Clonal transformation of human leukocytes by Epstein-Barr virus in soft agar.

The B95-8 strain of Epstein-Barr virus (EBV) induced colony formation of human umbilical cord-blood leukocytes in soft agar medium. One-hit response relationship between the number of colonies and the virus dose was observed with high dilutions of the virus preparation. However, there was a presumed cell-killing effect with low dilutions of virus. The colonies were similarly induced, but with a lower efficiency, in adult peripheral blood leukocyte cultures infected with the virus. The colony-forming activity of EBV was neutralized by anti-EBV-positive but not by negative human sera. The cells in colonies were capable of growing continuously and carried EBV-associated nuclear antigen. Thus, it was evident that the colony formation was caused by clonal transformation by EBV.     

A quantitative analysis of the susceptibility of human leukocytes to transformation by epstein-barr virus

Susceptibility of lymphocyte-enriched cell fractions isolated from human umbilical cord blood and adult peripheral blood to transformation by the B95–8 strain of Epstein-Barr virus (EBV) was investigated quantitatively. Minimum multiplicity of input of virus (50% transforming dose) per cell (MOI) necessary to induce maximum level transformation of cord cells ranged from 0.02 to 0.2. The frequency of initially transformed cells (fraction of transformable cells) in the cord cell samples from two different individuals was estimated to be 2.6 to 6.2%. In this system, the appearance of cells positive for EBV-associated nuclear antigen (EBNA) paralleled the growth curve of transformed cells. About 70% of the latter were EBNA-positive. In adult cell preparations from two individuals, 1.8 and 0.03%, respectively, of the cells were transformable indicating larger individual variations in sensitivity to EBV than in cord cells. The EBV susceptibility was also determined by the transforming efficiency (TE) expressed as the negative log of the virus dilution which induces transformation in 50% of cell cultures infected at an MOI of 0.2. From the TE value, a minimum MOI which induces transformation could be calculated. Also by this test it was shown that the EBV susceptibility of adult cells was not only lower but also much more variable between individuals than that of cord cells. There was no correlation between the susceptibility of cells and the titer of anti-EBV antibody in donors' sera. In cultures of mixed cord cells and adult cells known to have low EBV susceptibility, the minimum MOI increased in proportion to the amount of adult cells.     

In vitro evaluation of cell-mediated immunity to Epstein-Barr herpesvirus by cell migration inhibition tests.

Migration of peripheral leukocytes in samples from sensitized [Epstein-Barr virus (EBV) antibody-positive] humans was greatly inhibited when challenged by antigen prepared from EBV-producing P3HR-1 cells but not by antigen prepared from EBV-nonproducing RAJI cells, EBV-negative human fibroblasts, or epithelial cells. Such inhibition was not observed when peripheral leuocytes from subjects or neonates not sensitized to EBV were challenged. Similar results were obtained in a two-stage test when the same leukocyte samples were challenged in vitro by antigen prepared from P3HR-1 cells and the cell-free supernatant was assayed for migration inhibition factor (MIF) in the guinea pig macrophage migration inhibition test; migration of guinea pig peritoneal exudate cells was greatly inhibited by the supernatant filtrates of leukocyte cultures only from subjects positive for EBV-antibody. Furthermore, this inhibitory effect was not observed if supernatant filtrates from leukocyte cultures challenged by antigens prepared from RAJI cells, fibroblasts, or epithelial cells were used. The EBV antigen transformed peripheral leukocytes and induced early antigen production in RAJI cells; however, a "killed" preparation (by UV irradiation) was sufficient for eliciting MIF production.     

