Eitan Yefenof

Relationships between complement activation, complement binding, and EBV absorption by human hematopoietic cell lines

Abstract Complement consumption (C.C.) and C3 deposition on the cell membrane, visualized by membrane fluorescence (CMF), were compared in a collection of established human lymphoid lines. C.C. was independent of the presence of C3 receptors. A positive CMF reaction was seen only in lines that expressed C3 receptors, however. Trypsin treatment abolished CMF and EAC rosetting but had virtually no influence on C.C. EBV absorptive capacity correlated with both C3-receptor expression, as measured by EAC rosetting, and CMF but not with C.C. This is in line with our previous finding on the association of EBV and C3 receptors.

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) receptors, complement receptors, and EBV infectibility of different lymphocyte fractions of human peripheral blood. I. Complement receptor distribution and complement binding by separated lymphocyte subpopulations.

Abstract Human peripheral lymphocytes were fractionated into a variety of B-, T-, and O-cell fractions and were characterized with regard to several surface receptors. There was a strong correlation between the frequency of EAC receptor-positive cells and the percentage of complement membrane fluorescence (CMF)-stained cells following exposure to fresh human serum and subsequent staining with an anti-C3 conjugate. CMF staining did not diminish in C4-deficient or hypogammaglobulinemic serum, or in the presence of EDTA or EGTA-Mg 2+ , but was completely negative with C3-depleted normal human serum. In all likelihood, the staining is therefore due to the direct binding of C3 to preformed receptors on the lymphocyte surface. In addition to the surface Ig-positive B-cell fractions, C3 receptors were also detected on part of the O-cell population and on a proportion of the Fc receptor-positive T cells.

C3-Activating proteases on human lymphoblastoid cells superinfected with epstein-barr virus☆

Abstract Mouse spleen lymphocytes to which uncleaved human C3 had been attached were interacted with two EBV− lines (RAMOS and BJAB) and six EBV+ sublines (A.W-RAMOS, 11.W.A.RAMOS, EHR.A.RAMOS, EHR.B.RAMOS; GC-BJAB, BJAB/HRIK), which were obtained by superinfection of the EBV− lines with EBV. Depending on the presence of C3, the EBV− sublines except for EHR-A.RAMOS formed rosettes with the mouse spleen lymphocytes. The interaction was inhibitable by DFP. The experiments are interpreted as indicating the presence of proteolytic, C3-cleaving activity on the surface of the EBV+ sublines. The expression of this activity on the EBV+ lines only, correlates it to the transformation of EBV− lines into EBV+ lines.

Stimulation of human peripheral blood lymphocytes by autologous EBV-infected B cells

EBV carrying lymphoblastoid cell lines (LCL) strongly stimulate DNA synthesis in normal autologous peripheral T lymphocytes in vitro (autologous stimulation—AS), and generate non-specifically cytotoxic T cells. The AS reaction was explored by replacing the stimulating LCLs with recently infected B cells from normal individuals. Autologous B cells infected one day earlier with EBV induced significant DNA stimulation but generated no killer cells. The ability to generate cytotoxic T cells appeared 6 to 8 days after EBV infection. Cytotoxicity was not EBV-specific. The ability to activate the alternate complement pathway appeared at approximately the same time. B-cells of convalescent infectious mononucleosis (IM) patients could generate autologous cytotoxicity already 1 day after EBV-infection. The difference between the AS reactions induced by normal and convalescent IM donors is discussed.

Burkitt's lymphoma cells: Membrane properties and surface morphology as seen by scanning electron microscopy

Abstract Membrane properties and surface morphology were studied in cells from 13 different cultured Burkitts lymphoma cell lines in an attempt to determine whether there was any structure-function correlation for these cells. Burkitts lymphoma cells were all bone marrow (B)-derived and most were spherical in shape with moderate to markedly villous surfaces. There was no obvious correlation between the number of surface microvilli seen under the scanning electron microscope and the degree of expression of the different B-cell surface markers and other membrane properties.

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.

The fate of IgM and of anti IgM on antibody-coated Daudi cells

Publisher Summary This chapter discusses an experiment to show the fate of immunoglobulin M (IgM) and anti-immunoglobulin M (anti- IgM) antibodies on antibody-coated Daudi cells. In the experiment, antihuman IgM (aIgM) antibodies were purified by the affinity chromatography of a sheep aIgM antiserum on IgM-conjugated sepharose columns. Daudi cells were coated with 125I labeled aIgM (125I-algM) and incubated under culture conditions at a temperature of 37°C. It resulted that more than 80% of the cell-bound radioactivity was shed from the cells into the culture medium within 10 hours. From the experiment, it was found that 125I-aIgM shed from Daudi cells could rebind to fresh Daudi cells but less efficiently than native 125I-aIgM. The shedding of aIgM from Daudi cells resulted in the disappearance of the IgM molecules from the cell membrane and in the formation of IgM–aIgM complexes. Shed aIgM antibodies were partially degraded during the binding to the cell membrane and shedding.