Protein LMP1 Research Articles

PARP-1 expression against Epstein-Barr virus LMP-1 and BZLF-1 in undifferentiated nasopharyngeal carcinoma

Objective: This study aims to determine the poly(ADP-ribose) polymerase (PARP)-1 expression levels against the Epstein-Barr virus (EBV) latent membrane protein (LMP-1) and BamHI-Z leftward reading frame (BZLF)-1 of nasopharynx tissues and serum of nasopharyngeal carcinoma (NPC) patients and characterize the histopathology of necrosis cells of NPC tissues. Methods: 32 tissues sections consisting 24 NPC and 12 polyp tissues, and also blood serum of patients were taken from Ulin General Hospital, Banjarmasin. Tissue samples were analyzed by using immunofluorescence staining in order to examine LMP-1 and PARP-1 expression. Analyses were done by using immunoblotting with anti-LMP-1, anti-BZLF-1, anti-PARP-1, and anticaspase-3 antibodies. Histopathological profiles of necrosis cells were analyzed by using hematoxylin-eosin staining and necrotic cells were counted by using the standard software of the microscope. Results: LMP-1, BZLF-1 and PARP-1 proteins have high levels of expression in patients with NPC. LMP-1 expression of NPC patients and control sera are equal. Interestingly we found that 35 kDa of BZLF-1 and 55 kDa PARP-1 only expressed in NPC patients, meanwhile caspase-3 expression was negative. Histopathologically, NPC tissues were more dominated by karyorrhexis than pyknosis and karyolysis. Conclusions: This study showed that the expression level of PARP-1 in NPC tissues was increased against LMP-1 and BZLF-1 EBV to protect DNA damage of the recipient cells. This mechanism probably stimulated cell metamorphosis and consequently provoked necrosis. © 2013 GESDAV

RNA Interference: Systemic RNAi SIDes with Endosomes

Systemic RNAi, the intercellular spreading of RNAi silencing, requires SID-1 and SID-3 to import silencing signals in Caenorhabditis elegans. How are these signals exported? SID-5, an endosome-associated protein, is a candidate for the job.

SID-5 is an endosome-associated protein required for efficient systemic RNAi in C. elegans.

In the nematode C. elegans, RNAi silencing signals are efficiently taken up from the environment and transported between cells and tissues. Previous studies implicating endosomal proteins in systemic RNAi lack conclusive evidence. Here, we report the identification and characterization of SID-5, a C. elegans endosome-associated protein that is required for efficient systemic RNAi in response to both ingested and expressed double-stranded RNA (dsRNA). SID-5 is detected in cytoplasmic foci that partially colocalize with GFP fusions of late endosomal proteins RAB-7 and LMP-1. Furthermore, knockdown of various endosomal proteins similarly relocalizes both SID-5 and LMP-1::GFP. Consistent with a non-cell-autonomous function, intestine-specific SID-5 expression restored body wall muscle (bwm) target gene silencing in response to ingested dsRNA. Finally, we show that sid-5 is required for the previously described sid-1-independent transport of ingested RNAi triggers across the intestine. Together, these data demonstrate that an endosome-associated protein, SID-5, promotes the transport of RNAi silencing signals between cells. Furthermore, SID-5 acts differently than the previously described SID-1, SID-2, and SID-3 proteins, thus expanding the systemic RNAi pathway.

Persistence of Epstein-Barr Virus in Self-Reactive Memory B Cells

Epstein-Barr virus infection has been epidemiologically associated with the development of multiple autoimmune diseases, particularly systemic lupus erythematosus and multiple sclerosis. Currently, there is no known mechanism that can account for these associations. The germinal-center (GC) model of EBV infection and persistence proposes that EBV gains access to the memory B cell compartment via GC reactions by driving infected cells to differentiate using the virus-encoded LMP1 and LMP2a proteins, which act as functional homologues of CD40 and the B cell receptor, respectively. The ability of LMP2a, when expressed in mice, to allow escape of autoreactive B cells suggests that it could perform a similar role in infected GC B cells, permitting the survival of potentially pathogenic autoreactive B cells. To test this hypothesis, we cloned and expressed antibodies from EBV(+) and EBV(-) memory B cells present during acute infection and profiled their self- and polyreactivity. We find that EBV does persist within self- and polyreactive B cells but find no evidence that it favors the survival of pathogenic autoreactive B cells. On the contrary, EBV(+) memory B cells express lower levels of self-reactive and especially polyreactive antibodies than their uninfected counterparts do. Our work suggests that EBV has only a modest effect on the GC process, which allows it to access and persist within a subtly unique niche of the memory compartment characterized by relatively low levels of self- and polyreactivity. We suggest that this might reflect an active process where EBV and its human host have coevolved so as to minimize the virus's potential to contribute to autoimmune disease.

