Differential Effect of IL-4 and IL-13 on CD44 Expression in the Burkitt's Lymphoma B Cell Line BL30/B95-8 and in Epstein–Barr Virus (EBV) Transformed Human B Cells: Loss of IL-13 Receptors on Burkitt's Lymphoma B Cells

When human peripheral blood lymphocytes (PBLs) from Epstein-Barr virus (EBV)-seropositive donors are injected intraperitoneally into SCID mice, EBV+ B cell tumors develop within weeks. A preliminary report (Mosier, D. E., R. J. Gulizia, S. M. Baird, D. D. Richman, D. B. Wilson, R. I. Fox, and T. J. Kipps, 1989. Blood. 74(Suppl. 1):52a) has suggested that such tumors resemble the EBV-positive malignancy, Burkitt's lymphoma. The present work shows that generally the human (hu) PBL-SCID tumors are distinct from Burkitt's lymphoma and instead resemble lymphoblastoid cell lines (LCLs) generated by EBV-infection of normal B cells in vitro in terms of: (a) their cell surface phenotype, with expression of B cell activation antigens and adhesion molecules, (b) normal karyotype, and (c) viral phenotype, with expression of all the transformation-associated EBV latent proteins and, in a minority of cells, productive cycle antigens. Indeed, in vitro-transformed LCLs also grow when inoculated into SCID mice, the frequency of tumor outgrowth correlating with the in vitro growth phenotype of the LCL which is itself determined by the identity of the transforming virus (i.e., type 1 or type 2 EBV). Histologically the PBL-derived hu-SCID tumors resemble the EBV+ large cell lymphomas that develop in immuno-suppressed patients and, like the human tumors, often present at multiple sites as individual monoclonal or oligoclonal foci. The remarkable efficiency of tumor development in the hu-SCID model suggests that lymphomagenesis involves direct outgrowth of EBV-transformed B cells without requirement for secondary genetic changes, and that selection on the basis of cell growth rate alone is sufficient to explain the monoclonal/oligoclonal nature of tumor foci. EBV+ large cell lymphoma of the immunosuppressed may arise in a similar way.

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Regression of experimental Burkitt's lymphoma induced by Epstein-Barr virus-immortalized human B cells

Blood ◽

10.1182/blood.v83.3.776.776 ◽

1994 ◽

Vol 83 (3) ◽

pp. 776-784 ◽

Cited By ~ 26

Author(s):

G Tosato ◽

C Sgadari ◽

K Taga ◽

KD Jones ◽

SE Pike ◽

...

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Athymic Mice ◽

Lymphoma Cells ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr ◽

Burkitt’S Lymphoma Cells

Abstract Epstein-Barr virus (EBV)-immortalized human B cells survive only transiently when injected subcutaneously into athymic mice, whereas Burkitt's lymphoma cells give rise to progressively growing subcutaneous tumors. In this study, we tested whether these Burkitt's tumors could be induced to regress via a bystander effect induced by EBV-immortalized B cells. Simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in the same subcutaneous site resulted in tumors that regressed with necrosis and scarring. Similarly, simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in separate subcutaneous sites resulted in regression of a proportion of the Burkitt's tumors. Furthermore, most of the established human Burkitt's tumors regressed with necrosis and scarring after intratumor inoculations with EBV-immortalized B cells. The EBV-immortalized B cells continued to exert this antitumor effect even when killed with irradiation. The experimental approach to Burkitt's lymphoma treatment described here exploits the ability of athymic mice to reject EBV-immortalized B cells to target an effective antitumor response to malignant cells normally incapable of eliciting it.

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Regression of experimental Burkitt's lymphoma induced by Epstein-Barr virus-immortalized human B cells

Blood ◽

10.1182/blood.v83.3.776.bloodjournal833776 ◽

1994 ◽

Vol 83 (3) ◽

pp. 776-784 ◽

Cited By ~ 2

Author(s):

G Tosato ◽

C Sgadari ◽

K Taga ◽

KD Jones ◽

SE Pike ◽

...

