Spontaneous Lymphoblastoid Cell Lines Research Articles

Combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Epstein-Barr virus (EBV) manipulates the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and survival of the host cells through the expression of viral proteins in distinct latency patterns. We postulate that the combination of bortezomib (proteasome inhibitor) and venetoclax (Bcl-2 inhibitor) [bort/venetoclax] will cause synergistic killing of post-transplant lymphoproliferative disorder (PTLD) through targeting the pro-survival function of latent viral proteins such as latent membrane protein-1 (LMP-1) and EBV nuclear antigen-3C (EBNA-3C). Bort/venetoclax could synergistically kill spontaneous lymphoblastoid cell lines (sLCLs) derived from patients with PTLD and EBV-associated hemophagocytic lymphohistiocytosis by inducing DNA damage response, apoptosis and G1-S cell cycle arrest in a ROS-dependent manner. Bortezomib potently induced the expression of Noxa, a pro-apoptotic initiator and when combined with venetoclax, inhibited Mcl-1 and Bcl-2 simultaneously. Bortezomib prevented LMP-1 induced proteasomal degradation of IκBα leading to the suppression of the NF-κB signaling pathway. Bortezomib also rescued Bcl-6 from EBNA-3C mediated proteasomal degradation thus maintaining the repression of cyclin D1 and Bcl-2 causing G1-S arrest and apoptosis. Concurrently, venetoclax inhibited Bcl-2 upregulated by either LMP-1 or EBNA-3C. Bort/venetoclax decreased the expression of phosphorylated p65 and Bcl-2 at serine 70 thereby suppressing the NF-κB signaling pathway and promoting apoptosis, respectively. These data corroborated the marked suppression of the growth of xenograft of sLCL in SCID mice (p<0.001). Taken together, the combination of bortezomib and venetoclax targets the pro-survival function of LMP-1 and EBNA-3C of Epstein-Barr virus in spontaneous lymphoblastoid cell lines.

Multiple sclerosis patient-derived spontaneous B cells have distinct EBV and host gene expression profiles in active disease.

Epstein-Barr virus (EBV) is an aetiologic risk factor for the development of multiple sclerosis (MS). However, the role of EBV-infected B cells in the immunopathology of MS is not well understood. Here we characterized spontaneous lymphoblastoid cell lines (SLCLs) isolated from MS patients and healthy controls (HC) ex vivo to study EBV and host gene expression in the context of an individual's endogenous EBV. SLCLs derived from MS patient B cells during active disease had higher EBV lytic gene expression than SLCLs from MS patients with stable disease or HCs. Host gene expression analysis revealed activation of pathways associated with hypercytokinemia and interferon signalling in MS SLCLs and upregulation of forkhead box protein 1 (FOXP1), which contributes to EBV lytic gene expression. We demonstrate that antiviral approaches targeting EBV replication decreased cytokine production and autologous CD4+ T cell responses in this ex vivo model. These data suggest that dysregulation of intrinsic B cell control of EBV gene expression drives a pro-inflammatory, pathogenic B cell phenotype that can be attenuated by suppressing EBV lytic gene expression.

EBNA1 Inhibitors Block Proliferation of Spontaneous Lymphoblastoid Cell Lines From Patients With Multiple Sclerosis and Healthy Controls.

Epstein-Barr virus (EBV) is a ubiquitous herpesvirus that establishes lifelong latency in memory B cells and has been identified as a major risk factor of multiple sclerosis (MS). B cell depletion therapies have disease-modifying benefit in MS. However, it is unclear whether this benefit is partly attributable to the elimination of EBV+ B cells. Currently, there are no EBV-specific antiviral therapies available for targeting EBV latent infection in MS and limited experimental models to study EBV in MS. In this study, we describe the establishment of spontaneous lymphoblastoid cell lines (SLCLs) generated ex vivo with the endogenous EBV of patients with MS and controls and treated with either an Epstein-Barr virus nuclear antigen 1 (EBNA1) inhibitor (VK-1727) or cladribine, a nucleoside analog that eliminates B cells. We showed that a small molecule inhibitor of EBNA1, a critical regulator of the EBV life cycle, blocks the proliferation and metabolic activity of these SLCLs. In contrast to cladribine, a highly cytotoxic B cell depleting therapy currently used in MS, the EBNA1 inhibitor VK-1727 was cytostatic rather than cytotoxic and selective for EBV+ cells, while having no discernible effects on EBV- cells. We validate that VK-1727 reduces EBNA1 DNA binding at known viral and cellular sites by ChIP-qPCR. This study shows that patient-derived SLCLs provide a useful tool for interrogating the role of EBV+ B cells in MS and suggests that a clinical trial testing the effect of EBNA1 inhibitors in MS may be warranted.

