A Dual Role for Talin in NK Cell Cytotoxicity: Activation of LFA-1-Mediated Cell Adhesion and Polarization of NK Cells

Abstract Background Myalgic Encephalomyelitis/Chronic Fatigue Syndrome (ME/CFS) is a serious multifactorial disorder. The origin remains ambiguous, however reduced natural killer (NK) cell cytotoxicity is a consistent immunological feature of ME/CFS. Impaired transient receptor potential melastatin 3 (TRPM3), a phosphatidylinositol dependent channel, and impaired calcium mobilisation have been implicated in ME/CFS pathology. This investigation aimed to examine the localisation of TRPM3 at the NK cell plasma membrane and co-localisation with phosphatidylinositol 4,5-bisphosphate (PIP2). The effect of IL-2 priming and treatment using pregnenolone sulfate (PregS) and ononetin on TRPM3 co-localisation and NK cell cytotoxicity in ME/CFS patients and healthy controls (HC) was also investigated. Methods NK cells were isolated from 15 ME/CFS patients and 15 age- and sex-matched HC. Immunofluorescent technique was used to determine co-localisation of TRPM3 with the NK cell membrane and with PIP2 of ME/CFS patients and HC. Flow cytometry was used to determine NK cell cytotoxicity. Following IL-2 stimulation and treatment with PregS and ononetin changes in co-localisation and NK cell cytotoxicity were measured. Results Overnight treatment of NK cells with PregS and ononetin resulted in reduced co-localisation of TRPM3 with PIP2 and actin in HC. Co-localisation of TRPM3 with PIP2 in NK cells was significantly reduced in ME/CFS patients compared with HC following priming with IL-2. A significant increase in co-localisation of TRPM3 with PIP2 was reported following overnight treatment with ononetin within ME/CFS patients and between groups. Baseline NK cell cytotoxicity was significantly reduced in ME/CFS patients; however, no changes were observed following overnight incubation with IL-2, PregS and ononetin between HC and ME/CFS patients. IL-2 stimulation significantly enhanced NK cell cytotoxicity in HC and ME/CFS patients. Conclusion Significant changes in co-localisation suggest PIP2-dependent TRPM3 function may be impaired in ME/CFS patients. Stimulation of NK cells with IL-2 significantly enhanced cytotoxic function in ME/CFS patients demonstrating normal function compared with HC. A crosstalk exists between IL-2 and TRPM3 intracellular signalling pathways which are dependent on Ca2+ influx and PIP2. While IL-2R responds to IL-2 binding in vitro, Ca2+ dysregulation and impaired intracellular signalling pathways impede NK cell function in ME/CFS patients.

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α-Pinene Enhances the Anticancer Activity of Natural Killer Cells via ERK/AKT Pathway

International Journal of Molecular Sciences ◽

10.3390/ijms22020656 ◽

2021 ◽

Vol 22 (2) ◽

pp. 656

Author(s):

Hantae Jo ◽

Byungsun Cha ◽

Haneul Kim ◽

Sofia Brito ◽

Byeong Mun Kwak ◽

...

Keyword(s):

Nk Cells ◽

Cancer Cells ◽

Natural Killer ◽

Nk Cell ◽

Granzyme B ◽

Akt Pathway ◽

Cell Cytotoxicity ◽

Anticancer Effects ◽

Nk Cell Cytotoxicity

Natural killer (NK) cells are lymphocytes that can directly destroy cancer cells. When NK cells are activated, CD56 and CD107a markers are able to recognize cancer cells and release perforin and granzyme B proteins that induce apoptosis in the targeted cells. In this study, we focused on the role of phytoncides in activating NK cells and promoting anticancer effects. We tested the effects of several phytoncide compounds on NK-92mi cells and demonstrated that α-pinene treatment exhibited higher anticancer effects, as observed by the increased levels of perforin, granzyme B, CD56 and CD107a. Furthermore, α-pinene treatment in NK-92mi cells increased NK cell cytotoxicity in two different cell lines, and immunoblot assays revealed that the ERK/AKT pathway is involved in NK cell cytotoxicity in response to phytoncides. Furthermore, CT-26 colon cancer cells were allografted subcutaneously into BALB/c mice, and α-pinene treatment then inhibited allografted tumor growth. Our findings demonstrate that α-pinene activates NK cells and increases NK cell cytotoxicity, suggesting it is a potential compound for cancer immunotherapy.

