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Cutaneous lupus erythematosus (CLE) is an autoimmune skin disorder that is characterized by an anti-epidermal lymphocytic infiltrate invading the dermo-epidermal junction, causing an interface dermatitis (ID). Pathogenesis of CLE has been linked to activation of innate immunity. NKG2D is an innate immune receptor on NK cells and distinct T-cell populations. The NKG2D ligands MHC class I polypeptide-related sequence A and B (MICA, MICB) have been associated to CLE susceptibility. Our gene microarray analyses of chronic discoid lupus erythematosus (CDLE) skin lesions, separated in epidermal, junctional and dermal skin areas via laser microdissection, revealed a high expression of NKG2D in the lymphocytic infiltrate and led us to further investigate the role of NKG2D in CLE. Pathway analyses showed a strong "interferon (IFN) signature" and vast activation of innate immune response pathways (TLR, RIG-I, cytosolic DNA sensing, JAK/STAT) in CDLE, that expressed the high NKG2D signal. Immunohistochemistry (IHC) confirmed the presence of NKG2D and its ligand MICB in CDLE and subacute cutaneous lupus erythematosus (SCLE) lesions. Finally, HaCaT cells were stimulated with nucleic acids and extracted RNA was sequenced with Illumina HiSeq and showed that stressed keratinocytes express typical NKG2D ligands MICA/B and ULBP2. This study provides first evidence that NKG2D is present in CDLE and SCLE skin lesions and could be relevant for cytotoxicity in IFN-driven skin lesions with upregulated innate immune response pathways present in CLE. It could furthermore play a role in CLE inflammation promoted by keratinocytes under cell stress.

Staphylococcus aureus is among the leading causes of bacterial infections worldwide. The pathogenicity and establishment of S. aureus infections are tightly linked to its ability to modulate host immunity. Persistent infections are often associated with mutant staphylococcal strains that have decreased susceptibility to antibiotics; however, little is known about how these mutations influence bacterial interaction with the host immune system. Here, we discovered that clinical S. aureus isolates activate human monocytes, leading to cell-surface expression of immune stimulatory natural killer group 2D (NKG2D) ligands on the monocytes. We found that expression of the NKG2D ligand UL16-binding protein 2 (ULBP2) is associated with bacterial degradability and phagolysosomal activity. Moreover, S. aureus-induced ULBP2 expression was linked to altered host cell metabolism, including increased cytoplasmic (iso)citrate levels, reduced glycolytic flux, and functional mitochondrial activity. Interestingly, we found that the ability of S. aureus to induce ULBP2 and proinflammatory cytokines in human monocytes depends on a functional ClpP protease in S. aureus These findings indicate that S. aureus activates ULBP2 in human monocytes through immunometabolic mechanisms and reveal that clpP inactivation may function as a potential immune evasion mechanism. Our results provide critical insight into the interplay between the host immune system and S. aureus that has evolved under the dual selective pressure of host immune responses and antibiotic treatment. Our discovery of a immune stimulatory pathway consisting of human monocyte-based defense against S. aureus suggests that targeting the NKG2D pathway holds potential for managing persistent staphylococcal infections.

Cervical cancer (CC) remains a large burden in the developing countries. The tumor inhibitory role of miR-873 has been verified in a variety of cancers, however, whether miR-873 has a suppressive effect on CC remains unclear. The purpose of this study was to investigate the functional role of miR-873 in CC, as well as explore the underlying molecular mechanism. The prognostic values of miR-873 were assessed by Kaplan–Meier methods and cox regression models using the data which were downloaded from TCGA database. The expression of miR-873 was measured by RT-qPCR. Cell counting Kit-8, clone formation, and Transwell assays were used to assess the cell viability and metastasis, appropriately. The targeting relationship between miR-873 and ULBP2 was predicted by biological software and confirmed by dual luciferase reporter assay. Rescue assays were conducted to investigate whether miR-873 affects the phenotype of CC cells via regulating ULBP2. We observed that miR-873 was low-expressed in CC. Up-regulation of miR-873 notably restrained the proliferation, invasion and migration of C33a cells. Meanwhile, down-regulation of miR-873 in SiHa cells presented the opposite outcomes. ULBP2 was forecasted and certified as a target of miR-873. The results of rescue assays showed that overexpression of ULBP2 could restore the proliferation and motility of CC cells that inhibited by miR-873. MiR-873 suppressed the CC cells proliferation, invasion and migration via negatively regulating ULBP2, suggesting that miR-873 could serve as a valuable therapeutic target for CC therapy.

