Transformation of BCR-deficient germinal-center B cells by EBV supports a major role of the virus in the pathogenesis of Hodgkin and posttransplantation lymphomas

Blood ◽  
2005 ◽  
Vol 106 (13) ◽  
pp. 4345-4350 ◽  
Author(s):  
Dörte Bechtel ◽  
Julia Kurth ◽  
Claus Unkel ◽  
Ralf Küppers
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Germinal Center ◽  
Somatic Hypermutation ◽  
Cell Receptor ◽  
Lymphoproliferative Disease ◽  
Barr Virus ◽  
Classic Hodgkin Lymphoma ◽  
Epstein Barr

In classic Hodgkin lymphoma (HL) and posttransplantation lymphoproliferative disease (PTLD), 2 malignancies frequently associated with Epstein-Barr virus (EBV), the tumor cells often appear to derive from B-cell receptor (BCR)–deficient and therefore preapoptotic germinal center (GC) B cells. To test whether EBV can rescue BCR-less GC B cells, we infected human tonsillar CD77+ GC B cells in vitro with EBV. More than 60 monoclonal lymphoblastoid cell lines (LCLs) were established. Among these, 28 cell lines did not express surface immunoglobulin (sIg). Two of the sIg-negative cell lines carry obviously destructive mutations that have been introduced into originally functional VH gene rearrangements during the process of somatic hypermutation. Quantitative reverse transcriptase–polymerase chain reaction (RT-PCR) showed that in most other lines the sIg deficiency was not simply the result of transcriptional down-regulation, but it was rather due to posttranscriptional defects. These findings strongly support the idea that EBV plays a central role in the pathogenesis of classic HL and PTLD by rescuing BCR-deficient, preapoptotic GC B cells from apoptosis, and that EBV infection renders the cells independent from survival signals normally supplied by a BCR. The monoclonal LCLs represent valuable models for early stages of lymphoma development in classic HL and PTLD.

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 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 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.

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 Germinal Center B Cells Latently Infected with Epstein-Barr Virus Proliferate Extensively but Do Not Increase in Number

2009 ◽  
Vol 84 (2) ◽  
pp. 1158-1168 ◽  
Author(s):  
Jill E. Roughan ◽  
Charles Torgbor ◽  
David A. Thorley-Lawson
Keyword(s):  
B Cells ◽  
Germinal Center ◽  
Epstein Barr Virus ◽  
Acute Infection ◽  
Absolute Number ◽  
Barr Virus ◽  
Infected Cells ◽  
The Absolute ◽  
Epstein Barr

ABSTRACT In this study we show that in long-term persistent infection, Epstein-Barr virus (EBV)-infected cells undergoing a germinal center (GC) reaction in the tonsils are limited to the follicles and proliferate extensively. Despite this, the absolute number of infected cells per GC remains small (average of 3 to 4 cells per germinal center; range, 1 to 9 cells), and only about 38 to 55% (average, 45%) of all GCs carry infected cells. The data fit a model where, on average, cells in the GC divide approximately three times; however, only one progeny cell survives to undergo a further three divisions. Thus, a fraction of cells undergo multiple rounds of division without increasing in numbers; i.e., they die at the same rate that they are dividing. We conclude that EBV-infected cells in the GC undergo the extensive proliferation characteristic of GC cells but that the absolute number is limited either by the immune response or by the availability of an essential survival factor. We suggest that this behavior is a relic of the mechanism by which EBV establishes persistence during acute infection. Lastly, the expression of the viral latent protein LMP1 in GC B cells, unlike in vitro, does not correlate directly with the expression of bcl-2 or bcl-6. This emphasizes our claim that observations made regarding the functions of EBV proteins in cell lines or in transgenic mice should be treated with skepticism unless verified in vivo.

scholarly journals c-Myc and Rel/NF-κB Are the Two Master Transcriptional Systems Activated in the Latency III Program of Epstein-Barr Virus-Immortalized B Cells

2009 ◽  
Vol 83 (10) ◽  
pp. 5014-5027 ◽  
Author(s):  
Nathalie Faumont ◽  
Stéphanie Durand-Panteix ◽  
Martin Schlee ◽  
Sebastian Grömminger ◽  
Marino Schuhmacher ◽  
...  
Keyword(s):  
B Cells ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Expression Patterns ◽  
Dominant Negative ◽  
Barr Virus ◽  
Conditional Expression ◽  
Epstein Barr