Combination of Bortezomib and Venetoclax Induces Synergistic Killing of Epstein-Barr Virus-Driven Lymphoproliferative Diseases By Targeting the Pro-Survival Function of Latent Membrane Protein-1 and Epstein-Barr Nuclear Antigen-3C

Epstein-Barr virus (EBV) is strongly associated with lymphoproliferative diseases (LPDs), in particular B cell, in both immunocompetent and immunocompromised hosts. The virus is able to manipulate host cell machineries such as the ubiquitin-proteasome system and regulators of Bcl-2 family to enable the persistence of the virus and the survival of the host cells through expression of various viral proteins in distinct latency patterns. Latent membrane protein-1 (LMP-1) is a constitutively active CD40 receptor homolog and activates NF-κB signaling pathways to induce Bcl-2 expression whilst Epstein-Barr nuclear antigen-3C (EBNA-3C) interacts with and mediates proteasomal degradation of Bcl-6 protein which, in turn, increases Bcl-2 expression. We hypothesize that combining a proteasome inhibitor, bortezomib, with a Bcl-2 inhibitor, venetoclax, will induce synergistic killing of EBV-driven LPDs expressing both LMP-1 and EBNA-3C in the latency III pattern. Isobologram showed that combination of bortezomib and venetoclax could synergistically induce potent apoptosis of spontaneous lymphoblastoid cell lines (sLCL), derived from patients with post-transplant lymphoproliferative disorder (PTLD), expressing LMP-1 and EBNA-3C proteins. The mechanism of killing was related to the suppression of NF-κB signaling pathway induced by LMP-1. The phosphorylation of serine 70 of Bcl-2, which enhances the anti-apoptotic activity of Bcl-2 through stabilization of its interaction with other pro-apoptotic proteins such as Bak and Bim, was decreased in the sLCL, but not in the LMP-1 or EBNA-3C knockdown LCL, upon treatment with the drug combination. Activation of DNA damage response and production of reactive oxygen species were observed in the sLCL, contributing to the synergistic cell death. Bortezomib induced the expression of pro-apoptotic initiator, NOXA, to enhance the susceptibility of the sLCL to apoptosis upon treatment with venetoclax whilst knockdown of NOXA in the sLCL led to the resistance of the cells to apoptosis induced by the drug combination. In-vivo study demonstrated that the drug combination significantly inhibited the growth of xenograft of sLCL in SCID mice (p&amp;lt;0.001). Taken together, we conclude that the combination of bortezomib and venetoclax induces synergistic killing of EBV-driven LPDs such as PTLD by targeting the pro-survival function of LMP-1 and EBNA-3C. Disclosures No relevant conflicts of interest to declare.

Spontaneous lymphoblastoid cell lines from patients with Epstein-Barr virus infection show highly variable proliferation characteristics that correlate with the expression levels of viral microRNAs

The Epstein-Barr virus (EBV) induces B-cell proliferation with high efficiency through expression of latent proteins and microRNAs. This process takes place in vivo soon after infection, presumably to expand the virus reservoir, but can also induce pathologies, e.g. an infectious mononucleosis (IM) syndrome after primary infection or a B-cell lymphoproliferation in immunosuppressed individuals. In this paper, we investigated the growth characteristics of EBV-infected B-cells isolated from transplant recipients or patients with IM. We found that these cells grew and withstood apoptosis at highly variable rates, suggesting that the expansion rate of the infected B-cells widely varies between individuals, thereby influencing the size of the B-cell reservoir and the ability to form tumors in infected individuals. All viruses investigated were type 1 and genetically close to western strains. EBV-infected B-cells expressed the transforming EBV latent genes and microRNAs (miRNAs) at variable levels. We found that the B-cell growth rates positively correlated with the BHRF1 miRNA levels. Comparative studies showed that infected B-cells derived from transplant recipients with iEBVL on average expressed higher levels of EBV miR-BHRF1 miRNAs and grew more rapidly than B-cells from IM patients, suggesting infection by more transforming viruses. Altogether, these findings suggest that EBV infection has a highly variable impact on the B-cell compartment that probably reflects the genetic diversity of both the virus and the host. It also demonstrates the unexpected finding that B-cells from different individuals can grow at different speed under the influence of the same virus infection.

