scholarly journals Distinct Ex Vivo Susceptibility of B-Cell Subsets to Epstein-Barr Virus Infection According to Differentiation Status and Tissue Origin

2008 ◽  
Vol 82 (9) ◽  
pp. 4400-4412 ◽  
Author(s):  
Marcus Dorner ◽  
Franziska Zucol ◽  
Christoph Berger ◽  
Rahel Byland ◽  
Gregory T. Melroe ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Ex Vivo ◽  
Memory B Cells ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr ◽  
Differentiation Status ◽  
B Cell Subsets

ABSTRACT Epstein-Barr virus (EBV) uses tonsils as the portal of entry to establish persistent infection. EBV is found in various B-cell subsets in tonsils but exclusively in memory B cells in peripheral blood. The in vitro susceptibilities of B-cell subsets to EBV infection have been studied solely qualitatively. In this work, we examined quantitatively the in vitro susceptibilities of various B-cell subsets from different tissue origins to EBV infection. First, we established a centrifugation-based inoculation protocol (spinoculation) that resulted in a significantly increased proportion of infected cells compared to that obtained by conventional inoculation, enabling a detailed susceptibility analysis. Importantly, B-cell infection occurred via the known EBV receptors and infected cells showed EBV mRNA expression patterns similar to those observed after conventional inoculation, validating our approach. Tonsillar naïve and memory B cells were infected ex vivo at similar frequencies. In contrast, memory B cells from blood, which represent B cells from various lymphoid tissues, were infected at lower frequencies than their naïve counterparts. Immunoglobulin A (IgA)-positive or IgG-positive tonsillar memory B cells were significantly more susceptible to EBV infection than IgM-positive counterparts. Memory B cells were transformed with lower efficiency than naïve B cells. This result was paralleled by lower proliferation rates. In summary, these data suggest that EBV exploits the B-cell differentiation status and tissue origin to establish persistent infection.

scholarly journals Epstein-Barr Virus Induces Adhesion Receptor CD226 (DNAM-1) Expression during Primary B-Cell Transformation into Lymphoblastoid Cell Lines

mSphere ◽  
2017 ◽  
Vol 2 (6) ◽  
Author(s):  
Lisa Grossman ◽  
Chris Chang ◽  
Joanne Dai ◽  
Pavel A. Nikitin ◽  
Dereje D. Jima ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Human Herpesvirus ◽  
Barr Virus ◽  
Ebv Infection ◽  
Cellular Aggregation ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out. Epstein-Barr virus (EBV), an oncogenic herpesvirus, infects and transforms primary B cells into immortal lymphoblastoid cell lines (LCLs), providing a model for EBV-mediated tumorigenesis. EBV transformation stimulates robust homotypic aggregation, indicating that EBV induces molecules that mediate cell-cell adhesion. We report that EBV potently induced expression of the adhesion molecule CD226, which is not normally expressed on B cells. We found that early after infection of primary B cells, EBV promoted an increase in CD226 mRNA and protein expression. CD226 levels increased further from early proliferating EBV-positive B cells to LCLs. We found that CD226 expression on B cells was independent of B-cell activation as CpG DNA failed to induce CD226 to the extent of EBV infection. CD226 expression was high in EBV-infected B cells expressing the latency III growth program, but low in EBV-negative and EBV latency I-infected B-lymphoma cell lines. We validated this correlation by demonstrating that the latency III characteristic EBV NF-κB activator, latent membrane protein 1 (LMP1), was sufficient for CD226 upregulation and that CD226 was more highly expressed in lymphomas with increased NF-κB activity. Finally, we found that CD226 was not important for LCL steady-state growth, survival in response to apoptotic stress, homotypic aggregation, or adhesion to activated endothelial cells. These findings collectively suggest that EBV induces expression of a cell adhesion molecule on primary B cells that may play a role in the tumor microenvironment of EBV-associated B-cell malignancies or facilitate adhesion in the establishment of latency in vivo. IMPORTANCE Epstein-Barr virus (EBV) is a common human herpesvirus that establishes latency in B cells. While EBV infection is asymptomatic for most individuals, immune-suppressed individuals are at significantly higher risk of a form of EBV latent infection in which infected B cells are reactivated, grow unchecked, and generate lymphomas. This form of latency is modeled in the laboratory by infecting B cells from the blood of normal human donors in vitro. In this model, we identified a protein called CD226 that is induced by EBV but is not normally expressed on B cells. Rather, it is known to play a role in aggregation and survival signaling of non-B cells in the immune system. Cultures of EBV-infected cells adhere to one another in “clumps,” and while the proteins that are responsible for this cellular aggregation are not fully understood, we hypothesized that this form of cellular aggregation may provide a survival advantage. In this article, we characterize the mechanism by which EBV induces this protein and its expression on lymphoma tissue and cell lines and characterize EBV-infected cell lines in which CD226 has been knocked out.

