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Epstein-Barr virus (EBV) persistently infects 95% of adults worldwide and is associated with multiple human lymphomas that express characteristic EBV latency programs used by the virus to navigate the B-cell compartment. Upon primary infection, the EBV latency III program, comprised of six Epstein-Barr Nuclear Antigens (EBNA) and two Latent Membrane Protein (LMP) antigens, drives infected B-cells into germinal center (GC). By incompletely understood mechanisms, GC microenvironmental cues trigger the EBV genome to switch to the latency II program, comprised of EBNA1, LMP1 and LMP2A and observed in GC-derived Hodgkin lymphoma. To gain insights into pathways and epigenetic mechanisms that control EBV latency reprogramming as EBV-infected B-cells encounter microenvironmental cues, we characterized GC cytokine effects on EBV latency protein expression and on the EBV epigenome. We confirmed and extended prior studies highlighting GC cytokine effects in support of the latency II transition. The T-follicular helper cytokine interleukin 21 (IL-21), which is a major regulator of GC responses, and to a lesser extent IL-4 and IL-10, hyper-induced LMP1 expression, while repressing EBNA expression. However, follicular dendritic cell cytokines including IL-15 and IL-27 downmodulate EBNA but not LMP1 expression. CRISPR editing highlighted that STAT3 and STAT5 were necessary for cytokine mediated EBNA silencing via epigenetic effects at the EBV genomic C promoter. By contrast, STAT3 was instead necessary for LMP1 promoter epigenetic remodeling, including gain of activating histone chromatin marks and loss of repressive polycomb repressive complex silencing marks. Thus, EBV has evolved to coopt STAT signaling to oppositely regulate the epigenetic status of key viral genomic promoters in response to GC cytokine cues., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Liao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Epstein-Barr virus (EBV) is an oncogenic herpesvirus implicated in the pathogenesis of several malignant and non-malignant conditions. However, a number of fundamental aspects about the biology of EBV and the mechanism(s) by which this virus induces pathology remain unknown. One major obstacle has been the lack of a suitable animal model for EBV infection. In this study, using our recently established rabbit model of EBV infection, we examined the early events following primary EBV infection. We show that, both immunocompetent and immunosuppressed animals were readily susceptible to EBV infection. However, immunosuppressed animals showed marked splenomegaly and widespread infection. Following EBV infection, the virus primarily targeted naïve IgM + , CD20 + , CD21 + and CD79a + B cells. Infected cells expressed varying sets of viral latent/lytic gene products. Notably, co-expression of latent and lytic proteins in the same cell was not observed. Infected cells in type 0/1 latency (EBERs + ), were small and proliferating (Ki67 + ). By contrast, cells in type 2/3 latency (LMP1 + ), were large, non-proliferating (Ki-67 - ) and p53 + . Although infected B-cells were widely present in splenic follicles, they did not express germinal center marker, BCL-6. Taken together, this study shows for the first time, some of the early events following primary EBV infection., (© 2021. The Author(s).)

The Epstein-Barr virus (EBV) is a well-adapted human virus, and its infection is exclusive to our species, generally beginning in the childhood and then persisting throughout the life of most of the affected adults. Although this infection generally remains asymptomatic, EBV can trigger life-threatening conditions under unclear circumstances. The EBV lifecycle is characterized by interactions with other viruses or bacteria, which increases the probability of awakening its pathobiont capacity. For instance, EBV infects B cells with the potential to alter the germinal center reaction (GCR)-an adaptive immune structure wherein mutagenic-driven processes take place. HIV- and Plasmodium falciparum -induced B cell hyperactivation also feeds the GCR. These agents, along with the B cell tropic KSHV, converge in the ontogeny of germinal center (GC) or post-GC lymphomas. EBV oral transmission facilitates interactions with local bacteria and HPV, thereby increasing the risk of periodontal diseases and head and neck carcinomas. It is less clear as to how EBV is localized in the stomach, but together with Helicobacter pylori , they are known to be responsible for gastric cancer. Perhaps this mechanism is reminiscent of the local inflammation that attracts different herpesviruses and enhances graft damage and chances of rejection in transplanted patients. In this review, we discussed the existing evidence suggestive of EBV possessing the potential to synergize or cooperate with these agents to trigger or worsen the disease.

