scholarly journals Selective Gene Expression of Latent Murine Gammaherpesvirus 68 in B Lymphocytes

2003 ◽  
Vol 77 (13) ◽  
pp. 7308-7318 ◽  
Author(s):  
Sofia Marques ◽  
Stacey Efstathiou ◽  
K. G. Smith ◽  
Matthias Haury ◽  
J. Pedro Simas
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Critical Role ◽  
Murine Gammaherpesvirus ◽  
Virus Transcription ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Germinal Center B Cells

ABSTRACT Intranasal infection of mice with murine gammaherpesvirus 68 (MHV-68), a virus genetically related to the human pathogen Kaposi's sarcoma-associated herpesvirus, results in a persistent, latent infection in the spleen and other lymphoid organs. Here, we have determined the frequency of virus infection in splenic dendritic cells, macrophages, and several B-cell subpopulations, and we quantified cell type-dependent virus transcription patterns. The frequencies of virus genome positive cells were maximal at 14 days postinfection in all splenic cell populations analyzed. Marginal zone and germinal center B cells harbored the highest frequency of infection and the former population accounted for approximately half the total number of infected B cells. Analysis of virus transcription during the establishment of latency revealed that virus gene expression in B cells was restricted and dependent on the differentiation stage of the B cell. Notably, transcription of ORF73 was detected in germinal center B cells, a finding in agreement with the predicted latent genome maintenance function of ORF73 in dividing cells. At late times after infection, virus DNA could only be detected in newly formed and germinal center B cells, which suggests that B cells play a critical role in facilitating life-long latency.

scholarly journals B Cell-Intrinsic Expression of Interferon Regulatory Factor 1 Supports Chronic Murine Gammaherpesvirus 68 Infection

2020 ◽  
Vol 94 (13) ◽  
Author(s):  
C. N. Jondle ◽  
K. E. Johnson ◽  
A. A. Uitenbroek ◽  
P. A. Sylvester ◽  
C. Nguyen ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Chronic Infection ◽  
Latent Reservoir ◽  
B Cell Differentiation ◽  
B Cell Lymphomas ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Gammaherpesviruses are ubiquitous pathogens that are associated with cancers, including B cell lymphomas. These viruses are unique in that they infect naive B cells and subsequently drive a robust polyclonal germinal center response in order to amplify the latent reservoir and to establish lifelong infection in memory B cells. The gammaherpesvirus-driven germinal center response in combination with robust infection of germinal center B cells is thought to precipitate lymphomagenesis. Importantly, host and viral factors that selectively affect the gammaherpesvirus-driven germinal center response remain poorly understood. Global deficiency of antiviral tumor-suppressive interferon regulatory factor 1 (IRF-1) selectively promotes the murine gammaherpesvirus 68 (MHV68)-driven germinal center response and expansion of the viral latent reservoir. To determine the extent to which antiviral effects of IRF-1 are B cell intrinsic, we generated mice with conditional IRF-1 deficiency. Surprisingly, B cell-specific IRF-1 deficiency attenuated the establishment of chronic infection and the germinal center response, indicating that MHV68 may, in a B cell-intrinsic manner, usurp IRF-1 to promote the germinal center response and expansion of the latent reservoir. Further, we found that B cell-specific IRF-1 deficiency led to reduced levels of active tyrosine phosphatase SHP1, which plays a B cell-intrinsic proviral function during MHV68 infection. Finally, results of this study indicate that the antiviral functions of IRF-1 unveiled in MHV68-infected mice with global IRF-1 deficiency are mediated via IRF-1 expression by non-B cell populations. IMPORTANCE Gammaherpesviruses establish lifelong infection in over 95% of all adults and are associated with B cell lymphomas. The virus’s manipulation of the germinal center response and B cell differentiation to establish lifelong infection is thought to also precipitate malignant transformation, through a mechanism that remains poorly understood. The host transcription factor IRF-1, a well-established tumor suppressor, selectively attenuates MHV68-driven germinal center response, a phenotype that we originally hypothesized to occur in a B cell-intrinsic manner. In contrast, in testing, B cell-intrinsic IRF-1 expression promoted the MHV68-driven germinal center response and the establishment of chronic infection. Our report highlights the underappreciated multifaceted role of IRF-1 in MHV68 infection and pathogenesis.

