Measurement Of Natural Killer Cell Activity Research Articles

Assessment of NK Cell Activity Based on NK Cell-Specific Receptor Synergy in Peripheral Blood Mononuclear Cells and Whole Blood.

Natural killer (NK) cells are cytotoxic innate lymphocytes endowed with a unique ability to kill a broad spectrum of cancer and virus-infected cells. Given their key contribution to diverse diseases, the measurement of NK cell activity (NKA) has been used to estimate disease prognosis or the effect of therapeutic treatment. Currently, NKA assays are primarily based on cumbersome procedures related to careful labeling and handling of target cells and/or NK cells, and they require a rapid isolation of peripheral blood mononuclear cells (PBMCs) which often necessitates a large amount of blood. Here, we developed an ELISA-based whole blood (WB) NKA assay involving engineered target cells (P815-ULBP1+CD48) providing defined and synergistic stimulation for NK cells via NKG2D and 2B4. WB collected from healthy donors (HDs) and patients with multiple myeloma (MM) was stimulated with P815-ULBP1+CD48 cells combined with IL-2. Thereafter, it utilized the serum concentrations of granzyme B and IFN-γ originating in NK cells as independent and complementary indicators of NKA. This WB NKA assay demonstrated that MM patients exhibit a significantly lower NKA than HDs following stimulation with P815-ULBP1+CD48 cells and had a good correlation with the commonly used flow cytometry-based PBMC NKA assay. Moreover, the use of P815-ULBP1+CD48 cells in relation to assessing the levels of NKG2D and 2B4 receptors on NK cells facilitated the mechanistic study and led to the identification of TGF-β1 as a potential mediator of compromised NKA in MM. Thus, our proposed WB NKA assay facilitates the reliable measurement of NKA and holds promise for further development as both a clinical and research tool.

A purified, fermented, extract of Triticum aestivum has lymphomacidal activity mediated via natural killer cell activation.

Non-Hodgkin lymphoma (NHL) affects over 400,000 people in the United States; its incidence increases with age. Treatment options are numerous and expanding, yet efficacy is often limited by toxicity, particularly in the elderly. Nearly 70% patients eventually die of the disease. Many patients explore less toxic alternative therapeutics proposed to boost anti-tumor immunity, despite a paucity of rigorous scientific data. Here we evaluate the lymphomacidal and immunomodulatory activities of a protein fraction isolated from fermented wheat germ. Fermented wheat germ extract was produced by fermenting wheat germ with Saccharomyces cerevisiae. A protein fraction was tested for lymphomacidal activity in vitro using NHL cell lines and in vivo using mouse xenografts. Mechanisms of action were explored in vitro by evaluating apoptosis and cell cycle and in vivo by immunophenotyping and measurement of NK cell activity. Potent lymphomacidal activity was observed in a panel of NHL cell lines and mice bearing NHL xenografts. This activity was not dependent on wheat germ agglutinin or benzoquinones. Fermented wheat germ proteins induced apoptosis in NHL cells, and augmented immune effector mechanisms, as measured by NK cell killing activity, degranulation and production of IFNγ. Fermented wheat germ extract can be easily produced and is efficacious in a human lymphoma xenograft model. The protein fraction is quantifiable and more potent, shows direct pro-apoptotic properties, and enhances immune-mediated tumor eradication. The results presented herein support the novel concept that proteins in fermented wheat germ have direct pro-apoptotic activity on lymphoma cells and augment host immune effector mechanisms.

Evaluation of a Flow Cytometry‐Based Assay for Natural Killer Cell Activity in Clinical Settings

Natural killer (NK) cells are not only important in first line defence against viral and bacterial infections, but also in immune surveillance of malignant cells and thus NK cell cytotoxicity is primary indicator of immune function. Although chromium release assay is recognized as 'gold standard' for measuring NK cell activity, it has disadvantages like use of radioactive compounds, poor loading and high spontaneous release. It is difficult to perform this assay in clinical laboratory because of difficulties with disposal of radioactive waste and standardization problems. We describe a flow cytometry-based assay for the measurement of NK cell activity by incorporating fluorescent dye, DiO, into membranes of target cells. NK cell activity was measured at baseline, 1 and 4 weeks follow-up in 20 normal healthy individuals on a dietary supplement immunomodulator to enhance NK cell function. Mean baseline NK cell activity percentage (21.5; SD = 9.3) increased significantly to a maximum level at 1 week (31.3%; SD = 7.9; P = 0.007) and then returned to baseline level at 4 weeks (21.5; SD = 8.3). An important feature of flow cytometry-based assays is its ability to discriminate effector cells from target cells, and potential for explaining molecular interactions underlying target cell lysis. Under clinical settings, this assay will be of interest for frequently monitoring immunological status of patients on treatment for various diseases that affect their immune status. The assay is easy to perform without using radioactive material and thus could become a tool for monitoring pathogenesis and immune reconstitution.