Expression of the latent membrane protein-1 (LMP1) of Epstein-Barr virus (EBV) was investigated in 153 cases of EBV+ classic Hodgkin lymphoma (cHL); 120 cases were pediatric patients (< 14 years of age) from Iraq, and 33 cases were adult patients from Italy. We describe for the first time the presence of LMP1 protein in EBV-encoded RNA (EBER)-negative follicular dendritic cells (FDCs) of reactive germinal centers (GC) associated with EBV+ cHL. Presence of LMP1+ GCs was independent of geographic region and age of patients. Variable numbers of reactive GCs were present in 22.2% of cases (34 of 153), whereas LMP1 staining of FDCs was present in about a third of cases (10 of 34) with reactive GC. Most cases with LMP1+ GC were mixed-cellularity (MC) subtype, but some nodular sclerosis (NS) was also present. GC cells with LMP1+ FDCs were surrounded by numerous EBV-infected cells which were positive for EBER, LMP1, and CD30. Double immunolocalization analysis revealed that LMP1 was associated with CD63, an exosomal marker, and with CD21. The possibility is discussed that peri-follicular EBV-infected cells release LMP1 protein, perhaps through exosomes, and that the protein is then captured by FDCs and is presented to EBER-negative GC B cells.

During unresolved infections, some viruses escape immunological control and establish a persistant reservoir in certain cell types, such as human immunodeficiency virus (HIV), which persists in follicular helper T cells (TFH cells), and Epstein-Barr virus (EBV), which persists in B cells. Here we identified a specialized group of cytotoxic T cells (TC cells) that expressed the chemokine receptor CXCR5, selectively entered B cell follicles and eradicated infected TFH cells and B cells. The differentiation of these cells, which we have called 'follicular cytotoxic T cells' (TFC cells), required the transcription factors Bcl6, E2A and TCF-1 but was inhibited by the transcriptional regulators Blimp1, Id2 and Id3. Blimp1 and E2A directly regulated Cxcr5 expression and, together with Bcl6 and TCF-1, formed a transcriptional circuit that guided TFC cell development. The identification of TFC cells has far-reaching implications for the development of strategies to control infections that target B cells and TFH cells and to treat B cell-derived malignancies.

Considerable data implicate the thymus as the main site of autosensitization to the acetylcholine receptor in myasthenia gravis (MG), a B-cell-mediated autoimmune disease affecting the neuromuscular junction. We recently demonstrated an active Epstein-Barr virus (EBV) infection in the thymus of MG patients, suggesting that EBV might contribute to the onset or maintenance of the autoimmune response within MG thymus, because of its ability to activate and immortalize autoreactive B cells. EBV has been reported to elicit and modulate Toll-like receptor (TLR) 7- and TLR9-mediated innate immune responses, which are known to favor B-cell dysfunction and autoimmunity. Aim of this study was to investigate whether EBV infection is associated with altered expression of TLR7 and TLR9 in MG thymus. By real-time PCR, we found that TLR7 and TLR9 mRNA levels were significantly higher in EBV-positive MG compared to EBV-negative normal thymuses. By confocal microscopy, high expression levels of TLR7 and TLR9 proteins were observed in B cells and plasma cells of MG thymic germinal centers (GCs) and lymphoid infiltrates, where the two receptors co-localized with EBV antigens. An increased frequency of Ki67-positive proliferating B cells was found in MG thymuses, where we also detected proliferating cells expressing TLR7, TLR9 and EBV antigens, thus supporting the idea that EBV-associated TLR7/9 signaling may promote abnormal B-cell activation and proliferation. Along with B cells and plasma cells, thymic epithelium, plasmacytoid dendritic cells and macrophages exhibited enhanced TLR7 and TLR9 expression in MG thymus; TLR7 was also increased in thymic myeloid dendritic cells and its transcriptional levels positively correlated with those of interferon (IFN)-β. We suggested that TLR7/9 signaling may be involved in antiviral type I IFN production and long-term inflammation in EBV-infected MG thymuses. Our overall findings indicate that EBV-driven TLR7- and TLR9-mediated innate immune responses may participate in the intra-thymic pathogenesis of MG., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

