Natural Killer Group 2 Member D Expression Research Articles

Effect of Spatholobi Caulis extract from ethyl acetate on immune killing function of NK cells

This study aims to investigate the regulatory effect of the Spatholobi Caulis extract from ethyl acetate(SEA) on natural killer(NK) cells under physiological conditions and elucidate the underlying mechanism. The C57BL/6 mice were randomized into NC and SEA groups, and NK-92 cells were respectively treated with 0, 25, 50, and 100 μg·mL~(-1) SEA. The body weight and immune organ index of the mice were compared between groups. The lactate dehydrogenase(LDH) assay was employed to examine the cytotoxicity of NK-92 cells treated with SEA and the killing activity of mouse NK cells against YAC-1 cells. The cell-counting kit-8(CCK-8) was used to examine the impact of SEA on the proliferation of NK-92 cells. Flow cytometry was employed to measure the number of NK cells in the peripheral blood as well as the expression levels of natural killer group 2 member A(NKG2A) and natural killer group 2 member D(NKG2D). The enzyme-linked immunosorbent assay(ELISA) was performed to determine the interferon(IFN)-γ secretion in the serum. Semi-quantitative PCR was conducted to determine the mRNA levels of NKG2A, NKG2D, and IFN-γ in spleen cells. Western blot was employed to investigate the involvement of phosphoinositide 3-kinase(PI3K)/extracellular regulated protein kinase 1(ERK1) signaling pathway. The results showed that SEA exhibited no adverse effects on the body, while significantly enhance the number of NK cells and augment the cytotoxicity of NK-92 cells against YAC-1 cells. Moreover, it suppressed the expression of NKG2A, enhanced the expression of NKG2D, promoted IFN-γ secretion, and upregulated the protein levels of PI3K and ERK. The findings suggest that SEA has the potential to enhance the immune recognition and effector function of NK cells by increasing the cell number, modulating the expression of functional receptors, and promoting IFN-γ secretion via the PI3K/ERK signaling pathway.

IL-17a-producing γδT cells and NKG2D signaling mediate bacterial endotoxin-induced neonatal lung injury: implications for bronchopulmonary dysplasia.

Bronchopulmonary dysplasia (BPD) is a chronic lung disease in preterm birth survivors characterized by inflammation, impaired alveolarization and dysmorphic vasculature. Activated IL-17A+ lymphocytes are key drivers of inflammation in preterm infants. We have shown that in immature mice chronic airway exposure to lipopolysaccharide (LPS) induces pulmonary inflammation, increased IL-17a expression, and hypoalveolarization, a BPD-like phenotype. The source of IL-17a and contribution to lung pathology is unknown. The natural-killer group 2, member D (NKG2D) receptor mediates activation and IL-17a production in γδ T cells by binding to stress molecules. LPS induces NKG2D ligand expression, including Rae-1 and MULT1. We hypothesized that IL-17a+ γδ T cells and NKG2D signaling mediate neonatal LPS-induced lung injury. Immature C57BL/6J (wild type), Nkg2d-/- or Tcrd-/- (lacking γδ T cells) mice were inoculated with 3ug/10ul of LPS from E. coli O26:B6 or 10ul of PBS intranasally on day of life 3, 5, 7, and 10. Selected mice were treated with neutralizing antibodies against IL-17a, or NKG2D intraperitoneally. Lung immune cells were assessed by flow cytometry and gene expression was analyzed by qPCR. Alveolar growth was assessed by lung morphometry. We established that anti-IL-17a antibody treatment attenuated LPS-induced hypoalveolarization. We found that LPS induced the fraction of IL-17a+NKG2D+ γδ T cells, a major source of IL-17a in the neonatal lung. LPS also induced lung mRNA expression of NKG2D, Rae-1, MULT1, and the DNA damage regulator p53. Anti-NKG2D treatment attenuated the effect of LPS on γδ T cell IL-17a expression, immune cell infiltration and hypoalveolarization. LPS-induced hypoalveolarization was also attenuated in Nkg2d-/- and Tcrd-/- mice. In tracheal aspirates of preterm infants IL-17A and its upstream regulator IL-23 were higher in infants who later developed BPD. Also, human ligands of NKG2D, MICA and MICB were present in the aspirates and MICA correlated with median FiO2. Our novel findings demonstrate a central role for activated IL-17a+ γδ T cells and NKG2D signaling in neonatal LPS-induced lung injury. Future studies will determine the role of NKG2D ligands and effectors, other NKG2D+ cells in early-life endotoxin-induced lung injury and inflammation with a long-term goal to understand how inflammation contributes to BPD pathogenesis.

Down-regulation of PR/SET domain 10 underlies natural killer cell dysfunction in hepatocellular carcinoma.