Epstein-Barr virus genetic variation in lymphoblastoid cell lines derived from Kenyan pediatric population.

Epstein-Barr virus (EBV) is associated with Burkitt’s lymphoma (BL), and in regions of sub-Saharan Africa where endemic BL is common, both the EBV Type 1 (EBV-1) and EBV Type 2 strains (EBV-2) are found. Little is known about genetic variation of EBV strains in areas of sub-Saharan Africa. In the present study, spontaneous lymphoblastoid cell lines (LCLs) were generated from samples obtained from Kenya. Polymerase chain reaction (PCR) amplification of the EBV genome was done using multiple primers and sequenced by next-generation sequencing (NGS). Phylogenetic analyses against the published EBV-1 and EBV-2 strains indicated that one sample, LCL10 was closely related to EBV-2, while the remaining 3 LCL samples were more closely related to EBV-1. Moreover, single nucleotide polymorphism (SNP) analyses showed clustering of LCL variants. We further show by analysis of EBNA-1, BLLF1, BPLF1, and BRRF2 that latent genes are less conserved than lytic genes in these LCLs from a single geographic region. In this study we have shown that NGS is highly useful for deciphering detailed inter and intra-variations in EBV genomes and that within a geographic region different EBV genetic variations can co-exist, the implications of which warrant further investigation. The findings will enhance our understanding of potential pathogenic variants critical to the development and maintenance of EBV-associated malignancies.

Autoimmune Hemolytic Anemia Accompanied by Reactivation of an Epstein-Barr Virus Infection with Suppressed CTL Response to EBV-infected Cells in an Elderly Man

An 88-year-old man with autoimmune hemolytic anemia (AIHA) who had been treated with low dose prednisolone developed a sudden worsening of his anemia accompanied by reactivation of Epstein-Barr virus (EBV). We established EBV-infected spontaneous lymphoblastoid cell lines (LCL), performed an enzyme-linked immunosorbent spot assay, and confirmed a significantly suppressed EBV-specific cytotoxic T-cell (CTL) response to the LCL. EBV reactivation might have been brought about by suppressed CTL activity which could have been due to low dose PSL administration or aging. Since the EBV-DNA titer decreased as AIHA improved, we concluded that EBV might have played a role in the development of anemia.

EBV+ B Lymphoma Cell Lines from Patients with Post-Transplant Lymphoproliferative Disease Are Resistant to TRAIL-Induced Apoptosis

Lymphomas associated with post-transplant lymphoproliferative disease (PTLD) represent a significant complication of immunosuppression in transplant recipients. In immunocompetent individuals, EBV-specific cytotoxic T lymphocytes (CTL) prevent the outgrowth of activated B lymphoblasts through apoptosis induction. Soluble versions of TNF-related apoptosis-inducing ligand/Apo2 ligand (TRAIL) can induce apoptosis in numerous tumor cell types. Given the therapeutic potential of TRAIL, we examined the sensitivity of EBV+ spontaneous lymphoblastoid cell lines (SLCL) derived from patients with PTLD to treatment with soluble TRAIL. Despite abundant expression of TRAIL receptors (TRAIL-R), resistance to TRAIL-induced apoptosis was observed in all SLCL examined. This resistance could not be overcome by concomitant treatment with several pharmacological agents. Unlike BJAB positive control cells, for each SLCL tested, cleavage and activation of caspase 8 was inhibited due to failed recruitment of FADD and caspase 8 to TRAIL receptors upon stimulation. Further indicative of a proximal defect, TRAIL receptor aggregation could not be detected on the cell surface of SLCL following ligand engagement. These results suggest that the use of TRAIL for eliminating PTLD-associated tumors may be of limited clinical utility, and illustrate another mechanism by which EBV+ B lymphoma cells can evade tumor surveillance at the level of death receptor signaling.