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Hypoxia Induced Impairment of NK Cell Cytotoxicity against Multiple Myeloma Can Be Overcome by IL-2 Activation of the NK Cells

PLoS ONE ◽

10.1371/journal.pone.0064835 ◽

2013 ◽

Vol 8 (5) ◽

pp. e64835 ◽

Cited By ~ 64

Author(s):

Subhashis Sarkar ◽

Wilfred T. V. Germeraad ◽

Kasper M. A. Rouschop ◽

Elisabeth M. P. Steeghs ◽

Michel van Gelder ◽

...

Keyword(s):

Multiple Myeloma ◽

Nk Cells ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

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PRESENCE OF FcR FOR IgG AND IgM ON HUMAN NK CELLS: THE ROLE OF IMMUNE COMPLEXES IN THE REGULATION OF NK CELL CYTOTOXICITY

NK Cells and Other Natural Effector Cells ◽

10.1016/b978-0-12-341360-4.50099-8 ◽

1982 ◽

pp. 631-637 ◽

Cited By ~ 2

Author(s):

Jean E. Merrill ◽

Sidney Golub ◽

Mikael Jondal ◽

Fred Lanefeldt ◽

Bertil Fredholm

Keyword(s):

Nk Cells ◽

Immune Complexes ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

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Umbilical Mesenchymal Stem Cell-Derived Extracellular Vesicle Conditioning Has an Immunosuppressive Effect on NK Cells

Stem Cells and Regenerative Medicine - Biomedical and Health Research ◽

10.3233/bhr210028 ◽

2021 ◽

Author(s):

G. Dostert ◽

V. Jouan-Hureaux ◽

H. Louis ◽

É. Velot

Keyword(s):

Flow Cytometry ◽

Stem Cell ◽

Mesenchymal Stem Cell ◽

Nk Cells ◽

Cell Activation ◽

Nk Cell ◽

Cell Communication ◽

K562 Cells ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

Background: In peripheral blood, human natural killer (NK) cells are immunological cells that nearly don’t express the ectonucleotidase CD73 on their plasma membrane. When exposed to mesenchymal stem cells (MSCs), NK cells are able to acquire CD73. MSCs are known to be CD73-positive (CD73+) and also to modulate the immune system, e.g. through adenosynergic pathway by ectonucleosidases, such as CD73. Extracellular vesicles (EVs) are involved in cell-to-cell communication. Mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) have emerged as paracrine mediators that are part of MSC immunomodulatory effects including immunosuppressive properties and immune privilege. Objective: The aim of our work was to study if CD73 could be acquired by NK cells through cell-to-cell communication with MSC-EVs as cell culture additives. We also hypothesised that MSC-EVs would act as tolerance inducers to attenuate NK cell cytotoxicity. Methods: Cell isolation was made from human umbilical cords for MSCs and from human peripheral blood for NK cells. MSC-EVs were isolated by ultracentrifugation and filtration, then characterized by nanoparticle tracking assay and flow cytometry (CD9, 63, 81 and 73). MSC-EV interaction with NK cells was monitored by PKH67 staining. NK cell activation was followed by measuring the expression of CD73 and NK-activating receptor natural-killer group 2, member D (NKG2D) by flow cytometry. The cytotoxicity of NK cells or EV-conditioned NK cells was evaluated after co-culture with K562 cells. Results: We showed that MSC-EVs are nanoparticles able to express CD73 and interact with NK cells. MSC-EV conditioned NK cells seem to increase CD73 and decrease NKG2D through an EV-mediated mechanism. MSC-EVs have an immunosuppressive effect on NK cells by preventing NK cell activation and NK cell cytotoxicity towards K562 cells. Conclusions: Our results demonstrate that MSC-EVs could influence NK cell behaviour and act as immunosuppressant cell-based products.

