Epstein-Barr virus strain-specific differences in transformed cell lines demonstrated in growth characteristics, induction of viral antigens and ADCC susceptibility

Paired lymphoid cell lines were established by transformation with both B95-8 (B) and QIMR-WIL (Q) EBV strains, of cells either from cord blood or from peripheral blood or spleens of patients with leukemias or lymphomas. The morphologies of the transformed cell clumps differed consistently between B-transformed lines (B-lines) and Q-transformed lines (Q-lines) even after 1 year in culture. When the B- and Q-lines were compared for superinfection with P3HR-1 EBV, B-lines had a higher frequency of EA induction than did the Q-lines. The shape of dose-response curves for superinfection indicated that a much higher multiplicity of infection by P3HR-1 EBV was required for EA expression in Q-lines than in B-lines. This difference in superinfection was independent of the EBV receptor concentration on the two types of lines and reflected apparent control of the level of EA induction by the resident EBV genome of the transformed line. Transforming EBV could be rescued by P3HR-1 EBV superinfection of both B- and Q-lines originating from cells of patients with Hodgkin's disease and hairy cell leukemia but not from cord blood. The cord blood lines transformed with virus produced spontaneously from these B- or Q-lines, showed that the cell lines contained antigen-determining information of the resident genome in the original B- or Q-lines, respectively. Superinfected B-lines were more susceptible to ADCC with anti-EBV-positive sera than were superinfected Q-lines. These experiments demonstrate that distinct biology. differences exist in paired cell lines transformed by different EBV strains.     

Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.     

Cytokine signatures of transformed B cells with distinct Epstein-Barr virus latencies as a potential diagnostic tool for B cell lymphoma

Immunocompromised individuals, including those infected with human immunodeficiency virus (HIV), are at increased risk of Epstein–Barr virus (EBV)‐associated aggressive B cell malignancies such as Burkitt’s lymphoma (BL) or diffuse large B cell lymphoma (DLBCL). Differential diagnosis of these lymphomas requires histopathological, immunohistochemical and cytogenetic assessments. Rapid, less invasive approaches to the diagnosis of EBV‐associated B cell lymphomas are needed. Here, high‐throughput cytokine profiling of BL cell lines and EBV‐transformed B lymphoblastoid cell lines (B‐LCL), representing DLBCL, was carried out. By monitoring the production of 42 different cytokines, unique cytokine signatures were identified for BL and B‐LCL/DLBCL. The BL cells produced interleukin (IL)‐10, 10 kDa interferon gamma‐induced protein (IP‐10)/CXCL10, macrophage‐derived chemokine (MDC)/CCL22, macrophage inflammatory protein (MIP)‐1α/CCL3 and MIP‐1β/CCL4. In addition to these five cytokines, the cytokine signature of B‐LCL/DLBCL cells included IL‐8/CXCL8, IL‐13, platelet‐derived growth factor (PDGF)‐AA, and regulated upon activation, normal T cell expressed and secreted (RANTES)/CCL5. Epstein–Barr virus latency was responsible for the increased production of IL‐10, MDC/CCL22 and MIP‐1α/CCL3 in BL cells, suggesting that EBV‐mediated BL‐genesis involves these three cytokines. These results suggest that high‐throughput cytokine profiling might be a valuable tool for the differential diagnosis and might deepen our understanding of the pathogenesis of EBV‐associated B cell malignancies. (Cancer Sci 2011; 102: 1236–1241)     

Biologic and antigenic characteristics of Epstein-Barr virus-related Herpesviruses of chimpanzees and baboons.

Leukocyte-transforming agents were isolated in baboon leukocytes inoculated with oral excretions from immunosuppressed chimpanzees. The transformed lymphoblasts had B cell surface markers and harbored herpes-type virus particles; 5-10% of the cells contained cytoplasmic antigens reactive with Epstein-Barr virus (EBV)-antibody-positive chimpanzee, human and baboon sera. These sera also neutralized the transforming activity of the chimpanzee virus. Long-term lymphoid cell lines were established from circulating lymphocytes of normal baboons: two from Papio cynocephalus and three from P. hamadryas. The cells had B cell surface markers, contained herpes-type virus particles and produced virus with leukocyte-transforming activity. No virus-associated nuclear antigen was detectable with reference baboon and chimpanzee sera; however, the cells reacted with selected human sera containing antibodies to EBV nuclear antigen (EBNA). Absorption experiments confirmed the specificity of this reaction. Baboon lymphoblasts produced baboon virus-associated soluble complement-fixing (CF/S) antigen. Baboon sera had CF antibodies to viral (CF/V) antigen derived from EBV but failed to react with EBV-associated CF/S antigen. Chimpanzee and baboon herpesviruses had similar in vitro host cell ranges but were different from those of EBV. Inoculation of baboons, rhesus monkeys and cottontop marmosets failed to produce detectable illness or palpable tumors.     

