After the primary infection, that may or may not cause infectious mononucleosis, the ubiquitous Epstein–Barr virus (EBV) is carried for lifetime. The great majority of adult humans are virus carriers. EBV was discovered in a B-cell lymphoma (Burkitt lymphoma). EBV infection in humans is the example for the power of immune surveillance against virus transformed, potentially malignant cells. Although the virus can transform B lymphocytes in vitro into proliferating lines, it induces malignancy directly only in immunosuppressed hosts. EBV-induced growth transformation occurs only in B lymphocytes. It is the result of a complex interaction between virally encoded and cellular proteins. Different forms of the virus–cell and the cell–host interactions have evolved during a long period of coexistence between the virus and all Old World (but not New World) primates. The asymptomatic carrier state is based on a viral-strategy that downregulates the expression of the transforming proteins in the virus-carrying cell. In addition to the silent viral-gene carriers and the expressors of the nine virus-encoded genes that drive the growth program, virus carrying cells exist that show other patterns of gene expression, depending on the differentiated state of the host cell. Certain combinations contribute to malignant transformation, but only in conjunction with additional cellular changes. These are induced by direct or cytokine-mediated interactions with normal cells of the immune system.
With rare exceptions, all humans harbor the Epstein–Barr virus (EBV). Although this virus was shown to induce proliferation of B lymphocytes, the virus carrying state is largely harmless. The harmonious host–virus coexistence is the result of a long history with mutual adaptation, based on variation in the viral gene expression in different types of infected cells and the finely tuned immune response of the host (Klein, 1994).
EBV shows a high degree of B-cell tropism. It binds to a B lymphocyte-specific surface molecule, CD21 (receptor for the C3d fragment of complement). The virus–receptor interaction induces activation of the cell. Infected cells can enter into the mitotic cycle, leading to the emergence of proliferating lines (Rickinson and Kieff, 2001). These are referred to as lymphoblastoid cell lines (LCLs). In these cells, the viral genomes are maintained in episomal form and express nine proteins. Experimental infection of epithelial, T and natural killer (NK) cells can be achieved only by manipulation of the virus or the host cell, e.g. by using virus with selection markers or target cells with inserted viral receptors. However, the occurrence of EBV-carrying neoplasms originating from cell lineages other than B cells shows that they are occasionally infected in vivo.
In the following, we restrict our discussion to the latent EBV–lymphocyte interaction and to the EBV-carrying haematopoetic malignancies.
The expression of EBV-encoded proteins differs depending on the type, differentiation and activation status of the target cell. The growth transformation program is based on the expression of six nuclear and three membrane proteins. Six of these are essential for the activating and proliferation driving effect of the virus. Latently infected virus-carrying B cells in healthy individuals are found in the resting, memory compartment in a silent state (Chen et al., 1995; Babcock et al., 1998). Only one virally encoded nuclear protein, the long-lived EBNA-1, is expressed in these cells (Hochberg et al., 2004). EBNA-1 is required for the maintenance of the viral episomes.
EBV was first seen in Burkitt lymphoma (BL), a B-cell-derived tumor (Epstein et al., 1964). The transforming capacity for B lymphocytes in vitro was its first biological effect demonstrated (Pope et al., 1968). This finding was first thought of as the in vitro correlate of the EBV-carrying malignancy BL. However, it was soon shown that BL cells and LCLs are very different phenotypically (Nilsson and Ponten, 1975). The former resemble non-activated cells, whereas the latter are similar to activated lymphoblasts. Discovery of the Ig/myc translocation in both EBV-positive and EBV-negative BLs in different geographical areas revealed the common critical denominator for the development of these lymphomas and left the role of EBV unanswered.
Primary EBV infection of adolescents and young adults induces a self-limiting disease, infectious mononucleosis (IM) in about half of the individuals. The clinical picture is highly variable, with mild to serious symptoms. In the absence of typical symptoms, as mostly in children, seroconversion is the indicator for infection.