scholarly journals Chromosomal rearrangements after ex vivo Epstein–Barr virus (EBV) infection of human B cells

Oncogene ◽  
2009 ◽  
Vol 29 (4) ◽  
pp. 503-515 ◽  
Author(s):  
S Lacoste ◽  
E Wiechec ◽  
A G dos Santos Silva ◽  
A Guffei ◽  
G Williams ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Ex Vivo ◽  
Chromosomal Rearrangements ◽  
Barr Virus ◽  
Ebv Infection ◽  
Human B Cells ◽  
Epstein Barr

scholarly journals Epstein-Barr Virus LMP2A Alters In Vivo and In Vitro Models of B-Cell Anergy, but Not Deletion, in Response to Autoantigen

2005 ◽  
Vol 79 (12) ◽  
pp. 7355-7362 ◽  
Author(s):  
Michelle A. Swanson-Mungerson ◽  
Robert G. Caldwell ◽  
Rebecca Bultema ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Nuclear Translocation ◽  
Cell Receptor ◽  
Barr Virus ◽  
Low Levels ◽  
Epstein Barr

ABSTRACT A significant percentage of the population latently harbors Epstein-Barr virus (EBV) in B cells. One EBV-encoded protein, latent membrane protein 2A (LMP2A), is expressed in tissue culture models of EBV latent infection, in human infections, and in many of the EBV-associated proliferative disorders. LMP2A constitutively activates proteins involved in the B-cell receptor (BCR) signal transduction cascade and inhibits the antigen-induced activation of these proteins. In the present study, we investigated whether LMP2A alters B-cell receptor signaling in primary B cells in vivo and in vitro. LMP2A does not inhibit antigen-induced tolerance in response to strong stimuli in an in vivo tolerance model in which B cells are reactive to self-antigen. In contrast, LMP2A bypasses anergy induction in response to low levels of soluble hen egg lysozyme (HEL) both in vivo and in vitro as determined by the ability of LMP2A-expressing HEL-specific B cells to proliferate and induce NF-κB nuclear translocation after exposure to low levels of antigen. Furthermore, LMP2A induces NF-κB nuclear translocation independent of BCR cross-linking. Since NF-κB is required to bypass tolerance induction, this LMP2A-dependent NF-κB activation may complete the tolerogenic signal induced by low levels of soluble HEL. Overall, the findings suggest that LMP2A may not inhibit BCR-induced signals under all conditions as previously suggested by studies with EBV immortalized B cells.

scholarly journals In VitroMeasles Virus Infection of Human Lymphocyte Subsets Demonstrates High Susceptibility and Permissiveness of both Naive and Memory B Cells

2018 ◽  
Vol 92 (8) ◽  
pp. e00131-18 ◽  
Author(s):  
Brigitta M. Laksono ◽  
Christina Grosserichter-Wagener ◽  
Rory D. de Vries ◽  
Simone A. G. Langeveld ◽  
Maarten D. Brem ◽  
...  
Keyword(s):  
T Cells ◽  
B Cells ◽  
B Cell ◽  
Immune Suppression ◽  
Peripheral Blood ◽  
Measles Virus ◽  
Memory B Cells ◽  
T And B Cells ◽  
B Cell Subsets

