Michael Steinitz

Epstein-Barr virus (EBV) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood: II. Epstein-Barr virus studies

Abstract In B-cell fractions isolated from human peripheral blood, the frequency of surface immunoglobulin-positive and of complement receptor-positive cells showed a good correlation with the frequency of EBV-binding cells, as detected by membrane fluorescence or by a quantitative bioassay for infectious virus in the absorbed supernatant fluid. There was a close relationship between all three parameters mentioned, the frequency of EBNA-positive cells 2 or 3 days after the infection, and the stimulation of cellular DNA synthesis. So-called O-cell fractions remaining after the removal of nylon adherent and E-rosetting cells contained a certain frequency of complement receptor-positive cells and absorbed EBV to a limited extent, but did not respond to EBV infection with EBNA induction or stimulation of DNA synthesis. None of the T-cell fractions absorbed EBV to a detectable extent. This includes the T ea + fraction that contained a certain proportion of complement receptor-positive cells. It is concluded that the previously demonstrated relationship between EBV receptors and complement receptors on B-lymphoblastoid lines also holds for peripheral B lymphocytes. In these cells, virus absorption is followed by an intracellular infectious process, signaled by the appearance of EBNA and cellular DNA synthesis. O cells carry complement receptors and absorb EBV to a certain extent, but do not respond with EBNA synthesis or DNA stimulation, presumably due to intracellular restrictions. T cells do not bind EBV, and the complement receptors present on some cells of the T ea + fraction do not function as EBV receptors.

Epstein-Barr virus (EBV)-induced change in the saturation sensitivity and serum dependence of established, EBV-negative lymphoma lines in vitro.

Abstract Superinfection with EBV was found to have made two EBV-negative cell lines, BJAB and Ra#1, more independent of some serum factor or factors. The converted cells become also more resistant to saturation conditions. This effect of the superinfection differs from the serum effect.

Further studies on the differences in serum dependence in EBV negative lymphoma lines and their in vitro EBV converted, virus-genome carrying sublines☆

Abstract Independently EBV-converted, viral genome carrying sublines of the originally EBV negative Ramos and BJAB lymphoma lines showed decreased serum dependence, in comparison with the progenitor lines. The virus negative lines could not grow in 10% dialysed FCS, unless reconstituted with the dialysate. The converted lines grew well on dialysed serum. Seeding of the EBV negative lines with larger inocula enabled them to “take off” on dialysed serum as well. The EBV converted lines were able to form colonies in soft agar, whereas the original negative lines failed to do so.

Stimulation of peripheral human lymphocytes by autologous EBV genome-carrying lymphoblastoid cell lines.

Abstract EBV-carrying lymphoblastoid cell lines (LCL) can stimulate lymphocytes of the autologous donor [autologous stimulation (AS) assay] to blast transformation and generation of killer cells of broad-range cytotoxicity. We have tested the possibility of developing an EBV-specific AS assay for use in the demonstration of EBV-specific memory cells in the peripheral blood of normal donors. For this purpose, the stimulating lines were treated by heat and protein synthesis inhibitors to prevent the release of possible nonspecifically mitogeneic factors. Moreover, an extensive purification of the effector cells was achieved in the hope of removing a possible cellular contributor to the observed nonspecific cytotoxicity. None of these approaches was able to narrow down this nonspecific cytotoxicity to an EBV-specific response. We have shown that AS reaction (1) is not related to the release of lymphocyte mitogeneic factors by stimulating LCL, (2) is mediated by Fc receptor-negative T cells, and (3) does not require macrophage nor any other non-T helper cells.

Establishment of a human lymphoblastoid cell line with specific antibody production against group A streptococcal carbohydrate.

A human lymphoblastoid cell line, secreting specific antibody against Group A carbohydrate (A-CHO) was established by pre-selection of antigen binding normal human lymphocytes, followed by Epsetin Barr virus (EBV) induced immortalization. Culture supernatants were assayed for anti A-CHO antibodies by radioimmunoassay, N-acetyl-glucosamine-coupled T4-phage plaque inhibition tests and passive hemagglutination. As a rule, the supernatants contained about 10 micrograms/ml anti-A-CHO antibodies of the IgM-kappa type. The antibody was fractionated and partially purified on an N-acetyl glucosamine Sepharose 4B column with a recovery of about 3 micrograms/ml of supernatant.

Inhibition of DNA Metabolism in Human B Lymphocytes by a Substrain of Epstein-Barr Virus (P3HR-1): A Method for Virus Quantitation

Pretreatment with the P3HR-1 substrain of Epstein-Barr virus (EBV) inhibited approximately 85% of the DNA stimulation induced by Staphylococcus aureus in human B lymphocytes. In parallel experiments, the DNA stimulation induced by the transforming B95-8 substrain of EBV was almost completely inhibited by prior exposure to P3HR-1 virus. Phytohemagglutinin stimulation was only slightly inhibited, whereas the DNA synthesis of continuously growing Raji cells was inhibited to a considerable extent. The degree of DNA inhibition was correlated with the induction of the EBV-determined antigens (membrane, early, and nuclear antigens) in sensitive indicator cells.

Superinfection of EBV-carrying lines with Herpes virus papio (HVP): Induction of early antigen (EA) and inhibition of cellular DNA synthesis

Abstract An Epstein-Barr virus (EBV) related herpes virus H. papio (HVP) derived from 9B cell line was shown to inhibit the DNA synthesis in B-cell mitogen stimulated human lymphocytes, in B95-8 virus exposed human lymphocytes, and the DNA metabolism of EBV-receptor positive cell lines. The inhibition could be abolished by a Burkitt serum known to have a high anti-EBV titer. Similar inhibition activities were shown for P3HR-1 virus. The relationship between the inability to immortalize normal cells, the ability to induce early antigen (EA) in certain cell lines and the DNA inhibitory effect of both 9B and P3HR-1 viruses is discussed.

Low-molecular-weight (61K) mu chain in P3HR-1 cells and various derived somatic hybrids

The expression of a truncated 61K mu chain in the Burkitt lymphoma lien P3HR-1 and a derived ouabain and TG-resistant subline, PUT, and in various somatic cell hybrids with PUT as one of their parents is described. Both PUT and P3HR-1 contain intracellular mu and kappa chains, but express no membrane immunoglobulin. Immunoprecipitation of 14C-labeled amino acid or [3H]glucosamine-labeled P3HR-1 extracts with anti-mu serum brought down the same 61K mu chain. Anti-light-chain sera did not precipitate the truncated mu chain. P3HR-1 is a clonal derivative of the Burkitt lymphoma (BL) line Jijoye. The parental Jijoye line is membrane-IgM positive and contains two normal-sized mu chains. Both are precipitable by anti-mu and anti-kappa sera. In addition, anti-mu also precipitated a 61K mu chain. A 61K mu chain was also identified in the following somatic hybrids: PICATPO, an autohybrid of two different P3HR-1 sublines, PUTRAL and PUT/ARH-77, derived from the fusion of PUT with the membrane-IgG-positive BL line Rael and the lymphoblastoid cell line (LCL) ARH-77, respectively, and the HP-1 (PUT/HL-60) hybrid, derived from the fusion of PUT with the granulocytic leukemia line, HL-60. The 61K mu chain could not be detected in some other BL/BL hybrid combinations, namely RAMPUT (PUT/Ramos) and NAMPUT (PUT/Namalva). The anti-light-chain serum (lambda or kappa) had no detectable effect on the truncated 61K mu chain in any of the cases tested, suggesting a lack of assembly between the 61K mu chain and the light chain.