Genetic variability of three common NK and γδ T cell receptor genes (FCγ3R, NCR3, and DNAM-1) and their role in Polish patients with rheumatoid arthritis and ankylosing spondylitis.

Various lymphocyte subpopulations, including NK cells as well as γδ T cells, have been considered an important element in the pathogenesis of autoimmune, inflammatory, rheumatic diseases, such as rheumatoid arthritis (RA) and ankylosing spondylitis (AS). The aim of this study was to assess the potential role of polymorphic variations in the genes coding for three NK and γδ T cell receptors: NCR3, FCγR3A, and DNAM-1 (rs1052248, rs396991, and rs763361, respectively) in the disease susceptibility and the efficacy of treatment with TNF inhibitors. The study included 461 patients with RA, 168 patients with AS, and 235 voluntary blood donors as controls. The NCR3 rs1052248 AA homozygosity prevailed in RA in patients lacking rheumatoid factor (p = 0.044) as well as in those who manifested the disease at a younger age (p = 0.005) and had higher CRP levels after 12 weeks of anti-TNF therapy (p = 0.021). The FCγR3A rs396991 polymorphism was associated with pain visual analogue scale (VAS) values before the initiation of anti-TNF treatment. Lower VAS values were observed in the GG homozygous RA patients (p = 0.024) and in AS patients with the TT genotype (p = 0.012). Moreover, AS heterozygous patients with the TG genotype presented higher CRP levels in the 12th week of anti-TNF treatment (p = 0.021). The findings suggest that the NCR3 rs1052248 AA homozygosity may have an adverse effect on RA, while the T allele potentially plays a protective role in the development of AS. Moreover, the rs1052248 T allele and TT genotype appear to have a favorable impact on the response to anti-TNF therapy in RA patients.

Altered NCR3 Splice Variants May Result in Deficient NK Cell Function in Renal Cell Carcinoma Patients.

BACKGROUND/AIM: The natural killer (NK) cell function of patients with malignant tumours may be suppressed by deficiency, and the poor prognosis of renal cell carcinoma (RCC) patients may be due to escape from NK cell cytotoxicity, especially with respect to natural cytotoxicity receptors (NCRs) on the NK cell surface. However, the specific mechanism remains unclear. Therefore, in this study, we sought to explore the role of NCR, especially NCR3 splice variants, in the process of NK cell deficiency in RCC patients. MATERIALS AND METHODS: We used flow cytometry to analyse the phenotype of NK cells from the peripheral blood and kidney tumour tissue of RCC patients. The NKp30-mediated NK cell killing function was measured by antibody-dependent cell-mediated cytotoxicity (ADCC) in NK and RCC cell coincubation. We extracted RNA from the peripheral blood mononuclear cells (PBMCs) of RCC patients and renal carcinoma tissue and carried out real-time quantitative PCR to detect the mRNA levels of NKp30a, NKp30b and NKp30c. mRNA expression levels of cytokines (IL-6, IL-8, IL-10, IL-18 and TGF-ß) based on RNA extracted from renal carcinoma tissue and adjacent normal kidney tissues were also measured by real-time quantitative PCR. RESULTS: Regarding the phenotype of NK cells in RCC patients, the proportion of NK cells in tumour tissue was significantly reduced, with changes in the NK cell proportion being most obvious in NKp30+ NK cells. Furthermore, the results of the ADCC function assay showed limited NKp30+ NK cell-mediated cytotoxicity in RCC patients. Through real-time quantitative PCR, we found lower expression of NKp30a and NKp30b, the immunostimulatory splice variants of NCR3 encoding NKp30, in RCC patients. Moreover, expression of activating cytokines (IL-6 and IL-8) in renal cancer tissue was decreased, though inhibitory cytokine (TGF-ß) expression remained unchanged, which may result in an immunosuppressive cytokine microenvironment. CONCLUSION: Decreased expression of immunostimulatory NCR3 splice variants and the inhibitory cytokine microenvironment in RCC patients may contribute to deficient NK cell cytotoxicity and renal carcinoma cell immune escape from NK cell killing, which may provide a theoretical basis for finding new immunotherapeutic targets for RCC.

