Establishment and application of a highly sensitive coupled luminescent method (CLM) to study natural killer cell cytolytic activity

Natural killer (NK) cells are white blood lymphocytes of the innate immune system that have diverse biological functions, including recognition and destruction of certain microbial infections and neoplasms [1]. NK cells comprise ~ 10% of all circulating lymphocytes and are also found in peripheral tissues including the liver, peritoneal cavity and placenta. Resting NK cells circulate in the blood, but, following activation by cytokines, they are capable of extravasation and infiltration into most tissues that contain pathogen-infected or malignant cells [2-5]. NK cells discriminate between normal and abnormal cells (infected or transformed) through engagement and dynamic integration of multiple signaling pathways, which are initiated by germline-encoded receptors [6-8]. Healthy cells are protected from NK cell-mediated lysis by expression of major histocompatibility complex (MHC) class I ligands for NK cell inhibitory receptors [6, 9]. The MHC is a group of highly polymorphic glycoproteins that are expressed by every nucleated cell of vertebrates, and that are encoded by the MHC gene cluster. The human MHC molecules are termed human leucocyte antigen (HLA)-A, B and C molecules. Every NK cell expresses at least one inhibitory receptor that recognizes a self-MHC class I molecule. So, normal cells that express MHC class I molecules are protected from self-NK cells, but transformed or infected cells that have down-regulated MHC class I expression are attacked by NK cells [10]. There are 2 distinct subsets of human NK cells identified mainly by cell surface density of CD56. The majority (approximately 90%) of human NK cells are CD56dimCD16bright and express high levels of FcγRIII (CD16), whereas a minority (approximately 10%) are CD56brightCD16dim/- [11]. Resting CD56dim NK cells are more cytotoxic against NK-sensitive targets than CD56bright NK cells [12]. However, after activation with interleukin (IL)-2 or IL-12, CD56bright cells exhibit similar or enhanced cytotoxicity against NK targets compared to CD56dim cells [12-14]. The functions of NK cells are regulated by a balance of signals (Fig. 1.1). These are transmitted by inhibitory receptors, which bind MHC class I molecules, and activating receptors, which bind ligands on tumors and virus-infected cells [15]. These receptors are completely encoded in the genome, rather than being generated by somatic recombinations, like T- and B-cell receptors.Schnelle, empfindliche und material-sparende Methoden zur Messung der zytolytischen Aktivität natürlicher Killerzellen (NK), eine wichtige Determinante der NK Zell Funktion, sind entscheidend für die Analyse der physiologischen Funktion, als auch für die pathologisch veränderten Zustände des Immunsystems. Den Goldstandard für die Messung zellvermittelter Zytotoxizität stellt das Chromium (51Cr) oder Europium (Eu3+) release assay dar. Allerdings stellt die Messung der Zielzelllyse mittels dem 51Cr oder Eu3+ release assay eine zeitaufwendige Methode dar und die notwendige Markierung der Zielzellen mit radioaktiven Substanzen bedeutet eine drastische Manipulation. Darüber hinaus kommt es aufgrund der vielen Zellen, die für den Test benötigt werden, zu hohen Hintergrundwerten. Verschiedene andere Methoden zur Messung von Zell Zytotoxizität sind widersprüchlich und weisen zahlreiche Mängel auf. Im Verlauf dieser Forschungsarbeit wurde zur Messung der zytolytischen Aktivität von Interleukin (IL)-2-aktivierten NK Zellen gegen die NK Zell sensitive erythroleukämische Zelllinie K562 erstmals die hoch empfindliche gekoppelte Lumineszenz Methode (coupled luminescent method (CLM)) etabliert. Diese Methode basiert auf der Messung der Glyceraldehyd-3-phosphat-dehydrogenasefreisetzung (G3PDH) aus lysierten Zielzellen. Zur Validierung der CLM wurden NK-resistente Neuroblastom (NB) Zellen verwendet. Alle getesteten NB Zelllinien (UKF-NB-2, UKF-NB-3, UKF-NB-4 and UKF-NB-2rVCR10) wurden durch IL-2 aktivierte NK Zellen lysiert. Im Gegensatz zu herkömmlichen Methoden wie 51Cr oder Eu3+, ist es bei der CLM nicht nötig eine Vorbehandlung der Zielzellen mit radioaktiven oder toxischen Substanzen vorzunehmen. CLM stellt daher eine hochsensible, sichere und materialsparende Methode zur Messung von NK Zellen dar

