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20 pages, 7374 KiB

Open AccessArticle

Spontaneous EBV-Reactivation during B Cell Differentiation as a Model for Polymorphic EBV-Driven Lymphoproliferation

by Matthew A. Care, Sophie Stephenson, Roger Owen, Gina M. Doody and Reuben M. Tooze

Cancers 2023, 15(12), 3083; https://doi.org/10.3390/cancers15123083 - 07 Jun 2023

Viewed by 1373

Epstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder (LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation [...] Read more.

Epstein-Barr virus (EBV)-driven B cell neoplasms arise from the reactivation of latently infected B cells. In a subset of patients, EBV was seen to drive a polymorphous lymphoproliferative disorder (LPD) in which B cell differentiation was retained. In this work, spontaneous EBV reactivation following B cell mitogen stimulation was shown to provide a potential model of polymorphic EBV-driven LPD. Here, we developed an in vitro model of plasma cell (PC) differentiation from peripheral blood memory B cells. To assess the frequency and phenotypes of EBV-associated populations derived during differentiation, we analysed eight differentiations during the PC stage with a targeted single-cell gene expression panel. We identified subpopulations of EBV-gene expressing cells with PC and/or B cell expression features in differentiations from all tested donors. EBV-associated cells varied in frequency, ranging from 3–28% of cells. Most EBV-associated cells expressed PC genes such as XBP1 or MZB1, and in all samples these included a quiescent PC fraction that lacked cell a cycle gene expression. With increasing EBV-associated cells, populations with B cell features became prominent, co-expressing a germinal centre (GC) and activating B cell gene patterns. The presence of highly proliferative EBV-associated cells was linked to retained MS4A1/CD20 expression and IGHM and IGHD co-expression, while IGHM class-switched cells were enriched in quiescent PC fractions. Thus, patterns of gene expression in primary EBV reactivation were shown to include features related to GC B cells, which was also observed in EBV-transformed lymphoblastoid cell lines. This suggests a particular association between spontaneously developing EBV-expansions and IgM+ IgD+ non-switched B cells. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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18 pages, 4551 KiB

Open AccessEditor’s ChoiceArticle

Follicular Helper and Regulatory T Cells Drive the Development of Spontaneous Epstein–Barr Virus Lymphoproliferative Disorder

by Elshafa Hassan Ahmed, Mark Lustberg, Claire Hale, Shelby Sloan, Charlene Mao, Xiaoli Zhang, Hatice Gulcin Ozer, Sarah Schlotter, Porsha L. Smith, Frankie Jeney, Wing Keung Chan, Bonnie K. Harrington, Christoph Weigel, Eric Brooks, Haley L. Klimaszewski, Christopher C. Oakes, Tamrat Abebe, Muntaser E. Ibrahim, Lapo Alinari, Gregory K. Behbehani, Polina Shindiapina, Michael A. Caligiuri and Robert A. Baiocchi Show full author list Hide full author list

Cancers 2023, 15(11), 3046; https://doi.org/10.3390/cancers15113046 - 03 Jun 2023

Cited by 1 | Viewed by 2538

Abstract

Epstein–Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a [...] Read more.

Epstein–Barr virus (EBV) is a ubiquitous herpes virus associated with various cancers. EBV establishes latency with life-long persistence in memory B-cells and can reactivate lytic infection placing immunocompromised individuals at risk for EBV-driven lymphoproliferative disorders (EBV-LPD). Despite the ubiquity of EBV, only a small percentage of immunocompromised patients (~20%) develop EBV-LPD. Engraftment of immunodeficient mice with peripheral blood mononuclear cells (PBMCs) from healthy EBV-seropositive donors leads to spontaneous, malignant, human B-cell EBV-LPD. Only about 20% of EBV+ donors induce EBV-LPD in 100% of engrafted mice (High-Incidence, HI), while another 20% of donors never generate EBV-LPD (No-Incidence, NI). Here, we report HI donors to have significantly higher basal T follicular helper (Tfh) and regulatory T-cells (Treg), and depletion of these subsets prevents/delays EBV-LPD. Transcriptomic analysis of CD4+ T cells from ex vivo HI donor PBMC revealed amplified cytokine and inflammatory gene signatures. HI vs. NI donors showed a marked reduction in IFNγ production to EBV latent and lytic antigen stimulation. In addition, we observed abundant myeloid-derived suppressor cells in HI donor PBMC that decreased CTL proliferation in co-cultures with autologous EBV+ lymphoblasts. Our findings identify potential biomarkers that may identify individuals at risk for EBV-LPD and suggest possible strategies for prevention. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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14 pages, 1045 KiB

