scholarly journals An EBNA3A-Mutated Epstein-Barr Virus Retains the Capacity for Lymphomagenesis in a Cord Blood-Humanized Mouse Model

2020 ◽  
Vol 94 (10) ◽  
Author(s):  
James C. Romero-Masters ◽  
Makoto Ohashi ◽  
Reza Djavadian ◽  
Mark R. Eichelberg ◽  
Mitchell Hayes ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Mouse Model ◽  
Cord Blood ◽  
Wild Type ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) causes B cell lymphomas and transforms B cells in vitro. The EBV protein EBNA3A collaborates with EBNA3C to repress p16 expression and is required for efficient transformation in vitro. An EBNA3A deletion mutant EBV strain was recently reported to establish latency in humanized mice but not cause tumors. Here, we compare the phenotypes of an EBNA3A mutant EBV (Δ3A) and wild-type (WT) EBV in a cord blood-humanized (CBH) mouse model. The hypomorphic Δ3A mutant, in which a stop codon is inserted downstream from the first ATG and the open reading frame is disrupted by a 1-bp insertion, expresses very small amounts of EBNA3A using an alternative ATG at residue 15. Δ3A caused B cell lymphomas at rates similar to their induction by WT EBV but with delayed onset. Δ3A and WT tumors expressed equivalent levels of EBNA2 and p16, but Δ3A tumors in some cases had reduced LMP1. Like the WT EBV tumors, Δ3A lymphomas were oligoclonal/monoclonal, with typically one dominant IGHV gene being expressed. Transcriptome sequencing (RNA-seq) analysis revealed small but consistent gene expression differences involving multiple cellular genes in the WT EBV- versus Δ3A-infected tumors and increased expression of genes associated with T cells, suggesting increased T cell infiltration of tumors. Consistent with an impact of EBNA3A on immune function, we found that the expression of CLEC2D, a receptor that has previously been shown to influence responses of T and NK cells, was markedly diminished in cells infected with EBNA3A mutant virus. Together, these studies suggest that EBNA3A contributes to efficient EBV-induced lymphomagenesis in CBH mice. IMPORTANCE The EBV protein EBNA3A is expressed in latently infected B cells and is important for efficient EBV-induced transformation of B cells in vitro. In this study, we used a cord blood-humanized mouse model to compare the phenotypes of an EBNA3A hypomorph mutant virus (Δ3A) and wild-type EBV. The Δ3A virus caused lymphomas with delayed onset compared to the onset of those caused by WT EBV, although the tumors occurred at a similar rate. The WT EBV and EBNA3A mutant tumors expressed similar levels of the EBV protein EBNA2 and cellular protein p16, but in some cases, Δ3A tumors had less LMP1. Our analysis suggested that Δ3A-infected tumors have elevated T cell infiltrates and decreased expression of the CLEC2D receptor, which may point to potential novel roles of EBNA3A in T cell and NK cell responses to EBV-infected tumors.

scholarly journals Latent Membrane Protein 1 (LMP1) and LMP2A Collaborate To Promote Epstein-Barr Virus-Induced B Cell Lymphomas in a Cord Blood-Humanized Mouse Model but Are Not Essential