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Transient receptor potential melastatin 2 channels are overexpressed in myalgic encephalomyelitis/chronic fatigue syndrome patients

Journal of Translational Medicine ◽

10.1186/s12967-019-02155-4 ◽

2019 ◽

Vol 17 (1) ◽

Author(s):

Cassandra Balinas ◽

Helene Cabanas ◽

Donald Staines ◽

Sonya Marshall-Gradisnik

Keyword(s):

Nk Cells ◽

Transient Receptor Potential ◽

Nk Cell ◽

Receptor Potential ◽

Surface Expression ◽

Chronic Fatigue ◽

Cell Cytotoxicity ◽

Fatigue Syndrome ◽

Transient Receptor Potential Melastatin ◽

Nk Cell Cytotoxicity

Abstract Background Myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS) is hallmarked by a significant reduction in natural killer (NK) cell cytotoxicity, a mechanism tightly regulated by calcium (Ca2+). Interestingly, interleukin-2 (IL-2) increases NK cell cytotoxicity. Transient receptor potential melastatin 2 (TRPM2) ion channels are fundamental for Ca2+ signalling in NK cells. This pilot investigation aimed to characterise TRPM2 and CD38 surface expression in vitro on NK cells in ME/CFS patients. This investigation furthermore examined the pharmaceutical effect of 8-bromoadenosine phosphoribose (8-Br-ADPR) and N6-Benzoyladenosine-3′,5′-cyclic monophosphate (N6-Bnz-cAMP) on TRPM2 and CD38 surface expression and NK cell cytotoxicity between ME/CFS and healthy control (HC) participants. Methods Ten ME/CFS patients (43.45 ± 12.36) and 10 HCs (43 ± 12.27) were age and sex-matched. Isolated NK cells were labelled with fluorescent antibodies to determine baseline and drug-treated TRPM2 and CD38 surface expression on NK cell subsets. Following IL-2 stimulation, NK cell cytotoxicity was measured following 8-Br-ADPR and N6-Bnz-cAMP drug treatments by flow cytometry. Results Baseline TRPM2 and CD38 surface expression was significantly higher on NK cell subsets in ME/CFS patients compared with HCs. Post IL-2 stimulation, TRPM2 and CD38 surface expression solely decreased on the CD56DimCD16+ subset. 8-Br-ADPR treatment significantly reduced TRPM2 surface expression on the CD56BrightCD16Dim/− subset within the ME/CFS group. Baseline cell cytotoxicity was significantly reduced in ME/CFS patients, however no changes were observed post drug treatment in either group. Conclusion Overexpression of TRPM2 on NK cells may function as a compensatory mechanism to alert a dysregulation in Ca2+ homeostasis to enhance NK cell function in ME/CFS, such as NK cell cytotoxicity. As no improvement in NK cell cytotoxicity was observed within the ME/CFS group, an impairment in the TRPM2 ion channel may be present in ME/CFS patients, resulting in alterations in [Ca2+]i mobilisation and influx, which is fundamental in driving NK cell cytotoxicity. Differential expression of TRPM2 between NK cell subtypes may provide evidence for their role in the pathomechanism involving NK cell cytotoxicity activity in ME/CFS.

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A research-driven approach to the identification of novel natural killer cell deficiencies affecting cytotoxic function

Blood ◽

10.1182/blood.2019000925 ◽

2020 ◽

Vol 135 (9) ◽

pp. 629-637

Author(s):

Michael T. Lam ◽

Emily M. Mace ◽

Jordan S. Orange

Keyword(s):

Natural Killer Cell ◽

Nk Cells ◽

Natural Killer ◽

Nk Cell ◽

Killer Cell ◽

Cell Development ◽

Impact Testing ◽

Primary Immune Deficiency ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity

Abstract Natural killer cell deficiencies (NKDs) are an emerging phenotypic subtype of primary immune deficiency. NK cells provide a defense against virally infected cells using a variety of cytotoxic mechanisms, and patients who have defective NK cell development or function can present with atypical, recurrent, or severe herpesviral infections. The current pipeline for investigating NKDs involves the acquisition and clinical assessment of patients with a suspected NKD followed by subsequent in silico, in vitro, and in vivo laboratory research. Evaluation involves initially quantifying NK cells and measuring NK cell cytotoxicity and expression of certain NK cell receptors involved in NK cell development and function. Subsequent studies using genomic methods to identify the potential causative variant are conducted along with variant impact testing to make genotype-phenotype connections. Identification of novel genes contributing to the NKD phenotype can also be facilitated by applying the expanding knowledge of NK cell biology. In this review, we discuss how NKDs that affect NK cell cytotoxicity can be approached in the clinic and laboratory for the discovery of novel gene variants.