Epstein-Barr virus: transformation of non-human primate lymphocytes in vitro

Continuous lymphoblastoid cell cultures were established from marmoset (Saguinus sp.), squirrel (Saimiri sciureus), owl (Aotus trivirgatus) and cebus (Cebus apella) monkeys after culturing their peripheral lymphocytes with lethally X-irradiated cells carrying Epstein-Barr virus (EBV). Transformation also was achieved by exposing simian lymphocytes to infectious, cell-free EBV derived from the simian lymphoblastoid cell cultures. Simian lymphocytes were not transformed after exposure to cell-free EBV derived from HR-1 cells. The simian cell cultures were similar to cell cultures derived from Burkitt's lymphoma or infectious mononucleosis patients. EBV-induced early, viral capsid and membrane antigens, intranuclear inclusion bodies and herpesvirus virions were demonstrable in most cultures. Seven cultures were insusceptible to superinfection with EBV and treatment of the cultures with halogenated pyrimidines was relatively ineffective for inducing synthesis of early or viral capsid antigens. All cell cultures had B-cell characteristics: they produced immunoglobulins but did not form spontaneous rosettes with sheep erythrocytes. Four of six marmoset monkeys, inoculated with EBV-transformed marmoset lymphocytes, developed antibodies to EB viral capsid antigens and one marmoset inoculated with autochthonous transformed cells also developed heterophile antibodies. Seven marmosets, inoculated with cell-free EBV derived from HR-1 cell cultures, developed no detectable levels of antibodies to EBV-specified antigens or heterophile antibodies. No overt clinical abnormalities were detected in any of the marmosets inoculated with HR-1 or Kaplan EBV but one of five marmosets inoculated with B95hyphen;8 EBV developed a lymphoma.     

Epstein-Barr virus-related herpesvirus from a rhesus monkey (Macaca mulatta) with malignant lymphoma

A herpesvirus (RhEBV) was isolated from a lymphoblastoid cell line (LCL) that became established from a malignant lymphoma in a rhesus monkey. The predominant cell marker in the LCL was that of B lymphocytes. RhEBV-induced viral capsid (VCA) and nuclear antigens (NA) in the LCL were serologically related to similar antigens known to be induced by human Epstein-Barr virus (EBV). RhEBV was of nonhuman primate origin and was clearly differentiated from EBV in the anti-complement immunofluorescence reaction using human and non-human primate sera with antibodies to the NA induced by the respective viruses. While human sera reacted with NA induced by both EBV and RhEBV, monkey sera failed to recognize the NA induced by EBV. RhEBV-induced NA was present in nearly all the cells of a suspension prepared from the tumor tissue mass, but not in the monolayer fibroblasts derived from the tumor tissue or in the blood and lymph-node lymphocytes of clinically healthy animals. RhEBV induced in vitro transformation and establishment of LCLs from peripheral blood lymphocytes of normal rhesus and cynomolgus monkeys but not from those of 6 other non-human primate species tested. The LCLs, with predominant B-lymphocyte markers, established after treatment with RhEBV, all had evidence of the virus infection since nearly all cells in the culture expressed the virus-induced NA.     

HTLV-I infection of T and B cells of a patient with adult T-cell leukemia-lymphoma (ATLL) and transmission of HTLV-I from B cells to normal T cells

We analysed the DNA of different tissues of a patient (HS) with adult T-cell leukemia/lymphoma virus (HTLV-I). We detected viral sequences in fresh specimens from spleen, thymus, liver, skin and peripheral blood neoplastic lymphocytes. The pattern of HTLV-I intergration is identical in the leukemic cells and in all other tissues analysed, but the signal intensity is strongest in the leukemic cells, indicating the source of HTLV-I proviral sequences was the leukemic T-cells which had infiltrated these tissues. In fact, the cultured skin fibroblasts of the patient did not contain HTLV-I sequence. However, cultured lymphocytes of this patient was consistently an immortalized B-cell line containing HTLV-I sequences in a manner indicative of a polyclonal infection. This cell line was also infected with the Epstein-Barr virus (EBV). In order to determine whether HTLV-I alone was sufficient for B-cell immortalization, we obtained single cell clones by limiting dilution. The DNA of all the cell clones that we analysed contained both the HTLV-I and EBV genomes, suggesting that immortalization of the B-cell was more likely due to the EBV rather than HTLV-I. Infectious HTLV-I viruses produced by the B-cell line still had the propensity to infect and transform T-lymphocytes in normal human umbilical cord blood. Unlike the parental B cells, the transformed T lymphocytes were clonally selected. Our results indicate that although the predominant infected cell population of the patient was his leukemic T lymphocytes, some of his EBV-positive B-lymphocytes were also polyclonally infected. The latter had a growth advantage in culture over the T lymphocytes but the virus produced by these immortalized B cells has not been adapted and has maintained its tropism for T cells.     

