Expression Of ULBP1 Research Articles

MiR-17–5p/STAT3/H19: A novel regulatory axis tuning ULBP2 expression in young breast cancer patients

Background and aimUL-16 binding protein 2 (ULBP2) is a highly altered ligand for the activating receptor, NKG2D in breast cancer (BC). However, the mechanism behind its de-regulation in BC patients remains to be explored. The sophisticated crosstalk between miR-17–5p, the lncRNA H19, and STAT3 as a possible upstream regulatory loop for ULBP2 in young BC patients and cell lines remains as an unexplored area. Therefore, this study aimed at unravelling the ncRNA circuit regulating ULBP2 in young BC patients and cell lines. Patients and methodsA total of 30 BC patients were recruited for this study. The expression levels of miR-17–5p, lncRNA H19, and STAT3 were examined in 30 BC tissues compared to their normal counterparts. In addition, the expression signatures of those transcripts were compared in young (<40 years) and old BC (≥40 years) patients. miR-17–5p oligonucleotides, STAT3 and H19 siRNAs were transfected in MDA-MB-231 cells using HiPerfect® Transfection Reagent. miR-17–5p and the transcripts of the target genes quantified using RT-qPCR. Their relative expression was calculated using the 2–ΔΔCT method. ResultsThrough acting as a ceRNA circuit that antagonizes the function of miR-17–5p, H19 prevented the miR-17–5p-induced downregulation of STAT3; this mechanism further contributes to the pathogenesis of BC. Ectopic expression of miR-17–5p in MDA-MB-231 cells displayed its prominent role as an indirect potential activator of NK cells by significantly repressing the expression levels of the oncogenic mediator STAT3 and the oncogenic lncRNA H19 and inducing ULBP2 expression level by 3 folds in TNBC cell lines compared to mock cells. Furthermore, knocking down of STAT3 repressed the lncRNA H19 and increased ULBP2 expression levels, whereas siRNAs against H19 increased the expression levels of ULBP2. ConclusionThis study highlighted the crosstalk between the novel regulatory network composed of miR-17–5p, H19 and STAT3, and their impact on ULBP2 in BC. Moreover, this study underscored the potential role of miR-17–5p in counteracting the immune evasion tactics, particularly the shedding of ULBP2 in young BC patients, through the modulation of the STAT3/H19/ULBP2 regulatory axis. Thus, targeting this novel regulatory network could potentially enhance our understanding and advance the future application of the innate system-mediated immunotherapy in BC.

Human esophageal cancer stem-like cells escape the cytotoxicity of natural killer cells via down-regulation of ULBP-1

BackgroundCancer stem-like cells (CSCs) play an important role in initiation and progression of aggressive cancers, including esophageal cancer. Natural killer (NK) cells are key effector lymphocytes of innate immunity that directly attack a wide variety of cancer cells. NK cell-based therapy may provide a new treatment option for targeting CSCs. In this study, we aimed to investigate the sensitivity of human esophageal CSCs to NK cell-mediated cytotoxicity.MethodsCSCs were enriched from human esophageal squamous cell carcinoma cell lines via sphere formation culture. Human NK cells were selectively expanded from the peripheral blood of healthy donors. qRT-PCR, flow cytometry and ELISA assays were performed to examine RNA expression and protein levels, respectively. CFSE-labeled target cells were co-cultured with human activated NK cells to detect the cytotoxicity of NK cells by flow cytometry.ResultsWe observed that esophageal CSCs were more resistant to NK cell-mediated cytotoxicity compared with adherent counterparts. Consistently, esophageal CSCs showed down-regulated expression of ULBP-1, a ligand for NK cells stimulatory receptor NKG2D. Knockdown of ULBP-1 resulted in significant inhibition of NK cell cytotoxicity against esophageal CSCs, whereas ULBP-1 overexpression led to the opposite effect. Finally, the pro-differentiation agent all-trans retinoic acid was found to enhance the sensitivity of esophageal CSCs to NK cell cytotoxicity.ConclusionsThis study reveals that esophageal CSCs are more resistant to NK cells through down-regulation of ULBP-1 and provides a promising approach to promote the activity of NK cells targeting esophageal CSCs.

UCA1 Inhibits NKG2D-mediated Cytotoxicity of NK Cells to Breast Cancer.