DNA Methyltransferase 1 Contributes to Epigenetic Signatures and Biological Phenotype during Normal B-Cell Differentiation and Lymphomagenesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 685-685 ◽  
Author(s):  
Rita Shaknovich ◽  
Leandro Cerchietti ◽  
Maria E. Figueroa ◽  
Ari Melnick
Keyword(s):  
Gene Expression ◽  
Dna Methylation ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Dna Methyltransferase ◽  
Critical Role ◽  
Epigenetic Programming ◽  
Differential Digestion ◽  
Epigenetic Signatures

Abstract Normal hematopoiesis requires incremental changes in gene expression in order to establish cellular phenotypes with specialized functions. We are particularly interested in the transcriptional and epigenetic programming of germinal center (GC) B-cells, which acquire unusual biological features normally associated with cancer. Specifically, GC B-cells (i.e. centroblasts - CB) undergo rapid DNA replication while at the same time undergoing genetic recombination, and give rise to a majority of B-cell lymphomas. We hypothesized that epigenetic programming would play a critical role in the CB stage of development, and that gene-specific and genome-wide DNA methyltransferase activity is critical for these cells. We first examined the CpG methylation levels of 24,000 gene promoters in five sets of primary human B-cells just prior to (i.e. naïve B-cells - NBC) and upon entering the GC reaction (i.e. CBs). This was achieved using the HELP (HpaII tiny fragment Enrichment by Ligation-mediated PCR) assay, which relies on differential digestion of genomic DNA by the isoschizomer enzymes HpaII and Msp. HELP is a robust and reproducible method that provides accurate and quantitative measurement of DNA methylation levels throughout the genome. Remarkably, we found that the DNA methylation profile of B-cells undergoes a significant shift as readily appreciated by hierarchical clustering. The epigenetic signatures of NBC and CB are differentiation-stage dependent and do not vary significantly between individuals. The coefficient of correlation between individuals was 0.98, as compared to the NBC vs. CB fractions 0.92–0.95. Supervised analysis demonstrated that 266 genes (P<0.001) were differentially methylated upon entry of NB-cells into the GC reaction. We further correlated the methylation status of these genes with their gene expression level. The most heavily affected pathways by differential methylation and concordant expression in naïve B-cells were the Jak/STAT and MAP3K signaling pathways, while in CBs the p38 MAPK pathway and Ikaros family of genes were most affected. Given the epigenetic reprogramming observed in CBs vs. NBCs, along with the need for maintenance of methylation during rapid replication, we predicted that DNA methyltransferase (DNMT) enzymes play a critical role in centroblasts. By performing QPCR and Western blots on isolated fractions of human tonsilar lymphocytes and anatomical localization by immunohistochemistry, we found that DNMTs have a complex temporal and combinatorial expression pattern whereby DNMT1 was the main methyltransferase detectable in centroblasts. Additionally we studied 10 DLBCL cell lines and a panel of primary DLBCL (n=176 for mRNA and 70 for protein) for DNMTs expression. Spearman Rank correlation analysis revealed that DNMT1 was preferentially highly expressed in GCB vs. ABC primary DLBCLs, as well as in BCR vs. OxPhos DLBCLs. Taken together, our data suggest that i) dynamic changes in epigenetic programming contribute to formation of GCs, ii) that DNMT1 may play both a de novo and maintenance methylation role in GC cells, iii) that DNMT1 is markedly upregulated in normal centroblasts and in DLBCLs with the BCR or GCB gene expression profiles and iv) specific therapeutic targeting of DNMT1 rather than non-specific global inhibition of DNA methylation could be a useful anti-lymphoma strategy for germinal center-derived DLBCLs.

