Wolfgang Leibold

Biological differences between Epstein-Barr virus (EBV) strains with regard to lymphocyte transforming ability, superinfection and antigen induction.

Abstract EB virus (EBV) preparations derived from various producing lymphoblastoid cell lines (LCL) differed in their biological properties, as judged by the following four tests: (1) cord blood lymphocyte (CBL) transformation into EBV-carrying LCL; (2) early antigen (EA) induction in Raji cells; (3) inhibition of Raji cell growth; (4) induction of the EBV-determined nuclear antigen (EBNA) in CBL. B95-8 virus transformed and induced EBNA in CBL but did not induce EA in Raji cells, nor did it inhibit their growth. P3HR-1 virus did not transform CBL, induced no EBNA or EA in CBL, but induced EA in Raji cells and inhibited their growth. EBV isolated from the QIMR-WIL, 833L, F137 and cb-8-7 LCL resembled the B95-8 virus with regard to its biological activity (CBL transformation, EA induction in and growth inhibition of Raji cells). Transformation of CBL as contrasted to EA induction in, and growth inhibition of Raji cells thus appear as mutually exclusive viral functions.

Infection of EBV-Genome-Negative and – Positive Human Lymphoblastoid Cell Lines with Biologically Different Preparations of EBV

Epstein-Barr virus (EBV) derived from the B95-8 line has transforming activity for cord blood cells, whereas virus derived from the P3HR-1 line lacks such activity. When the two viral preparations were compared for their ability to infect the same EBV-genome-negative lymphoblastoid cell line, BJA-B, they induced approximately the same number of EBV-determined nuclear antigen (EBNA)-positive cells. EBNA is compatibile with continued cell proliferation. No early antigen (EA)-positive cells appeared in the B95-8 virus-infected cultures, whereas P3HR-1 virus-infected cells went on to express EA. EA signals the entry of the cell into the lytic cycle. No late viral antigen (VCA) appeared and, as a consequence, the P3HR-1 virus infection became abortive. In contrast, the EBNA-positive cells induced by the B95-8 virus continued to divide over several weeks. These findings show that different EBV isolates may differ in their biological activity, probably due to their having different degrees of viral dependence on restrictive host cell controls.

Stimulation of Normal Lymphocytes with Autologous Lymphoid Cell Lines: Properties of Derived Killer Cells

Lymphocytes from normal adults, with or without serological signs of previous Epstein‐Barr virus (EBV) infection, could be stimulated to proliferate and produce killer cells by incubation with autologous EBV‐genome‐positive lymphoid cell lines (LCLs). The stimulated cells were most probably of T‐cell origin, although at the peak of stimulation many of them lacked the sheep erythrocyte marker. Direct effector‐target cell contact was necessary for lysis to occur The cytotoxicity of autologously stimulated (AS) lymphocytes was not restricted to EBV‐genome‐positive LCLs, nor to cell lines of hematopoietic origin It was equally broad if cells carrying complement receptor had been removed before stimulation Fresh lymphocytes, blasts induced by phytohemagglutinin or concanavalin A. and Burkitts lymphoma biopsy cells were resistant or considerably less sensitive Mouse cells‐even cell lines‐were resistant The sensitivity of target cells to lysis correlated positively with their capacity to block AS lymphocyte lysis of autologous LCLs in competition experiments The cytotoxicity of AS lymphocytes was blocked by EBV‐genome‐positive and‐negative cell lines, whereas the EBV‐related cytotoxicity of T cells from acute cases of infectious mononucleosis was blocked by EBV‐genome‐positive LCL only.

Insolubilization of protein antigens on polyacrylic plastic beads using poly-L-lysine.

A new model system for quantitation of immunofluorescence on single cells is described using poly-L-lysine (PLL) coated polyacrylic plastic beads of approximately cell sizes as carriers for protein antigen. By increasing PLL concentration on the beads increased amounts of 125I labeled antigen were attached to the particles. Excess binding sites of PLL could be completely blocked by unrelated proteins. After staining with FITC-conjugated antibodies and quantitative fluorescence measurements of individual beads using a microspectrofluorimeter, strong correlations were found between antibody and antigen concentration on the beads. Neither repeated washings with PBS nor storage of the beads for two months caused detectable shedding of antigen-antibody complexes. There was a strong linear correlation between fluorescence intensity and the volume of beads, but the correlation between surface area and fluorescence was nonlinear. The described procedure was shown to be a simple method for quantitative and stable coating of particles with proteins. It can be applied as a useful model system for quantitative immunofluorescence studies on intracellular antigens.