The symptomatology of IM reflects the involvement of the immune system. The appearance of the heterophile antibodies in the serum, which is one of the critical diagnostical markers, is the result of B-cell activation. Detection of IgM antibodies against the viral capsid antigen and by IgM and IgA antibodies against the early antigen expressed in the lytically infected cells is the diagnostic sign of the newly acquired infection. These antibodies are helpful in distinguishing IM from other diseases with lymphocytosis.
Antibodies specific for other EBV-encoded proteins expressed in cells with lytic and latent infection appear subsequently in regular sequence. The antibody profile was shown to be characteristic for the different EBV-associated diseases (Henle et al., 1979).
IM patients excrete regularly transforming virus in the saliva. Healthy virus carriers do so intermittently. Explanted lymphocyte populations from virus-carrying individuals can yield LCLs provided that the EBV-specific memory T cells are inhibited.
Expression of EBV-encoded proteins in lymphocytes with latent infection
The function of the EBV-encoded proteins in the latently infected cells has been characterized in the LCLs. Only rare cells enter the lytic cycle in such cultures. Initially, attention was focused mainly on the analysis of B-cell transforming capacity of the virus.
Type III latency – growth program
Expression of the full set of EBV-encoded proteins detected in the LCLs is designated as ‘growth program’ (Thorley-Lawson, 2001) and is referred to as type III latency (Rowe et al., 1992) (Figure 1). In these cells, expression of six nuclear proteins (EBNAs) is regulated by one of two alternative viral promoters (designated Wp and Cp). The spliced products of a giant message are translated to six nuclear proteins (EBNA-1 to 6). In addition, the virus encodes three cell membrane associated proteins (LMP1, LMP2a and LMP2b). The function of these nine proteins is only incompletely known. They interact with each other and with cellular transcription factors and co-activators and by this activate a large number of cellular genes. Five of the nine proteins are required for the induction and maintenance of the transformed phenotype. The type III, growth program is expressed only in B lymphocytes.
LMP-1 has a strong impact on the B cell phenotype. It induces activation markers and co-stimulatory molecules. These contribute to the immunogenicity of the cells. Therefore, cells expressing Type III latency can exist only during the acute phase of primary infection, before the EBV-specific T-cell response develops, and in patients with impaired immune functions, such as transplant recipients who are treated with immunosuppressive agents (Young et al., 1989). Consequently, these patients are at high risk for the development of EBV-positive lymphoproliferative disease (PTLD). Correction of the immune deficient state by adoptive transfer of CTLs can lead to the regression of the malignancy.
Type I latency program
Only one nuclear protein, EBNA-1, is expressed in the type I cells. The giant Wp- or Cp-initiated transcript is not made in these cells. The monocistronic EBNA-1 mRNA is transcribed from the Q promoter (Schaefer et al., 1995). The type I program does not induce proliferation and the phenotype corresponds to non-activated B cells. Type I cells are found among memory B cells in healthy individuals. In the absence of the expression of co-stimulatory molecules, these are not recognized by EBV-specific T-cell immunity. EBNA-1 does not serve as a target for CD8+ CTLs, because its long glycine–alanine repeat prevents the ubiquitin-proteosome-dependent processing required to generate peptides that associate with MHC class I molecules (Levitskaya et al., 1995).
This viral strategy secures the persistence of EBV, firstly through maintenance of virus-carrying cells in healthy individuals, secondly by not endangering the life of the virus-carrying individual.
Type IIa latency program
This EBV gene expression program is restricted to EBNA-1, LMP-1 and LMP2. It was first seen in nasopharyngeal carcinoma (NPC) (Fahraeus et al., 1988) (thus in a malignancy of epithelial origin), and is referred to as type II latency program. EBNA-2 (along with EBNA-3, -4, -5 and -6) is not expressed in these cells, nor in any other non-B cells. This is due to the lack of specific transcription factors, which are only present in B lymphocytes. The EBV-positive Hodgkin-, T- and NK-lymphomas show the same viral expression phenotype (Deacon et al., 1993; Chiang et al., 1996). In order to accommodate an additional restricted expression of the viral genes with different assortment of the viral proteins, we renamed it as type IIa.