ABSTRACT The Epstein-Barr virus (EBV) latency III program imposed by EBNA2 and LMP1 is directly responsible for immortalization of B cells in vitro and is thought to mediate most immunodeficiency-related posttransplant lymphoproliferative diseases in vivo. To answer the question whether and how this proliferation program is related to c-Myc, we have established the transcriptome of both c-Myc and EBV latency III proliferation programs using a Lymphochip specialized microarray. In addition to EBV-positive latency I Burkitt lymphoma lines and lymphoblastoid cell lines (LCLs), we used an LCL expressing an estrogen-regulatable EBNA2 fusion protein (EREB2-5) and derivative B-cell lines expressing a constitutively active or tetracycline-regulatable c-myc gene. A total of 897 genes were found to be fourfold or more up- or downregulated in either one or both proliferation programs compared to the expression profile of resting EREB2-5 cells. A total of 661 (74%) of these were regulated similarly in both programs. Numerous repressed genes were known targets of STAT1, and most induced genes were known to be upregulated by c-Myc and to be involved in cell proliferation. In keeping with the gene expression patterns, inactivation of c-Myc by a chemical inhibitor or by conditional expression of dominant-negative c-Myc and Max mutants led to proliferation arrest of LCLs. Most genes differently regulated in both proliferation programs corresponded to genes induced by NF-κB in LCLs, and many of them coded for immunoregulatory and/or antiapoptotic molecules. Thus, c-Myc and NF-κB are the two main transcription factors responsible for the phenotype, growth pattern, and biological properties of cells driven into proliferation by EBV.

Epstein-Barr virus persistence in the absence of conventional memory B cells: IgM+IgD+CD27+ B cells harbor the virus in X-linked lymphoproliferative disease patients

Blood ◽  
2008 ◽  
Vol 112 (3) ◽  
pp. 672-679 ◽  
Author(s):  
Sridhar Chaganti ◽  
Cindy S. Ma ◽  
Andrew I. Bell ◽  
Debbie Croom-Carter ◽  
Andrew D. Hislop ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Epstein Barr Virus ◽  
Cell System ◽  
Lymphoproliferative Disease ◽  
Memory B Cells ◽  
Cell Compartment ◽  
Barr Virus ◽  
Epstein Barr

AbstractEpstein-Barr virus (EBV) persists in healthy virus carriers within the immunoglobulin (Ig)D−CD27+ (class-switched) memory B-cell compartment that normally arises through antigen stimulation and germinal center transit. Patients with X-linked lymphoproliferative disease (XLP) lack such class-switched memory B cells but are highly susceptible to EBV infection, often developing fatal symptoms resembling those seen in EBV-associated hemophagocytic syndrome (EBV-AHS), a disease caused by aberrant virus entry into the NK- or T-cell system. Here we show that XLP patients who survive primary EBV exposure carry relatively high virus loads in the B-cell, but not the NK- or T-cell, compartment. Interestingly, in the absence of conventional class-switched memory B cells, the circulating EBV load was concentrated within a small population of IgM+IgD+CD27+ (nonswitched) memory cells rather than within the numerically dominant naive (IgM+IgD+CD27−) or transitional (CD10+CD27−) subsets. In 2 prospectively studied patients, the circulating EBV load was stable and markers of virus polymorphism detected the same resident strain over time. These results provide the first definitive evidence that EBV can establish persistence in the B-cell system in the absence of fully functional germinal center activity and of a class-switched memory B-cell compartment.

scholarly journals CD4+ T-Cell Effectors Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation

2001 ◽  
Vol 75 (8) ◽  
pp. 3740-3752 ◽  
Author(s):  
Sarah Nikiforow ◽  
Kim Bottomly ◽  
George Miller
Keyword(s):  
Cell Proliferation ◽  
T Cells ◽  
T Cell ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Epstein Barr Virus ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT In immunodeficient hosts, Epstein-Barr virus (EBV) often induces extensive B-cell lymphoproliferative disease and lymphoma. Without effective in vitro immune surveillance, B cells infected by the virus readily form immortalized cell lines. In the regression assay, memory T cells inhibit the formation of foci of EBV-transformed B cells that follows recent in vitro infection by EBV. No one has yet addressed which T cell regulates the early proliferative phase of B cells newly infected by EBV. Using new quantitative methods, we analyzed T-cell surveillance of EBV-mediated B-cell proliferation. We found that CD4+ T cells play a significant role in limiting proliferation of newly infected, activated CD23+ B cells. In the absence of T cells, EBV-infected CD23+ B cells divided rapidly during the first 3 weeks after infection. Removal of CD4+ but not CD8+ T cells also abrogated immune control. Purified CD4+ T cells eliminated outgrowth when added to EBV-infected B cells. Thus, unlike the killing of EBV-infected lymphoblastoid cell lines, in which CD8+ cytolytic T cells play an essential role, prevention of early-phase EBV-induced B-cell proliferation requires CD4+ effector T cells.

