Early life exposure to nicotine modifies lung gene response after elastase-induced emphysema.

BACKGROUND: Chronic obstructive pulmonary disease (COPD) is among the top 5 causes of mortality in the world and can develop as a consequence of genetic and/or environmental factors. Current efforts are focused on identifying early life insults and how these contribute to COPD development. In line with this, our study focuses on the influence of early life nicotine exposure and its potential impact on (a) lung pulmonary functions, and (b) elastase-induced emphysema in adulthood. METHODS: To address this hypothesis, we developed a model of 2 hits, delivered at different time points: mouse pups were first exposed to nicotine/placebo in utero and during lactation, and then subsequently received elastase/placebo at the age of 11 weeks. The effect of nicotine pretreatment and elastase instillation was assessed by (a) measurement of pulmonary function at post-elastase day (ped) 21, and (b) transcriptomic profiling at ped3 and 21, and complementary protein determination. Statistical significance was determined by 3- and 2-way ANOVA for pulmonary functions, and RNAseq results were analyzed using the R project. RESULTS: We did not observe any impact of nicotine pre- and early post-natal exposure compared to control samples on lung pulmonary functions in adulthood, as measured by FLEXIVENT technology. After elastase instillation, substantial lung damage was detected by x-ray tomography and was accompanied by loss in body weight at ped3 as well as an increase in cell numbers, inflammatory markers in BAL and lung volume at ped21. Lung functions showed a decrease in elastance and an increase in deep inflation volume and pressure volume (pv) loop area in animals with emphysema at ped21. Nicotine had no effect on elastance and deep inflation volume, but did affect the pv loop area in animals with emphysema at ped21. Extensive transcriptomic changes were induced by elastase at ped3 both in the nicotine-pretreated and the control samples, with several pathways common to both groups, such as for cell cycle, DNA adhesion and DNA damage. Nicotine pretreatment affected the number of lymphocytes present in BAL after elastase instillation and some of the complement pathway related proteins, arguing for a slight modification of the immune response, as well as changes related to general body metabolism. The majority of elastase-induced transcriptomic changes detected at ped3 had disappeared at ped21. In addition, transcriptomic profiling singled out a common gene pool that was independently activated by nicotine and elastase. CONCLUSIONS: Our study reports a broad spectrum of transient transcriptomic changes in mouse emphysema and identifies nicotine as influencing the emphysema-associated immune system response.

HLA-E and HLA-F Are Overexpressed in Glioblastoma and HLA-E Increased After Exposure to Ionizing Radiation.

BACKGROUND/AIM: Glioblastoma (GBM) is one of the deadliest human cancers responding very poorly to therapy. Although the central nervous system has been traditionally considered an immunologically privileged site with an enhanced immune response, GBM appears to benefit from this immunosuppressive milieu. Immunomodulatory molecules play an important role in immune tumor-host interactions. Non-classical human leukocyte antigens (HLA) class Ib molecules HLA-E, HLA-F, and HLA-G have been previously described to be involved in protecting semi-allogeneic fetal allografts from the maternal immune response and in transplant tolerance as well as tumoral immune escape. Unfortunately, their role in GBM remains poorly understood. Our study, therefore, aimed to characterize the relationship between the expression of these molecules in GBM on the transcriptional level and clinicopathological and molecular features of GBM as well as the effect of ionizing radiation. MATERIALS AND METHODS: We performed the analysis of HLA-E, HLA-F, and HLA-G mRNA expression in 69 GBM tissue samples and 21 non-tumor brain tissue samples (controls) by reverse transcription polymerase chain reaction. Furthermore, two primary GBM cell cultures had been irradiated to identify the effect of ionizing radiation on the expression of non-classical HLA molecules. RESULTS: Analyses revealed that both HLA-E and HLA-F are significantly up-regulated in GBM samples. Subsequent survival analysis showed a significant association between low expression of HLA-E and shorter survival of GBM patients. The dysregulated expression of both molecules was also observed between patients with methylated and unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter. Finally, we showed that ionizing radiation increased HLA-E expression level in GBM cells in vitro. CONCLUSION: HLA-E and HLA-F play an important role in GBM biology and could be used as diagnostic biomarkers, and in the case of HLA-E also as a prognostic biomarker.

lncRNA HLA Complex Group 18 (HCG18) Facilitated Cell Proliferation, Invasion, and Migration of Prostate Cancer Through Modulating miR-370-3p/DDX3X Axis.