Keyword(s):

B Cells ◽

Epstein Barr Virus ◽

Burkitt’S Lymphoma ◽

Burkitt's Lymphoma ◽

Athymic Mice ◽

Lymphoma Cells ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr ◽

Burkitt’S Lymphoma Cells

Epstein-Barr virus (EBV)-immortalized human B cells survive only transiently when injected subcutaneously into athymic mice, whereas Burkitt's lymphoma cells give rise to progressively growing subcutaneous tumors. In this study, we tested whether these Burkitt's tumors could be induced to regress via a bystander effect induced by EBV-immortalized B cells. Simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in the same subcutaneous site resulted in tumors that regressed with necrosis and scarring. Similarly, simultaneous inoculation of EBV-immortalized B cells and Burkitt's lymphoma cells in separate subcutaneous sites resulted in regression of a proportion of the Burkitt's tumors. Furthermore, most of the established human Burkitt's tumors regressed with necrosis and scarring after intratumor inoculations with EBV-immortalized B cells. The EBV-immortalized B cells continued to exert this antitumor effect even when killed with irradiation. The experimental approach to Burkitt's lymphoma treatment described here exploits the ability of athymic mice to reject EBV-immortalized B cells to target an effective antitumor response to malignant cells normally incapable of eliciting it.

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Rewiring of B cell receptor signaling by Epstein–Barr virus LMP2A

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.2007946117 ◽

2020 ◽

Vol 117 (42) ◽

pp. 26318-26327

Author(s):

Kamonwan Fish ◽

Federico Comoglio ◽

Arthur L. Shaffer ◽

Yanlong Ji ◽

Kuan-Ting Pan ◽

...

Keyword(s):

Cell Cycle ◽

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Cell Receptor ◽

B Cell Receptor ◽

Barr Virus ◽

Human B Cells ◽

Bcr Signaling ◽

Epstein Barr

Epstein–Barr virus (EBV) infects human B cells and reprograms them to allow virus replication and persistence. One key viral factor in this process is latent membrane protein 2A (LMP2A), which has been described as a B cell receptor (BCR) mimic promoting malignant transformation. However, how LMP2A signaling contributes to tumorigenesis remains elusive. By comparing LMP2A and BCR signaling in primary human B cells using phosphoproteomics and transcriptome profiling, we identified molecular mechanisms through which LMP2A affects B cell biology. Consistent with the literature, we found that LMP2A mimics a subset of BCR signaling events, including tyrosine phosphorylation of the kinase SYK, the calcium initiation complex consisting of BLNK, BTK, and PLCγ2, and its downstream transcription factor NFAT. However, the majority of LMP2A-induced signaling events markedly differed from those induced by BCR stimulation. These included differential phosphorylation of kinases, phosphatases, adaptor proteins, transcription factors such as nuclear factor κB (NF-κB) and TCF3, as well as widespread changes in the transcriptional output of LMP2A-expressing B cells. LMP2A affected apoptosis and cell-cycle checkpoints by dysregulating the expression of apoptosis regulators such as BCl-xL and the tumor suppressor retinoblastoma-associated protein 1 (RB1). LMP2A cooperated with MYC and mutant cyclin D3, two oncogenic drivers of Burkitt lymphoma, to promote proliferation and survival of primary human B cells by counteracting MYC-induced apoptosis and by inhibiting RB1 function, thereby promoting cell-cycle progression. Our results indicate that LMP2A is not a pure BCR mimic but rather rewires intracellular signaling in EBV-infected B cells that optimizes cell survival and proliferation, setting the stage for oncogenic transformation.

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Low expression of lymphocyte function-associated antigen (LFA)-1 and LFA-3 adhesion molecules is a common trait in Burkitt's lymphoma associated with and not associated with Epstein-Barr virus

Blood ◽

10.1182/blood.v75.9.1827.1827 ◽

1990 ◽

Vol 75 (9) ◽

pp. 1827-1833 ◽

Cited By ~ 38

Author(s):

M Billaud ◽

F Rousset ◽

A Calender ◽

M Cordier ◽

JP Aubry ◽

...