Determining virological, serological and immunological parameters of EBV infection in the development of PTLD.

Post-transplant lymphoproliferative disease (PTLD) in Epstein-Barr virus (EBV) seronegative solid organ transplant recipients remains a significant problem, particularly in the first year post-transplant. Immune monitoring of a cohort of high-risk patients indicated that four EBV seronegative transplant recipients developed early-onset PTLD prior to evidence of an EBV humoral response. EBV status has been classically defined serologically, however these patients demonstrated multiple parameters of EBV infection, including the generation of EBV-specific CTL, outgrowth of spontaneous lymphoblastoid cell lines, and elevated EBV DNA levels, despite the absence of a classic EBV antibody response. As EBV serology is influenced by both immunosuppression and cytomegalovirus immunoglobulin treatment, both the EBV-specific CTL response and elevated EBV levels are more reliable indicators of EBV infection post-transplant.

Constitutive activation of Jak/STAT proteins in Epstein-Barr virus-infected B-cell lines from patients with posttransplant lymphoproliferative disorder.

Posttransplant lymphoproliferative disease (PTLD) is a major complication after bone marrow and solid organ transplantation. The disease encompasses a spectrum of abnormal, Epstein-Barr virus (EBV)-associated B-cell proliferations. We have previously shown that EBV-infected, spontaneous lymphoblastoid cell lines (SLCL) derived from PTLD patients require autocrine interleukin (IL)-10 to proliferate. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of the Jak/STAT signaling pathway proteins were analyzed in three different SLCL, termed JB7, MF4, and VB4. The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblot. Activated STAT dimer formation was determined by electromobility shift assays. All three SLCL, but not the Daudi Burkitt's lymphoma B-cell line, expressed the four known Jak kinases constitutively tyrosine phosphorylated, with particularly high levels of P-tyr Jak1 in the JB7 line. STAT1 and STAT3, but not STAT2 or STAT5, are also constitutively activated in all SLCL. The ability of the activated STAT proteins to form DNA-binding dimers was confirmed by electromobility shift assay. The SLCL, but not the Daudi line, express activated STAT complexes composed of STAT1 and STAT3. Another EBV-infected B-cell line, isolated from a lymph node biopsy after kidney transplantation, is phenotypically similar to the other SLCL in both surface antigen and activated STAT1 and STAT3 expression. These data support the presence of a constitutively active autocrine signaling pathway consistent with IL-10 in the SLCL.

Resistance to Fas-mediated apoptosis in EBV-infected B cell lymphomas is due to defects in the proximal Fas signaling pathway.

Post-transplant lymphoproliferative disorder is characterized by the outgrowth of EBV-infected B cell lymphomas in immunosuppressed transplant recipients. Using a panel of EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from post-transplant lymphoproliferative disorder patients, we assessed the sensitivity of such lymphomas to Fas-mediated cell death. Treatment with either an agonist anti-Fas mAb or Fas ligand-expressing cells identifies two subsets of SLCL based on their sensitivity or resistance to Fas-driven apoptosis. Fas resistance in these cells cannot be attributed to reduced Fas expression or to mutations in the Fas molecule itself. In addition, all SLCL are sensitive to staurosporine-induced cell death, indicating that there is no global defect in apoptosis. Although all SLCL express comparable levels of Fas signaling molecules including Fas-associated death domain protein, caspase 8, and caspase 3, Fas-resistant SLCL exhibit a block in Fas-signaling before caspase 3 activation. In two SLCL, this block results in impaired assembly of the death-inducing signaling complex, resulting in reduced caspase 8 activation. In a third Fas-resistant SLCL, caspase 3 activation is hindered despite intact death-inducing signaling complex formation and caspase 8 activation. Whereas multiple mechanisms exist by which tumor cells can evade Fas-mediated apoptosis, these studies suggest that the proximal Fas-signaling pathway is impeded in Fas-resistant post-transplant lymphoproliferative disorder-associated EBV(+) B cell lymphomas.

Gene-modified spontaneous Epstein-Barr virus-transformed lymphoblastoid cell lines as autologous cancer vaccines: mutated p21 ras oncogene as a model.