Human NCR3 gene variants rs2736191 and rs11575837 alter longitudinal risk for development of pediatric malaria episodes and severe malarial anemia.

BACKGROUND: Plasmodium falciparum malaria is a leading cause of pediatric morbidity and mortality in holoendemic transmission areas. Severe malarial anemia [SMA, hemoglobin (Hb) < 5.0 g/dL in children] is the most common clinical manifestation of severe malaria in such regions. Although innate immune response genes are known to influence the development of SMA, the role of natural killer (NK) cells in malaria pathogenesis remains largely undefined. As such, we examined the impact of genetic variation in the gene encoding a primary NK cell receptor, natural cytotoxicity-triggering receptor 3 (NCR3), on the occurrence of malaria and SMA episodes over time. METHODS: Susceptibility to malaria, SMA, and all-cause mortality was determined in carriers of NCR3 genetic variants (i.e., rs2736191:C > G and rs11575837:C > T) and their haplotypes. The prospective observational study was conducted over a 36 mos. follow-up period in a cohort of children (n = 1,515, aged 1.9-40 mos.) residing in a holoendemic P. falciparum transmission region, Siaya, Kenya. RESULTS: Poisson regression modeling, controlling for anemia-promoting covariates, revealed a significantly increased risk of malaria in carriers of the homozygous mutant allele genotype (TT) for rs11575837 after multiple test correction [Incidence rate ratio (IRR) = 1.540, 95% CI = 1.114-2.129, P = 0.009]. Increased risk of SMA was observed for rs2736191 in children who inherited the CG genotype (IRR = 1.269, 95% CI = 1.009-1.597, P = 0.041) and in the additive model (presence of 1 or 2 copies) (IRR = 1.198, 95% CI = 1.030-1.393, P = 0.019), but was not significant after multiple test correction. Modeling of the haplotypes revealed that the CC haplotype had a significant additive effect for protection against SMA (i.e., reduced risk for development of SMA) after multiple test correction (IRR = 0.823, 95% CI = 0.711-0.952, P = 0.009). Although increased susceptibility to SMA was present in carriers of the GC haplotype (IRR = 1.276, 95% CI = 1.030-1.581, P = 0.026) with an additive effect (IRR = 1.182, 95% CI = 1.018-1.372, P = 0.029), the results did not remain significant after multiple test correction. None of the NCR3 genotypes or haplotypes were associated with all-cause mortality. CONCLUSIONS: Variation in NCR3 alters susceptibility to malaria and SMA during the acquisition of naturally-acquired malarial immunity. These results highlight the importance of NK cells in the innate immune response to malaria.

Non-fitness status of peripheral NK cells defined by decreased NKp30 and perforin, and increased soluble B7H6, in cervical cancer patients.

The NKp30 receptor is one of the three natural cytotoxic receptors reported in NK cells. This receptor is codified by the NCR3 gene, which encodes three isoforms, a consequence of the alternative splicing of exon 4. A greater expression of the three isoforms (A, B, and C), along with low levels of the NKp30 ligand B7H6, has been reported as a positive prognostic factor in different cancer types. Here, in patients with cervical cancer and precursor lesions, we report an altered immune-phenotype, characterized by non-fitness markers, that correlated with increased disease stage, from CIN 1 to FIGO IV. While overall NK cell numbers increased, loss of NKp30+ NK cells, especially in the CD56dim subpopulation, was found. Perforin levels were decreased in these cells. Decreased expression of the NKp30 C isoform and overexpression of soluble B7H6 was found in cervical cancer patients when compared against healthy subjects. PBMCs from healthy subjects downregulated NKp30 isoforms after co-culture with B7H6-expressing tumour cells. Taken together, these findings describe a unique down-modulation or non-fitness status of the immune response in cervical cancer, the understanding of which will be important for the design of novel immunotherapies against this disease.

Suppression of breast cancer cells resistant to a pure anti-estrogen with CAR-transduced natural killer cells.