Restrictions upon Epstein-Barr virus infection of the leukemic cell are demonstrated in patients with hairy cell leukemia

We tested the hypothesis that Epstein-Barr virus (EBV) might actually infect leukemic hairy cells in vivo by examining those cells for the EBV-receptor, EBV nuclear antigen (EBNA) and membrane antigen (MA), for spontaneous transformation and rescue of infectious virus and for presence of EBV genome. EBV-receptors were found on subpopulations of leukemic cells from each of 7 patients with hairy cell leukemia (HCL) tested. MA was present on low numbers (1-5 per cent) of fresh leukemic cells of 7 patients and in some instances occurred with a greater frequency after 3 to 5 days in culture, with or without 12-O-tetradecanoylphorbol-13-acetate. In 11 fresh leukemic cell preparations from 8 HCL patients, no EBNA was demonstrated. However, 2 samples after 4 days in culture expressed low frequencies of EBNA-positive cells. Spontaneous, EBV-positive cell lines were established with a high transformation efficiency from 3 HCL blood samples but not from 8 other specimens. Infectious EBV could be rescued from some hairy leukemic cell preparations by co-cultivation with cord blood lymphocytes. These results demonstrated that leukemic cell populations harbored infectious EBV, that the leukemic cells expressed virus receptors and suggested that a small subpopulation of leukemic cells might become infected in vivo at least transiently and possibly transformed in vitro by EBV. To test for the extent of occult in vivo infection of leukemic cells with EBV, Southern type hybridization studies were performed with a probe for EBV genome (Bam HI W). At a sensitivity level of 0.1 genome per cell, EBV genome was not detected in the leukemic cell populations of 7 patients. We conclude that host defence mechanisms protecting these individuals from EBV also prevent infections of the leukemic cell and/or most hairy leukemic cells are not suitable targets for both infection and transformation.     

Growth in semisolid agar medium of human cord leukocytes freshly transformed by Epstein-Barr virus.

The semisolid agar method for the selective growth of transformed cells was applied to investigations of Epstein-Barr virus (EBV) transformation. When 1 X 10(5) HUMAn umbilical-cord blood leukocytes were seeded in agar medium immediately after EBV exposure, about 100 colonies developed in each dish. The occurrence of colonies correlated well with dilutions of EBV inoculum. These colonies were composed of lymphoblasts, were positive for EBV-determined nuclear antigen immunofluorescence, and consistently resulted in the establishment of cell lines. In cord leukocyte cultures exposed to EBV, cells capable of growing in semisolid agar medium appeared early and increased rapidly, though the total numbers of leukocytes and control cultures decreased during the first several days.     

Mechanisms of enhancement of Epstein-Barr virus-induced transformation of peripheral blood lymphocytes by a tumor promoter, TPA, with special reference to lowering of cytotoxicity of T cells

The mechanism of the previously observed enhancement of Epstein-Barr virus (EBV)-induced cell transformation of human lymphocytes by 12-0-tetradecanoyl-phorbol-13-acetate (TPA) was studied. A concentration of TPA (0.5 ng/ml) was used. In cultures of human cord blood lymphocytes infected with EBV in the presence of TPA, a larger number of EBV-associated nuclear antigen (EBNA)-positive and/or DNA synthesizing cells was observed than in the absence of TPA. In the virus-infected lymphocyte cultures of EBV-seropositive adult donors, in vitro regression of transformation, which is known to be caused by T lymphocytes, was suppressed by TPA, EBV-specific and -non-specific cytotoxicity of T cells generated in mixed cultures of peripheral blood lymphocytes (PBL) of seropositive adults and autologous lymphoblastoid cell line (LCL) cells was markedly lowered by the presence of TPA in the cultures, However, TPA had little effect on proliferation of T cells in stimulated cultures, and addition of TPA to the reaction mixture for the cytotoxicity test did not lower the cytotoxicity. These results suggest that TPA has an inhibitory effect on T cells which suppress EBV-transformation. THe effect on T cells, together with the direct promoting effect on proliferation of EBV-transformed cells, may explain the outcome of enhancement of EBV-induced LCL establishment from PBL of seropositive donors by TPA previously observed.     