The transfer of genes coding for tumor antigens (Ags) into Ag-presenting cells is a promising strategy to develop cancer vaccines for patients not limited by major histocompatibility complex restriction. To test whether autologous lymphoblastoid cell lines (LCL) can be used as an alternative to dendritic cells for Ag presentation, we established LCL by spontaneous growth in cyclosporin-containing medium in vitro (SP-LCL). SP-LCL, which have an advantage over B95-8-transfected LCL in that they carry no risk of introducing a new infectious agent when used for vaccination, served as a permanent source for transfection with an episomal Epstein-Barr virus-based expression vector encoding the Ki-ras p21 oncogene carrying a point mutation at codon 12 (muRas) as a model tumor Ag. The ability of muRas-transfected SP-LCL (muRas-LCL) to induce immunoreactivity was determined in vitro. After electroporation with an optimized protocol, muRas-LCL expressed mutated ras peptides for a considerable period of time (at least 8 weeks) on the cell surface. The transfection procedure did not affect the expression of the costimulatory molecules B7.1, intercellular adhesion molecule-1, and leukocyte function associated Ag-3 by SP-LCL. muRas-LCL were able to induce muRas-specific cytotoxic T lymphocytes from three of three healthy donors and one of one patient with pancreatic carcinoma. Our results suggest that the gene transfer of muRas sequences to SP-LCL leads to an endogenous processing of this Ag in the major histocompatibility complex class I pathway and to functional presentation of antigenic peptides to CD8+ T lymphocytes. Autologous SP-LCL can serve as an unlimited renewable source for autologous cellular vaccines and appear to be good candidates as presenters for a wide range of human tumor Ags.

Increased usage of TCR V-beta8 in kidney transplant recipients with aberrant immune reconstitution and clinical complications.

The efficiency of immunosuppressive drugs prescribed after organ transplantation is mostly monitored through clinical and biological signs of organ rejection or infection. However, it may be expected that some patients develop subtle alterations of their reconstituting immune system, not immediately associated with clinical events. Identification of such anomalies could be useful to alert clinicians for possible future complications. A systematic follow-up of peripheral lymphocyte subsets, performed in a cohort of 89 kidney transplant recipients, identified severely skewed CD4/CD8 ratios in 32 patients. For 19 patients, the expression of specific T cell receptor fragments was examined using a panel of 10 monoclonal antibodies. Abnormal control of spontaneously Epstein Barr virus-infected B cells was tested by investigating for the generation of spontaneous lymphoblastoid cell lines in 17 cases. The incidence of rejection and infectious episodes was monitored. A bias in T cell receptor fragments usage was detected in 14/19 cases, involving Vbeta8 in all cases. Spontaneous lymphoblastoid cell lines of Epstein Barr positive B blasts developed in 9 of 17 cases. Eleven patients had early rejection episodes and 16 presented with viral primo-infection or reactivation. The incidence of rejection and infectious episodes was higher in the group of 32 patients who developed such abnormal patterns than in the 57 who did not. Transient bias in the T cell receptor repertoire may be observed during immune reconstitution after kidney transplantation, perhaps related to abnormal lymphocyte functions and associated to an impaired control of rejection and/or infectious agents.

AUTONOMOUS GROWTH OF EBV TRANSFORMED B CELL LYMPHOMAS IN PATIENTS WITH PTLD MAY INVOLVE CONSTITUTIVELY ACTIVATED CYTOKINE SIGNALING PATHWAYS