New Groups and Segregant Series Among B-Cell Alloantigens of the Merrit System A Study of Leukemia Cells, Peripheral B Cells, and Lymphoblastoid Cell Lines

On the basis of reactions with a chronic lymphatic leukemia cell panel, evidence for 6 new specificities of the Merrit B‐cell alloantigenic system in man is presented, bringing the number of provisionally defined specificities to 19. These can he roughly divided into two segregrant series. The system is well represented on both homozygous and heterozygous B‐type lymphoblastoid cell lines. In the cell lines some specificities show a suggestive but inexact correlation with HLA‐D locus factors. This correlation is represented also on peripheral blood B cells, on the non‐T variety of acute lymphatic leukemia cells, and on acute mycloid leukemia cells. Although great similarities exist, each of the cell populations may manifest differences other than or in addition to mere differences in B‐cell antigen frequencies.

Single and dual labelling of cytotoxic target cells Comparison of three radioactive tracers, [35S]methionine, [75Se]selenomethionine, and chromium-51

[75Se]selenomethionine (75SeM) has been shown to provide several advantages over Na(2)51CrO4 (51Cr) labelling of metabolizing target cells: high labelling efficiency and low spontaneous release of 75SeM-labelled target cells permit improved monitoring of cytotoxicity due to extended effector/target ratios in short- and long-term assays. Unfortunately, 75SeM will soon be difficult to obtain. Therefore we studied the suitability of [35S]methionine (35SM) as a substitute for 75SeM. Furthermore, we explored the potential of dual labelling of suspension target cells applying combinations of 35SM and 51Cr or 75SeM and 51Cr. 35SM is a suitable substitute for 75SeM retaining most of the advantages of 75SeM labelling. Although considerably higher labelling of cells is possible we obtained the most efficient labelling with 100-400 kBq/ml of 35SM or 75SeM resulting in a relatively high uptake (3-15 cpm/cell) and very low spontaneous release (1-2%/h) up to 24 h. This permits short- and long-term cytotoxic assays and the use of low numbers of target cells (1 x 10(3)) providing increased cytotoxic sensitivity with reduced amounts of effector cells. Suitable dual labelling of target cells with 35SM plus 51Cr or 75SeM plus 51Cr documented convincingly identical release kinetics for 35SM and 75SeM but partially discordant ones for 51Cr. Depending on the target cell used dual labelling permits discrimination and monitoring of different cytotoxic or release mechanisms in cellular cytotoxicity.

T-Cell regulation of B-lymphoblastoid cell line function.

Abstract Seventeen B-lymphoblastoid cell lines (B-LCLs) have been studied for their ability to intereact with normal T cells and produce IgG and IgM in culture. All B-LCLs were HLA homozygous, having been derived from consanguineous donors by in vitro transformation with Epstein-Barr virus, 1 × 10 4 B-LCLs were cultured with 0, 5, 10, or 20 times as many normal peripheral blood T cells in a 0.2-ml culture. Culture supernatants were removed after 3 and 6 days and assayed for IgG and IgM by a radioimmunoassay. Thirteen of the cell lines were able to secrete immunoglobulin (50–6000 ng/ml), primarily IgG, when cultured without T cells. Addition of T cells (sheep erythrocyte rosette-forming cells) modulated immunoglobulin production, causing either marked enhancement or suppression depending upon the B-cell line. T cells cultured without the B-LCLs did not secrete immunoglobulin above the background level of the immunoassays (6.25 ng/ml). Cell lines which did not secrete IgM when cultured alone could frequently be induced to do so when T cells from select donors were added. Under these conditions, IgM was generally found only in the supernatant fluid removed after 6 days. Taken together, these results suggest that B-LCLs contain cells of at least two stages, those that secrete IgG and resting cells capable of secreting IgM. Furthermore, cells at both stages can be regulated by normal T cells.