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.

The immune system plays important roles in pancreatic cancer. MHC class I-chain-related proteins A and B (MICA/B) and UL16-binding proteins (ULBPs) are known natural killer group 2D (NKG2D) ligands. Soluble NKG2D ligands can inhibit the activation of Natural killer (NK) cells. In pancreatic cancer, soluble ULBPs are relatively unstudied in contrast to soluble MICA/B. We examined the significance of soluble ULBPs, especially ULBP2, in pancreatic cancer. Soluble ULBP2 but neither soluble ULBP1 nor soluble ULBP3, was etected in the supernatants of pancreatic cancer cells. Soluble ULBP2 derived from pancreatic cancer cells could reduce the cytotoxicity of NK cells. Multivariate analysis demonstrated that serum soluble ULBP2 was a significant independent factor associated with poor overall survival (OS) in all pancreatic cancer patients, specifically in stage IV patients. In conclusion, pancreatic cancer-derived soluble ULBP2 might affect the prognosis in pancreatic cancer.

Background. The distinction between right-sided and left-sided colon adenocarcinoma has recently received considerable. This study aims to identify key MicroRNA (miRNA) and mRNAs in right-sided colon adenocarcinoma (RSCOAD) and left-sided colon adenocarcinoma (LSCOAD) by TCGA integration analysis.Methods. The miRNA and mRNA expression profiles of a large group of patients with RSCOAD and LSCOAD were obtained from TCGA. The differentially expressed miRNAs (DEmiRNAs) and mRNAs (DEmRNAs) were identified by TCGA integration analysis. The optimal diagnostic miRNA biomarkers for RSCOAD and LSCOAD were identified by Boruta algorithm. We established classification models to distinguish RSCOAD and LSCOAD. Protein-protein interaction (PPI) network analysis, DEmiRNA-DEmRNA interaction analysis, and functional annotation were performed. The expression of selected DEmiRNAs and DEmRNAs was validated by qRT-PCR.Results. A total of 2534 DEmRNAs (940 downregulated and 1594 upregulated mRNAs) and 54 DEmiRNAs (22 downregulated and 32 upregulated miRNAs) between RSCOAD and LSCOAD were identified. The feature selection procedure was to obtain 22 optimal diagnostic miRNAs biomarkers in RSCOAD compared to LSCOAD. The AUC of the random forests model was 0.869 and the specificity and sensitivity of this model were 79% and 84.6%, respectively. Three DEmiRNAs (hsa-miR-224-5p, hsa-miR-155-5p, and hsa-miR-31-5p) and five DEmRNAs (CXCR4, SMAD4, KRAS, FITM2, and PLAGL2) were identified key DEmiRNAs and DEmRNAs in RSCOAD compared to LSCOAD. The qRT-PCR results of CXCR4, FITM2, TFAP2A, ULBP2, hsa-miR-224-5p, and hsa-miR-155-5p were consistent with our integrated analysis.Conclusion. A total of three DEmiRNAs (hsa-miR-224-5p, hsa-miR-155-5p, and hsa-miR-31-5p) and five DEmRNAs (CXCR4, SMAD4, KRAS, FITM2, and PLAGL2) may be involved in the pathogenesis of RSCOAD and LSCOAD which may make a contribution for understanding mechanisms and developing therapeutic strategies for RSCOAD and LSCOAD.

