Natural Cytotoxicity Triggering Receptor Research Articles

Deciphering Immune Landscape Remodeling Unravels the Underlying Mechanism for Synchronized Muscle and Bone Aging.

Evidence from numerous studies has revealed the synchronous progression of aging in bone and muscle; however, little is known about the underlying mechanisms. To this end, human muscles and bones are harvested and the aging-associated transcriptional dynamics of two tissues in parallel using single-cell RNA sequencing are surveyed. A subset of lipid-associated macrophages (triggering receptor expressed on myeloid cells 2, TREM2+ Macs) is identified in both aged muscle and bone. Genes responsible for muscle dystrophy and bone loss, such as secreted phosphoprotein 1 (SPP1), are also highly expressed in TREM2+ Macs, suggesting its conserved role in aging-related features. A common transition toward pro-inflammatory phenotypes in aged CD4+ T cells across tissues is also observed, activated by the nuclear factor kappa B subunit 1 (NFKB1). CD4+ T cells in aged muscle experience Th1-like differentiation, whereas, in bone, a skewing toward Th17 cells is observed. Furthermore, these results highlight that degenerated myocytes produce BAG6-containing exosomes that can communicate with Th17 cells in the bone through its receptor natural cytotoxicity triggering receptor 3 (NCR3). This communication upregulates CD6 expression in Th17 cells, which then interact with TREM2+ Macs through CD6-ALCAM signaling, ultimately stimulating the transcription of SPP1in TREM2+ Macs. The negative correlation between serum exosomal BCL2-associated athanogene 6 (BAG6) levels and bone mineral density further supports its role in mediating muscle and bone synchronization with aging.

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

BackgroundPlasmodium 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.MethodsSusceptibility 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.ResultsPoisson 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.ConclusionsVariation 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.

Differential modulation of natural killer cell cytotoxicity by 17β‑estradiol and prolactin through the NKG2D/NKG2DL axis in cervical cancer cells

Natural killer (NK) cells play a crucial role in cervical cancer (CC). As estrogens and prolactin (PRL) have been reported to be involved in CC, the present study attempted to elucidate the effects of both hormones on NK cells in CC. For this purpose, NKL cells, as well as CC-derived cell lines (HeLa, SiHa and C33A) and non-tumorigenic keratinocytes (HaCaT cells) were stimulated with 17β-estradiol (E2; 10 nM), PRL (200 ng/ml), or both (E2 and PRL) for 48 h. The expression of hormone receptors (estrogen receptor α and β, G protein-coupled estrogen receptor 1 and PRL receptor) and NK cell activating receptors [natural killer group 2D (NKG2D), natural cytotoxicity triggering receptor 3, natural cytotoxicity triggering receptor 2 and natural cytotoxicity triggering receptor 1] were measured using western blot analysis and flow cytometry, respectively. In the HeLa, SiHa, C33A and HaCaT cells stimulated with the hormones, the expression of NKG2D ligands [MHC class I polypeptide-related sequence A/B (MICA/B)] on the membrane and the soluble form of MICA was evaluated using flow cytometry and ELISA. Cytotoxicity assay was performed using GFP-transfected K562 cells as target cells. E2 reduced NKL cell-mediated cytotoxicity, while PRL exerted the opposite effect. NKL cells expressed different hormone receptor forms, of which PRL only induced a decrease in NKG2D expression compared to the untreated control NKL cells. PRL increased MICA/B expression in HeLa cells and E2 and PRL reversed this effect. However, in SiHa cells, the concurrent incubation with the two hormones decreased MICA/B expression. E2 and PRL, either alone or in combination, decreased soluble MICA secretion in all CC cell lines, while E2 solely increased soluble MICA secretion in SiHa cells. On the whole, the present study provides evidence that E2 and PRL mediate the mechanisms through which NK and CC cells mediate a cytotoxic response and these have an antagonistic effect on NK cell-mediated cytotoxicity.