The interaction of non-transforming Epstein-Barr virus (EBV) with cell-associated EBV DNA in superinfected lymphoblastoid cell lines

Two human lymphoblastoid cell lines were established by the transformation of human cord-blood lymphocytes with transforming Epstein-Barr virus (EBV). One cell line (HLB-R1) was established with EBV obtained after the superinfection of Raji cells with HR-1 EBV and the other (HLB-Bl) was established from B95-8 EBV-infected human cord-blood lymphocytes. Both the HLB-R1 and HLB-B1 lines were susceptible to superinfection with HR-1 EBV. We found that EBV DNA was replicated in the superinfected cell lines and that transforming EBV was produced in both the HLB-B1 and HLB-R1 cells. The average titer of transforming EBV obtained in the HR-1 EBV superinfected HLB-B1 and HLB-R1 cell lines was 10(4) transforming units (TU)/ml, whereas the average titers of transforming EBV obtained by the superinfection of Raji cells was 10(1) TU/ml. Epstein-Barr virus capable of inducing early antigen (EA) in superinfected Raji cells (lytic virus) was not detected in any transforming virus preparation. Restriction enzyme digestion patterns of virus DNA isolated from HR-1 and B95-8 cells, as well as from superinfected cells, were compared. The EBV DNA that was replicated in the superinfected HLB-R1 and HLB-B1 cell lines showed a more complex pattern. Our data suggest that recombination between input HR-1 EBV DNA and latent cell-associated EBV DNA occurs. Presumably this recombination results in a change in the biological properties of the newly synthesized virus.     

A natural HIV p17 protein variant up‐regulates the LMP‐1 EBV oncoprotein and promotes the growth of EBV‐infected B‐lymphocytes: Implications for EBV‐driven lymphomagenesis in the HIV setting

Human immunodeficiency virus p17 matrix protein is released by infected cells and may accumulate within lymphoid tissues where it may deregulate the biological activities of different cell populations by binding to CXCR1 and CXCR2 cellular receptors. S75X, a natural p17 variant, was recently shown to enhance the malignant properties of lymphoma cells. We investigated a reference p17 protein and the S75X variant for their ability to bind to Epstein–Barr virus (EBV)‐infected primary and fully transformed B‐lymphocytes and trigger downstream effects of potential pathogenic relevance. We demonstrate that EBV infection of primary B‐lymphocytes or the ectopic expression of the latent membrane protein‐1 viral oncoprotein in EBV‐negative B‐cells up‐regulates CXCR2, but not CXCR1. Multispectral imaging flow cytometry showed that EBV‐infected primary B‐cells more efficiently bind and internalize p17 proteins as compared with activated B‐lymphocytes. The S75X variant bound more efficiently to EBV‐infected primary and fully transformed B‐lymphocytes compared with reference p17, because of a higher affinity to CXCR2, and enhanced the proliferation of these cells, an effect associated with cyclin D2 and D3 up‐regulation and increased interleukin‐6 production. Notably, the S75X variant markedly up‐regulated latent membrane protein‐1 expression at both mRNA and protein levels and enhanced the activation of Akt, ERK1/2 and STAT3 signaling, thereby contributing to EBV⁺ B‐cell growth promotion. These results indicate that EBV infection sensitizes B‐lymphocytes to CXCR2‐mediated effects of p17 proteins and provide evidence supporting a possible contribution of natural p17 variants to EBV‐driven lymphomagenesis in the human immunodeficiency virus setting.     

