Comparison of the different anti-CD16 antibody clones in the activation and expansion of peripheral blood NK cells.

Natural killer (NK) cells are promising tool for cancer treatment. Methods have been developed for large-scale NK cell expansion, including feeder cell-based methods or methods involving stimulation with NK cell activating signals, such as anti-CD16 antibodies. Different clones of anti-CD16 antibodies are available; however, a comprehensive comparison of their differential effects on inducing NK cell activation and expansion has not been conducted among these various clones under the same experimental conditions. Herein, we found that the NK cell expansion rate differed depending on the various anti-CD16 antibodies (CB16, 3G8, B73.1, and MEM-154) coated on microbeads when stimulated with genetically engineered feeder cells, K562­membrane-bound IL­18, and mbIL­21 (K562­mbIL­18/-21). Only the CB16 clone combination caused enhanced NK cell expansion over K562­mbIL­18/-21 stimulation alone with similar NK cell functionality. Treatment with the CB16 clone once on the initial day of NK cell expansion was sufficient to maximize the combination effect. Overall, we developed a more enhanced NK expansion system by merging a feeder to effectively stimulate CD16 with the CB16 clone.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Natural Killer Cell Expansion and Cytotoxicity Differ Depending on the Culture Medium Used.

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results: The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

Expansion of cytotoxic natural killer cells in multiple myeloma patients using K562 cells expressing OX40 ligand and membrane-bound IL-18 and IL-21.

BACKGROUND: Natural killer (NK) cell-based immunotherapy is a promising treatment approach for multiple myeloma (MM), but obtaining a sufficient number of activated NK cells remains challenging. Here, we report an improved method to generate ex vivo expanded NK (eNK) cells from MM patients based on genetic engineering of K562 cells to express OX40 ligand and membrane-bound (mb) IL-18 and IL-21. METHODS: K562-OX40L-mbIL-18/-21 cells were generated by transducing K562-OX40L cells with a lentiviral vector encoding mbIL-18 and mbIL-21, and these were used as feeder cells to expand NK cells from peripheral blood mononuclear cells of healthy donors (HDs) and MM patients in the presence of IL-2/IL-15. Purity, expansion rate, receptor expression, and functions of eNK cells were determined over four weeks of culture. RESULTS: NK cell expansion was enhanced by short exposure of soluble IL-18 and IL-21 with K562-OX40L cells. Co-culture of NK cells with K562-OX40L-mbIL-18/-21 cells resulted in remarkable expansion of NK cells from HDs (9,860-fold) and MM patients (4,929-fold) over the 28-day culture period. Moreover, eNK cells showed increased expression of major activation markers and enhanced cytotoxicity towards target K562, U266, and RPMI8226 cells. CONCLUSIONS: Our data suggest that genetically engineered K562 cells expressing OX40L, mbIL-18, and mbIL-21 improve the expansion of NK cells, increase activation signals, and enhance their cytolytic activity towards MM cells.

A Flow Cytometry-Based Whole Blood Natural Killer Cell Cytotoxicity Assay Using Overnight Cytokine Activation.

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.

Phenotypic and Functional Analysis of Human NK Cell Subpopulations According to the Expression of FcεRIγ and NKG2C.

Human memory-like NK cells are commonly defined by either a lack of FcεRIγ or gain of NKG2C expression. Here, we investigated the heterogeneity of human CD56dim NK cell subpopulations according to the expression of FcεRIγ and NKG2C in a large cohort (n = 127). Although the frequency of FcεRIγ- and NKG2C+ NK cells positively correlated, the FcεRIγ- and NKG2C+ NK cell populations did not exactly overlap. The FcεRIγ+NKG2C+, FcεRIγ-NKG2C+, and FcεRIγ-NKG2C- NK cell populations were only evident after HCMV infection, but each had distinct characteristics. Among the subpopulations, FcεRIγ-NKG2C+ NK cells exhibited the most restricted killer immunoglobulin-like receptor repertoire, suggesting clonal expansion. Moreover, FcεRIγ-NKG2C+ NK cells exhibited the lowest Ki-67 and highest Bcl-2 expression, indicating the long-lived quiescent memory-like property. Functionally, FcεRIγ-NKG2C+ NK cells had weak natural effector function against K562 but strong effector functions by CD16 engagement, whereas FcεRIγ+NKG2C+ NK cells had strong effector functions in both settings. Anatomically, the FcεRIγ+NKG2C+, FcεRIγ-NKG2C+, and FcεRIγ-NKG2C- NK cell populations were present in multiple human peripheral organs. In conclusion, we demonstrate the heterogeneity of memory-like NK cells stratified by FcεRIγ and NKG2C and suggest both markers be utilized to better define these cells.