Long non-coding RNAs (lncRNAs) have been reported to exert critical functions in the malignant development of many cancers. lncRNA HLA complex group 18 (HCG18) has been confirmed to have a promoting effect on various cancers. However, whether HCG18 functions in PC is still unclear. Therefore, the current study aimed at unveiling the role of HCG18 in PC progression and its regulatory mechanism on the biological behaviors of PC. Here, RT-qPCR was utilized to detect HCG18 expression, and then, functional experiments were conducted to verify the effects of HCG18 on PC cell proliferation, migration, invasion, and apoptosis. According to the results, HCG18 was significantly up-regulated in PC cells and it facilitated cell proliferation, migration, and invasion in PC. Furthermore, a series of mechanism experiments were carried out to verify the relationship among HCG18, miR-370-3p, and DEAD-box helicase 3 X-linked(DDX3X) in PC cells. Final rescue assays showed that DDX3X overexpression could reverse the inhibitory function of silencing HCG18 on PC progression. In summary, our study showed that lncRNA HCG18 accelerated cell proliferation, invasion, and migration of PC via up-regulating DDX3X through sponging miR-370-3p, providing a novel finding about PC-related regulatory mechanism.

Neuronal ApoE upregulates MHC-I expression to drive selective neurodegeneration in Alzheimer's disease.

Selective neurodegeneration is a critical causal factor in Alzheimer's disease (AD); however, the mechanisms that lead some neurons to perish, whereas others remain resilient, are unknown. We sought potential drivers of this selective vulnerability using single-nucleus RNA sequencing and discovered that ApoE expression level is a substantial driver of neuronal variability. Strikingly, neuronal expression of ApoE-which has a robust genetic linkage to AD-correlated strongly, on a cell-by-cell basis, with immune response pathways in neurons in the brains of wild-type mice, human ApoE knock-in mice and humans with or without AD. Elimination or over-expression of neuronal ApoE revealed a causal relationship among ApoE expression, neuronal MHC-I expression, tau pathology and neurodegeneration. Functional reduction of MHC-I ameliorated tau pathology in ApoE4-expressing primary neurons and in mouse hippocampi expressing pathological tau. These findings suggest a mechanism linking neuronal ApoE expression to MHC-I expression and, subsequently, to tau pathology and selective neurodegeneration.

MHC heterogeneity and response of metastases to immunotherapy.

In recent years, immunotherapy has proven to be an effective treatment against cancer. Cytotoxic T lymphocytes perform an important role in this anti-tumor immune response, recognizing cancer cells as foreign, through the presentation of tumor antigens by MHC class I molecules. However, tumors and metastases develop escape mechanisms for evading this immunosurveillance and may lose the expression of these polymorphic molecules to become invisible to cytotoxic T lymphocytes. In other situations, they may maintain MHC class I expression and promote immunosuppression of cytotoxic T lymphocytes. Therefore, the analysis of the expression of MHC class I molecules in tumors and metastases is important to elucidate these escape mechanisms. Moreover, it is necessary to determine the molecular mechanisms involved in these alterations to reverse them and recover the expression of MHC class I molecules on tumor cells. This review discusses the role and regulation of MHC class I expression in tumor progression. We focus on altered MHC class I phenotypes present in tumors and metastases, as well as the molecular mechanisms responsible for MHC-I alterations, emphasizing the mechanisms of recovery of the MHC class I molecules expression on cancer cells. The individualized study of the HLA class I phenotype of the tumor and the metastases of each patient will allow choosing the most appropriate immunotherapy treatment based on a personalized medicine.

The NKG2D ligand ULBP4 is not expressed by human monocytes.