scholarly journals Brg1 Supports B Cell Proliferation and Germinal Center Formation Through Enhancer Activation

2021 ◽  
Vol 12 ◽  
Author(s):  
Dominik Schmiedel ◽  
Hadas Hezroni ◽  
Amit Hamburg ◽  
Ziv Shulman
Keyword(s):  
Gene Expression ◽  
Cell Proliferation ◽  
B Cells ◽  
B Cell ◽  
Transcriptional Activation ◽  
Germinal Center ◽  
Cell Activation ◽  
Critical Role ◽  
B Cell Activation ◽  
Germinal Center Formation

Activation and differentiation of B cells depend on extensive rewiring of gene expression networks through changes in chromatin structure and accessibility. The chromatin remodeling complex BAF with its catalytic subunit Brg1 was previously identified as an essential regulator of early B cell development, however, how Brg1 orchestrates gene expression during mature B cell activation is less clear. Here, we find that Brg1 is required for B cell proliferation and germinal center formation through selective interactions with enhancers. Brg1 recruitment to enhancers following B cell activation was associated with increased chromatin accessibility and transcriptional activation of their coupled promoters, thereby regulating the expression of cell cycle-associated genes. Accordingly, Brg1-deficient B cells were unable to mount germinal center reactions and support the formation of class-switched plasma cells. Our findings show that changes in B cell transcriptomes that support B cell proliferation and GC formation depend on enhancer activation by Brg1. Thus, the BAF complex plays a critical role during the onset of the humoral immune response.

scholarly journals Establishment and Maintenance of Long-Term Murine Gammaherpesvirus 68 Latency in B Cells in the Absence of CD40

2005 ◽  
Vol 79 (5) ◽  
pp. 2891-2899 ◽  
Author(s):  
David O. Willer ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
B Cell ◽  
Virus Replication ◽  
Receptor Expression ◽  
Cell Phenotype ◽  
Memory B Cell ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (γHV68), like Epstein-Barr virus (EBV), establishes a chronic infection in its host by gaining access to the memory B-cell reservoir, where it persists undetected by the host's immune system. EBV encodes a membrane protein, LMP1, that appears to function as a constitutively active CD40 receptor, and is hypothesized to play a central role in EBV-driven differentiation of infected naive B cells to a memory B-cell phenotype. However, it has recently been shown that there is a critical role for CD40-CD40L interaction in B-cell immortalization by EBV (K.-I. Imadome, M. Shirakata, N. Shimizu, S. Nonoyama, and Y. Yamanashi, Proc. Natl. Acad. Sci. USA 100:7836-7840, 2003), indicating that LMP1 does not adequately recapitulate all of the necessary functions of CD40. The role of CD40 receptor expression on B cells for the establishment and maintenance of γHV68 latency is unclear. Data previously obtained with a competition model, demonstrated that in the face of CD40-sufficient B cells, γHV68 latency in CD40-deficient B cells waned over time in chimeric mice (I.-J. Kim, E. Flano, D. L. Woodland, F. E. Lund, T. D. Randall, and M. A. Blackman, J. Immunol. 171:886-892, 2003). To further investigate the role of CD40 in γHV68 latency in vivo, we have characterized the infection of CD40 knockout (CD40−/−) mice. Here we report that, consistent with previous observations, γHV68 efficiently established a latent infection in B cells of CD40−/− mice. Notably, unlike the infection of normal C57BL/6 mice, significant ex vivo reactivation from splenocytes harvested from infected CD40−/− mice 42 days postinfection was observed. In addition, in contrast to γHV68 infection of C57BL/6 mice, the frequency of infected naive B cells remained fairly stable over a 3-month period postinfection. Furthermore, a slightly higher frequency of γHV68 infection was observed in immunoglobulin D (IgD)-negative B cells, which was stably maintained over a period of 3 months postinfection. The presence of virus in IgD-negative B cells indicates that γHV68 may either directly infect memory B cells present in CD40−/− mice or be capable of driving differentiation of naive CD40−/− B cells. A possible explanation for the apparent discrepancy between the failure of γHV68 latency to be maintained in CD40-deficient B cells in the presence of CD40-sufficient B cells and the stable maintenance of γHV68 B-cell latency in CD40−/− mice came from examining virus replication in the lungs of infected CD40−/− mice, where we observed significantly higher levels of virus replication at late times postinfection compared to those in infected C57BL/6 mice. Taken together, these findings are consistent with a model in which chronic virus infection of CD40−/− mice is maintained through virus reactivation in the lungs and reseeding of latency reservoirs.