Analysis of immune cell components and immune-related gene expression profiles in peripheral blood of patients with type 1 diabetes mellitus

BackgroundType 1 diabetes mellitus (T1DM) is a chronic autoimmune disease caused by severe loss of pancreatic β cells. Immune cells are key mediators of β cell destruction. This study attempted to investigate the role of immune cells and immune-related genes in the occurrence and development of T1DM.MethodsThe raw gene expression profile of the samples from 12 T1DM patients and 10 normal controls was obtained from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified by Limma package in R. The least absolute shrinkage and selection operator (LASSO)—support vector machines (SVM) were used to screen the hub genes. CIBERSORT algorithm was used to identify the different immune cells in distribution between T1DM and normal samples. Correlation of the hub genes and immune cells was analyzed by Spearman, and gene-GO-BP and gene-pathway interaction networks were constructed by Cytoscape plug-in ClueGO. Receiver operating characteristic (ROC) curves were used to assess diagnostic value of genes in T1DM.ResultsThe 50 immune-related DEGs were obtained between the T1DM and normal samples. Then, the 50 immune-related DEGs were further screened to obtain the 5 hub genes. CIBERSORT analysis revealed that the distribution of plasma cells, resting mast cells, resting NK cells and neutrophils had significant difference between T1DM and normal samples. Natural cytotoxicity triggering receptor 3 (NCR3) was significantly related to the activated NK cells, M0 macrophages, monocytes, resting NK cells, and resting memory CD4+ T cells. Moreover, tumor necrosis factor (TNF) was significantly associated with naive B cell and naive CD4+ T cell. NCR3 [Area under curve (AUC) = 0.918] possessed a higher accuracy than TNF (AUC = 0.763) in diagnosis of T1DM.ConclusionsThe immune-related genes (NCR3 and TNF) and immune cells (NK cells) may play a vital regulatory role in the occurrence and development of T1DM, which possibly provide new ideas and potential targets for the immunotherapy of diabetes mellitus (DM).

Vogt–Koyanagi–Harada patients show higher frequencies of circulating NKG2Dpos NK and NK T cells

Vogt-Koyanagi-Harada (VKH) is an autoimmune disease characterized by inflammation in tissues that contain melanocytes. We aimed to increase the knowledge regarding immunological pathways deregulated in VKH disease. We compared the percentages of circulating natural killer (NK), NK T and T cells expressing the activatory markers: CD16, CD69, NK group 2D (NKG2D), natural cytotoxicity triggering receptor 3 (Nkp30), natural cytotoxicity triggering receptor 1 (Nkp46) and the inhibitory marker: NK group 2 member A (NKG2A) in 10 active VKH patients, 20 control subjects (CTR) and seven patients with Behçet disease (BD) by flow cytometry. Cytotoxic potential of NK cells was determined through the degranulation marker CD107a expression after contact with K562 cells by flow cytometry. Moreover, plasmatic levels of 27 cytokines were determined with a multiplex bead-based assay. VKH patients showed higher percentages of NKG2Dpos NK and NK T cells versus CTR. The cytotoxic potential of NK cells induced by K562 cells was comparable between VKH patients and CTR. Finally, higher concentrations of interleukin (IL)-4, IL-5, IL-7, IL-17 and platelet-derived growth factor-subunits B (PDGF-bb) were detected in plasma of VKH patients versus CTR. The immune profile of VKH patients was similar to that of BD patients.

Oral cancer cell‑derived exosomes modulate natural killer cell activity by regulating the receptors on these cells.

Oral cancer (OC) is the most common type of head and neck malignant tumor. Tumor-derived exosomes induce a complex extracellular environment that affects tumor immunity. In the present study, exosomes were isolated from OC cell lines (WSU-HN4 and SCC-9) by ultrafiltration and the protein content of these oral cancer-derived exosomes (OCEXs) was analyzed by mass spectrometry, which revealed the enrichment of transforming growth factor (TGF)-β1. Natural killer (NK) cells were examined by flow cytometry following co-culture with OCEXs. The expression of killer cell lectin like receptor K1 (KLRK1; also known as NKG2D, as used herein) and natural cytotoxicity triggering receptor 3 (NCR3; also known as NKp30, as used herein) in NK cells was found to be significantly upregulated following co-culture with the OCEXs for 1 day, whereas this expression decreased at 7 days. Killer cell lectin like receptor C1 (KLRC1; also known as NKG2A; as used herein) expression exhibited an opposite trend at 1 day. In addition, NK cell cytotoxicity against the OC cells was enhanced at 1 day, but was attenuated at 7 days. TGF-β1 inhibited the function of NK cells at 7 days, whereas it had no obvious effects at 1 and 3 days. On the whole, the findings of the present study reveal changes in NK cell function and provide new insight into NK cell dysfunction.