Hepatocellular carcinoma (HCC) is the world's leading cause of tumor-related mortalities. Natural killer (NK) cells play a critical role at the first immunological defense line against HCC initiation and progression. NK cell dysfunction is therefore an important mechanism for immune evasion of HCC cells. In the present study using a murine HCC model, we revealed the down-regulation of PR/SET Domain 10 (PRDM10) in hepatic NK cells that were phenotypically and functionally exhausted. PRDM10 silencing diminished the expression of natural killer group 2 member D (NKG2D) and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), augmented Tcellimmunoglobulin and ITIM domain (TIGIT) expression, and decreased the expression of interferon (IFN)-γ, perforin and granzyme B in normal hepatic NK cells in vitro. Consistently, PRDM10-deficient NK cells exhibited impaired cytotoxicity on target cells. In contrast, PRDM10 over-expression promoted NKG2D and Fas ligand(FasL) expression, reduced CD96 expression and enhanced transcripts of IFN-γ, perforin and granzyme B in NK cells in vivo. Moreover, PRDM10 silencing and PRDM10 over-expression down-regulated and up-regulated Eomesodermin (Eomes) expression, respectively. In summary, this study reveals PRDM10 down-regulation as a novel mechanism underlying NK cell dysfunction and identifies PRDM10 as a supporting factor of NK cell function.

Erroneous expression of NKG2D on granulocytes detected by phycoerythrin-conjugated clone 149810 antibody.

The activating Natural killer group 2 member D (NKG2D) receptor is typically expressed on NK cells, CD8 T lymphocytes, γδ T cells and small subsets of CD4 T lymphocytes. During the course of an extensive flow cytometry phenotyping of immune cells in the peripheral blood of patients with glioblastoma multiforme (GBM) we noticed an unexpected expression of NKG2D receptor on granulocytes using the phycoerythrin (PE)-conjugated clone 149810 antibody. Peripheral blood samples from 35 patients with GBM and 22 age-matched healthy control (HC) donors were analyzed using flow cytometry, imaging cytometry and real-time quantitative reverse transcription PCR to validate the observed expression of NKG2D receptor on myeloid cells. Reactivity with PE-149810 was mostly observed on granulocytes from GBM patients on dexamethasone treatment where it correlated with inferior survival rates. Surprisingly, such NKG2D expression on granulocytes was not observed using the allophycocyanin (APC)-conjugate of the same clone 149810 antibody or an indirect staining procedure with unconjugated clone 149810 antibody. Moreover, the PE-conjugate of a different anti-NKG2D clone (1D11) also did not stain granulocytes. Imaging cytometry indicated cell surface and intracellular localization of PE-149810 but not of PE-1D11 in granulocytes. Our results uncover an erroneous and false positive reactivity of PE-labeled (but not of APC-labeled or unconjugated) anti-NKG2D antibody 149810 on granulocytes from dexamethasone-treated GBM patients and raise a note of caution for studies of NKG2D expression on non-lymphoid cells.

The killing effect of Tanshinol on breast cancer cells: insight into the reversion of TGF-𝜷1-mediated suppression of NK cell functions

Background: Natural killer (NK) cells play an indispensable role in anti-tumor immunity. TGF-β1 is the main accomplice of tumor immune escape, inhibiting tumor immunity mediated by NK cells. It is reported that Salvia miltiorrhiza can promote the immune killing effect of NK cells. In this study, Tanshinol, a water-soluble active component of Salvia miltiorrhiza, was used to investigate its effect on the inhibition of NK cell functions mediated by TGF-β1 in breast cancer. Methods: We constructed a mouse model of breast cancer by tail vein injection, H&E staining and ELISA were used to verify the role of TGF-β1 and the effects of Tanshinol on breast cancer and NK cells. In vitro, we used CCK8 and cytotoxicity assays to preliminarily evaluate the effect of Tanshinol on the anti-tumor effect of NK cells intervention by TGF-β1. We explored the killing activity of NK cells and related signal pathways by immunofluorescence imaging technology, RT-PCR, ELISA and flow cytometry. Also, Western blot, RT-PCR and immunofluorescence experiments were applied to investigate the expression level of the natural killer group 2 member D (NKG2D)-NKG2D ligands (NKG2DL) signal axis, and combined with immunoprecipitation, to detect the formation of NKG2D-DNAX-activating protein of 10 kD (DAP10) complex. Results: TGF-β1 played a role in promoting lung metastasis of breast cancer and inhibiting the secretion of cytotoxic mediators from NK cells, but Tanshinol could reverse it. High-dose Tanshinol also significantly optimized the survival rate of tumor-bearing mice. TGF-β1 could destroy the NKG2D-NKG2DL axis, down-regulate the expression and nuclear accumulation of p-smad2/3. Moreover, TGF-β1 inhibited the activation of PI3K-ERK1/2-PLCγ2 signaling pathway that is related to the degranulation of NK cells, and diminished the expression of degranulation marker CD107a and the release of anti-tumor cytotoxic killing medium of NK cells. However, Tanshinol was able to interfere with the negative regulation of TGF-β1 on the functions of NK cells, mainly through promoting the expression of NKG2D and its molecular chaperone DAP10, thereby propelling the formation of NKG2D-DAP10 complex. Conclusions: Collectively, Tanshinol enables NK cells to activate and release multiple killing mediators to carry out immune attacks on tumor cells.