Natural killer cells play important roles in tumor immune surveillance, and cancer cells must resist this surveillance in order to progress and metastasise. The study aimed to explore the mechanism of how breast cancer cells become resistant to the cytotoxicity of NK cells. We established NK-resistant breast cancer cells by exposing MDA-MB-231 cells and MCF-7 cells to NK92 cells. Profiles of lncRNA were compared between the NK-resistant and parental cell lines. Primary NK cells were isolated by MACS, and the NK attacking effect was tested by non-radioactive cytotoxicity. The change in lncRNAs was analyzed by Gene-chip. The interaction between lncRNA and miRNA was displayed by Luciferase assay. The regulation of the gene was verified by QRT-PCR and WB. The clinical indicators were detected by ISH, IH, and ELISA, respectively. UCA1 was found to be significantly up-regulated in both NK-resistant cell lines, and we confirmed such up-regulation on its own to be sufficient to render parental cell lines resistant to NK92 cells. We found that UCA1 up-regulated ULBP2 via the transcription factor CREB1, while it up-regulated ADAM17 by "sponging" the miR-26b-5p. ADAM17 facilitated the shedding of soluble ULBP2 from the surface of breast cancer cells, rendering them resistant to killing by NK cells. UCA1, ADAM17, and ULBP2 were found to be expressed at higher levels in bone metastases of breast cancer than in primary tumors. Our data strongly suggest that UCA1 up-regulates ULBP2 expression and shedding, rendering breast cancer cells resistant to killing by NK cells.

Involvement of KLRK1 in immune infiltration of head and neck squamous cell carcinoma correlates with favorable prognosis.

Head and neck squamous cell carcinoma (HNSCC) is a malignancy commonly found in the head and neck region, with a low 5-year survival rate. Although immunotherapy has made significant progress, its efficacy in HNSCC treatment remains unsatisfactory. Killer cell lectin-like receptor K1 (KLRK1), a marker highly expressed in immune cells, can bind to its ligands expressed by cancer cells to exert its antitumor effect. However, the role of KLRK1 in HNSCC has yet to be studied extensively. This study aimed to explore the involvement of KLRK1 in immune infiltration of HNSCC and its correlation with prognosis. We analyzed KLRK1 expression data from the Cancer Genome Atlas database. The relationship between KLRK1 and immune cell infiltration has also been investigated. Finally, we analyzed the association between the expression of KLRK1 and its ligands and the prognosis of patients with HNSCC. We found that KLRK1 was highly expressed in HNSCC and correlated with better prognosis. KLRK1 expression was correlated with age, histological grade, HPV infection, pT, pN, pTNM stage, primary site, and survival status. High expression levels of KLRK1 have been linked to high levels of immune cell infiltration, particularly CD4/8 (+) T lymphocytes. Among the ligands of KLRK1, UL16 binding protein (ULBP) 1-3 showed high expression, which was associated with an increased risk of death. Notably, the expression of KLRK1 was negatively correlated with ULBP1-3. Patients with high levels of ULBP2/3 expression in tonsil carcinoma had poorer prognosis than those with low levels (P < .01), whereas ULBP1 expression levels had no significant effect on tonsil carcinoma prognosis (P = .770). The expression levels of ULBP1/3 were correlated with worse prognosis in patients with laryngeal cancer (P < .05), whereas there was no significant correlation between ULBP2 expression levels and overall survival (P = .269). Our study revealed that KLRK1 is highly expressed in HNSCC and is associated with a better prognosis and immune infiltration. Patients with high expression of KLRK1 ligands exhibited worse prognoses, possibly because of the expression of more soluble ligands.

ULBP2 is a biomarker related to prognosis and immunity in colon cancer.

The study aimed to determine whether ULBP2 was associated with prognosis and immune infiltration in colon cancer (CC) and provided important molecular basis inorderto early non-invasive diagnosis and immunotherapy of CC. Using The Cancer Genome Atlas database (TCGA) and ImmPort database, we extracted messenger RNA (mRNA) data of CC and immune-related genes, then we used "limma" package, "survival" package, and Venn overlap analysis to obtain the differentially expressed mRNA (DEmRNA) associated with prognosis and immunity of CC patients. "pROC" package was used to analyze receiver operating characteristics (ROC) of target gene. We used chi-square test and two-class logistics model to identify clinicopathological parameters that correlated with target gene expression. In order to determine the effects of target gene expression and clinicopathological parameters on survival, univariate and multivariate cox regression analyses were performed. We analyzed the related functions and signaling pathways of target gene by enrichment analysis. Finally, the correlation between target gene and tumor immune infiltrating was explored by ssGSEA and spearman correlation analysis. Results showed that ULBP2 was a target gene associated with immunity and prognosis in CC patients. CC patients with higher ULBP2 expression had poor outcomes. In terms of ROC, ULBP2 had an area under the curve (AUC) of 0.984. ULBP2 was associatedwith T stage, N stage, and pathologic stage of CC patients, and served as an independent predictor of overall survival in CC patients. Functional enrichment analysis revealed ULBP2 was obviously enriched in pathways connected with carcinogenesis and immunosuppression. The expression of ULBP2 was significantly associated with tumor immune cells and immune checkpoints according to ssGSEA and spearman correlation analysis. To conclude, our study suggested that ULBP2 was associated with tumor immunity, and might be a biomarker associated with the diagnosis and prognosis of CC patients, and a potential target of CC immunotherapy.

