J. H. Pope

Reactions of sera from patients with rheumatoid arthritis, systemic lupus erythematosus and infectious mononucleosis to Epstein-Barr virus-induced polypeptides.

P3HR-1 and Ramos cells induced with sodium butyrate and 12-O-tetradecanoylphorbol 13-acetate were used in the protein immunoblot technique to identify Epstein-Barr virus (EBV)-specific antibodies present in sera from clinically normal individuals and patients with systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). Sixteen EBV-specific polypeptides were detected ranging in mol. wt. from 22,000 (22K) to 140K. Many of the sera contained antibodies to different subsets of these antigens, and a high proportion expressed autoantibodies which reacted with cellular components from an EBV genome-negative cell line. About 50% of the sera from each category reacted with the 44K to 48K and 36K and 38K early antigen (EA) components. A high proportion of the SLE sera (64%) were found to contain anti-EA antibodies, suggesting an association between EBV and SLE. Almost all of the EBV-seropositive sera examined contained antibodies against a 22K late antigen, but none of the sera from IM patients reacted with this polypeptide.

Factors influencing the human cytotoxic T cell response to autologous lymphoblastoid cell lines in vitro

Epstein-Barr virus (EBV)- and fetal calf serum (FCS)-specific cytotoxic human T cells can be generated in vitro, and have been shown to be HLA-antigen restricted. In the present work, peripheral blood mononuclear cells were stimulated with the gamma-irradiated autologous lymphoblastoid cell line (LCL) grown previously in FCS, human serum, or without serum. The induction and generation of cytotoxic T cells was carried out exclusively in culture medium containing autologous serum. With EBV-seronegative responders, FCS-grown stimulator LCL generated a FCS-specific cytotoxic T cell response. AB serum-grown LCL generated only a weak response, except at a high stimulatory dose, where the response tended to be essentially nonspecific. EBV-seropositive responders, in contrast, gave a typical secondary EBV-specific response regardless of the serum in which the LCL had been grown previously; no FCS response was detected. Dose-response and cold target inhibition studies confirmed these results. EBV immunity obviously plays a major role in the T cell response to the autologous LCL, which can no longer be viewed simply as a form of autologous mixed leucocyte reaction.

Epstein-Barr Virus Nuclear Antigens 1 and 2 in Burkitt Lymphoma Cell Lines Containing either ‘A’- or ‘B’-Type Virus

Epstein-Barr virus (EBV) has previously been classified into two different types according to the organization of the EB nuclear antigen 2 (EBNA2) gene region. Type A virus hybridizes with probes from B95-8 or M-ABA viruses and the B type virus with probes from the Jijoye virus strain. The substituted region in EBV type B codes for a different, but related EBNA2 antigen, named EBNA2B as opposed to EBNA2A. In this study Burkitt lymphoma cell lines, previously typed according to the EBV viral genomes they carry, as well as some matching lymphoblastoid cell lines were examined by immunoblotting for the expression of both EBNA1 and EBNA2 antigens. Variation in the molecular weight of EBNA1 indicated that both A and B virus types contained a variety of different virus isolates. EBNA2A was identified in all lines carrying A type viral genomes, but was not observed in any of the lines harboring B type virus. EBNA2B was identified in 4 of 10 Burkitt lymphoma lines carrying EBV type B.

Evidence for the involvement of HLA-DR antigens in restricted cytotoxicity by fetal calf serum-specific human T cells

Fetal calf serum (FCS) generated at least two distinct populations of human cytotoxic cells in vitro. One population expressed natural killer (NK) cell-like activity and lysed K562 and HSB-2 targets more effectively than autologous or allogeneic lymphoblastoid cell lines (LCLs). The other population contained FCS-specific cytotoxic T cells which preferentially lysed the autologous LCLs and showed minimal lysis of K562. E-rosette separation and cold target competition experiments clearly established that NK cells were not involved in the self-reactive lysis. Moreover, the lytic activity of the E-rosetted T cells was reduced by up to 95% when autologous target cells were grown in human AB serum rather than FCS, showing that FCS-associated determinants on targets were essential in the cytolytic phase. Autologous LCLs grown in FCS were also considerably stronger competitors than human serum-grown LCLs. The consistent self-preferred lysis suggested that HLA antigen-related restriction was involved, but the patterns of lysis did not implicate HLA-A or B antigens, and monoclonal antibody (W6/32) to an A, B, and C monomorphic determinant failed to block FCS-specific lysis. In contrast, monoclonal antibody (DA.2) to a monomorphic determinant of DR effectively blocked FCS-specific lysis. Cytotoxicity tests with a small panel of DR-typed donors indicated that strong cross-reactions were invariably associated with sharing of DR antigens, particularly DR2, and to a lesser but significant extent DR7. Although DR antigen sharing did not always result in lysis of allogeneic targets, the overall evidence strongly suggests that FCS-specific T-cell cytotoxicity in humans is restricted by products encoded by or associated with the DR genes.

