Expression Of ULBP1 Research Articles

MICA/B and ULBP1 NKG2D ligands are independent predictors of good prognosis in cervical cancer.

BackgroundNKG2D (natural killer group 2, member D) is thought to play an important role in mediating the activation of anticancer immune response. Expression of NKG2D ligands (NKG2DLs) is pronounced in malignancies and the heterogeneity of NKG2DL expression remains unclear. Here, we investigate the expression and clinical significance of NKG2DLs in cervical cancer.MethodsImmunohistochemical analyses of MICA/B, ULBP1, ULBP2, ULBP3, RAET1E, and RAET1G were performed using tissue microarray analysis of 200 cervical cancers, 327 high-grade cervical intraepithelial neoplasias (CINs), 99 low-grade CINs, and 541 matched nonadjacent normal cervical epithelial tissues and compared the data with clinicopathologic variables, including the survival of cervical cancer patients.ResultsMICA/B, ULBP1, and RAET1E expression was higher in cervical cancer than in low-grade CIN (p < 0.001, p = 0.012, p = 0.013, respectively) and normal cervix (all p < 0.001). Among these markers, expression of ULBP1 was significantly different depending on patient tumor stage (p = 0.010) and tumor size (p = 0.045). ULBP1 expression was correlated with MICA/B (p < 0.001) and ULBP2 (p = 0.002) expression in cervical cancer. While MICA/B+ or ULBP1+ patients had improved disease-free survival time (p = 0.027 and p = 0.009, respectively) relative to that of the low expression group, RAET1E+ or RAET1G+ was correlated with shorter survival time (p = 0.018 and p = 0.029, respectively). However, in terms of overall survival, the ULBP1+ group had significantly longer survival time than the low expression group (p = 0.009). Multivariate analysis indicated that MICA/B+/ULBP1+ (HR = 0.16, p = 0.015) and ULBP1+ (HR = 0.31, p = 0.024) are independent prognostic factors of disease-free survival in cervical cancer.ConclusionsHigh expression of either ULBP1 or MICA/B and ULBP1 combined is an indicator of good prognosis in cervical cancer, suggesting their potential utility as prognostic tests in clinical assessment.

Abstract 3669: IDH mutant glial cell resistance to natural killer cell cytotoxicity

Abstract Mutations in IDH 1 and 2 are common in gliomas. Methylation-related epigenetic repression of gene expression is a feature of IDH mutant (IDHmut) tumors. Epigenetic repression of NK cell ligands is a common occurrence in cancer, but it is unknown if this occurs in gliomas. Here, we demonstrate transcriptional repression of NKG2D activating ligands (NKG2DLs) in IDHmut tumors, and correlate this with decreased susceptibility to natural killer (NK) cell-mediated cytolysis in vitro We compared IDHmut and IDHwt gene expression values for annotated immune response genes and examined NKG2DL HM450 mean methylation values in glioma patients using TCGA data. cDNA was synthesized from cultured IDHwt or IDHmut astrocytes. NKG2DL expression was assayed using primers for NKG2D ligands ULBP1, ULBP2, ULBP3 and RAET1G. IDHwt astrocytes, U87 cells and IDHmut astrocytes were cultured with activated NK cells. Cytotoxicity was measured by flow cytometry. mRNA expression values for NKG2DLs ULBP1 and ULBP3 were significantly reduced in IDHmut gliomas. Conversely, methylation values were significantly increased. NKG2DL hierarchical clustering of gene expression of shows genotype-based expression clusters. IDHmt astrocytes expressed a two to eight-fold reduction in the relative gene expression of NKG2DLs compared with IDHwt astrocyte. Cytotoxicity is significantly lower in IDHmut astrocytes/NK cell co-cultures compared with IDHmut/NK or U87/NK co-cultures. Our data demonstrates evidence for the transcriptional repression of NKG2DLs in IDHmut gliomas in silico and in vitro. Reduced NKG2D levels in IDHmut cells correlates with impaired NK-mediated cytotoxicity and suggests a role for NK cell resistance in IDH mutant tumors. Further studies are in progress to utilize HSV vectors that express ULBP3 to determine whether restoration of ULBP3 expression in cancer cells will enhance susceptibility to NK activation and tumor cell killing. Results of these experiments will be reported. Citation Format: Xiaoran Zhang, Yigang Chang, Aofei Li, Joseph C. Glorioso, Paola Grandi, Mike Lotze, Nduka Amankulor. IDH mutant glial cell resistance to natural killer cell cytotoxicity. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3669. doi:10.1158/1538-7445.AM2014-3669