An Anti-MICA/B Antibody and IL-15 Rescue Altered NKG2D-Dependent NK Cell Responses in Hepatocellular Carcinoma.

Simple SummaryPrimary liver cancer is one of the most common causes of cancer-related deaths worldwide. The present study shows that the cytokine IL-15 and a humanized antibody specific for a ligand recognized by a major activating receptor (NKG2D) expressed by a subset of lymphocytes named natural killer (NK) cells can restore dysfunctional NK cell responses in patients affected by the most common liver cancer hepatocellular carcinoma. This study emphasizes the importance of NK cells for immune responses to hepatocellular carcinoma and identifies the NKG2D activating receptor/ligand axis as a possible target for immunotherapeutic interventions. More importantly, building upon previously published studies, these findings provide a basis for the future treatment of tumors unresponsive to current therapies.Natural killer (NK) cells play a pivotal role in cancer immune surveillance, and activating the receptor/ligand interaction may contribute to control the development and evolution of hepatocellular carcinoma (HCC). We investigated the role of the natural killer group 2 member D (NKG2D) activating receptor and its ligand, the major histocompatibility complex class I chain-related protein A and B (MICA/B) in patients with cirrhosis and HCC subjected to surgical resection, patients with cirrhosis and no HCC, and healthy donors (HD). The NKG2D-mediated function was determined in peripheral blood (PB), in tumor-infiltrating lymphocytes (NK-TIL), and in matched surrounding liver tissue (NK-LIL). A group of patients treated with sorafenib because of clinically advanced HCC was also studied. A humanized anti-MICA/B monoclonal antibody (mAb) was used in in vitro experiments to examine NK cell-mediated antibody-dependent cellular cytotoxicity. Serum concentrations of soluble MICA/B were evaluated by ELISA. IL-15 stimulation increased NKG2D-dependent activity which, however, remained dysfunctional in PB NK cells from HCC patients, in line with the reduced NKG2D expression on NK cells. NK-TIL showed a lower degranulation ability than NK-LIL, which was restored by IL-15 stimulation. Moreover, in vitro IL-15 stimulation enhanced degranulation and interferon-γ production by PB NK from patients at month one of treatment with sorafenib. Anti-MICA/B mAb associated with IL-15 was able to induce PB NK cytotoxicity for primary HCC cells in HD and patients with HCC, who also showed NK-TIL degranulation for autologous primary HCC cells. Our findings highlight the key role of the NKG2D-MICA/B axis in the regulation of NK cell responses in HCC and provide evidence in support of a potentially important role of anti-MICA/B mAb and IL-15 stimulation in HCC immunotherapy.

Down-regulation of the human major histocompatibility complex class I chain-related gene A (MICA) and its receptor is mediated by microRNA-146b-5p and is a potential mechanism of immunoediting in papillary thyroid carcinoma

Immune escape is one of the main reasons for the rapid progression of cancer and the poor efficacy of immunotherapy. Papillary thyroid cancer (PTC) is usually accompanied by intra-tumoral lymphocytic infiltration. The mechanisms regulating this tumor associated immune response or its evasion are not well understood. The major histocompatibility complex class I chain-related proteins A (MICA) and its receptor the natural killer group 2 member D (NKG2D) are major executers of the anti-tumor defense. This work aimed to study the expression and regulation of MICA-NKG2D and its association with the lymphocytic infiltration and miRNAs in PTC. Expression of MICA and NKG2D in thyroid tissues, and in cultured primary thyroid cancer cells and lymphocytes transfected with miR-146b-5p inhibitor/mimic was tested by RT-PCR. Results were confirmed by immunofluorescence staining and confocal microscopy. MICA is expressed in malignant and benign thyroid tissues with no association with aggressive behavior. Expression of MICA and NKG2D in PTC is concomitant with the presence of tumor associated lymphocytic response and is regulated by miR-146b-5p. MiR-146b-5p indirectly downregulates NKG2D expression in cancer cells and in lymphocytes. Overexpression of miR-146b-5p in PTC down-regulates MICA expression possibly to reduce the immunogenicity of the tumor cells. Targeting of the MICA-NKG2D axis by miR-146b-5p might be one of the ways adopted by thyroid cancer cells to aid the tumor in evading the immune response. The importance of our findings resides in the potential therapeutic use of MICA, NKG2D and miRNA-146b-5p as targets or modulators to enable the immune response against cancer.