Effective control of Epstein–Barr virus infection following pediatric liver transplantation by monitoring of viral DNA load and lymphocyte surface markers

EBV-associated PTLD is a serious complication of liver transplantation. We performed periodical molecular EBV monitoring in 140 consecutive pediatric patients who had living-related liver transplantation in the National Center for Child Health and Development, Tokyo. Sixty-three of the 140 patients showed elevation of EBV DNA level to >10(2) copies/μg DNA and were further examined immunologically by flow cytometry, and the dose of tacrolimus and/or cyclosporine A was adjusted according to the results. The decrease in CD4/CD8 ratio and the increase in the number of HLA-DR(+) CD8(+) cells were observed in parallel with the decrease in EBV DNA load and in the number of CD19(+) CD23(+) cells following the reduction in immunosuppressive drugs. Analysis with HLA tetramers in a patient demonstrated a dramatic increase in the number of CD8(+) T cells specific to the EBV latent protein LMP2 accompanying the decline of EBV DNA load, suggesting that T cells of this specificity were actually involved in the control of EBV infection. No clinically apparent PTLD has developed in the 140 recipients, suggesting that our program of EBV control by molecular EBV monitoring coupled with lymphocyte phenotype analyses is effective in controlling EBV infection in pediatric liver transplant recipients.

Epstein-Barr Virus Infection Is Common in Inflamed Gastrointestinal Mucosa

Epstein-Barr virus (EBV) is present in the malignant epithelial cells of 10% of all gastric adenocarcinomas; however, localization of the virus in normal gastrointestinal mucosa is largely unexplored. In the present study, we measured EBV DNA and localized viral gene products in gastritis specimens (n = 89), normal gastric and colonic mucosa (n = 14), Crohn's disease (n = 9), and ulcerative colitis (n = 11) tissues. A battery of sensitive and specific quantitative polymerase chain reactions targeted six disparate regions of the EBV genome: BamH1 W, EBNA1, LMP1, LMP2, BZLF1, and EBER1. EBV infection was localized by EBV-encoded RNA (EBER) in situ hybridization and by immunohistochemical stains for viral latent proteins LMP1 and LMP2 and for viral lytic proteins BMRF1 and BZLF1. B lymphocytes were identified using CD20 immunostains. EBV DNA was essentially undetectable in normal gastric mucosa but was present in 46% of gastritis lesions, 44% of normal colonic mucosa, 55% of Crohn's disease, and 64% of ulcerative colitis samples. Levels of EBV DNA exceeded what would be expected based on the numbers of B lymphocytes in inflamed tissues, suggesting that EBV is preferentially localized to inflammatory gastrointestinal lesions. Histochemical staining revealed EBER expression in lymphoid cells of some PCR-positive lesions. The viral lytic viral proteins, BMRF1 and BZLF1, were expressed in lymphoid cells of two ulcerative colitis tissues, both of which had relatively high viral loads by quantitative PCR. EBV-infected lymphocytes are frequently present in inflamed gastric and colonic mucosa. Active viral replication in some lesions raises the possibility of virus-related perpetuation of gastrointestinal inflammation.

Assessment of Epstein-Barr virus in blood from patients with multiple sclerosis

Viruses such as Epstein-Barr virus (EBV) which can establish latent infections in the central nervous system or the immune system have been associated with chronic neurological disorders, including multiple sclerosis. Results vary, therefore the aim of this study was to investigate the presence of EBV using both viral DNA and antibody screening techniques, using PCR and ELISA assays respectively, to evaluate viral presence in blood from control subjects and patients with multiple sclerosis. Viral gene sequences for latent proteins EBNA-1 and LMP-1 and lytic gene BamH1-W were present equally in both patients and controls (<7%). Anti-EBV-VCA IgG positive cases were present in >99% of all study subjects, and anti-EBV-VCA IgG immune status ratio showed a near-significant positive correlation with the EDSS in patients with multiple sclerosis. In contrast, Anti-EBV-VCA IgM positive cases were significantly increased in patients (controls: 23.3%; patients; 41.9%; P = 0.046). The IgM to IgG immune status ratio was near-significantly higher in patients with relapse episodes in the year preceding blood sampling (P = 0.058). Results from this and previous studies have shown higher prevalence rates for EBV evaluating anti-EBV IgM positive cases against viral DNA positive cases. Also, IgM, an innate immune response, showed an association with relapse episodes, suggesting viral re-activation as a contributing factor to these relapses.

