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The EBV carrying lines MEC1 and MEC2 were established earlier from explants of blood derived cells of a chronic lymphocytic leukemia (CLL) patient at different stages of progression to prolymphocytoid transformation (PLL). This pair of lines is unique in several respects. Their common clonal origin was proven by the rearrangement of the immunoglobulin genes. The cells were driven to proliferation in vitro by the same indigenous EBV strain. They are phenotypically different and represent subsequent subclones emerging in the CLL population. Furthermore they reflect the clinical progression of the disease. We emphasize that the support for the expression of the EBV encoded growth program is an important differentiation marker of the CLL cells of origin that was shared by the two subclones. It can be surmised that proliferation of EBV carrying cells in vitro, but not in vivo, reflects the efficient surveillance that functions even in the severe leukemic condition. The MEC1 line arose before the aggressive clinical stage from an EBV carrying cell within the subclone that was in the early prolymphocytic transformation stage while the MEC2 line originated one year later, from the subsequent subclone with overt PLL characteristics. At this time the disease was disseminated and the blood lymphocyte count was considerably elevated. The EBV induced proliferation of the MEC cells belonging to the subclones with markers of PLL agrees with earlier reports in which cells of PLL disease were infected in vitro and immortalized to LCL. They prove also that the expression of EBV encoded set of proteins can be determined at the event of infection. This pair of lines is particularly important as they provide in vitro cells that represent the subclonal evolution of the CLL disease. Furthermore, the phenotype of the MEC1 cells shares several characteristics of ex vivo CLL cells.

Altered DNA methylation occurs ubiquitously in human cancer from the earliest measurable stages. A cogent approach to understanding the mechanism and timing of altered DNA methylation is to analyze it in the context of carcinogenesis by a defined agent. Epstein-Barr virus (EBV) is a human oncogenic herpesvirus associated with lymphoma and nasopharyngeal carcinoma, but also used commonly in the laboratory to immortalize human B-cells in culture. Here we have performed whole-genome bisulfite sequencing of normal B-cells, activated B-cells, and EBV-immortalized B-cells from the same three individuals, in order to identify the impact of transformation on the methylome. Surprisingly, large-scale hypomethylated blocks comprising two-thirds of the genome were induced by EBV immortalization but not by B-cell activation per se. These regions largely corresponded to hypomethylated blocks that we have observed in human cancer, and they were associated with gene-expression hypervariability, similar to human cancer, and consistent with a model of epigenomic change promoting tumor cell heterogeneity. We also describe small-scale changes in DNA methylation near CpG islands. These results suggest that methylation disruption is an early and critical step in malignant transformation.

Epstein Barr virus (EBV) is carried by almost all adults, mostly without clinical manifestations. Latent virus infection of B lymphocytes induces activation and proliferation that can be demonstrated in vitro. In healthy individuals, generation of EBV induced malignant proliferation is avoided by continuous immunological surveillance. The proliferation inducing set of the virally encoded genes is expressed exclusively in B cells in a defined differentiation window. It comprises nine EBV encoded nuclear proteins, EBNA 1-6, and three cell membrane associated proteins, LMP-1, 2A and 2B, designated as latency Type III. Outside this window the expression of the viral genes is limited. Healthy carriers harbor a low number of B lymphocytes in which the viral genome is either silent or expresses one virally encoded protein, EBNA-1, latency Type I. In addition, EBV genome carrying B cells can lack either EBNA-2 or LMP-1, latency Type IIa or Type IIb respectively. These cells have no inherent proliferation capacity. Detection of both EBNA-2 and LMP-1 can identify B cells with growth potential. We devised therefore a method for their simultaneous detection in cytospin deposited cell populations. Simultaneous detection of EBNA-2 and LMP-1 was reported earlier in tissues derived from infectious mononucleosis (IM), postransplantation lymphoproliferative disorders (PTLD) and from "humanized" mice infected with EBV. We show for the first time the occurrence of Type IIa and Type IIb cells in cord blood lymphocyte populations infected with EBV in vitro. Further, we confirm the variation of EBNA-2 and LMP-1 expression in several Type III lines and that they vary independently in individual cells. We visualize that in Type III LCL, induced for plasmacytoid differentiation by IL-21 treatment, EBV protein expression changes to Type IIa (EBNA-2 negative LMP-1 positive). We also show that when the proliferation of EBV infected cord blood lymphocyte culture is inhibited by the immunomodulator, PSK, the majority of the cells express latency Type IIa pattern. These results show that by modifying the differentiation state, the proliferating EBV positive B cells can be "curbed". Type IIa expression is a characteristic for EBV positive Reed-Sternberg (R/S) cells in EBV positive Hodgkin's lymphomas. For survival and proliferation, the R/S cells require the contribution of the in vivo microenvironment. Consequently, Type IIa lines could not be established from Hodgkin's lymphoma in vitro. We propose that these experimental cultures can be exploited for study of the Type IIa cells., (Copyright © 2012 Elsevier B.V. All rights reserved.)

We report that type I interferons (IFNs) upregulate latent membrane protein 1 (LMP-1) expression by direct activation of the ED-L1 promoter in several Epstein-Barr virus (EBV)-carrying Burkitt's lymphoma lines. In EBV-infected primary B cells, IFN-α transiently upregulates LMP-1 mRNA, but not protein levels, followed by downregulation of both, suggesting a novel antiproliferative mechanism of type I IFNs. Furthermore, our results may explain the expression of LMP-1 in memory B cells of systemic lupus erythematosus patients.

Type I interferons (IFN) exert multiple effects on both the innate and adaptive immune system in addition to their antiviral and antiproliferative activities. Little is known, however about the direct effects of type I IFNs on germinal center (GC) B cells, the central components of adaptive B cell responses. We used Burkitt's lymphoma (BL) lines, as a model system of normal human GC B cells, to examine the effect of type I IFNs on the expression of BCL-6, the major regulator of the GC reaction. We show that type I IFNs, but not IFNγ, IL-2 and TNFα rapidly down-regulate BCL-6 protein and mRNA expression, in cell lines derived from endemic, but not from sporadic BL. IFNα-induced down-regulation is specific for BCL-6, independent of Epstein-Barr virus and is not accompanied by IRF-4 up-regulation. IFNα-induced BCL-6 mRNA down-regulation does not require de novo protein synthesis and is specifically inhibited by piceatannol. The proteasome inhibitor MG132 non-specifically prevents, while inhibitors of alternate type I IFN signaling pathways do not inhibit IFNα-induced BCL-6 protein downregulation. We validate our results with showing that IFNα rapidly down-regulates BCL-6 mRNA in purified mouse normal GC B cells. Our results identify type I IFNs as the first group of cytokines that can down-regulate BCL-6 expression directly in GC B cells., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Background: Epstein-Barr virus (EBV) encodes six nuclear transformation-associated proteins that induce extensive changes in cellular gene expression and signaling and induce B-cell transformation. The role of HIF1A in EBV-induced B-cell immortalization has not been previously studied., Methods and Findings: Using Western blotting and Q-PCR, we found that HIF1A protein is stabilized in EBV-transformed lymphoblastoid cells. Western blotting, GST pulldown assays, and immunoprecipitation showed that EBV-encoded nuclear antigens EBNA-5 and EBNA-3 bind to prolylhydroxylases 1 and 2, respectively, thus inhibiting HIF1A hydroxylation and degradation. Immunostaining and Q-PCR showed that the stabilized HIF1A translocates to the nucleus, forms a heterodimer with ARNT, and transactivates several genes involved in aerobic glycolysis. Using biochemical assays and Q-PCR, we also found that lymphoblastoid cells produce high levels of lactate, lactate dehydrogenase and pyruvate., Conclusions: Our data suggest that activation of the aerobic glycolytic pathway, corresponding to the Warburg effect, occurs in EBV-transformed lymphoblastoid cells, in contrast to mitogen-activated B-cells.