Inhibition of apoptosis prevents West Nile virus induced cell death

We found that WNV infection induces cell death in the brain-derived tumour cell line T98G by apoptosis under involvement of constituents of the extrinsic as well as the intrinsic apoptotic pathways. Our results illuminate the molecular mechanism of WNV-induced neural cell death

Histone deacetylase inhibitors suppress natural killer cell cytolytic activity.

Treatment of transformed cells from leukemia or solid tumors with histone deacetylase inhibitors (HDACi) was shown to increase their sensitivity to NK cell lysis. In this study, treatment of IL-2-activated NK cells with HDACi including suberoylanilide hydroxamic acid and valproic acid was studied. Both drugs at therapeutic concentrations inhibited NK cell cytotoxicity on human leukemic cells. This inhibition was associated with decreased expression and function of NK cell activating receptors NKp46 and NKp30 as well as impaired granule exocytosis. NFkappaB activation in IL-2-activated NK cells was inhibited by both HDACi. Pharmacologic inhibition of NFkappaB activity resulted in similar effects on NK cell activity like those observed for HDACi. These results demonstrate for the first time that HDACi prevent NK cytotoxicity by downregulation of NK cell activating receptors probably through the inhibition of NFkappaB activation

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

The anti-tumoral drug enzastaurin inhibits natural killer cell cytotoxicity via activation of glycogen synthase kinase-3?.

Enzastaurin is a selective protein kinase C? inhibitor which is shown to have direct antitumor effect as well as suppress glycogen synthase kinase-3? (GSK-3?) phosphorylation (resulting in its activation) in both tumor tissues and peripheral blood mononuclear cells (PBMC). It is currently used in phase II trials for the treatment of colon cancer, refractory glioblastoma and diffuse large B cell lymphoma. In this study, the direct effect of enzastaurin on effector function of human natural killer (NK) cells was investigated. The results obtained showed that enzastaurin suppressed both natural and antibody-dependent cellular cytotoxicity (ADCC) of NK cells against different tumor targets. This inhibition was associated with a specific down-regulation of surface expression of NK cell activating receptor NKG2D and CD16 involved in natural cytotoxicity and ADCC respectively, as well as the inhibition of perforin release. Analysis of signal transduction revealed that enzastaurin activated GSK-3? by inhibition of GSK-3? phosphorylation. Treatment of NK cells with GSK-3?-specific inhibitor TDZD-8 prevented enzastaurin-induced inhibition of NK cell cytotoxicity. Apart from the known antitumor and antiangiogenic effects, these results demonstrate that enzastaurin suppresses NK cell activity and may therefore interfere with NK cell-mediated tumor control in enzastaurin-treated cancer patients

Measurement of cytotoxic T lymphocyte activity of human cytomegalovirus seropositive individuals by a highly sensitive coupled luminescent method.

A coupled luminescent method (CLM) based on glyceraldehyde-3-phosphate dehydrogenase released from injured target cells was used to evaluate the cytotoxicity of antigen-specific HLA class I-restricted CTLs. In contrast to established methods, CLM does not require the pretreatment of target cells with radioactive or toxic labeling substances. CTLs from healthy HLA-A2 positive donors were stimulated by autologous dendritic cells (DCs) pulsed with HLA-A2 restricted HCMV-pp65 nonamer peptides. HLA-A2 positive T2 cells or autologous monocytes pulsed with HCMV-pp65 nonamer peptide served as target cells. Lysis was detected only in HCMV-pp65-pulsed target cells incubated with CTLs from seropositive donors stimulated by HCMV-pp65-pulsed DCs. After 3 days, stimulation 38% of T2 cells and 17% of monocytes were lysed at an effector to target ratio of 8:1. In conclusion, CLM represents a highly sensitive, fast, material-saving and non-toxic/non-radioactive method for the measurement of antigen-specific CTL cytotoxic activity

Are Helicobacter pylori and other Helicobacter species infection associated with human biliary lithiasis? A meta-analysis.