Open AccessArticle

Development of a Duplex Serological Multiplex Assay for the Simultaneous Detection of Epstein-Barr Virus IgA and IgG Antibodies in Nasopharyngeal Carcinoma Patients

by Jennifer Schieber, Miranda Pring, Andy Ness, Zhiwei Liu, Wan-Lun Hsu, Nicole Brenner, Julia Butt, Tim Waterboer and Julia Simon

Cancers 2023, 15(9), 2578; https://doi.org/10.3390/cancers15092578 - 30 Apr 2023

Cited by 1 | Viewed by 1612

Abstract

Epstein-Barr virus (EBV) IgA and IgG antibodies in serum from nasopharyngeal carcinoma (NPC) patients are well-established markers for EBV-positive NPC. Luminex-based multiplex serology can analyze antibodies to multiple antigens simultaneously; however, the detection of both IgA and IgG antibodies requires separate measurements. Here [...] Read more.

Epstein-Barr virus (EBV) IgA and IgG antibodies in serum from nasopharyngeal carcinoma (NPC) patients are well-established markers for EBV-positive NPC. Luminex-based multiplex serology can analyze antibodies to multiple antigens simultaneously; however, the detection of both IgA and IgG antibodies requires separate measurements. Here we describe the development and validation of a novel duplex multiplex serology assay, which can analyze IgA and IgG antibodies against several antigens simultaneously. Secondary antibody/dye combinations, as well as serum dilution factors, were optimized, and 98 NPC cases matched to 142 controls from the Head and Neck 5000 study (HN5000) were assessed and compared to data previously generated in separate IgA and IgG multiplex assays. EBER in situ hybridization (EBER-ISH) data available for 41 tumors was used to calibrate antigen-specific cut-offs using receiver operating characteristic (ROC) analysis with a prespecified specificity of ≥90%. A directly R-Phycoerythrin-labeled IgG antibody in combination with a biotinylated IgA antibody and streptavidin-BV421 reporter conjugate was able to quantify both IgA and IgG antibodies in a duplex reaction in a 1:1000 serum dilution. The combined assessment of IgA and IgG antibodies in NPC cases and controls from the HN5000 study yielded similar sensitivities as the separate IgA and IgG multiplex assays (all > 90%), and the duplex serological multiplex assay was able to unequivocally define the EBV-positive NPC cases (AUC = 1). In conclusion, the simultaneous detection of IgA and IgG antibodies provides an alternative for the separate IgA/IgG antibody quantification and may present a promising approach for larger NPC screening studies in NPC endemic areas. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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15 pages, 3365 KiB

Open AccessArticle

Exploring Spatial Heterogeneity of Immune Cells in Nasopharyngeal Cancer

by Aastha Sobti, Christina Sakellariou, Johan S. Nilsson, David Askmyr, Lennart Greiff and Malin Lindstedt

Cancers 2023, 15(7), 2165; https://doi.org/10.3390/cancers15072165 - 05 Apr 2023

Cited by 2 | Viewed by 2193

Abstract

Nasopharyngeal cancer (NPC) is a malignant tumor. In a recent publication, we described the presence and distribution of CD8⁺ T cells in NPC and used the information to identify ‘inflamed’, ‘immune-excluded’, and ‘desert’ immune phenotypes, where ‘inflamed’ and ‘immune-excluded’ NPCs were correlated [...] Read more.