We report that MDM2, a negative regulator of p53, can bind to EBNA-5. Using GST pull-down assay, immunoprecipitation, surface plasmon resonance and immunostaining of lymphoblastoid cells, we found that trimolecular complexes are formed between EBNA-5, MDM2 and p53, where MDM2 serves as a bridge. The EBNA-5 binding to MDM2 counteracted destabilizing effect of the latter on the p53. In ubiquitination and degradation assays in vitro, EBNA-5 inhibited p53 polyubiquitination (but not monoubiquitination) in a concentration-dependent manner. This resembles the effect of p14ARF on p53. Moreover, EBNA-5 was found to inhibit the degradation of p53 in vitro. High levels of p53 expression were maintained in LCLs. The binding of EBNA-5 to MDM2 also could impair the functional activity of p53. The p53-dependent genes P21 and VDR were not induced in EBV-infected, in contrast to mitogen-activated cells. This may explain the tolerance of established LCLs to high levels of p53 without undergoing apoptosis., (Copyright © 2010 UICC.)

Epstein-Barr virus, EBV, and humans have a common history that reaches back to our primate ancestors. The virus co-evolved with man and has established a largely harmless and highly complex co-existence. It is carried as silent infection by almost all human adults. A serendipitous discovery established that it is the causative agent of infectious mononucleosis. Still, EBV became known first in 1964, in a rare, geographically prevalent malignant lymphoma of B-cell origin, Burkitt lymphoma BL. Its association with a malignancy prompted intensive studies and its capacity to immortalize B-lymphocytes in vitro was soon demonstrated. Consequently EBV was classified therefore as a potentially tumorigenic virus. Despite of this property however, the virus carrier state itself does not lead to malignancies because the transformed cells are recognized by the immune response. Consequently the EBV induced proliferation of EBV carrying B-lymphocytes is manifested only under immunosuppressive conditions. The expression of EBV encoded genes is regulated by the cell phenotype. The virus genome can be found in malignancies originating from cell types other than the B-lymphocyte. Even in the EBV infected B-cell, the direct transforming capacity is restricted to a defined window of differentiation. A complex interaction between virally encoded proteins and B-cell specific cellular proteins constitute the proliferation inducing program. In this short review we touch upon aspects which are the subject of our present work. We describe the mechanisms of some of the functional interactions between EBV encoded and cellular proteins that determine the phenotype of latently infected B-cells. The growth promoting EBV encoded genes are not expressed in the virus carrying BL cells. Still, EBV seems to contribute to the etiology of this tumor by modifying events that influence cell survival and proliferation. We describe a possible growth promoting mechanism in the genesis of Burkitt lymphoma that depends on the presence of EBV., (2010 Elsevier Inc. All rights reserved.)

The Epstein-Barr virus (EBV) is carried by more than 90% of the adult world population and has been implicated in several human malignancies. Its ability to induce unlimited in vitro proliferation of B cells is frequently used to generate lymphoblastoid cell lines (LCLs). In this study, we have investigated the evolution of two LCLs up to 25 weeks after EBV infection. LCLs were karyotyped once a month by spectral karyotyping (SKY). LCLs but not mitogen-activated B cells showed evidence of DNA damage and DNA damage response within the first 2 weeks. After 4 weeks, the former, but not the latter, showed a high level of non-clonal structural aberrations, mainly deletions, fragments, dicentric chromosomes and unbalanced translocations. Genomic instability decreased thereafter over time. Nonrandom aneuploidy 12 weeks after infection showed clonal evolution in culture. After 25 weeks post-infection, most cells exhibited karyotypic stability. Chromosomal aberrations were compatible with telomere dysfunction, although in the absence of telomere shortening. The telomere capping protein TRF2 was partially displaced from telomeres in EBV-infected cells, suggesting an EBV-mediated uncapping problem. In conclusion, this study suggests that DNA damage and telomere dysfunction contribute to EBV-related chromosomal instability in early LCLs.

Epstein-Barr virus (EBV) is associated with a variety of human tumors. Although the EBV-infected normal B cells in vitro and the EBV-carrying B cell lymphomas in immunodeficient patients express the full set of latent proteins (type III latency), the majority of EBV-associated malignancies express the restricted type I (EBNA-1 only) or type II (EBNA-1 and LMPs) viral program. The mechanisms responsible for these different latent viral gene expression patterns are only partially known. IL-21 is a potent B cell activator and plasma cell differentiation-inducer cytokine produced by CD4(+) T cells. We studied its effect on EBV-carrying B cells. In type I Burkitt lymphoma (BL) cell lines and in the conditional lymphoblastoid cell line (LCL) ER/EB2-5, IL-21 potently activated STAT3 and induced the expression of LMP-1, but not EBNA-2. The IL-21-treated type I Jijoye M13 BL line ceased to proliferate, and this was paralleled by the induction of IRF4 and the down-regulation of BCL6 expression. In the type III LCLs and BL lines, IL-21 repressed the C-promoter-derived and LMP-2A mRNAs, whereas it up-regulated the expression of LMP-1 mRNAs. The IL-21-treated type III cells underwent plasma cell differentiation with the induction of Blimp-1, and high levels of Ig and Oct-2. IL-21 might be involved in the EBNA-2-independent expression of LMP-1 in EBV-carrying type II cells. In light of the fact that IL-21 is already in clinical trials for the treatment of multiple malignancies, the in vivo modulation of EBV gene expression by IL-21 might have therapeutic benefits for the EBV-carrying malignancies.

Chromosomal translocations that juxtapose one of the three immunoglobulin loci to the c-myc protooncogene are the hallmark of Burkitt lymphomas (BLs), whether they carry the Epstein Barr Virus (EBV) or not. Ig/myc translocations occur as accidents of normal B lymphocyte differentiation. Unless protected, the translocation carrying cells are apoptosis prone. However, the high B cell stimulatory cytokine level found in the two BL prone conditions, in chronic hyperendemic malaria and HIV infection, may rescue them. X-linked lymphoproliferative disease (XLP) is due to the lack of functional SAP protein, a consequence of mutation or deletion of the SAP gene. We and others have shown that SAP is pro-apoptotic. Here we summarize our finding that 8 of 10 EBV carrying, but none of 9 EBV negative BL lines express SAP. We suggest that the apoptosis prone Ig/myc translocation carrying EBV negative precursors of BL can only grow into lymphomas if they do not express SAP. However, their EBV positive counterparts are permissive for SAP expression, due to the anti-apoptotic function of EBNA-1.