ABSTRACTMeasles is characterized by a transient immune suppression, leading to an increased risk of opportunistic infections. Measles virus (MV) infection of immune cells is mediated by the cellular receptor CD150, expressed by subsets of lymphocytes, dendritic cells, macrophages, and thymocytes. Previous studies showed that human and nonhuman primate memory T cells express higher levels of CD150 than naive cells and are more susceptible to MV infection. However, limited information is available about the CD150 expression and relative susceptibility to MV infection of B-cell subsets. In this study, we assessed the susceptibility and permissiveness of naive and memory T- and B-cell subsets from human peripheral blood or tonsils toin vitroMV infection. Our study demonstrates that naive and memory B cells express CD150, but at lower frequencies than memory T cells. Nevertheless, both naive and memory B cells proved to be highly permissive to MV infection. Furthermore, we assessed the susceptibility and permissiveness of various functionally distinct T and B cells, such as helper T (TH) cell subsets and IgG- and IgA-positive memory B cells, in peripheral blood and tonsils. We demonstrated that TH1TH17 cells and plasma and germinal center B cells were the subsets most susceptible and permissive to MV infection. Our study suggests that both naive and memory B cells, along with several other antigen-experienced lymphocytes, are important target cells of MV infection. Depletion of these cells potentially contributes to the pathogenesis of measles immune suppression.IMPORTANCEMeasles is associated with immune suppression and is often complicated by bacterial pneumonia, otitis media, or gastroenteritis. Measles virus infects antigen-presenting cells and T and B cells, and depletion of these cells may contribute to lymphopenia and immune suppression. Measles has been associated with follicular exhaustion in lymphoid tissues in humans and nonhuman primates, emphasizing the importance of MV infection of B cellsin vivo. However, information on the relative susceptibility of B-cell subsets is scarce. Here, we compared the susceptibility and permissiveness toin vitroMV infection of human naive and memory T- and B-cell subsets isolated from peripheral blood or tonsils. Our results demonstrate that both naive and memory B cells are more permissive to MV infection than T cells. The highest infection levels were detected in plasma cells and germinal center B cells, suggesting that infection and depletion of these populations contribute to reduced host resistance.

scholarly journals Epstein-Barr virus transformation of human pre-B cells.

1983 ◽  
Vol 158 (2) ◽  
pp. 616-622 ◽  
Author(s):  
M Hansson ◽  
K Falk ◽  
I Ernberg
Keyword(s):  
Bone Marrow ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Bone Marrow Cells ◽  
Fetal Bone ◽  
Barr Virus ◽  
Epstein Barr

In vitro infection of human B lymphocytes with Epstein-Barr virus (EBV) results in establishment of B lymphoblastoid cell lines that reflect normal B cell phenotypes. In this study we have investigated whether immature B cells from fetal bone marrow and liver can serve as targets for EBV. The fetal bone marrow cells were readily transformed by EBV. Among the resulting cell lines, five were surface Ig (sIg)-negative. Three B cell-associated antigens defined by monoclonal antibodies were expressed to the same extent on the fetal cell lines, whether they belonged to the sIg- or sIg+ group. The various differentiation stages that these cell lines may represent are discussed.

scholarly journals Optimisation of ex vivo memory B cell expansion/differentiation for interrogation of rare peripheral memory B cell subset responses

2018 ◽  
Vol 2 ◽  
pp. 97 ◽  
Author(s):  
Luke Muir ◽  
Paul F. McKay ◽  
Velislava N. Petrova ◽  
Oleksiy V. Klymenko ◽  
Sven Kratochvil ◽  
...  
Keyword(s):  
Cell Culture ◽  
B Cells ◽  
B Cell ◽  
Cell Expansion ◽  
Ex Vivo ◽  
Cell Subset ◽  
Human Memory ◽  
Memory B Cells ◽  
Memory B Cell ◽  
B Cell Subsets