The Natural killer (NK) cells' ability to destroy cancerous cells is predominantly focused on the activation of the co-stimulatory and natural killer group two with receptor of member-D also called NKGD2/NKG2D. These identifies ligands that are MHC-Class1 structural homologs like that of the UL16 protein-binding type 2. The ULBP2 has been shown to mediate-natural resistance against the tumors mechanism in the condition of in-vitro, in-vivo, making it a possible target for producing the immune-therapeutic drugs for the diagnosis of the cancers and certain other viral diseases. In this present research, we created a stable and high-quality 3-D structure of the ULBP-2 protein through SWISS-Model also visualized by using UCSF-Chimera Tool. Moreover, the ULBP2 protein was prognosticated to be acts as antigenic, 11-discontinuous-B-Cell epitopes, 05 ULBP2 proteins antibody-based epitopes, and possible predicted the top hits of six linear B-cell epitopes. The ULBP2 protein carried seven cytotoxic-T lymphocytes (CTLs), two helper-T lymphocytes (HTLs), the LGKKLNVTTAWAQN is a promiscuous epitope MHC bounded to the T cells and with LRDIQLENY highest antigen scores in MHC molecule. Finally, the promising epitopes that could be successful in producing B-cell and T-cell mediated immunity against the required immune reaction to tumorigenesis were expected.

The expression of NKG2D ligands (NKG2DLs), induced by stress or malignant transformation, is considered to mark dysfunctional cells for elimination by NK cells or cytotoxic lymphocytes via NKG2D/NKG2DLs pathway. ULBP1, ULBP2, and ULBP3 (ULBP1-3), three members of NKG2DLs, are common expressed in breast cancer. We analyzed the expression of ULBP1-3 in breast cancer and healthy control tissues with several databases, and found that breast cancer had a higher mRNA level of ULBP1 and ULBP2, and a higher protein level of ULBP1-3. Analysis with the bc-GenExMiner database showed that the expression of ULBP1-3 were down-regulated by the wild type P53, PR, and HER2+ in breast cancer. Except for ULBP1, ULBP2 and ULBP3 were associated with poor prognosis in breast cancer. The analysis of the correlated genes suggested that ULBP1-3 have some common pathways including NK cell-mediated cytotoxicity, microRNA in cancer, and IL-17 signaling pathway, whereas they also have their own pathways. But ULBP1-3 expression were also a significantly negative correlation with few immune markers on NK and central memory CD8+ T. The correlations and co-occurrence between ULBP1-3 and the other ligands by using TIMER and cbioportal databases showed the same form proteins or the proteins in the same family were more likely to change at the same time. Together with all these findings, increased ULBP2 and ULBP3 were correlated with poor prognosis and various markers on immune cells. These conclusions indicated that ULBP2 and ULBP3 could serve as potential biomarkers to assess prognosis and they were strong correlation with various markers in immune cells.

Background: Colorectal cancer (CRC) is the most common type of gastrointestinal malignant tumour. Colorectal adenocarcinoma (COAD) – the most common type of CRC – is particularly dangerous. The role of the immune system in the development of tumour-associated inflammation and cancer has received increasing attention recently. Methods: In the present study, we compiled the expression profiles of 262 patients with complete follow-up data from The Cancer Genome Atlas (TCGA) database as an experimental group and selected 65 samples from the Gene Expression Omnibus (GEO) dataset (of which 46 samples were with M0) as a verification group. First, we screened the immune T helper 17 (Th17) cells related to the prognosis of COAD. Subsequently, we identified Th17 cells-related hub genes by utilising Weighted Gene Co-expression Network Analysis (WGCNA) and Least Absolute Shrinkage and Selector Operation (LASSO) regression analysis. Six genes associated with the prognosis in patients with COAD were identified, including: KRT23, ULBP2, ASRGL1, SERPINA1, SCIN, and SLC28A2. We constructed a clinical prediction model and analysed its predictive power. Results: The identified hub genes are involved in developing many diseases and closely linked to digestive disorders. Our results suggested that the hub genes could influence the prognosis of COAD by regulating Th17 cells’ infiltration. Conclusions: These newly discovered hub genes contribute to clarifying the mechanisms of COAD development and metastasis. Given that they promote COAD development, they may become new therapeutic targets and biomarkers of COAD.