Splenic NKG2D confers resilience versus susceptibility in mice after chronic social defeat stress: beneficial effects of (R)-ketamine

The spleen is a large immune organ that plays a key role in the immune system. The precise molecular mechanisms underlying the relationship between the spleen and stress-related psychiatric disorders are unknown. Here we investigated the role of spleen in stress-related psychiatric disorders. FACS analysis was applied to determine the contribution of the spleen to susceptibility and resilience in mice that were subjected to chronic social defeat stress (CSDS). We found a notable increase in splenic volume and weight in CSDS-susceptible mice compared to control (no CSDS) mice and CSDS-resilient mice. The number of granulocytes, but not of T cells and B cells, in the spleen of susceptible mice was higher than in the spleen of both control and resilient mice. Interestingly, NKG2D (natural killer group 2, member D) expression in the spleen of CSDS-susceptible mice was higher than that in control mice and CSDS-resilient mice. In addition, NKG2D expression in the spleen of patients with depression was higher than that in controls. Both increased splenic weight and increased splenic NKG2D expression in CSDS-susceptible mice were ameliorated after a subsequent administration of (R)-ketamine. The present findings indicate a novel role of splenic NKG2D in stress susceptibility versus resilience in mice subjected to CSDS. Furthermore, abnormalities in splenic functions in CSDS-susceptible mice were ameliorated after subsequent injection of (R)-ketamine. Thus, the brain–spleen axis might, at least in part, contribute to the pathogenesis of stress-related psychiatric disorders such as depression.

Blockade of NKG2D/NKG2D ligand interaction attenuated cardiac remodelling after myocardial infarction.

Accumulating evidence demonstrates that cardiomyocyte death contributes to the onset and progression of heart failure (HF) after myocardial injury. Recent studies revealed that immune/inflammatory reactions play important roles in cardiovascular diseases. However, it remains unclear whether immunosurveillance system, which eliminates cytopathic cells, including infected or malignant cancer cells, is involved in cardiomyocyte death, though cardiomyocytes are exposed to pathological stresses during post-infarct remodelling. The aim of this study is to clarify the pathophysiological significance of Natural Killer Group 2 member D (NKG2D)/NKG2D ligand (NKG2DL)-mediated cell death in HF after myocardial infarction (MI). MI was generated by ligating left anterior descending artery in mice. The expression of NKG2D, NKG2DLs, especially Retinoic acid early induced transcript-1ɛ (Rae-1ɛ), perforin and granzyme B was concomitantly up-regulated after MI. Immunohistological analysis revealed that Rae-1 was expressed on the membranes of injured cardiomyocytes in the infarct and border area. The MI-induced increase of Rae-1 expression was suppressed in p53-/- mice and Rae-1 was induced by the overexpression of p53. We identified p53-binding sites in Rae-1ɛ gene promoter, by chromatin immunoprecipitation assay, indicating that Rae-1 expression was mediated partially through p53. Flow cytometric analysis indicated that NKG2D-expressing immune cells in the post-infarct myocardium were mainly γδT cells. The co-culture with γδT cells increased the frequency of apoptotic cells in the cultured cardiomyocytes. The blockade of NKG2D/NKG2DL interaction by intraperitoneal injection of anti-Rae-1ɛ antibody after MI reduced the frequency of apoptotic cardiomyocytes, accompanied by suppression of cardiac fibrosis, attenuating cardiac dysfunction. Finally, tamoxifen-inducible cardiomyocyte-specific Rae-1ɛ overexpressing mice exhibited the susceptibility to post-infarct remodelling with increased cardiomyocyte apoptosis and severer cardiac dysfunction. The interaction between immune cells and cardiomyocytes via NKG2D/NKG2DL induces cardiomyocyte death, exacerbating cardiac remodelling after MI. The blockade of NKG2D/NKG2DL interaction could be a promising therapeutic strategy against HF.

Functional Characterisation and Analysis of the Soluble NKG2D Ligand Repertoire Detected in Umbilical Cord Blood Plasma.