163 Post transplant lymphoproliferative disease (PTLD) is a major complication of the immunosuppression required for human organ transplantation. The disease encompasses a spectrum of abnormal, EBV-associated B cell proliferations. We have previously shown that EBV-infected spontaneous lymphoblastoid cell lines (SLCL) derived from patients with PTLD secrete IL-10, IL-6, TNF-α and LT-α, and more significant, require autocrine IL-10 for sustained growth. Using a ribonuclease protection assay, we found that these cells also express RNA for IL-9, IL-13, IL-15, and IFN-γ. To determine if cytokine signal transduction is involved in the autonomous growth of the SLCL, the activation states of proteins of the Jak/STAT cytokine signaling pathway were analyzed in three different SLCL, termed JB7, MF4 and VB4. The tyrosine phosphorylation (P-tyr) states of the Janus kinases (Jaks) and signal transducers and activators of transcription (STAT) proteins were examined by immunoprecipitation and immunoblotting. The JB7 line expresses a high level of constitutively P-tyr Jak1. All three SLCL, but not the Daudi Burkitt's lymphoma B cell line also express Jak2, Jak3 and Tyk2 constitutively phosphorylated on tyrosine residues. STAT1 and STAT3, but not STAT2 or STAT5, were also found to be constitutively phosphorylated in each of the SLCL. Activation of the STAT proteins was confirmed by electromobility shift assay. All three SLCL, but not the Daudi line, express DNA-binding STAT complexes that were identified as STAT1 and STAT3 by antibody supershift experiments. These data support the presence of a constitutively active IL-10 autocrine signaling pathway in the SLCL. Many cytokines are known to induce cellular proliferation in different cell types. The constitutive activation of proteins of the Jak/STAT pathway is likely due to autocrine cytokine signaling. Furthermore, this constitutively activated pathway found in all SLCL, but not in the EBV-infected Burkitt's lymphoma B cell line Daudi, may be critical for the autonomous growth of SLCL. Understanding the signal transduction molecules that participate in the cytokine dependent growth of EBV B cell lymphomas in PTLD may aid in the treatment of this disease. Supported by NIH-AI-41769 and the Stanford Immunology Program Fellowship 5T32-AI-07290.

THE CHIMERIC MONOCLONAL ANTIBODY C2B8 INHIBITS GROWTH OF EBV INFECTED B CELLS FROM PATIENTS WITH PTLD

543 Post transplant lymphoproliferative disorder (PTLD) is a major complication of organ transplantation, and is associated with significant morbidity and mortality. Current therapeutic strategies for the treatment of PTLD are limited. The "humanized" mAb C2B8, which recognizes the CD20 molecule on human B cells has been used successfully to treat non-Hodgkin's B cell lymphomas and shows promise in the treatment of PTLD. The mechanism of C2B8 however remains largely unknown. The purpose of this study is to determine the effect of C2B8 on Epstein-Barr virus (EBV) infected B cells derived from PTLD patients. CD20 expression was determined on four EBV infected spontaneous lymphoblastoid cell lines (SLCL) from PTLD patients, as well as the Burkitt's lymphoma cell lines Daudi and Raji by indirect immunofluoresence and flow cytometry using the C2B8 parent murine mAb 2B8. All cell lines express CD20, although to varying degrees. To determine the effect of C2B8 on cell growth, C2B8 (10 ug/ml) or control mAb was added to SLCL cultures and proliferation assessed at 3-5 days (Figure 1). C2B8 inhibited proliferation in all SLCL tested (range 23-61%) as well as the Raji cell line (42%). In contrast C2B8 had a modest effect on Daudi (16%). Interestingly, JB7 had the highest degree of CD20 expression and inhibition by C2B8. To test the possibility that C2B8 induces apoptosis, SLCL were cultured with C2B8 or anti-Fas mAb (CH11) and stained with propidium iodide. Cell cycle analysis after 18 hrs. of anti-Fas exposure revealed apoptosis in 13% of MF4 cells and 17% of VB5 cells. Daudi was resistant to Fas mediated apoptosis, while JB7 and JC62 were minimally affected (2%). All SLCL express Fas on the cell surface as determined by staining with anti-Fas mAb. Importantly, all SLCL undergo apoptosis when treated with staurosporine, which acts independent of the Fas pathway. In addition, the JB7 cell line undergoes apoptosis when treated with C2B8 (1 ug/ml). Thus, EBV infected B cells have differential sensitivity to apoptotic signals and further studies are underway to dissect the pathways involved. Furthermore, C2B8 inhibits proliferation and may induce apoptosis in EBV infected B cells from PTLD patients.Figure 1

The major immunogenic epitopes of Epstein-Barr virus (EBV) nuclear antigen 1 are encoded by sequence domains which vary among nasopharyngeal carcinoma biopsies and EBV-associated cell lines.