COX-2- and endoplasmic reticulum stress-independent induction of ULBP-1 and enhancement of sensitivity to NK cell-mediated cytotoxicity by celecoxib in colon cancer cells

In the present study, we investigated whether celecoxib could induce the expression of NKG2D ligands in clonogenic colon cancer cells, and increase their susceptibility to NK cell-mediated cell death. Celecoxib and its non-coxib analog, 2,5-dimethyl celecoxib, induced ULBP-1 and DR5 in both COX-2 negative HCT-15 cells and COX-2 positive HT-29 cells. Celecoxib increased their susceptibility to NK92 cells in both DELFIA assay and soft agar colony forming assay. The inducibility of ULBP-1 and DR5 by celecoxib was not different between CD44- and CD44+ HCT-15 cells, and CD133- and CD133+ HT-29 cells. Celecoxib increased the susceptibility of highly clonogenic CD44+ HCT-15 and CD133+ HT-29 cells to NK92 cells, at least comparable to less clonogenic CD44- HCT-15 and CD133- HT-29 cells, respectively. In addition, celecoxib induced CHOP, and thapsigargin, an inducer of ER (endoplasmic reticulum) stress, induced DR5 but not ULBP1 in HCT-15. Taken together, these findings suggest that celecoxib induces the expression of ULBP-1 as well as DR5 in clonogenic colon cancer cells via COX-2 and ER stress-independent pathways, and increases their susceptibility to NK cells.

Up-regulation of NKG2D ligand ULBP2 by matrine in K562 cells and the underlying molecular mechanisms

To probe matrine acting on natural killer cell (NK) activating receptor NKG2D ligands expression in CML cell line K562 and its underlying molecular mechanism. The expression of NKG2D ligands (major histocompatibility complex class I chain-related molecule A or B (MICA/B), UL16-binding proteins (ULBP) 1, 2, and 3 on K562 cells were analyzed before and after treated with matrine by FCM. The cytotoxic sensitivity of K562 to NK cell was detected by FCM after CFSE staining at different effect-to-target (E/T) cell ratios. The expression of signal transduction and transcriptional activator 3 (STAT3) protein as well as phosphorylated STAT3 (p-STAT3) were detected by western blot. After treatment with matrine, ULBP1 and ULBP2 expression, especially ULBP2 on K562 cells significantly increased, with mean fluorescence intensity (MFI) increasing to 615 and 1614 by 220 and 615 in the untreated cells, respectively. There was no significant change for MICA or ULBP3 expression. Matrine enhanced the susceptibility of K562 cells to NK-mediated cell lysis. At the ratio of E/T with 5:1, the proportion of the killed K562 cells increased to 32.8%, 38.1% and 40.5%, respectively (after 0.2, 0.5 and 0.8 mg/ml matrine treatment) by 29.2% in the untreated cells. The phosphorylated STAT3 protein, but not STAT3 protein, was significantly inhibited by matrine treatment in K562 cells. Matrine induced the expression of NKG2D ligands in K562cells and enhanced the cytotoxicity of NK cells against K562, which was closely related to the inhibition of STAT3 activity in K562 cell.

Recognition of adult and pediatric acute lymphoblastic leukemia blasts by natural killer cells

In this study, we aimed to investigate the pathways of recognition of acute lymphoblastic leukemia blasts by natural killer cells and to verify whether differences in natural killer cell activating receptor ligand expression among groups defined by age of patients, or presence of cytogenetic/molecular aberrations correlate with the susceptibility to recognition and killing. We analyzed 103 newly diagnosed acute lymphoblastic leukemia patients: 46 adults and 57 children. Pediatric blasts showed a significantly higher expression of Nec-2 (P=0.03), ULBP-1 (P=0.01) and ULBP-3 (P=0.04) compared to adult cells. The differential expression of these ligands between adults and children was confined to B-lineage acute lymphoblastic leukemia with no known molecular alterations. Within molecularly defined subgroups of patients, a high surface expression of NKG2D and DNAM1 ligands was found on BCR-ABL(+) blasts, regardless of patient age. Accordingly, BCR-ABL(+) blasts proved to be significantly more susceptible to natural killer-dependent lysis than B-lineage blasts without molecular aberrations (P=0.03). Cytotoxic tests performed in the presence of neutralizing antibodies indicated a pathway of acute lymphoblastic leukemia cell recognition in the setting of the Nec-2/DNAM-1 interaction. These data provide a biological explanation of the different roles played by alloreactive natural killer cells in pediatric versus adult acute lymphoblastic leukemia and suggest that new natural killer-based strategies targeting specific subgroups of patients, particularly those BCR-ABL(+), are worth pursuing further.

