Epstein-Barr Virus-induced Gene 3 Expression Research Articles

Epstein-Barr Virus-induced Gene 3 as a Novel Biomarker in Metastatic Melanoma With Infiltrating CD8+ T Cells: A Study Based on The Cancer Genome Atlas (TCGA).

Epstein-Barr virus-induced gene 3 (EBI3) is an immunomodulatory protein-coding gene. So far, the prognostic role of EBI3 in human metastatic melanoma has been unclear. This study aimed to evaluate the EBI3 expression as a potential biomarker using the public database with tumor-infiltrating lymphocytes (TILs) data. Survival analyses were performed in the database of The Cancer Genome Atlas (TCGA) and GSE65904, GSE19234, GSE22153, and GSE22154. The mRNA levels, the distribution pattern of TILs, and the estimated fractions of TILs from the TCGA database were integrated. Higher EBI3 expression in tumors was significantly associated with longer overall survival in TCGA and the other independent cohorts. Interestingly, the patients with high EBI3 expression had a brisk pattern of TILs and increased CD8+ T cells over regulatory T cells with less pigmentation-related gene expressions. EBI3 could serve as a novel biomarker in metastatic melanoma with a favorable TILs profile.

A Chaperone-Like Role for EBI3 in Collaboration With Calnexin Under Inflammatory Conditions.

The interleukin-6 (IL-6)/IL-12 family of cytokines plays critical roles in the induction and regulation of innate and adaptive immune responses. Among the various cytokines, only this family has the unique characteristic of being composed of two distinct subunits, α- and β-subunits, which form a heterodimer with subunits that occur in other cytokines as well. Recently, we found a novel intracellular role for one of the α-subunits, Epstein-Barr virus-induced gene 3 (EBI3), in promoting the proper folding of target proteins and augmenting its expression at the protein level by binding to its target protein and a well-characterized lectin chaperone, calnexin, presumably through enhancing chaperone activity. Because calnexin is ubiquitously and constitutively expressed but EBI3 expression is inducible, these results could open an avenue to establish a new paradigm in which EBI3 plays an important role in further increasing the expression of target molecules at the protein level in collaboration with calnexin under inflammatory conditions. This theory well accounts for the heterodimer formation of EBI3 with p28, and probably with p35 and p19 to produce IL-27, IL-35, and IL-39, respectively. In line with this concept, another β-subunit, p40, plays a critical role in the assembly-induced proper folding of p35 and p19 to produce IL-12 and IL-23, respectively. Thus, chaperone-like activities in proper folding and maturation, which allow the secretion of biologically active heterodimeric cytokines, have recently been highlighted. This review summarizes the current understanding of chaperone-like activities of EBI3 to form heterodimers and other associations together with their possible biological implications.

EBV-induced gene 3 augments IL-23Rα protein expression through a chaperone calnexin.

Epstein-Barr virus-induced gene 3 (EBI3) is a subunit common to IL-27, IL-35, and IL-39. Here, we explore an intracellular role of EBI3 that is independent of its function in cytokines. EBI3-deficient naive CD4+ T cells had reduced IFN-γ production and failed to induce T cell-dependent colitis in mice. Similarly reduced IFN-γ production was observed in vitro in EBI3-deficient CD4+ T cells differentiated under pathogenic Th17 polarizing conditions with IL-23. This is because the induction of expression of one of the IL-23 receptor (IL-23R) subunits, IL-23Rα, but not another IL-23R subunit, IL-12Rβ1, was selectively decreased at the protein level, but not the mRNA level. EBI3 augmented IL-23Rα expression via binding to the chaperone molecule calnexin and to IL-23Rα in a peptide-dependent manner, but not a glycan-dependent manner. Indeed, EBI3 failed to augment IL-23Rα expression in the absence of endogenous calnexin. Moreover, EBI3 poorly augmented the expression of G149R, an IL-23Rα variant that protects against the development of human colitis, because binding of EBI3 to the variant was reduced. Taken together with the result that EBI3 expression is inducible in T cells, the present results suggest that EBI3 plays a critical role in augmenting IL-23Rα protein expression via calnexin under inflammatory conditions.