scholarly journals Role for MyD88 Signaling in Murine Gammaherpesvirus 68 Latency

2008 ◽  
Vol 82 (8) ◽  
pp. 3853-3863 ◽  
Author(s):  
Lisa M. Gargano ◽  
Janice M. Moser ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Response ◽  
Wild Type ◽  
Tlr Signaling ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
B Cell Response

ABSTRACT Toll-like receptors (TLRs) are known predominantly for their role in activating the innate immune response. Recently, TLR signaling via MyD88 has been reported to play an important function in development of a B-cell response. Since B cells are a major latency reservoir for murine gammaherpesvirus 68 (MHV68), we investigated the role of TLR signaling in the establishment and maintenance of MHV68 latency in vivo. Mice deficient in MyD88 (MyD88−/−) or TLR3 (TLR3−/−) were infected with MHV68. Analysis of splenocytes recovered at day 16 postinfection from MyD88−/− mice compared to those from wild-type control mice revealed a lower frequency of (i) activated B cells, (ii) germinal-center B cells, and (iii) class-switched B cells. Accompanying this substantial defect in the B-cell response was an approximately 10-fold decrease in the establishment of splenic latency. In contrast, no defect in viral latency was observed in TLR3−/− mice. Analysis of MHV68-specific antibody responses also demonstrated a substantial decrease in the kinetics of the response in MyD88−/− mice. Analysis of wild-type × MyD88−/− mixed-bone-marrow chimeric mice demonstrated that there is a selective failure of MyD88−/− B cells to participate in germinal-center reactions as well as to become activated and undergo class switching. In addition, while MHV68 established latency efficiently in the MyD88-sufficient B cells, there was again a ca. 10-fold reduction in the frequency of MyD88−/− B cells harboring latent MHV68. This phenotype indicates that MyD88 is important for the establishment of MHV68 latency and is directly related to the role of MyD88 in the generation of a B-cell response. Furthermore, the generation of a B-cell response to MHV68 was intrinsic to B cells and was independent of the interleukin-1 receptor, a cytokine receptor that also signals through MyD88. These data provide evidence for a unique role for MyD88 in the establishment of MHV68 latency.

scholarly journals Ablation of STAT3 in the B Cell Compartment Restricts Gammaherpesvirus LatencyIn Vivo