The Diverse Functions of the Ubiquitous Fcγ Receptors and Their Unique Constituent, FcRγ Subunit.

Fc gamma receptors (FcγRs) are widely expressed on a variety of immune cells and play a myriad of regulatory roles in the immune system because of their structural diversity. Apart from their indispensable role in specific binding to the Fc portion of antibody subsets, FcγRs manifest diverse biological functions upon binding to their putative ligands. Examples of such manifestation include phagocytosis, presentation of antigens, mediation of antibody-dependent cellular cytotoxicity, anaphylactic reactions, and the promotion of apoptosis of T cells and natural killer cells. Functionally, the equilibrium between activating and inhibiting FcγR maintains the balance between afferent and efferent immunity. The γ subunit of the immunoglobulin Fc receptor (FcRγ) is a key component of discrete immune receptors and Fc receptors including the FcγR family. Furthermore, FcγRs exert a key role in terms of crosslinking the innate and adaptive workhorses of immunity. Ablation of one of these receptors might positively or negatively influence the immune response. Very recently, we discovered that FcRγ derived from natural cytotoxicity triggering receptor 1 (NCR1) curtails CD8+ T cell expansion and thereby turns an acute viral infection into a chronic one. Such a finding opens a new avenue for targeting the FcγRs as one of the therapeutic regimens to boost the immune response. This review highlights the structural heterogeneity and functional diversity of the ubiquitous FcγRs along with their featured subunit, FcRγ.

High Serum Levels of Cholesterol Increase Antitumor Functions of Nature Killer Cells and Reduce Growth of Liver Tumors in Mice

The relationship between serum cholesterol level and development of hepatocellular carcinoma (HCC) remains unclear. We investigated the effects of serum cholesterol level on development of liver tumors in mice. We performed studies with C57BL/6J mice, mice with disruption of the low-density lipoprotein receptor gene (Ldlr-/-mice), and mice with conditional deletion of nature killer (NK) cells (NKdele mice). Some C57BL/6J and NKdele mice were given injections of diethylinitrosamine to induce liver tumor formation. Mice were placed on a normal diet (ND) or high-cholesterol diet (HCD) to induce high serum levels of cholesterol. We also studied mice with homozygous disruption of ApoE (ApoE-/- mice), which spontaneously develop high serum cholesterol. C57BL/6J and NKdele mice on the ND or HCD were implanted with Hep1-6 (mouse hepatoma) cells and growth of xenograft tumors and lung metastases were monitored. Blood samples were collected from mice and analyzed by biochemistry and flow cytometry; liver and tumor tissues were collected and analyzed by histology, immunohistochemistry, and RNA-sequencing analysis. NK cells were isolated from mice and analyzed for cholesterol content, lipid raft formation, immune signaling, and changes in functions. We obtained matched tumor tissues and blood samples from 30 patients with HCC and blood samples from 40 healthy volunteers; levels of cholesterol and cytotoxicity of NK cells were measured. C57BL/6J mice on HCD and ApoE-/- mice with high serum levels of cholesterol developed fewer and smaller liver tumors and lung metastases after diethylinitrosamine injection or implantation of Hep1-6 cells than mice on ND. Liver tumors from HCD-fed mice and ApoE-/- mice had increased numbers of NK cells compared to tumors from ND-fed mice. NKdele mice or mice with antibody-based depletion for NK cells showed similar tumor number and size in ND and HCD groups after diethylinitrosamine injection or implantation of Hep1-6 cells. NK cells isolated from C57BL/6J mice fed with HCD had increased expression of NK cell-activating receptors (natural cytotoxicity triggering receptor 1 and natural killer group 2, member D), markers of effector function (granzyme B and perforin), and cytokines and chemokines compared with NK cells from mice on ND; these NK cells also had enhanced cytotoxic activity against mouse hepatoma cells, accumulated cholesterol, increased lipid raft formation, and immune signaling activation. NK cells isolated from HCD-fed Ldlr-/- mice did not have increased cholesterol content or cytotoxic activity against mouse hepatoma cells compared with ND-fed Ldlr-/- mice. Serum levels of cholesterol correlated with number and activity of NK cells isolated from human HCCs. Mice with increased serum levels of cholesterol due to an HCD or genetic disruption of ApoE develop fewer and smaller tumors after injection of hepatoma cells or a chemical carcinogen. We found cholesterol to accumulate in NK cells and activate their effector functions against hepatoma cells. Strategies to increase cholesterol uptake by NK cells can be developed for treatment of HCC.