605 Phototherapy-induced IFNκ drives type I IFN induced anticancer responses in CTCL

Study of Stage I-II mycosis fungoides (MF) skin lesions from 14 patients by gene expression profiling demonstrated reduced expression of six type I IFNs compared to healthy human skin. Psoralen plus UVA therapy (PUVA) induced increased levels of type I IFNs that were associated with CD8 T cell recruitment into tumors, expression of antigen specific T cell activation genes and depletion of malignant T cells. MF skin lesions expressed significantly higher levels of two negative regulators of IFN production, IRF2 and IFI35; IRF2 levels correlated with the number of malignant T cells in skin. IRF2 is known to antagonize epithelial production of type I IFNs. Indeed, we found reduced expression of ULBP2, a marker of epithelial type I interferon production. Immunostaining confirmed reduced epidermal production of type I alpha IFNs that was reversed by PUVA therapy. IFNκ is an epithelial type I interferon induced by UVB that triggers production of other type I IFNs and is responsible for the flaring of cutaneous lupus after sun exposure. We studied human keratinocytes treated with UVA with or without psoralen. IFNκ enhanced keratinocyte production of type I IFNs and downstream interferon genes in response to UVA. IFNκ knockout keratinocytes had markedly reduced type I IFNs induction after UVA, illustrating the key role of epithelial IFNκ in boosting type I interferon production. In summary, we find that type I IFNs are markedly reduced in MF lesions, that phototherapy induces epithelial type I interferon production via INFκ and that this increase is associated with effective tumor clearance. Our results suggest other medications that induce epithelial type I IFNs, including MEK or EGFR inhibitors, may be effective therapies for CTCL.

DAP10 integration in CAR-T cells enhances the killing of heterogeneous tumors by harnessing endogenous NKG2D

Although chimeric antigen receptor T (CAR-T) cells have achieved remarkable successes in hematological malignancies, the efficacies of CAR-T cells against solid tumors remains unsatisfactory. Heterogeneous antigen expression is one of the obstacles on its effective elimination of solid cancer cells. DNAX-activating protein 10 (DAP10) interacts with natural killer group 2D (NKG2D), acting as an adaptor that targets various malignant cells for surveillance. Here, we designed a DAP10 chimeric receptor that utilized native NKG2D on T cells to target NKG2D ligand-expressing cancer cells. We then tandemly incorporated it with anti-glypican 3 (GPC3) single-chain variable fragment (scFv) to construct a dual-antigen-targeting system. T cells expressing DAP10 chimeric receptor (DAP10-T cells) displayed with an enhancement on both cytotoxicity and cytokine secretion against solid cancer cell lines, and its tandem connection with anti-GPC3 scFv (CAR GPC3-DAP10-T cells) exhibited a dual-antigen-targeting capacity on eliminating heterogeneous cancer cells in vitro and suppressing the growth of heterogeneous cancer in vivo. Thus, this novel dual-targeting system enabled a high efficacy on killing cancer cells and extended the recognition profile of CAR-T cells toward tumors, which providing a potential strategy on treatment of solid cancer clinically.

The metabolism of cells regulates their sensitivity to NK cells depending on p53 status

Leukemic cells proliferate faster than non-transformed counterparts. This requires them to change their metabolism to adapt to their high growth. This change can stress cells and facilitate recognition by immune cells such as cytotoxic lymphocytes, which express the activating receptor Natural Killer G2-D (NKG2D). The tumor suppressor gene p53 regulates cell metabolism, but its role in the expression of metabolism-induced ligands, and subsequent recognition by cytotoxic lymphocytes, is unknown. We show here that dichloroacetate (DCA), which induces oxidative phosphorylation (OXPHOS) in tumor cells, induces the expression of such ligands, e.g. MICA/B, ULBP1 and ICAM-I, by a wtp53-dependent mechanism. Mutant or null p53 have the opposite effect. Conversely, DCA sensitizes only wtp53-expressing cells to cytotoxic lymphocytes, i.e. cytotoxic T lymphocytes and NK cells. In xenograft in vivo models, DCA slows down the growth of tumors with low proliferation. Treatment with DCA, monoclonal antibodies and NK cells also decreased tumors with high proliferation. Treatment of patients with DCA, or a biosimilar drug, could be a clinical option to increase the effectiveness of CAR T cell or allogeneic NK cell therapies.