Cloning and Expression of EBI3 and p28 Subunits of Human Interleukin 27 in E. coli

Background: Interleukin-27 is a heterodimeric cytokine belonging to IL-12 and IL-23 families, secreted by Antigen Presenting Cells( APCs ) .The IL-27 is composed of 2 subunits: Epstein-Barr virus induced gene 3 (EBI3) and p28. IL-27 is an anti-inflammatory cytokine which has an inhibitory effect on Th17 population and suppress the IL-17 expression. It is suggested that IL-27 could be a potent drug candidate for treating auto immune diseases. Methods: The IL-27 subunit genes were constructed into plasmid vectors, they sub-cloned into pETDeut-1an expression vector in restriction sites of BamHI, SacI and NotI. Subsequently the induction was done by IPTG and the recombinant proteins were confirmed by SDS-PAGE and Western Blot analysis using anti His-tag antibody. Results: The 28kDa and 25kDa protein bands were observed on SDS-PAGE and finally confirmed by Western blot technique.

Interleukin-35 is associated with the tumorigenesis and progression of prostate cancer.

Interleukin (IL)-35 is a novel member of the IL-12 cytokine family, which exhibits a unique immune regulatory function. Previously, it was demonstrated that IL-35 expression is significantly increased in a wide range of tumor tissues, promoting angiogenesis and inhibiting the anti-tumor immune response. In the present study, the IL-35 protein expression levels were measured in the plasma and tumor tissue of patients with prostate cancer (PCA), and its role was determined in the occurrence and progression of PCA. Plasma IL-35 expression levels were measured using ELISA, and the associations of plasma IL-35 and clinicopathological parameters were analyzed. Receiver operating characteristic curves were plotted to analyze the role of IL-35 as a clinical biomarker for the diagnosis of PCA. Survival curve analysis demonstrated a significant decrease in the survival time in months in patients with PCA and increased expression levels of IL-35 compared with the participants with relatively lower IL-35 protein expression levels. IL-35 expression was detected using immunohistochemical staining of a human PCA tissue microarray. Plasma IL-35 expression levels in the patients with PCA were significantly increased compared with the patients with benign prostatic hyperplasia and the healthy controls. An increase in the plasma concentration of IL-35 was associated with progression of PCA stage and an increase in the Gleason score. Significant differences in AUCs for IL-35 and prostate-specific antigen were observed with regard to the presence of lymph node and distant metastases in patients with PCA. The expression levels of Epstein-barr virus-induced gene 3 (EBI3) and IL-12A (p35; the subunits which together form IL-35) were significantly increased in the tumor tissue compared with the adjacent normal tissue. An association was identified between the Gleason score and the expression of EBI3 and p35. Therefore, increased expression of IL-35 in the plasma and tumor tissues may contribute to the progression and metastasis of PCA, and IL-35 may serve as a novel prognostic biomarker or therapeutic target for the treatment of PCA.

MEK-ERK signaling diametrically controls the stimulation of IL-23p19 and EBI3 expression in epithelial cells by IL-36γ.

Interleukin (IL)-36 cytokines are important regulators of mucosal homeostasis and inflammation. We previously established that oral epithelial cells strongly upregulate IL-36γ expression in response to the bacterial pathogen Porphyromonas gingivalis. Here, we have established that IL-36γ stimulates the expression of the IL-12 cytokine family members, IL-23p19 and Epstein-Barr Virus-Induced Gene 3 (EBI3), by oral epithelial cells; their expression was also selectively stimulated by IL-36α. Notably, IL-23p19 and EBI3 expression was not stimulated by P. gingivalis, thus suggesting that their expression by the oral epithelium in response to P. gingivalis is likely to be mediated in an autocrine manner by IL-36γ. The IL-36γ-inducible expression of IL-23p19 and EBI3 was found to be diametrically regulated by the mitogen-activated protein kinase/extracellular signal regulated kinase (MEK)-extracellular signal-regulated kinase 1/2 (ERK1/2) pathway, whereby the activation of MEK-ERK signaling likely functions as a negative feedback mechanism to limit EBI3 expression. Furthermore, epidermal growth factor receptor (EGFR) signaling, which is important for mucosal homeostasis, was demonstrated to modulate, in a MEK-ERK-dependent manner, the stimulation of IL-23p19 and EBI3 expression by IL-36γ. IL-23p19 and EBI3 have recently been shown to heterodimerize to form the novel cytokine IL-39 and promote neutrophil expansion. EBI3 has been shown to also have IL-12 cytokine family independent functions (e.g. mediating IL-6 trans-signaling). Thus, this study not only advances our understanding of how IL-36 cytokines may control mucosal inflammation, but also establishes EGFR signaling as a potentially important modulator of IL-36 cytokine function.