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Impaired natural and CD16-mediated NK cell cytotoxicity in patients with WAS and XLT: ability of IL-2 to correct NK cell functional defect

Blood ◽

10.1182/blood-2003-07-2621 ◽

2004 ◽

Vol 104 (2) ◽

pp. 436-443 ◽

Cited By ~ 102

Author(s):

Angela Gismondi ◽

Loredana Cifaldi ◽

Cinzia Mazza ◽

Silvia Giliani ◽

Silvia Parolini ◽

...

Keyword(s):

Nk Cells ◽

Molecular Mechanisms ◽

Nk Cell ◽

Interleukin 2 ◽

Target Cells ◽

Cell Cytotoxicity ◽

Antibody Dependent Cellular Cytotoxicity ◽

Functional Defect ◽

Nk Cell Cytotoxicity ◽

Syndrome Protein

Abstract In this study we show that Wiskott-Aldrich syndrome protein (WASp), a critical regulator of actin cytoskeleton that belongs to the Scar/WAVE family, plays a crucial role in the control of natural killer (NK) cell cytotoxicity. Analysis of NK cell numbers and cytotoxic activity in patients carrying different mutations in the WASP coding gene indicated that although the percentage of NK cells was normal or increased, natural cytotoxicity and antibody-mediated NK cell cytotoxicity were inhibited in all patients with the classical WAS phenotype and in most patients carrying mutations associated with the X-linked thrombocytopenia (XLT) phenotype. The inhibition of NK cell-mediated cytotoxicity was associated with the reduced ability of WAS and XLT NK cells to form conjugates with susceptible target cells and to accumulate F-actin on binding. Treatment with interleukin-2 (IL-2) corrected the functional defects of NK cells by affecting their ability to bind to sensitive target cells and to accumulate F-actin. In addition, we provide information on the molecular mechanisms that control WASp function, demonstrating that binding of NK cells to sensitive targets or triggering through CD16 by means of reverse antibody-dependent cellular cytotoxicity (ADCC) rapidly activates Cdc42. We also found that WASp undergoes tyrosine phosphorylation upon CD16 or β2-integrin engagement on NK cells. (Blood. 2004;104:436-443)

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Enhancing NK cell-mediated cytotoxicity to cisplatin-resistant lung cancer cells via MEK/Erk signaling inhibition

Scientific Reports ◽

10.1038/s41598-017-08483-z ◽

2017 ◽

Vol 7 (1) ◽

Cited By ~ 22

Author(s):

Li Yang ◽

MingJing Shen ◽

Li Jun Xu ◽

Xiaodong Yang ◽

Ying Tsai ◽

...

Keyword(s):

Nk Cells ◽

Nk Cell ◽

Death Receptor ◽

Neutralizing Antibody ◽

Erk Signaling ◽

Cytotoxic Action ◽

Cell Cytotoxicity ◽

Nk Cell Cytotoxicity ◽

Resistant Cells

Abstract Major progress has been made clinically in inhibiting the programmed death receptor 1 (PD-1)/PD-L1 interaction to enhance T cell-mediated immune function, yet the effectiveness of anti-PD-L1/PD-1 agents in enhancing natural killer (NK) cell’s function remains largely unknown. Susceptibilities of cisplatin-resistant A549CisR and H157CisR cells vs. parental cells to the cytotoxic action of NK cells were examined. We found cisplatin-resistant cells more resistant to NK cell cytotoxicity than parental cells. There were constitutively higher expressions of PD-L1 in A549CisR and H157CisR cells than in parental cells in vitro, as well as in H157CisR cell-derived tumors than H157P cell-derived tumors. In contrast, we observed that the expression of PD-1 in NK cells was induced after co-culture with cisplatin-resistant cells. We also observed increased susceptibility of cisplatin-resistant cells to NK cell cytotoxicity when neutralizing antibody of PD-1 or PD-L1 was added. Further, we found that the NK group 2, member D (NKG2D) ligand levels were lower in A549CisR and H157CisR cells than in parental cells. Meanwhile, we discovered that the MEK/Erk signaling pathway played a significant role in this regulation, and the addition of a MEK/Erk pathway inhibitor significantly enhanced the PD-L1 Ab effect in enhancing NK cell cytotoxicity to cisplatin-resistant cells.