Natural Killer Cell-Mediated Shedding of ULBP2

UL16 binding proteins (ULBPs) are a family of cell surface proteins that are present in transformed and stressed cells and ligands for NKG2D. Soluble NKG2D ligands have been found in sera from cancer patients with their protein concentrations correlated with poor cancer prognosis. Here we show, for the first time, that human tumor cells lost their surface expression of ULBP2, but not ULBP1 and ULBP3, during NK cell-mediated cytolysis. In contrast to spontaneous shedding of NKG2D ligands, NK cytolysis-mediated shedding of ULBP2 was linked to target cell apoptosis, although both resulted from metalloproteinase cleavages. Inhibition of ULBP2 shedding by a metalloproteinase inhibitor BB-94 lead to reduced NK cell-mediated cytotoxicity and cytokine production. These results illustrate a regulation of NK cell effector functions through cytolysis-induced NKG2D ligand shedding. Consequently, compounds inhibiting NKG2D ligand shedding may offer therapeutic means to reduce excessive pathogenic NK cell activities.

A disintegrin and metalloproteinases 10 and 17 modulate the immunogenicity of glioblastoma-initiating cells

There are emerging reports that the family of a disintegrin and metalloproteinases (ADAM) are involved in the maintenance of the malignant phenotype of glioblastomas. Notably, ADAM proteases 10 and 17 might impair the immune recognition of glioma cells via the activating immunoreceptor NKG2D by cleavage of its ligands from the cell surface. Glioblastoma-initiating cells (GIC) with stem cell properties have been identified as an attractive target for immunotherapy. However, GIC immunogenicity seems to be low. Here,we show that ADAM10 and ADAM17 are expressed on the cell surface of GIC and contribute to an immunosuppressive phenotype by cleavage of ULBP2. The cell surface expression of ULBP2 is enhanced upon blocking ADAM10 and ADAM17, and treatment with ADAM10 and ADAM17specific inhibitors leads to enhanced immunerecognition of GIC by natural killer cells. Therefore, ADAM10 and ADAM17 constitute suitable targets to boost an immune response against GIC.

CEBPA Gene Different Mutants Play Distinct Impacts On Tumor Suppressor Function Of C/EBP-Alpha and Bone Marrow Stromal Cells