The C-type lectin-like receptor NKG2D contributes to the immunosurveillance of virally infected and malignant cells by cytotoxic lymphocytes. A peculiar and puzzling feature of the NKG2D-based immunorecognition system is the high number of ligands for this single immunoreceptor. In humans, there are a total of eight NKG2D ligands (NKG2DL) comprising two members of the MIC (MICA, MICB) and six members of the ULBP family of glycoproteins (ULBP1 to ULBP6). While MICA has been extensively studied with regard to its biochemistry, cellular expression and function, very little is known about the NKG2DL ULBP4. This is, at least in part, due to its rather restricted expression by very few cell lines and tissues. Recently, constitutive ULBP4 expression by human monocytes was reported, questioning the view of tissue-restricted ULBP4 expression. Here, we scrutinized ULBP4 expression by human peripheral blood mononuclear cells and monocytes by analyzing ULBP4 transcripts and ULBP4 surface expression. In contrast to MICA, there was no ULBP4 expression detectable, neither by freshly isolated monocytes nor by PAMP-activated monocytes. However, a commercial antibody erroneously indicated surface ULBP4 on monocytes due to a non-ULBP4-specific binding activity, emphasizing the critical importance of validated reagents for life sciences. Collectively, our data show that ULBP4 is not expressed by monocytes, and likely also not by other peripheral blood immune cells, and therefore exhibits an expression pattern rather distinct from other human NKG2DL.

Genetic variants in NKG2D axis and susceptibility to Epstein-Barr virus-induced nasopharyngeal carcinoma.

BACKGROUND: Nasopharyngeal carcinoma (NPC) is a rare epithelial carcinoma arising from the nasopharyngeal region. The pathogenesis of NPC is linked to Epstein-Barr virus (EBV) infection, although genetics and lifestyle factors appears to be also implicated. NKG2D is an immunoreceptor expressed by NK and T-cell subsets that recognizes MICA protein and other ligands on tumor cells. NKG2D interaction with MICA plays a role in the immunosurveillance to viruses and cancer. METHODS: We investigated potential associations between functional polymorphisms in NKG2D and MICA genes with NPC susceptibility. We conducted a case-control study including 255 Vietnamese patients with EBV + non-differentiated NPC and 220 healthy controls. RESULTS: We observed a significant association between the LNK/LNK genotype of rs1049174 (a variant associated with lower NKG2D receptor expression and reduced NK cell cytotoxicity) and increased susceptibility to NPC (adjusted OR = 1.66, 95% CI 1.07-2.59; p = 0.024). Similarly, the AA genotype of MICA rs2596542 was significantly associated with NPC (adjusted OR = 2.12; 95% CI 1.22-3.81; p = 0.009). In addition, tumor specimens of NPC patients with the AA genotype displayed a higher expression level of MICA proteins and showed higher EBV titers compared with tumor tissues from patients with the GG or GA genotypes. Higher EBV copy numbers were also observed in tumors with the A allele of MICA rs1051792 (also known as MICA-129 Met/Val) compared with those with the G allele; however, MICA rs1051792 variants were not associated with NPC susceptibility. These results suggest that genetic variants in components of the NKG2D axis may influence the individual susceptibility to EBV-induced NPC.

Brain astrocytes and microglia express functional MR1 molecules that present microbial antigens to mucosal-associated invariant T (MAIT) cells.

It is unknown whether brain astrocytes and microglia have the capacity to present microbial antigens via the innate immune MR1/MAIT cell axis. We have detected MAIT cells in the normal mouse brain and found that both astrocytes and microglia are MR1+. When we stimulated brain astrocytes and microglia with E. coli, and then co-cultured them with MAIT cells, MR1 surface expression was upregulated and MAIT cells were activated in an antigen-dependent manner. Considering the association of MAIT cells with inflammatory conditions, including those in the CNS, the MR1/MAIT cell axis could be a novel therapeutic target in neuroinflammatory disorders.

Neuronal NLRC5 regulates MHC class I expression in Neuro-2a cells and also during hippocampal development.