Induction of lymphangiogenesis through vascular endothelial growth factor-C/vascular endothelial growth factor receptor 3 axis and its correlation with lymph node metastasis in nasopharyngeal carcinoma

The contribution of the lymphatic system to tumor metastasis is being increasingly appreciated through studies of human cancers. As the biological behavior of nasopharyngeal carcinoma (NPC) depends on its nodal status, patients with advanced nodal status show a higher tendency toward a poor outcome. Here, we examined the role of lymphangiogenesis on lymphatic spread of NPC. We also evaluated the involvement of vascular endothelial growth factor (VEGF)-C/VEGF receptor 3 (VEGFR3) signaling pathway on lymphangiogenesis in NPC. Furthermore, we tested whether Epstein-Barr virus (EBV)-latent membrane protein (LMP) 1 induces VEGF-C. Forty-one patients with NPC were evaluated for expressions of VEGF-C and its receptor, VEGFR3, and LMP1 proteins and lymphatic vessel counts (LVC) highlighted by anti-podoplanin employing immunohistochemistry. The VEGF-C induction by LMP1 was then tested with Western blotting and enzyme-linked immunosorbent assay in vitro. The LVC and VEGF-C expression were significantly higher in cases with advanced regional lymph node metastasis (N2,3) than those with no or limited lymph node involvement (N0,1) (p=0.0380 and p=0.0109, respectively). In VEGF-C/VEGFR3-positive cases, the LVC were significantly increased compared with VEGF-C/VEGFR3-negative cases (p=0.0007). However, LMP1 expression did not show significant associations with LVC and VEGF-C-expression scores (p=0.1210 and p=0.1324, respectively). Induction of VEGF-C protein by LMP1 was not detected in vitro. These results suggest the involvement of the VEGF-C/VEGFR3 axis in the induction of lymphangiogenesis which results in lymphatic spread of NPC. However, EBV-LMP1 was not associated with the mechanism.

Identification and Characterization of CCAAT Enhancer-binding Protein (C/EBP) as a Transcriptional Activator for Epstein-Barr Virus Oncogene Latent Membrane Protein 1

Epstein-Barr virus LMP1, a major oncoprotein expressed in latent infection, is critical for primary B cell transformation, functioning as a TNFR family member by aggregation in the plasma membrane resulting in constitutive activation of cellular signals, such as NF-κB, MAPK, JAK/STAT, and AKT. Although transcription of LMP1 in latent type III cells is generally under the control of the viral coactivator EBNA2, little is known about EBNA2-independent LMP1 expression in type II latency. We thus screened a cDNA library for factors that can activate the LMP1 promoter in an EBNA2-independent manner, using a reporter assay system. So far, we have screened >20,000 clones, and here identified C/EBPε as a new transcriptional activator. Exogenous expression of C/EBPα, -β, or -ε efficiently augmented LMP1 mRNA and protein levels in EBV-positive cell lines, whereas other members of the C/EBP family exhibited modest or little activity. It has been demonstrated that LMP1 gene transcription depends on two promoter regions: proximal (ED-L1) and distal (TR-L1). Interestingly, although we first used the proximal promoter for screening, we found that C/EBP increased transcription from both promoters in latent EBV-positive cells. Mutagenesis in reporter assays and EMSA identified only one functional C/EBP binding site, through which activation of both proximal and distal promoters is mediated. Introduction of point mutations into the identified C/EBP site in EBV-BAC caused reduced LMP1 transcription from both LMP1 promoters in epithelial cells. In conclusion, C/EBP is a newly identified transcriptional activator of the LMP1 gene, independent of the EBNA2 coactivator.