Curcumin Boosts Natural Killer Cell-Based Immunotherapy in Impeding Progression of Hepatocellular Carcinoma Through Androgen Receptor/UL16 Binding Protein-2 Signal

Hepatocellular carcinoma (HCC) is hardly curable with present treatment modalities, especially for those in advanced disease stage. Several chronic hepatic disorders, including alcoholic liver disease, cirrhotic and viral hepatitis, can ultimately lead to occurrence of HCC. This study investigated the effects of curcumin on natural killer (NK) cell-based immunotherapies being applied to restrain the progress of HCC, and disclose valid message for better clinical application of curcumin. After receiving curcumin treatment, the HCC cells were co-cultured with NK cells to study cytotoxicity of NK cells. After thein vitrocytotoxicity assay, we determined the function of curcumin in modulating the cytotoxicity of NK cells. Additionally, the mechanism of curcumin on HCC cells was investigated by utilizing the following techniques: lentiviral infection/transfection, qPCR and Western-blot, thereby exploring the genetic alterations of differently treated HCC cells. HCC cell lines (SK-Hep1 and SUN423) were studied, and findings confirmed that curcumin significantly strengthened the cytotoxic NK cells to kill HCC cells. This effect was mainly achieved through regulating androgen receptor (AR)/UL162 signal, which led to efficacy enhancement of NK cell-based immunotherapy. Moreover, secretion of interferon-γwhich is an immunoregulator executing an anti-tumor effect was also elevated. In addition, curcumin strengthened the expression of ULBP2 in SK-Hep1 and SUN423 cells, while relatively weakening the expression of androgen receptor. The findings from this study can provide a theoretical base for further research and development of curcumin as a beneficial suppressor of HCC progression. We found that the cytotoxicity of NK cells towards HCC cells was gradually elevated as the HCC cells were treated with increased concentration of curcumin. Meanwhile, the secretion of interferon-γ, which was an immunoregulator exerting an anti-tumor effect, was also gradually elevated. In addition, the expression levels of ULBP2 in SK-Hep1 and SUN423 cells were continuously enhanced with increased concentration of curcumin in the pre-treatment of HCC cells, while the expression of androgen receptor was relatively weakened. Similar results were also obtained after the overexpression or knockdown of androgen receptor (AR) gene in SK-Hep1 and SUN423 cells. Therefore, this study found that curcumin can boost the NK cell-based immunotherapy in impeding progression of HCC through the androgen receptor/ULBP2 signal.

TLR3 Mediates Senescence and Immunosurveillance of Hepatic Stellate Cells

Background: Activation of hepatic stellate cells (HSCs) is an important driver of liver fibrosis, which is a health problem of global concern, and there is no effective solution for it at the present. Senescent activated HSCs are preferentially killed by natural killer cells (NK cells) to promote the regression of hepatic fibrosis. Objectives: The purpose of this study was to investigate the effect of polyinosinic-polycytidylic acid (poly I:C) on HSCs’ senescence, a trigger for NK cell-induced cytotoxicity. Methods: The senescence of HSCs was assessed by western blot, qRT-PCR, and flow cytometry, and NK cell cytotoxicity was assessed in a co-culture of NK cells with poly I:C-treated HSCs by measuring CD107a expression. Results: The expression of p16, p21, SA-β-gal, MICA/MICB, and ULBP2 increased in poly I:C-treated HSCs, rendering them significantly susceptible to NK cell cytotoxicity. Conclusions: Poly I:C induces cellular senescence in HSCs and triggers NK cell immunosurveillance, suggesting that the role of poly I:C in HSC senescence may promote fibrosis regression.