scholarly journals Cytolytic CD4+-T-Cell Clones Reactive to EBNA1 Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation

2003 ◽  
Vol 77 (22) ◽  
pp. 12088-12104 ◽  
Author(s):  
Sarah Nikiforow ◽  
Kim Bottomly ◽  
George Miller ◽  
Christian Münz
Keyword(s):  
T Cells ◽  
T Cell ◽  
B Cells ◽  
Lymphoproliferative Disease ◽  
T Cell Clones ◽  
Barr Virus ◽  
Cell Clones ◽  
Epstein Barr

ABSTRACT In the absence of immune surveillance, Epstein-Barr virus (EBV)-infected B cells generate neoplasms in vivo and transformed cell lines in vitro. In an in vitro system which modeled the first steps of in vivo immune control over posttransplant lymphoproliferative disease and lymphomas, our investigators previously demonstrated that memory CD4+ T cells reactive to EBV were necessary and sufficient to prevent proliferation of B cells newly infected by EBV (S. Nikiforow et al., J. Virol. 75:3740-3752, 2001). Here, we show that three CD4+-T-cell clones reactive to the latent EBV antigen EBNA1 also prevent the proliferation of newly infected B cells from major histocompatibility complex (MHC) class II-matched donors, a crucial first step in the transformation process. EBNA1-reactive T-cell clones recognized B cells as early as 4 days after EBV infection through an HLA-DR-restricted interaction. They secreted Th1-type and Th2-type cytokines and lysed EBV-transformed established lymphoblastoid cell lines via a Fas/Fas ligand-dependent mechanism. Once specifically activated, they also caused bystander regression and bystander killing of non-MHC-matched EBV-infected B cells. Since EBNA1 is recognized by CD4+ T cells from nearly all EBV-seropositive individuals and evades detection by CD8+ T cells, EBNA1-reactive CD4+ T cells may control de novo expansion of B cells following EBV infection in vivo. Thus, EBNA1-reactive CD4+-T-cell clones may find use as adoptive immunotherapy against EBV-related lymphoproliferative disease and many other EBV-associated tumors.

scholarly journals Toll-Like Receptor Agonists Synergistically Increase Proliferation and Activation of B Cells by Epstein-Barr Virus

2010 ◽  
Vol 84 (7) ◽  
pp. 3612-3623 ◽  
Author(s):  
Stefanie Iskra ◽  
Markus Kalla ◽  
Henri-Jacques Delecluse ◽  
Wolfgang Hammerschmidt ◽  
Andreas Moosmann
Keyword(s):  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Epstein Barr Virus ◽  
Lymphoproliferative Disease ◽  
Cpg Dna ◽  
Cpg Dinucleotides ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) efficiently drives proliferation of human primary B cells in vitro, a process relevant for human diseases such as infectious mononucleosis and posttransplant lymphoproliferative disease. Human B-cell proliferation is also driven by ligands of Toll-like receptors (TLRs), notably viral or bacterial DNA containing unmethylated CpG dinucleotides, which triggers TLR9. Here we quantitatively investigated how TLR stimuli influence EBV-driven B-cell proliferation and expression of effector molecules. CpG DNA synergistically increased EBV-driven proliferation and transformation, T-cell costimulatory molecules, and early production of interleukin-6. CpG DNA alone activated only memory B cells, but CpG DNA enhanced EBV-mediated transformation of both memory and naive B cells. Ligands for TLR2 or TLR7/8 or whole bacteria had a weaker but still superadditive effect on B-cell transformation. Additionally, CpG DNA facilitated the release of transforming virus by established EBV-infected lymphoblastoid cell lines. These results suggest that the proliferation of EBV-infected B cells and their capability to interact with immune effector cells may be directly influenced by components of bacteria or other microbes present at the site of infection.

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