Major histocompatibility Complex class I (MHC I) molecules are ubiquitously expressed, being found in most nucleated cells, where they are central mediators of both the adaptive and innate immune responses. Recent studies have shown that MHC I are also expressed in the developing brain where they participate in synapse elimination and plasticity. Up-regulation of MHC I within the developing brain has been reported, however, the mechanism(s) regulating this developmental up-regulation of neuronal MHC I remains unknown. Here, we show NLR family CARD domain containing 5 (NLRC5), a newly identified member of the NLR family, is widely expressed in hippocampal neurons, and the expression pattern of NLRC5 coincides with increased MHC I mRNA in the developing hippocampus. Using a luciferase assay in Neuro-2a cells we demonstrate that NLRC5 can induce the activation of MHC I and this induction requires the W/S-X-Y motif. Further studies show that transcription factors regulatory factor X (RFX) and CREB1, which bind to X1 and X2 box, are crucial for NLRC5-mediated induction. Moreover immunoprecipitation experiments reveal that NLRC5 interacts with RFX subunits RFX5 and RFXANK. Knockout of Nlrc5 dramatically impairs basal expression of MHC I in mouse hippocampus. Taken together, our findings identify NLRC5 as a key regulator of MHC I up-regulation in the developing hippocampus and suggest an important role for NLRC5 in neurons. Cover Image for this issue: doi: 10.1111/jnc.14729.

What the HLA-I!-Classical and Non-classical HLA Class I and Their Potential Roles in Type 1 Diabetes.

PURPOSE OF REVIEW: Hyperexpression of classical HLA class I (HLA-I) molecules in insulin-containing islets has become a widely accepted hallmark of type 1 diabetes pathology. In comparison, relatively little is known about the expression, function and role of non-classical subtypes of HLA-I. This review focuses on the current understanding of the non-classical HLA-I subtypes: HLA-E, HLA-F and HLA-G, within and outside the field of type 1 diabetes, and considers the possible impacts of these molecules on disease etiology. RECENT FINDINGS: Evidence is growing to suggest that non-classical HLA-I proteins are upregulated, both at the RNA and protein levels in the pancreas of individuals with recent-onset type 1 diabetes. Moreover, associations between non-classical HLA-I genotypes and age at onset of type 1 diabetes have been reported in some studies. As with classical HLA-I, it is likely that hyperexpression of non-classical HLA-I is driven by the release of diffusible interferons by stressed ß cells (potentially driven by viral infection) and exacerbated by release of cytokines from infiltrating immune cells. Non-classical HLA-I proteins predominantly (but not exclusively) transduce negative signals to immune cells infiltrating at the site of injury/inflammation. We propose a model in which the islet endocrine cells, through expression of non-classical HLA-I are fighting back against the infiltrating immune cells. By inhibiting the activity and function on NK, B and select T cells, the non-classical HLA-I, proteins will reduce the non-specific bystander effects of inflammation, while at the same time still allowing the targeted destruction of ß cells by specific islet-reactive CD8+ T cells.

HLA Upregulation During Dengue Virus Infection Suppresses the Natural Killer Cell Response.

Dengue virus (DENV) is the most prevalent mosquito-borne virus in the world and a major cause of morbidity in the tropics and subtropics. Upregulation of HLA class I molecules has long been considered a feature of DENV infection, yet this has not been evaluated in the setting of natural infection. Natural killer (NK) cells, an innate immune cell subset critical for mounting an early response to viral infection, are inhibited by self HLA class I, suggesting that upregulation of HLA class I during DENV infection could dampen the NK cell response. Here we addressed whether upregulation of HLA class I molecules occurs during in vivo DENV infection and, if so, whether this suppresses the NK cell response. We found that HLA class I expression was indeed upregulated during acute DENV infection across multiple cell lineages in vivo. To better understand the role of HLA class I upregulation, we infected primary human monocytes, a major target of DENV infection, in vitro. Upregulation of total HLA class I is dependent on active viral replication and is mediated in part by cytokines and other soluble factors induced by infection, while upregulation of HLA-E occurs in the presence of replication-incompetent virus. Importantly, blocking DENV-infected monocytes with a pan-HLA class I Fab nearly doubles the frequency of degranulating NK cells, while blocking HLA-E does not significantly improve the NK cell response. These findings demonstrate that upregulation of HLA class I during DENV infection suppresses the NK cell response, potentially contributing to disease pathogenesis.