We previously reported that cord blood plasma (CBP) contains significantly more soluble NKG2D ligands (sNKG2DLs), such as sMICB and sULBP1, than healthy adult plasma. Viral infection or malignant transformation upregulates expression of NKG2D ligand on affected cells, leading to NK group 2, member D (NKG2D)-mediated natural killer (NK) cell lysis. Conversely, sNKG2DL engagement of NKG2D decreases NK cell cytotoxicity leading to viral or tumour immune escape. We hypothesised that sNKG2DLs detected in CBP may represent an additional fetal–maternal tolerance mechanism. To further understand the role of sNKG2DL in pregnancy and individual contributions of the various ligand types, we carried out functional analysis using 181 CBP samples. To test the ability of CBP to suppress the function of NK cells in vitro, we measured expression of NKG2D, CD107a, and IFN-γ in NK cells from control donors after exposure to 181 individual CBP samples and characterised the sMICA, sMICB, and sULBP1 content of each one. Furthermore, to detect possible allelic differences between samples that may also affect function, we carried out umbilical cord blood typing for MHC class I-related chain A (MICA) and MHC class I-related chain B (MICB) coding and promoter allelic types. Strongest functional correlations related to increasing concentration of exosomal sULBP1, which was present in all CBP samples tested. In addition, common MICB alleles, such as MICB*005:02, resulted in increased concentration of sMICB. Interestingly, MICB*005:02 uniquely associated with eight different promoter types. Among promoter polymorphisms, P2 resulted in the highest expression of sMICB and P9 the least and was confirmed using luciferase reporter assays. Higher levels of sMICB associated with lower IFN-γ production, indicating that sMICB also suppressed NK cell function. We also examined the MICA functional dimorphism encoding methionine (met) or valine (val) at residue 129 associated with strong or weak NKG2D binding, respectively. Most sMICA associated with val/val, some with met/val but none with met/met and, counter-intuitively, the presence of sMICA in CBP increased NK cell cytotoxicity. We propose a model for fetal–maternal tolerance, whereby NK cell activity is limited by sULBP1 and sMICB in CBP. The release of 129val sMICA with weak NKG2D signalling may reduce the overall net suppressive signal and break tolerance thus allowing fetal NK cells to overcome immunological threats in utero.

The NKG2D/NKG2DL Axis in the Crosstalk Between Lymphoid and Myeloid Cells in Health and Disease

Natural killer group 2, member D (NKG2D) receptor is a type II transmembrane protein expressed by both innate and adaptive immune cells, including natural killer (NK) cells, CD8+ T cells, invariant NKT cells, γδ T cells, and some CD4+ T cells under certain pathological conditions. NKG2D is an activating NK receptor that induces cytotoxicity and production of cytokines by effector cells and supports their proliferation and survival upon engagement with its ligands. In both innate and T cell populations, NKG2D can costimulate responses induced by other receptors, such as TCR in T cells or NKp46 in NK cells. NKG2D ligands (NKG2DLs) are remarkably diverse. Initially, NKG2DL expression was typically attributed to stressed, infected, or transformed cells, thus signaling “dysregulated-self.” However, many reports indicated their expression under homeostatic conditions, usually in the context of cell activation and/or proliferation. Myeloid cells, including macrophages and dendritic cells (DCs), are among the first cells sensing and responding to pathogens and tissue damage. By secreting a plethora of soluble mediators, by presenting antigens to T cells and by expressing costimulatory molecules, myeloid cells play vital roles in inducing and supporting responses of other immune cells in lymphoid organs and tissues. When activated, both macrophages and DCs upregulate NKG2DLs, thereby enabling them with additional mechanisms for regulating lymphocyte responses. In this review, we will focus on the expression of NKG2D by innate and adaptive lymphocytes, the regulation of NKG2DL expression on myeloid cells, and the contribution of the NKG2D/NKG2DL axis to the crosstalk of myeloid cells with NKG2D-expressing lymphocytes. In addition, we will highlight pathophysiological conditions associated with NKG2D/NKG2DL dysregulation and discuss the putative involvement of the NKG2D/NKG2DL axis in the lymphocyte/myeloid cell crosstalk in these diseases.

Analysis of the Expression of Surface Receptors on NK Cells and NKG2D on Immunocytes in Peripheral Blood of Patients with Nasopharyngeal Carcinoma