IntroductionCEBPA gene encodes CCAAT/enhancer-binding protein-alpha (C/EBPα), a crucial granulocytic differentiation factor and tumor suppressor in hematologic and non-hematologic malignancies. CEBPA gene is mutated in approximately 5–14% of acute myeloid leukemia (AML) patients and exhibited 3 different types of mutations, germ-line N-terminal mutation, N-terminal frameshift mutation and C-terminal mutation. Although murine models provide the functional consequences of CEBPA gene mutations in human hematopoiesis, the impacts of different mutations on abrogating C/EBPα functions and those of germ-line N-terminal mutations on bone marrow microenvironment have been received less attention. MethodsIn our previous research, we have provided the first report of multiple mutations of CEBPA contributing to the transformation of donor cells to the leukemic phenotype after allogeneic hematopoietic stem cell transplantation and identified 3 different CEBPA gene mutant forms which disrupted 3 major functional domains of C/EBPα respectively (H Xiao, Blood 2011; 117: 5257-5260). The patient and his donor-sister both harbored the N-terminal germ-line mutation (584_589dup disrupting the TAD2 domain of protein). Susceptible donor hematopoietic cells evolved to overt AML by developing two somatic CEBPA mutations, the N-terminal frameshift mutation (247dupC causing overproduction of truncated 30-KDa isoform (C/EBPα- p30) lacking the TAD1 domain) and the C-terminal mutation (914_916dup disrupting the bZIP domain), in the patient's microenvironment. We used these 3 mutant forms, as well as CEBPA gene wild type to subclone into pLenti6.3-MSC vector. Human leukemia cell lines (NB4, Kasumi-1, HL60, K562) and mouse myeloid progenitor cell line (32Dcl3) were transduced by different mutant forms to assess the impacts on blockage the major functions of C/EBPα including antileukemia effect and inducing granulocyte differentiation. To further discover previously unkown tumor suppressor genes dysregulated by C/EBPα mutant forms, the gene expression profiles of NB4 cells stably transfected with CEBPA gene different mutant forms were compared with that of CEBPA gene wild type vector by using Affymetrix PrimeView human gene expression microarray analysis. On the other hand, human normal bone marrow mesenchymal stromal cell ( MSC ) was transduced by N-terminal germ-line mutation to assess the impacts on the capacity of MSC to differentiate towards the osteogenic/adipogenic lineages, migrate and protect to leukemia cells. Results(1) The N-terminal germ-line mutation retains the functions of inducing apoptosis in leukemic cells and granulocyte differentiation of C/EBPα.The truncated C/EBPα-p30 protein mutant, produced from the N-terminal frameshift mutation, abrogates the effect of inducing apoptosis in leukemia cells. The C-terminal mutation (914_916 dup) abrogates both the effects of inducing apoptosis in leukemic cells and of promoting G-CSF-induced differentiation of 32Dcl3 cells into mature neutrophile granulocyte. (2) Gene expression microarray profiling analysis showed that compared with CEBPA gene wild type, leukemia-associated CEBPA somatic mutations, the N-terminal frameshift mutation and the C-terminal mutation, significantly inhibited the expression of ULBP2, an innate surface ligand of the natural killer (NK) cell receptor NKG2D, ultimately contributed to resistance to NK cell-mediated cytotoxicity. (3) MSCs harboring CEBPA N-terminal germ-line mutation showed impaired differentiation potential to osteogenic lineage by downregulation the expression of osteogenic genes (BSP1 and Runx2) , however similar in differentiation potential to adipogenic lineage, migration and the protection to leukemia cells, compared with MSCs with CEBPA wild type. ConclusionsOur data provide clues to support that the N-terminal frameshift mutation of CEBPA works as a class I mutation, while the C-terminal mutation works as both of class I and class II mutations in inducing leukemia. Furthermore, those leukemia-associated CEBPA somatic mutations abrogate the tumor suppressor function of C/EBPα by inhibiting NKG2D-mediated NK cell cytotoxicity. The N-terminal germ-line mutation retains the major functions of C/EBPα, however impaired the osteogenic differentiation potential of bone marrow stromal cells, which is important to support hematopoiesis. Disclosures:No relevant conflicts of interest to declare.

Enhanced interaction between natural killer cells and lung cancer cells: involvement in gefitinib-mediated immunoregulation

BackgroundNatural killer (NK) cells can kill tumor cells in a non-MHC-restricted manner. However, cancer cells frequently escape from the attack of NK cells by multiple ways. In this study, we investigated the effect of gefitinib on the interaction between NK cells and lung cancer cells.Methods51Cr release assay, CD107a assay, and IFN-γ secretion assay were performed to detect the sensitivity of lung cancer cell lines A549 and H1975 to NK cells cytotoxicity in the presence of gefitinib. Human NK cells were co-cultured with A549 and H1975 cell lines in the presence of gefitinib. NKG2D ligands, ULBP1, ULBP2, MICA, and MHC-I on tumor cells, and NKG2D, NKp44 and NKp46 on NK cells were evaluated with flow cytometry. 51Cr release assay was performed when NKG2D antibody were added into the co-culture system. Expressions of stat3 and LC3 I/II on tumor cells were determined with western blot after co-cultured with NK cells. After treated with gefitinib, mannose-6-phosphate receptor (MPR) on H1975 cells was evaluated by flow cytometry. 51Cr release assay were performed when MPR antagonist were used.ResultsGefitinib increased cytotoxicity of NK cells to human lung cancer H1975 cells with EGFR L858R + T790M mutations, while not in A549 cells with wild type EGFR. Gefitinib could block the immune escape by up-regulating the expression of NKG2D ligands ULBP1, ULBP2 or MICA on tumor cells and NKG2D on NK cells in the co-culture system. Gefitinib and NK cells up-regulated MHC-I expression in A549 while not in H1975 cells. NKG2D antibody blocked the enhanced NK cytotoxicity by gefitinib. The combination of NK cells and gefitinib could significantly down-regulate stat3 expression. Furthermore, NK cells-mediated tumor cell autophagy was observed in A549 cells while not in H1975 cells. Notably, gefitinib increased autophagy and MPR expression in H1975 cells, which improved the sensitivity to NK cell-based immunotherapy.ConclusionsGefitinib greatly enhanced NK cell cytotoxicity to lung cancer cells with EGFR L858R + T790M resistance mutation. Combination of EGFR tyrokinase inhibitors and NK cells adoptive immunotherapy may represent a potentially effective strategy for patients with non-small cell lung cancer.