Keyword(s):

Cell Surface ◽

B Cell ◽

Adhesion Molecules ◽

Epstein Barr Virus ◽

Burkitt’S Lymphoma ◽

Receptor Activation ◽

Burkitt's Lymphoma ◽

Lymphocyte Function ◽

Barr Virus ◽

Epstein Barr

Abstract Lymphocyte function-associated antigens 1 and 3 (LFA-1, LFA-3) and intercellular adhesion molecule 1 (ICAM-1) are cell surface adhesion molecules necessary for immune processes requiring intercellular contact. It was recently proposed that malignant Burkitt's lymphoma cells (BL) may escape from immunosurveillance through the downregulation of LFA-1 (CD11a/CD18) or LFA-3 (CD58) and ICAM-1 (CD54) molecules. Expression of these three adhesion antigens was investigated in 19 BL lines. LFA-1 or LFA-3 expression was found to be absent or low in 8 of 11 Epstein-Barr virus (EBV) genome positive BL, but strongly expressed on all nonmalignant EBV genome positive lymphoblastoid cell lines (LCL). Negative or weak expression of LFA-1 and LFA-3 was also observed in 7 of 8 EBV genome negative BL. ICAM-1 was found to be expressed on the cell surface of the majority of BL lines. BL lines growing as individual cells did not express LFA-1, whereas clump- forming BL lines expressed this marker involved in B-cell homotypic aggregation. Expression of LFA-1 and LFA-3 was induced on in vitro infection of EBV-negative BL cells with the immortalizing EBV strain B95–8, and weakly with the nonimmortalizing EBV strain P3HR1. EBNA2 and LMP, two EBV encoded proteins expressed in LCL and in BL infected with B95–8 (BL/B95–8), are not expressed in P3HR1 infected BL cells (BL/P3HR1). Stable expression of EBNA2 after gene transfer in a BL/P3HR1 cell line did not restore the level of LFA-1 and LFA-3 found on BL/B95–8 cells. In EBV-positive BL cells expressing LFA-1 and LFA-3, LMP was found coexpressed, supporting the recent finding of the role of LMP in B-cell adhesion receptor activation. Consequently, diminished LFA-1 and LFA-3 expression appears to be a common characteristic of numerous EBV-positive BL as well as EBV-negative BL. These findings are discussed in the framework of BL pathogenesis.

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Compatibility of the gH homologues of Epstein–Barr virus and related lymphocryptoviruses

Journal of General Virology ◽

10.1099/vir.0.82949-0 ◽

2007 ◽

Vol 88 (8) ◽

pp. 2129-2136 ◽

Cited By ~ 7

Author(s):

Liguo Wu ◽

Lindsey M. Hutt-Fletcher

Keyword(s):

B Cells ◽

Epithelial Cells ◽

B Cell ◽

Cell Fusion ◽

Epstein Barr Virus ◽

Common Marmoset ◽

Barr Virus ◽

Human B Cells ◽

The Common ◽

Epstein Barr

Glycoprotein gH, together with its chaperone gL and a third glycoprotein gB, is essential for cell–cell fusion and virus–cell fusion mediated by herpesviruses. Epstein–Barr virus (EBV), the prototype human lymphocryptovirus, requires a fourth glycoprotein gp42 to support fusion with B cells in addition to epithelial cells. Two other lymphocryptoviruses, the rhesus lymphocryptovirus (Rh-LCV) and the common marmoset lymphocryptovirus (CalHV3), have been sequenced in their entirety and each has a gp42 homologue. Combinations of proteins from EBV, Rh-LCV and CalHV3 were able to mediate fusion of epithelial cells, but, even when complexed with EBV gp42, only Rh-LCV and not CalHV3 proteins were able to mediate fusion with human B cells. CalHV3 gL was also unable to function effectively as a chaperone for EBV or Rh-LCV gH. The Rh-LCV gH homologue supported more fusion than EBV gH with an epithelial cell and supported the highest levels of fusion with a B cell. Chimeric constructs made from Rh-LCV gH and EBV gH that have 85.4 % sequence identity should prove useful for mapping the regions of gH that are of importance to fusion as a whole and to B-cell fusion in particular.