B-cell development is a multistep process sustained by a highly coordinated transcriptional network under the control of a limited set of transcription factors. Epigenetic mechanisms, including DNA methylation, histone posttranslational modifications and microRNAs act in concert with transcription factors to promote lineage commitment, define and sustain cell identity and establish heritable cell-type- and stage-specific gene expression profiles. Epigenetic modifiers have recently emerged as key regulators of B-cell development and activation. Central to B-cell-mediated immunity are germinal centers, transient structures formed in secondary lymphoid organs where antigen-specific B cells undergo intense proliferation, immunoglobulin somatic hypermutation and isotype switching, to generate ultimately long-lived memory B cells and terminally differentiated plasma cells expressing high-affinity antibodies. Deregulation of one or more epigenetic axes represents a common feature of several B-cell disorders arising from germinal center B cells, including autoimmunity and lymphoma. Moreover, the hijacking of epigenetic determinants is central to the ability of the B-lymphotropic Epstein-Barr virus (EBV) to establish, via the germinal center reaction, life-long latency and occasionally contribute to malignant B-cell transformation. In the light of recent findings, this review will discuss the relevance of epigenetic deregulation in the pathogenesis of B-cell diseases. Understanding how specific epigenetic alterations contribute to the development of lymphomas, autoimmunity and EBV-associated disorders is instrumental to develop novel therapeutic interventions for the cure of these often fatal pathologies.

The tonsils are uncommonly affected by granulomatous inflammation. This study attempted to clarify the clinicopathological and immunohistochemical findings and presence or absence of Epstein-Barr virus (EBV) in tonsilar granulomatous inflammation. A total of 537 consecutive specimens from tonsillectomies performed at Dokkyo University School of Medicine between 1999 and March 2012 were reexamined. Using formalin-fixed, paraffin-embedded sections, histological, immunohistochemical, and in situ hybridization (ISH) studies were performed. Epithelioid granulomas (EPGs) were identified in the tonsils in 16 (3.0%) cases. There were 8 males and 8 females, aged 4 to 57 years (mean, 23). In 11 patients, EPGs were located in the germinal center (GC), whereas they were located in the interfollicular area as well as GC in the remaining 5 cases. Three types of EPG have been delineated : (i) poorly demarcated small epithelioid cell granulomas (n = 6) ; (ii) well-demarcated non-caseating sarcoid-like granulomas (n = 5) ; and (iii) EPGs within GC showing suppurations at the center (n = 5). An ISH study demonstrated EBV-encoded small RNA (EBER)(+) cells in 4 lesions. The present study demonstrated that the majority of EPGs were located in the GC and tonsilar EPGs showed histological variation.

Endoplasmic reticulum (ER) stress triggers a homeostatic cellular response in mammalian cells to ensure efficient folding, sorting, and processing of client proteins. In lytic-permissive lymphoblastoid cell lines (LCLs), pulse exposure to the chemical ER-stress inducer thapsigargin (TG) followed by recovery resulted in the activation of the EBV immediate-early (BRLF1, BZLF1), early (BMRF1), and late (gp350) genes, gp350 surface expression, and virus release. The protein phosphatase 1 a (PP1a)-specific phosphatase inhibitor Salubrinal (SAL) synergized with TG to induce EBV lytic genes; however, TG treatment alone was sufficient to activate EBV lytic replication. SAL showed ER-stress-dependent and -independent antiviral effects, preventing virus release in human LCLs and abrogating gp350 expression in 12-O-tetradecanoylphorbol-13-acetate (TPA)-treated B95-8 cells. TG resulted in sustained BCL6 but not BLIMP1 or CD138 expression, which is consistent with maintenance of a germinal center B-cell, rather than plasma-cell, phenotype. Microarray analysis identified candidate genes governing lytic replication in LCLs undergoing ER stress.