DNA methylation in nasal epithelial cells from smokers: identification of ULBP3-related effects

We previously demonstrated that, in nasal epithelial cells (NECs) from smokers, methylation of an antiviral gene was associated with impaired antiviral defense responses. To expand these findings and better understand biological mechanisms underlying cigarette smoke (CS)-induced modifications of host defense responses, we aimed to compare DNA methylation of genes that may play a role in antiviral response. We used a two-tiered analytical approach, where we first implemented a genome-wide strategy. NECs from smokers differed in the methylation levels of 390 genes, the majority (84%) of which showed decreased methylation in smokers. Secondly, we generated an a priori set of 161 antiviral response-related genes, of which five were differentially methylated in NEC from smokers (CCL2, FDPS, GSK3B, SOCS3, and ULBP3). Assessing these genes at the systems biology level revealed a protein interaction network associated with CS-induced epigenetic modifications involving SOCS3 and ULBP3 signaling, among others. Subsequent confirmation studies focused on SOCS3 and ULBP3, which were hypomethylated and hypermethylated, respectively. Expression of SOCS3 was increased, whereas ULBP3 expression was decreased in NECs from smokers. Addition of the demethylating agent 5-Aza-2-deoxycytidine enhanced ULBP3 expression in NECs from smokers. Furthermore, infection of differentiated NECs with influenza virus resulted in significantly lower levels of ULBP3 in cells from smokers. Taken together, our findings show that genomic DNA methylation profiles are altered in NECs from smokers and that these changes are associated with decreased antiviral host defense responses, indicating that epigenenic dysregulation of genes such as SOCS3 and ULBP3 likely impacts immune responses in the epithelium.

Viral protein R upregulates expression of ULBP2 on uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes

HIV-1 Vpr triggers NK cell-mediated lysis of infected cells by upregulating ULBP2, a ligand of the NKG2D receptor, through activation of the ATR-mediated DNA damage response. Herein, we demonstrate that Vpr augments ULBP2 expression on both infected and uninfected bystander cells during HIV-1 infection of primary CD4+ T lymphocytes. Indeed, the frequency of uninfected bystander cells expressing high levels of ULBP2 was elevated in a Vpr-dependent manner. Nevertheless, the same does not hold true for a Vpr mutant that is not packaged into virions, suggesting the involvement of virion-associated Vpr in this process. Additionally, we show that soluble Vpr has the ability to induce a DNA damage response and to augment cell-surface ULBP2 upon transducing target cells, including T cells, conditions known to promote NK cell-mediated killing. Overall, these findings suggest that Vpr could contribute to CD4+ T cell loss by rendering uninfected bystander cells susceptible to NK cell-mediated killing.

Preliminary study on the changes of NKG2D expression and its role in children with infectious mononucleosis

Objective To investigate the mechanism of disturbed immunological function induced by Epstein-Barr virus(EBV) infection through evaluating NKG2D expressions in NK cells and CD8+ T lymphocytes from children with infectious mononucleosis.Methods Twenty-nine children with infectious mononucleosis (IM) and twenty-five age-matched healthy children were enrolled in the study.NKG2D/NKG2A expressions in NK cells and CD8+T lymphocytes,and NKG2D ligand MHC Ⅰ chain-related molecules A(MICA) and UL-16binding proteins (ULBP-1) expressions on CD14+ mononuclear cells (MC) were analyzed by flow cytometry.The concentrations of cytokines such as IL-7,IL-12,IL-15,IFN-γ,TGF-β and soluble MICA(sMICA) in plasma were determined by enzyme-linked immunosorbent assay (ELISA).Results (1) Compared with the control group,NKG2D expression was significantly decreased in both NK cells and CD8+T lymphocytes from children with IM (P＜0.05),especially in the three cases of suspected EVB-HLH children.(2)There was no difference in the expression of MICA and ULBP-1 in CD14+ MC between the children with IM and the normal control(P＞0.05).(3)The concentrations of IL-15 and TGF-β in plasma were lower than those of the control group (P＜0.05),while the levels of IL-7,IL-12,IFN-γ and sMICA were up-regulated(P＜0.05) in children with IM.(4)NKG2A expression in NKcells in children with IM was significantly increased as compared with healthy controls(P＜0.05),however,there was no differences in the expression of NKG2A in CD8+ T lymphocytes between the two groups (P＞0.05).Conclusion Remarkable down-regulated expressions of NKG2D in NK cells and CD8+T lymphocytes might be one of the factors causing disturbed immunological function in children with EBV infection,which might have a close relationship with abnormal cytokine milieu of IL-15 and IL-12or high concentration of sMICA. Key words: Epstein-Barr virus; Infectious mononucleosis; NKG2D