In vitro transformation of human B-cell follicular lymphoma cells by Epstein-Barr virus

Human low-grade B-cell lymphoma cells cannot be readily maintained in long-term tissue culture. In an effort to obtain low-grade B-cell lymphoma cell lines for in vitro study, we used Epstein-Barr virus (EBV) as a transforming agent. T-cells were removed prior to EBV transformation by rosetting with sheep erythrocytes, followed by treatment with anti-T11 monoclonal antibody plus complement. The resulting cell population was cocultured with EBV prepared from tissue culture supernatants of marmoset cell line B95-8. Identical immunoglobulin gene rearrangements of tumor cells and EBV-transformed cells were the criteria used to determine that the transformed cells were of tumor origin. DNA was prepared from both biopsy tissue and EBV cell lines and digested with restriction endonucleases, and Southern blots were prepared by standard methods. B-cells isolated from biopsies of four low-grade B-cell lymphomas of follicular, small cleaved cell type and one of follicular, mixed cell type were transformed by EBV into rapidly growing in vitro tissue culture lines. Two of the five transformed cell lines had immunoglobulin heavy chain and light chain gene rearrangements which were present in cells from the original tumor biopsy, indicating that these EBV-transformed cell lines are of tumor origin.     

Analysis of two human monoclonal antibodies against melanoma.

B cells derived from peripheral-blood lymphocytes (PBL) and tumor-infiltrating lymphocytes (TIL) from a patient with a high serum antibody titer to autologous melanoma were transformed with Epstein-Barr virus (EBV) and evaluated for reactivity against autologous tumor. B cells producing antibody reactive with autologous tumor and unreactive with normal fibroblasts were detected both in TIL and in PBL. One cell line derived from PBL and another derived from TIL sustained production of tumor-reactive antibody for 10 weeks and over 15 months respectively. The cell line derived from PBL, 2D11, produced an antibody reactive with a trypsin-resistant antigen expressed on the cell membrane of autologous and allogeneic melanoma cell lines. The cell line derived from TIL, 1F6, produced an antibody reactive with a cell-surface glycoprotein expressed by 5 autologous melanoma cell lines derived from 5 different metastases and 16/19 allogeneic melanoma cell lines. 1F6 also showed reactivity with cell lines derived from a blue nevus, a congenital nevus, an astrocytoma, and 1/4 renal-cell carcinomas; but it was not reactive with 5 foreskin melanocyte cell lines, 2 normal fibroblast lines, 5 leukemia/lymphoma lines, 8 lung-cancer lines, 8 glioblastoma lines, or lines derived from 1 ovarian carcinoma, 1 colon carcinoma, 1 vulvar carcinoma, 1 fibrosarcoma, 1 murine melanoma, or 4 murine leukemia/lymphomas. We describe here an antibody that detects a new melanoma specificity obtained by EBV transformation of tumor-infiltrating B cells.     

Colonies of EBNA-positive cells in soft agar from peripheral leukocytes of infectious mononucleosis patients.

Epstein-Barr virus (EBV)-associated nuclear antigen (EBNA)-positive lymphoblastoid cells grew as colonies in soft agar after seeding of leukocytes from the peripheral blood of four patients with infectious mononucleosis serologically determined to be caused by EBV. In individual cases more colonies were obtained from blood specimens during the acute phase of the disease than during the convalescent phase. Incorporation of human umbilical cord serum, which contained neutralizing antibody to EBV, into the agar medium did not reduce the number of colonies developing. Our observations indicate that colony-forming cells were originally present in the blood samples, and that they were not infected and subsequently transformed in vitro. Cells from less than 20% of the EBNA-positive colonies grew to form lymphoblastoid cell lines, which were EBNA-positive and had B lymphocyte surface markers. However, the majority (over 80%) of the EBNA-positive colonies failed to form immortalized cell lines. No colonies were obtained from 91 blood samples from healthy young adults and from five patients with an IM-like disease unrelated to EBV infections. The present results strongly suggest that already transformed cells or cells very easily transformed by EBV are present in the blood of IM patients.     

Superinfection of epithelial hybrid cells (D98/HR-1, NPC-KT, and A2L/AH) with Epstein-Barr virus and the relationship to the C3d receptor