Lymphomas involving the central nervous system are recognized increasingly in immunocompetent as well as immunosuppressed individuals, and the majority of the cases are diffuse large B-cell lymphoma (DLBCL). The aim of this study was to compare the immunophenotype, clinicopathological features, and association with Epstein-Barr virus (EBV) of DLBCL of the central nervous system (CNS) in 3 different clinical situations: primary, in immunocompetent patients; "primary," in immunosuppressed patients; and in patients with secondary involvement by systemic lymphoma. The authors reviewed the clinicopathological features, morphology, immunophenotype (according to germinal-center B-cell-like and nongerminal B-cell-like subtypes), and association with EBV in 36 cases of DLBCL of the CNS, including 25 primary cases, 5 associated with immunosuppression, and 6 cases with secondary involvement. Survival was evaluated in 15 cases of primary CNS lymphomas. Of the 36 patients, 19 were male and 18 female. Only 2 cases of lymphomas were EBV-positive; both occurred in immunosuppressed patients. Separation into germinal-center and non-germinal center subtypes by an immunohistochemistry panel showed that 68% of primary, 80% of secondary, and 83% of the cases associated with immunosuppression were of non-germinal-center subtype, respectively. Patients with non-germinal-center immunophenotype showed significantly worse survival than those with CNS lymphomas of the germinal-center subtype.

Progressive transformation of germinal center (PTGC) usually affects the peripheral lymph nodes. Little is known about the extranodal PTGC. To clarify the clinicopathological and molecular findings of extranodal PTGC, we studied 14 such cases. Using formalin-fixed, paraffin-embedded sections, we carried out histological and immunohistochemical examinations, as well as in situ hybridization (ISH) and polymerase chain reaction (PCR). Eleven patients were female, and three were male. They were between 44 and 77 years old, with a mean age of 62 years. The large intestine (n=7) was the most frequently involved tissue, followed by skin (n=2) and subcutaneous soft tissue (n=2). Oral cavity, Waldeyer ring, and orbit were affected in one case each. Histologically, 13 cases contained both early stage PTGC and late stage PTGC. The remaining 14th case contained only late stage PTGC. Expansion of the marginal zone was identified in three cases. Immunohistochemical study demonstrated the reactive nature of the B-cells in all 14 lesions. However, PCR study revealed immunoglobulin heavy chain (IgH) gene rearrangement in one of our 14 cases. There was no development of B-cell lymphoma in one lesion with IgH rearrangement. ISH study demonstrated Epstein-Barr virus-encoded small RNA+ cells in three lesions. Compared with PTGC of the peripheral lymph node, PTGC of extranodal sites was characterized by a female predominance, an older age group, and the presence of numerous PTGC at the affected sites. However, the histological findings of extranodal PTG were similar to those of lymph node PTGC. The clinicopathological findings of the extranodal PTGCs appeared to be different from those of lymph node PTGC., (Copyright 2010 Elsevier GmbH. All rights reserved.)

Background: Complete resection seemed to be curative in patients with Castleman disease of any location but the disease is likely to be reactive in its pathogenesis. The relation between Epstein-Barr virus and Castleman disease has not been elucidated. We tried to define the role of Epstein-Barr virus in the pathogenesis of Castleman disease., Methods: 20 cases of Castleman disease were retrospectively reviewed from 1993 to 2006. At least 2 to 4 representative sections of formalin-fixed, paraffin-embedded specimens from each patient were obtained to examine the presence of EBV and its localization by hematoxylin-eosin stain, immunohistochemistry, polymerase chain reaction and In-situ hybridization, Results: Hyaline-vascular type was diagnosed in 18 cases, plasma cell type in 1 and mixed type in 1 case. All of them were positive for Epstein-Barr virus confirmed by PCR. For tumors that EBER(Epstein-Barr early region) signals mainly localized in the germinal centers have increased vascularity than cases with EBER detected in inter-follicular areas., Conclusion: There is a strong association between Castleman disease and Epstein-Barr virus. EBV may have a potential role in angiogenesis of Castleman disease. For smaller lesion with high activity of angiogenesis but not amenable for curative resection, anti-angiogenesis medications may have a potential role to control the disease.