Soluble ligands for the NKG2D receptor are released during HIV‐1 infection and impair NKG2D expression and cytotoxicity of NK cells

In humans, the interaction of the natural killer group 2 member D (NKG2D)-activating receptor on natural killer (NK) and CD8(+) T cells with its major histocompatibility complex class I-related chain (MIC) and UL16 binding protein (ULBP) ligands (NKG2DLs) promotes recognition and elimination of stressed cells, such as tumor or infected cells. Here, we investigated the capacity of HIV-1 to modulate NKG2DL expression and escape NGK2D-mediated immunosurveillance. In CD4(+) T lymphocytes, both cell surface expression and release of MICA, MICB, and ULBP2 were up-regulated >2-fold by HIV-1 infection. In HIV-infected CD4(+) T lymphocytes or Jurkat T-cell lines, increased shedding of soluble NKG2DLs (sNKG2DLs) was impaired by a matrix metalloproteinase inhibitor (MMPI). Moreover, naive HIV(+) patients displayed increased plasma sMICA and sULBP2 levels and reduced NKG2D expression on NK and CD8(+) T cells compared to patients receiving highly active antiretroviral therapy (HAART) or healthy donors. In individual patients, HAART uptake resulted in the drop of sNKG2DL and recovery of NKG2D expression. Finally, sNKG2DLs in patients' plasma down-regulated NKG2D on NK and CD8(+) T cells and impaired NKG2D-mediated cytotoxicity of NK cells. Thus, NKG2D detuning by sNKG2DLs may promote HIV-1 immune evasion and compromise host resistance to opportunistic infections, but HAART and MMPI have the potential to avoid such immune dysfunction.

Valproic acid upregulates NKG2D ligand expression and enhances susceptibility of human renal carcinoma cells to NK cell-mediated cytotoxicity

IntroductionWe aimed to investigate the effect of valproic acid (VPA) on NKG2D ligand expression in human renal carcinoma cell lines and to investigate the mechanisms.Material and methodsDifferent concentrations of VPA from 0.5 mM to 8.0 mM were applied to 786-O and ACHN cell lines, respectively. Cell viability after treatment with VPA was determined by flow cytometry (FCM). Real-time PCR and FCM were used to detect the changes of mRNA and protein level of NKG2D ligands (MICA/B and ULBPs) in the two cell lines treated with 4 mM VPA. The cytotoxicity assay and CD107a mobilization assay were carried out to detect the cytotoxicity changes of NK cells against renal carcinoma cell lines after the same treatment.ResultsValproic acid can efficiently upregulate MICA/B, ULBP1 and ULBP2 expression in the renal carcinoma cell lines at the mRNA and protein level (p < 0.05). 786-O and ACHN cells treated with VPA were more susceptible to killing by NK cells than untreated cells and the enhanced cytotoxicity of NK cells was blocked by the pretreatment of NK cells with anti-NKG2D monoclonal antibodies (p < 0.05).ConclusionsValproic acid can clearly induce the expression of NKG2D ligands of renal carcinoma cell lines, thereby enhancing the cytotoxicity of NK cells against renal carcinoma cell lines.

Valproic Acid Upregulates NKG2D Ligand Expression through an ERK-dependent Mechanism and Potentially Enhances NK Cell-mediated Lysis of Myeloma

Modulation of the antitumor immune response through the engagement of NKG2D receptors with their ligands (L) on targets represents a promising therapeutic approach against cancer. In this study, we tested the effect of valproic acid (VPA), a histone deacetylase (HDAC) inhibitor, on the expression of NKG2D ligands in myeloma cells. We demonstrated that VPA was able to upregulate both protein and mRNA expression of major histocompatibility complex class I-related chain (MIC) A/B and UL16-binding protein (ULBP) 2 without any significant effect on the expression of ULBP1, ULBP3, and ULBP4 or induction of other natural killer (NK) cell ligands, such as NKp30-L, NKp44-L, and NKp46-L in myeloma cells. A (51)Cr release assay and degranulation assay indicated that the induction of MICA/B and ULBP2 augmented NK cell-mediated lysis of myeloma cells, which was abolished by the addition of a blocking NKG2D antibody. Activation of constitutively phosphorylated extracellular signal-regulated kinase (ERK) by VPA is essential for the up-regulation of MICA/B and ULBP2 expressions. Inhibition of ERK using ERK inhibitor PD98059 decreased both MICA/B and ULBP2 expressions and NK cell cytotoxicity. Furthermore, overexpression of constitutively active ERK in ARK resulted in increased MICA/B and ULBP2 expressions and enhanced NK cell lysis. These data indicate that increased sensitivity of VPA-treated myeloma cells to NK cell lysis is caused by higher NKG2D ligand expression, resulting from more active ERK signaling pathway. Our results provide evidence that targeting ERK signaling pathway may be an additional mechanism supporting the antimyeloma activity of HDAC inhibitors and suggest its possible immunotherapeutic value for myeloma treatment.