mBio ◽  
2016 ◽  
Vol 7 (4) ◽  
Author(s):  
Sandeep Steven Reddy ◽  
Hui-Chen Chang Foreman ◽  
Thubten Ozula Sioux ◽  
Gee Ho Park ◽  
Valeria Poli ◽  
...  
Keyword(s):  
B Cells ◽  
B Cell ◽  
Cell Function ◽  
Acute Infection ◽  
Therapeutic Benefit ◽  
Therapeutic Interventions ◽  
Murine Gammaherpesvirus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACTA challenging property of gammaherpesviruses is their ability to establish lifelong persistence. The establishment of latency in B cells is thought to involve active virus engagement of host signaling pathways. Pathogenic effects of these viruses during latency or following reactivation can be devastating to the host. Many cancers, including those associated with members of the gammaherpesvirus family, Kaposi’s sarcoma-associated herpesvirus and Epstein-Barr virus, express elevated levels of active host signal transducer and activator of transcription-3 (STAT3). STAT3 is activated by tyrosine phosphorylation in response to many cytokines and can orchestrate effector responses that include proliferation, inflammation, metastasis, and developmental programming. However, the contribution of STAT3 to gammaherpesvirus pathogenesis remains to be completely understood. This is the first study to have identified STAT3 as a critical host determinant of the ability of gammaherpesvirus to establish long-term latency in an animal model of disease. Following an acute infection, murine gammaherpesvirus 68 (MHV68) established latency in resident B cells, but establishment of latency was dramatically reduced in animals with a B cell-specific STAT3 deletion. The lack of STAT3 in B cells did not impair germinal center responses for immunoglobulin (Ig) class switching in the spleen and did not reduce either total or virus-specific IgG titers. Although ablation of STAT3 in B cells did not have a global effect on these assays of B cell function, it had long-term consequences for the viral load of the host, since virus latency was reduced at 6 to 8 weeks postinfection. Our findings establish host STAT3 as a mediator of gammaherpesvirus persistence.IMPORTANCEThe insidious ability of gammaherpesviruses to establish latent infections can have detrimental consequences for the host. Identification of host factors that promote viral latency is essential for understanding latency mechanisms and for therapeutic interventions. We provide the first evidence that STAT3 expression is needed for murine gammaherpesvirus 68 to establish latency in primary B cells during an active immune response to infection. STAT3 deletion in B cells does not impair adaptive immune control of the virus, but loss of STAT3 in B cells has a long-lasting impact on viral persistence. These results indicate a potential therapeutic benefit of STAT3 inhibitors for combating gammaherpesvirus latency and, thereby, associated pathologies.

UCH-L1 Cooperates with BCL6 and Identifies Patients with Aggressive Germinal Center Diffuse Large B-Cell Lymphoma

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2673-2673
Author(s):  
Tibor Bedekovics ◽  
Sajjad Hussain ◽  
Andrew L Feldman ◽  
Paul J. Galardy
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
B Cell ◽  
Germinal Center ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Substantial Impact ◽  
Large B Cell Lymphoma ◽  
Large B Cell ◽  
Germinal Center B Cells

Abstract Gene expression profiling has identified two major subclasses of diffuse large B-cell lymphoma. Cases resembling germinal center B-cells (GCB-DLBCL) generally occur in younger patients, have a distinct molecular pathophysiology, and have improved outcomes compared with those similar to activated post-germinal center cells (ABC-DLBCL). We previously found that the ubiquitin hydrolase UCH-L1 is frequently overexpressed in mature B-cell malignancies and is a potent oncogene in mice. The cause for its overexpression in lymphoma, and whether it impacts the outcome of patients with DLBCL is unknown. Here we show that UCH-L1 reflects germinal center lineage in lymphoma and is an oncogenic biomarker of aggressive GCB-DLBCL. We find that UCH-L1 is specifically induced in germinal center B-cells in mice and humans, and that its expression correlates highly with the GCB subtype in DLBCL. Despite the typically good outcomes of GCB-DLBCL, increased UCHL1 identifies a subgroup with early relapses independent of MYC expression, suggesting biologic diversity in this subset of disease. We also find that UCH-L1 synergizes with BCL6 in a mouse model of germinal center B-cell lymphoma, but not with the development of multiple myeloma derived from post-germinal center cells. Consistent with this, forced Uchl1 overexpression had a substantial impact on gene expression in germinal center B-cells including pathways of cell cycle progression, cell death and proliferation, and DNA replication. These data demonstrate a novel role for UCH-L1 outside of the nervous system and suggest its potential use as a biomarker and therapeutic target in DLBCL. Disclosures Galardy: Mission Therapeutics: Research Funding.

scholarly journals Lung Epithelial Cells Are a Major Site of Murine Gammaherpesvirus Persistence

1998 ◽  
Vol 187 (12) ◽  
pp. 1941-1951 ◽  
Author(s):  
James P. Stewart ◽  
Edward J. Usherwood ◽  
Alan Ross ◽  
Heather Dyson ◽  
Tony Nash
Keyword(s):  
B Cells ◽  
Epithelial Cells ◽  
B Cell ◽  
Virus Genome ◽  
Lung Epithelial Cells ◽  
Murine Gammaherpesvirus ◽  
Latently Infected ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68 ◽  
Deficient Mice