The EBNA-2 protein is required for the induction of proliferation of EBV-infected B cells (Hammerschmidt and Sugden, 1989). Type IIa cells are not induced to proliferate, unless additional cellular changes occur in the cell, and growth-promoting signals are provided by the microenvironment (discussed below).
Type IIb latency program
In type IIb latency, all EBNAs are expressed, but not LMP-1. On the basis of the behavior of the cells with restricted expression of EBV proteins that do not induce proliferation, the type IIa and the type IIb latencies represent a group. They lack one or the other pivotal proteins needed for transformation: EBNA-2 in the type IIa and LMP-1 in the type IIb cells. The latter was first seen in B-chronic lymphocytic leukemia (B-CLL) cells infected with EBV in vitro (Takada et al., 1980; Rickinson et al., 1982; Walls et al., 1989; Doyle et al., 1993).
B-CLL is the clonal expansion of long-lived resting (G0) B lymphocytes (Caligaris-Cappio and Hamblin, 1999). Its pathogenesis is not clarified. EBV is not involved in the pathogenesis of B-CLL and only rare EBV-positive cells were detected in the lymph nodes and in bone marrow (Kanzler et al., 2000; Tsimberidou et al., 2006). We have encountered an exceptional case that harbored an EBV-carrying subclone in vivo and gave rise, on repeated occasions, to immortalized EBV-positive lines with identical cytogenetic and Ig rearrangement as the B-CLL clone (Lewin et al., 1991).
The B-CLL cells collected from blood could be infected in vitro, but in the overwhelming majority of such experiments the EBV-positive cells did not turn into proliferating lines (Takada et al., 1980; Rickinson et al., 1982; Walls et al., 1989; Teramoto et al., 2000).
The lack of LMP-1 expression in the EBNA-2-positive B-CLL cells is noteworthy because normally, EBNA-2 is responsible for activation of the LMP-1 promoter in B cells (Abbot et al., 1990; Fahraeus et al., 1990; Wang et al., 1990). The molecular events that would explain the inadequacy of EBNA-2 in the infected CLL cells has not yet been clarified. The message for the nuclear proteins is initiated from the C promoter (Fu Chen personal communication).
In addition to the in vitro-infected B-CLL cells, type IIb protein expression have been detected in the lymphoid tissues of IM patients (Kurth et al., 2003) and in post-transplant lympho-proliferations, PTLD (Oudejans et al., 1995). No tumors have been encountered with type IIb expression. We have therefore, studied the characteristics of the type IIb program in the in vitro EBV-infected CLL cells. It differed considerably from in vitro-infected normal B-cell populations. The immediate-early genes c-myc, activating transcription factor (ATF)-2 and c-Jun were not activated and there were no phenotypic signs of B-cell activation. Furthermore, the typical blastoid transformation did not occur and the nuclear chromatin remained dense (Figure 2). Critical steps in B-cell transformation such as phosphorylation of Rb and decline of p27 expression did not take place (Maeda et al., 2001; Bandobashi et al., 2005). The CLL cells were not refractory to activation however, as shown by their response to CD40 ligation that induced c-myc expression and entry to the S phase. Although exposure to CD40-ligand (CD40L) overcame the inability of EBV to induce activation of the CLL cells, LMP-1 was not induced and cell lines emerged only in occasional experiments. These rare LCLs (proven to be derived from the CLL clone) emerged with delayed latency, were type III. In our hands, one such line lost the CD5 marker (a hallmark of the B-CLL cell) after prolonged cultivation, whereas another line maintained it.
We found thus blocks in the EBV-infection-induced steps in the CLL cultures. The first block affected the activation of the cell. This could be overcome by exposure of the cells to CD40L. A second block affected LMP-1 expression.