Metabolism of benzo(a)pyrene by human epithelial cells in vitro.

Primary cell cultures derived from human skin epithelium metabolized benzo(a)pyrene to three classes of compounds: phenols, quinones, and dihydrodiols. The relative proportions of metabolites varied according to the skin donor but differed from the pattern of metabolites in rat liver microsome preparations. While appreciable amounts of 7,8- and 9,10-dihydrodiol; 1,6-, 3,6-, and 6,12-quinone; and 3- and 9-hydroxy derivatives were found in the medium, no 4,5 (K-region)-dihydrodiol or epoxide was detected. Reduced amounts of quinones were produced when the cultures were pretreated with hydrocortisone before exposure to the hydrocarbon. The cultures did not require a period of enzyme induction for efficient metabolism of the hydrocarbon. Cultures of fibroblasts derived from the same skin samples as the epithelial cells metabolized the hydrocarbon but to a much different extent. Preexposure of the epithelial cell cultures to mixtures of polycyclic hydrocarbons resulted in a decrease in the amounts of carcinogen metabolized to phenols and dihydrodiols. These findings suggest that the prevalence of carcinomatous disease in humans is due to the differential capacity of the epithelial cells to metabolize potential carcinogens to active forms, a capacity reduced in fibroblasts or other nonepithelial cells. This suggestion is supported by the observations that supposedly normal prostate cells also efficiently metabolize polycyclic hydrocarbons in a manner similar to that of epidermal cells. No evidence of neoplastic transformation was seen in cytological preparations of cells exfoliated into the medium.     

Studies on chemical carcinogen enhancement of Epstein-Barr virus induced transformation of human neonatal and adult peripheral blood lymphocytes

The effects of a wide range of selected chemical carcinogens on the frequency of Epstein-Barr virus (EBV)-induced transformation have been investigated. The carcinogens tested included direct-acting chemicals and chemicals requiring either activation via reactions with nucleophiles, or cell-mediated enzyme activation. Treatment with some but not all chemicals suspected of being carcinogens resulted in enhanced EBV-induced transformation of neonatal or adult peripheral blood lymphocytes (PBLs). The temporal relationship between carcinogen exposure and EBV infection could dramatically influence the results of the chemical carcinogen-cellular interaction as measured by the cells' ability to subsequently undergo morphologic transformation. This relationship was particularly evident when cells were treated with alkylating agents such as dimethylsulfonate (DMS) or N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Beginning at 24 h, and at later times following EBV-infection, cellular transformation became more resistant to the cytotoxic effects of DMS and, in contrast, more sensitive to the cytotoxic effects of MNNG. These diametrically opposed results clearly demonstrate the ability of EBV infection to alter the response of lymphocytes to chemical carcinogens as measured by transformation. The lymphoblastoid cell lines (LCLs) established from carcinogen-treated PBLs had increased cloning efficiency. Furthermore, using radiolabelled, molecularly cloned subgenomic fragments of EBV DNA and DNA-DNA hybridization, we have been able to detect an increased number of EBV genome equivalents in whole-cell and high-molecular-weight cellular DNA extracted from LCLs established from MNNG as well as DMS-treated PBLs. We propose that carcinogen enhancement of EBV-induced transformation is an example of a two-step mechanism of oncogenic transformation in primary human lymphoid cells. The possible significance of these findings in relation to potential development of lymphomas following EBV exposure will be discussed.     