The Anion Gap is a Predictive Clinical Marker for Death in Patients with Acute Pesticide Intoxication.

Pesticide formulation includes solvents (methanol and xylene) and antifreeze (ethylene glycol) whose metabolites are anions such as formic acid, hippuric acid, and oxalate. However, the effect of the anion gap on clinical outcome in acute pesticide intoxication requires clarification. In this prospective study, we compared the anion gap and other parameters between surviving versus deceased patients with acute pesticide intoxication. The following parameters were assessed in 1,058 patients with acute pesticide intoxication: blood chemistry (blood urea nitrogen, creatinine, glucose, lactic acid, liver enzymes, albumin, globulin, and urate), urinalysis (ketone bodies), arterial blood gas analysis, electrolytes (Na(+), K(+), Cl(-) HCO3 (-), Ca(++)), pesticide field of use, class, and ingestion amount, clinical outcome (death rate, length of hospital stay, length of intensive care unit stay, and seriousness of toxic symptoms), and the calculated anion gap. Among the 481 patients with a high anion gap, 52.2% had a blood pH in the physiologic range, 35.8% had metabolic acidosis, and 12.1% had acidemia. Age, anion gap, pesticide field of use, pesticide class, seriousness of symptoms (all P < 0.001), and time lag after ingestion (P = 0.048) were significant risk factors for death in univariate analyses. Among these, age, anion gap, and pesticide class were significant risk factors for death in a multiple logistic regression analysis (P < 0.001). In conclusions, high anion gap is a significant risk factor for death, regardless of the accompanying acid-base balance status in patients with acute pesticide intoxication.

The effects of nonyl phenoxypolyethoxyl ethanol on cell damage pathway gene expression in SK-NSH cells.

BACKGROUND/AIMS: Most pesticide formulations contain both chief and additive ingredients. But, the additives may not have been tested as thoroughly as the chief ingredients. The surfactant, nonyl phenoxypolyethoxylethanol (NP40), is an additive frequently present in pesticide formulations. We investigated the effects of NP40 and other constituents of a validamycin pesticide formulation on cell viability and on the expression of genes involved in cell damage pathways. METHODS: The effects of validamycin pesticide ingredients on cell viability and of NP40 on the mRNA expression of 80 genes involved in nine key cellular pathways were examined in the human neuroblastoma SK-N-SH cell line. RESULTS: The chemicals present in the validamycin pesticide formulation were cytotoxic to SK-N-SH cells and NP40 showed the greatest cytotoxicity. A range of gene expression changes were identified, with both up- and down-regulation of genes within the same pathway. However, all genes tested in the necrosis signaling pathway were down-regulated and all genes tested in the cell cycle checkpoint/arrest pathway were up-regulated. The median fold-change in gene expression was significantly higher in the cell cycle checkpoint/arrest pathway than in the hypoxia pathway category (p = 0.0064). The 70 kDa heat shock protein 4 gene, within the heat shock protein/unfolded protein response category, showed the highest individual increase in expression (26.1-fold). CONCLUSIONS: NP40 appeared to be particularly harmful, inducing gene expression changes that indicated genotoxicity, activation of the cell death (necrosis signaling) pathway, and induction of the 70 kDa heat shock protein 4 gene.

Common Pesticides Used in Suicide Attempts Following the 2012 Paraquat Ban in Korea.

To determine the change in pesticides used during suicide attempts after the 2012 paraquat (PQ) ban, we evaluated the annual number of suicide attempts by pesticide ingestion between 2011 and 2014. We extracted demographic, clinical outcome, and pesticide class data from the medical records of 1,331 patients that attempted suicide by pesticide ingestion. Pesticides were sorted into 5 groups: herbicides, insecticides, fungicides, other pesticides, and combined pesticides. Each group was subdivided into various classes based on publications by the respective Resistance Action Committees. The chi-square test for trends was used to compare the annual incidence of categorical variables. The total number of suicide attempts decreased each year, from 399 in 2011 to 245 in 2014. Simultaneously, PQ ingestion decreased from 253 patients in 2011 to 60 in 2014. The proportion of PQ to pesticides also decreased from 63.4% in 2011 to 24.5% in 2014. Furthermore, the rate of decrease in the proportion of PQ to all herbicide categories increased by calendar year. In conclusion, there is a significant trend in increased annual number of suicides and proportion of suicides using glyphosates and glufosinates versus total herbicides. However, the number of suicide attempts using glyphosate and glufosinate is lower than that using PQ. The ratio of persons completing suicide to those attempting suicide after pesticide ingestion has decreased every year after the PQ ban.

Epigenetic modification and antibody-dependent expansion of memory-like NK cells in human cytomegalovirus-infected individuals.

