Cytotoxicity Triggering Receptor Research Articles

Plasma Gelsolin Inhibits Natural Killer Cell Function and Confers Chemoresistance in Epithelial Ovarian Cancer.

Plasma gelsolin (pGSN) overexpression in ovarian cancer (OVCA) disarms immune function, contributing to chemoresistance. The aim of this study was to investigate the immunoregulatory effects of pGSN expression on natural killer (NK) cell function in OVCA. OVCA tissues from primary surgeries underwent immunofluorescent staining of pGSN and the activated NK cell marker natural cytotoxicity triggering receptor 1 to analyze the prognostic impact of pGSN expression and activated NK cell infiltration. The immunoregulatory effects of pGSN on NK cells were assessed using apoptosis assay, cytokine secretion, immune checkpoint-receptor expression, and phosphorylation of STAT3. In OVCA tissue analyses, activated NK cell infiltration provided survival advantages to patients. However, high pGSN expression attenuated the survival benefits of activated NK cell infiltration. In the in vitro experiment, pGSN in OVCA cells induced NK cell death through cell-to-cell contact. pGSN increased T-cell immunoglobulin and mucin-domain-containing-3 expression (TIM-3) on activated NK cells. Further, it decreased interferon-γ production in activated TIM-3+ NK cells, attenuating their anti-tumor effects. Thus, increased pGSN expression suppresses the anti-tumor functions of NK cells. The study provides insights into why immunotherapy is rarely effective in patients with OVCA and suggests novel treatment strategies.

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.

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.

B7H6 Serves as a Negative Prognostic Marker and an Immune Modulator in Human Pancreatic Cancer.

Pancreatic cancer (PC), the third leading cause of cancer-related death in the U.S., is frequently found too late to be cured by traditional chemotherapy. Expression of B7 homolog 6 (B7H6), a member of the B7 family of immunoreceptors, has been found in PC and several other cancers. B7H6 is a ligand for cytotoxicity triggering receptor 3 (NKp30), which is expressed on NK cells. Here, we demonstrate that B7H6 can be detected in PC tissues but not normal organs. Its expression in patients associated significantly with tumor differentiation grade and lymphatic metastasis. The soluble form of B7H6 was detected in the PC patients’ sera, and its concentration associated with tumor differentiation grade and tumor, node, metastasis (TNM) stages. Also, higher levels of B7H6 in PC patients’ malignant tissues or serum correlated with shorter overall survival. In vitro, downregulation of B7H6 by CRISPR/Cas9 or siRNA technology had no significant impact on the viability or mobility of PC cells. Instead, knocking out B7H6 sensitized PC cells to NK-mediated cytotoxicity and cytokine production. These results indicate that B7H6 not only serves as a negative prognostic marker but also acts as an immune modulator in PC.

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.

Dual role of B7-H6 as a novel prognostic marker in hepatocellular carcinoma.

B7 homolog 6 (B7-H6), a new member of the B7 family, is identified as an activating ligand for cytotoxicity triggering receptor 3 (NKp30) expressing on natural killer cells. The purpose of this study was to investigate the clinical significance of B7-H6 in hepatocellular carcinoma (HCC). We evaluated B7-H6 expression by immunohistochemistry in a cohort of 90 HCC tumors with clinical follow-up, the potential relationship between the B7-H6 expression and the clinicopathological characteristics of HCC patients was also analyzed. Stable B7-H6 knockdown in hepatoma cell line was established to explore the function and mechanism of B7-H6 in HCC. This study showed that high expression of B7-H6 was significantly associated with smaller tumor size, single tumor number in HCC, but no significant association was found between B7-H6 overexpression and other clinicopathological parameters. Moreover, Kaplan-Meier survival analysis showed that high expression of B7-H6 was significantly correlated with better survival of HCC patients. Knockdown of B7-H6 inhibited tumor cell proliferation and induced cell apoptosis. However, it also impaired the sensitivityof tumor cells to NK-mediated lysis together with significantly decreased degranulation and IFN-γ release of NK cells. These results indicated that B7-H6 has a dual role in HCC. It could be an independent indicator for better survival of HCC and maybe a potential target for future cancer treatment.

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.

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.

Chronic Myeloid Leukemia Patients with Deep Molecular Responses to Tyrosine Kinase Inhibitors Have Increased Effector Natural Killer and Cytotoxic T Cell Immune Responses to Leukaemia-Associated Antigens and Concomitant Reduced Immune Suppressors