2017 ◽  
Vol 91 (7) ◽  
Author(s):  
Shi-Dong Ma ◽  
Ming-Han Tsai ◽  
James C. Romero-Masters ◽  
Erik A. Ranheim ◽  
Shane M. Huebner ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cord Blood ◽  
B Cell ◽  
Cell Receptor ◽  
B Cell Lymphomas ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Tumor Onset ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) infection is associated with B cell lymphomas in humans. The ability of EBV to convert human B cells into long-lived lymphoblastoid cell lines (LCLs) in vitro requires the collaborative effects of EBNA2 (which hijacks Notch signaling), latent membrane protein 1 (LMP1) (which mimics CD40 signaling), and EBV-encoded nuclear antigen 3A (EBNA3A) and EBNA3C (which inhibit oncogene-induced senescence and apoptosis). However, we recently showed that an LMP1-deleted EBV mutant induces B cell lymphomas in a newly developed cord blood-humanized mouse model that allows EBV-infected B cells to interact with CD4 T cells (the major source of CD40 ligand). Here we examined whether the EBV LMP2A protein, which mimics constitutively active B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A delays the onset of EBV-induced lymphomas but does not affect the tumor phenotype or the number of tumors. The simultaneous deletion of both LMP1 and LMP2A results in fewer tumors and a further delay in tumor onset. Nevertheless, the LMP1/LMP2A double mutant induces lymphomas in approximately half of the infected animals. These results indicate that neither LMP1 nor LMP2A is absolutely essential for the ability of EBV to induce B cell lymphomas in the cord blood-humanized mouse model, although the simultaneous loss of both LMP1 and LMP2A decreases the proportion of animals developing tumors and increases the time to tumor onset. Thus, the expression of either LMP1 or LMP2A may be sufficient to promote early-onset EBV-induced tumors in this model. IMPORTANCE EBV causes human lymphomas, but few models are available for dissecting how EBV causes lymphomas in vivo in the context of a host immune response. We recently used a newly developed cord blood-humanized mouse model to show that EBV can cooperate with human CD4 T cells to cause B cell lymphomas even when a major viral transforming protein, LMP1, is deleted. Here we examined whether the EBV protein LMP2A, which mimics B cell receptor signaling, is required for EBV-induced lymphomas in this model. We find that the deletion of LMP2A alone has little effect on the ability of EBV to cause lymphomas but delays tumor onset. The deletion of both LMP1 and LMP2A results in a smaller number of lymphomas in infected animals, with an even more delayed time to tumor onset. These results suggest that LMP1 and LMP2A collaborate to promote early-onset lymphomas in this model, but neither protein is absolutely essential.

scholarly journals An EBNA3C-deleted Epstein-Barr virus (EBV) mutant causes B-cell lymphomas with delayed onset in a cord blood-humanized mouse model

2018 ◽  
Vol 14 (8) ◽  
pp. e1007221 ◽  
Author(s):  
James C. Romero-Masters ◽  
Makoto Ohashi ◽  
Reza Djavadian ◽  
Mark R. Eichelberg ◽  
Mitch Hayes ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cord Blood ◽  
B Cell ◽  
Epstein Barr Virus ◽  
B Cell Lymphomas ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Delayed Onset ◽  
Epstein Barr

scholarly journals PD-1/CTLA-4 Blockade Inhibits Epstein-Barr Virus-Induced Lymphoma Growth in a Cord Blood Humanized-Mouse Model

2016 ◽  
Vol 12 (5) ◽  
pp. e1005642 ◽  
Author(s):  
Shi-Dong Ma ◽  
Xuequn Xu ◽  
Richard Jones ◽  
Henri-Jacques Delecluse ◽  
Nicholas A. Zumwalde ◽  
...  
Keyword(s):  
Mouse Model ◽  
Cord Blood ◽  
Epstein Barr Virus ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Epstein Barr

scholarly journals Epstein-Barr Virus BNRF1 Protein Allows Efficient Transfer from the Endosomal Compartment to the Nucleus of Primary B Lymphocytes

2006 ◽  
Vol 80 (19) ◽  
pp. 9435-9443 ◽  
Author(s):  
R. Feederle ◽  
B. Neuhierl ◽  
G. Baldwin ◽  
H. Bannert ◽  
B. Hub ◽  
...  
Keyword(s):  
B Cells ◽  
Epstein Barr Virus ◽  
Wild Type ◽  
Barr Virus ◽  
Fold Reduction ◽  
Viral Particles ◽  
Viral Mutant ◽  
Tumor Virus ◽  
Epstein Barr

