Epstein-Barr Virus: Evasive Maneuvers in the Development of PTLD

Authors

  • Andrew L. Snow,

    1. Program in Immunology
    2. Department of Surgery/Division of Transplantation, Stanford University School of Medicine, Stanford, California, USA
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  • Olivia M. Martinez

    Corresponding author
    1. Program in Immunology
    2. Department of Surgery/Division of Transplantation, Stanford University School of Medicine, Stanford, California, USA
      * Corresponding author: Dr. Olivia M. Martinez, omm@stanford.edu
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* Corresponding author: Dr. Olivia M. Martinez, omm@stanford.edu

Abstract

Epstein-Barr virus (EBV) infection is linked to ∼90% of B-cell lymphomas associated with posttransplant lymphoproliferative disease (PTLD), a serious complication for immunosuppressed transplant recipients. In this paper, we review the myriad ways by which EBV can inadvertently drive the genesis and persistence of B-cell lymphomas, particularly when the antiviral immune response is compromised. Probing the basic mechanisms by which EBV infection proceeds and contributes to malignancy in such cases will hopefully improve our understanding and treatment of PTLD and other EBV-associated malignancies.

B-cell lymphomas associated with posttransplant lymphoproliferative disease (PTLD) continue to pose a serious problem for immunosuppressed transplant recipients. Depending on the organ transplanted, ∼1–20% of transplant recipients will develop such tumors (1). Over 90% of all PTLD cases are linked to Epstein-Barr virus (EBV), which plays a prominent role in the development of several types of B-cell malignancy. EBV was first discovered in 1964 by Epstein and colleagues, who identified a unique herpesvirus present in cultured tumor biopsies derived from Burkitt's lymphoma (BL) patients (2). As such, EBV became the first tumor virus candidate ever described in humans. Since its initial discovery, we now know that EBV is a B-lymphotropic, gammaherpesvirus that benignly infects over 95% of the human population for life, making it arguably one of the most successful viruses known to man (3). In recent years, numerous studies have shed light on how EBV persists in the face of a dedicated and robust host cellular immune response. EBV has evolved several immune evasion strategies designed to ensure that the virus coexists benignly with the immune system, but may inadvertently contribute to B-cell oncogenesis, including Burkitt's, Hodgkin's, PTLD and AIDS-related lymphomas. By investigating the nature of benign versus pathogenic EBV infection, basic scientists and clinicians can apply this knowledge to correctly describe the mechanisms underlying PTLD development, and thereby, identify meaningful therapeutic targets.

Immunological Detente: the Normal EBV Life Cycle

The life cycle of EBV in human B cells reveals how a successful herpesvirus has coevolved with the host immune system to exploit normal processes of B-cell development while maintaining a balance with the T-cell response directed against the virus (4). EBV establishes a lytic infection of naïve, tonsillar B cells in the follicular mantle, which also serves as the site for the production of infectious viral particles. Although a small number of infected B cells remain permissive for viral replication, EBV exists primarily as a latent infection within the host B cell. Following viral entry via CD21 on the B-cell surface, the EBV genome circularizes within 12–16 h, and the first of three possible latent gene transcription programs is initiated, known as type III latency or the “growth program” (Table 1) (5). Type III latency is characterized by the expression of the entire set of EBV latent genes, including Epstein Barr nuclear antigens (EBNAs) 1, 2, 3A, 3B, 3C, LP, latent membrane protein (LMP1, LMP2A, LMP2B), and the polyadenylated viral RNAs (EBERs 1 and 2). Expression of these genes promotes survival and expansion of infected naïve B-cell hosts that resemble activated B lymphoblasts. The unrestricted growth program also facilitates transformation of resting B cells in vitro and generation of immortalized lymphoblastoid cell lines (LCL). However, the outgrowth of transformed EBV+ B cells is normally controlled in vivo by EBV-specific cytotoxic T lymphocytes (CTL).