Nasopharyngeal cancer (NPC) is a malignant tumor. In a recent publication, we described the presence and distribution of CD8⁺ T cells in NPC and used the information to identify ‘inflamed’, ‘immune-excluded’, and ‘desert’ immune phenotypes, where ‘inflamed’ and ‘immune-excluded’ NPCs were correlated with CD8 T cell infiltration and survival. Arguably, more detailed and, in particular, spatially resolved data are required for patient stratification and for the identification of new treatment targets. In this study, we investigate the phenotype of CD45⁺ leukocytes in the previously analyzed NPC samples by applying multiplexed tissue analysis to assess the spatial distribution of cell types and to quantify selected biomarkers. A total of 47 specified regions-of-interest (ROIs) were generated based on CD45, CD8, and PanCK morphological staining. Using the GeoMx^® Digital Spatial Profiler (DSP), 49 target proteins were digitally quantified from the selected ROIs of a tissue microarray consisting of 30 unique NPC biopsies. Protein targets associated with B cells (CD20), NK cells (CD56), macrophages (CD68), and regulatory T cells (PD-1, FOXP3) were most differentially expressed in CD45⁺ segments within ‘immune-rich cancer cell islet’ regions of the tumor (cf. ‘surrounding stromal leukocyte’ regions). In contrast, markers associated with suppressive populations of myeloid cells (CD163, B7-H3, VISTA) and T cells (CD4, LAG3, Tim-3) were expressed at a higher level in CD45⁺ segments in the ‘surrounding stromal leukocyte’ regions (cf. ‘immune-rich cancer cell islet’ regions). When comparing the three phenotypes, the ‘inflamed’ profile (cf. ‘immune-excluded’ and ‘desert’) exhibited higher expression of markers associated with B cells, NK cells, macrophages, and myeloid cells. Myeloid markers were highly expressed in the ‘immune-excluded’ phenotype. Granulocyte markers and immune-regulatory markers were higher in the ‘desert‘ profile (cf. ‘inflamed’ and ‘immune-excluded’). In conclusion, this study describes the spatial heterogeneity of the immune microenvironment in NPC and highlights immune-related biomarkers in immune phenotypes, which may aid in the stratification of patients for therapeutic purposes. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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Review

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16 pages, 908 KiB

Open AccessReview

Targeting Metabolic Vulnerabilities in Epstein–Barr Virus-Driven Proliferative Diseases

by Nicole Yong Ting Leung and Liang Wei Wang

Cancers 2023, 15(13), 3412; https://doi.org/10.3390/cancers15133412 - 29 Jun 2023

Viewed by 1647

Abstract

The metabolism of cancer cells and Epstein–Barr virus (EBV) infected cells have remarkable similarities. Cancer cells frequently reprogram metabolic pathways to augment their ability to support abnormal rates of proliferation and promote intra-organismal spread through metastatic invasion. On the other hand, EBV is [...] Read more.

The metabolism of cancer cells and Epstein–Barr virus (EBV) infected cells have remarkable similarities. Cancer cells frequently reprogram metabolic pathways to augment their ability to support abnormal rates of proliferation and promote intra-organismal spread through metastatic invasion. On the other hand, EBV is also capable of manipulating host cell metabolism to enable sustained growth and division during latency as well as intra- and inter-individual transmission during lytic replication. It comes as no surprise that EBV, the first oncogenic virus to be described in humans, is a key driver for a significant fraction of human malignancies in the world (~1% of all cancers), both in terms of new diagnoses and attributable deaths each year. Understanding the contributions of metabolic pathways that underpin transformation and virus replication will be important for delineating new therapeutic targets and designing nutritional interventions to reduce disease burden. In this review, we summarise research hitherto conducted on the means and impact of various metabolic changes induced by EBV and discuss existing and potential treatment options targeting metabolic vulnerabilities in EBV-associated diseases. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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23 pages, 1129 KiB

Open AccessReview

EBV and Lymphomagenesis

by Daniel G. Sausen, Ayeman Basith and Syed Muqeemuddin

Cancers 2023, 15(7), 2133; https://doi.org/10.3390/cancers15072133 - 04 Apr 2023

Cited by 11 | Viewed by 2572

Abstract

The clinical significance of Epstein–Barr virus (EBV) cannot be understated. Not only does it infect approximately 90% of the world’s population, but it is also associated with numerous pathologies. Diseases linked to this virus include hematologic malignancies such as diffuse large B-cell lymphoma, [...] Read more.

