These findings form a basis for the development of MAIT cells as a platform for adoptive immunotherapy.
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