Downregulation of Epstein-Barr virus-induced gene 3 is associated with poor prognosis of hepatocellular carcinoma after curative resection.

Epstein-Barr virus-induced gene 3 (EBI3) encodes a secretory glycoprotein, and has previously been identified as upregulated in various types of tumors. The presnet study examined the clinical significance of EBI3 expression for predicting tumor recurrence and survival after resection in hepatocellular carcinoma (HCC). EBI3 expression in various HCC cell lines and in 20 pairs of tumor and peritumor tissue samples were detected using western blot analysis. Immunohistochemical staining using tissue microarray with 312 samples from randomly selected patients with HCC who underwent surgery. Survival analysis was performed using univariate and multivariate analyses. EBI3 protein level was higher in L-02 cells and in peri-tumor tissues compared with tumor tissues. Immunohistochemical staining of EBI3 was reduced in HCC tissues in comparison with adjacent normal tissues and significantly associated with tumor invasive characteristics, including tumor thrombus, poor differentiation and large size. Notably, the results suggested that EBI3 was a predictor for tumor recurrence and patient survival, and multivariate analysis indicated EBI3 to be an independent prognostic factor. Even in early-stage disease, low EBI3 expression was also independently associated with increased tumor recurrence and shortened survival. Downregulation of EBI3 in HCC indicated aggressive tumor behaviors and predicted a more severe clinical outcome, which suggests that EBI2 may be a useful biomarker to identify patients at high risk of post-operative recurrence.

Upregulated EBI3 Correlates with Poor Outcome and Tumor Progression in Breast Cancer

Background and Aim: So far, the understanding of the role of Epstein-Barr virus-induced gene 3 (EBI3) in breast cancer has been limited. This study uncovers the functional role and clinical significance of EBI3 in breast cancer patients. Patients and Methods: The expression levels of EBI3, IL-27p28, and IL-12p35 were measured by quantitative real-time reverse transcription (RT)-polymerase chain reaction (PCR). Correlations of EBI3 expression with IL-27p28 and IL-12p35 expression were analyzed using Pearson's correlation assay. The prognostic performance of EBI3 was assessed via Kaplan-Meier survival assay and Cox regression analysis. Results:EBI3 expression was increased in cancerous tissues compared with the controls (P < 0.05). This overexpression of EBI3 was correlated with lymph node metastasis and clinical stage (both P < 0.05). Besides, elevated expression of EBI3 was usually found in patients with positive lymph node metastasis (P < 0.05), and similar results were obtained in advanced clinical-stage breast cancer cases (P < 0.05). Increases in both IL-27p28 and IL-12p35 expression were identified in breast cancer tissues (all P < 0.05), and IL-12p35 expression was found to be associated with EBI3 expression (R = 0.888, P < 0.001). Survival curves revealed that high EBI3 expression was correlated with poor overall survival (log-rank P < 0.05). The Cox analysis indicated that EBI3 was an independent prognostic factor in breast cancer. Conclusion: Taken together, overexpression of EBI3 was associated with poor prognosis and might be involved in the progression of breast cancer.

Expression and significance of interleukin-35 in lesion tissue of oral lichen planus patients