Ataxia-telangiectasia (A-T) is a multisystem disorder caused by biallelic pathogenic variants in the gene encoding A-T mutated (ATM) kinase, a master regulator of the DNA damage response (DDR) pathway. Most A-T patients show cellular and/or humoral immunodeficiency that has been associated with cancer risk and reduced survival, but NK cells have not been thoroughly studied. Here we investigated NK cells of A-T patients with a special focus on the NKG2D receptor that triggers cytotoxicity upon engagement by its ligands (NKG2DLs) commonly induced via the DDR pathway on infected, transformed, and variously stressed cells. Using flow cytometry, we examined the phenotype and function of NK cells in 6 A-T patients as compared with healthy individuals. NKG2D expression was evaluated also by western blotting and RT-qPCR; plasma soluble NKG2DLs (sMICA, sMICB, sULBP1, ULBP2) were measured by ELISA. Results showed that A-T NK cells were skewed towards the CD56neg anergic phenotype and displayed decreased expression of NKG2D and perforin. NKG2D was reduced at the protein but not at the mRNA level and resulted in impaired NKG2D-mediated cytotoxicity in 4/6 A-T patients. Moreover, in A-T plasma we found 24-fold and 2-fold increase of sMICA and sULBP1, respectively, both inversely correlated with NKG2D expression. Overall, NK cells are disturbed in A-T patients showing reduced NKG2D expression, possibly caused by persistent engagement of its ligands, that may contribute to susceptibility to cancer and infections and represent novel targets for therapeutic interventions.

Background & Aims 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. Methods 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. Results 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. Conclusions 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. Lay summary 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.

Background Growing evidence are demonstrating the therapeutic efficacy of immune checkpoint inhibitors (ICI) in mesothelioma; however, a limited percentage of patients benefits from this therapeutic approach. Epigenetic modifications play a relevant role in negatively regulating the cross-talk between neoplastic and immune cells, and in contributing to the highly immunosuppressive mesothelioma microenvironment. A better understanding of mesothelioma epigenetic landscape could open the path to novel and potentially more effective approaches combining ICI and epigenetic drugs. We investigated the immunomodulatory potential of epigenetic agents by comparing the activity of DNA hypomethylating agents (DHA) with histone deacetylases inhibitors (HDACi) and EZH2 inhibitors (EZH2i), alone or combined with DHA, in mesothelioma cells. Methods Four mesothelioma cell lines were treated with the DHA guadecitabine 1μM, or with the HDACi, Valproic Acid (VPA) 1mM, or the EZH2i, EPZ-6438 1μM, alone or combined with guadecitabine. We investigated the expression of HLA class I molecules by flow-cytometry and of PD-L1, cancer testis antigens (CTA: NY-ESO, MAGE-A1), Natural Killer Group 2 member D Ligands (NKG2DLs: MIC-A, MIC-B, ULBP2) and EMT-regulating cadherins (CDH1, CDH2) by quantitative Real-Time PCR. Fold change (FC) expression for each treatment vs untreated cells was reported as mean values (FCm) among investigated cell lines. A positive modulation of the expression was considered if FCm>1.5. Results Guadecitabine upregulated the expression of HLA class I antigens (FCm=1.75), PD-L1 (FCm=2.38), NKG2DLs (MIC-A FCm=1.96, MIC-B FCm=2.57, and ULBP2 FCm=3.56), and upregulated/induced CTA expression. Similarly, VPA upregulated HLA class I antigens (FCm=1.67), PD-L1 (FCm=3.17), NKG2DLs (MIC-A FCm=1.78, MIC-B FCm=3.04, and ULBP2 FCm=3.75) expression; however, CTA expression was modulated only in 1 mesothelioma cell line. Conversely, EPZ-6438 up-regulated only NY-ESO-1 and MIC-B expression in 1 mesothelioma cell line. The addition of both VPA and EPZ-6483 to guadecitabine strengthened its immunomodulatory activity. Specifically, guadecitabine plus VPA or EPZ-6438 upregulated the expression of HLA class I antigens FCm=2.55 or 2.69, PD-L1 FCm=8.04 or 2.65, MIC-A FCm=3.81 or 2.26, MIC-B FCm=8.00 or 3.03, ULBP2 FCm=6.24 or 4.53, respectively. Higher levels of CTA upregulation/induction were observed with combination treatments vs guadecitabine alone. Cadherins modulation was mesothelioma histotype-related: CDH1 expression was induced in the 2 constitutively-negative sarcomatoid mesothelioma cells by guadecitabine alone or combined with VPA or EPZ-6438; CDH2 expression was upregulated by VPA alone (FCm=1.53) or plus guadecitabine (FCm=2.54). Conclusions Combination of DHA-based immunotherapies with other classes of epigenetic drugs could be an effective strategy to be pursued in the mesothelioma clinic.