Table 1.  EBV latent gene transcription programs. EBV utilizes three distinct latent gene transcription programs over the course of its life cycle. Following initial lytic infection, naïve B cells in the tonsil exhibit type III latency (growth program), during which the expression of all nine latent genes promotes proliferation and survival without further differentiation. Once these cells exit the cell cycle, EBNA2 is downregulated and EBV switches to type II latency (default program) as cells migrate into follicles and differentiate into a germinal center cell phenotype. LMP2A and LMP1 are thought to functionally mimic the B-cell receptor and CD40, respectively, providing survival signals typically associated with antigen binding and T-cell help. Cells that survive this stage pass into the memory B-cell compartment, where EBV lies dormant by shutting down all latent genes except EBNA1, which is expressed during division to ensure maintenance of the EBV episome. This type I latent phenotype (latency program) allows EBV to escape CTL surveillance and persist in resting memory B cells for the lifetime of the host.
ProgramInfected B cellViral genes expressed
I (Latency)MemoryNone (EBNA1)
II (Default)Germinal centerEBNA1, LMP1, LMP2A
III (Growth)NaiveEBNA1-EBNA6 LMP1, LMP2A, LMP2B

From this stage, latently infected B lymphoblasts can undergo differentiation normally associated with a germinal center (GC) reaction. Infection switches to type II latency (‘default program’) by turning off expression of EBNA2, a master transactivator for latent gene transcription that mimics Notch signaling in its ability to block B-cell differentiation (6, 7). During type II latency, LMP1 and LMP2A expression provides the necessary signals required for B-cell differentiation independent of antigen-driven interactions with follicular dendritic cells (FDCs) or T helper (TH) lymphocytes. LMP2A actively suppresses signaling through the B-cell receptor to curtail growth, while simultaneously providing the tonic signals required for B-cell survival by co-opting Syk and Src-family kinases (8). LMP1 is a functional mimic of CD40, delivering potent survival signals to B cells normally provided through T cell help (CD154 on TH cells) (9). Both viral proteins are constitutively active and signal independently of any ligands, utilizing several transmembrane domains to aggregate in lipid rafts. Hence EBV encodes two mediators for the survival of infected B cells through differentiation into a pool of long-lived memory B cells, where EBV persists for the lifetime of the host. In peripheral memory B cells, no latent genes are expressed (type I latency or ‘latency program’) allowing the host cells to escape immunosurveillance by EBV-specific CTL, explaining the nonpathogenic nature of EBV infection in most healthy carriers. Only the poorly immunogenic EBNA1 is expressed periodically in memory B cells to ensure passage of the viral episome during cell division. Expression of LMP1 and LMP2A has been detected in recirculating tonsillar memory B cells, however, which may assist in ensuring long-term survival of this latently infected memory B-cell pool (10). The exact mechanism for how EBV switches between different latent programs, however, remains unknown.

Unintended Consequences: EBV in Disease and Malignancy

Despite the balance struck between EBV persistence and the immune response in healthy individuals, EBV is associated with several diseases in which this detente is disrupted (Table 2). EBV has only been proven to play a definitive causative role in the etiology of two diseases: X-linked lymphoproliferative (XLP) syndrome and infectious mononucleosis (IM). In XLP, affected males are unable to combat proliferation of EBV-infected B cells due to a mutation in the gene encoding SAP (signaling lymphocyte activation molecule [SLAM]-associated molecule), a T-cell surface protein thought to be required for normal T–B cell interactions (11). IM is a short-lived lymphoproliferative disease associated with primary EBV infection during adolescence. A huge proportion of memory B cells become infected with EBV (∼50%), countered later by an overwhelming proliferation of EBV-specific T cells that can also approach 50% of the patient's peripheral T-cell pool (12). Our understanding of both diseases underscores the importance of a dedicated EBV-specific T-cell response in keeping the virus in check throughout the life of healthy carriers.

Table 2.  EBV-associated diseases. EBV is associated with several diseases characterized by lymphoproliferation or overt malignancy. Immunosuppression or immunodeficiency can drive XLP-, PTLD- and AIDS-associated B-cell lymphomas, which debilitates the CTL response and allows infected cells to constitutively express all latent genes. Other malignancies may be representative of inappropriate infection (NPC) or infection at the wrong stage of B-cell development (IM), when the target cell cannot exit the cell cycle.
DiseaseEBV latencyCellular origin
Infectious mononucleosis (IM)III, lyticNaïve, memory B cells (tonsil)
XLPIIINaïve
Burkitt's lymphomaIGC or memory B cells
Hodgkin's lymphomaIIGC B cells
PTLDIIIVariable
AIDS-associated B cell lymphomaIIIVariable
Nasopharyngeal carcinoma (NPC)I or IIOropharyngeal epithelium

EBV is also strongly associated with several malignancies. Indeed, EBV was first unveiled in tumor cells derived from patients with BL, a malignancy endemic to equatorial Africa where EBV is associated with 95–100% of all cases (13). The hallmark of BL is a reciprocal chromosomal translocation for which the c-myc oncogene is positioned near one of the immunoglobulin (Ig) loci, resulting in dysregulated c-myc expression. Most EBV+ BLs resemble a ‘centroblast’ phenotype (based on Ig hypermutation) and exhibit type I viral latency, where only EBNA1 is expressed. The role of EBV in the development of BL remains unresolved.

