Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

Allegra, Séverine; Deleine, Cécile; Michael-Jubely, Rime; Gryson, Céline; Boirie‌, Yves; Kantakamalakul, Wannee; Vasson, Marie‐Paule

doi:10.1002/cyto.a.20301

Cited by 13 publications

(7 citation statements)

References 37 publications

(42 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The number of dead CFSE‐labelled target cells was determined by analyzing CFSE and PI double‐positive cells (emission at 509 and 625 nm, respectively) by flow cytometry. As reported previously (Allegra et al, ; Lamas et al, ), the percent of lytic activity was calculated with the following equation:

lytic a c t i v i t y (%) = [(% C F S {normalE}^{+} P {normalI}^{+} t \arg e t s) - (% s p o n \tan e o u s C F S {normalE}^{+} P {normalI}^{+} t \arg e t s)] / [100 - (% s p o n \tan e o u s C F S {normalE}^{+} P {normalI}^{+} t \arg e t s)] .

…”

Section: Methodsmentioning

confidence: 99%

“…The number of dead CFSE-labelled target cells was determined by analyzing CFSE and PI double-positive cells (emission at 509 and 625 nm, respectively) by flow cytometry. As reported previously (Allegra et al, 2006;Lamas et al, 2013), the percent of lytic activity was calculated with the following equation: After spleen cell isolation, cells were treated depending on five conditions. Condition 1: Assays immediately carried out on effective cells (splenocytes and NK cells) after seeding.…”

Section: Lytic Assaysmentioning

confidence: 99%

See 1 more Smart Citation

Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes

et al. 2016

Self Cite

View full text Add to dashboard Cite

Leptin, a pleiotropic adipokine, is known as a regulator of food intake, but it is also involved in inflammation, immunity, cell proliferation, and survival. Leptin receptor is integrated inside cholesterol-rich microdomains called lipid rafts, which, if disrupted or destroyed, could lead to a perturbation of lytic mechanism. Previous studies also reported that leptin could induce membrane remodeling. In this context, we studied the effect of membrane remodeling in lytic activity modulation induced by leptin. Thus, primary mouse splenocytes were incubated with methyl-β-cyclodextrin (β-MCD), a lipid rafts disrupting agent, cholesterol, a major component of cell membranes, or ursodeoxycholic acid (UDCA), a membrane stabilizer agent for 1 h. These treatments were followed by splenocyte incubation with leptin (absence, 10 and 100 ng/ml). Unlike β-MCD or cholesterol, UDCA was able to block leptin lytic induction. This result suggests that leptin increased the lytic activity of primary spleen cells against syngenic EO771 mammary cancer cells independently from lipid rafts but may involve membrane fluidity. Furthermore, natural killer cells were shown to be involved in the splenocyte lytic activity. To our knowledge it is the first publication in primary culture that provides the link between leptin lytic modulation and membrane remodeling. J. Cell. Physiol. 232: 101-109, 2017. © 2016 Wiley Periodicals, Inc.

show abstract

lytic a c t i v i t y (%) = [(% C F S {normalE}^{+} P {normalI}^{+} t \arg e t s) - (% s p o n \tan e o u s C F S {normalE}^{+} P {normalI}^{+} t \arg e t s)] / [100 - (% s p o n \tan e o u s C F S {normalE}^{+} P {normalI}^{+} t \arg e t s)] .

…”

Section: Methodsmentioning

confidence: 99%

Section: Lytic Assaysmentioning

confidence: 99%

Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes

et al. 2016

Self Cite

View full text Add to dashboard Cite

show abstract

“…Lytic activity of NK cells was assessed as described [42] with slight modifications. Available samples of PBMC (n = 16) were extensively washed to remove any trace of EDTA from extraction tubes and monocytes/macrophages were removed by plastic adherence for 1 h at 37°C (5% CO 2 ), this treatment removed 82.4% of monocytes from PBMC samples (n = 13).…”