The clinical picture and histopathology of BL is unique. EBV-positive BLs occur in the high endemic regions, whereas the majority of sporadic BLs is EBV-negative. In vivo BL cells have Type I EBV expression (EBNA-1 only) (Rowe et al., 1987). Constitutive activation of c-myc owing to a reciprocal chromosomal translocation between chromosome 8 and either chromosome 14, 2 or 22, that juxtaposes the proto-oncogene to one of the three immunoglobulin loci provides the drive for proliferation. Cells with Ig-myc translocation have been detected in healthy individuals (Roschke et al., 1997). Constitutive activation of myc can drive cells into proliferation or apoptosis, depending on whether these cells do or do not receive concomitant growth-promoting signals. Survival factors, such as growth-stimulatory cytokines, may rescue them from apoptosis. Accordingly, the two conditions associated with Ig/myc tranlocation-carrying BL, chronic hyper-endemic malaria and HIV infection have high levels of B-cell stimulatory cytokines. In addition to cellular factors, EBV-expressed genes may counter-balance the proapoptotic function of myc, as shown in vitro for EBERs (Komano et al., 1999) and EBNA-1 (Kennedy et al., 2003).
Although BL patients are immunocompetent, the EBV-carrying BLs escape from rejection. This escape is partially due to their phenotype, as they lack co-stimulatory surface molecules. In addition EBNA-1, the only EBV-encoded protein they express, does not provide MHC class I-associated peptides that could be recognized by CD8-positive CTLs.
The HL tissue is a complex granuloma, made up by T and B lymphocytes, macrophages, eosinophils, plasma cells and less than 2% H-RS cells (Kuppers, 2002). In the EBV-positive cases (40–50% of the classical HL type) the viral-gene expression pattern corresponds to Type IIa, with uniformly abundant LMP-1 protein expression (Pallesen et al., 1991; Deacon et al., 1993). Based on the clonal rearrangements of the immunoglobulin genes, the cells were assigned to the B-lymphoid lineage (Kuppers et al., 1994); however, they do not express several of the B-cell-specific transcription factors and markers, such as CD19, CD20 and surface Ig (Hertel et al., 2002; Schwering et al., 2003). Detailed analysis of the gene expression profile of the HL lines and the immunohistochemical analysis of tumor samples showed considerable deviations from the B lymphocyte pattern. This indicates that the B-cell differentiation program of the HRS cell is impaired.
Characteristics of the HL tissue are similar in the EBV-negative and -positive cases. Apart from the expression of EBV-encoded proteins, special properties that could be ascribed to the presence of the virus in the H-RS cells have not been discovered.
In spite of considerable effort, B-cell lines with type IIa EBV-latency have not been established from HLs (Staratschek-Jox et al., 2000). With the exception of one EBV-positive line, L591, that is Type III (Vockerodt et al., 2002), the few available HL-derived cell lines are all EBV-negative. The L591 line is not representative for HD as type III cells do not occur in the HLs.
We and others established EBV-positive sublines of the HL-derived-EBV-negative KMH2 by in vitro infection (Baumforth et al., 2005; Kis et al., 2005). The converted cells, called KMH2-EBV, expressed only EBNA-1 and LMP2a. The in vitro established EBV carrier KMH2 cells thus failed to mimic the viral gene expression of HRS cells in vivo. However, we induced LMP-1 expression by exposing the cells to CD40L and interleukin (IL)-4, and by this we generated in vitro for the first time the type IIa EBV gene expression in a HL-derived cell line (Kis et al., 2005). This in vitro finding led us to assume that cytokine exposure may occur in vivo, and induces modifications in the expression of EBV genes, especially LMP-1. In the lymphogranuloma, H-RS cells are surrounded by activated CD4-positive T-cells that express CD40L (Carbone et al., 1995), and are known to produce IL-13 (Atayar et al., 2006).