Chemokines and chemokine receptors are likely to play important roles in the pathogenesis of Epstein-Barr virus (EBV) -associated disease. The primary EBV infection occurs in the oropharynx where the virus infects mainly tonsillar B cells. We have previously shown that CXCR4 expression on tonsillar B cells is modulated by EBV. Here, CXCR5 and CCR7 expression, which is important for migration into lymphoid tissue, was followed for 14 days after EBV infection of tonsillar B cells. Early after infection (2 days) there were only minor changes in CXCR5 and CCR7 expression. However, at day 7 the expression of CXCR5, as well as of CCR7, was decreased and by day 14 these molecules were no longer present at the cell surface. Furthermore, EBV infection affects the chemotactic response to CXCL13 and CCL21 (the ligands for CXCR5 and CCR7, respectively) with a reduction of ligand-induced migration at day 2. Using gene expression profiling, we identified an additional set of chemokines and chemokine receptors that were changed upon EBV infection in comparison with non-infected tonsillar B cells. In particular, messenger RNA expression for CCR9 and the complement receptor C5AR1 was increased. Both receptors mediate homing to mucosal tissue. The alterations of the expression of these molecules may lead to retention of EBV-infected tonsillar B cells in the interfollicular region of the tonsil.

Epstein - Barr virus (EBV) is a lymphotropic virus that infects more than 90% of the human population, and targets B cells for infection. Infection of human B cells leads to the malignant transformation and eventual immortalization. In latency III infection six EBV-encoded nuclear antigens (EBNAs) and three latent membrane proteins (LMPs) are expressed in the transformed cells that can grow as a lymphoblastoid cell lines in vitro . These proteins hijack the normal B cell growth pathways by activating the constitutive growth promotion and external survival signals. We have determined a set of the nuclear receptors that are up- (and down-) regulated in the latency III infected cells at the mRNA level. In the present paper we discussed the possible role of these receptors in B cell transformation upon EBV infection based on the literature data.

Background: Infection of human B cells with Epstein - Barr virus (EBV) induces metabolic activation, morphological transformation, cell proliferation and eventual immortalization., Aim: To identify the nuclear receptors, which are the cellular interaction partners of EBNAs, that will help to elucidate the mechanism of B cell transformation., Methods: We have compared the nuclear receptor profile in the naïve and EBV-transformed B-lymphocytes, using TaqMan LDA microfluidic card technology., Results: Out of 48 nuclear receptor, 17 showed differential expression at the mRNA level. The expression of 5 genes was elevated in EBV-transformed cells, whereas 12 genes were downregulated in lymphoblastoid cells (LCLs). 7 genes were studied at the protein level; 2 genes were up regulated (Nr2F2 and RARA) and 4 genes were down regulated (ERB, NUR77, PPARG, and VDR) in LCLs., Conclusion: The nuclear receptor profiling on EBV infected B cells showed alterations of nuclear receptors expression at both mRNA and protein levels compared with non infected peripheral blood cells. Further analysis on a possible role of each nuclear receptor in EBV induced cell transformation should be performed.

Epstein-Barr virus (EBV)-specific cellular memory is not transferred from mother to child. Therefore, EBV-induced B-cell proliferation in in vitro-infected cord blood mononuclear cell cultures is not inhibited. However, by addition of immunomodulators, polysaccharide K (PSK) or truncated thioredoxin (Trx80) that activate monocytes, EBV-specific T-cell response could be generated in such cultures. Presently, we demonstrate that leukotriene B(4) (LTB(4)) is involved in the effect of the immunomodulators. LTB(4) was detected in the medium, and T-cell activation was compromised by addition of leukotriene biosynthesis inhibitors. Moreover, we found that LTB(4) added to infected cultures, which did not receive the immunomodulators, induced functional activation of the T cells. LTB(4) activated the monocytes and acted directly on the T cells. In consequence, addition of LTB(4) inhibited the EBV-induced proliferation of B lymphocytes. Specific cytotoxicity could be generated by restimulation of the T cells. The experiments showed successive stages of T-cell activation in acquisition of their immunologic effector function. This is orchestrated by complex cellular interactions, and autocrine loops mediated by soluble factors-here interferon (IFN)-gamma, interleukin (IL)-15, IL-12, and LTB(4). Importantly, the results indicate that endogenous LTB(4) can induce T-cell activation that inhibits the EBV-induced proliferation of B lymphocytes.

In the in vitro infected B-cells six EBV-encoded nuclear antigens (EBNA-1-6) and three latent membrane proteins (LMP-1, -2A, -2B) are expressed (type III latency). In addition, other restricted forms of latency occur in the EBV-carrying malignancies. In Burkitt lymphoma (BL) only EBNA-1 is expressed (type I), while in Hodgkin lymphoma (HL), T-, and NK-lymphoma, and nasopharyngeal carcinoma EBNA-1 and LMPs are expressed (type II). B-cells with these three expression patterns have been detected in healthy virus carriers. While in type III latency two viral transcriptional activators, EBNA-2 and -5, are responsible for LMP-1 expression, the mechanism that controls the expression of LMP-1 in type II latent cells is not known. In order to study the interaction of EBV- and HL-derived cells, we studied the in vitro EBV-converted subline of the KMH2 cells that express only EBNA-1 and LMP-2A. Interestingly, exposure of the KMH2-EBV cells to CD40-ligand and IL-4 induced LMP-1 expression, in the absence of EBNA-2. In BL cell lines lacking EBNA-2 another cytokine, IL-10, could induce LMP-1 expression. IL-10 induced LMP-1 also in tonsillar B-cells infected with the EBNA-2-deleted virus strain P3HR-1. Our results show that cytokines are responsible for the expression of LMP-1 in type II latent B-cells. These signals are available in the germinal center environment and in the granulation tissue of HLs. Based on these results we propose that LMP-1 expression is induced by extracellular signals and is not a constitutive characteristic of the EBV-carrying type II B-cells. Cytokine mediated induction of LMP-1 may also explain the heterogeneous expression of this viral gene seen in normal and malignant cells.

EBV-positive nasopharyngeal carcinoma and Hodgkin, T, and natural killer (NK) lymphomas express EBNA-1 and the latent membrane proteins (LMP1-2; type II latency). In contrast to type III EBV-transformed lymphoblastoid cell lines, in these cells the LMPs are expressed in the absence of EBNA-2. We have previously reported that exposure to CD40 ligand and IL-4 could induce LMP-1 in an in vitro EBV-infected Hodgkin lymphoma-derived cell line, which expressed only EBNA-1. We show now that both human and EBV-encoded IL-10 can induce LMP-1 in the absence of EBNA-2 in the Daudi, P3HR1, and other BL cell lines. Interestingly, induction of LMP-1 was not accompanied by the downregulation of BCL-6. IL-10 could also induce LMP-1 in the conditional lymphoblastoid cell line ER/EB2-5 where EBNA-2 was downregulated in the absence of estrogen. Moreover, IL-10 could induce the expression of LMP-1 in tonsillar B cells infected with the nontransforming, EBNA-2-deficient EBV strain P3HR1 and enhance LMP-1 expression in 2 EBV-positive NK lymphoma lines. The demonstration that IL-10 can induce the expression of LMP-1 in an EBNA-2-independent manner shows that the major transforming EBV gene LMP-1 can be induced by extracellular signals in lymphoid cells, and IL-10 might contribute to the establishment of type II EBV latency.