ABSTRACT Epstein-Barr virus (EBV) is a tumor virus with marked B lymphotropism. After crossing the B-cell membrane, the virus enters cytoplasmic vesicles, where decapsidation takes place to allow transfer of the viral DNA to the cell nucleus. BNRF1 has been characterized as the EBV major tegument protein, but its precise function is unknown. We have constructed a viral mutant that lacks the BNRF1 gene and report here its in vitro phenotype. A recombinant virus devoid of BNRF1 (ΔBNRF1) showed efficient DNA replication and production of mature viral particles. B cells infected with the ΔBNRF1 mutant presented viral lytic antigens as efficiently as B cells infected with wild-type or BNRF1 trans-complemented ΔBNRF1 viruses. Antigen presentation in B cells infected with either wild-type (EBV-wt) or ΔBNRF1 virus was blocked by leupeptin addition, showing that both viruses reach the endosome/lysosome compartment. These data were confirmed by direct observation of the mutant virus in endosomes of infected B cells by electron microscopy. However, we observed a 20-fold reduction in the number of B cells expressing the nuclear protein EBNA2 after infection with a ΔBNRF1 virus compared to wild-type infection. Likewise, ΔBNRF1 viruses transformed primary B cells much less efficiently than EBV-wt or BNRF1 trans-complemented viruses. We conclude from these findings that BNRF1 plays an important role in viral transport from the endosomes to the nucleus.

scholarly journals Pembrolizumab Therapy Exacerbates EBV-Induced Infections and Tumors in a Long-Term Fully Humanized Mouse Model

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2405-2405
Author(s):  
Renata Stripecke ◽  
Simon Danisch ◽  
Constanze Slabik ◽  
Reinhard Zeidler ◽  
Wolfgang Hammerschmidt ◽  
...  
Keyword(s):  
T Cells ◽  
T Cell ◽  
Cd8 T Cells ◽  
Epstein Barr Virus ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

Abstract INTRODUCTION: A promising rich pipeline of combination therapies targeting checkpoint molecules expressed on T cells and/or tumor cells is currently being developed to abrogate tumor-induced immunosuppression. Novel in vivo models suitable for validating these immunotherapies and predict safety issues are warranted to accelerate their translation to patients. AIM: Epstein Barr virus (EBV) is a type 1 carcinogen that is directly associated with the development of human B cell neoplasms. We modelled EBV infection and tumor progression in long-term humanized mice and investigated the activation of T cells with PD-1 expression. Further, we performed studies evaluating the effects of an anti-PD-1 antibody (pembrolizumab/ keytruda) in on EBV infections and/or tumor growth. METHODS: Humanized mice transplanted with human cord-blood CD34+ stem cells and showing long-term (15 weeks) human T cell reconstitution were infected with an oncogenic recombinant Epstein Barr Virus (EBV), encoding enhanced firefly luciferase (fLuc) and green fluorescent protein (GFP). EBV infections were monitored by optical imaging analyses and PCR. CD8+ and CD4+ T cell subtypes (PD-1+, naïve, central memory, effector memory and terminal effector) were sequentially monitored in blood by longitudinal flow cytometry analyses and in organs at experimental endpoint. Histopathological analyses were performed to characterize EBV infection (EBER+) and PD-1+ T cell-rich infiltrates in tissues and tumors. We used the model to evaluate the effects of pembrolizumab administered after EBV challenge at low dose (first dose 1.65mg/kg and then 3.30 mg/kg, every other week, n=3) or high dose (first dose 5.00 mg/kg and then 10.00 mg/kg every other week, n=3) in respect to EBV infected controls (n=2). RESULTS: EBV-fLuc was detectable one week after infection by non-invasive optical imaging in the spleen, from where it spread rapidly and systemically. Among the EBV-infected mice, 8/18 (=44%) developed macroscopically visible tumors in the spleen. For further analyses of the data, we then compared EBV-infected mice with ("EBV-Tumor") or without ("EBV") macroscopic tumors. At 6 weeks post-infection, the relative CD8+ T cell frequencies increased significantly and constantly (control Vs. EBV p=0.0021, control Vs. EBV-Tumor p=<0.0001, EBV Vs. EBV-Tumor p=0.0072). For absolute cell counts in tissues, CD8+ T cell increases were more dramatic in mice infected with EBV and developing tumors. These differences amounted to approximately tenfold relative to controls and 3-fold relative to mice not developing tumors. Mice infected with EBV showed 90-100% of the CD4+ and CD8+ T cells in lymphatic tissues expressing PD-1. Mice with EBV-tumors showed twice as many PD-1+ CD4+ and three times as many PD-1+ CD8+ T cells as infected mice without tumors. Histopathology combined with EBER in situ hybridization, showed foci of EBV infected cells in close association with PD-1+ infiltrating lymphocytes, often in perivascular regions. This model was then used to evaluate dose-dependent effects of pembrolizumab. The check-point inhibitor controlled EBV-fLUC spread for 2 weeks, but later prompted increased levels of infections. At endpoint analyses, mice receiving pembrolizumab showed larger dissemination of tumors. CONCLUSIONS: We are currently performing additional experiments in order to elucidate this mechanism of EBV rebound. This humanized mouse model contributes to risk assessment prior to clinical trials of the use of checkpoint inhibitors in patients after transplantations at high risk of EBV infections. Disclosures Ganser: Novartis: Membership on an entity's Board of Directors or advisory committees.