Objective: To investigate the expression of Epstein-Barr virus-induced gene 3 (EBi3) and interleukin-12p35 (IL-12p35) in two subunits of interleukin-35 (IL-35) in oral lichen planus (OLP) lesions, and to explore the role of IL-35 played in the formation and development of OLP lesions. Methods: Totally 41 samples of OLP lesions and 15 samples of normal tissues were collected from patients of the Department of Periodontology and Oral Medicine, Affiliated Stomatological Hospital of Guizhou Medical University from October 2010 to December 2016. The expression levels of EBi3 mRNA and IL-12p35 mRNA in the samples were detected by quantitative real-time PCR (qPCR) and the distribution and expression of protein EBi3 and IL-12p35 were detected by immunohistochemistry. The potential relationship between IL-35 and clinicopathological features of OLP was analyzed. Results: The expression [M(Q(25), Q(75))] of EBi3 [3.38 (1.63, 11.25)] and IL-12p35 mRNA [6.39 (2.55, 14.30)] in OLP lesion tissues were significantly higher than those in normal control group [1.41 (0.33, 3.16), 2.47 (1.10, 5.14)] (Z=-2.806, P=0.005; Z=-2.276, P=0.023), respectively. The positive expression rates of EBi3 and IL-12p35 were 66% (27/41) and 39% (16/41), respectively, were significantly higher in OLP lesion tissues comparing with that in normal oral mucosa tissues [0%(0/15)] (P<0.05). The relative expressions of EBi3 and IL-12p35 were positively correlated (r=0.404, P=0.009). A significant correlation was found between EBi3 protein over expression and the degeneration of base cells in OLP lesions (χ(2)=9.172, P=0.010). The positive expression rate of IL-12p35 protein in erosive type lesions was higher than that in non-erosive type lesions (χ(2)=7.220, P=0.007). The positive expression rate of IL-35 protein in OLP lesions [34% (14/41)] was higher than that in normal control group (χ(2)=6.829, P=0.009). The expression rate of IL-35 in erosive type lesions (10/20) was significantly higher than that in eruption type lesions (4/21) (χ(2)=4.364, P=0.037). Conclusions: The expression of IL-35 in OLP localized lesions was up-regulated, suggesting that IL-35 might play an important role in OLP lesion formation.

Epstein-Barr Virus-Induced Gene 3 (EBI3) Blocking Leads to Induce Antitumor Cytotoxic T Lymphocyte Response and Suppress Tumor Growth in Colorectal Cancer by Bidirectional Reciprocal-Regulation STAT3 Signaling Pathway.

Epstein-Barr virus-induced gene 3 (EBI3) is a member of the interleukin-12 (IL-12) family structural subunit and can form a heterodimer with IL-27p28 and IL-12p35 subunit to build IL-27 and IL-35, respectively. However, IL-27 stimulates whereas IL-35 inhibits antitumor T cell responses. To date, little is known about the role of EBI3 in tumor microenvironment. In this study, firstly we assessed EBI3, IL-27p28, IL-12p35, gp130, and p-STAT3 expression with clinicopathological parameters of colorectal cancer (CRC) tissues; then we evaluated the antitumor T cell responses and tumor growth with a EBI3 blocking peptide. We found that elevated EBI3 may be associated with IL-12p35, gp130, and p-STAT3 to promote CRC progression. EBI3 blocking peptide promoted antitumor cytotoxic T lymphocyte (CTL) response by inducing Granzyme B, IFN-γ production, and p-STAT3 expression and inhibited CRC cell proliferation and tumor growth to associate with suppressing gp130 and p-STAT3 expression. Taken together, these results suggest that EBI3 may mediate a bidirectional reciprocal-regulation STAT3 signaling pathway to assist the tumor escape immune surveillance in CRC.

Expression of Epstein-Barr virus-induced gene 3 in cervical cancer: Association with clinicopathological parameters and prognosis.

Epstein-Barr virus-induced gene 3 (EBI3) encodes a secretory glycoprotein, and has previously been identified as upregulated in a series of cancers. However, the clinical significance of EBI3 in cervical cancer and the potential of EBI3 as a therapeutic target for this disease have not been elucidated. In the present study, EBI3 expression was analyzed by immunohistochemistry in 90 clinicopathologically characterized cervical cancer tissue samples. The association between EBI3 expression and survival of patients with cervical cancer was also analyzed. The expression level of EBI3 in cervical cancer tissues was found to be significantly increased compared with the expression levels in the normal squamous epithelium. In addition, EBI3 expression was significantly correlated with the clinical stage and size of tumors (P<0.05). Furthermore, the presence of EBI3 expression was associated with a poor prognosis compared with patients without EBI3 expression. Multivariate analysis revealed that EBI3 expression was an independent predictor of overall survival (hazard ratio, 4.032; 95% confidence interval, 1.538-7.436; P=0.035). To the best of our knowledge, the present results indicate for the first time that EBI3 expression is significantly associated with the progression and poor prognosis of cervical cancer. EBI3 may be a potential prognostic marker and a therapeutic target in cervical cancer.