The primary Epstein-Barr virus (EBV) infection occurs in the oropharynx, where the virus infects B cells and subsequently establishes latency in the memory B-cell compartment. EBV has previously been shown to induce changes in the cell surface expression of several chemokine receptors in cell lines and the transfection of EBNA2 or LMP1 into a B-cell-lymphoma-derived cell line decreased the expression of CXCR4. We show that in vitro EBV infection reduces the expression of CXCR4 on primary tonsil B cells already 43 hr after infection. Furthermore, EBV infection affects the chemotactic response to stromal cell-derived factor (SDF-1)alpha/CXCL12, the ligand for CXCR4, with a reduction of SDF-1alpha-induced migration. To clarify whether this reduced migration is EBV-specific or a consequence of cell activation, tonsillar B cells were either infected with EBV, activated with anti-CD40 and interleukin-4 (IL-4) or kept in medium. Activation by anti-CD40 and IL-4 decreased the CXCR4 expression but the CD40 + IL-4-stimulated cells showed no reduction of chemotactic efficacy. Our finding suggests that changing the SDF-1alpha response of the EBV-infected B cells may serve the viral strategy by directing the infected cells into the extrafollicular areas, rather than retaining them in the lymphoepithelium.

B cell type chronic lymphocytic leukaemia (B-CLL) cells carry the Epstein-Barr virus (EBV) receptor CD21 and can be infected in vitro with the virus. The infected cells exhibit an unusual EBV program, they express the nuclear proteins but not latent membrane protein 1 (LMP-1). Similar cells were encountered in lymphoid tissues of infectious mononucleosis (IM) patients and in lymphoproliferations of immunosuppressed patients. EBV infected B-CLL cells can be regarded as model for this viral program. In B cells the regulation of LMP-1 is executed mainly by EBV encoded nuclear antigen 2 (EBNA-2), interacting with several cellular proteins and these complexes bind to specific sequences in the LMP-1 promoter. ATF2 and c-Jun were shown to be among the interacting partners of EBNA-2. These molecules can be detected in experimentally infected B lymphocytes. We found c-Jun and/or phosphorylated ATF-2 (p-ATF-2) expression in some B-CLL ex vivo samples. They disappeared or their expression declined promptly in explanted cells, even if they were infected with EBV in vitro. Activation of the infected B-CLL cells by exposure to CD40L was accompanied by p-ATF-2 and c-Jun but not by LMP-1 expression. In one of three clones tested, subsequent treatment with histone deacetylase inhibitors (HDACi), TSA or n-butyrate, could induce LMP-1. Treatment with phorbol-12, 13-dibutyrate (PDB) induced LMP-1 expression in three of four clones. Neither the HDACi nor the PDB treated cells survived.

Epstein-Barr virus (EBV)-specific immunologic memory is not transferred from mother to child. In vitro infection of cord blood cells can therefore readily lead to the outgrowth of transformed B lymphocytes. We found that the immunomodulator polysaccharide K (PSK) or the mitogenic cytokine truncated thioredoxin (Trx80) inhibited the EBV-induced B-cell proliferation. Using signaling lymphocytic activation molecule (SLAM)-associated protein (SAP) induction as a sign for T- and natural killer (NK) cell activation, we could follow it without any need for cell separation because neither macrophages nor B lymphocytes express SAP. The results suggest the following scenario: EBV infected and activated B lymphocytes. Upon interacting with these cells, T cells became posed for responding to cytokines produced by monocytes. Both PSK and Trx80, which is a secreted C-terminally truncated thioredoxin, activated the monocytes, which then produced cytokines in the presence of the primed T cells. PSK induced interleukin-15 (IL-15), while Trx80 induced IL-12 production. Both cytokines activated the T cells for function. Phosphatidylinositol 3-(PI 3)-kinase and reactive oxygen species (ROSs) were involved in the PSK-induced activation of monocytes. Restimulation of the cultures with EBV-transformed B cells generated specific cytotoxic activity.

Epstein-Barr virus (EBV) transforms resting human B cells into immortalized immunoblasts. EBV-encoded nuclear antigens EBNA-5 (also called EBNA-LP) is one of the earliest viral proteins expressed in freshly infected B cells. We have recently shown that EBNA-5 binds p14ARF, a nucleolar protein that regulates the p53 pathway. Here, we report the identification of another protein with partially nucleolar localization, the v-fos transformation effector Fte-1 (Fte-1/S3a), as an EBNA-5 binding partner. In transfected cells, Fte-1/S3a and EBNA-5 proteins showed high levels of colocalization in extranucleolar inclusions. Fte-1/S3a has multiple biological functions. It enhances v-fos-mediated cellular transformation and is part of the small ribosomal subunit. It also interacts with the transcriptional factor CHOP and apoptosis regulator poly(ADP-ribose) polymerase (PARP). Fte-1/S3a is regularly expressed at high levels in both tumors and cancer cell lines. Its high expression favors the maintenance of malignant phenotype and undifferentiated state, whereas its down-regulation is associated with cellular differentiation and growth arrest. Here, we show that EBV-induced B cell transformation leads to the up-regulation of Fte-1/S3a. We suggest that EBNA-5 through binding may influence the growth promoting, differentiation inhibiting, or apoptosis regulating functions of Fte-1/S3a.

Previous work has demonstrated that chromosome 11 is trisomic or shows a duplicated region encompassing the E1/E2 band of chromosome 11 in 90% of v-abl/myc-induced plasmacytomas (Wiener et al. 1995). In the present report, we have studied BALB/c PreB lymphocytes that were immortalized by v-abl and stably transfected with a conditional MycER vector (Mai et al. 1999). These cells, termed PreB ABL/MYC, showed changes in the E1/E2 bands of chromosome 11 that are similar to those reported previously for v-abl/myc-induced plasmacytomas. This was shown by the use of chromosome painting, SKY, FISH and mBAND. Our findings suggest that the Pre-B ABL/MYC cells may be used to analyse the genetic changes affecting chromosome 11 that are associated with v-abl/myc-dependent tumorigenesis in mouse B cells.

Epstein-Barr virus (EBV) carrying lymphoblastoid cells of normal origin express the full program of all 9 virus-encoded, growth transformation associated proteins. They have an intact p53 pathway as a rule. This raises the question of whether any of the viral proteins impair the pathway functionally. Using a yeast 2-hybrid system, we have shown that EBNA-5 but not the other EBNAs interacts with the p14ARF protein, a regulator of the p53 pathway. The interaction was confirmed in vitro using a GST pull-down assay. Moreover, expression of EBNA-5 increased the survival of p14ARF-transfected cells. EBV infection of resting B cells induced the expression of p14ARF mRNA without increased level of the protein. A fraction of the p14ARF localized to the nucleoli but the bulk of the protein accumulated in nuclear but extranucleolar inclusions. Formation of the extranucleolar inclusions led to complete relocalization of EBNA-5 from nucleoplasm to these structures. The inclusions also contained p53 and HDM2, and were surrounded by PML bodies and proteasomes, which suggests that these inclusions could be targets for proteasome dependent protein degradation., (Copyright 2003 Wiley-Liss, Inc.)