Abstract 1770: Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice

Abstract ULBP2 is one of the ligands for NKG2D (NKG2DLs). ULBP2 expression is induced in transformed cells and is recognized by immune effector cells via the activating NKG2D immunoreceptor. Previously, we reported high concentrations of serum-soluble ULBP2 (sULBP2) were correlated with poor prognosis in NSCLC patients. As a possible mechanism, we demonstrated that sULBP2 directly suppressed the cytolytic activity of peripheral blood mononuclear cells (PBMCs). However, little is known about the in vivo function of human NKG2DLs on tumor cells. The present study investigates the function of human NKG2DLs in the syngeneic C57BL/6 mice. We generated ULBP2 stably expressing B16 melanoma cell lines (ULBP2-B16). As previously reported, 4-hour 51Cr releasing assay showed NK cell cytotoxicity against the tumor cells was enhanced by ectopic expression of ULBP2 on the tumor cells. IFN-gamma production in NK cells was decreased by non-contact co-culture with ULBP2-B16. To investigate the in vivo function of ULBP2, C57BL/6 mice were inoculated s.c. with mock-B16 cells or ULBP2-B16 cells. ULBP2-B16 tumors grew rapidly compared to mock-B16 tumors. NKG2D blockade by anti-NKG2D antibody injected i.p. suppressed tumor growth of ULBP2-B16 tumors. Spleen cells and tumor-infiltrating lymphocytes (TILs) were analyzed by flow cytometry. NKG2D was expressed on splenic NK cells, while it was rarely expressed on splenic CD8+ T cells. NKG2D was downregulated on NK cells in TILs (NK-TILs) from ULBP2-B16. NKG2D was highly expressed on CD8+ T cells in TILs (CD8+-TILs) from mock-B16 tumors, while its expression was suppressed on these from ULBP2-B16 tumors. Degranulation analysis revealed that ULBP2-B16 reduced activity of NK-TILs and CD8+-TILs compared to mock-B16. In conclusion, ectopic expression of ULBP2 on tumor cells promotes tumor progression via suppression of the activity of NK cells and CD8+ T cells. These findings suggest that neutralization of NKG2DLs may be a promising approach for the treatment of NKG2DLs-expressing tumors. Citation Format: Kosuke Yamaguchi, Naomi Miyake, Yasuhiko Teruya, Kohei Yamane, Naoki Kinoshita, Tomohiro Sakamoto, Masahiro Kodani, Hiroki Chikumi, Akira Yamasaki. Ectopic expression of ULBP2 promotes tumor progression in syngeneic mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr 1770.

Natural Killer Receptor Ligand Expression By AML Blasts Determines Survival and Relapse Following Induction Chemotherapy

IntroductionNatural killer (NK) cells contribute to the immune surveillance of cancer and are capable of controlling acute myeloid leukemia (AML) in vitro and following allogeneic hematopoietic stem cell transplantation. NK cell activation is dependent on the balance of activating and inhibitory receptor-ligand interactions. The prognostic impact of the expression of NK receptor ligands (NKRL) on AML blasts at diagnosis has not been described. We investigated the impact of NKRL expression by AML on the outcome of newly diagnosed patients undergoing induction chemotherapy. We hypothesized that patients with AML that expressed NKRL that facilitated NK cell activation (low inhibitory and high activating ligand expression) have improved immune surveillance and a reduced relapse rate.MethodsCryopreserved bone marrow cells and clinical data of 66 AML patients who underwent induction chemotherapy between 2001 and 2013 were obtained from the Australasian Leukemia Lymphoma Group tissue bank. Patients were stratified by cytogenetics (CG) and mutation status (European LeukemiaNet criteria, ELN). Expression (by relative fluorescence intensity - RFI) of 6 activating (MICA, MICAB, CD155, CD112, ULBP1, ULBP2/5/6) and 3 inhibitory (HLA class I, PD-L1 and PD-L2) NKRL on AML blasts was analyzed by flow cytometry. A net score of activating minus inhibitory ligands was calculated, which stratified patients into 2 groups: G0 (inhibitory pattern) and G1 (activating pattern).ResultsThe median age of patients was 56 years. Cytogenetic risk was adverse (13), intermediate (46) and favorable (7); ELN risk incorporating mutational profile was adverse (25), intermediate (15), and favorable (20). 79% of patients received an anthracycline in combination with cytarabine as induction therapy. Among activating ligands, the MICA, CD112 and ULBP1 were frequently expressed by AML blasts (79%, 80% and 82% of patients respectively), and also demonstrated the greatest intensity of expression (mean RFI of 3, 4.2 and 6.5 respectively). When analyzed individually, only ULBP1 expression was significantly associated with improved overall survival (OS) (p<0.05), relapse free survival (RFS) (p<0.05) and lower relapse incidence (RI) (p<0.005). The intensity of ULBP1 expression was significantly associated with improved OS, RFS and RI in a stepwise fashion. Patients with the highest ULBP1 expression had a reduced hazard ratio of death (HR 0.20, p<0.05) or relapse (HR 0.24, p<0.05) compared with non-expressors. PD-L2 expression on AML cells was higher and more frequent than PD-L1 (85% vs 5%). All AML variably expressed HLA class I. According to RFI, 32 patients were assigned to G0 and 34 to G1. There was no difference in patient age or distribution of CG or ELN risk categories between patients in G0 or G1 subgroups. G1 was significantly associated with lower relapse incidence than G0 (p<0.005, 2-year-RI: G1 28% vs G0 71%, Fig. 1). G1 was associated with higher RFS (log-rank p<0.05; at 2 years: G1 52% vs G0 20%; Fig 2) and improved OS (log rank p<0.05; at 2 years: G1 56% vs G0 29%; Fig 3). The G1 phenotype remained significantly associated with lower RI when taking into account either CG or ELN risk.ConclusionsWe have identified an immunological risk stratification method based on NKRL expression which is independent of established cytogenetic and molecular prognostic factors. Expression of activating NK cell receptor ligands by AML blasts is associated with superior survival and reduced relapse following induction chemotherapy. Our findings suggest that AML blasts are effective targets for NK cell-mediated immune surveillance, and support investigation into therapeutic strategies to enhance NK cell activation as a means of maintaining remission. [Display omitted] DisclosuresRitchie:Amgen Inc.: Honoraria.