The simultaneous occurrence of a Hodgkin lymphoma (HL) and a non-Hodgkin lymphoma (NHL) is a rare event, and single cell analyses of such composite lymphomas revealed that NHL and Hodgkin/Reed-Sternberg (HRS) tumor cells are frequently descendants of the same tumor clone precursors. Here we present a composite lymphoma consisting of a mantle cell lymphoma (MCL) and an HL with EBV- and EBV+ HRS cells. Analysis of rearranged V genes of single cells revealed a clonal relationship between MCL and HL tumor cells. Although V gene rearrangements of the MCL were unmutated, mutations were observed in HRS cells. Besides mutations shared by all HRS cells, the EBV+ HRS cells carried identical additional mutations. These findings show that both lymphomas derive from a common precursor, most likely a pre germinal center (GC) B cell that already carried some transforming event(s). However, the presence of mutations in the V genes of the HRS cells further corroborates the importance of the GC reaction for the pathogenesis of HL. Importantly, the finding that only a subclone of the HRS clone, defined by a particular mutation pattern, was EBV infected represents a strong indication that EBV infection of the HRS cell precursor happened in the GC.

Infection by the Epstein-Barr virus (EBV) in immunocompetent individuals seems mainly confined to antigen-experienced memory B cells. However, a recent report shows that EBV(+) post-transplant lymphoproliferative disease might arise not only from memory B cells but also from nai;ve and germinal center (GC) B cells. Intriguingly, some of the EBV-positive B-cell clones seem to carry non-functional Ig-V-region genes as a result of deleterious somatic mutations acquired during the GC reaction. Given that such GC B cells are destined to die by apoptosis in the absence of EBV, these findings suggest that transformation by EBV might bypass negative selection of B cells within GCs.

Burkitt's lymphoma (BL) and Hodgkin's disease (HD) occurring in developing regions are frequently associated with Epstein-Barr virus (EBV) infection and have a high incidence in childhood. Recent genotyping studies indicate that the tumour cells of both neoplasms represent B cells that contain somatically mutated immunoglobulin heavy chain genes. This implies that the precursors of these neoplasms have participated in the germinal centre (GC) reaction. We therefore presumed that normal lymphoid tissues from children living in developing regions would harbour an increased number of EBV-infected cells within the GC, when compared with children living in industrialized nations. To test this hypothesis, hyperplastic tonsils from 40 children living in Bahia (Brazil) and 40 from German children were analysed for the presence of EBV-encoded small nuclear RNA (EBER) and EBV-encoded proteins by in situ hybridization and immunohistology, respectively. Although the overall EBV infection rate was similar in both groups (50 per cent of Bahian vs. 45 per cent of German cases), a significantly higher number of EBER-positive lymphoid cells were found in the GCs of 8/20 EBV-positive tonsils from Brazil (9-89 cells/GC; mean: 14.5 cells/GC per case), while only 3/18 tonsils from Germany displayed a few EBER positive cells (1-9 cells/GC; mean: 0.5 cell/GC per case) in this compartment (p < 0.007). In addition, the EBV-infected GC cells in Bahian samples resembled centroblasts, exhibited mitotic activity, and in two cases showed expression of EBV-encoded latent membrane protein (LMP)-1, findings not present in German samples. These data show that latently EBV-infected cells participate more frequently in GC reactions in developing regions than in industrialized countries and may abnormally express the oncogenic protein LMP-1. This could in part explain the higher incidence in this region of EBV association with lymphomas related to GC cells or their progeny, such as BL and HD.