Epstein-Barr virus (EBV) drives the proliferation of human B cells in vitro and during primary infection in vivo. The transformed immunoblasts express nuclear proteins EBNA1-6, transcribed from the Cp/Wp promoter, and the membrane proteins LMP-1, -2A and -2B (lymphoblastoid type of latency). EBV persists through life in resting memory B cells with a restricted type of latency in the absence of the Cp/Wp promoter activity. Since CD40 crosslinking can reportedly inhibit the growth of EBV-transformed lymphoblastoid cell lines (LCLs), we have examined the effect of CD40 ligation on the expression of EBNAs and LMP-1 and on Cp EBV promoter activity together with several phenotypic markers. CD40 crosslinking led to a partial downregulation of EBNA-2, EBNA3-6 and LMP-1 in LCLs, paralleled by downregulation of Cp promoter activity. It also induced upregulation of the germinal center marker CD77 on the LCL cells. Our findings suggest that the encounter of proliferating EBV-transformed immunoblasts with CD40L, as would occur when normal B cells generate memory cells in germinal centers, may switch the viral transcription program from the full lymphoblastoid to a more restricted latency program in a proportion of cells. This would permit virus persistence in the B-cell memory compartment., (Copyright 2002 Wiley-Liss, Inc.)

Objectives: To understand the mechanism for the refractoriness of B-chronic lymphocytic leukemia (B-CLL) cells for Epstein-Barr virus (EBV)-induced immortalization., Study Design/methods: Cultures were initiated with EBV-infected tonsillar B and B-CLL cells. Expression of EBNA-2 and some of the key players regulating G1/S phase transition such as c-myc expression, phosphorylation of Rb protein, expression of G1 cyclins, and the cyclin-dependent kinase inhibitor p27 were followed., Results: In line with earlier studies, EBV infection induced c-myc expression, pRb phosphorylation, D2 and D3 expression, and disappearance of p27 in normal B cells. In contrast, EBV-infected B-CLL cells remained resting and they did not express c-myc; cyclin D2, ppRb and cyclin D3 were seen only in occasional cells. Importantly, p27 expression was maintained., Conclusions: In B-CLL cells, the expression of the EBV-encoded nuclear proteins EBNAs is not followed by entrance to the cell cycle. Thus, the difference in the interaction of EBV-normal B cells and EBV-B-CLL cells is already apparent early after infection.

Three types of virus-host cell interactions have been described in cells latently infected with EBV: EBNA 1 expression in type I Burkitt's lymphoma cell lines (BL), EBNA 1, LMP1 and 2 expression in most nasopharyngeal carcinomas (NPC) and EBNA 1-6 with LMP 1 and 2 expression in group III BL-lines as well as lymphoblastoid cell lines (LCL). Two group I BL lines that express only EBNA 1 were found to initiate their EBNA 1 mRNA transcription from a promoter in the Bam HI Q-fragment. They use a sequence at +210 bp relative to the Fp transcription initiation site in group I BL cell lines. The Fp promoter-region seems to be activated in the lytic cycle. LCLs initiate their transcription from one of several upstream sites, usually the Cp promoter or, less frequently, one of several Wp-promoters. Using RNA-reverse transcription polymerase chain reaction (RT-PCR), we have now shown that EBV carrying cells that do not express EBNA 2-6 always splice their EBNA mRNA at the Q-exon, while EBNA 2-6 positive cells use either the Cp or one of the Wp promotors. When EBNA 2-6 are downregulated by somatic cell hybridisation between EBNA 1-6 positive B-cell lines and non B-cells of hematopoetic, epithelial or fibroblastic origin that express the phenotype of the non-B cell parent, the parental usage of Cp/Wp is switched off, and the Q-exon is activated. NPC cells show the same pattern of promoter usage as the hybrids with non-B phenotype. Group III BL cells use both promoter regions. Thus, the virus can use two alternative programs, depending on the cell phenotype. The "EBNA-1 only" program is activated from the Q-promoter. In cells with an immunoblastic (LCL or BL group III) phenotype, the upstream Cp/Wp promoters generate a 100 kb. long pre-mRNA, from which all the EBNAs are spliced. As a rule, only one of the two programs is used for each phenotype, except for the BL group III cells that began as group I but subsequently developed into a more LCL-like cell. Such cells used both promoter regions, with or without activation of the lytic cycle.

Enzymatically mediated rearrangement of Ig and T-cell receptor genes is essential for generating the huge molecular repertoire of the mammalian immune system, but it also carries a danger for the organism in the form of high risk zones for illegitimate juxtaposition of DNA from other areas of the genome. Translocation-dependent activation of oncogenes, transcription factors or developmental genes can trigger the development of neoplasia in a lineage-specific fashion. These events are not sufficient for tumorigenesis, however, since some of the most prominent tumor-associated translocations, such as Ig/myc and Ig/bcl-2, have been detected in normal individuals who did not develop tumors. Tumor development must, therefore, require subsequent genetic changes. Among them, the increased expression of genes that protect against apoptosis or, alternatively, mutations that cripple apoptosis-activating genes play a prominent role. Some of the translocations associated with T-cell leukemia, myeloid leukemia, and a variety of sarcomas act by generating fusion proteins. The participating genes encode transcription factors and/or developmental regulators. Fusion protein-expressing cells may serve as targets for specific interference with abnormal signaling pathways or for targeted immune attack. Using PCR to detect cells carrying such translocations is useful for tumor diagnosis, prognosis, and choice of therapy.

The Epstein-Barr virus (EBV) encoded latent membrane protein of B cell origin, B-LMP1 (B95-8 prototype) and nasopharyngeal carcinoma (NPC) derived C-LMP1 (CAO prototype) were transfected individually in S6C adenocarcinoma cells of ACA (H-2f) origin. We have shown previously that inoculation of B-LMP1 expressing S6C cells led to tumor rejection in pre-immunized, immunocompetent syngeneic ACA mice, whereas the C-LMP1 transfectants were not immunogenic. Furthermore, B-LMP1 but not C-LMP1 expressing S6C cells grew with necrosis and extensive skin damage in non-immunized mice. A study was carried out to determine whether the in vivo growth pattern of S6C cells expressing two different LMP1 isolates could be correlated to any immunomodulatory mechanism. An increased infiltration of CD45+ leukocytes was found in B-LMP1 expressing S6C tumors originating in non-immunized, syngeneic ACA mice. The C-LMP1 expressors, vector transfectants and untransfected parental tumors had significantly lower number of infiltrating leukocytes. The immunoaccessory molecules ICAM-1, B7-1 and MHC Class I and II expression was unaltered in both B- and C-LMP1 transfectants. The data suggest that B-LMP1 but not C-LMP1 induce anti-tumor immune response., (Copyright 2000 Wiley-Liss, Inc.)