It is currently believed that latently infected, resting B lymphocytes are central to gammaherpesvirus persistence, whereas mucosal epithelial cells are considered nonessential. We have readdressed the question of nonlymphoid persistence using murine gammaherpesvirus 68 (MHV-68). To dissect lymphoid from nonlymphoid persistence, we used μMT transgenic mice that are defective in B cells. MHV-68 DNA persisted in the lungs of intact and B cell–deficient mice. Both episomal and linear forms of the virus genome were present in lungs, implying the presence of both latency and productive replication. In situ hybridization for virus tRNA transcripts revealed latent MHV-68 in pulmonary epithelial cells. Infectious virus was recovered from the lungs of μMT mice after T cell depletion, showing that the persisting virus DNA was reactivatable. Finally, using adoptive transfer of B cells into B cell–deficient mice, it was shown that virus persisting in lungs seeded splenic B cells, and virus resident in the spleen seeded the lungs. These results show that mucosal epithelia can act as a nonlymphoid reservoir for gammaherpesvirus persistence, and that there is a two-way movement of virus between lymphoid and nonlymphoid compartments during persistence.

scholarly journals Systematic Mutagenesis of the Murine Gammaherpesvirus 68 M2 Protein Identifies Domains Important for Chronic Infection

2008 ◽  
Vol 82 (7) ◽  
pp. 3295-3310 ◽  
Author(s):  
Jeremy H. Herskowitz ◽  
Andrea M. Siegel ◽  
Meagan A. Jacoby ◽  
Samuel H. Speck
Keyword(s):  
B Cells ◽  
Inbred Mice ◽  
Critical Role ◽  
Small Animal ◽  
M2 Protein ◽  
Experimental Conditions ◽  
Murine Gammaherpesvirus ◽  
Barr Virus ◽  
Gammaherpesvirus 68 ◽  
Murine Gammaherpesvirus 68

ABSTRACT Murine gammaherpesvirus 68 (MHV68) infection of inbred mice represents a genetically tractable small-animal model for assessing the requirements for the establishment of latency, as well as reactivation from latency, within the lymphoid compartment. By day 16 postinfection, MHV68 latency in the spleen is found in B cells, dendritic cells, and macrophages. However, as with Epstein-Barr virus, by 3 months postinfection MHV68 latency is predominantly found in isotype-switched memory B cells. The MHV68 M2 gene product is a latency-associated antigen with no discernible homology to any known cellular or viral proteins. However, depending on experimental conditions, the M2 protein has been shown to play a critical role in both the efficient establishment of latency in splenic B cells and reactivation from latently infected splenic B cells. Inspection of the sequence of the M2 protein reveals several hallmarks of a signaling molecule, including multiple PXXP motifs and two potential tyrosine phosphorylation sites. Here, we report the generation of a panel of recombinant MHV68 viruses harboring mutations in the M2 gene that disrupt putative functional motifs. Subsequent analyses of the panel of M2 mutant viruses revealed a functionally important cluster of PXXP motifs in the C-terminal region of M2, which have previously been implicated in binding Vav proteins (P. A. Madureira, P. Matos, I. Soeiro, L. K. Dixon, J. P. Simas, and E. W. Lam, J. Biol. Chem. 280:37310-37318, 2005; L. Rodrigues, M. Pires de Miranda, M. J. Caloca, X. R. Bustelo, and J. P. Simas, J. Virol. 80:6123-6135, 2006). Further characterization of two adjacent PXXP motifs in the C terminus of the M2 protein revealed differences in the functions of these domains in M2-driven expansion of primary murine B cells in culture. Finally, we show that tyrosine residues 120 and 129 play a critical role in both the establishment of splenic latency and reactivation from latency upon explant of splenocytes into tissue culture. Taken together, these analyses will aide future studies for identifying M2 interacting partners and B-cell signaling pathways that are manipulated by the M2 protein.

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