Epstein–Barr virus-induced gene 3 (EBI3) polymorphisms and expression are associated with susceptibility to pulmonary tuberculosis

Tuberculosis (TB) remains a major global health problem and host genetic factors play a critical role in susceptibility and resistance to TB. The aim of this study was to identify novel candidate genes associated with TB susceptibility. We performed a population-based case-control study to genotype 13 tag SNPs spanning Epstein-Barr virus-induced gene 3 (EBI3), colony stimulating factor 2 (CSF2), IL-4, interferon beta 1 (IFNB1), chemokine (C-X-C motif) ligand 14 (CXCL14) and myeloid differentiation primary response gene 88 (Myd88) genes in 435 pulmonary TB patients and 375 health donors from China. We observed that EBI3 gene rs4740 polymorphism was associated with susceptibility to pulmonary tuberculosis (PTB) and the allele G was associated with a protective effect against PTB. Furthermore, EBI3 deficiency led to reduced bacterial burden and histopathological impairment in the lung of mice infected with Mycobacterium bovis BCG. Meanwhile, higher abundance of EBI3 was observed in the granuloma of PTB patients and in the lung tissue of BCG-infected mice. Of note, the expression of EBI3 in macrophages was remarkably induced by mycobacteria infection at both mRNA and protein level. In conclusion, EBI3 gene rs4740 polymorphism is closely associated with susceptibility to PTB and the elevation and enrichment of EBI3 in the lung which at least partially derived from macrophages may contribute to the exacerbation of mycobacterial infection.

P7 Investigating the role of the novel cytokine IL-35 in the regulation of anti-tumour immunity against head and neck cancer

Background Cancer cells have the ability to induce immune tolerance, in order to escape the host’s immune cells, which aids in tumour survival. The mechanism by which immune tolerance occurs is not completely known; however, it has been suggested that cytokines produced by cancer cells may serve a key role. Interleukin (IL)-35 is a novel immune suppression cytokine forming part of the IL-12 family and is a heterodimeric secreting protein composed of two subunits: Epstein-Barr virus-Induced gene 3 (EBI3) and IL-12p35. Previous studies have demonstrated that IL-35 is expressed in cancer cells. Immune cells in tumour tissue include CD4+ and CD8+ T cells and inflammatory macrophages (M1) that produce pro-inflammatory cytokines including IFNγ, TNF α and IL-17A for tumour rejection. The aim of this study was to explore whether IL-35 was expressed in head and neck cancer cells, and whether those cytokines produced by anti-tumour T-cells and macrophages are able to up-regulate IL-35 expression in head and neck cancer cells. Material and Methods Five head and neck cancer cell lines (H357, H376, FADU, C1 and VB6) were cultured and stimulated with or without IFNγ. The FADU cells were also stimulated with TNF α and IL-17A. The expression of IL-35 subunits was investigated by real-time quantitative polymerase chain reaction and the protein expression confirmed by Western blotting. Results All five head and neck cancer cell lines demonstrated expression of IL-35 subunits: EBI3 and IL-12p35. EBI3 expression was increased in α V integrin (C1) and α V β 6 integrin (VB6) over-expressed cell lines. IFN? stimulation up-regulated the expression of the IL-12p35 subunit in all head and neck cancer cell lines. TNF α up-regulated EBI3 expression in FADU cells; co-stimulation with IFNγ and TNF α synergistically up-regulated EBI3 expression in FADU cells. IL-17A had no effect on IL-35 expression in FADU cells. Conclusion Anti-tumour immunity mediated by pro-inflammatory cytokines stimulates head and neck cancer cells to produce more of the immune suppressing cytokine IL-35. This may serve an important role in cancer cells inducing immune-tolerance in tumour tissue; thus, further research is required to provide insights into IL-35 function in head and neck cancer.

Increased expression of interleukin (IL)-35 and IL-17, but not IL-27, in gingival tissues with chronic periodontitis.