Qianggan extract improved nonalcoholic steatohepatitis by modulating lncRNA/circRNA immune ceRNA networks

BackgroundThe traditional Chinese medicine prescription, Qianggan formula have been confirmed to be effective on non-alcoholic steatohepatitis (NASH), however, the underlying molecular mechanisms remain obscure.MethodsThirty-six male C57BL/6 mice were randomly divided into three groups: normal chow diet group; methionine-and-choline-deficient diet (MCD) group, and Qianggan extract (QG) intervention group (0.4 g/kg daily) that fed with MCD. The efficacy of QG was biochemically and histologically evaluated. The expression profiles of messenger ribonucleic acids (mRNAs), long non-coding RNAs (lncRNAs) and circular RNAs (circRNAs) were examined using microarray and verified by RT-qPCR.ResultsQG significantly improved the phenotypic characteristics of NASH, as serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and lactate dehydrogenase (LDH) levels and liver inflammatory cytokines were significantly decreased. By the cutoff of a 1.5-fold change and P < 0.05, 6193 mRNAs, 5692 lncRNAs and 4843 circRNAs were identified as differentially expressed between the MCD and normal groups, and 514 mRNAs, 1182 lncRNAs and 443 circRNAs were identified as differentially expressed between the QG and MCD groups. The intersections (244 mRNAs, 259 lncRNAs and 98 circRNAs) among the three groups were chosen for analysis. Gene Ontology (GO) terms and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment revealed that most overlapping mRNAs were related to immune functions such as natural-killer-cell-mediated cytotoxicity, intestinal immune network for IgA production, and T cell receptor signaling pathway. Pathway interactions, protein-protein interactions and molecular complex detection (MCODE) analysis identified numerous immune-related hub genes e.g. natural cytotoxicity triggering receptor 1(Ncr1), C-X-C motif chemokine ligand 9 (Cxcl9), Klra1, and Cd28. Finally, two lncRNAs (Sngh1 and Slc36a3os) and four circRNAs (circ_0009029, circ_0004572, circ_0009212 and circ_0009453) in competing endogenous RNA (ceRNA) networks were constructed by Cytoscape, and immune-related mRNAs (e.g., Cd28, Cd8a, Il15, and Klrk1) were involved in the ceRNA networks.ConclusionsLncRNA and circRNA-associated immune ceRNA networks might be the targets of QG in alleviating NASH, and our work may provide valuable clues for exploring the mechanisms underlying the effect of QG.

NCR- group 3 innate lymphoid cells orchestrate IL-23/IL-17 axis to promote hepatocellular carcinoma development.

BackgroundInnate lymphoid cells (ILCs) are a newly discovered family of immune cells that have similar cytokine-secreting profiles as T helper cell subsets. Although ILCs are critical for host defense against infections and tissue homeostasis, their roles in tumor development are not well established.MethodsWe studied the function of ILC3 cells in the liver for the development of hepatocellular carcinoma (HCC) in murine HCC models using flow cytometry, adoptive transfer, and in vitro functional assays.FindingsWe found that ILC3 lacking the natural cytotoxicity-triggering receptor (NCR−ILC3) promoted the development of HCC in response to interleukin 23 (IL-23). IL-23 serum level is elevated in HCC patients and its high expression is associated with poor clinical outcomes. We found that IL-23 could promote tumor development in murine HCC tumor models. IL-23 promoted the expansion of NCR−ILC3 and its differentiation from group 1 ILCs (ILC1s). Furthermore, NCR−ILC3 initiated IL-17 production upon IL-23 stimulation and directly inhibited CD8+ T cell immunity by promoting lymphocyte apoptosis and limiting their proliferation.InterpretationTogether, our findings suggest that NCR−ILC3 initiates the IL-17-rich immunosuppressive tumor microenvironment and promotes the development of HCC, thus may serve as a promising target for future cancer immunotherapy.FundThis work was supported by grants from National Natural Science Foundation of China (81471586, 81571556), the Priority Academic Program Development of Jiangsu Higher Education Institutions, the collaborative Innovation Center of Hematology, start-up grant from National University of Singapore, the Cancer Prevention and Research Institute of Texas CPRIT (RR180017), and the National Cancer Institute's Cancer Center Support (Core) Grant CA016672 (to The University of Texas MD Anderson Cancer Center).