The Pneumoviridae family includes human metapneumovirus (HMPV) and human orthopneumovirus, which is also known as a respiratory syncytial virus (HRSV). These are large enveloped, negative single-strand RNA viruses. HMPV and HRSV are the human members, which commonly infect children. HMPV, which was discovered in 2001, infects most children until the age of five, which causes an influenza-like illness. The interaction of this virus with immune cells is poorly understood. In this study, we show that HMPV evades natural killer (NK) cell attack by downregulating stress-induced ligands for the activating receptor NKG2D including: Major histocompatibility complex (MHC) class I polypeptide-related sequences A and B (MICA, MICB), UL16 binding proteins ULBP2, and ULBP3, but not ULBP1. Mechanistically, we show that the viral protein G is involved in the downregulation of ULBP2 and that the viral protein M2.2 is required for MICA and MICB downregulation. These findings emphasize the importance of NK cells, in general, and NKG2D, in particular, in controlling HMPV infection, which opens new avenues for treating HMPV.

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.

We have developed a chimeric antigen receptor (CAR) platform that functions as a modular system to address limitations of traditional CAR therapies. An inert form of the human NKG2D extracellular domain (iNKG2D) was engineered as the ectodomain of the CAR to generate convertibleCARTM-T cells. These cells were specifically directed to kill antigen-expressing target cells only in the presence of an activating bispecific adapter comprised of an iNKG2D-exclusive ULBP2-based ligand fused to an antigen-targeting antibody (MicAbodyTM). Efficacy against Raji tumors in NSG mice was dependent upon doses of both a rituximab-based MicAbody and convertibleCAR-T cells. We have also demonstrated that the exclusive ligand-receptor partnering enabled the targeted delivery of a mutant form of IL-2 to selectively promote the expansion of convertibleCAR-T cells in vitro and in vivo. By altering the Fv domains of the MicAbody or the payload fused to the orthogonal ligand, convertibleCAR-T cells can be readily targeted or regulated., Landgraf et al. developed a chimeric antigen receptor (CAR) platform that functions as a modular system. convertibleTM-T cells are designed to kill antigen-expressing target cells only in the presence of ligands fused to antigen-targeting antibodies (MicAbodyTM). This method provides a wide dosing window while minimizing toxicity.

Pancreatic cancer (PC) is a global health problem that features a very high mortality rate. The UL16 binding protein 2 (ULBP2) is a new biomarker for PC detection. This study develops a simple, reliable, and inexpensive immunosensor for the detection of the ULBP2 antigen while also investigating the effects of an array configuration of connected sensors and zinc oxide (ZnO) nanoparticles on the immunosensor&rsquo, s sensitivity. The ULBP2 antibody was immobilized onto the screen-printed carbon electrode (SPCE) surfaces of three different sensors: a simple SPCE (ULBP2-SPCE), an SPCE array, which is a series of identical SPCE connected to each other at different arrangements of rows and columns (ULBP2-SPCE-1x2 and ULBP2-SPCE-1x3), and an SPCE combined with ZnO nanoparticles (ULBP2-ZnO/SPCE). Impedance spectrum measurements for the immunosensors to ULBP2 antigen were conducted and compared. According to the result, the array configurations (ULBP2-SPCE-1x2 and ULBP2-SPCE-1x3) show an improvement of sensitivity compared to the ULBP2-SPCE alone, but the improvement is not as significant as that of the ULBP2-ZnO/SPCE configuration (ULBP2-ZnO/SPCE &gt, ULBP2-SPCE: 18 times larger). The ULBP2-ZnO/SPCE immunosensor has a low limit of detection (1 pg/mL) and a high sensitivity (332.2 &Omega, /Log(pg/mL)), excellent linearity (R2 = 0.98), good repeatability (coefficients of variation = 5.03%), and is stable in long-term storage (retaining 95% activity after 28 days storage). In an array configuration, the immunosensor has an increased signal-to-noise ratio (ULBP2-SPCE-1x3 &gt, ULBP2-SPCE: 1.5-fold) and sensitivity (ULBP2-SPCE-1x3 &gt, ULBP2-SPCE: 2.6-fold). In conclusion, either the modification with ZnO nanoparticles onto the sensor or the use of an array configuration of sensors can enhance the immunosensor&rsquo, s sensitivity. In this study, the best immunosensor for detecting ULBP2 antigens is the ULBP2-ZnO/SPCE immunosensor.