The lack of EBNA-2 expression in HL is consistent with the impaired differentiation of the HRS cells, particularly with regard to the absence of B cell-specific transcription factors, such as BSAP (PAX-5) and Oct-2. These proteins are known to activate the viral-Wp and Cp promoters (Contreras-Brodin et al., 1996; Tierney et al., 2000) and therefore required to generate the giant mRNA from which all six EBNA messages are spliced in type III latency. The need for B-cell-specific factors for type III latent gene expression was first demonstrated in work with somatic cell hybrids (Contreras-Brodin et al., 1991). Whenever an EBV-carrying LCL or the Type III BL line Raji were fused with non-B-cell partners such as HeLa (human cervical carcinoma) or A9HT (mouse fibrosarcoma), respectively, the B-cell phenotype eclipsed. Concomitantly, they lost EBNA-2 expression. On the other hand, the two LCL–HeLa hybrids maintained the LMP-1 expression (Contreras-Brodin et al., 1991). The downregulation of EBNA-2 in these hybrids occurred at the transcriptional level, as detected by the lack of Cp-initiated transcripts (Altiok et al., 1992). The only hybrid that maintained the B-cell phenotype (a P3HR1-HL-60 hybrid) also maintained the expression of the high molecular EBNAs (EBNA-3, -4 and 6) driven from the Wp (similarly to the parental P3HR1 BL cells). When P3HR1 was fused with K562 cells, in parallel with the loss of B-cell phenotype, the Wp-promoter was inactivated and hence the expression of high molecular EBNAs was turned off (Contreras-Brodin et al., 1991; Altiok et al., 1992). These early experiments have shown that the expression of type III latency requires a B-cell phenotype, and when it is lost the activity of Cp and Wp is silenced. Such scenarios of changes in EBV gene expression from type III to type I/II latency might apply for the pathogenesis of the H-RS cell that has lost its B-cell phenotype during its evolution.
The impairment of B-cell differentiation is proposed to be decisive for the development of HL (Kuppers, 2002). According to the scheme of Kuppers, the faulty B cells that arise in the germinal center by somatic hypermutation are the progenitors of H-RS cells. Although the correctly differentiated cells exit the GC, these ‘crippled’ surface Ig-negative B lymphocytes are eliminated by apoptosis (Kanzler et al., 1996). They may be rescued, however, by EBV-infection for which the functions of LMP-1 and LMP-2a are responsible.
LMP-1 contributes also to the establishment of the HL granulomatous tissue. For this, the known LMP-1-induced activation of the nuclear-factor kappa B (NF-κB) pathway is important. This is a hallmark of the H-RS cells (Bargou et al., 1997). In the EBV-carrying cases LMP-1, in the EBV-negative cases mutations in the NF-κB inhibitors (IkB-alpha) (Cabannes et al., 1999), amplification of the REL gene (encodes an NF-κB family member) (Martin-Subero et al., 2002) or signals acting on surface receptors CD30 and CD40 (Carbone et al., 1995; Horie et al., 2002), may activate NF-κB.
Further contribution of LMP-1 to the development of HL may arise by the induction of CCL17/TARC and CCL22/MDC chemokines that can attract the CCR4-expressing Th2 and regulatory T cells (van den Berg et al., 1999; Nakayama et al., 2004). Through such a mechanism, the LMP-1-positive H-RS precursors could receive additional survival signals from the infiltrating Th2 cells; moreover, the regulatory T cells would inhibit the potential damaging effect of the EBV-specific immune response.
Emphasizing the contribution of cytokines and cell contacts, we envisage the following steps in the development of EBV-positive H-RS cells (Figure 3): in a certain maturation window, the EBV-infected B cells express the type I pattern. Type IIa cells may arise from these under the influence of the GC environment. Cell contacts and cytokines would induce LMP-1 expression (Kis et al., 2006a, 2006b). When the EBV genome-carrying cells follow the dynamics of B lymphocyte traffic and exit from the GC to become resting memory cells, the expression of all viral proteins would be downregulated. Doomed, ‘crippled’ B cells (Figure 3) would succumb. Some of them may be saved by LMP-1 and if they enter in a mutual stimulatory interaction with the normal cellular components of the lymphoid tissue an HL granuloma can develop.