Gene expression of the liver of vaccination-protected mice in response to early patent infections of Plasmodium chabaudi blood-stage malaria

BackgroundThe role of the liver for survival of blood-stage malaria is only poorly understood. In experimental blood-stage malaria with Plasmodium chabaudi, protective vaccination induces healing and, thus, survival of otherwise lethal infections. This model is appropriate to study the role of the liver in vaccination-induced survival of blood-stage malaria.MethodsFemale Balb/c mice were vaccinated with a non-infectious vaccine consisting of plasma membranes isolated in the form of erythrocyte ghosts from P. chabaudi-infected erythrocytes at week 3 and week 1 before infection with P. chabaudi blood-stage malaria. Gene expression microarrays and quantitative real-time PCR were used to investigate the response of the liver, in terms of expression of mRNA and long intergenic non-coding (linc)RNA, to vaccination-induced healing infections and lethal P. chabaudi malaria at early patency on day 4 post infection, when parasitized erythrocytes begin to appear in peripheral blood.ResultsIn vaccination-induced healing infections, 23 genes were identified to be induced in the liver by > tenfold at p < 0.01. More than one-third were genes known to be involved in erythropoiesis, such as Kel, Rhag, Ahsp, Ermap, Slc4a1, Cldn13 Gata1, and Gfi1b. Another group of > tenfold expressed genes include genes involved in natural cytotoxicity, such as those encoding killer cell lectin-like receptors Klrb1a, Klrc3, Klrd1, the natural cytotoxicity-triggering receptor 1 Ncr1, as well as the granzyme B encoding Gzmb. Additionally, a series of genes involved in the control of cell cycle and mitosis were identified: Ccnb1, Cdc25c, Ckap2l were expressed > tenfold only in vaccination-protected mice, and the expression of 22 genes was at least 100% higher in vaccination-protected mice than in non-vaccinated mice. Furthermore, distinct lincRNA species were changed by > threefold in livers of vaccination-protected mice, whereas lethal malaria induced different lincRNAs.ConclusionThe present data suggest that protective vaccination accelerates the malaria-induced occurrence of extramedullary erythropoiesis, generation of liver-resident cytotoxic cells, and regeneration from malaria-induced injury in the liver at early patency, which may be critical for final survival of otherwise lethal blood-stage malaria of P. chabaudi.

Candidate gene molecular markers as tools for analyzing genetic susceptibility to morbillivirus infection in stranded Cetaceans.

Morbilliviruses, such as Cetacean morbillivirus (CeMV) or Phocine distemper virus (PDV), represent a growing threat for marine mammals on both hemispheres. Because free-ranging animal populations strongly rely on natural resistance mechanisms, innate immunity-related genes and virus cell entry receptor genes may represent key factors involved in susceptibility to CeMV in Cetaceans. Using the next generation sequencing technology, we have sequenced 11 candidate genes in two model species, Stenella coeruleoalba and Phocoena phocoena. Suitable single nucleotide polymorphism markers of potential functional importance, located in genes coding for basigin (BSG, CD147), the signaling lymphocyte activating molecule (SLAMF1), the poliovirus-related receptor-4 (NECTIN4, PVRL4), toll-like receptors 3, 7, 8 (TLR3, TLR7, TLR8), natural resistance-associated macrophage protein (SLC11A1) and natural cytotoxicity triggering receptor 1 (NCR1), were identified in each model species, along with MHC-DQB haplotypes unique for each species. This set of molecular markers represents a potentially useful tool for studying host genetic variation and susceptibility to morbillivirus infection in Cetaceans as well as for studying functionally important genetic diversity of selected Cetacean populations.

Clinical significance of novel costimulatory molecule B7-H6 in human breast cancer.