Background:Human memory B cells play a vital role in the long-term protection of the host from pathogenic re-challenge. In recent years the importance of a number of different memory B cell subsets that can be formed in response to vaccination or infection has started to become clear. To study memory B cell responses, cells can be culturedex vivo,allowing for an increase in cell number and activation of these quiescent cells, providing sufficient quantities of each memory subset to enable full investigation of functionality. However, despite numerous papers being published demonstrating bulk memory B cell culture, we could find no literature on optimised conditions for the study of memory B cell subsets, such as IgM+memory B cells.Methods:Following a literature review, we carried out a large screen of memory B cell expansion conditions to identify the combination that induced the highest levels of memory B cell expansion. We subsequently used a novel Design of Experiments approach to finely tune the optimal memory B cell expansion and differentiation conditions for human memory B cell subsets. Finally, we characterised the resultant memory B cell subpopulations by IgH sequencing and flow cytometry.Results:The application of specific optimised conditions induce multiple rounds of memory B cell proliferation equally across Ig isotypes, differentiation of memory B cells to antibody secreting cells, and importantly do not alter the Ig genotype of the stimulated cells. Conclusions:Overall, our data identify a memory B cell culture system that offers a robust platform for investigating the functionality of rare memory B cell subsets to infection and/or vaccination.

scholarly journals Highly efficient CRISPR-Cas9-mediated gene knockout in primary human B cells for functional genetic studies of Epstein-Barr virus infection

2021 ◽  
Vol 17 (4) ◽  
pp. e1009117
Author(s):  
Ezgi Akidil ◽  
Manuel Albanese ◽  
Alexander Buschle ◽  
Adrian Ruhle ◽  
Dagmar Pich ◽  
...  
Keyword(s):  
Cell Cycle ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Gene Editing ◽  
Barr Virus ◽  
Ebv Infection ◽  
Highly Efficient ◽  
Human B Cells ◽  
Epstein Barr

Gene editing is now routine in all prokaryotic and metazoan cells but has not received much attention in immune cells when the CRISPR-Cas9 technology was introduced in the field of mammalian cell biology less than ten years ago. This versatile technology has been successfully adapted for gene modifications in human myeloid cells and T cells, among others, but applications to human primary B cells have been scarce and limited to activated B cells. This limitation has precluded conclusive studies into cell activation, differentiation or cell cycle control in this cell type. We report on highly efficient, simple and rapid genome engineering in primary resting human B cells using nucleofection of Cas9 ribonucleoprotein complexes, followed by EBV infection or culture on CD40 ligand feeder cells to drive in vitro B cell survival. We provide proof-of-principle of gene editing in quiescent human B cells using two model genes: CD46 and CDKN2A. The latter encodes the cell cycle regulator p16INK4a which is an important target of Epstein-Barr virus (EBV). Infection of B cells carrying a knockout of CDKN2A with wildtype and EBNA3 oncoprotein mutant strains of EBV allowed us to conclude that EBNA3C controls CDKN2A, the only barrier to B cell proliferation in EBV infected cells. Together, this approach enables efficient targeting of specific gene loci in quiescent human B cells supporting basic research as well as immunotherapeutic strategies.

Epstein-Barr virus colonization of tonsillar and peripheral blood B-cell subsets in primary infection and persistence

Blood ◽  
2009 ◽  
Vol 113 (25) ◽  
pp. 6372-6381 ◽  
Author(s):  
Sridhar Chaganti ◽  
Emily M. Heath ◽  
Wolfgang Bergler ◽  
Michael Kuo ◽  
Maike Buettner ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Primary Infection ◽  
Epstein Barr Virus ◽  
Quantitative Polymerase Chain Reaction ◽  
Viral Loads ◽  
Barr Virus ◽  
Memory Pool ◽  
Epstein Barr ◽  
B Cell Subsets

AbstractEpstein-Barr virus (EBV) persists in the immune host by preferentially colonizing the isotype-switched (IgD−CD27+) memory B-cell pool. In one scenario, this is achieved through virus infection of naive (IgD+CD27−) B cells and their differentiation into memory via germinal center (GC) transit; in another, EBV avoids GC transit and infects memory B cells directly. We report 2 findings consistent with this latter view. First, we examined circulating non–isotype-switched (IgD+CD27+) memory cells, a population that much evidence suggests is GC-independent in origin. Whereas isotype-switched memory had the highest viral loads by quantitative polymerase chain reaction, EBV was detectable in the nonswitched memory pool both in infectious mononucleosis (IM) patients undergoing primary infection and in most long-term virus carriers. Second, we examined colonization by EBV of B-cell subsets sorted from a unique collection of IM tonsillar cell suspensions. Here viral loads were concentrated in B cells with the CD38 marker of GC origin but lacking other GC markers CD10 and CD77. These findings, supported by histologic evidence, suggest that EBV infection in IM tonsils involves extrafollicular B cells expressing CD38 as an activation antigen and not as a marker of ectopic GC activity.