B lymphocytes have been identified as the main reservoir of latent Epstein-Barr virus (EBV) in healthy virus carriers. We have established a semi-quantitative PCR method to estimate the EBV genome load in the blood B-cell subpopulation in healthy individuals. EBV DNA was detected in subfractionated IgM-, IgG- and IgA-positive B cells. Between 80% and 90% of the viral DNA was found in the IgA-positive compared with the IgA-negative fraction., (Copyright 1999 Wiley-Liss, Inc.)

Infection of human B lymphocytes with Epstein-Barr virus (EBV) in vitro induces a G0 to G1 transition followed by DNA synthesis and cell division. The virus activation of the cell cycle closely mimics the antigen-dependent normal B cell activation pathway. Infected B cells undergo blast transformation followed by the emergence of immortalized lymphoblastoid cell lines. Numerous cellular proteins are switched on in the infected cells, including p53. In view of the frequent association of wild-type p53 (wtp53) expression with growth arrest and apoptosis, p53 expression, cell viability (absence of apoptosis) and cell cycle progression at the single cell level during the first week after EBV infection were assessed. The rate of EBV infection was scored by EBNA-5 staining between 20 and 72 h after infection and varied between 20 and 25% of the cell population. All EBNA-5-positive blasts were p53-positive as well. Double staining for p53 and for DNA ends (TUNEL) revealed that p53-positivity and apoptosis were mutually exclusive. Quantification of the DNA content by Hoechst staining and computer-assisted image analysis showed that a fraction of the p53-positive blasts had a DNA content higher than 2N, indicating entry into the S/G2 phases. Double p53 and BrdU staining of the cells, pulse-labelled with BrdU, revealed that 65% of the p53-positive blasts were in S phase 3 days after infection. Similarly, B cell activation by CD40L and IL-4 induced p53 expression without any adverse effect on cell cycle progression. Therefore, the phenomenon is not EBV-specific but correlates with immunoblast activation.

A body cavity lymphoma-derived cell line (BC1), known to carry both Epstein-Barr virus (EBV) and human herpes virus type 8 (HHV-8; or Kaposi's sarcoma-associated herpesvirus, KSHV), was analysed for the expression of EBV-encoded, growth transformation-associated antigens and cellular phenotype by immunofluorescence staining, Western blotting, RT-PCR and flow cytometry. A similar phenotypic analysis was also performed on another body cavity lymphoma line, BCBL1, that is singly infected with HHV-8. Phenotypically, the two lines were closely similar. Although both lines are known to carry rearranged immunoglobulin genes, they were mostly negative for B-cell surface markers. Both expressed the HHV-8-encoded nuclear antigen (LNA1). Similarly to Epstein-Barr nuclear antigen type 1 (EBNA1), LNA1 was associated with the chromatin in interphase nuclei and the mitotic chromosomes in metaphase. It accumulated in a few well-circumscribed nuclear bodies that did not co-localize with EBNA1. BC1 cells expressed EBNA1, LMP2A and EBV-encoded small RNAs but not EBNA2-6, LMP1 and LMP2B. They were thus similar to type I Burkitt's lymphoma cells and latently infected peripheral B-cells. Analysis of the splicing pattern of the EBNA1-encoding message by RT-PCR showed that BC1 cells used the QUK but not the YUK splice, indicating that the mRNA was initiated from Qp and not from Cp or Wp.

We examined the patterns of viral gene expression in acute infectious mononucleosis (IM) patients and the clonality of the directly growing EBV-carrying cell lines. Both low- and high-density EBV-carrying B cells obtained from the patients' tonsils expressed EBNA1, EBNA2 and LMP1. Like LCLs and immunoblastic B-cell lymphomas, the in vivo EBV-carrying low-density cells used only the latency III program for viral gene expression. The in vivo EBV-carrying high-density B cells used both the latency I program, as indicated by the QUK-, and the latency III program, as indicated by the YUK-EBNA1. This suggests that the lymphoid tissues contained not only proliferating immunoblasts but also cells programmed for latent viral persistence in vivo. EBV-carrying cells that grew directly into permanent cell lines in the presence of virus-neutralizing antibody and a late viral inhibitor were polyclonal, as indicated by JH rearrangement. Two of the high-density-derived lines had identical JH and TR patterns, indicating a common parental origin. Our investigation indicates that EBV-carrying cells divide and survive in a fully competent immune system during the outbreak of acute IM.

Exposure of normal resting B lymphocytes to EBV in vitro leads to activation, subsequent immortalization, and the establishment of lymphoblastoid cell lines (LCLs). The endemic form of Burkitt's lymphoma (BL) is associated with EBV. EBV-positive BL lines maintain the original tumor phenotype (group I BL) initially and express only one EBV-encoded protein, EBV nuclear antigen (EBNA)-1. Most of them drift toward a LCL-like phenotype during in vitro culturing and express all nine EBV-encoded growth transformation-associated proteins (group III BL). Cyclin D2 and D3 have been found previously to differ in their mRNA expression in BL and LCL. Cyclin D2 expression has been attributed to EBV gene expression and to EBNA-2 and EBNA-5 in particular. We have studied cyclin D2/D3 expression in larger series of LCLs, BL lines, and freshly EBV-infected peripheral blood B lymphocytes, both at the mRNA and protein levels. The predominant cyclin D2 expression in the LCLs and group III BLs correlated with an activated B-cell phenotype. In contrast, only cyclin D3 was expressed in the group I BL lines. EBV-carrying group II BL lines that retained the BL-associated CD10 did not express cyclin D2. A collection of in vitro EBV-converted sublines of originally EBV-negative BLs that expressed a full set of the growth transformation-associated EBV proteins maintained their CD10 and cyclin D3 expression. Blood B lymphocytes expressed no D2 or D3 as judged by immunostaining. Cyclin D2 could be detected by immunostaining in a proportion of the activated blasts 40 h postinfection. Cyclin D3 that is expressed at a low level in the established LCLs was stained easily in B blasts at this time. The level of cyclin D3 at 72 h postinfection was two to three times higher than in the exponentially growing LCLs, as detected by immunoblotting. It was still 5-10 times lower than in group I BLs. Mitogen stimulation of primary B cells induced cyclin D3 at a similar level as in the LCLs. Our data are consistent with the notion of cell lineage-specific D-type cyclin expression. They also indicate that B cells may switch their D-type cyclin expression during differentiation.

We have studied the activity of reporter plasmids, carrying the Epstein-Barr virus (EBV) nuclear antigen (EBNA) promoters Wp and Cp, in a group of somatic cell hybrids obtained by fusing EBV-positive lymphoblastoid cell lines or group II/III Burkitt's lymphoma cell lines with non-B cell lines. In B/non-B cell hybrids of this type, B cell markers are extinguished as a rule, in parallel with the inactivation of Cp or Wp and down-regulation of EBNA-2-6 expression. A Wp-carrying reporter construct was active in non-B cell lines. Only cells with a B cell phenotype could support the activity of Cp-carrying plasmids. EBNA-2 transactivated Cp only in B cells. Our data suggest that while Wp can be used for EBNA transcription in B and non-B cells, Cp activity is restricted to B cells. The inability of EBNA-2 to transactivate Cp in non-B cells indicates that other factors present in B cells might be involved in Cp transactivation.