Epstein-Barr virus Latent Membrane Protein LMP1 reduces p53 protein levels independent of the PI3K-Akt pathway

BackgroundNasopharyngeal carcinoma (NPC) is an epithelial malignancy, which commonly occurs in Southern China, Taiwan, North Africa and Southeast Asia. Nasopharyngeal carcinoma is strongly associated with Epstein-Barr virus infection. The p53 tumour suppressor protein is rarely mutated in NPC suggesting that the inactivation of p53 pathway in NPC could be due to the presence of EBV proteins. The aim of this work was to determine the effects of EBV proteins namely LMP1 and LMP2A on the expression levels of p53 protein.FindingsIn this work we found that LMP1, but not LMP2A, decreased p53 protein levels. Overexpression of LMP1 resulted in increased ubiquitination of p53 suggesting that the decreased p53 protein levels by LMP1 was due to increased degradation of the protein. The reduction of p53 protein levels was independent of the PI3K-Akt pathway.ConclusionsLMP1, but not LMP2A, reduced p53 protein levels through the increase in the polyubiquitination of p53 protein and was independent of the PI3K-Akt pathway.

Epstein-Barr virus BART9 miRNA modulates LMP1 levels and affects growth rate of nasal NK T cell lymphomas.

Nasal NK/T cell lymphomas (NKTCL) are a subset of aggressive Epstein-Barr virus (EBV)-associated non-Hodgkin's lymphomas. The role of EBV in pathogenesis of NKTCL is not clear. Intriguingly, EBV encodes more than 40 microRNAs (miRNA) that are differentially expressed and largely conserved in lymphocryptoviruses. While miRNAs play a critical role in the pathogenesis of cancer, especially lymphomas, the expression and function of EBV transcribed miRNAs in NKTCL are not known. To examine the role of EBV miRNAs in NKTCL, we used microarray profiling and qRT-PCR to identify and validate expression of viral miRNAs in SNK6 and SNT16 cells, which are two independently derived NKTCL cell lines that maintain the type II EBV latency program. All EBV BART miRNAs except BHRF-derived miRNAs were expressed and some of these miRNAs are expressed at higher levels than in nasopharyngeal carcinomas. Modulating the expression of BART9 with antisense RNAs consistently reduced SNK6 and SNT16 proliferation, while antisense RNAs to BARTs-7 and -17-5p affected proliferation only in SNK6 cells. Furthermore, the EBV LMP-1 oncoprotein and transcript levels were repressed when an inhibitor of BART9 miRNA was transfected into SNK6 cells, and overexpression of BART9 miRNA increased LMP-1 protein and mRNA expression. Our data indicate that BART9 is involved in NKTCL proliferation, and one of its mechanisms of action appears to be regulating LMP-1 levels. Our findings may have direct application for improving NKTCL diagnosis and for developing possible novel treatment approaches for this tumor, for which current chemotherapeutic drugs have limited effectiveness.

LMP1 expression is positively associated with metastasis of nasopharyngeal carcinoma: evidence from a meta-analysis

ObjectiveLatent membrane protein 1 (LMP1) is an oncogene of Epstein–Barr virus (EBV). Although EBV is associated with 90% of nasopharyngeal carcinoma (NPC), the reported detectable expression rates of LMP1 determined...

A phase I trial of recombinant modified vaccinia ankara (MVA) vaccine encoding Epstein-Barr virus (EBV) antigens.

2592 Background: EBV+ve nasopharyngeal carcinoma (NPC) selectively expresses the EBV-coded nuclear antigen EBNA1 and latent membrane protein LMP2. A recombinant vaccinia virus MVA-EL, encodes an EBNA1/LMP2 fusion, is designed to boost T cell immunity to these tumour-associated antigens. It was tested in Hong Kong, an area endemic for NPC, in a phase I dose escalation trial to determine a safe and immunogenic dose for phase II. Methods: MHC I/II-typed NPC patients (pts), all >12 weeks after primary therapy, received three intra-dermal vaccinations of MVA-EL at 3-weekly intervals, with dose escalations of 5x107, 1x108, 2x108, 3.3x108 and 5x108 pfu providing dose limiting toxicity (DLT) had not been observed on the previous cohort. Blood samples were taken during pre-screening, immediately before and 1 week after each cycle of vaccination, and at 11, 14, 26 and 52 weeks post-vaccination. Cryo-preserved peripheral blood mononuclear cells (PBMC) were later tested for spot-forming cells (sfc) in interferon-gamma ELIspot assays against complete EBNA1 and LMP2 15-mer peptide mixes and against defined epitope peptides selected according to MHC-type. Results: Eighteen pts were treated, three per dose level 1-4 and six at the highest dose. No DLT occurred. Pre-existing immunity to EBNA1 and/or LMP2 was observed in 16/18 pts with mean frequencies of 181 (EBNA1) and 81 (LMP2) sfc/106 PBMC. Immunity to one or both antigens was significantly increased in 15/18 pts, with mean sfc/106 PBMC among responders reaching 411 and 237 after the second, and 353 and 213 after the third vaccination, while immunity to control antigens (a non-vaccine EBV antigen and influenza) showed no such change. Furthermore, the size of EBNA1/LMP2 responses strongly suggested a linear relationship with vaccine dose. Screening on defined peptides identified individuals mounting both MHC I and II-restricted responses to known epitopes in one or both target antigens. Conclusions: MVA-EL is safe and immunogenic at all doses tested. The recommended dose for phase II is 5x108pfu because this dose induced the strongest responses. Acknowledgement: Supported by Cancer Research UK, Research Grant Council of Hong Kong (CUHK 460708) and Hong Kong Cancer Fund.