Monoclonal anti-programmed cell death 1 (PD1) antibodies are successful cancer therapeutics, but it is not well understood why individual antibodies should have idiosyncratic side-effects. As the humanized antibody SHR-1210 causes capillary hemangioma in patients, a unique toxicity amongst anti-PD1 antibodies, we performed human receptor proteome screening to identify nonspecific interactions that might drive angiogenesis. This screen identified that SHR-1210 mediated aberrant, but highly selective, low affinity binding to human receptors such as vascular endothelial growth factor receptor 2 (VEGFR2), frizzled class receptor 5 and UL16 binding protein 2 (ULBP2). SHR-1210 was found to be a potent agonist of human VEGFR2, which may thereby drive hemangioma development via vascular endothelial cell activation. The v-domains of SHR-1210’s progenitor murine monoclonal antibody ‘Mab005ʹ also exhibited off-target binding and agonism of VEGFR2, proving that the polyspecificity was mediated by the original mouse complementarity-determining regions (CDRs), and had survived the humanization process. Molecular remodelling of SHR-1210 by combinatorial CDR mutagenesis led to deimmunization, normalization of binding affinity to human and cynomolgus PD1, and increased potency in PD1/PD-L1 blockade. Importantly, CDR optimization also ablated all off-target binding, rendering the resulting antibodies fully PD1-specific. As the majority of changes to the paratope were found in the light chain CDRs, the germlining of this domain drove the ablation of off-target binding. The combination of receptor proteome screening and optimization of the antibody binding interface therefore succeeded in generating novel, higher-potency, specificity-enhanced therapeutic IgGs from a single, clinically sub-optimal progenitor. This study showed that highly-specific off-target binding events might be an under-appreciated phenomenon in therapeutic antibody development, but that these unwanted properties can be fully ameliorated by paratope refinement.

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.

In pilot HIV-1 eradication studies, patients' immune responses were ineffective at killing viral reservoirs reactivated through latency reversing agents (LRAs) like suberoylanilide hydroxamic acid (SAHA). We hypothesized that T cells harboring reactivated HIV-1 express MIC and ULBP ligands for the activating NKG2D receptor of natural killer (NK) cells. Here, we demonstrated that MICA/B and ULBP2 are induced by SAHA on primary T cells harboring reactivated virus. Using latently HIV-1-infected J-Lat 6.3/8.4/9.2 and J1.1 cell lines, we showed that SAHA reverts latency and, simultaneously, up-regulates MICA/B and ULBP2 acting at the transcriptional level and through ATR activation, thus sensitizing T cells with reactivated virus to NKG2D-mediated killing by NK cells. Moreover, IL-2 and IL-15 potently boosted NKG2D expression and cytotoxicity of NK cells against SAHA-reactivated p24+ target cells. Therefore, immunotherapy with cytokines enhancing NKG2D-mediated NK-cell cytotoxicity combined with administration of LRAs up-modulating NKG2D ligands, represents a promising approach towards HIV-1 eradication.