Interleukin (IL)-35 plays an important role in immune regulation through the suppression of effector T-cell populations, including T-helper 17 (Th17) cells. Although Th17 cells and IL-17 are involved in the pathogenesis of periodontitis, the level of IL-35 in inflamed periodontal tissues is unclear. Here, IL-35, IL-17, and IL-27 production/expression in gingival crevicular fluid (GCF) and human gingival tissue were investigated. GCF samples were collected from buccal (mesial, center, and distal) sites of teeth from patients with chronic periodontitis (CP) and healthy controls and were analyzed by enzyme-linked immunosorbent assay for IL-35 (periodontitis, n = 36; healthy, n = 30) and IL-17 (periodontitis, n = 16; healthy, n = 13). Gingival tissue, including sulcus/pocket epithelium and underlying connective tissue, was collected from an additional 10 healthy participants and 10 patients with CP and were analyzed by quantitative polymerase chain reaction (qPCR) for Epstein Barr virus-induced gene 3 (EBI3), IL12A, and IL17A. IL27p28 was also tested by qPCR. IL-35 and IL-17 were significantly higher in GCF from patients with periodontitis than healthy participants (P <0.01, P <0.05, respectively). In both healthy participants and those with periodontitis, positive correlations were found among IL-35 and probing depth and clinical attachment level (CAL) as well as between IL-17 and CAL. EBI3, IL12A (components of IL-35), and IL17A messenger RNA expression levels were significantly higher in inflamed gingival tissue than in healthy control tissues (P <0.05). IL27p28 was not detected in any sample, suggesting that IL-27 is not produced in large quantities in periodontal tissue. IL-35 and IL-17, but not IL-27, may play important roles in the pathogenesis of periodontitis.

Interleukin 35 is an independent prognostic factor and a therapeutic target for nasopharyngeal carcinoma.

Aim of the studyInterleukin (IL)-35 is composed of two subunits: Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. Recently, overexpression of IL-35 has been found in several types of cancers. However, its clinical significance in nasopharyngeal carcinoma is still obscure. We have studied the clinical significance of IL-35 expression and its correlation with outcome of nasopharyngeal carcinoma patients.Material and methodsInterleukin 35 expression was investigated in 80 nasopharyngeal carcinoma cases by immunohistochemistry. Moreover, Fisher's exact test, Kaplan-Meier plots, and Cox proportional hazards regression were utilized to analyse these results.ResultsIn the present study, IL-35 is highly expressed in the majority of nasopharyngeal carcinoma samples. EBI3 and p35 immunoreactivity in nasopharyngeal carcinoma samples was 67.5% and 51.3%, respectively. Both EBI3 and p35 expressions were significantly associated with advancement of tumour stage. In addition, EBI3 expression was also correlated with lymph node metastasis. Further analysis showed that EBI3 or p35 staining indicated unfavourable prognosis (p < 0.05). Multivariate analysis suggested EBI3 was an independent prognostic predictor (p < 0.05).ConclusionsOur results indicate for the first time that IL-35 is correlated with progression of nasopharyngeal carcinoma. Therefore, IL-35 may be a useful target for the treatment of nasopharyngeal carcinoma.

Lipopolysaccharide-Induced M2 to M1 Macrophage Transformation for IL-12p70 Production Is Blocked by Candida albicans Mediated Up-Regulation of EBI3 Expression