A UK-based phase I trial of recombinant modified vaccinia ankara (MVA) vaccine encoding Epstein-Barr virus (EBV) antigens.

2592 Background: EBV+ve nasopharyngeal carcinoma (NPC) selectively expresses the EBV-coded nuclear antigen EBNA1 and latent membrane protein LMP2. A recombinant vaccinia virus MVA-EL, encodes an EB...

Epstein-Barr Virus Latent Membrane Protein 1 Increases Calcium Influx through Store-operated Channels in B Lymphoid Cells

Ca(2+) signaling plays an important role in B cell survival and activation and is dependent on Ca(2+) trapped in the endoplasmic reticulum (ER) and on extracellular Ca(2+). Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis. Previously, we showed that the ER Ca(2+) content of Burkitt lymphoma cell lines was increased following infection with immortalization-competent virus expressing the full set of EBV latency genes (B95-8). In contrast, infection with an immortalization-deficient virus (P3HR-1) not expressing LMP-1 is without effect. LMP-1 protein expression was sufficient to increase the ER Ca(2+) content and to increase the cytosolic Ca(2+) concentration ([Ca(2+)](cyt)). In this follow-up study, we showed that the resting [Ca(2+)](cyt) of P3HR-1-infected cells was decreased, implying that EBV not only modified the ER homeostasis but also affected the cytosolic Ca(2+) homeostasis. Furthermore, even if the store-operated calcium entry (SOCE) of these cells was normal, the [Ca(2+)](cyt) increase after thapsigargin + CaCl(2) stimulation was blunted. In contrast, the resting [Ca(2+)](cyt) of B95-8 infected cells was not changed, even if their SOCE was increased significantly. When expressed alone, LMP-1 induced an increase of the SOCE amplitude and the expression of the protein allowing this influx, Orai1, showing the effect of EBV on SOCE of B cells are mediated by LMP-1. However, other hitherto unidentified EBV processes, unmasked in P3HR-1 infected cells, counteract this LMP-1-dependent increase of SOCE amplitude to impair a general and potentially toxic increase of [Ca(2+)](i). Thus, EBV infection modifies the cellular Ca(2+) homeostasis by acting on the ER and plasma membrane transporters.

Mechanism study of osteoinductive LMP-1 inhibitory effect on nitric oxide production in pre-osteoclasts

Objective To investigate the inhibitory effect of osteoinductive protein LMP-1 on synthesis of nitric oxide(NO) in pre-osteoclasts and its molecular mechanism.Methods TAT-LMP-1 was previously constructed for transduction of LMP-1 into cells.After transduction of TAT-LMP-1,pre-osteoclasts (RAW 264.7 cells) were stimulated by tumor necrosis factor-α (TNF-α) or lipopolysaccharide (LPS).Greiss assay was used to detect the synthesis of NO.Real-time RT-PCR and Western blot were used to detect themRNA and protein of inducible NO synthase (iNOS),respectively.Western blot was used to detect the phosphorylation and translocation of IκB and p65 in the activation of NF-κB pathway.Lucfferase reporter assay was used to determine p65 transcriptional activity.Results LMP-1 was observed to decrease the synthesis of NO induced by TNF-α or LPS in a concentration-dependent manner.LMP-1 also effectively inhibited the expression of iNOS.It potently suppressed the transcriptional activity and nuclear translocation of p65,and the phosphorylation of IκB.Conclusion LMP-1 can inhibit the synthesis of NO in pre-osteoclasts due to its inhibitive effects on phosphorylation of IκB,p65 translocation,and iNOS transcription partly. Key words: Nitric oxide; Osteoclasts; NF-κB