Three Epstein-Barr virus (EBV) genome-positive epithelial/hybrid cell lines (D98/HR-1, NPC-KT, and A2L/AH) were superinfected with EBV derived from P3HR-1 (HR-1), NPC-KT, and B95-8 cells. The HR-1 virus is lytic and induces early antigen in superinfected Raji cells; the virus is not capable of transforming B-lymphocytes. Virus preparations from NPC-KT cells have both transforming and early antigen-inducing properties, while B95-8 virus can only transform B-lymphocytes. It was possible to demonstrate EBV antigens after superinfection of D98/HR-1 cells with both HR-1 virus and NPC-EBV. The NPC-KT hybrid cells, which were originally prepared by fusing human adenoid epithelial cells (Ad-AH) and EBV genome-positive NPC explanted epithelial cells, were susceptible to superinfection with HR-1 virus but not to NPC-EBV. The A2L/AH hybrid cells, which were prepared by fusion between Ad-AH cells and lymphocytes transformed by B95-8 virus, could not be superinfected with any of the virus preparations. In order to further investigate the nature of the EBV receptor as it relates to other cell membrane components, we examined cell surface markers on Ad-AH, NPC-KT, A2L/AH, and D98/HR-1 cells using monoclonal antibodies and by rosette formation. We found that the NPC-KT, A2L/AH, and Ad-AH cell lines express the OKB2 antigen and that the common acute lymphoblastic leukemia antigen is expressed on the A2L/AH cells. We also found that NPC-KT parental cells and a clone of NPC-KT cells express erythrocyte antibody complement b and erythrocyte antibody complement d, as determined by rosette formation, but were negative for C3b and C3d when monoclonal antibodies against these two markers were used. The D98/HR-1 cells were also confirmed to be negative for C3b and C3d. The data suggest that the C3d receptor may be part of the EBV receptor but that the C3d receptor, by itself, is not the only receptor to which EBV can bind.     

Progenitor and pre-B lymphocytes transformed by Epstein-Barr virus

By rosetting with SRBC coupled to rabbit-anti-human IgM, the surface IgM-negative cells of human fetal bone marrow were enriched, and subsequently infected and transformed by Epstein-Barr virus (EBV). Single clones of the transformed cells were obtained. Ninety percent of the resulting cell clones were surface-immunoglobulin-negative, and of 8 clones which were further studied, 5 lacked intracellular, cytoplasmic Ig as measured by immunofluorescence. Control cell clones derived from the same material without pre-selection expressed surface Ig and also secreted Ig. Utilization of a panel of B-cell-specific monoclonal antibodies (MAbs) showed no difference between the cell clones expressing surface Ig and those that did not. The progenitor B-cell lines did not show a phenotype resembling that of cell lines derived from B-cell malignancies, such as high agarose clonability. In spite of their immature Ig-phenotype, these clones showed rearrangement of at least one heavy chain Ig-allele. Efforts to induce differentiation in these clones were unsuccessful. These clones may represent progenitor B cells, or B cells with faulty heavy-chain rearrangement. EBV can apparently be used as a tool to derive cell lines representing different levels of B-cell differentiation, and can also transform immature B cells, which may be useful in the analysis of B-cell differentiation.     

Cell-killing by Epstein-Barr virus: analysis by colony inhibition procedure.

The P3HR-1 and B95-8 strains of Epstein-Barr virus (EBV) were cytocidal for EBV-carrier human cell lines, as revealed by a colony inhibition procedure. The cytocidal activity was proportional to virus dose added. The cell killing was neutralized by anti-EBV antibody-positive but not -negative human sera. When the relative sensitivity to ultraviolet light of EBV activities was examined, the cytocidal actitivy was much more resistant than the viral infectivity as assayed by early antigen-forming activity (P3HR-1 virus) or leukocyte-transforming activity (B95-8 virus), but it closely paralleled the ability to adsorb to cells.     

Transformation of lymphocytes by Herpesvirus papio.

Cotton-topped (CT) or white-lipped (WL) marmoset lymphocytes were transformed in vitro with herpesvirus papio (HVP) into permanently growing lymphoblastoid cell lines (LCL). Five of 9 HVP-transformed CT cell lines contained cells with antigens reacting with antibodies to Epstein-Barr virus (EBV) capsid antigen (VCA) and/or to EBV-induced early antigens (EA). None of 12 WL LCL revealed such antigen-producing cells. Cells from both groups of cultures failed to react with antibodies to the EBV-specified nuclear antigen (EBNA). Exposure of baboon circulating lymphocytes to X-irradiated HVP or EBV-carring cells, or to suspensions of EBV resulted in establishment of LCL which all contained VCA and/or EA-positive, but no EBNA-positive cells. Nuclear antigens were undetectable also with anti-VCA-positive sera from baboons, chimpanzees, or other non-human primates. DNA-complementary RNA (cRNA) filter hybridization with EBV cRNA showed that with one exception transformed CT or WL marmoset cells contained at least 1-2 virus genome equivalents per cell, while at least 12-25 virus genome equivalents per cell were detected in transformed baboon cells. These data need confirmation by DNA-DNA reassociation kinetics.     