Epstein-Barr Virus (EBV) LMP2A induces alterations in gene transcription similar to those observed in Reed-Sternberg cells of Hodgkin lymphoma

Blood ◽  
2003 ◽  
Vol 102 (12) ◽  
pp. 4166-4178 ◽  
Author(s):  
Toni Portis ◽  
Patricia Dyck ◽  
Richard Longnecker
Keyword(s):  
B Cells ◽  
B Cell ◽  
Hodgkin Lymphoma ◽  
Cell Lines ◽  
Gene Transcription ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Expression Of Genes ◽  
Epstein Barr

AbstractEpstein-Barr virus (EBV) is associated with the development of a variety of malignancies, including Hodgkin lymphoma. One of the few viral transcripts expressed in EBV-positive Hodgkin/Reed-Sternberg (HRS) cells of Hodgkin lymphoma is latent membrane protein 2A (LMP2A). This viral protein blocks B-cell receptor (BCR)-signaling in vitro. Furthermore, expression of LMP2A in developing B cells in vivo induces a global down-regulation of genes necessary for proper B-cell development. In this study we have analyzed gene transcription in primary B cells from LMP2A transgenic mice, LMP2A-expressing human B-cell lines, and LMP2A-positive and -negative EBV-infected lymphoblastoid cell lines (LCLs). We demonstrate that LMP2A increases the expression of genes associated with cell cycle induction and inhibition of apoptosis, alters the expression of genes involved in DNA and RNA metabolism, and decreases the expression of B-cell-specific factors and genes associated with immunity. Furthermore, many alterations in gene expression induced by LMP2A are similar to those recently described in HRS cells of Hodgkin lymphoma and activated, proliferating germinal center centroblasts/centrocytes. These correlations suggest that LMP2A expression in EBV-infected B cells may lead to the induction and maintenance of an activated, proliferative state that could ultimately result in the development of Hodgkin lymphoma. (Blood. 2003;102: 4166-4178)

scholarly journals Manipulation of the Toll-Like Receptor 7 Signaling Pathway by Epstein-Barr Virus

2007 ◽  
Vol 81 (18) ◽  
pp. 9748-9758 ◽  
Author(s):  
Heather J. Martin ◽  
Jae Myun Lee ◽  
Dermot Walls ◽  
S. Diane Hayward
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Cell Activation ◽  
B Cell Activation ◽  
Toll Like Receptor ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infection of primary B cells causes B-cell activation and proliferation. Activation of B cells requires binding of antigen to the B-cell receptor and a survival signal from ligand-bound CD40, signals that are provided by the EBV LMP1 and LMP2A latency proteins. Recently, Toll-like receptor (TLR) signaling has been reported to provide a third B-cell activation stimulus. The interaction between the EBV and TLR pathways was therefore investigated. Both UV-inactivated and untreated EBV upregulated the expression of TLR7 and downregulated the expression of TLR9 in naive B cells. UV-inactivated virus transiently stimulated naive B-cell proliferation in the presence of the TLR7 ligand R837, while addition of the TLR7 antagonist IRS 661 impaired cell growth induced by untreated EBV. Interferon regulatory factor 5 (IRF-5) is a downstream mediator of TLR7 signaling. IRF-5 was induced following EBV infection, and IRF-5 was expressed in B-cell lines with type III latency. Expression of IRF-5 in this setting is surprising since IRF-5 has tumor suppressor and antiviral properties. B-cell proliferation assays provided evidence that EBV modulates TLR7 signaling responses. Examination of IRF-5 transcripts identified a novel splice variant, V12, that was induced by EBV infection, was constitutively nuclear, and acted as a dominant negative form in IRF-5 reporter assays. IRF-4 negatively regulates IRF-5 activation, and IRF-4 was also present in type III latently infected cells. EBV therefore initially uses TLR7 signaling to enhance B-cell proliferation and subsequently modifies the pathway to regulate IRF-5 activity.

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