B7 homolog 6 (B7-H6), a member of the B7 family, is as a cell-surface ligand for natural cytotoxicity triggering receptor 3, which is expressed on natural killer cells. It has previously been reported that B7-H6 is undetectable in normal human tissues but is expressed on tumor cells. However, there are few studies focusing on the clinical significance of B7-H6 expression in human carcinoma, with the exception of three studies on ovarian, lung and gastric cancer. The present study investigated the expression of B7-H6 protein in pathologic tissue samples from 305 patients with breast cancer using immunohistochemistry. A high B7-H6 expression level was identified in tissues from 32.13% of patients with breast cancer. These patients were revealed to also exhibit a high expression level of human epidermal growth factor receptor 2, a shorter survival time and a higher rate of lymph node metastasis. Furthermore, the expression level of B7-H6 was not associated with patient age, breast cancer subtype, tumor size, tumor location or estrogen receptor expression. The results of the present study revealed that higher B7-H6 expression level in breast cancer tissues was positively associated with tumor progression. This indicates that B7-H6 is associated with the progression and immunoevasion of human breast cancer; however, the molecular mechanisms underlying this potential effect require further investigation.

Human decidua mesenchymal stem cells regulate decidual natural killer cell function via interactions between collagen and leukocyte‑associated immunoglobulin‑like receptor 1.

The development of maternal tolerance to the fetal allograft in critical for the maintenance of the pregnancy, and it is accompanied by the development of a special decidual natural killer (dNK) cell tolerance phenotype. To understand the factors that influence dNK cells during early pregnancy, the present study aimed to identify mesenchymal stem cells (MSCs) from human first‑trimester deciduas, termed decidual MSCs (DMSCs), and to investigate the effect of DMSCs on the regulation of dNK cells via collagen. Decidual samples were collected from women with normal pregnancy that had undergone elective vaginal surgical terminations at 6‑9weeks gestation. DMSCs derived from human decidual tissues were cultured under differentiation conditions to examine their multipotent differentiation capacities, and the expression of MSC‑specific markers, including cluster of differentiation (CD)44, CD73, CD105, CD90, CD34, CD31, CD14, CD45, CD11b and human leukocyte antigen‑antigen D related, was determined. dNK cells were co‑cultured with DMSCs in order to examine the effect of DMSCs on the tolerance phenotype of dNK cells. The expression of cell surface molecules, natural cytotoxicity triggering receptor 3 and killer cell immunoglobulin‑like receptor (KIR) 2DL1, and the secretion of cytokines, including interferon‑γ, tumor necrosis factor (TNF)‑α, interleukin (IL)‑10, IL‑4 and perforin, were examined by flow cytometry analysis. To determine whether the regulation of dNK cells by DMSCs was mediated by collagen, DMSCs were pre‑treated with human recombinant leukocyte‑associated immunoglobulin‑like receptor (LAIR)‑2 and transfected with pScoR‑GFP‑hP4H to inhibit the interaction between LAIR‑1 and collagen. The present results demonstrated that collagen produced by DMSCs increased the expression of KIR2DL1 and IL‑4, decrease the expression of NKp30 and TNF‑α. In conclusion, the results of the present study demonstrated that DMSCs may be cultured invitro for prolonged periods, whilst retaining the ability to differentiate into different cell lineages. In addition, DMSCs may modulate the function of dNK cells via the interaction between collagen and LAIR‑1.

CD1d-mediated activation of group 3 innate lymphoid cells drives IL-22 production.

Innate lymphoid cells (ILCs) are a heterogeneous family of immune cells that play a critical role in a variety of immune processes including host defence against infection, wound healing and tissue repair. Whether these cells are involved in lipid‐dependent immunity remains unexplored. Here we show that murine ILCs from a variety of tissues express the lipid‐presenting molecule CD1d, with group 3 ILCs (ILC3s) showing the highest level of expression. Within the ILC3 family, natural cytotoxicity triggering receptor (NCR)− CCR6+ cells displayed the highest levels of CD1d. Expression of CD1d on ILCs is functionally relevant as ILC3s can acquire lipids in vitro and in vivo and load lipids on CD1d to mediate presentation to the T‐cell receptor of invariant natural killer T (iNKT) cells. Conversely, engagement of CD1d in vitro and administration of lipid antigen in vivo induce ILC3 activation and production of IL‐22. Taken together, our data expose a previously unappreciated role for ILCs in CD1d‐mediated immunity, which can modulate tissue homeostasis and inflammatory responses.