Translocation of the c-myc gene to an immunoglobulin locus is a rate-limiting step in the genesis of three B-cell derived tumors: Burkitt lymphoma (BL) in humans, mouse plasmacytoma (MPC) and rat immunocytoma. Its consequences have been best analyzed in BL. They involve a non-immunological and an immunological component. The former acts by preventing the B cell from leaving the cycling compartment and entering the resting stage when programmed to do so. The latter acts by the down regulation of certain HLA class I polymorphic specificities, adhesion molecules and EBV encoded proteins. According to our interpretation, the translocation fixes the BL cell in a phenotypic window that can be referred to as "a resting cell that is not resting". The linking of c-myc to immunoglobulin sequences in a B cell leads to constitutive myc expression. In spite of its switch to a "resting phenotype", the cell is therefore unable to leave the cycling compartment. Normally, EBV-carrying B-blasts face immune controls. They can only proliferate in immunodefectives, as in XLP or transplant lymphoma. Due to its "resting" rather than B-blast phenotype and the correlated defective expression of certain EBV, HLA and adhesion molecules, the BL cell is not rejected by the EBV-specific immune response, however. The down-regulation of EBNA 2-6 in the BL cell may be also considered in relation to viral latency. The exclusive expression of EBNA 1 in the BL cell reflects the adaptation of the virus to prolonged persistence in long lived resting B cells, an important if not exclusive reservoir of the latent virus. Normal B cells that fail to be activated by appropriate mitogens or antigens within a limited period of time undergo apoptosis, as a rule. EBV may protect resting B cells. The role of the only virally encoded protein they are known to express, EBNA 1, remains to be explored in this respect. EBV infected immunoblasts are protected from apoptosis by the LMP 1 induced elevation of bcl-2 expression. During the lytic cycle when cellular protein synthesis is switched off by the early viral proteins, a member of the early protein group, a bcl-2 homologous protein encoded by the BHRF1 gene, takes over the function to protect against untimely apoptosis.

The effect of heat-shock on the expression of c-myc genes in different chromosomal contexts was investigated in a panel of human B-lymphoid cell lines. Burkitt's lymphoma cell lines with c-myc translocation breakpoints upstream of the first exon, within the exon itself, or in the first intron showed downregulation of c-myc levels as did a cell line without any translocation. The c-myc mRNA of cell lines with translocation breakpoints within the c-myc gene have previously been reported to have prolonged half-lives. After heat shock, the levels of these mRNA species were reduced with similar kinetics as the normal c-myc mRNA. An exception was an RNA species where the only c-myc sequences are derived from exon 1, showing that sequences from this part of the c-myc gene are not sufficient to mediate the rapid downregulation. Nuclear run-on analysis did not show reduced transcription of c-myc after heat shock and a comparison of cytoplasmic and total RNA did not indicate accumulation of longer, unspliced c-myc mRNA species. These observations suggest a posttranscriptional, cytoplasmic downregulation targeting exons 2 and/or 3. B-lymphoma lines transfected with a hsp70 promoter-linked c-myc gene were deficient in their ability to reinitiate proliferation after heat shock, providing a physiological rationale for the normal downregulation of c-myc after this type of physical stress.

Background: The developmental stage from which stems the malignant B cell population in Burkitt's lymphoma (BL) is unclear. An approach to answering this question is provided by the sequence analysis of rear-ranged immunoglobulin (Ig) variable region (V) genes from BL for evidence of somatic mutations, together with a phenotypic characterization. As somatic hypermutation of Ig V region genes occurs in germinal center B cells, somatically mutated Ig genes are found in germinal center B cells and their descendents., Materials and Methods: Rearranged V kappa region genes from 10 kappa-expressing sporadic and endemic BL-derived cell lines (9 IgM and 1 IgG positive) and three kappa-expressing endemic BL biopsy specimens were amplified by polymerase chain reaction and sequenced. In addition, VH region gene sequences from these cell lines were determined., Results: All BL cell lines and the three biopsy specimens carried somatically mutated V region genes. The average mutation frequency of rearranged V kappa genes from eight BL cell lines established from sporadic BL was 1.8%. A higher frequency (6%) was found in five endemic cases (three biopsy specimens and two BL cell lines)., Conclusions: The detection of somatic mutations in the rearranged V region genes suggests that both sporadic and endemic BL represent a B-cell malignancy originating from germinal center B cells or their descendants. Interestingly, the mutation frequency detected in sporadic BL is in a range similar to that characteristic for IgM-expressing B cells in the human peripheral blood and for mu chain-expressing germinal center B cells, whereas the mutation frequency found in endemic BL is significantly higher.

Using reverse transcription of whole cellular RNA and nested PCR, we have performed experiments mixing different proportions of Epstein-Barr virus (EBV)-carrying and EBV-negative cells. Based on the results, a method that detects viral transcripts for EBNA-1, EBNA-2, LMP1, and LMP2a from less than one positive cell among 10(5) negative cells was developed. With this method we have shown that the EBV DNA positive cells among small, high-density peripheral blood B-lymphocytes of normal healthy persons express EBNA-1-mRNA but not EBNA-2 or LMP1. A similar EBV expression pattern is found in type I Burkitt lymphoma cells. We suggest that the expression pattern in the lymphoma cells reflects the viral strategy in normal resting B cells and meets the requirements of latent persistence.

Using immunofluorescence technique we have analysed the Rb, p53, EBNA-2 and EBNA-5 expression pattern in EBV infected human B-cells and established lymphoblastoid cell lines (LCL-s). Resting B-cells showed only a faint Rb and no p53 immunostaining. The expression of both Rb and p53 increased after EBV infection. The change was first detectable 6 h after infection. The frequency of brilliantly Rb positive cells increased more rapidly than p53 positives. EBNA-2 and EBNA-5 became first detectable 12 h after infection. The frequency of EBNA positive cells in the freshly infected cultures was concordant with the proportion of CD23 and PCNA positives, but remained consistently below the frequency of Rb and p53 positive cells. Double immunofluorescence staining showed that all EBNA-5 positive cells were strongly Rb and p53 positive. LCL-s did not stain for p53, whereas the Rb staining was maintained at a high level. The EBNA-5 staining pattern changed from brilliant almost homogeneous nuclear staining in the freshly infected B-cells, to a nonhomogeneous pattern with a small number of strongly fluorescent nuclear bodies in established LCL-s. There was no change in the EBNA-2 staining pattern. Our findings indicate that the immortalization of B-cells by EBV may initially involve a high expression of EBNA-5, p53 and Rb, but only cells with low p53 and focal expression of EBNA-5 in nuclear bodies have the selective advantage required to grow into immortalized lines.