Background:The aberrant expression of surface receptors on immunocytes may represent potential markers of tumor escape for nasopharyngeal carcinoma (NPC). The aim of this study was to investigate the expression of representative receptors on natural killer (NK) cells and NK group 2, member D (NKG2D) on immunocytes in the peripheral blood of patients with NPC.Methods:Patients (n = 64) with NPC prior to initiation of treatment were defined as the study group. Healthy volunteers (n = 31) served as the control group. The expression of NK cells and NKT cells; the triggering receptors NKp30, NKp44, and NKp46 on NK cells; the activating receptor NKG2D on NK cells, CD4+ T cells, and CD8+ T cells; and the inhibitory receptors CD158b and CD159a on NK cells were analyzed by flow cytometry in the two groups.Results:Here, our study showed that no differences were observed in terms of the numbers of NK cells or NKT cells, or the expression of CD158b and CD159a on the surface of NK cells between the two groups. Nevertheless, the expression levels of NKp30 and NKp46 on NK cells in the NPC patients were significantly lower than in the healthy individuals (P < 0.05). No differences existed in the expression of NKG2D on NK cells, but NKG2D on CD8+ T cells showed a markedly lower expression in the study group (P < 0.001).Conclusions:Our findings may reflect a possible mechanism of immune evasion for NPC. The enhancement of immunotherapy concerning NKp30, NKp46, and NKG2D may be an innovative treatment strategy for patients with NPC.

Expression levels of UL16 binding protein 1 and natural killer group 2 member D affect overall survival in patients with gastric cancer following gastrectomy.

UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), cluster of differentiation (CD)8+ T, and γ δ T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. The present study investigated the associations between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The levels of ULBP1 and NKG2D expression were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-74 cells expressed ULBP1 with ULBP2, −5, or −6. NKG2D was expressed at a higher level following activation of T cells and NK cells. Among the tissue sections positive for NKG2D expression, 6 patients were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors exhibited significantly longer overall survival (OS) compared with patients without NKG2D expression in tumors (P=0.0217). The longest OS was observed in patients positive for ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer, and may have applications in immunotherapy for the induction of adaptive immunity in patients with cancer. Additionally, ULBP1 and NKG2D may be useful as prognostic biomarkers in gastric cancer.

Abstract 5733: Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer

Abstract UL16 binding protein 1 (ULBP1) expressed on the tumor cell surface binds to the natural killer group 2 member D (NKG2D) receptor presenting on natural killer (NK), CD8+ T, and gamma delta T cells. However, the roles of ULBP1 and NKG2D expression and associated immune responses in gastric cancer are unclear. Here, we investigated the relationships between ULBP1 and NKG2D expression and clinical outcomes in patients with gastric cancer. The expression levels of ULBP1 and NKG2D were examined in human gastric cancer cell lines and gastric cancer tissues from 98 patients who underwent surgery from 2004 to 2008. MKN-72 cells expressed ULBP1 with ULBP2, -5, or -6. NKG2D was expressed at a higher level after activation on T cells and NK cells. Among tissue sections positive for NKG2D expression, six cases were positive for CD8 and CD56. In all tissues, NKG2D-expressing cells were typically aCD8+ T cells. Patients with NKG2D expression in tumors had significantly longer overall survival (OS) than patients without NKG2D expression in tumors (p = 0.0217). The longest OS was observed in patients positive for both ULBP1 and NKG2D, whereas the shortest OS was observed in patients negative for both ULBP1 and NKG2D. The interaction between ULBP1 and NKG2D may improve OS in patients with gastric cancer and may have applications in immunotherapy for induction of adaptive immunity in patients with cancer. Moreover, ULBP1 and NKG2D may be useful as prognostic biomarker in gastric cancer. Citation Format: Kiyoshi Yoshimura, Moeko Inoue, Tetsuhiko Asao, Masanori Fuse, Satoshi Wada, Atsuo Kuramasu. Expression levels of UL16 binding protein 1 and natural killer group 2 member D in patients with gastric cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5733. doi:10.1158/1538-7445.AM2017-5733

On-treatment decrease of NKG2D correlates to early emergence of clinically evident hepatocellular carcinoma after interferon-free therapy for chronic hepatitis C.

Background and aimsInterferon (IFN)- free direct antiviral agents (DAAs) with rapid HCV eradication might evoke immunological reconstitutions, and some early recurrences of HCC after IFN-free DAAs have been reported. This study aimed to investigate whether natural killer group 2, member D (NKG2D) predicts early emergence of HCC after IFN-free DAAs.MethodsWe conducted a clinical practice-based observational study of 101 patients infected with genotype 1 HCV who received IFN-free (DAAs), and stratified them into those who did or did not develop early (i.e., during the 6-month surveillance period following treatment.) recurrence or occurrence of clinically evident HCC. We also analyzed the peripheral blood mononuclear cells, both before treatment and at end of treatment (EOT), of 24 of the patients who received IFN-free DAAs, and 16 who received IFN-combined protease inhibitor.ResultsWe found early emergence of clinically evident HCC after IFN-free DAAs in 12 (12%) patients. Higher pre-treatment NKG2D expression, higher FIB-4 score, previous HCC history and failure to achieve sustained viral response were significant factors correlating to early HCC emergence. After IFN-free DAAs, a rapid decrease of NKG2D at EOT correlated with early HCC emergence in the IFN-free DAA-treated patients, but not in patients treated with the IFN-combined regimen. The decrease of NKG2D until EOT was predictive of early HCC emergence at a cut-off of -52% (AUC = 0.92).ConclusionsOn-treatment decrease of NKG2D may be a useful predictor of early emerging HCC in patients treated with IFN-free DAAs.