Anti-estrogens as hormone therapy are the mainstay treatment for estrogen receptor (ER)-positive breast cancer. ER inhibitors through modulating the transcriptional function of ER have been the frontline anti-estrogens to which refractory phenotype often developed in advanced cancer. The anti-estrogen fulvestrant is currently the only clinically approved pure anti-estrogen which causes ER degradation. However, resistance to fulvestrant still occurs and unfortunately it leaves few choices other than chemotherapy as the later-line treatments to fulvestrant-resistant tumors. Here we show that fulvestrant resistance was accompanied by increased expression of a number of innate immune response genes including the natural killer (NK) cell ligand B7-H6 on the cell surface. In an attempt to overcome the drug resistance phenotype, a NK-based molecular approach taking advantage of a chimeric antigen receptor (CAR) system targeting B7-H6 was established and tested in cells with acquired resistance to fulvestrant. The results demonstrate that the cell therapy approach as a single agent can effectively induce cell death of the resistant cancer cells which is enhanced by the increased expression of cell surface B7-H6. This approach departs from the traditional strategies of conquering anti-estrogen resistant breast cancer and offers a new avenue to eradicate hormone-refractory malignant solid tumors.

Sevoflurane impedes glioma progression via regulating circ_0000215/miR-1200/NCR3LG1 axis.

Sevoflurane has been reported to have anti-tumorigenic effects in glioma. Circ_0000215 was found to play vital functions in the pathological progressions of glioma. However, whether circ_0000215 mediates the inhibitory effects of sevoflurane on glioma cells remains unclear. In vitro assays were performed using cell counting kit-8, flow cytometry, transwell and Western blot assays. The expression levels of circ_0000215, microRNA (miR)-1200 and NCR3LG1 (Natural Killer Cell Cytotoxicity Receptor 3 Ligand 1) were detected using quantitative real-time polymerase chain reaction (qRT-PCR) and/or Western blot. The dual-luciferase reporter assay and pull-down assay were used to investigate the relationship between miR-1200 and circ_0000215 or NCR3LG1. In vivo assay was conducted using xenograft nude mice model. In vitro assays suggested that sevoflurane repressed glioma cell proliferation, metastasis and induced apoptosis as well as hindered tumor growth in vivo, which were reversed by circ_0000215 overexpression. Mechanically, circ_0000215 was confirmed to directly target miR-1200, and NCR3LG1 was a target of miR-1200 in glioma cells. Importantly, circ_0000215 could regulate NCR3LG1 expression via miR-1200. Besides that, rescue assay suggested that circ_0000215 attenuated the inhibitory effects of sevoflurane on glioma cell growth and metastasis, which were reversed by miR-1200 overexpression or NCR3LG1 knockdown. Our study revealed that sevoflurane could suppress glioma tumorigenesis by regulating circ_0000215/miR-1200/NCR3LG1 axis, suggesting a new insight into the therapeutic potential of sevoflurane in glioma treatment.

NCR3LG1 (B7-H6) is a potential prognostic factor for bladder cancer patients.

BACKGROUND: NCR3LG1 (B7-H6) protein is selectively overexpressed on tumour and is associated with fatal disease progression of various cancer. However, its prognostic value in bladder cancer (BCa) has not been well elaborated. METHODS: We examined the expression of NCR3LG1 in human BCa and analysed its clinical significance and prognostic value. Meanwhile, the expression of NCR3LG1 was intervened in human BCa cell line 253JBV to analyse subsequent effects on tumour. RESULTS: According to TCGA data, the disease-free survival rate was statistically significant between the NCR3LG1 high expression group and the low expression group (Log Rank p = 0.006). Immunohistochemical staining showed that the expression of NCR3LG1 in BCa tissue was significantly higher than that in adjacent tissues (p < 0.0001), which was positively correlated with TNM staging (p = 0.008), histological grade (p = 0.022), and lymphoma metastasis of BCa (p = 0.032). The proliferation (p < 0.0001), invasion (p < 0.001) and migration ability (p < 0.001) of 253JBV cells are significantly inhibited by knocking down the expression of NCR3LG1, and the cell cycle arrest is induced at the G1 phase, which accelerates the apoptosis of BCa cells (p < 0.005). CONCLUSION: Our findings indicate that NCR3LG1 is involved in the progression of human BCa and may become a potential prognostic biomarker for BCa.