Conserved 33-kb haplotype in the MHC class III region regulates chronic arthritis

Genome-wide association studies have revealed many genetic loci associated with complex autoimmune diseases. In rheumatoid arthritis (RA), the MHC gene HLA-DRB1 is the strongest candidate predicting disease development. It has been suggested that other immune-regulating genes in the MHC contribute to the disease risk, but this contribution has been difficult to show because of the strong linkage disequilibrium within the MHC. We isolated genomic regions in the form of congenic fragments in rats to test whether there are additional susceptibility loci in the MHC. By both congenic mapping in inbred strains and SNP typing in wild rats, we identified a conserved, 33-kb large haplotype Ltab-Ncr3 in the MHC-III region, which regulates the onset, severity, and chronicity of arthritis. The Ltab-Ncr3 haplotype consists of five polymorphic immunoregulatory genes: Lta (lymphotoxin-α), Tnf, Ltb (lymphotoxin-β), Lst1 (leukocyte-specific transcript 1), and Ncr3 (natural cytotoxicity-triggering receptor 3). Significant correlation in the expression of the Ltab-Ncr3 genes suggests that interaction of these genes may be important in keeping these genes clustered together as a conserved haplotype. We studied the arthritis association and the spliceo-transcriptome of four different Ltab-Ncr3 haplotypes and showed that higher Ltb and Ncr3 expression, lower Lst1 expression, and the expression of a shorter splice variant of Lst1 correlate with reduced arthritis severity in rats. Interestingly, patients with mild RA also showed higher NCR3 expression and lower LST1 expression than patients with severe RA. These data demonstrate the importance of a conserved haplotype in the regulation of complex diseases such as arthritis.

CD69 and CXCR6 Identify a Distinct Population of Human Bone Marrow and Spleen Resident NK Cells

In recent years, evidence has been provided that natural killer (NK) cells can function as anti-leukemic effector cells. In humans, two NK cell populations are usually distinguished: CD56dimCD16+ NK cells form the predominant population in blood while the CD56brightCD16- NK cell population is more prominent in tissues. However, little data regarding tissue specific characteristics of human NK cells are available, especially in bone marrow as an important localization of leukemic cells. Therefore, we evaluated the expression of chemokine receptors and adhesion molecules on NK cells in healthy donor blood, bone marrow and spleen by flow cytometry.Besides the two conventional NK cell subsets, a major third NK cell population was identified in bone marrow and spleen based on the combined expression of CD69 and CXCR6 (Figure 1). CD69+CXCR6+ NK cells represented 9-50% (mean 30%) of NK cells in marrow (n=15) and 26-57% (mean 43%) of NK cells in spleen (n=7). This CD69+CXCR6+ population was not detected in blood (n=15) nor in cord blood, neither mobilized into the blood stream after G-CSF treatment. The ratio and phenotype of the remaining conventional CD56bright and CD56dim NK cells in bone marrow and spleen were comparable to blood. Early after pediatric hematopoietic stem cell transplantation, CD69+CXCR6+ NK cells were absent in bone marrow, but gradually reached normal levels within the first year after transplantation.CD56 was expressed on marrow and spleen CD69+CXCR6+ NK cells at slightly lower levels than the conventional CD56bright NK cells and CD16 was expressed by 7-30% of CD69+CXCR6+ NK cells. CD69+CXCR6+ NK cells expressed high levels of the adhesion molecule CD54 (ICAM-1) as well as natural cytotoxicity triggering receptor NKp46 compared to the conventional CD56bright and CD56dim NK cell populations. CD69+CXCR6+ NK cells did not express the early differentiation markers CD117 (c-kit) and CD127 (IL7Rα), which are expressed by immature NK cells, type III innate lymphoid cells and, to some extent, by conventional CD56bright NK cells. The inhibitory receptor NKG2A, which is acquired early in maturation but lost during the differentiation of CD56dim NK cells, was expressed by 60% of CD69+CXCR6+ NK cells. Furthermore, CD69+CXCR6+ NK cells did not express markers acquired late in differentiation (KIRs, CD57, KLRG1, NKG2C).In functional experiments assessing cytokine producing capacity, CD69+CXCR6+ NK cells were comparable to the CD56dim subset, requiring the combined stimulation with IL12+IL15+IL18 to produce IFN-γ. However, with respect to cytotoxic potential, CD69+CXCR6+ NK cells more resembled the CD56bright NK cell subset; in resting state, CD69+CXCR6+ NK cells expressed perforin but not granzyme B, which was upregulated during overnight IL12+IL15 stimulation. Upon co-culture with K562 tumor cells, CD69+CXCR6+ NK cells degranulated (CD107a) at levels comparable to the conventional CD56bright and CD56dim NK cells.In summary, we identified a distinct NK cell population in human bone marrow and spleen. These cells were a) absent in blood, b) expressed tissue retention marker CD69, and c) were present alongside the conventional NK cell subsets in marrow and spleen. Together, these findings indicate that NK cells with the discriminative CD69+CXCR6+ phenotype constitute a tissue resident NK cell subset. Furthermore, CD69+CXCR6+ NK cells did not express CD49a+, distinguishing them from the recently described liver resident NK cells (Marquardt et al, 2015). Based on their surface receptor expression profile and functional characteristics, CD69+CXCR6+ NK cells have a mature signature that differs from both conventional NK cell subsets. Additional studies in healthy individuals and patients with immuno-hematological diseases are needed to investigate the immunological function of CD69+CXCR6+ NK cells. For example, these cells could have a local immunoregulatory or effector function. Alternatively, bone marrow and spleen may form a reservoir for effector cells waiting to be released into the blood stream. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Type I Interferons Protect T Cells against NK Cell Attack Mediated by the Activating Receptor NCR1