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

PLoS Pathogens ◽

10.1371/journal.ppat.1009117 ◽

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.

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Frequencies of the separate human B cell subsets activatable to Ig secretion by Epstein-Barr virus and pokeweed mitogen.

Journal of Experimental Medicine ◽

10.1084/jem.157.6.1808 ◽

1983 ◽

Vol 157 (6) ◽

pp. 1808-1814 ◽

Cited By ~ 42

Author(s):

O Martínez-Maza ◽

S Britton

Keyword(s):

T Cells ◽

B Cells ◽

B Cell ◽

Epstein Barr Virus ◽

Human Peripheral Blood ◽

Pokeweed Mitogen ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr ◽

B Cell Subsets

We have developed a microculture system suitable for limiting dilution analysis of Epstein-Barr virus (EBV)- and pokeweed mitogen (PWM)-induced activation of immunoglobulin secretion by human B cells. It was found that exogenous filler cells were not required to obtain optimal EBV-induced B cell precursor frequency (PF) estimates, although filler T cells were required for optimal PWM activation. In fact, when autologous T cells were used as filler cells, a marked decrease in the EBV-induced IgM PF was noted. Treatment of the T cells with cyclosporin A partially eliminated, and irradiation of the T cells completely eliminated, this decrease. The calculated PF of B cells activated by EBV was from 1/290 to 1/3,700 for IgM, and from 1/920 to 1/3,250 for IgG secretion. PWM activated from 1/140 to 1/3,200 B cells to IgM secretion. The results of experiments in which EBV and PWM were mixed, indicated that these two polyclonal activators operated on different B cell subpopulations. Therefore, both these agents seem to activate small, discrete subpopulations of human peripheral blood B cells to Ig secretion.

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Limiting dilution analysis of Epstein-Barr virus-induced immunoglobulin production by human B cells.

Journal of Experimental Medicine ◽

10.1084/jem.157.1.1 ◽

1983 ◽

Vol 157 (1) ◽

pp. 1-14 ◽

Cited By ~ 79

Author(s):

R Yarchoan ◽

G Tosato ◽

R M Blaese ◽

R M Simon ◽

D L Nelson

Keyword(s):

B Cells ◽

B Cell ◽

Peripheral Blood ◽

Epstein Barr Virus ◽

Cell Activation ◽

B Cell Activation ◽

Barr Virus ◽

Human B Cells ◽

Epstein Barr ◽

Limiting Dilution

The Epstein-Barr virus (EBV) is a herpes virus that has the capacity to infect human B cells and to induce them to secrete immunoglobulin (Ig). In the current experiments, Poisson analysis of limiting dilution cultures has been used to study the activation of human peripheral B cells by the B95-8 strain of EBV. Under the culture conditions used, 0.2-1% of peripheral blood B cells were activated by EBV to secrete IgM or IgG. In addition, when multiple replicate cultures containing limited numbers of B cells were tested for IgM and for IgG production, the precursors for IgM and IgG segregated independently; thus, individual B cell precursors matured into cells secreting IgM or IgG but not both classes of Ig. Additional experiments using limiting dilutions of EBV were undertaken to study the viral requirements for B cell activation. These studies indicated that B cell activation by EBV to produce Ig was consistent with a "one-hit" model and inconsistent with a "two-hit" model. Taken together, these results indicate that infection by one EBV virion is sufficient to induce a precursor peripheral blood B cell to secrete Ig and that only one isotype of Ig is then secreted.

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