We hypothesized that immune responses contribute to deep BCR-ABL molecular responses in chronic phase chronic myeloid leukaemia (CML) patients on tyrosine kinase inhibitors (TKI). We studied 32 CML patients; 16 at diagnosis, patients treated with imatinib (n=20), nilotinib (n=9) or dasatinib (n=3).Methodology: The effector immune responses of Natural Killer (NK) cells were characterized by flowcytometry and functional analysis by CD107a degranulation assay. Cytotoxic T lymphocyte (CTL) responses to leukaemia-associated antigens (LAAs) WT1, BMI-1, PR3 and PRAME were quantified by interferon-gamma ELISPOT using peptide libraries of 15-mer peptides overlapping by 11 amino acids spanning the entire protein, or HLA-A0201 specific peptides in HLA-A0201+ patients. Immune suppressor regulatory T cells (Treg), Myeloid Derived Suppressor Cells (MDSC), Programmed cell death-1 (PD-1) expression on T cells, NK cells, B cells and monocytes, and major B cell subsets were extensively characterized by flowcytometry.Results: Patients in deep molecular response (MR4.5; BCR-ABL <0.0032%) displayed increased antigen-specific CTL responses to LAAs, both in the number of positive LAAs and frequency of responses, compared to patients at diagnosis and major molecular response (MMR; BCR-ABL <0.1%). The most abundant LAA response was to PRAME [51% of patients in MR4.5 compared to 31% in MMR and 0% at diagnosis] and WT1 [31% of patients in MR4.5 compared to 28% in MMR and 0% at diagnosis]. PR3-specific immune responses were the least abundant, with no difference in response between MR4.5and MMR (both 3%) compared to 0% at diagnosis.Immunophenotypic analysis revealed a shift toward a more mature, cytolytic NK cell phenotype (CD57+, CD161+CD62L-) in MMR and MR4.5, consistent with up-regulation of the CD94/NKG2 family of inhibitory/activating receptors (NKG2A, NKG2C and NKG2D), the cytotoxicity triggering receptor NKp46 and a functional increase in NK cell cytotoxicity capacity against K562 target cells. The percentage of CD3-CD56dimCD16bright cytolytic NK cells as a proportion of total lymphocytes was significantly increased in MMR and MR4.5 [33.6% ± 6.6 p=0.0008 and 33.1% ± 4.1 p=0.01, respectively] compared to 7.8% ± 2.8 at diagnosis.The absolute Treg number/µl was significantly lower in patients in MMR and MR4.5 [13.9 ± 1.7 and 10 ± 1.1, respectively] compared to 32.7 ± 4.4 at diagnosis. Similarly, MDSC were significantly reduced in patients in MMR and MR4.5 [3.9 ± 0.9 and 1.9 ± 0.5 MDSC/µl] compared to diagnosis [18.3 ± 3.9]. A predominantly granulocytic (CD66b+CD15+) MDSC phenotype was seen in CML patients at diagnosis. PD-1 expression as a proportion of total lymphocytes was significantly decreased in cytotoxic CD8+ T cells in MR4.5 [5.7% ± 1.2] compared to MMR [12.3% ± 2.0, p=0.008] and patients at diagnosis [21.7% ± 5.2, p=0.0003]. PD-1 expression was decreased in CD4+ helper T cells in MR4.5 [7.5% ± 1.7] compared to MMR [11.4% ± 1.5, p=0.07] and diagnosis [17% ± 2.9, p=0.008]. Overall, PD-1 expression was lower in NK cells in CML patients, albeit significant in MMR and MR4.5 [0.24% ± 0.09, p=0.006 and 0.36 ± 0.07, p=0.02, respectively] compared to [1.42% ± 0.4] at diagnosis. No difference in PD-1 expression was seen in B cells or monocytes.No significant difference was observed in CD3-CD19+ B cells in MMR and MR4.5 or at diagnosis. Analysis of major B cell subsets revealed no difference in the proportion of transitional, naïve or memory B cells, plasma blasts or plasma cells.Conclusion: Enhanced effector immune responses of NK and LAA-specific CTLs are associated with concomitant reduction in immune suppressor activity, and may increase the rate of deep molecular responses to TKIs in CML. Methods to augment these responses may result in greater rate of success in TKI cessation studies. DisclosuresHughes:ARIAD: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Yong:Novartis: Honoraria, Research Funding.

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.

Innate Immunity in multiple sclerosis white matter lesions: expression of natural cytotoxicity triggering receptor 1 (NCR1)

BackgroundPathogenic or regulatory effects of natural killer (NK) cells are implicated in many autoimmune diseases, but evidence in multiple sclerosis (MS) and its murine models remains equivocal. In an effort to illuminate this, we have here analysed expression of the prototypic NK cell marker, NCR1 (natural cytotoxicity triggering receptor; NKp46; CD335), an activating receptor expressed by virtually all NK cells and therefore considered a pan-marker for NK cells. The only definitive ligand of NCR1 is influenza haemagglutinin, though there are believed to be others. In this study, we investigated whether there were differences in NCR1+ cells in the peripheral blood of MS patients and whether NCR1+ cells are present in white matter lesions.ResultsWe first investigated the expression of NCR1 on peripheral blood mononuclear cells and found no significant difference between healthy controls and MS patients. We then investigated mRNA levels in central nervous system (CNS) tissue from MS patients: NCR1 transcripts were increased more than 5 times in active disease lesions. However when we performed immunohistochemical staining of this tissue, few NCR1+ NK cells were identified. Rather, the major part of NCR1 expression was localised to astrocytes, and was considerably more pronounced in MS patients than controls. In order to further validate de novo expression of NCR1 in astrocytes, we used an in vitro staining of the human astrocytoma U251 cell line grown to model whether cell stress could be associated with expression of NCR1. We found up-regulation of NCR1 expression in U251 cells at both the mRNA and protein levels.ConclusionsThe data presented here show very limited expression of NCR1+ NK cells in MS lesions, the majority of NCR1 expression being accounted for by expression on astrocytes. This is compatible with a role of this cell-type and NCR1 ligand/receptor interactions in the innate immune response in the CNS in MS patients. This is the first report of NCR1 expression on astrocytes in MS tissue: it will now be important to unravel the nature of cellular interactions and signalling mediated through innate receptor expression on astrocytes.