Section: Methodsmentioning

confidence: 99%

Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome

et al. 2013

View full text Add to dashboard Cite

BackgroundChronic Fatigue Syndrome (CFS) is a debilitating neuro-immune disorder of unknown etiology diagnosed by an array of clinical manifestations. Although several immunological abnormalities have been described in CFS, their heterogeneity has limited diagnostic applicability.MethodsImmunological features of CFS were screened in 22 CFS diagnosed individuals fulfilling Fukuda criteria and 30 control healthy individuals. Peripheral blood T, B and NK cell function and phenotype were analyzed by flow cytometry in both groups.ResultsCFS diagnosed individuals showed similar absolute numbers of T, B and NK cells, with minor differences in the percentage of CD4+ and CD8+ T cells. B cells showed similar subset frequencies and proliferative responses between groups. Conversely, significant differences were observed in T cell subsets. CFS individuals showed increased levels of T regulatory cells (CD25+/FOXP3+) CD4 T cells, and lower proliferative responses in vitro and in vivo. Moreover, CD8 T cells from the CFS group showed significantly lower activation and frequency of effector memory cells. No clear signs of T-cell immunosenescence were observed. NK cells from CFS individuals displayed higher expression of NKp46 and CD69 but lower expression of CD25 in all NK subsets defined. Overall, T cell and NK cell features clearly clustered CFS individuals.ConclusionsOur findings suggest that alterations in T-cell phenotype and proliferative response along with the specific signature of NK cell phenotype may be useful to identify CFS individuals. The striking down modulation of T cell mediated immunity may help to understand intercurrent viral infections in CFS.

show abstract

“…Adherent MCF-7-EGFP and MDA-MB-231-EGFP cells were first incubated for 24 h to stick to the plate and then NK-92 cells, previously cultured in presence of leptin at different concentrations (absence, 10, 100, and 200 ng/ml), were added at the effector:target cell ratio 5:1 for 12 h. When the K562-EGFP cells were used as target cells, they were co-cultured with NK-92 cells for 4 h as previously reported (Allegra et al, 2006). Subsequently, to remove the cell mixture, adherent MCF-7-EGFP and MDA-MB-231-EGFP cells and NK-92 cells were recovered after accutase treatment (1X, Millipore, Molsheim, France).…”

Section: Cytotoxicity Assaysmentioning

confidence: 99%

Leptin modulates dose‐dependently the metabolic and cytolytic activities of NK‐92 cells

Lamas

Goncalves-Mendès

Nachat‐Kappes

et al. 2013

Journal Cellular Physiology

Self Cite

View full text Add to dashboard Cite

Leptin, a hormone-cytokine produced primarily in the adipose tissue, has pleiotropic effects on many biological systems and in several cell types, including immune cells. Hyperleptinemia is associated with immune dysfunction and carcinogenesis. Natural killer (NK) cells are critical mediators of anti-tumor immunity, and leptin receptor deficiency in mice leads to impaired NK function. It was thus decided to explore the in vitro effects of leptin on human NK cell function. NK-92 cells were cultured during 48 h with different leptin concentrations [absence, 10 (physiological), 100 (obesity), or 200 ng/ml (pharmacology)]. Their metabolic activity was assessed using the resazurin test. NK-92 cell cytotoxicity and intracellular IFN-γ production were analyzed by flow cytometry. NK-92 cell mRNA and protein expression levels of cytotoxic effectors were determined by RT-qPCR and Western blot. In our conditions, leptin exerted a dose-dependent stimulatory effect on NK-92 cell metabolic activity. In addition, high leptin concentrations enhanced NK-92 cell cytotoxicity against K562-EGFP and MDA-MB-231-EGFP target cells and inversely reduced cytotoxicity against the MCF-7-EGFP target. At 100 ng/ml, leptin up-regulated both NK cell granzyme B and TRAIL protein expressions and concomitantly down-regulated perforin expression without affecting Fas-L expression. In response to PMA/ionomycin stimulation, the proportion of IFN-γ expressing NK-92 cells increased with 100 and 200 ng/ml of leptin. In conclusion, leptin concentration, at obesity level, variably increased NK-92 cell metabolic activity and modulated NK cell cytotoxicity according to the target cells. The underlying mechanisms are partly due to an up-regulation of TRAIL and IFN-γ expression and a down-regulation of perforin.

show abstract

Implementation of the EGFP‐K562 flow cytometric NK test: Determination of NK cytotoxic activity in healthy elderly volunteers before and after feeding

Cited by 13 publications

References 37 publications

Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes

Enhancement of Lytic Activity by Leptin Is Independent From Lipid Rafts in Murine Primary Splenocytes

Screening NK-, B- and T-cell phenotype and function in patients suffering from Chronic Fatigue Syndrome

Leptin modulates dose‐dependently the metabolic and cytolytic activities of NK‐92 cells

Contact Info

Product

Resources

About