The clinical significance of Epstein–Barr virus (EBV) cannot be understated. Not only does it infect approximately 90% of the world’s population, but it is also associated with numerous pathologies. Diseases linked to this virus include hematologic malignancies such as diffuse large B-cell lymphoma, Hodgkin lymphoma, Burkitt lymphoma, primary CNS lymphoma, and NK/T-cell lymphoma, epithelial malignancies such as nasopharyngeal carcinoma and gastric cancer, autoimmune diseases such as multiple sclerosis, Graves’ disease, and lupus. While treatment for these disease states is ever evolving, much work remains to more fully elucidate the relationship between EBV, its associated disease states, and their treatments. This paper begins with an overview of EBV latency and latency-associated proteins. It will then review EBV’s contributions to select hematologic malignancies with a focus on the contribution of latent proteins as well as their associated management. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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18 pages, 3909 KiB

Open AccessReview

Contribution of Epstein–Barr Virus Lytic Proteins to Cancer Hallmarks and Implications from Other Oncoviruses

by Mike Dorothea, Jia Xie, Stephanie Pei Tung Yiu and Alan Kwok Shing Chiang

Cancers 2023, 15(7), 2120; https://doi.org/10.3390/cancers15072120 - 02 Apr 2023

Cited by 3 | Viewed by 3012

Abstract

Epstein–Barr virus (EBV) is a prevalent human gamma-herpesvirus that infects the majority of the adult population worldwide and is associated with several lymphoid and epithelial malignancies. EBV displays a biphasic life cycle, namely, latent and lytic replication cycles, expressing a diversity of viral [...] Read more.

Epstein–Barr virus (EBV) is a prevalent human gamma-herpesvirus that infects the majority of the adult population worldwide and is associated with several lymphoid and epithelial malignancies. EBV displays a biphasic life cycle, namely, latent and lytic replication cycles, expressing a diversity of viral proteins. Among the EBV proteins being expressed during both latent and lytic cycles, the oncogenic roles of EBV lytic proteins are largely uncharacterized. In this review, the established contributions of EBV lytic proteins in tumorigenesis are summarized according to the cancer hallmarks displayed. We further postulate the oncogenic properties of several EBV lytic proteins by comparing the evolutionary conserved oncogenic mechanisms in other herpesviruses and oncoviruses. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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21 pages, 3635 KiB

Open AccessReview

Functional Implications of Epstein-Barr Virus Lytic Genes in Carcinogenesis

by Lee Fah Yap, Anna Kang Chee Wong, Ian C. Paterson and Lawrence S. Young

Cancers 2022, 14(23), 5780; https://doi.org/10.3390/cancers14235780 - 24 Nov 2022

Cited by 10 | Viewed by 2240

Abstract

Epstein-Barr virus (EBV) is associated with a diverse range of tumors of both lymphoid and epithelial origin. Similar to other herpesviruses, EBV displays a bipartite life cycle consisting of latent and lytic phases. Current dogma indicates that the latent genes are key drivers [...] Read more.

Epstein-Barr virus (EBV) is associated with a diverse range of tumors of both lymphoid and epithelial origin. Similar to other herpesviruses, EBV displays a bipartite life cycle consisting of latent and lytic phases. Current dogma indicates that the latent genes are key drivers in the pathogenesis of EBV-associated cancers, while the lytic genes are primarily responsible for viral transmission. In recent years, evidence has emerged to show that the EBV lytic phase also plays an important role in EBV tumorigenesis, and the expression of EBV lytic genes is frequently detected in tumor tissues and cell lines. The advent of next generation sequencing has allowed the comprehensive profiling of EBV gene expression, and this has revealed the consistent expression of several lytic genes across various types of EBV-associated cancers. In this review, we provide an overview of the functional implications of EBV lytic gene expression to the oncogenic process and discuss possible avenues for future investigations. Full article

(This article belongs to the Special Issue Epstein-Barr Virus-Associated Cancers: From Pathogenesis to Treatment)

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