Infection of leukaemic B lymphocytes by Epstein Barr virus

Epstein-Barr virus (EBV) infection of B lymphocytes in vitro gives rise to immortalized lymphoblastoid cell lines. Previous reports have shown that chronic lymphocytic leukaemia (CLL) cells, although infectable by EBV, are resistant to immortalization (1-4), although a small number of CLL cell lines have been reported (5-7). In the present study we have analysed early events occurring after EBV infection in 16 CLL samples. Out of 16 samples, 15 could be infected by the virus and expressed the full EB viral nuclear antigen (EBNA) complex but only one out of 16 expressed the latent membrane protein (LMP). The five CLLs in which we could investigate the presence of viral episomes showed circularized EBV by 16 hours after infection. The sequence of EBNA expression and genome circularization mirrored that seen in normal B cells, although genome amplification was not detected. The only CLL sample which expressed LMP after EBV infection was induced to proliferate for 2-3 weeks, but no cell line was established. Immortalized cell lines were obtained from three out of 16 samples tested, but all were polyclonal for light chain expression and had arisen from the CD5-negative, normal B-cell population. Thus the inability of EBV to induce proliferation of most CLL cells correlated with the absence of LMP expression which is invariably expressed during immortalization of normal B cells. This novel type of restricted gene expression could be compatible with evasion of host immune responses and consequent long-term survival of the cell in vivo.     

Epstein-Barr virus contributes to the malignant phenotype and to apoptosis resistance in Burkitt's lymphoma cell line Akata

In the present study, we established an in vitro system representing the Burkitt's lymphoma (BL)-type Epstein-Barr virus (EBV) infection which is characterized by expression of EBV-determined nuclear antigen 1 (EBNA-1) and absence of EBNA-2 and latent membrane protein 1 (LMP1) expression. EBV-negative cell clones isolated from the EBV-positive BL line Akata were infected with an EBV recombinant carrying a selectable marker, and the following selection culture easily yielded EBV-infected clones. EBV-reinfected clones showed BL-type EBV expression and restored the capacity for growth on soft agar and tumorigenicity in SCID mice that were originally retained in parental EBV-positive Akata cells and lost in EBV-negative subclones. Moreover, it was found that EBV-positive cells were more resistant to apoptosis than were EBV-negative cells. EBV-infected cells expressed the bcl-2 protein, through which cells might become resistant to apoptosis, at a higher level than did uninfected cells. This is the first report that BL-type EBV infection confers apoptosis resistance even in the absence of expression of LMP1 and BHRF1, both of which are known to have an antiapoptotic function. Surprisingly, transfection of the EBNA-1 gene into EBV-negative Akata clones could not restore malignant phenotypes and apoptosis resistance, thus suggesting that EBNA-1 alone was not sufficient for conferring them. Our results suggest that the persistence of EBV in BL cells is required for the cells to be more malignant and apoptosis resistant, which underlines the oncogenic role of EBV in BL genesis.     

Epstein-Barr virus transmission from a blood donor to an organ transplant recipient with recovery of the same virus strain from the recipient's blood and oropharynx

A previous study (Savoie et al, Blood 83:2715, 1994) identified eight transplant patients who acquired Epstein-Barr virus (EBV) infection during the peritransplant period. Three of these patients subsequently developed B-cell lymphoproliferative disease within 4 months of transplantation. Among these, there was a 16-year-old liver transplant patient who was negative for EBV at the time of transplant and who received an EBV-negative organ. After transplant, this patient was transfused with 9 U of packed red blood cells. Eight of the donors were EBV-positive and one was EBV-negative. We succeeded in obtaining spontaneous lymphoblastoid cell lines (LCLs) from the blood of three of these donors, one of whom also yielded a cord-blood line established with his throat-wash EBV. Blood from a fourth donor did not yield an LCL, but his throat washing did have transforming activity when inoculated onto cord-blood leukocytes. We initially could establish spontaneous LCLs only from the recipient's blood. However, a throat-wash sample taken 11 weeks later did show transforming activity. The recipient was shown to have acquired the EBV infection from one of eight EBV-seropositive blood donors. Analysis of fragment length polymorphisms after polymerase chain reaction amplification of the EBV BamHI-K fragment was used to establish strain identity. Western blot analysis for existence of size polymorphisms in three classes of Epstein-Barr nuclear antigens (EBNA-1, EBNA-2, and EBNA-3) confirmed the DNA results. It is noteworthy that the blood donor responsible for transmitting his EBV strain to the recipient had experienced clinical infectious mononucleosis 15 months before donating blood. Our results may, thus, indicate a requirement for leukodepletion of blood destined for immunosuppressed EBV-negative patients. Finally, blood donors with a recent history of infectious mononucleosis should probably be identified so that their blood is not given to EBV-negative transplant patients.     