Regulation of NKG2D-ligand cell surface expression by intracellular calcium after HDAC-inhibitor treatment

In this study we demonstrate that histone deacetylase (HDAC)-inhibitor mediated cell surface expression of the structural different NKG2D-ligands MICA/B and ULBP2 is calcium-dependent. Treatment with the calcium chelator EGTA inhibited constitutive as well as HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression on melanoma cells and Jurkat T-cells. A NKG2D-dependent cytolytic assay and staining with a recombinant NKG2D-Fc fusion protein showed that calcium chelation impaired the functional ability of NKG2D-ligands induced by HDAC-inhibitor treatment.The HDAC-inhibitor induced cell surface expression of ULBP2, but not MICA/B, was sensitive to treatment calmidazolium and trifluoperazine, two agents known to block calcium signaling. siRNA-mediated knock-down of the calcium-regulated proteins calmodulin or calpain did however not affect NKG2D-ligand cell surface expression on Jurkat T-cells. We further show that secretion and cell surface binding of the calcium-regulating protein galectin-1 is enhanced upon HDAC-inhibitor treatment of melanoma cells. However, binding of galectin-1 to cell surface glycoproteins was not critical for constitutive or HDAC-inhibitor induced MICA/B and ULBP2 cell surface expression.We provide evidence that MICA/B and ULBP2 cell surface expression is controlled differently by calcium, which adds to the increasing perception that cell surface expression of MICA/B and ULBP2 is controlled by distinct signal transduction pathways.

Ras Activation Induces Expression of Raet1 Family NK Receptor Ligands

NK cells play a crucial role in innate immunity against tumors. In many human tumors, Ras is chronically active, and tumor cells frequently express ligands for the activating NK cell receptor NKG2D. In this study, we report that Ras activation upregulates the expression of Raet1 protein family members Rae1α and Rae1β in mouse and ULBP1-3 in human cells. In addition, Ras also induced MHC class I chain-related protein expression in some human cell lines. Overexpression of the constitutively active H-RasV12 mutant was sufficient to induce NKG2D ligand expression. H-RasV12-induced NKG2D ligand upregulation depended on Raf, MAPK/MEK, and PI3K, but not ATM or ATR, two PI3K-like kinases previously shown to induce NKG2D ligand expression. Analysis of the 5' untranslated regions of Raet1 family members suggested the presence of features known to impair translation initiation. Overexpression of the rate-limiting translation initiation factor eIF4E induced Rae1 and ULBP1 expression in a Ras- and PI3K-dependent manner. Upregulation of NKG2D ligands by H-RasV12 increased sensitivity of cells to NK cell-mediated cytotoxicity. In summary, our data suggest that chronic Ras activation is linked to innate immune responses, which may contribute to immune surveillance of H-Ras transformed cells.

NKG2D ligand tumor expression and association with clinical outcome in early breast cancer patients: an observational study