Macrophages are heterogeneous cell populations that are present in all tissues. Macrophages can be divided into classically activated inflammatory macrophages (M1) and alternatively activated anti-inflammatory macrophages (M2). It has been generally accepted that M1 macrophages are polarised in an inflammatory environment to produce pro-inflammatory cytokines, whilst M2 macrophages are involved in anti-inflammation and aid tissue repair in wound healing. Bacterial endotoxin (lipopolysaccharide; LPS) is a potent factor in infection, which induces M1 macrophages resulting in higher levels of iNOS, TNFα and IL-12p70 which dictate inflammatory T cell responses. M2 macrophages can be transformed into M1 macrophages following LPS stimulation to promote inflammation. Candida albicans is a commensal fungal microorganism, which has been suggested to induce immune tolerance; however, the mechanism of C. albicans-induced immune tolerance has not been investigated in detail. IL-35 is a recently identified anti-inflammatory cytokine which is a heterodimeric protein consisting of the Epstein-Barr virus-induced gene 3 (EBI3) and IL-12p35. IL-35 shares the protein subunit p35, with IL-12p70. IL-12p70 is the most potent cytokine to induce Th1 responses during inflammation. In this study, we demonstrate that heat-killed C. albicans (HKC) strongly suppressed LPS-induced IL-12p70 production in M2 macrophages. Candida albicans induced a high level of EBI3 expression in M2 macrophages, which served as a mechanism for IL-12p70 suppression by competitive binding of the common protein subunit (p35) of IL-35 and IL-12p70. To demonstrate that EBI3 expression had the ability to block IL-12p70 production intracellularly, a Chinese Hamster Ovary (CHO) cell line with biscistronic expression of IL-12p40 and p35 was constructed, followed by ectopic over-expression of EBI3. The over-expression of EBI3 in the IL-12p70 producing cell line effectively suppressed IL-12p70 production. IL-35 secretion was also detected in the cell line, with suppressed IL-12p70 production by immune-precipitation Western blotting. However, this secretion was not evident in M2 macrophages following stimulation by HKC. This can be explained by the constitutive expression of IL-35 receptors (gp130 and IL-12Rβ2) in M2 macrophages for cytokine consumption. Our results have indicated that C. albicans can suppress host inflammatory responses in mucosal skin by suppressing LPS-induced IL-12p70 production. Lower IL-12p70 production may avoid an unnecessary Th1 response in order to retain immune tolerance, which may be one of the mechanisms by which C. albicans achieves a successful commensal lifestyle without having a detrimental effect on the host’s health.

Abstract 2370: Epstein-Barr Virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer.

Abstract Lung cancer is the leading cause of cancer deaths in the world. The number of patients responding well to current standard therapies is still small, and no tumor marker has been entirely useful for screening curative stage cancers. To identify molecules which could be useful for the development of diagnostic biomarkers and novel drugs, we have established an effective screening strategy as follows; i) To identify up-regulated genes in 120 lung cancers by genome-wide screening using the cDNA microarray representing 27,648 genes and pure populations of tumor cells taken from cancer tissues by laser microdissection, ii) To verify the candidate genes for their very low level of expression in normal tissues by northern-blot analyses, iii) To validate the clinicopathological significance of its over-expression with tissue microarray containing hundreds of archived lung-cancers, iv) To verify whether the target gene is essential for the growth of cancer cells by RNAi and cell growth assays, v) To evaluate it for usefulness as a serum diagnostic biomarker by ELISA, if they are tumor-specific transmembrane or secretory proteins. As a result of this process, we identified 35 druggable oncoproteins. We further identified the Epstein-Barr virus-induced gene 3 (EBI3), which encodes a secretory glycoprotein. Immunohistochemical staining of EBI3 using tissue microarrays revealed that strong EBI3 expression was associated with a poor prognosis (P = 0.0014), and multivariate analysis confirmed it to be an independent prognostic factor (P = 0.0439; N=414). In addition, we established an ELISA to measure serum EBI3 and found that the proportion of serum EBI3-positive cases was 47.1% (73 of 155) for lung adenocarcinoma, 54.5% (24 of 44) for lung squamous cell carcinoma, and 41.3% (31 of 75) for small cell lung cancer, while 3 (2.4%) of 126 healthy volunteers were falsely diagnosed. The sensitivity of serum EBI3 for cancer detection was 44.4% for stage I-II lung adenocarcinoma, 50.0% for stage I-II lung squamous cell carcinoma, and 35.2% for small cell lung cancer with limited disease (LD) stage, thereby proving it was superior to conventional tumor markers of CEA and CYFRA21-1 in terms of both sensitivity and specificity. Validation analysis using another independent set of serum samples (132 lung cancers and 41 healthy volunteers) confirmed significant elevation of EBI3 in the sera of lung cancer patients. Furthermore, reduction in EBI3 expression by siRNA suppressed cancer cell proliferation, while induction of EBI3 conferred growth-promoting activity. EBI3 should be a biomarker for early cancer detection and for the prediction of prognosis, as well as being a suitable therapeutic target. Citation Format: Yataro Daigo, Atsushi Takano, Yusuke Nakamura. Epstein-Barr Virus-induced gene 3 as a novel serum and tissue biomarker and a therapeutic target for lung cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2370. doi:10.1158/1538-7445.AM2013-2370 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