CXCL14 suppresses human papillomavirus-associated head and neck cancer through antigen-specific CD8+ T-cell responses by upregulating MHC-I expression.

Evasion of the host immune responses is critical for both persistent human papillomavirus (HPV) infection and associated cancer progression. We have previously shown that expression of the homeostatic chemokine CXCL14 is significantly downregulated by the HPV oncoprotein E7 during cancer progression. Restoration of CXCL14 expression in HPV-positive head and neck cancer (HNC) cells dramatically suppresses tumor growth and increases survival through an immune-dependent mechanism in mice. Although CXCL14 recruits natural killer (NK) and T cells to the tumor microenvironment, the mechanism by which CXCL14 mediates tumor suppression through NK and/or T cells remained undefined. Here we report that CD8+ T cells are required for CXCL14-mediated tumor suppression. Using a CD8+ T-cell receptor transgenic model, we show that the CXCL14-mediated antitumor CD8+ T-cell responses require antigen specificity. Interestingly, CXCL14 expression restores major histocompatibility complex class I (MHC-I) expression on HPV-positive HNC cells downregulated by HPV, and knockdown of MHC-I expression in HNC cells results in loss of tumor suppression even with CXCL14 expression. These results suggest that CXCL14 enacts antitumor immunity through restoration of MHC-I expression on tumor cells and promoting antigen-specific CD8+ T-cell responses to suppress HPV-positive HNC.

Measuring Synthesis and Degradation of MHC Class I Molecules.

Major histocompatibility complex (MHC) class I molecules function to present pathogen derived peptides to cytotoxic T cells and act as ligands for Natural Killer cells, thus alerting the immune system to the presence of invading pathogens. However, some MHC class I molecules, most notably HLA-B27, can be strongly associated with autoimmune diseases. In addition, the MHC class I pathway is a target for numerous viral evasion strategies Understanding not only the antigen presenting functions, but also the biosynthesis and the degradation pathways of MHC class I molecules has therefore become important in determining their role in pathogen and autoimmune related diseases. Here, we describe how using epitope tagged MHC class I molecules can aid in the analysis of MHC class I molecule biosynthesis and degradation as well as complementary studies using conventional conformationally specific antibodies. Coupled together with pharmacological manipulation which can target both biosynthetic and degradative pathways, this offers a powerful tool in analyzing MHC class I molecules.

Cell surface expression of HLA I molecules as a marker of young platelets.

BACKGROUND: Accurate identification of the proportion of young platelets is important to distinguish peripheral thrombocytopenia from a deficit in platelet production. Young platelets are defined by their higher RNA content and are often assessed as thiazole orange bright (TObright ) by flow cytometry. In clinical practice, their proportion is estimated by automatic blood counter according to their greater RNA content, which identifies a so-called immature platelet fraction (IPF). However, the detected IPFs are not strictly identical to the young TObright platelet population observed by flow cytometry. OBJECTIVES: The aim of this study was to assess the reliability of HLA I/major histocompatibility I (MHC I) cell surface expression as a marker of young platelets. METHODS: The HLA I/MHC I expression was evaluated by flow cytometry after costaining blood with TO and antibodies directed against HLA I/MHC I molecules. RESULTS: We found that platelets with a higher expression of plasma membrane-localized MHC I molecules displayed an increased TO staining and a higher content in ribosomal P-antigen. Transfusion experiments in mice showed that the number of MHC I molecules expressed on the cell surface of young murine platelets decreased during platelet aging, reaching basal levels within 24 h. Finally, we demonstrated that for patients with thrombocytopenias, the identification of young platelets is better assessed by the flow cytometric determination of the level of HLA I expression than by TO staining or the use of hematological blood counter. CONCLUSION: Overall, our results highlight the relevance of MHC I/HLA I expression as a valuable parameter to identify young platelets.

Higher human lymphocyte antigen class I expression in early-stage cancer cells leads to high sensitivity for cytotoxic T lymphocytes.