Long-lived "memory-like" NK cells have been identified in individuals infected by human cytomegalovirus (HCMV), but little is known about how the memory-like NK cell pool is formed. Here, we have shown that HCMV-infected individuals have several distinct subsets of memory-like NK cells that are often deficient for multiple transcription factors and signaling proteins, including tyrosine kinase SYK, for which the reduced expression was stable over time and correlated with epigenetic modification of the gene promoter. Deficient expression of these proteins was largely confined to the recently discovered FcRγ-deficient NK cells that display enhanced antibody-dependent functional activity. Importantly, FcRγ-deficient NK cells exhibited robust preferential expansion in response to virus-infected cells (both HCMV and influenza) in an antibody-dependent manner. These findings suggest that the memory-like NK cell pool is shaped and maintained by a mechanism that involves both epigenetic modification of gene expression and antibody-dependent expansion.

Cutting edge: antibody-dependent memory-like NK cells distinguished by FcRγ deficiency.

Because NK cells lack gene-recombination machinery and are thought to be relatively short-lived, it is unclear whether NK cells can mount long-term effective recall responses to reinfections by diverse pathogens. In this article, we report that FcRγ-deficient NK cells, which we recently identified and termed g(-)NK cells, possess distinct memory features directed by FcR-mediated Ab-dependent target recognition. The presence of g(-)NK cells was associated with prior human CMV (HMCV) infection, yet g(-)NK cell responses were not restricted to HCMV-infected target cells. In the presence of virus-specific Abs, g(-)NK cells had greatly enhanced functional capabilities, superior to conventional NK cells, and were highly responsive to cells infected with either HCMV or HSV-1. Remarkably, the g(-)NK cell subset persisted long-term at nearly constant levels in healthy individuals. Therefore, FcRγ deficiency distinguishes an Ab-dependent memory-like NK cell subset with enhanced potential for broad antiviral responses.

Identification of human NK cells that are deficient for signaling adaptor FcRγ and specialized for antibody-dependent immune functions.

NK cells respond to tumor and virus-infected cells directly through several activation receptors, including natural cytotoxicity receptors, or indirectly through the activating Fc receptor CD16 for antibody-coated cells. Triggering of NK-cell effector functions through these receptors depends on physically associated transmembrane signaling adaptors, such as FcRγ (also known as FcεRIγ) and CD3ζ, both of which have been traditionally believed to be expressed by all mature NK cells. However, we have identified a distinct subset of human NK cells that are deficient for FcRγ expression but express normal levels of CD3ζ. FcRγ-deficient NK cells were readily detectable in about one-third of the healthy individuals examined. The deficiency was confined to the CD56(dim) population and was due to low FcRγ mRNA. FcRγ-deficient NK cells displayed dramatically reduced expression of the natural cytotoxicity receptors NKp46 and NKp30 but still expressed substantial levels of CD16. Compared to FcRγ-expressing NK cells, FcRγ-deficient NK cells showed poor direct reactivity toward tumor targets as measured by cytokine production and degranulation. Unexpectedly, however, FcRγ-deficient NK cells exhibited significantly more robust responsiveness upon stimulation through CD16, particularly for cytokine production, compared to FcRγ-expressing NK cells. Thus, our study reveals FcRγ-deficient NK cells as a novel subset of human NK cells that have remarkably potent responses toward antibody-coated targets. These findings also illustrate a differential contribution of FcRγ and CD3ζ for the expression and functional activity of their associated receptors.

Activation mechanisms of natural killer cells during influenza virus infection.

During early viral infection, activation of natural killer (NK) cells elicits the effector functions of target cell lysis and cytokine production. However, the cellular and molecular mechanisms leading to NK cell activation during viral infections are incompletely understood. In this study, using a model of acute viral infection, we investigated the mechanisms controlling cytotoxic activity and cytokine production in response to influenza (flu) virus. Analysis of cytokine receptor deficient mice demonstrated that type I interferons (IFNs), but not IL-12 or IL-18, were critical for the NK cell expression of both IFN-γ and granzyme B in response to flu infection. Further, adoptive transfer experiments revealed that NK cell activation was mediated by type I IFNs acting directly on NK cells. Analysis of signal transduction molecules showed that during flu infection, STAT1 activation in NK cells was completely dependent on direct type I IFN signaling, whereas STAT4 activation was only partially dependent. In addition, granzyme B induction in NK cells was mediated by signaling primarily through STAT1, but not STAT4, while IFN-γ production was mediated by signaling through STAT4, but not STAT1. Therefore, our findings demonstrate the importance of direct action of type I IFNs on NK cells to mount effective NK cell responses in the context of flu infection and delineate NK cell signaling pathways responsible for controlling cytotoxic activity and cytokine production.