EBV infection has also been connected to classical Hodgkin's lymphoma, where EBV DNA can be detected in 40–60% of Hodgkin and Reed–Sternberg tumor cells (14). Such tumor cells are thought to arise from ‘crippled’ EBV-infected GC B cells that fail somatic hypermutation, consistent with the expression of type II latency genes (EBNA1, LMP1, LMP2A) in this compartment (15). EBV infection is also implicated in the pathogenesis of nasopharyngeal carcinomas (NPCs) prevalent in Southeast Asia. Most NPCs present as undifferentiated epithelial carcinomas that usually exhibit a type II latent EBV infection (16). The exact role of EBV in the development of NPC, some gastric carcinomas, and rare cases of EBV+ natural killer (NK) and T cell lymphomas remains elusive.

PTLD is characterized by the development of EBV-infected B-cell lymphomas in immunosuppressed transplant recipients (17). PTLD represents a spectrum of disorders ranging from B-cell hyperplasia to monoclonal B-cell lymphoma, over 90% of which are EBV+. PTLD-associated lymphoma cells may derive from B cells other than naïve tonsillar cells that become infected but cannot exit the cell cycle after the growth program is expressed. The immunosuppressive drugs used to prevent graft rejection debilitate EBV-specific T cells and permit the proliferation and lymphomagenesis of these type III infected B cells. Therefore, PTLD-associated B-cell lymphomas display an activated B-cell phenotype and express the entire set of EBV latent genes and closely resemble EBV+ LCL derived in vitro. EBV infection is of particular concern in pediatric transplant recipients, since primary infection of seronegative transplant patients is more likely to lead to PTLD. Other risk factors include age after transplant, severity of immunosuppression and concurrent CMV infection (18). Currently, there are no laboratory assays that can predict development of PTLD in at-risk patients. Measurement of EBV load is the most commonly utilized laboratory test for diagnostic purposes. Emerging studies indicate that direct quantitation of EBV-specific T-cell responses via ELISPOT or tetramer staining may prove beneficial in monitoring for the onset of EBV disease (1). Treatment of PTLD primarily involves the reduction of immunosuppressive therapy to restore the CTL response, with the unfortunate consequence of increasing the risk of graft rejection. Conversely, potent immunosuppression may select for highly proliferative B-cell lymphomas that cannot be brought to regression later by competent EBV-specific CTL. Thus PTLD represents another dangerous consequence of disturbing the balance between EBV infection in B cells and the T-cell response. Not surprisingly, phenotypically identical EBV-associated B lymphoblastoid tumors have been described in AIDS patients as well. It is in the context of studying EBV-associated cancers, particularly those exhibiting type III latency, that we begin to understand how such lymphomas develop and persist when evasive strategies exercised by EBV are left unchecked.

The Balancing Act: EBV and Immune Evasion

Herpesviruses are particularly adept at evading immune responses, explained in part by their large genomes, which accommodate genes encoding functional homologs of cellular factors involved in cell-cycle regulation, inhibition of apoptosis and signal transduction. These proteins presumably help the virus to survive and replicate in the midst of a vigorous immune response, and may inadvertently contribute to host cell transformation in the process (Figure 1).

Figure 1.

EBV and immune evasion. Lytic cycle genes BCRF1 (viral IL-10) and BARF1 (sCSF-R) aid in blunting T-cell responses by suppressing antiviral cytokine production. BHRF1, a homolog of Bcl-2, preserves mitochondrial membrane potential and contributes to apoptosis resistance. Latent genes (EBNA1, EBNA2, LMP2A, LMP1) also protect the host B cell from multiple apoptotic stimuli, mediated by p53, Nur77, BCR and DR signals. For example, LMP1-mediated NF-κ B activation upregulates several antiapoptotic genes capable of blocking intrinsic and extrinsic cell death pathways.