Platelets impair natural killer cell reactivity and function in endometriosis through multiple mechanisms.

Do platelets have any role in the reduced cytotoxicity of natural killer (NK) cells in endometriosis? Platelets impair NK cell reactivity and function in endometriosis through multiple mechanisms. Platelets play an important role in the development of endometriosis, and platelet-derived transforming growth factor-β1 (TGF-β1) suppresses the expression of NK Group 2, Member D (NKG2D) on NK cells, resulting in reduced cytotoxicity in women with endometriosis. Experiments on mice with induced endometriosis in which either platelets, NK cells or both were depleted and controls (none depleted). In vitro experiments with NK cells, platelets and, as target cells, endometriotic epithelial cell and endometrial stromal cell lines. Immunohistochemistry analysis of ectopic endometrial tissues from mice with induced endometriosis receiving either platelet depletion (PD), NK cell depletion, or both or none. Immunofluorescence, flow cytometry and gene expression analysis for major histocompatibility complex class I (MHC-I) expression in target cells. Cytotoxicity and degranulation assays and the measurement of interferon (IFN)-γ secretion for the evaluation of NK cytotoxicity. Flow cytometry and gene expression for the expression of NK cell receptors. PD resulted in significantly reduced lesion weight in mice with induced endometriosis, but NK cell depletion as well as concomitant platelet and NK cell depletion increased the weight, suggesting that the anti-endometriosis effect of PD is mediated, at least in part, by increased NK cell cytotoxicity against endometriotic cells. Co-incubation of target cells with platelets resulted in rapid platelet coating as well as increased MHC-I expression in these cells, effectively providing a cloak of 'pseudo-self' to these cells to shield against NK cell lysis. It also reduced the expression of NKG2D ligands MICA and MICB and reduced the NK cell cytotoxicity. In addition, co-incubation of NK cells with platelets impaired the NK cell cytotoxicity as well. This impaired NK cell cytotoxicity was not due to the increased NK cell apoptosis, but, rather, through reduced NK cell degranulation and IFN-γ production, and reduced expression of activating receptors NKG2D and NKp46 and increased expression of inhibitory receptor KIR2DL1 in NK cells. Inhibition of TGF-β1 signaling partially restored the aberrant expression of NKG2D, NKp46 and KIR2DL1, and partially restored the impaired NK cell cytotoxicity induced by activated platelets and their releasate. Not applicable. This study is confined by the limitation of animal and in vitro experimentation and the lack of direct human data. Anti-platelet treatment holds promise in treating endometriosis. The National Natural Science Foundation of China (81471434 to S.W.G., 81270676 to S.W.G., 81370695 to X.S.L. and 81671436 to X.S.L). None of the authors has anything to disclose.

Bone Marrow-Derived Mesenchymal Stem Cells Attenuate Immune-Mediated Liver Injury and Compromise Virus Control During Acute Hepatitis B Virus Infection in Mice.

Mesenchymal stem cells (MSCs) have been used as therapeutic tools not only for their ability to differentiate toward different cells, but also for their unique immunomodulatory properties. However, it is still unknown how MSCs may affect immunity during hepatitis B virus (HBV) infection. This study was designed to explore the effect of bone marrow-derived MSCs (BM-MSCs) on hepatic natural killer (NK) cells in a mouse model of acute HBV infection. Mice were injected with 1 × 106 BM-MSCs, which stained with chloromethyl derivatives of fluorescein diacetate fluorescent probe, 24 h before hydrodynamic injection of viral DNA (pHBV1.3) through the tail vein. In vivo imaging system revealed that BM-MSCs were accumulated in the injured liver, and they attenuated immune-mediated liver injury during HBV infection, as shown by lower alanine aminotransferase levels, reduced proinflammatory cytokine production, and decreased inflammatory cell infiltration in the liver. Importantly, administration of BM-MSCs restrained the increased expression of natural-killer group 2, member D (NKG2D), an important receptor required for NK cell activation in the liver from HBV-infected mice. BM-MSCs also reduced NKG2D expression on NK cells and suppressed the cytotoxicity of NK cells in vitro. Furthermore, BM-MSC-derived transforming growth factor-β1 suppressed NKG2D expression on NK cells. As a consequence, BM-MSC treatment enhanced HBV gene expression and replication in vivo. These results demonstrate that adoptive transfer of BM-MSCs influences innate immunity and limits immune-mediated liver injury during acute HBV infection by suppressing NK cell activity. Meanwhile, the effect of BM-MSCs on prolonging virus clearance needs to be considered in the future.