// Aroa Baragano Raneros 1 , Alfredo Minguela Puras 2 , Ramon M. Rodriguez 1 , Enrique Colado 3 , Teresa Bernal 3 , Eduardo Anguita 4 , Adela Vasco Mogorron 2 , Alberto Chaparro Gil 4 , Jose Ramon Vidal-Castineira 1 , Leonardo Marquez-Kisinousky 1 , Paula Diaz Bulnes 1 , Amelia Martinez Marin 5 , Maria Carmen Garcia Garay 6 , Beatriz Suarez-Alvarez 1, * , Carlos Lopez-Larrea 1, * 1 Department of Immunology, Hospital Universitario Central de Asturias, Oviedo, Spain 2 Immunology Service, Instituto Murciano de Investigacion Biosanitaria (IMIB), Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 3 Department of Hematology, Hospital Universitario Central de Asturias, Oviedo, Spain 4 Hematology Department, Hospital Clinico San Carlos, Instituto de Investigacion Sanitaria San Carlos (IdISSC), Department of Medicine, Universidad Complutense de Madrid (UCM), Madrid, Spain 5 Hematology Service, Hospital Clinico Universitario Virgen de la Arrixaca, Murcia, Spain 6 Hematology Service, Hospital General Universitario Santa Lucia, Cartagena, Murcia, Spain * These authors have contributed equally to this work Correspondence to: Carlos Lopez-Larrea, email: inmuno@hca.es Beatriz Suarez-Alvarez, email: bsuarez@hca.es Keywords: acute myeloid leukemia (AML), DNA methylation, NKG2DL, NKG2D, TIMP3 Received: September 16, 2016 Accepted: March 16, 2017 Published: March 29, 2017 ABSTRACT Acute myeloid leukemia (AML) is a disease with great morphological and genetic heterogeneity, which complicates its prognosis and treatment. The hypomethylating agents azacitidine (Vidaza ® , AZA) and decitabine (Dacogen ® , DAC) have been approved for the treatment of AML patients, but their mechanisms of action are poorly understood. Natural killer (NK) cells play an important role in the recognition of AML blasts through the interaction of the activating NKG2D receptor with its ligands (NKG2DL: MICA/B and ULBPs1-3). However, soluble NKG2DL (sNKG2DL) can be released from the cell surface, impairing immune recognition. Here, we examined whether hypomethylating agents modulate the release of sNKG2DL from AML cells. Results demonstrated that AZA- and DAC-treated AML cells reduce the release of sNKG2DL, preventing downregulation of NKG2D receptor on the cell surface and promoting immune recognition mediated by NKG2D-NKG2DL engagement. We show that the shedding of MICA, MICB and ULBP2 is inhibited by the increased expression of TIMP3, an ADAM17 inhibitor, after DAC treatment. The TIMP3 gene is highly methylated in AML cells lines and in AML patients (25.5%), in which it is significantly associated with an adverse cytogenetic prognosis of the disease. Overall, TIMP3 could be a target of the demethylating treatments in AML patients, leading to a decrease in MICA, MICB and ULBP2 shedding and the enhancement of the lytic activity of NK cells through the immune recognition mediated by the NKG2D receptor.

UL16-binding protein (ULBP) 1-6 and MHC class I chain-related molecule A and B (MICA/B) are NK group 2, member D (NKG2D) ligands, which are specifically expressed in infected or transformed cells and are recognized by NK cells via NKG2D-NKG2D ligand interactions. We previously reported that MICA/B overexpression predicted improved clinical outcomes in patients with resected non-small cell lung cancer (NSCLC). However, the clinicopathological features and prognostic significance of ULBPs in NSCLC remain unclear. Here,ULBP1-6 expression was evaluated based on immunohistochemistry of 91 NSCLC samples from patients following radical surgery. ULBPs were expressed by the majority of NSCLC. Either ULBP1 or ULBP2/5/6 overexpression was associated with squamous-cell carcinoma histology, whereas ULBP4 overexpression was associated with younger age and adenocarcinoma histology. Although overexpression of ULBP1-6 did not impact clinical outcomes in NSCLC patients, integrative profiling with cluster analysis classified patients into 3 subgroups based on the expression pattern of NKG2D ligands. The subgroup characterized by ULBP1 or ULBP2/5/6 high expressing but ULBP4 low expressing tumors showed poor overall survival. Taken together with previous results, NSCLC histological subtype strongly correlates with NKG2D ligands expression pattern. NKG2D ligands expression levels assessed by multiple immune parameters could predict clinical outcomes of patients with NSCLC.