Epstein-Barr virus (EBV) nuclear antigen 1 (EBNA-1) is a protein expressed consistently in EBV-infected cells and EBV-associated malignant tissues. A panel of monoclonal antibodies (MAbs) was generated against the C terminus of EBNA-1 and evaluated for the detection of EBNA-1 in different cell lines. The epitopes recognized were mapped. Since sequence variations of EBNA-1 have been reported in nasopharyngeal carcinoma (NPC) tissues and in infected healthy individuals, the ability of these MAbs to recognize a recombinant protein derived from an NPC biopsy was also analysed. MAb 4H11 appeared to react with EBNA-1 sequences from different sources, whereas MAbs 5C11, 5F12 and 8F6 failed to recognize a recombinant EBNA-1 protein cloned from an NPC patient. Using different recombinant EBNA-1 fragments in an immunoblot format, this study demonstrates that the domain bounded by amino acids 408 and 498 is very immunogenic in mice in that epitopes in this region are recognized by various MAbs. Amino acid sequences of EBNA-1 were also deduced from nucleotide sequences amplified from three Burkitt's lymphoma cell lines, two spontaneous lymphoblastoid cell lines, two NPC biopsies and one NPC hybrid cell line, NPC-KT, and compared to the sequence from B95-8. The amino acid sequence of EBNA-1 in Akata is almost identical to that in an NPC biopsy, except for amino acid 585. The results of this study indicate that the immunogenic epitopes of EBNA-1 are highly variable.

CONSTITUTIVE ACTIVATION OF CYTOKINE JAK/STAT SIGNALING PATHWAY PROTEINS IN EBV-TRANSFORMED B CELL LINES FROM PATIENTS WITH PTLD

127 B cell lymphomas are a major complication of the drug induced immunosuppression required for solid organ transplantation. Epstein-Barr virus(EBV) is the etiologic agent of this post-transplant lymphoproliferative disorder (PTLD). The molecular mechanisms involved in the cellular proliferation of these lymphomas are not yet clearly defined. From PTLD patients, we previously generated EBV infected spontaneous lymphoblastoid cell lines (SLCL) with morphologic and phenotypic similarities to PTLD tumors. SLCL treatment with IL-10 or IL-6 neutralizing antibodies, or with soluble IL-10 receptor inhibits cellular proliferation, indicating that SLCL utilize IL-10 and IL-6 as autocrine growth factors that are required for sustained proliferation. Cytokine receptor binding leads to the autophosphorylation of proteins of the Janus kinase (Jak) family, which then recruit STAT (signal transducers and activators of transcription) cytoplasmic proteins. Jak activated STAT proteins dimerize and translocate to the nucleus to control various cytokine activated genes. To understand the mechanisms by which IL-10 and IL-6 stimulate SLCL we analyzed the expression levels and tyrosine phosphorylation states of the Jak/STAT signal transduction pathway proteins. Three different SLCL, termed JB7, MF4 and VB4 were examined by Western blot and immunoprecipitation. All three SLCL express Jak1, with high levels of this protein particularly evident in the JB7 cell line. Tyk2 was found to be expressed by each of the SLCL at equivalent levels. STAT proteins expressed by these cells include STAT1, STAT2, STAT3, STAT5, and STAT6. STAT4 expression was not detected. To determine which of these proteins may be actively utilized by the SLCL, the tyrosine phosphorylation states of Jak proteins were examined by western blotting with the 4G10 anti-phosphotyrosine antibody. The JB7 line expresses a high level of constitutively tyrosine phosphorylated Jak1. Additionally, all three SLCL, but not the Daudi Burkitt's lymphoma B cell line express a high level of constitutively tyrosine phosphorylated Tyk2. Thus, the cytokine mediated constitutive activation of Jak1 and Tyk2 may be critical for the autonomous growth of SLCL. An understanding of the signal transduction molecules that participate in the cytokine dependent growth of EBV B cell lymphomas may aid in the treatment of PTLD.