Explorers. Christopher Columbus and Leonardo da Vinci

Epstein-Barr virus (EBV) recently has been associated with Hodgkin's disease (HD) and the EBV genome was found in CD30-positive Reed-Sternberg cells. Therefore, tissue sections from 25 cases of HD, 35 cases of CD30-positive non-Hodgkin's lymphoma (NHL) (seven CD30-positive anaplastic large cell lymphomas [ALCLs] and 28 CD30-positive non-ALCLs), and 12 cases of CD30-negative NHL that previously had been screened for the presence of EBV by polymerase chain reaction and DNA in situ hybridization were studied by immunohistochemistry for the expression of the latent EBV proteins, latent membrane protein (LMP), and Epstein-Barr nuclear antigen-2 (EBNA-2). We also analyzed the expression of the B-cell activation molecule CD23 and the adhesion molecules LFA-1/CD11a and ICAM-1/CD54 because the upregulation of these molecules by LMP and/or EBNA-2 in vitro has been related to the EBV-induced lymphocyte growth. Latent membrane protein expression was found in Reed-Sternberg cells in nine of 25 cases (36%) of HD and in large, occasionally Reed-Sternberg-like tumor cells in six of 47 cases (12%) of NHL; these six tumors were CD30-positive, histologically high-grade NHL (one CD30-positive ALCL and five CD30-positive non-ALCLs). All the LMP-positive cases were also polymerase chain reaction EBV positive while LMP expression was not found in polymerase chain reaction EBV-negative HD and NHL. No staining for EBNA-2 was detected in our series. In view of the transforming potential of the LMP, these findings suggest that EBV may be associated with the development of some cases of HD and CD30-positive NHL. These findings also suggest a correlation between the expression of LMP and the detection of CD30 in tumor cells of HD and NHL. In contrast, no correlation was found between the expression of LMP and the detection of CD23, LFA-1/CD11a, and ICAM-1/CD54 in tumor cells of HD and NHL.     

Drug delivery system (DDS) for the use of antisense oligodeoxynucleotide phosphorothioate in controlling gene expression]

Subgroups of the B cell malignancies are known to be associated with Epstein-Barr virus (EBV) infection, especially in immunocompromised patients. These are fatal and refractory to conventional antineoplastic therapy. B cells are usually post-mitotic cells and even mitogen activated or transformed B cells have shown relative resistance against viral mediated gene transfer. To address this issue, we employed a replication-defective herpes simplex virus-1 (HSV-1) to mediate gene transfer into EBV-transformed B cells. The virus expresses the herpes simplex virus thymidine kinase (HSV-TK) and the E. coli lacZ reporter genes and is designated T0Z.1. We used the lymphoblastoid cell line SWEIG as a model for human EBV-related B cell malignancy. This cell line was established by in vitro EBV infection of primary human peripheral blood mononuclear cells. When SWEIG cells were infected with T0Z.1, X-gal staining revealed lacZ expression in more than 20% cells even at multiplicity of infection (MOI) as low as 1 and the expression persisted for at least one week. Ganciclovir (GCV) administration after T0Z.1 infection effectively decreased the number of the infected tumor cells in a dose-responsive manner. Viral toxicity was analyzed by cell proliferation assay (MTS assay) and found to be little even at 10 MOI infection. Three MOI of the virus yielded maximum antineoplastic effect and more than 50% tumor cells were killed by HSV-TK/GCV. These results suggest the potential utility of replication-defective HSV-1 for the treatment of EBV-related B cell malignancies.     

In vitro effects of retinoids on the proliferation and differentiation features of Epstein-Barr virus-immortalized B lymphocytes