The hexamethylene bisacetamide (HMBA) anticancer drug was dismissed due to limited efficacy in leukemic patients but it may re-enter into the clinics in HIV-1 eradication strategies because of its recently disclosed capacity to reactivate latent virus. Here, we investigated the impact of HMBA on the cytotoxicity of natural killer (NK) cells against acute T lymphoblastic leukemia (T-ALL) cells or HIV-1-infected T cells that exit from latency. We show that in T-ALL cells HMBA upmodulated MICB and ULBP2 ligands for the NKG2D activating receptor. In a primary CD4+ T cell-based latency model, HMBA did not reactivate HIV-1, yet enhanced ULBP2 expression on cells harboring virus reactivated by prostratin (PRO). However, HMBA reduced the expression of NKG2D and its DAP10 adaptor in NK cells, hence impairing NKG2D-mediated cytotoxicity and DAP10-dependent response to IL-15 stimulation. Alongside, HMBA dampened killing of T-ALL targets by IL-15-activated NK cells and impaired NK cell-mediated clearance of PRO-reactivated HIV-1+ cells. Overall, our results demonstrate a dominant detrimental effect of HMBA on the NKG2D pathway that crucially controls NK cell-mediated killing of tumors and virus-infected cells, providing one possible explanation for poor clinical outcome in HMBA-treated cancer patients and raising concerns for future therapeutic application of this drug.

Despite the great success of chimeric antigen receptor T (CAR-T)–cell therapy in the treatment of hematologic malignancies, CAR-T–cell therapy is limited in solid tumors, including hepatocellular carcinoma (HCC). NK group 2 member D (NKG2D) ligands (NKG2DL) are generally absent on the surface of normal cells but are overexpressed on malignant cells, offering good targets for CAR-T therapy. Indeed, analysis of The Cancer Genome Atlas and HCC tumor samples showed that the expression of most NKG2DLs was elevated in tumors compared with normal tissues. Thus, we designed a novel NKG2D-based CAR comprising the extracellular domain of human NKG2D, 4-1BB, and CD3ζ signaling domains (BBz). NKG2D-BBz CAR-T cells efficiently killed the HCC cell lines SMMC-7721 and MHCC97H in vitro, which express high levels of NKG2DLs, whereas they less efficiently killed NKG2DL-silenced SMMC-7721 cells or NKG2DL-negative Hep3B cells. Overexpression of MICA or ULBP2 in Hep3B improved the killing capacity of NKG2D-BBz CAR-T cells. T cells expressing the NKG2D-BBz CAR effectively eradicated SMMC-7721 HCC xenografts. Collectively, these results suggested that NKG2D-BBz CAR-T cells could potently eliminate NKG2DL-high HCC cells both in vitro and in vivo, thereby providing a promising therapeutic intervention for patients with NKG2DL-positive HCC.

The natural killer group 2 member D (NKG2D) receptor and its ligands are important mediators of immune responses to tumors. NKG2D ligands are overexpressed in several malignant tumor types; however, the prognostic value of these ligands is unclear. Here, we aimed to elucidate the role of NKG2D ligands in extrahepatic cholangiocarcinoma (EHCC). We therefore investigated the expression of the NKG2D receptor and its ligands MHC class I chain-related proteins A and B (MICA/B), unique long 16 binding protein (ULBP) 1, and ULBP2/5/6 in resected specimens from 82 patients with EHCC. All NKG2D ligands were highly expressed in EHCC. High expression of MICA/B or ULBP2/5/6 correlated with overall and disease-free survival. In contrast, high expression of ULBP1 was significantly associated with improved overall survival, but not disease-free survival. Concurrent high expression of multiple NKG2D ligands revealed significantly better overall and disease-free survival than that observed with the overexpression of any one NKG2D ligand. Co-expression of multiple NKG2D ligands was an independent prognostic indicator of improved survival. Furthermore, co-overexpression of multiple NKG2D ligands was significantly correlated with high expression of the NKG2D receptor. Inhibiting interactions between multiple NKG2D ligands and the NKG2D receptor might be a promising approach for controlling cancer progression and improving patient prognosis in EHCC.