Cytokine modulation

One such factor expressed during the lytic phase of EBV infection is BCRF1, a viral homolog of IL-10 (vIL-10) that is highly homologous to its human counterpart despite binding to the IL-10 receptor with reduced affinity. Significant amounts of vIL-10 can be detected in serum of PTLD and IM patients, although the consequences of systemic vIL-10 levels are not known (19, 20). Like human IL-10, vIL-10 can potently suppress Th1 immune responses through the inhibition of IL-12 and IFN-γin vitro (21). On the other hand, a single amino acid change in the vIL-10 sequence removes its capacity to stimulate certain immune cells, suggesting this tailored cytokine serves as an effective immunosuppressant against the cellular immune response (22). It remains unclear whether vIL-10 is necessary for potentiating B-cell transformation. Whether or not vIL-10 directly participates in potentiating B-cell transformation, we propose that local vIL-10 secretion suppresses T-cell responses during the transition from lytic to latent infection until cellular IL-10 is induced following B-cell transformation. Several studies have established that cellular IL-10 is induced in EBV-transformed cells through LMP1 signaling and/or the function of EBV-encoded RNAs. We have previously shown cellular IL-10 is a critical autocrine growth factor for EBV-transformed B-cell lymphomas in vitro (19), and the abundant presence of IL-10 in serum from PTLD patients also implicates it in EBV-mediated PTLD pathogenesis in vivo.

During lytic infection, EBV also expresses a soluble receptor for colony stimulating factor 1 (CSF-1) called BARF1. Although BARF1 appears to act as an oncogene when expressed ectopically in certain cell types, it is not clear if BARF1 contributes directly to EBV tumorigenicity in B cells, considering a BARF1-deletion mutant of EBV maintains the same transformation potential as wild-type virus (23). However, BARF1 represents another weapon for immune evasion in its ability to block the function of CSF-1 in stimulating monocyte proliferation and cytokine release. Thus BARF1 may blunt cellular immune responses by interfering with secretion of antiviral factors like IFN-α.

Apoptosis resistance

EBV has also evolved mechanisms for combating host-cell apoptosis following infection. For example, the lytic cycle gene BHRF1 encodes a homolog of Bcl-2 capable of inhibiting apoptosis induced by multiple stimuli, including Fas ligand (FasL) and TNF-related apoptosis-inducing ligand (TRAIL) (24). Earlier studies have established that EBV latent genes can also protect B cells from apoptosis in vitro in response to serum withdrawal (25). Much work on the transforming capability of EBV has focused on LMP1, an oncogene that is indispensable for EBV-mediated transformation of B cells (26). LMP1 upregulates the expression of several antiapoptotic genes implicated in protection from intrinsic apoptosis pathways, including bcl-2, bfl-1, mcl-1, A20, and cIAP2 (27). All of these genes represent downstream targets of NF-κB: indeed, interfering with LMP1-mediated NF-κB activation promotes spontaneous apoptosis in EBV-immortalized B cells (28, 29).

Other EBV latent proteins can also interfere with certain apoptotic stimuli. For example, EBNA2 can specifically block apoptosis triggered by Nur77, a nuclear hormone receptor that induces cytochrome C release from mitochondria upon translocation to the cytoplasm (30). Recent work indicates LMP2A signaling also promotes B-cell survival through constitutive activation of the Ras/PI3K/Akt signaling axis, which represents a potent prosurvival signal in B cells (31). Young and colleagues have demonstrated that LMP1 also activates the Akt pathway, and that LMP2A can enhance the function of LMP1 by prolonging its half-life, suggesting that coexpression of these molecules during type II/III latency provides a powerful antiapoptotic signal to the host B cell (32, 33). Even EBNA1, known primarily for its role in EBV episome maintenance, contributes to cell survival by binding ubiquitin-specific protease 7 (USP7) and preventing p53 stabilization by deubiquitination (34). This unique mode of interference with p53 function explains why EBNA1 inhibition induces apoptosis in BL (35).

EBV may also perturb extrinsic apoptosis stimuli triggered through death receptors (DRs). We recently discovered that EBV itself can protect latently infected BJAB B lymphoma cells from apoptosis induced through Fas and TRAIL receptors, mediated in part by LMP1-driven upregulation of cellular FLICE inhibitory protein (cFLIP), another NF-κB-responsive gene (36). This phenomenon may be particularly relevant to Hodgkin's lymphomagenesis, where LMP1/LMP2A may cooperate to rescue the crippled GC cells that are otherwise destined to die. Hitherto, work from our laboratory and others indicated that EBV-infected LCL show differential sensitivity to Fas-induced apoptosis (37–39). We have also determined that EBV+ LCLs derived from PTLD patients show universal resistance to TRAIL-induced apoptosis (40). Variable DR apoptosis sensitivity among EBV+ B cell lines may be explained by differential latent gene expression, natural sequence variants with differing intrinsic signaling properties (e.g. LMP1) or derivation from myriad sources of host B cells; parameters that remain to be addressed experimentally. Taken together, an expanding body of work indicates that latent EBV infection, and especially LMP1 expression, can subvert important immune effector pathways of target cell elimination by inhibiting apoptosis induced by numerous stimuli. Using MHC/peptide tetramers we previously established that solid organ transplant recipients have a significant proportion of EBV-specific CD8+ T cells comparable to healthy seropositive individuals (41). The fact that some PTLD lymphomas persist or relapse even in the presence of EBV-specific CTL may be due in part to apoptosis resistance, particularly if more potent cytotoxic effector molecules (perforin, granzymes) are also rendered ineffective (42, 43).