Identification of Epstein-Barr Virus-induced Polypeptides in P3HR-1 Cells by Protein Immunoblot

The protein immunoblot technique was used to identify Epstein-Barr virus-specific antigens present in sodium butyrate-induced P3HR-1 cells. Using sera from patients with either nasopharyngeal carcinoma or arthritis, 16 polypeptides were detected ranging in molecular weight from 22K to 140K. Each of the anti-EA-, anti-VCA-positive sera were found to contain antibodies to different subsets of the antigens. A 72K protein was identified which was consistent with the nuclear antigen (EBNA), and culturing cells in the presence of disodium phosphonoacetate allowed identification of 140K and 22K antigens as late viral products. Treatment of cells with sodium butyrate revealed that expression of some antigens increased in parallel with the time of incubation of the cells in butyrate while other antigens either appeared early and then decreased in intensity or were only present after a number of days of butyrate treatment. One of the antigens which decreased with the time cells were treated with butyrate was EBNA.

Inhibition of EB virus transformation of non-adherent human lymphocytes by co-cultivation with adult fibroblasts

Transformation of a special population of non-adherent human lymphocytes by EB virus (EBV) is reversibly inhibited by co-cultivation on adult human fibroblasts. Neither fluid from adult fibroblast cultures nor extracts of fibroblasts inhibited such transformation, and the growth of already transformed lymphocytes was not inhibited on adult fibroblasts. The EBV-associated nuclear antigen (EBNA) was detectable in inhibited cultures, with a maximum level of about 15% at 14 days after which it decreased gradually. Reversal of inhibition at 28 days, by either addition of phytohaemagglutinin or by removal of the lymphocytes from the adult fibroblasts, resulted in a prompt increase in the percentage of EBNA-positive cells and typical lymphoblastoid outgrowth. Transformation of EBV-infected non-adherent lymphocytes could be inhibited by the addition of adult fibroblasts up to 2–4 days after infection. The results indicate that, in the EBV-infection of non-adherent lymphocytes on adult fibroblasts, a block resulting in inhibition of transformation occurs between the production of EBNA and the onset of autonomous proliferation of the infected lymphocytes.

Distribution of Epstein-Barr Virus Strains with Different EBNA 2 Genotypes in Burkitt-Endemic Areas

The Epstein-Barr (EB) virus shows a striking association with two human tumours, Burkitt’s lymphoma (BL) and nasopharyngeal carcinoma (NPC), both of which exhibit an unusual geographical distribution. This is particularly interesting because the virus itself is ubiquitous, being found as a widespread and largely asymptomatic infection in all human communities. To date, there is little evidence to support the view that these different consequences of EB virus infection reflect the existence of different viral strains. Indeed virus isolates from different regions of the world, or more importantly from patients with different virus-associated diseases, have proved to be remarkably similar when their genomes have been compared by restriction enzyme analysis (1). Accordingly much more attention has been given to the identification of non-viral co-factors, which are clearly involved in the pathogenesis of both BL and NPC, as a means of explaining the unusual distribution of these tumours.

Identification of EBV-Induced Polypeptides with Sera from Patients with SLE, RA or IM

Healthy EBV-seropositive individuals usually have antibodies against EBNAl, membrane antigens (MA) and viral capsid antigens (VGA). In addition, antibodies against Epstein-Barr virus early antigen (EA) often can be found in the sera of patients with diseases such as Burkitt’s lymphoma (BL), nasopharyngeal carcinoma (NPC), systemic lupus erythematosus (SLE), rheumatoid arthritis (RA) and infectious mononucleosis (IM). The aim of this study was to examine reactions of sera from patients with SLE, RA and IM to EBV-induced polypeptides. Because of the nature of these diseases, a major problem in the detection of antibodies to the EBV antigens is the presence of autoantibodies in the sera of many patients. However, this problem can be overcome by the use of immunoblotting, employing extracts of both an EBV-positive (P3HR-1) and an EBV-negative (Ramos) cell line.

Variation in Epstein-Barr Virus Strains

EBV is associated with several diseases in humans. It causes most heterophile-positive cases of infectious mononucleosis (IM), and is associated with African Burkitt’s lymphoma (BL) and nasopharyngeal carcinoma (NPC)(Pearson, 1980). The diversity in geographical distribution, age range, and histological characteristics of these diseases may be a reflection of different pathogenic properties of a family of closely related viruses. To be able to effectively determine whether different EBV subtypes are associated with specific diseases, spontaneous EBV genome-positive lymphoblastoid cell lines (LCL) were established from patients with either rheumatiod arthritis (RA) or infectious mononucleosis (IM) or from healthy controls. Differences in virus strains were determined by variation in the EBV specific antigens expressed in each of the cell lines.

Identification of Multiple Epstein-Barr Virus Nuclear Antigens

Using the protein immunoblot technique, the Epstein-Barr virus (EBV) nuclear antigen (EBNA) was identified in a variety of EBV genome-positive cell lines. The antigen portrayed a molecular weight of between 70,000 and 75,000 daltons depending on the cell line examined. When a similar immunoblot was performed, employing sera from patients with rheumatoid arthritis, a number of antigens in addition to EBNA were identified. The most prominent of these antigens exhibited molecular weights of 92,000 and 110–115,000 daltons. Unlike EBNA, the 92,000 dalton protein maintained the same molecular weight in each of the cell lines in which it was present. The 92,000 dalton protein was present in all of the EBV genome- positive cell lines except QIMR-GOR and lines carrying the P3HR-1 virus.