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In Vitro and In Vivo Sensitization of Malignant Cells to Autologous Natural Killer Cell Cytotoxicity Following Exposure to Bortezomib.

Blood ◽

10.1182/blood.v108.11.925.925 ◽

2006 ◽

Vol 108 (11) ◽

pp. 925-925 ◽

Cited By ~ 1

Author(s):

Andreas Lundqvist ◽

Kristy Greeneltch ◽

Maria Berg ◽

Shivani Srivastava ◽

Nanae Harashima ◽

...

Keyword(s):

Tumor Growth ◽

Nk Cells ◽

Tumor Cells ◽

Nk Cell ◽

Cell Cytotoxicity ◽

Malignant Cells ◽

Nk Cell Cytotoxicity ◽

Tumor Death

Abstract Killer IgG like receptor (KIR) inactivation of NK cells by self HLA molecules has been proposed as a mechanism through which malignant cells evade host NK cell-mediated immunity. To overcome this limitation, we sought to develop a method to sensitize the patient’s tumor to autologous NK cell cytotoxicity. The proteasome inhibitor bortezomib has recently been shown to enhance the activity of tumor death receptors. We found that exposure of a variety of different leukemia, lymphoma and solid tumor cancer cell lines to sub-apoptotic doses of bortezomib sensitized tumor cells in vitro to lysis by allogeneic NK cells. Importantly, this sensitizing effect also occurs with autologous NK cells normally rendered inactive via tumor KIR ligands; NK cells expanded from patients with metastatic renal cell carcinoma were significantly more cytotoxic against the patient’s own autologous tumor cells when pretreated with bortezomib compared to untreated tumors. This sensitization to autologous NK cell killing was also observed in vivo in two different murine tumor models. A significant delay in tumor growth in C57BL/6 mice bearing LLC1 tumors (figure) and a delay in tumor growth and a significant prolongation (p<0.01) in survival were observed in RENCA tumor bearing Balb/c mice treated with bortezomib and syngeneic NK cell infusions compared to untreated mice or animals treated with bortezomib alone or NK cells alone. An investigation into the mechanism through which NK cell cytotoxicity was potentiated revealed bortezomib enhanced the activity of tumor death receptor-dependent and -independent apoptotic pathways. More specifically, bortezomib sensitized human and murine tumor cells to TRAIL and perforin/granzyme mediated NK cell cytotoxicity respectively. These observations suggest that pretreatment of malignant cells with bortezomib could be used as a strategy to override NK cell inhibition via tumor KIR ligands, thus potentiating the activity of adoptively infused autologous NK cells. A clinical trial evaluating the safety and anti-tumor efficacy of adoptively infused autologous NK cells in patients with advanced malignancies with and without tumor sensitization using bortezomib is currently being explored. Figure: Tumor growth in LLC1 bearing C57BL/6 mice. Fourteen days following s.c. injection of 3x105 LLC1 tumor cells, mice received 15μg (i.p) bortezomib and/or an adoptive infusion of 1x106 NK cells from C57BL/6 mice (i.v) given on day 15. Each dot represents the tumor volume of individual mice measured on day 28 post tumor injection. Tumors were significantly smaller in mice treated with bortezomib followed by NK cells compared to controls or mice that received either NK cells alone or bortezomib alone (p<0.04 for all groups). Figure:. Tumor growth in LLC1 bearing C57BL/6 mice. . / Fourteen days following s.c. injection of 3x105 LLC1 tumor cells, mice received 15μg (i.p) bortezomib and/or an adoptive infusion of 1x106 NK cells from C57BL/6 mice (i.v) given on day 15. Each dot represents the tumor volume of individual mice measured on day 28 post tumor injection. Tumors were significantly smaller in mice treated with bortezomib followed by NK cells compared to controls or mice that received either NK cells alone or bortezomib alone (p<0.04 for all groups).

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