The leukemic-cell population of one CLL patient, PG, was found to contain a sub-set of EBV-genome-carrying cells. It was detected directly by the expression of EBNA (EBV-encoded nuclear antigen) and by its capacity to grow in vitro. The proportion of EBNA-positive cells (0.1%) was maintained constantly during the period of this study, the final 3 years of the patient's life. EBV-carrying clonal sibling B-cell lines were established on 5 occasions. They had identically rearranged JH bands and chromosomal markers corresponding to the ex vivo CLL cells. Analysis of the viral episomes in the lines proved that they were the descendants of one cell. On the last occasion of blood sampling, 8 B-cell lines were established; 4 of these contained the same clonal markers as the previous lines, while 4 other lines belonged to another clone with identical JH rearrangement. Their abnormal karyotypes were different from the first clone. The chromosomal markers were only partly identical, suggesting secondary diversifications. The EBV sub-strain carried by this group of lines was different from the sub-strain of the first clone, as judged by the EBNA size distributions (EBNOtype) and EBV-DNA analysis. Analysis of the terminal repeat in the viral episomes also showed that the first and the second set of clones represented 2 independent EBV-infection events in vivo.

We have analysed the expression of transformation-associated viral antigens, the Epstein-Barr virus (EBV) DNA content and the phenotypic characteristics of two B95-8 virus-converted sublines of the EBV-negative Burkitt's lymphoma (BL) line BL28. The converted lines called E95A-BL28 and E95B-BL28, respectively, differed in their EBV gene expression. The E95B convertant expressed virus-encoded nuclear antigens EBNA1 to -6 and the membrane protein LMP1, but only EBNA2 and EBNA5 were detected by immunofluorescence and immunoblotting in the E95A convertant. Only the entire BamHI W, Y and H regions could be detected in the E95A convertant by hybridization of Southern blots with probes covering the BamHI C, W, Y, H, F, E, K and Nhet regions of the EBV genome. EBV episomes were found to be absent in the E95A convertant as seen by Gardella gels. The E95A convertant retained the phenotypic characteristics of the EBV-negative parental line, and remained highly clonable in agarose. In contrast, expression of EBNA1 to -6 and LMP1 was accompanied by a shift towards a more lymphoblastoid cell line-like phenotype and by loss of agarose clonability in the E95B convertant.

We tested the effect of TGF-beta 1 on the EBV-induced activation and immortalization of human B lymphocytes. In lymphocyte cultures of EBV-seropositive individuals, T cells can inhibit the EBV-induced transformation of B cells. We found that in the presence of TGF-beta 1 the transformation was more efficient, due to the inhibition of this function. TGF-beta 1 inhibited the EBV-induced proliferation of purified B-cell cultures. Its effect was strongest when added at the beginning of the culture. Added later, the inhibition gradually decreased and was lost by the 4th day. After 10 to 14 days, in the cultures initiated with TGF-beta 1, the number of cells was higher and they formed larger aggregates as compared with control cultures. Thus, TGF-beta 1 modifies EBV-induced transformation in a complex way. It inhibits the activation of B cells but does not affect those already activated. Once they acquire the immortalized state, the B cells are even stimulated by TGF-beta 1.

The expression of the transformation-associated Epstein-Barr virus (EBV)-encoded nuclear antigens (EBNAs) 1 to 6 and membrane protein LMP-1 was studied in a series of somatic cell hybrids derived from the fusion of the EBV-transformed lymphoblastoid cell line (LCL) KR-4, and the EBV-carrying Burkitt's lymphoma lines Daudi, P3HR-1 and Raji, with non-B cell lines of fibroblast, erythroid, myeloid and epithelial origin. Expression of EBNAs 2 to 6 was down-regulated in the hybrids in parallel with extinction of the B cell markers CD19, CD20, CD21, CD23, HLA class II, and surface or cytoplasmic immunoglobulin. LMP-1 was expressed independently of EBNA-2 in hybrids derived by the fusion of the LMP-1-positive KR-4 and P3HR-1 cell lines with epithelial and myeloid cells, respectively. LMP-1 was down-regulated in hybrids derived by the fusion of P3HR-1 with an erythroid cell line and in the hybrid between Raji and a mouse fibrosarcoma line. EBNA-1 was the only EBV antigen that was regularly expressed in the hybrids regardless of the dominating cellular phenotype. The autonomous expression of EBNA-1 suggests that its regulatory pathway is independent of phenotype-associated cellular or viral factors. In contrast, the expression of EBNAs 2 to 6 appears to require a B cell environment. EBNA-2 was shown to contribute to the regulation of LMP expression in B cells. We show that in LCL-carcinoma hybrids the dominating epithelial phenotype is permissive for LMP expression in the absence of EBNA-2.

We tested the effect of transforming growth factor (TGF)-beta 1 and TGF-beta 2 on the proliferation of human B cell lines. The panel was selected to give information whether (1) their origin, (2) their phenotype, (3) their Epstein-Barr virus (EBV) carrier state, influence their responsiveness. The growth of lymphoblastoid cell lines (LCL) was not inhibited by TGF-beta 1. The EBV-carrying Burkitt lymphoma (BL) lines, Daudi, Jijoye, Rael but not Raji were inhibited. Three EBV-negative BL lines and the majority of their converted sublines were sensitive. The cell lines tested expressed TGF-beta receptors and TGF-beta 1 transcripts. The proliferation of EBV-infected B cells was inhibited by TGF-beta, their sensitivity decreased, however, after 3 days. The results suggest that the activation state of the B cells is decisive for TGF-beta sensitivity and EBV influences it indirectly by changing the cell phenotype.

We assessed 33 lymphoid tissues from 15 patients, including 7 with X-linked lymphoproliferative disease (XLP) and 8 patients with sporadic fatal infectious mononucleosis (IM), to determine whether the cellular infiltrate had the immunophenotype and expressed Epstein-Barr virus (EBV)-encoded proteins characteristic of either EBV-immortalized lymphoblastoid cell lines (LCL) or EBV-carrying Burkitt lymphoma (BL) cells. The results of these studies revealed that in 13 cases the proliferating B cells were polyclonal, LCL-like, and in 2 cases they were monoclonal, malignant lymphoma-like.

In accordance with earlier studies, we detected higher numbers of Epstein-Barr virus (EBV)-carrying lymphocytes (B-EBV) in the blood of acute infectious mononucleosis (IM) patients and higher amounts of transforming EBV particles in the saliva compared to healthy seropositive individuals. B cells grew in cultures seeded with the low and high density IM lymphocytes. The majority of B cells which grew acquired the infection in vitro (2-step outgrowth), because addition of virus neutralizing antibodies considerably reduced the emergence of lymphoblastoid cell lines (LCLs). Only the minority of the explanted B-EBV cells proliferated. The antiviral drug phosphonoformate (PFA) did not influence the frequency of 2-step LCLs in the IM cultures. This may indicate that a large proportion of EBV carrying B cells have already entered the viral productive cycle in vivo and passed the PFA-sensitive stage at the time of explantation. Earlier experiments with blood of healthy seropositive individuals showed an inhibitory effect of PFA on the generation of LCLs. One healthy individual who entered this study as a control, probably had a reactivated EBV infection as judged by the anti-EA activity in his serum and the high level of virus in his saliva. He had antibodies against EBV nuclear antigens (EBNA), and therefore he did not have a primary infection at the time of the test. Judged by the number of wells with B cell growth, the frequency of virus-carrying B cells in his blood was low. It seems that anti-EBV immunity can control the number of infected B cells in the blood, but does not influence the virus load in the epithelial cells.