Low natural killer cell cytotoxic activity in autism: The role of glutathione, IL-2 and IL-15

Although many articles have reported immune abnormalities in autism, NK cell activity has only been examined in one study of 31 patients, of whom 12 were found to have reduced NK activity. The mechanism behind this low NK cell activity was not explored. For this reason, we explored the measurement of NK cell activity in 1027 blood samples from autistic children obtained from ten clinics and compared the results to 113 healthy controls. This counting of NK cells and the measurement of their lytic activity enabled us to express the NK cell activity/100 cells. At the cutoff of 15–50 LU we found that NK cell activity was low in 41–81% of the patients from the different clinics. This NK cell activity below 15 LU was found in only 8% of healthy subjects ( p < 0.001). Low NK cell activity in both groups did not correlate with percentage and absolute number of CD16 +/CD56 + cells. When the NK cytotoxic activity was expressed based on activity/100 CD16 +/CD56 + cells, several patients who had displayed NK cell activity below 15 LU exhibited normal NK cell activity. Overall, after this correction factor, 45% of the children with autism still exhibited low NK cell activity, correlating with the intracellular level of glutathione. Finally, we cultured lymphocytes of patients with low or high NK cell activity/cell with or without glutathione, IL-2 and IL-15. The induction of NK cell activity by IL-2, IL-15 and glutathione was more pronounced in a subgroup with very low NK cell activity. We conclude that that 45% of a subgroup of children with autism suffers from low NK cell activity, and that low intracellular levels of glutathione, IL-2 and IL-15 may be responsible.

NK sensitivity of neuroblastoma cells determined by a highly sensitive coupled luminescent method

The measurement of natural killer (NK) cells toxicity against tumor or virus-infected cells especially in cases with small blood samples requires highly sensitive methods. Here, a coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase release from injured target cells was used to evaluate the cytotoxicity of interleukin-2 activated NK cells against neuroblastoma cell lines. In contrast to most other methods, CLM does not require the pretreatment of target cells with labeling substances which could be toxic or radioactive. The effective killing of tumor cells was achieved by low effector/target ratios ranging from 0.5:1 to 4:1. CLM provides highly sensitive, safe, and fast procedure for measurement of NK cell activity with small blood samples such as those obtained from pediatric patients.

Effect of Maitake (Grifola frondosa) D-Fraction on the activation of NK cells in cancer patients.

Maitake D-Fraction, extracted from maitake mushroom, has been reported to exert its antitumor effect in tumor-bearing mice by enhancing the immune system through activation of macrophages, T cells, and natural killer (NK) cells. In a previous study, the combination of immunotherapy with the maitake D-Fraction and chemotherapy suggested that the D-Fraction may have the potential to decrease the size of lung, liver, and breast tumors in cancer patients. In the present study, we administered maitake D-Fraction to cancer patients without anticancer drugs, and at the same time NK cell activity was monitored to investigate whether the activity is closely related with disease progression. The numbers of CD4(+) and CD8(+) cells in the peripheral blood were measured in 10 patients, and NK cell activity was assessed using K-562 cells as target cells. Serum soluble interleukin-2 receptor (sIL-2R) levels in three patients and the expression of tumor markers in four patients were determined by enzyme-linked immunosorbent assay. The slight changes observed in the CD4(+) and CD8(+) cell numbers were independent of disease severity or stage as well as serum sIL-2R levels. In contrast, maitake D-Fraction hindered metastatic progress, lessened the expression of tumor markers, and increased NK cell activity in all patients examined. Thus maitake D-Fraction appears to repress cancer progression and primarily exerts its effect through stimulation of NK activity. In addition, we conclude that measurement of NK cell activity may be a useful clinical parameter in monitoring disease progression during and following immunotherapy with maitake D-Fraction.

Determination of natural killer cell function by flow cytometry

Natural killer cells (NK cells) are a subset of peripheral blood lymphocytes that mediate non-major histocompatibility complex-restricted cytotoxicity of foreign target cells. The "gold standard" assay for NK cell activity has been the chromium release assay. This method is not easily performed in the clinical laboratory because of difficulties with disposal of radioactive and hazardous materials, short reagent half-lives, expense, and difficulties with assay standardization. We describe a flow cytometric assay for the clinical measurement of NK cell activity. This study compared the chromium release assay and the flow cytometric assay by using clinically relevant specimens. There were no significant differences between the two assays in the measurement of lytic activity for 17 peripheral blood specimens or in reproducibility in repeated samplings of healthy individuals. We also established a normal range of values for NK activity in healthy adults and identified a small cluster of individuals who have exceptionally high or low levels of NK activity. The flow cytometric assay was validated by testing specimens from subjects expected to have abnormally low levels of NK activity (pregnant women) and specimens from healthy individuals in whom the activity of NK cells was enhanced by exposure to interleukin-2 or alpha interferon. Treatment with these agents was associated with a significant increase in NK activity. These results confirm and extend those of others, showing that the flow cytometric assay is a viable alternative to the chromium release assay for measuring NK cell activity.