Expression of NK Cell Receptor Ligands on Leukemic Cells Is Associated with the Outcome of Childhood Acute Leukemia.

Simple SummaryNatural killer cells (NK cells) of the innate immune system are suspected of playing an important role in eliminating residual leukemia cells during maintenance chemotherapy given to children with acute lymphoblastic leukemia for about two years. This study analyzes the expression of ligands for the receptors that regulate the function of NK cells on leukemic cells of more than one hundred children with acute lymphoid and myeloid leukemia. Our results show that the loss of expression of some molecules involved in the activation of NK cells is associated with poorer survival. In addition, a genetic combination of molecules that interact to regulate NK cell function seems to be associated with a higher relapse rate during/after chemotherapy and shorter patient survival. Children who carry this genetic combination are refractory to current chemotherapy treatments, and stem cell transplantation does not seem to contribute to their cure either, and therefore, they should be considered as candidates for alternative biological therapies that might offer better results.Acute leukemia is the most common malignancy in children. Most patients are cured, but refractory/relapsed AML and ALL are the first cause of death from malignancy in children. Maintenance chemotherapy in ALL has improved survival by inducing leukemic cell apoptosis, but immune surveillance effectors such as NK cells might also contribute. The outcome of B-ALL (n = 70), T-ALL (n = 16), and AML (n = 16) pediatric patients was evaluated according to leukemic cell expression of ligands for activating and inhibiting receptors that regulate NK cell functioning. Increased expression of ULBP-1, a ligand for NKG2D, but not that of CD112 or CD155, ligands for DNAM-1, was associated with poorer 5-year event-free survival (5y-EFS, 77.6% vs. 94.9%, p < 0.03). Reduced expression of HLA-C on leukemic cells in patients with the KIR2DL1/HLA-C*04 interaction was associated with a higher rate of relapse (17.6% vs. 4.4%, p = 0.035) and lower 5y-EFS (70.6% vs. 92.6%, p < 0.002). KIR2DL1/HLA-C*04 interaction was an independent predictive factor of events (HR = 4.795, p < 0.005) or death (HR = 6.731, p < 0.005) and might provide additional information to the current risk stratification. Children who carry the KIR2DL1/HLA-C*04 interaction were refractory to current chemotherapy treatments, including allogeneic stem cell transplantation; therefore, they should be considered as candidates for alternative biological therapies that might offer better results.

Investigation and verification of the clinical significance and perspective of natural killer group 2 member D ligands in colon adenocarcinoma

This study investigated and verified the diagnostic and prognostic values of natural killer group 2 member D ligand (NKG2DL) genes in colon adenocarcinoma (COAD). We downloaded NKG2DLs expression data and corresponding clinical parameters from The Cancer Genome Atlas (TCGA) and used bioinformatics techniques to investigate the values of NKG2DLs in COAD. Then, we used the GSE40967 cohort to verify the prognostic value of NKG2DLs. Finally, we verified the ULBP2 expression level in tissues, and also investigated the diagnostic and prognostic values of ULBP2 in COAD. The diagnostic receiver operating characteristic curves showed that ULBP1, ULBP2, ULBP3, and RAET1L had high diagnostic values in COAD [Area Under Curve (AUC) > 0.9]. In TCGA cohort, the univariate and multivariate survival analyses suggested that ULBP2 was correlated with the prognosis of COAD recurrence-free survival (RFS) and overall survival (OS). In GSE40967 cohort, ULBP2 was associated with CC RFS and OS. Reverse transcription-quantitative polymerase chain reaction and immunohistochemistry results showed that ULBP2 was highly expressed in COAD tumor tissues (P < 0.05) and both had diagnostic values (AUC > 0.7). Validated survival analysis showed that the high expression of ULBP2 had a worse prognosis in COAD OS and RFS. Thus, ULBP2 might be an independent diagnostic and prognostic biomarker of COAD.