Humoral immune responses to Epstein-Barr virus encoded tumor associated proteins and their putative extracellular domains in nasopharyngeal carcinoma patients and regional controls

Epstein-Barr virus (EBV) latency proteins EBNA1, LMP1, LMP2, and BARF1 are expressed in tumor cells of nasopharyngeal carcinoma (NPC). IgG and IgA antibody responses to these non-self tumor antigens were analyzed in NPC patients (n=125) and regional controls (n=100) by three approaches, focusing on the putative LMP1, LMP2 extracellular domains. Despite abundant IgG and IgA antibody responses to lytic antigens and EBNA1, patients had low titer (1:25-1:100) IgG to LMP1 (81.2%), LMP2 (95.6%), and BARF1 (84.8%), while immunoblot showed such reactivity in 24.2%, 12.5%, and 12.5% at 1:50 dilution, respectively. Few IgA responses were detected, except for EBNA1. Controls only showed IgG to EBNA1. ELISA using peptides from different domains of LMP1, LMP2, and BARF1 also yielded mostly negative results. When existing, low level IgG to intracellular C-terminus of LMP1 (62.9%) prevailed. Rabbit immunization with peptides representing extracellular (loop) domains yielded loop-specific antibodies serving as positive control. Importantly, these rabbit antibodies stained specifically extracellular domains of LMP1 and LMP2 on viable cells and mediated complement-driven cytolysis. Rabbit anti-LMP1 loop-1 and -3 killed 50.4% and 59.4% of X50/7 and 35.0% and 35.9% of RAJI cells, respectively, and 22% of both lines were lysed by anti-LMP2 loop-2 or -5 antibodies. This demonstrates that (extracellular domains of) EBV-encoded tumor antigens are marginally immunogenic for humoral immune responses. However, peptide-specific immunization may generate such antibodies, which can mediate cell killing via complement activation. This opens options for peptide-based tumor vaccination in patients carrying EBV latency type II tumors such as NPC.

STAT6 signaling pathway activated by the cytokines IL-4 and IL-13 induces expression of the Epstein-Barr virus–encoded protein LMP-1 in absence of EBNA-2: implications for the type II EBV latent gene expression in Hodgkin lymphoma

AbstractIn line with the B-lymphotropic nature of Epstein-Barr virus (EBV), the virus is present in several types of B-cell lymphomas. EBV expresses a different set of latent genes in the associated tumors, such as EBV nuclear antigen 1 (EBNA-1) and latent membrane proteins (LMPs; type II latency) in classical Hodgkin lymphomas (HLs). We previously reported that exposure of in vitro EBV-converted, HL-derived cell line KMH2-EBV to CD40-ligand and interleukin-4 (IL-4) induced the expression of LMP-1. Here, we show that exposure to IL-4 or IL-13 alone induced LMP-1 in the absence of EBNA-2. Induction of LMP-1 by IL-4 and IL-13 was mediated by the signal transducer signal transducer and activator of transcription 6 (STAT6) and a newly defined high-affinity STAT6-binding site in the LMP-1 promoter. IL-4 induced LMP-1 also in Burkitt lymphoma–derived lines and in tonsillar B cells infected with the EBNA-2–deficient EBV strain P3HR-1. Furthermore, coculture of EBV-carrying Burkitt lymphoma cells with activated CD4+ T cells resulted in the induction of LMP-1 in the absence of EBNA-2. Because Hodgkin/Reed-Sternberg cells are known to secrete IL-13, to have constitutively activated STAT6, and to be closely surrounded by CD4+ T cells, these mechanisms may be involved in the expression of LMP-1 in EBV-positive chronic HLs.

Inflammation and Epstein-Barr Virus Infection Are Common Features of Myasthenia Gravis Thymus: Possible Roles in Pathogenesis

The thymus plays a major role in myasthenia gravis (MG). Our recent finding of a persistent Epstein-Barr (EBV) virus infection in some MG thymuses, combined with data showing that the thymus is in a proinflammatory state in most patients, supports a viral contribution to the pathogenesis of MG. Aim of this study was to gain further evidence for intrathymic chronic inflammation and EBV infection in MG patients. Transcriptional profiling by low density array and real-time PCR showed overexpression of genes involved in inflammatory and immune response in MG thymuses. Real-time PCR for EBV genome, latent (EBER1, EBNA1, LMP1) and lytic (BZLF1) transcripts, and immunohistochemistry for LMP1 and BZLF1 proteins confirmed an active intrathymic EBV infection, further supporting the hypothesis that EBV might contribute to onset or perpetuation of the autoimmune response in MG. Altogether, our results support a role of inflammation and EBV infection as pathogenic features of MG thymus.