Cytotoxic activity of allogeneic natural killer cells on U251 glioma cells invitro.

The present study aimed to observe the cytotoxic activity of allogeneic natural killer (NK) cells on U251 glioma cells and to investigate their mechanism of action to establish an effective treatment strategy for neuroglioma. Cell survival curves, colony formation assays and karyotype analysis were performed to investigate the characteristics of U251glioma cells. The present study demonstrated that natural killer group2, memberD (NKG2D)‑major histocompatibility complex class I‑related chainA/B (MICA/B) interactions contributed to the cytotoxic effect of NKcells on K562 and U251cells. In antibody‑blocking assays to inhibit NKG2D ligands, the cytotoxic activity was not completely attenuated, which suggested that other signaling pathways contribute to the cytotoxic activity of NKcells on tumor cells in addition to the NKG2D‑mediated activity. The present study identified that the expression levels of NKG2D ligands on the surface of target cells influenced the strength of the NKcell immune response. Furthermore, allogeneic NKcells were observed to kill glioma cells invitro, and this anticancer activity is associated with the rate of NKG2D expression on the surface of glioma cells.

Aberrant NKG2D expression with IL-17 production of CD4+ T subsets in patients with type 2 diabetes

Type 2 diabetes (T2D) is a systemic inflammatory disease. Although the natural killer group 2, member D (NKG2D) receptor, was not expressed normally on CD4+ T cells, the aberrant expression was found in pathological conditions such as in auto-immune diseases. However, the involvement of NKG2D in pathogenesis of T2D is unclear. We hypothesize that there is an inflammatory CD4+ T cell subpopulation expressing NKG2D and producing interleukin (IL)-17 in T2D. NKG2D expression on CD4+ T cells and their subsets were analyzed by multi-color staining using flow cytometry. Lymphocytes were activated by phorbol-12-myristate-13-acetate (PMA) and ionomycin, and were stained for intracellular IL-17. To investigate the mechanism of IL-17 production, patients’ lymphocytes were stimulated using specific anti-T cell receptor (TCR) alone, anti-NKG2D alone or a combination of the two antibodies. CD4+ T cells and particularly, CD4+CD28nullT subset of T2D patients were highly expressed NKG2D and more prevalent compared to non-diabetic individuals (ND) (P=0.039 and P=0.022, respectively). Significantly higher percentages of CD4+CD28nullNKG2D+T cells of patients produced IL-17 when compared to those of ND (P=0.024) and were positively correlated with the level of glycated hemoglobin A1c (HbA1c) (R2=0.386, P=0.041). Additionally, this cell population could be stimulated by specific monoclonal anti-NKG2D to produce IL-17. In conclusion, CD4+CD28nullNKG2D+T cells were expanded in T2D, especially in patients with poor glycemic control. NKG2D may be one of the surrogate co-stimulatory receptors leading to irregular inflammatory function producing IL-17. An IL-17 producing CD4+CD28nullNKG2D+T cells may potentially be involved in pathogenesis and drive severity of the disease with the glycemic dependence. This particular cell type could be targeted for prognostic or therapeutic purposes.

NKG2D-H60a interaction via homotypic T cell contact delays NOD diabetes development.

Abstract Type I diabetes is an autoimmune disorder mediated by T cells that destroy the insulin-secreting β-islet cells in the pancreas. Both human and murine studies implicate the immune receptor natural killer group 2 member D (NKG2D) in enhancing type 1 diabetes development. However, a mechanism for NKG2D influence in type 1 diabetes development is not clear. Utilizing NKG2D-deficient non-obese diabetic (NOD) mice, we demonstrate that NKG2D expression affects diabetes development via multiple mechanisms in multiple anatomical locations, with both pro- and anti-diabetic effects. First, we found NKG2D influences NOD diabetes development via alteration in the microbiota composition. When we eliminated this microbiome effect, NKG2D-deficient mice had enhanced diabetes development, demonstrating a protective role for NKG2D in this disease. Our in vitro studies suggest this protection is mediated via a previously undescribed interaction between NKG2D and the NKG2D ligand H60a, both expressed by activated NOD CD8+ T cells, during diabetogenic cytotoxic T cell (CTL) generation. We show that during CD8+ T cell differentiation into CTLs, NKG2D-H60a interaction generally decreases the subsequent CTL effector cytokine response. Taken together, these findings demonstrate there is a protective role for NKG2D during diabetes development that is likely mediated by NKG2D-ligand interaction during diabetogenic CTL differentiation.