Calcium concentration defines two stages in transformation of lymphocytes by Epstein-Barr virus

Lymphocytes from Epstein-Barr virus (EBV) seronegative donors were either stimulated with Protein A or infected with EBV and cultured in growth medium containing a range of calcium levels (700 microM to less than 2 microM available calcium). Three calcium end-points for the proliferation of B lymphocytes were defined: 18 microM for mitogenically-stimulated B cells; 8 microM for a pre-12 h event in EBV transformation and less than 2 microM for a post-12 h event in EBV transformation resembling the end-point for the emergent lymphoblastoid cell line. EBV-infected lymphocytes cultured in calcium-depleted medium expressed the EB virus nuclear antigen and proliferated after addition of calcium chloride. The present study should be useful both in defining the sequence of events in EBV transformation and in studying properties specific to the transformed phenotype.     

EBNA promoter usage in EBV-negative Burkitt lymphoma cell lines converted with a neomycin-resistant EBV strain

Latent Epstein-Barr virus (EBV) uses two alternative strategies to express the Epstein-Barr nuclear antigens (EBNAs). Resting normal B cells harboring latent virus and Burkitt's lymphoma (BL) cells use monocistronic messages generated from the Q promoter (restricted strategy). EBV-transformed immunoblasts express all EBNAs by using giant messages generated from the W/C promoter (full program). Whether the virus establishes the restricted program on primary infection of a BL cell (or its progenitor) or, alternatively, whether such cells are generated by phenotypic down-regulation from the immunoblast is unclear. We found previously that conversion of EBV-negative BL lines to EBV-positive sublines required repeated exposure to large virus doses. The converted sublines used the full program. However, the possibility that cells with a full program had a selective advantage during the long period of in vitro passage could not be excluded. We therefore infected EBV-negative BL lines with recombinant EBV carrying a neomycin resistance marker. Most convertants of the 12 lines tested were positive for YUK splicing, indicative of the full program, but some were also positive for the restricted QUK splice program. One convertant DG75 line showing both YUK and QUK was cloned and gave rise to stable QUK users. We conclude that EBV infection of established BL lines can give rise to subclones with either the full or the restricted program. The fact that all EBVs carrying BL lines use the restricted program in vitro may be a consequence of immunoselection.     

Restricted expression of EBV encoded proteins in in vitro infected CLL cells

CLL is not associated with EBV. CLL cells separated from blood express CR2, the complement receptor that serves also as EBV receptor. Thus CLL cells can be infected in vitro with the virus, however, in contrast to normal B lymphocytes, only rare CLL clones yield transformed lines. This is due to a restricted EBV encoded protein expression in the CLL cells, they express EBNAs, the virus encoded proteins that are localized in the nucleus, but not the cell membrane associated LMP-1, that is also pivotal for the virus induced transformation of B lymphocytes. This expression pattern seems to be unique to a defined B cell maturation window that is represented by the CLL cells. We named this restricted viral expression as Type IIb. Such B lymphocytes have been encountered in lymphoid tissues of infectious mononucleosis (IM) and in post transplant lymphoproliferative disease (PTLD). Moreover, they were shown in tissues of EBV infected "humanized" mice. The EBV encoded protein expression pattern may serve as a marker for the B cell differentiation stage from which CLL clones can develop.     

Difference in viral binding between two Epstein-Barr virus substrains to a spectrum of receptor-positive target cells

Radio-labelled Epstein-Barr virus (EBV) was utilized in a direct binding assay to detect the presence of EBV receptors. The sensitivity of this method was affirmed by the detection of EBV-receptors on three EV-carrying cell lines that have previously been reported as receptor negative. Two laboratory substrains of EBV, derived from the cell lines B95-8 and P3HR-I (designated B and P virus respectively), were tested in the binding assay. The main repcptor prototype adsorbed both viral strains without apparent distinction. In contrast, two lines, a Swedish EBV-negative B-cell lymphoma (U698) and a virus non-producer subline of the receptor-negative P3HR-1 line, adsorbed P virus selectively but failed to adsorb B virus.