Human lymphocyte antigen (HLA) class I molecules play a central role in cytotoxic T lymphocytes (CTL)-based antitumor immunity. However, the expression rate of HLA class I in cancer cells remains a topic of discussion. We compared HLA class I expression levels between cancer cells and surrounding non-tumorous hepatocytes in 20 early-stage hepatocellular carcinoma (HCC) patients by immunohistochemistry using EMR 8-5. The expression levels of HLA class I were classified as negative, incomplete positive or complete positive. Similarly, for various types of solid cancers, HLA class I expression was examined. For the HLA class I expression in cancer cells, among 20 HCC patients, 13 were complete positive, 3 were incomplete positive, and 4 were negative. In addition, 15 (75.0%) had higher expression levels of HLA class I in cancer cells compared with that in surrounding non-tumorous hepatocytes. An interferon-γ (IFN-γ) enzyme-linked immunospot (ELISPOT) assay indicated that cancer cells with positive expression of HLA class I had strong sensitivity to antigen-specific CTL. We suggested that HLA class I expression in cancer cells could be involved in the clinical prognosis of HCC patients. Similarly, 66.7%, 100.0%, 66.7% and 62.5% of patients with early-stage pancreatic, gallbladder, esophageal and breast cancers, respectively, had higher expression levels of HLA class I in cancer cells than in surrounding normal tissue cells. We suggest that in several early-stage solid cancers, including HCC, HLA class I expression levels in cancer cells are higher than that in surrounding normal tissue cells, which could result in the anti-tumor effect of CTL-based cancer immunotherapy.

Structure of the IFNγ receptor complex guides design of biased agonists.

The cytokine interferon-γ (IFNγ) is a central coordinator of innate and adaptive immunity, but its highly pleiotropic actions have diminished its prospects for use as an immunotherapeutic agent. Here, we took a structure-based approach to decoupling IFNγ pleiotropy. We engineered an affinity-enhanced variant of the ligand-binding chain of the IFNγ receptor IFNγR1, which enabled us to determine the crystal structure of the complete hexameric (2:2:2) IFNγ-IFNγR1-IFNγR2 signalling complex at 3.25 Å resolution. The structure reveals the mechanism underlying deficits in IFNγ responsiveness in mycobacterial disease syndrome resulting from a T168N mutation in IFNγR2, which impairs assembly of the full signalling complex. The topology of the hexameric complex offers a blueprint for engineering IFNγ variants to tune IFNγ receptor signalling output. Unexpectedly, we found that several partial IFNγ agonists exhibited biased gene-expression profiles. These biased agonists retained the ability to induce upregulation of major histocompatibility complex class I antigen expression, but exhibited impaired induction of programmed death-ligand 1 expression in a wide range of human cancer cell lines, offering a route to decoupling immunostimulatory and immunosuppressive functions of IFNγ for therapeutic applications.

Ribosomal Proteins Regulate MHC Class I Peptide Generation for Immunosurveillance.

The MHC class I antigen presentation system enables T cell immunosurveillance of cancers and viruses. A substantial fraction of the immunopeptidome derives from rapidly degraded nascent polypeptides (DRiPs). By knocking down each of the 80 ribosomal proteins, we identified proteins that modulate peptide generation without altering source protein expression. We show that 60S ribosomal proteins L6 (RPL6) and RPL28, which are adjacent on the ribosome, play opposite roles in generating an influenza A virus-encoded peptide. Depleting RPL6 decreases ubiquitin-dependent peptide presentation, whereas depleting RPL28 increases ubiquitin-dependent and -independent peptide presentation. 40S ribosomal protein S28 (RPS28) knockdown increases total peptide supply in uninfected cells by increasing DRiP synthesis from non-canonical translation of "untranslated" regions and non-AUG start codons and sensitizes tumor cells for T cell targeting. Our findings raise the possibility of modulating immunosurveillance by pharmaceutical targeting ribosomes.

Distinct Roles of Extracellular Domains in the Epstein-Barr Virus-Encoded BILF1 Receptor for Signaling and Major Histocompatibility Complex Class I Downregulation.