BACKGROUND: Since the isolation of Helicobacter species in biliary system, a hypothetical question was raised about the role of these agents in the development of cholelithiasis. This meta-analysis is to explore the association between the Helicobacter infection and biliary lithiasis. METHODOLOGY/PRINCIPAL FINDINGS: A systematic literature search was performed to identify all eligible articles. Meta-analysis which was carried out using odds ratio and random effect model, 95% confidence intervals for odds ratio was calculated. Quantitative assessment of heterogeneity was explored by chi-square test with significance set at P value 0.10 and was measured using I(2) statistic. Eighteen studies published between 1998 and 2011 were finally eligible for meta-analysis. H. pylori, H. bilis, H. hepaticus, H. pullorum and H. ganmani were studied. With heterogeneity (I(2) = 69.5%, P<0.0001), significantly higher pooled infection rates of H. pylori (OR: 2.59, 35.82% versus 26.75%, P = 0.01) and H. hepaticus (OR: 3.13, 31.30% versus 12.12%, P = 0.02) were observed in lithiasis group. Higher prevalence of H. pylori in cholelithiasis patients were reported by studies from East Asia, South Asia and South America. Evidences supporting the higher presence of H. pylori in cholelithiasis patients could be found by PCR for detecting 16s rRNA in bile, 26 kDa protein gene in biliary tissue and immunohistochemistry. Using multiple detection tests could increase the detection rate of H. pylori. CONCLUSIONS/SIGNIFICANCES: Our meta-analysis suggests a trend of higher presence of H. pylori in cholelithiasis patients than control group and this trend was significant in the regions with higher prevalence of this agent. Evidences supporting the association between Helicobacter and cholelithiasis could be found by using different tests but the gold standard for the identification of these bacteria in biliary system has yet to be established. Considering obvious heterogeneity, a large multi-center study will facilitate us to further clarify the association between the Helicobacter infection and cholelithiasis

Tumor cells infected with oncolytic influenza A virus prime natural killer cells for lysis of resistant tumor cells.

Tumor resistance to lysis by resting natural killer (NK) cells may be overcome by priming of NK cells with cytokines or by binding of NK activating receptors to ligands expressed on target cells. In this study, major histocompatibility complex class I (MHC-I)-negative LNCaP and MHC-I-positive DU145 cells were infected with genetically modified influenza A virus lacking the non-structural gene 1 (NS1 IAV). The cells were used to investigate the influence of NS1 IAV infection on NK cell lysis of tumor cells as well as to prime NK cells for lysis of LNCaP and DU145 cells. While LNCaP cells infected with DeltaNS1 IAV showed enhanced lysis when compared with mock-infected cells (93% +/- 1.47 vs. 52% +/- 0.74), both mock-infected and DeltaNS1 IAV-infected DU145 cells were resistant to NK cell lysis. Moreover, NK cells primed with DeltaNS1 IAV-infected LNCaP/DU145 cells effectively lysed resistant DU145 and sensitive LNCaP cells to a greater extent than NK cells primed with mock-infected LNCaP/DU145 or non-primed NK cells. Also, NK cell priming with DeltaNS1 IAV-infected tumor cells enhanced extracellular signal-regulated kinase phosphorylation and increased granule release in NK cells. The increased granule release was specifically mediated by NKp46, which eventually potentiated NK cells primed with DeltaNS1 IAV-infected tumor cells to overcome the inhibitory effects posed by MHC-I expression on DU145 cells. These findings show that in addition to direct lytic activity of NK cells, DeltaNS1 IAV may influence anti-tumoral responses by priming NK cells