BackgroundCell surface NKG2D ligands (NKG2DL) bind to the activating NKG2D receptor present on NK cells and subsets of T cells, thus playing a role in initiating an immune response. We examined tumor expression and prognostic effect of NKG2DL in breast cancer patients.MethodsOur study population (n = 677) consisted of all breast cancer patients primarily treated with surgery in our center between 1985 and 1994. Formalin-fixed paraffin-embedded tumor tissue was immunohistochemically stained with antibodies directed against MIC-A/MIC-B (MIC-AB), ULBP-1, ULBP-2, ULBP-3, ULBP-4, and ULBP-5.ResultsNKG2DL were frequently expressed by tumors (MIC-AB, 50% of the cases; ULBP-1, 90%; ULBP-2, 99%; ULBP-3, 100%; ULBP-4, 26%; ULBP-5, 90%) and often showed co-expression: MIC-AB and ULBP-4 (p = 0.043), ULBP-1 and ULBP-5 (p = 0.006), ULBP-4 and ULBP-5 (p < 0.001). MIC-AB (p = 0.001) and ULBP-2 (p = 0.006) expression resulted in a statistically significant longer relapse free period (RFP). Combined expression of these ligands showed to be an independent prognostic parameter for RFP (p < 0.001, HR 0.41). Combined expression of all ligands showed no associations with clinical outcome.ConclusionsWe demonstrated for the first time that NKG2DL are frequently expressed and often co-expressed in breast cancer. Expression of MIC-AB and ULBP-2 resulted in a statistically significant beneficial outcome concerning RFP with high discriminative power. Combination of all NKG2DL showed no additive or interactive effect of ligands on each other, suggesting that similar and co-operative functioning of all NKG2DL can not be assumed. Our observations suggest that among driving forces in breast cancer outcome are immune activation on one site and tumor immune escape on the other site.

Tumor Suppressive MicroRNAs miR-34a/c Control Cancer Cell Expression of ULBP2, a Stress-Induced Ligand of the Natural Killer Cell Receptor NKG2D

Malignant cells express ligands for the natural killer cell immunoreceptor NKG2D, which sensitizes to early recognition and elimination by cytotoxic lymphocytes and provides an innate barrier against tumor development. However, the mechanisms that control NKG2D ligand (NKG2DL) expression in tumor cells remain unknown. We recently identified the NKG2DL ULBP2 as strong prognostic marker in human malignant melanoma. Here, we provide evidence that the tumor-suppressive microRNAs (miRNA) miR-34a and miR-34c control ULBP2 expression. Reporter gene analyses revealed that both miRNAs directly targeted the 3'-untranslated region of ULBP2 mRNA and that levels of miR-34a inversely correlated with expression of ULBP2 surface molecules. Accordingly, treatment of cancer cells with miRNA inhibitors led to upregulation of ULBP2, whereas miR-34 mimics led to downregulation of ULBP2, diminishing tumor cell recognition by NK cells. Treatment with the small molecule inhibitor Nutlin-3a also decreased ULBP2 levels in a p53-dependent manner, which was due to a p53-mediated increase in cellular miR-34 levels. Taken together, our study shows that tumor-suppressive miR-34a and miR-34c act as ULBP2 repressors. These findings also implicate p53 in ULBP2 regulation, emphasizing the role of the specific NKG2DL in tumor immune surveillance.

Dynamic Shift from CD85j/ILT-2 to NKG2D NK Receptor Expression Pattern on Human Decidual NK during the First Trimester of Pregnancy

During the first trimester of human pregnancy, Natural Killer (NK) cells of the maternal uterine mucosa (e.g. decidua) have a unique phenotype and are involved in crucial physiological processes during pregnancy. We investigated whether modifications of the NK receptor repertoire occur during the first trimester of pregnancy. We found significantly decreased expression of KIR2DL1/S1 and KIR2DL2/L3/S2 receptors, NKp30 and NKp44 activatory receptors, and the CD85j (ILT-2) inhibitory receptor. We also observed significantly increased expression of the NKG2D activatory receptor at the decidual NK cell surface. By flow cytometry, we further highlighted an evolution of NK subsets between 8 and 12 weeks of gestation, with a shift from the KIR2DL1/S1+/KIR2DL2/L3/S2+ subset towards the double negative subset, coupled with a decrease of the CD85j+/NKG2D− subset in favour of the CD85j−/NKG2D+ subset. Furthermore, cell surface expression of NK receptor ligands, including CD85j and NKG2D ligands, has been characterized by flow cytometry on decidual immune CD14+ and CD3+ cells. HLA-G, the high affinity ligand of CD85j, was detected on both cell types. In contrast, NKG2D ligands ULBP-2 ULBP-3 and MICA/B were not expressed on CD14+ and CD3+ cells, however a variable expression of ULBP-1 was observed. The ligand expression of KIR2DL1/S1 and KIR2DL2/L3/S2 was also analyzed: the HLA-C molecule was expressed at a low level on some CD14+ cells whereas it was not detected on CD3+ cell surface. NK receptor ligands are known to be also expressed on the invading placental trophoblast cells. Thus, the phenotypic evolutions of decidual NK cells described in this present study may preserve their activation/inhibition balance during the first trimester of pregnancy.