In vitro infection of human B lymphocytes with adult T-cell leukemia virus

Experimental transmission of adult T-cell leukemia (ATL) virus (ATLV) into human B lymphocytes was attempted. Cocultivation of B-cell rich fraction of peripheral blood from a healthy adult with X-ray irradiated ATLV producer MT-2 cells resulted in the establishment of OKA(B) cell line co-infected with both Epstein-Barr virus (EBV) and ATLV. OKA(B) cells and its subclones contained: (1) B cell markers exclusively; (2) both EBV-specific antigen, EBNA and ATLV-specific antigen, ATLA detected by immunofluorescence test; (3) ATLV-specific polypeptides, p24 and p19; (4) ATLV-specific mRNA in ATLA-positive clones; (5) ATLV and EBV particles detectable by electron microscopy. These data clearly show that human B lymphocytes are susceptible to ATLV infection.     

Epstein-Barr virus-transformation of B-cell lines in ovarian cancer patients: feasibility of genomic storage for unlimited use

Objective

The aim of the current study is to test whether immortalized B-lymphocyte cell line via Ebstein-Barr virus (EBV) transformation is feasible and can be an unlimited source of genome wide study.

Methods

We obtained peripheral whole blood from 5 ovarian cancer patients and immortalized the B-cell lines using EBV transformation. The success rate was analyzed and the bio-identity of the genome was performed using human leukocyte antigen (HLA) identity test.

Results

EBV transformation was successful in all 5 cases (95% confidence interval, 46.3% to 100%). After cryopreservation of EBV-transformed B-cell lines and subsequent thawing, we observed that all cell lines were viable and proliferative. To check bio-identity, HLA-A, B, and DR were tested between the genome of the original samples and the transformed samples. The HLA typing revealed that all observed HLA-A, B, and DR type was identical in 5 cases before and after EBV-transformation.

Conclusion

The current results suggest that EBV-transformation of peripheral blood is an efficient tool in genome banking. The EBV-transformed B-cell lines may be a valuable resource of genome in multi-center translational research by the Korean Gynecologic Oncology Group.     

Intrinsic radiosensitivity of normal human fibroblasts and lymphocytes after high- and low-dose-rate irradiation.

The existence of heritable radiosensitivity syndromes and clinical observations in radiotherapy patients suggests that human cellular radiosensitivity differs among individuals. We report here an in vitro study of radiosensitivity in 30 fibroblast and 29 lymphocyte cultures obtained from cancer patients and controls. In 25 cases, both fibroblasts and lymphocytes were obtained from the same donors. Fibroblasts were cultured from skin biopsy samples, and peripheral T-cell lymphocytes were cultured from blood. Clonogenic survival assays were performed by using high- and low-dose-rate irradiation; lymphocytes were in G0 phase and fibroblasts in confluent plateau phase. Various end points were calculated and compared (i.e., surviving fraction at 2 Gy, initial slope of the survival curve, and doses resulting in 10 and 1% survival, respectively). Depending on the end point, the coefficient of variation of the survival parameters ranged from 31 to 68% for lymphocytes and 21 to 41% for fibroblasts following high-dose-rate irradiation. Similar ranges were obtained after low-dose-rate irradiation. Variance analysis performed on replicate assays in cultures derived from the same patient showed that variation due to technical or sampling errors was significantly lower than variation between individuals (P = 0.00034 and 0.014 for fibroblasts and lymphocytes, respectively). No correlation was observed between the radiosensitivity of lymphocyte and fibroblast cultures derived from the same donors. We conclude that there is significant variation in normal cell radiosensitivity among individuals. On the other hand, comparisons of lymphocyte and fibroblast radiosensitivities suggest that tissue-specific characteristics, such as differentiation status, may variably modulate radiosensitivity.     