The Epstein-Barr virus (EBV) BILF1 gene encodes a constitutively active G protein-coupled receptor (GPCR) that downregulates major histocompatibility complex (MHC) class I and induces signaling-dependent tumorigenesis. Different BILF1 homologs display highly conserved extracellular loops (ECLs) including the conserved cysteine residues involved in disulfide bridges present in class A GPCRs (GPCR bridge between transmembrane helix 3 [TM-3] and ECL-2) and in chemokine receptors (CKR bridge between the N terminus and ECL-3). In order to investigate the roles of the conserved residues in the receptor functions, 25 mutations were created in the extracellular domains. Luciferase reporter assays and flow cytometry were used to investigate the G protein signaling and MHC class I downregulation in HEK293 cells. We find that the cysteine residues involved in the GPCR bridge are important for both signaling and MHC class I downregulation, whereas the cysteine residues in the N terminus and ECL-3 are dispensable for signaling but important for MHC class I downregulation. Multiple conserved residues in the extracellular regions are important for the receptor-induced MHC class I downregulation, but not for signaling, indicating distinct structural requirements for these two functions. In an engineered receptor containing a binding site for Zn+2 ions in a complex with an aromatic chelator (phenanthroline or bipyridine), a ligand-driven inhibition of both the receptor signaling and MHC class I downregulation was observed. Taken together, this suggests that distinct regions in EBV-BILF1 can be pharmacologically targeted to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation.IMPORTANCE G protein-coupled receptors constitute the largest family of membrane proteins. As targets of >30% of the FDA-approved drugs, they are valuable for drug discovery. The receptor is composed of seven membrane-spanning helices and intracellular and extracellular domains. BILF1 is a receptor encoded by Epstein-Barr virus (EBV), which evades the host immune system by various strategies. BILF1 facilitates the virus immune evasion by downregulating MHC class I and is capable of inducing signaling-mediated tumorigenesis. BILF1 homologs from primate viruses show highly conserved extracellular domains. Here, we show that conserved residues in the extracellular domains of EBV-BILF1 are important for downregulating MHC class I and that the receptor signaling and immune evasion can be inhibited by drug-like small molecules. This suggests that BILF1 could be a target to inhibit the signaling-mediated tumorigenesis and interfere with the MHC class I downregulation, thereby facilitating virus recognition by the immune system.

Expression and characterization of soluble epitope-defined major histocompatibility complex (MHC) from stable eukaryotic cell lines.

MHC class I-specific reagents such as fluorescently-labeled multimers (e.g., tetramers) have greatly advanced the understanding of CD8+ T cells under normal and diseased states. However, recombinant MHC class I components (comprising MHC class I heavy chain and ß2 microglobulin) are usually produced in bacteria following a lengthy purification protocol that requires additional non-covalent folding steps with exogenous peptide for complete molecular assembly. We have provided an alternative and rapid approach to generating soluble and fully-folded MHC class I molecules in eukaryotic cell lines (such as CHO cells) using a Sleeping Beauty transposon system. Importantly, this method culminates in generating stable cell lines that reliably secrete epitope-defined MHC class I molecules into the tissue media for convenient purification and eventual biotinylation/multimerization. Additionally, MHC class I components are covalently linked, providing the opportunity to produce a diverse set of CD8+ T cell-specific reagents bearing peptides with various affinities to MHC class I.

Production of soluble pMHC-I molecules in mammalian cells using the molecular chaperone TAPBPR.

Current approaches for generating major histocompatibility complex (MHC) Class-I proteins with desired bound peptides (pMHC-I) for research, diagnostic and therapeutic applications are limited by the inherent instability of empty MHC-I molecules. Using the properties of the chaperone TAP-binding protein related (TAPBPR), we have developed a robust method to produce soluble, peptide-receptive MHC-I molecules in Chinese Hamster Ovary cells at high yield, completely bypassing the requirement for laborious refolding from inclusion bodies expressed in E.coli. Purified MHC-I/TAPBPR complexes can be prepared for multiple human allotypes, and exhibit complex glycan modifications at the conserved Asn 86 residue. As a proof of concept, we demonstrate both HLA allele-specific peptide binding and MHC-restricted antigen recognition by T cells for two relevant tumor-associated antigens. Our system provides a facile, high-throughput approach for generating pMHC-I antigens to probe and expand TCR specificities present in polyclonal T cell repertoires.