Expression of NKG2D ligands is downregulated by β-catenin signalling and associates with HCC aggressiveness

The NKG2D system is a potent immunosurveillance mechanism in cancer, wherein the activating NK cell receptor (NKG2D) on immune cells recognises its cognate ligands on tumour cells. Herein, we evaluated the expression of NKG2D ligands in hepatocellular carcinoma (HCC), in both humans and mice, taking the genomic features of HCC tumours into account. The expression of NKG2D ligands (MICA, MICB, ULBP1 and ULBP2) was analysed in large human HCC datasets by Fluidigm TaqMan and RNA-seq methods, and in 2 mouse models (mRNA and protein levels) reproducing the features of both major groups of human tumours. We provide compelling evidence that expression of the MICA and MICB ligands in human HCC is associated with tumour aggressiveness and poor patient outcome. We also found that the expression of ULBP1 and ULBP2 was associated with poor patient outcome, and was downregulated in CTNNB1-mutated HCCs displaying low levels of inflammation and associated with a better prognosis. We also found an inverse correlation between ULBP1/2 expression levels and the expression of β-catenin target genes in patients with HCC, suggesting a role for β-catenin signalling in inhibiting expression. We showed in HCC mouse models that β-catenin signalling downregulated the expression of Rae-1 NKG2D ligands, orthologs of ULBPs, through TCF4 binding. We demonstrate that the expression of NKG2D ligands is associated with aggressive liver tumorigenesis and that the downregulation of these ligands by β-catenin signalling may account for the less aggressive phenotype of CTNNB1-mutated HCC tumours. The NKG2D system is a potent immunosurveillance mechanism in cancer. However, its role in hepatocellular carcinoma development has not been widely investigated. Herein, we should that the expression of NKG2D ligands by tumour cells is associated with a more aggressive tumour subtype.

Clinical Impact of Natural Killer Group 2D Receptor Expression and That of Its Ligand in Ovarian Carcinomas: A Retrospective Study.

PurposeNatural killer (NK) cells are innate immune cells with antitumor activity. NKG2D is the most important activating receptor expressed on the NK cell surface; this receptor binds to the ligands MICA/B and ULBPs to activate NK cells. The current study aimed to evaluate the expression of NKG2D by NK cells, and to the evaluate expression of its ligands in ovarian carcinomas; it also examined the clinical relevance of NK receptor/ligand expression by analyzing the relationship between expression, clinicopathological parameters, and prognosis.Materials and MethodsFormalin-fixed paraffin-embedded archival ovarian high-grade serous carcinoma (HGSC, n=79) tissue samples were used for tissue microarray analysis. The expressions of NK cell markers (CD56 and NKG2D) and NKG2D ligands (MICA/B, ULBP1, ULBP3, and ULBP2/5/6) in carcinoma tissues were evaluated by immunohistochemical staining, and the association between these results and clinical prognostic parameters was analyzed statistically.ResultsULBP1 was highly expressed in 51 cases (64.6%), and ULBP2/5/6 was highly expressed in 56 cases (70.9%) of HGSC. High expression of ULBP1 and ULBP2/5/6 was significantly associated with lower recurrence of HGSC, whereas high expression of ULBP3 was significantly associated with higher recurrence. Multivariate Cox regression analysis revealed that high expression of ULBP1 was associated with increased overall survival and a decreased hazard ratio (0.150, p=0.044), suggesting that it is an independent predictor of better survival.ConclusionHigh expression of ULBP1 predicts a better prognosis for HGSC, suggesting that ULBP1 expression could be a novel prognostic indicator in this subset of carcinomas.

Effects and Mechanism of PARP Inhibitor Olaparib on the Expression of NKG2D Ligands in HL-60 Cells