Cytotoxic T Cells Specific for Adenovirus, BK Virus, Cytomegalovirus, Epstein Barr Virus and Varicella Zoster Virus Produced for Clinical Use In Immune Reconstitution Post Allogeneic Stem Cell Transplantation

AbstractAbstract 830 Introduction:Adoptive immunotherapy using donor-derived cellular effectors can restore immunity and prevent virus related disease after haemopoietic stem cell transplantation (HSCT) as shown by trials targeting EBV, CMV and adenovirus. It is therefore desirable to expand the repertoire of T cells for adoptive immunotherapy to include other viruses that cause disease in this patient group. We have developed a method for the production of a T cell product specific for multiple viruses that does not rely on EBV transformed cells for stimulation. In this study we assessed the efficacy and reproducibility of the method for generating antigen specific T cells with broad specificity. Methods:Monocyte-derived dendritic cells (mo-DC) were used to present antigens from 5 viruses to T cells of normal donors. Presentation of CMV, EBV and human adenovirus (HadV) antigens was achieved by transfection with recombinant adenovirus vectors encoding the entire CMV pp65 gene (Ad5F35pp65) or epitopes derived from EBV proteins EBNA1, LMP1 and LMP2a (AdE1-LMPpoly). Additionally, mo-DC were infected with a clinically approved cell free vaccine preparation of the live attenuated Varicella Zoster virus (VZV) OKA strain or incubated with overlapping peptide mixes covering the BK virus (BKV) genes LTA, STA, VP1, VP2 and VP3.Activated antigen-specific T cells were preferentially expanded with two dendritic cell stimulations on day one and seven with the addition of increasing doses of IL-2 from day 7 to day 21. Methods were initially established for stimulation with individual (VZV and BKV) or dual viruses (EBV/Hadv and CMV/Hadv). In subsequent cultures, responding cells were combined at various time-points to optimise the total number of cells in the final product. Cultures were assessed by cell count, viability and phenotype. Specificity for antigens was demonstrated with multimer staining where available (EBV and CMV) and by intracellular cytokine detection (IL-2, IFN-g and TNF) after restimulation with antigens. The quality of antigen specific T cells was assessed by analysis of the production of multiple cytokines. Results:The final multi-virus cell products from 7 cultures increased in cell number from baseline (mean increase in total cell number was 8-fold), were CD3 positive (mean 92.1%), with variation between individuals in CD4:CD8 ratio. Cells were CD45RO+ (mean 87.7%) and a variable number expressed CD62L+ (mean 27.12%, range 12.1 to 41.0%). The total percentage of T cells in the final product that produced cytokines in response to restimulation with the various antigens ranged from 2.6 to 52% (mean 14.63). This was composed of both CD4 (mean 12.9%, range 5.5 to 44.5%) and CD8 cells (mean 20.3%, range 0.9 to 61.5%). Fold increase in MHC multimer positive cells compared to baseline was as follows: CMVpp65 HLA-A2 NLV 1742-fold, CMVpp65 HLA-B7 TPR 1689-fold, EBV LMP2a HLA-A2 CLG 124-fold, EBV LMP2a HLA-A2 FLY 180-fold. The quality of T cell responses in combined cultures was commensurate with the responses of cultures of cells specific for individual viruses. T cells responding to VZV, CMV and BKV antigens consistently co-produced up to 3 cytokines, IFN-γ, TNF and IL-2 indicative of the multi-functional quality of the expanded T cell product. Discussion:To our knowledge, this is the first report of a methodology for production of T cells specific for 5 viruses for use in adoptive immunotherapy in HSCT. We have been able to generate cells with multiple TH1 cytokine functions in all of 7 cultures from 5 normal donors. There are several advantages to our methodology: it can be tailored to individual serostatus, it is not HLA-dependent, it is rapid and it potentially enhances the protection available to immunocompromised transplant recipients. The use of antigen and reagent sources that are consistent with Good Manufacturing Practice will allow for rapid clinical translation. Safety and efficacy data from upcoming clinical trial of this product are awaited. Disclosures:No relevant conflicts of interest to declare.