In the HL tissue, the immune response seems to be inhibited locally (Frisan et al., 1995). In addition to the above mentioned immunoregulatory T cells that surround the H-RS cells, this may be achieved by their production of immunosuppressive cytokines, such as IL-10 and transforming growth factor (TGF)-β (Marshall et al., 2004; Ishida et al., 2006). Furthermore, secreted LMP-1 has been shown to be immunosuppressive (Dukers et al., 2000).
Extranodal nasal NK/T-cell lymphoma
This rare and highly malignant lymphoma occurs mainly in Asia. The tumor cells are intermixed with inflammatory cells in the tissue that is ischemic and necrotic (Harabuchi et al., 1990). These lymphomas are regularly EBV-positive, containing cells with type I and type II expression with high variation in the level of LMP-1. EBV-negative and EBV-positive cell lines have been established from such tumors and except for one, the EBV carriers maintain the Type II EBV-gene expression pattern, with abundant LMP-1 expression (Tsuge et al., 1999; Zhang et al., 2003). Importantly, these tumor-derived lines are not autonomous. They require IL-2 for in vitro proliferation. In this respect they resemble normal NK cells. We found that LMP-1 expression in these lines depended on the supply of IL-2, thus it was coupled to the proliferation of the cells (Takahara et al., 2006). Importantly, other cytokines, such as IL-10 and interferon (IFN)-γ, could also induce LMP-1 expression, but they did not sustain cell proliferation. Concomitantly with LMP-1, the expression of CD25, the high affinity component of the IL-2 receptor, was increased. This may lead to a lower IL-2 concentration requirement for proliferation.
The following sequence of events can contribute to the development of NK/T lymphoma. The tumor occurs in sites with frequent infection. The inflammatory tissue may activate EBV genome carrying B cells for virus production. Through inter-cellular contact neighbouring T and NK cells may be infected. Activated T cells produce IL-2 and macrophages produce the related cytokine IL-15. In addition, the stroma tissue provides IL-10 that induces elevated expression of LMP-1 and of CD25 (IL-2R-α). As a consequence the IL-2-induced proliferation of the cells can be potentiated through efficient exploitation of the available IL-2 and IL-15 growth-promoting factors. This mechanism assigns an important role to LMP-1 in the development of the lymphoma that is always EBV-positive. It is important to note that unlike normal B lymphocytes in which LMP-1, together with other EBV-encoded proteins, drives cell proliferation, the role of EBV in the NK/T cells is confined to potentiate the response to the growth promoting cytokines.
In EBV-carrying lymphoid cells the expression of virally-encoded proteins occurs in various assortments that is regulated by the differentiation and maturation of the cell. The expression of these proteins determines the fate of the EBV-harbouring cell. Among the various types of expression strategies only type III cells (with six nuclear and three membrane-associated proteins) have autonomous proliferative potential. This occurs only in B lymphocytes.
EBV infection is ubiquitous in humans. In spite of the proliferation-inducing capacity of the virus in B cells, the viral carrier state is mostly harmless. This is possible because the immune system recognizes the cells that express the growth-promoting proteins. Normally, therefore such cells are eliminated but immunosuppressed individuals have a high risk for EBV-induced immunoblastic proliferations.
EBV may contribute, however, to other types of malignancies that arise in other cell types. These EBV-carrying cells are not driven to proliferation by the virus but their phenotype is changed. They acquire alterations in cellular interactions, production and response to cytokines that impose avoidance of apoptosis and enhanced response to growth stimulating signals.
Supported by funds from the Swedish Cancer Society, Sweden. LLK is recipient of Cancer Research Fellowship of Cancer Research Institute (New York)/Concern Foundation (Los Angeles).
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The nucleotide polymorphisms within the Epstein–Barr virus C and Q promoters from nasopharyngeal carcinoma affect transcriptional activity in vitro
European Archives of Oto-Rhino-Laryngology (2012)