To investigate the regulatory effects of Olaparib on natural killer cell activating receptor (NKG2D) ligands expression on human acute myeloid leukemia (AML) cell line HL-60, and to explore the molecular mechanism of Olaparib on HL-60 cells. After HL-60 cells in logarithmic growth phase were treated with Olaparib at different concentrations for different times (24, 48 h), the expression of NKG2D ligand on the surface of HL-60 cells was detected by flow cytometry. Western blot was used to dectect the expression of ERK expression in HL-60 cells. The killing effect of NK cells to HL-60 cells was detected by CFSE/PI method. 10 μmol/L Olaparib could upregulate the expression of NKG2D ligand on the surface of HL-60 cell at 24 and 48 hours, while 5 μmol/L Olaparib could induce up-regulation of the expression of ULBP-2 and ULBP-3 at 48 hours. Western blot analysis showed that ERK phosphorylation of HL-60 cells was enhanced after treating with Olaparib. The killing effect of NK cells to HL-60 cells could be enhanced by Olaparib, however, ERK inhibitor could suppress the killing effect of NK cells to HL-60 cells. Olaparib can upregulate NKG2D ligands expression on the surface of HL-60 cells and enhance the cytotoxicity of NK cell to HL-60 cells. The mechanism may be related to Olaparib promoting ERK phosphorylation expression.

B7-H3 confers resistance to Vγ9Vδ2 T cell-mediated cytotoxicity in human colon cancer cells via the STAT3/ULBP2 axis.

Immunotherapy based on γδT cells has limited efficiency in solid tumors, including colon cancer (CC). The immune evasion of tumor cells may be the main cause of the difficulties of γδT cell-based treatment. In the present study, we explored whether and how B7-H3 regulates the resistance of CC cells to the cytotoxicity of Vγ9Vδ2 (Vδ2) T cells. We observed that B7-H3 overexpression promoted, while B7-H3 knockdown inhibited, CC cell resistance to the killing effect of Vδ2 T cells in vitro and in vivo. Mechanistically, we showed that B7-H3-mediated CC cell resistance to the cytotoxicity of Vδ2 T cells involved a molecular pathway comprising STAT3 activation and decreased ULBP2 expression. ULBP2 blockade or knockdown abolished the B7-H3 silencing-induced increase in the cytotoxicity of Vδ2 T cells to CC cells. Furthermore, cryptotanshinone, a STAT3 phosphorylation inhibitor, reversed the B7-H3 overexpression-induced decrease in ULBP2 expression and attenuated the killing effect of Vδ2 T cells on CC cells. Moreover, there was a negative correlation between the expression of B7-H3 and ULBP2 in the tumor tissues of CC patients. Our results suggest that the B7-H3-mediated STAT3/ULBP2 axis may be a potential candidate target for improving the efficiency of γδT cell-based immunotherapy in CC.

Effect of Chidamide on the Killing Acitivity of NK Cells Targeting K562 Cells and Its Related Mechanism In Vitro

To investigate the effect of chidamide on the killing activity of NK (Natural killer cell, NK) cells targeting K562 cells and its related mechanism. K562 cells were pretreated with chidamide at different concentrations and cocultured with NK cells at different effect-target ratios. The killing effect of chidamide on K562 cells by NK cells, the expression of natural killer group 2 member D (NKG2D) ligands and apoptosis rate of K562 cells were detected by flow cytometry. The killing sensitivity of NK cells to K562 cells could be enhanced by chidamide. The expression of ULBP2 on K562 cell surface could be up-regulate, however, the expression of ULBP1 and MICA/MICB showed no statistically difference as compared with control group. Chidamide showed no obvious cytotoxicity to K562 cells. Chidamide can significantly improve killing efficiency of NK cells on K562 cells, which may be related to the up-regulation of ULBP2 expression.

Entinostat augments NK cell functions via epigenetic upregulation of IFIT1-STING-STAT4 pathway.

Histone deacetylase inhibitors (HDACi) are an emerging cancer therapy; however, their effect on natural killer (NK) cell-mediated anti-tumor responses remain unknown. Here, we evaluated the impact of a benzamide HDACi, entinostat, on human primary NK cells as well as tumor cell lines. Entinostat significantly upregulated the expression of NKG2D, an essential NK cell activating receptor. Independently, entinostat augmented the expression of ULBP1, HLA, and MICA/B on both rhabdomyosarcoma and Ewing sarcoma cell lines. Additionally, entinostat increased both cytotoxicity and IFN-γ production in human NK cells following coculture with these tumor cells. Mechanistically, entinostat treatment resulted in increased chromatin accessibility to the promoter region for interferon-induced protein with tetratricopeptide repeats 1 (IFIT1) gene and thereby increasing the transcript and protein levels of IFIT1 that augmented the IFIT1-mediated IRF1, STAT4, and STING pathways. Corresponding transcriptome analysis revealed enrichment of IRF1 and STAT4 and gene sets responsible for NK cell-mediated IFN-γ production and cytotoxicity, respectively. Our results show a novel mechanism by which entinostat initiates an IFIT1-STING-mediated potentiation of STAT4 via IRF1 to augment NK cell-mediated anti-tumor responses.