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 An Epstein-Barr Virus (EBV) Mutant with Enhanced BZLF1 Expression Causes Lymphomas with Abortive Lytic EBV Infection in a Humanized Mouse Model

2012 ◽  
Vol 86 (15) ◽  
pp. 7976-7987 ◽  
Author(s):  
S.-D. Ma ◽  
X. Yu ◽  
J. E. Mertz ◽  
J. E. Gumperz ◽  
E. Reinheim ◽  
...  
Keyword(s):  
Mouse Model ◽  
Epstein Barr Virus ◽  
Humanized Mouse ◽  
Humanized Mouse Model ◽  
Barr Virus ◽  
Ebv Infection ◽  
Epstein Barr

scholarly journals An Epstein-Barr virus-associated superantigen.

1996 ◽  
Vol 184 (3) ◽  
pp. 971-980 ◽  
Author(s):  
N Sutkowski ◽  
T Palkama ◽  
C Ciurli ◽  
R P Sekaly ◽  
D A Thorley-Lawson ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
Mhc Class Ii ◽  
T Cell Activation ◽  
Epstein Barr Virus ◽  
Cell Activation ◽  
Lytic Cycle ◽  
Barr Virus ◽  
Epstein Barr

More than 90% of adults are latently infected with Epstein-Barr virus (EBV), the causative agent of infectious mononucleosis, a self-limiting lymphoproliferative disease characterized by extensive T cell activation. Reactivation of this herpesvirus during immunosuppression is often associated with oncogenesis. These considerations led us to analyze the early events that occur after exposure of the immune system to EBV. Strong major histocompatibility complex (MHC) class II-dependent but not MHC-restricted, T cell proliferation was observed in vitro in response to autologous, lytically infected EBV-transformed B cells. By measuring the appearance of the early activation marker CD69 on individual T cell V beta subsets, we could demonstrate selective activation of human V beta 13- T cells. This was confirmed with murine T cell hybridomas expressing various human BV genes. While EBV- Burkitt's lymphoma cells were nonstimulatory, they induced V beta-restricted T cell activation after EBV infection. EBV specific activation was also demonstrated in cord blood cells, excluding a recall-antigen response. Thus, all of the characteristics of a superantigen-stimulated response are seen, indicating that induction of the EBV lytic cycle is associated with the expression of a superantigen in B cells. A model is presented proposing a role for the superantigen in infection, latency, and oncogenesis.

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