EFFECT OF CYCLOSPORINE AND TACROLIMUS ON THE GROWTH OF EPSTEIN-BARR VIRUS-TRANSFORMED B-CELL LINES1,2

Transplant patients receiving immunosuppressive drugs are at increased risk for Epstein-Barr virus (EBV)-associated disorders including posttransplant lymphoproliferative disorder. The function of T lymphocytes, which are critical to preventing the expansion of EBV-infected B cells, is inhibited by immunosuppressive drugs. The purpose of this study was to determine whether immunosuppressive drugs have direct effects on EBV-infected B cells. The growth and proliferation of EBV-infected spontaneous lymphoblastoid cell lines (SLCLs), cultured in the presence or absence of cyclosporine (CsA) and tacrolimus (TAC), were measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and [3H]thymidine incorporation assays. The effect of CsA and TAC on the viability of SLCLs was determined by cell counts with trypan blue. Apoptosis of SLCLs was induced with an anti-Fas agonist monoclonal antibody in the presence or absence of CsA and TAC and measured by flow cytometry after terminal deoxynucleotidyl transferase end-labeling and propidium iodide staining. CsA and TAC, but not sirolimus, increased the growth of SLCLs. The increased growth in the presence of CsA and TAC was attributable to enhanced cell viability and not increased cell division of SLCLs. In addition, CsA and TAC inhibited Fas-mediated apoptosis of SLCLs. CsA and TAC enhance the survival of EBV-transformed B-cell lines. CsA and TAC promote or augment SLCL growth through protection from cell death but do not affect cell division. The inhibition of cell death by CsA and TAC may contribute to the expansion of EBV-infected cells in immunosuppressed individuals.

Selection of a diverse TCR repertoire in response to an Epstein-Barr virus-encoded transactivator protein BZLF1 by CD8+ cytotoxic T lymphocytes during primary and persistent infection.

We investigated the CD8+ cytotoxic T lymphocyte (CTL) repertoire to an HLA B8-restricted peptide, RAKFKQLLQ, located in the Epstein-Barr virus (EBV) immediate-early protein, BZLF1. Repertoire selection was monitored by determining the TCR beta chain sequences of RAKFKQLLQ-specific CTL established from primary infected and healthy virus carriers. PCR analysis of spontaneous EBV-transformed lymphoblastoid cell lines (LCL) from three individuals with primary infection showed that two were infected with type A and one with type B EBV. Polyclonal and clonal CTL that were generated by stimulating peripheral blood mononuclear cells with an HLA B8+ homozygous LCL lysed T cell blasts pulsed with the peptide, RAKFKQLLQ; lysis of certain HLA B8+ LCL targets was associated with the abundance of BZLF1 transcripts. TCR beta analysis showed that while there was loop length restriction in the putative peptide contact site of all responding beta chains, diverse and unique (non-recurrent) TCR beta clonotypes were selected in individuals during primary infection and continued to emerge after long-term virus exposure. TCR-contact site heterogeneity was excluded as the selective force in diversity generation since the epitope-encoded sequences were found to be identical within endogenous virus isolates. In this first study of TCR repertoire selection for an EBV lytic antigen, a BZLF1-reactive component of diverse clonotypes was identified in primary type A or type B EBV infection which was sustained in the EBV-specific memory response throughout life-long infection. This diversity selection is likely to play a critical role in maintaining a balanced viral load throughout EBV persistence.

Involvement of IL-10 in the autonomous growth of EBV-transformed B cell lines.

Immunocompromised individuals have an increased incidence of EBV-associated B cell lymphomas. The growth factors responsible for the unrestrained proliferation of these lymphomas have not yet been determined. In this study, spontaneous lymphoblastoid cell lines (SLCL) were derived without the addition of growth factors or virus from four patients with EBV-associated lymphoproliferative disorder. These cell lines were EBV transformed in vivo, and infection was verified through amplification of the viral gp220 gene. SLCL have an activated B cell phenotype (CD19+, CD21+, CD23+, CD38+, and CD40+) and produce IL-6, IL-10, TNF-alpha, and lymphotoxin-alpha. To determine whether these cytokines contribute to autonomous growth, neutralizing Abs for IL-10, IL-6, and TNF-alpha, a soluble TNFR:Fc fusion protein, and soluble IL-10R were used. These experiments established that, of the cytokines produced by SLCL, only IL-10 is an autocrine factor. IL-10 was produced by the majority of cells within each SLCL, and IL-10 secretion was concomitant with SLCL growth. Our findings demonstrate that IL-10 is utilized in the autonomous growth of EBV-related lymphomas and may be crucial in the development of lymphoproliferative disorder.