Applying our Knowledge: Implications for Treatment of PTLD

Given the evasive tactics employed by EBV as part of its interaction with the immune system, it is imperative that therapeutic options for PTLD be evaluated with these aspects of basic virology in mind. At present, several strategies for the treatment of PTLD are being implemented in the clinic. As mentioned earlier, removal of immunosuppression is often the first approach utilized to combat PTLD lymphomas, which allows the CTL response to rebound. However, risk of graft rejection and selection for highly proliferative monoclonal lymphoma cells leave this approach far from ideal. An alternative may be found in using immunosuppressive drugs that protect the graft without promoting lymphoma outgrowth, such as rapamycin or everolimus. Both of these drugs effectively retard the growth of PTLD-associated lymphomas in vitro and in xenografted SCID mice (44–46). It remains to be determined if the potential antitumor benefits of mTOR inhibitors are manifest in clinical transplantation.

Cytokine- and antibody-based immunotherapy

Immunotherapy directly targeting B cells and associated cytokines must also be considered. Regimens involving the addition of cytokines like IFN-α or the neutralization of B-cell growth factors like IL-6 have shown some promise, although caution must be exercised in disturbing cytokine balances systemically. Notwithstanding the potential salutary effect on graft survival, the importance of IL-10 to proliferation of EBV+ B cell lymphomas and suppression of antiviral CTL responses suggests neutralization or inhibition of IL-10 may also prove beneficial. Our own work indicating rapamycin inhibits the growth of PTLD-associated lymphomas in part by decreasing IL-10 secretion underscores IL-10 as a pertinent target (45). Clinicians have also turned to the humanized anti-CD20 mAb rituximab, which nonspecifically depletes virtually all B cells and can elicit strong anti-B-cell tumor responses. Although a thorough description of long-term effects arising from systemic B-cell depletion using this reagent awaits further study, rituximab represents an important tool for eliminating CD20+ lymphoma cells in PTLD patients.

Cellular immunotherapy

Recently, much attention has focused on cellular immunotherapy for combating PTLD-associated tumors by infusing EBV-specific CTL. Although posthuman stem cell transplant patients receiving polyclonal EBV-specific CTL infusions prophylactically or in response to overt PTLD have fared well, efficacy is mixed in solid organ transplant patients, perhaps due to the presence of debilitating immunosuppressive drugs and the limited survival and expansion of the infused CTL in lymphoid-replete recipients (47). In the context of EBV latency, infused CTL may also fail to control EBV+ lymphomas due to local cytokine-based suppression, resistance to CTL-induced apoptotic stimuli and/or downregulation of relevant EBV protein epitopes. Hence we suggest that subversion of T-cell immunity by EBV not only contributes to lymphomagenesis, but can also foil attempts to regain control of EBV+ lymphoma growth using autologous or allogeneic CTL therapy.

The multipronged approach: Targeting EBV itself

Indeed, effective treatment of PTLD-associated tumors may not be achieved with monotherapies. For instance, our own findings prompt careful evaluation of the utility of recombinant TRAIL a promising anticancer reagent, as a potential single therapeutic for EBV-associated B-cell lymphomas (40). As with other malignancies, PTLD therapy may require a multipronged approach involving a combination of surgery, radiation, chemotherapy (e.g. CHOP), and reagents that target host B cells or boost T-cell immunity. More importantly, basic research informs us that successful approaches to eradicating PTLD-associated lymphomas must also focus on EBV infection itself. Antiviral agents such as acyclovir only target the lytic phase of viral replication, but recent work reminds us that the importance of lytic gene expression to the establishment of PTLD should not be ignored (48). Specifically disrupting the function of latent genes like LMP1 and LMP2A will likely prove most efficacious in combating PTLD and associated EBV+ type II/III latent cancers. The propensity of reagents that silence latent gene expression or block relevant signaling pathways (i.e. NF-κB and proteosome inhibitors) to successfully kill such lymphomas in vitro has already been established (28, 29, 49). In conclusion, such studies demonstrate that we must redouble efforts to introduce such EBV-specific therapies into the clinic and regain the upper hand against such a ‘clever’ virus in eradicating PTLD.

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