Circulating innate immune markers and outcomes in treatment-naïve advanced non-small cell lung cancer patients.

INTRODUCTION: Innate immunity represents the first step of activation of the immune system and dictates the quality of adaptive immune responses. Studies have reported links between systemic inflammatory or innate immune markers and prognosis in patients with lung cancer. To our knowledge, the prospective and concomitant study of these systemic markers has never been performed. METHODS: Advanced treatment-naive non-small cell lung cancer (NSCLC) patients eligible for first-line platinum-based chemotherapy were prospectively included from December 2012 to July 2015 (N = 148). Blood samples of patients were collected before the first cycle for fresh NK cell phenotyping. Peripheral blood mononuclear cells were cryopreserved for natural cytotoxicity receptor (NCR) genotyping as well as sera for NCR's ligand quantification. Data on leukocytes, neutrophils and monocyte counts and lactate dehydrogenase (LDH) levels were extracted from electronic medical records. RESULTS: Among all studied markers, monocytosis, neutrophilia, leucocytosis, high LDH and sBAG6 levels and reduced levels of NCR3 transcripts were associated with poor overall survival (OS) in univariate analysis. The levels of NCR3 transcripts was linked to age, number of metastatic sites, monocyte counts, LDH and sBAG6 levels. Neutrophilia was associated to high sBAG6 levels. NCR3 was the unique innate immune parameter that remained as an independent factor associated with both OS (P = 0.003) and progression-free survival (P = 0.009) in the multivariate analysis. CONCLUSION: This study brought evidence that these biomarkers are entangled; parameters associated with an inflammatory process were related to reduced levels of NCR3 transcripts. Finally, the level of NCR3 transcripts was independently associated with outcomes in treatment-naive patients with advanced NSCLC.

NKp30 isoforms and NKp30 ligands are predictive biomarkers of response to imatinib mesylate in metastatic GIST patients.

Despite effective targeted therapy acting on KIT and PDGFRA tyrosine kinases, gastrointestinal stromal tumors (GIST) escape treatment by acquiring mutations conveying resistance to imatinib mesylate (IM). Following the identification of NKp30-based immunosurveillance of GIST and the off-target effects of IM on NK cell functions, we investigated the predictive value of NKp30 isoforms and NKp30 soluble ligands in blood for the clinical response to IM. The relative expression and the proportions of NKp30 isoforms markedly impacted both event-free and overall survival, in two independent cohorts of metastatic GIST. Phenotypes based on disbalanced NKp30B/NKp30C ratio (ΔBClow) and low expression levels of NKp30A were identified in one third of patients with dismal prognosis across molecular subtypes. This ΔBClow blood phenotype was associated with a pro-inflammatory and immunosuppressive tumor microenvironment. In addition, detectable levels of the NKp30 ligand sB7-H6 predicted a worse prognosis in metastatic GIST. Soluble BAG6, an alternate ligand for NKp30 was associated with low NKp30 transcription and had additional predictive value in GIST patients with high NKp30 expression. Such GIST microenvironments could be rescued by therapy based on rIFN-α and anti-TRAIL mAb which reinstated innate immunity.

Homo-oligomerization of the activating natural killer cell receptor NKp30 ectodomain increases its binding affinity for cellular ligands.

The natural cytotoxicity receptors, comprised of three type I membrane proteins NKp30, NKp44, and NKp46, are a unique set of activating proteins expressed mainly on the surface of natural killer (NK) cells. Among these, NKp30 is a major receptor targeting virus-infected cells, malignantly transformed cells, and immature dendritic cells. To date, only few cellular ligands of NKp30 have been discovered, and the molecular details of ligand recognition by NKp30 are poorly understood. Within the current study, we found that the ectodomain of NKp30 forms functional homo-oligomers that mediate high affinity binding to its corresponding cellular ligand B7-H6. Notably, this homo-oligomerization is strongly promoted by the stalk domain of NKp30. Based on these data, we suggest that homo-oligomerization of NKp30 in the plasma membrane of NK cells, which might be favored by IL-2-dependent up-regulation of NKp30 expression, provides a way to improve recognition and lysis of target cells by NK cells.