T-cell-mediated inhibition of EBV-induced B-cell transformation: recognition of virus particles

In order to analyze the components of EBV-specific immunological memory that contribute to the T-cell-mediated inhibition of EBV-induced B-cell transformation, we have investigated the growth inhibitory potential of T cells cultured for 3 days with B cells exposed to transforming and non-transforming EBV preparations. T cells cultured for 3 days with EBV-infected autologous B cells inhibited the transformation of newly infected B lymphocytes. T cells cultured with the virus, with autologous uninfected B cells, or in the presence of 10 micrograms/ml Con-A had a significantly weaker inhibitory capacity. The inhibitory capacity was induced by co-cultivation with B cells infected with the transforming B95-8 virus strain, with the non-transforming P3HRI virus strain, and also with UV-inactivated B95-8 virus. B-cells infected with transforming B95-8 virus induced the strongest growth inhibitory activity. Activation of the T cells was shown by their IL-2 production, proliferation, and generation of non-specific cytotoxicity. These results suggest that the initial event of the T-cell response in EBV-infected cultures is induced by antigens associated with the viral particles presented by B lymphocytes.     

Lymphoblastoid transformation and kinetics of appearance of viral nuclear antigen (EBNA) in cord-blood lymphocytes infected by Epstein-Barr Virus (EBV).

Human cord-blood lymphocytes were infected with B95.8 Epstein-Barr virus (EBV) before and after separation into B- and T-cell populations. Lymphoblastoid cells exhibiting B-cell characteristics appeared after 2 to 3 days of culture in the total population and in the separated B-cell subpopulation but not in the T-cell subpopulation. EBV nuclear antigen (EBNA) was detected concurrently with the appearance of lymphoblastoid cells. The proportion of EBNA-positive cells corresponded to that of lymphoblastoid cells, and reached 50% after 4 days. EBNA was present only in cells with B-cell markers. These observations indicate that only B-cells are susceptible to EBV infection, that the transformation occurs within a few days and that EBNA is a valid early marker for susceptibility to EBV transformation.     

Cellular localization of an Epstein-Barr virus (EBV)-associated complement-fixing antigen in producer and non-producer lymphoblastoid cell lines

Anti-complement immunofluorescence (ACIF) was used to study the complementfixing antigens of human lymphoblastoid cell lines. These cell lines carry the Epstein-Barr virus (EBV) genome although only producer cultures synthetize EBV-specific antigens (virus capsid antigen, VCA and early antigen, EA) detectable by direct and indirect immunofluorescence, usually in less than 5% of the cells. The ACIF test revealed an antigen localized in the nucleus of the lymphoblastoid cells. In contrast to EA and VCA, this antigen was present in over 90% of the cells of both producer and non-producer cultures. The antigen was shown to be specific for EBV by comparing the reactions of 52 sera in the ACIF test. Sera giving the nuclear reaction contained antibodies to VCA, EA or antigens detectable by complement fixation tests on cell extracts, but sera without EBV antibodies failed to give the reaction. Weak, equivocal or discordant reactions occurred with six sera with low titres in VCA, EA or complement fixation tests. Cell lines derived by transformation of human and primate lymphocytes by EBV gave the nuclear reaction. Control cells with no known association with EBV were non-reactive. These included foetal lymphocytes transformed by phytohaemagglutinin, cell lines derived from breast cancer, glioma, normal glia, pleuritis maligna and myeloma, and two marmoset lymphoid lines carrying Herpesvirus saimiri (HVS). In preliminary experiments, the ACIF test was used as a tool to trace the EBV genome at the cellular level. Cells from two Burkitt lymphoma biopsies, one tested after biopsy and one after passaging in nude mice, contained an EBV-specific antigen. Three clones of cells derived from hybrids of mouse somatic cells and a human lymphoblastoid cell line also contained such an antigen, but the number of reactive cells varied from clone to clone. A fourth clone was non-reactive.     