Direct type I interferon (IFN) signaling on Tcells is necessary for the proper expansion, differentiation, and survival of responding Tcells following infection with viruses prominently inducing type I IFN. The reasons for the abortive response of Tcells lacking the type I IFN receptor (Ifnar1(-/-)) remain unclear. We report here that Ifnar1(-/-) Tcells were highly susceptible to natural killer (NK) cell-mediated killing in a perforin-dependent manner. Depletion of NK cells prior to lymphocytic choriomeningitis virus (LCMV) infection completely restored the early expansion of Ifnar1(-/-) Tcells. Ifnar1(-/-) Tcells had elevated expression of natural cytotoxicity triggering receptor 1 (NCR1) ligands upon infection, rendering them targets for NCR1 mediated NK cell attack. Thus, direct sensing of type I IFNs by Tcells protects them from NK cell killing by regulating the expression of NCR1 ligands, thereby revealing a mechanism by which Tcells can evade the potent cytotoxic activity of NK cells.

NCR2 (natural cytotoxicity triggering receptor 2)

Review on NCR2 (natural cytotoxicity triggering receptor 2), with data on DNA, on the protein encoded, and where the gene is implicated.

Altered endometrial immune gene expression in beef heifers with retarded embryos

The aim of the present study was to compare endometrial gene expression profiles in a group of beef heifers yielding viable or retarded embryos on Day 7 after oestrus as a means of potentially explaining differences in embryo survival rates. Heifers were classified as either: (1) viable, when the embryo collected on Day 7 after oestrus was at the correct developmental stage (i.e. morula/early blastocyst); or (2) retarded, when the embryo was arrested at the 2-16-cell stage. The focus of the present study was on genes that were associated with either the pro- or anti-inflammatory immune response. Endometrial gene expression was determined using quantitative real-time polymerase chain reaction analysis. Expression of the β-defensin (DEFB1), interferon (IFN)-α (IFNA), IFN-γ (IFNG), interleukin (IL)-6 (IL6), IL-10 (IL10), forkhead box P3 (FOXP3) and natural cytotoxicity triggering receptor 1 (NCR1) genes was lower in endometria from viable than retarded heifers. Expression of the nuclear factor of kappa light polypeptide gene enhancer in B cells 1 (NKFB1), transforming growth factor (TGF)-β (TGFB), IFN-γ-inducible protein 16 (IFI16) and IL-21 (IL21) genes was higher in viable than retarded heifers. We propose that small disturbances in the expression of immune genes in the endometrium on Day 7 after oestrus can have detrimental effects on embryo survival.