Retinoids have been shown to be effective in the chemoprevention and treatment of certain human malignancies. In this review, we will summarize our recent results concerning the effects of retinoids on the proliferation and differentiation of Epstein-Barr virus (EBV)-immortalized lymphoblastoid B-cell lines (LCLs), an in vitro model of EBV-related lymphoproliferative disorders arising in immunosuppressed hosts. Retinoids proved to be powerful inhibitors of the proliferation of EBV-infected LCLs in vitro, with 13-cis-retinoic acid (RA), all-trans-RA, and 9-cis-RA being the most effective compounds. Of note, retinoid-induced growth arrest in vitro appears irreversible at drug concentrations (10(-6) mol/L) which may be reached in man after oral systemic therapy. The antiproliferative activity exerted by retinoids on LCLs is a generalized phenomenon usually associated with a progressive accumulation in G0/G1 phases of treated cells. The strong upregulation of p27Kip1 invariably observed in cells exposed to retinoids may contribute to the decreased number of cycling cells, probably by inhibiting the transition from the G1 to S phase. Moreover, we obtained evidence indicating that the antiproliferative effects of retinoids are not dependent on the induction of terminal differentiation of EBV-immortalized B lymphocytes. In fact, the modifications induced by retinoids relative to LCL morphology, phenotype (downregulation of CD19, HLA-DR, and s-Ig, and upregulation of CD38 and c-Ig), and IgM production were highly variable among the lines tested and often only slightly relevant. Finally, the antiproliferative activity exerted by retinoids on LCLs is not mediated by a direct modulation of viral latent antigens, since EBNA-2 and LMP- downregulation was a late event detected only in some cell lines. These results indicate that retinoids may be useful in the medical treatment of EBV-related lymphoproliferative disorders of immunosuppressed patients, particularly in the earlier phases of these diseases.     

CD4+ T cells inhibit growth of Epstein-Barr virus-transformed B cells through CD95-CD95 ligand-mediated apoptosis

Greater than 90% of the human population acquire Epstein-Barr virus (EBV) in infancy and retain a lifelong latent infection without any clinical consequences. Nevertheless EBV has been identified as the causal agent of infectious mononucleosis, and is associated with several tumours including endemic Burkitt's lymphoma and B cell lymphomas in immunosupressed patients. B cells infected with EBV are transformed in vitro and grow continuously as lymphoblastoid cell lines. The growth of EBV-transformed B cells in vivo is controlled by the immune system. Studies on immunity to EBV have mainly focused on MHC class I-restricted CD8+ cytotoxic T cells specific for viral latent antigens. Here it is reported that in vitro stimulation of peripheral blood lymphocytes by autologous EBV-infected B cells, which have been induced to express lytic cycle antigens, gives rise to a predominantly CD4+ T cell response. Furthermore, the growth of EBV-infected B cells can also be regulated by these activated CD4+ T cells through apoptosis mediated by CD95-CD95 ligand (CD95L). CD95-CD95L-mediated apoptosis is an important mechanism of normal B cell growth regulation. As EBV-transformed B cells remain susceptible to this mechanism, the control of EBV in vivo may be not only by virus-specific CD8+ cytotoxic T cell immunity but also by normal mechanisms of immune regulation of B cell growth.     

Phenotype-dependent differences in proteasome subunit composition and cleavage specificity in B cell lines

We have compared the subunit composition and enzymatic activity of purified 26S proteasomes from Burkitt's lymphoma (BL) cells and in vitro EBV-transformed lymphoblastoid cell lines (LCLs) of normal B cell origin. Low expression of the IFN-gamma-regulated beta low molecular mass polypeptide (Lmp)2, Lmp7, and MECL-1 was demonstrated in a panel of seven BL lines that express the germinal center cell phenotype of the original tumor. Coexpression of Lmp2 and Lmp7 with the constitutively expressed subunits delta and MB1 was demonstrated in the BL lines by immunoprecipitation and two-dimensional gel fractionation of the 20S proteasomes. Coexpression of these subunits correlated with reduced levels of chymotrypsin- and trypsin-like activities detected by the cleavage of fluorogenic substrates. Down-regulation of Lmp2 and Lmp7 and decreased chymotrypsin- and trypsin-like activities were also observed in purified proteasomes from a c-myc-transfected subline of the ER/EB2-5 LCL that has adopted a BL-like phenotype. A synthetic peptide analogue of the immunodominant epitope from the EBV nuclear Ag 4 (E4416-424Y) was cleaved by proteasomes from BLs and A1, while proteasomes from LCLs were inactive. Cleavage of the E4416-424Y peptide was not affected by treatment of the BL cells with IFN-gamma despite both significant up-regulation of Lmp2 and Lmp7 and reconstitution of chymotrypsin and trypsin-like activities against fluorogenic substrates to LCL-like levels. The results demonstrate that B cell lines representing different stages of B cell activation and differentiation express proteasomes with different subunit compositions and enzymatic activity. This may result in the generation of a distinct set of endogenous peptides and influence the immunogenicity of these cells.