Retinoic acid and steroids inhibit Epstein-Barr virus-induced nuclear antigen, DNA synthesis and lymphocyte transformation

Retinoic acid, dexamethasone and prednisolone were evaluated for their effects on Epstein-Barr virus (EBV)-induced nuclear antigen (EBNA), DNA synthesis and transformation of human thymus-independent, B lymphocytes. It was found that continuous treatment of target cells with these agents completely inhibited EBV-induced transformation events. However, discontinuous treatment of the virus-infected cultures with these agents resulted in the recovery of DNA synthesis, and the appearance of EBNA and transformation. When the cells were treated with these agents 8 days after the virus infection, the inhibitors had no effect. These results show that only continuous treatment with these agents inhibited EBV-mediated transformation; these inhibitors had no effect once the EBNA and EBV-induced DNA synthesis were initiated in target lymphocytes.     

Effects of cyclosporine on human B-cell lymphoma development in vivo.

Cyclosporine (CsA) is a potent immunosuppressive agent primarily affecting T-lymphocyte function. Patients receive CsA following organ transplantation to prevent rejection. These patients are at high risk for developing Epstein-Barr virus (EBV)-induced lymphoproliferative disease (LPD) or B-cell lymphoma (BCL). Severe Combined Immunodeficient (SCID) mice reconstituted with human peripheral blood leukocytes (PBL) develop fatal B-cell lymphomas of human origin following latent or active infection with EBV. This model was utilized to determine the role of CsA in the development of human BCL. SCID mice were reconstituted with PBL, latently or actively infected with EBV, and treated with CsA. Following active EBV infection, mice developed human BCL with or without CsA treatment. In contrast, treatment with CsA prevented the development of BCL in mice latently infected with EBV. This suggests a T-cell interaction with latently infected B-cells which is perturbed by CsA. Further understanding of this interaction and the occurrence of human BCL may allow the development of strategies to prevent, detect, or treat malignancies associated with immunosuppression.     

Interleukin 15-mediated induction of cytotoxic effector cells capable of eliminating Epstein-Barr virus-transformed/immortalized lymphocytes in culture.

BACKGROUND: Interleukin 15 (IL-15) activates cytotoxic lymphocytes and drives the expansion of memory T cells. Its role in immune control of virus-transformed cells and other tumor cells remains to be elucidated. We investigated the role of IL-15 in controlling Epstein-Barr virus (EBV)-transformed/immortalized lymphocytes in culture. EBV is a highly potent lymphocyte-transforming and opportunistic oncogenic herpesvirus associated with several human tumors. METHODS: Peripheral blood mononuclear cells (PBMCs) from healthy donors were infected with EBV and cultured with either IL-15 or IL-15 plus anti-IL-15 antibodies for 3-4 weeks. We monitored EBV-induced transformation by assessing the clearly visible cell clusters by microscopy and analyzing the expression of EBV-encoded latent membrane oncoprotein-1 (LMP-1) and the EBV nuclear antigen (EBNA) complex by immunoblotting and immunofluorescence techniques, respectively. We depleted EBV-infected cultures of PBMCs of specific effector cell populations to investigate the effector cells involved in mediating IL-15 effect. RESULTS: The presence of IL-15 resulted in the complete elimination of EBV-transformed cells in PBMC cultures. Western blot and immunofluorescence analyses performed 3-4 weeks after infection showed no detectable levels of LMP-1 and EBNA in IL-15-treated EBV-infected cultures, whereas IL-15-untreated EBV-infected cultures and IL-15/anti-IL-15-treated cultures expressed both proteins. IL-15 mediated its anti-EBV effect through early and late response mechanisms, i.e., by first activating natural killer (NK) cells and subsequently inducing cytolytic NK-T cells. The presence of anti-IL-15 neutralizing antibodies abrogated IL-15's effect on both mechanisms. CONCLUSION: In vitro, IL-15 mediated complete elimination of EBV-infected/transformed lymphocytes via successive activation of NK and NK-T cytotoxic effectors. If these in vitro findings reflect in vivo mechanisms, then IL-15 might be considered for cytokine-based immunotherapy in patients with EBV-associated lymphoproliferative disorders/malignancies.