Decreased Expression of EBI3 and Foxp3 in CD4+CD25+ Regulatory T Cells in Murine Experimental Allergic Rhinitis

Background: Epstein-Barr virus-induced gene 3 (EBI3) is a widely expressed interleukin-12-related protein that plays a critical role in the regulation of effector T cells and inflammatory responses. Methods: Allergic rhinitis (AR) was induced in mice by both intraperitoneal injection and intranasal administration of ovalbumin. By using immunohistochemical techniques, we investigated EBI3 expression in mouse spleens. The expressions of EBI3 and forkhead transcription factor 3 (Foxp3) in CD4+CD25+ regulatory T (T_reg) cells isolated from spleens were assessed by flow cytometry. RNA extraction and RT-PCR were used to measure EBI3 and Foxp3 mRNA expression. Results: EBI3 and Foxp3 were expressed mainly in the white pulp of the spleens. Flow cytometry analysis showed that EBI3 and Foxp3 coexpressed in mouse CD4+CD25+ T_reg cells. Furthermore, RT-PCR and flow cytometry analysis showed that expression of both Foxp3 and EBI3 was reduced in CD4+CD25+ T_reg cells in an AR mouse model compared with controls, but there was no difference in the frequency of CD4+CD25+ T_reg cells. Conclusions: The deficiency in CD4+CD25+ T_reg cell suppressive activity in AR is associated with the molecular mediators EBI3 and Foxp3.

Epstein-Barr Virus-Induced Gene 3 (EBI3): A Novel Diagnosis Marker in Burkitt Lymphoma and Diffuse Large B-Cell Lymphoma

The distinction between Burkitt lymphoma (BL) and diffuse large B-cell lymphoma (DLBCL), two types of mature aggressive B-cell lymphomas that require distinct treatments, can be difficult because of forms showing features intermediate between DLBCL and BL (here called BL/DLBCL). They can be discriminated by the presence of c-myc translocations characteristic of BL. However, these are not exclusive of BL and when present in DLBCL are associated with lower survival. In this study, we show that Epstein-Barr virus-induced gene 3 (EBI3) is differentially expressed among BL and DLBCL. Analysis of gene expression data from 502 cases of aggressive mature B-cell lymphomas available on Gene Expression Omnibus and immunohistochemical analysis of 184 cases of BL, BL/DLBCL or DLBCL, showed that EBI3 was not expressed in EBV-positive or -negative BL cases, whereas it was expressed by over 30% of tumoral cells in nearly 80% of DLBCL cases, independently of their subtypes. In addition, we show that c-myc overexpression represses EBI3 expression, and that DLBCL or BL/DLBCL cases with c-myc translocations have lower expression of EBI3. Thus, EBI3 immunohistochemistry could be useful to discriminate BL from DLBCL, and to identify cases of BL/DLBCL or DLBCL with potential c-myc translocations.

Enhanced expression of IL‐27 mRNA in human newborns

Interleukin (IL)-27, a heterodimer composed of Epstein-Barr virus-induced gene 3 (EBI3) and p28, is an early product of activated dendritic cells (DC). Binding of IL-27 to the WSX-1 receptor initiates Th1 (Thelper 1) responses in naïve T cells. In order to assess the Th1 responses in human neonates with high susceptibility to infectious diseases, expression of EBI3-, p28- and WSX-1-mRNA in response to Toll-like receptor ligands was compared in neonate and adult monocyte-derived (m)DC. Only the combined addition of ligands induced expression of IL-27p28 mRNA. Surprisingly, neonatal mDC produced significantly more IL-27p28 mRNA than that obtained from adults. Furthermore, there was enhanced expression of EBI3 mRNA in cord blood as compared with adult blood. In addition, the secretion of WSX-1 mRNA in neonatal mDC and T cells was also significantly increased. Taken together, these findings indicate that the restricted Th1 responses in human newborns owing to deficient IL-12 production may be compensated for, in part, by enhanced IL-27 secretion.