R. Grunow

The high efficiency, human B cell immortalizing heteromyeloma CB-F7. Production of human monoclonal antibodies to human immunodeficiency virus.

This paper describes the construction of a new heteromyeloma cell line designated CB-F7. The cell line was derived from xenogeneic somatic cell hybridization between normal human B lymphocytes and the murine HAT-sensitive P3X63Ag8/653 cell line. CB-F7 cells were characterized by rapid cell growth (doubling time about 16 h) and high cloning efficiencies in culture medium supplemented with 10% or 5% fetal calf serum, respectively. The karyotype of the cells consists of about 75-78 chromosomes as well as two chromosomal fragments. Fusions of the cells with human peripheral blood cells resulted in approximately 2-6 clones per 10(5) seeded lymphocytes. Furthermore, the cells are ouabain resistant and therefore suitable for fusions with EBV-transformed lymphoblastoid cell lines. Using CB-F7 as the parental cell line a number of specific human mAb producing hybrids were established. For the first time, we describe here the generation of hybrids secreting human monoclonal antibodies to human immunodeficiency virus (HIV). Two monoclonal antibodies of IgG type and one of IgM type reacted with the major core protein p25 and one IgG antibody reacted with the transmembrane protein gp41.

A rapid and sensitive enzyme immunoassay for Cu/Zn superoxide dismutase with polyclonal and monoclonal antibodies.

An enzyme immunoassay for the quantification of human Cu/Zn SOD in serum, urine and erythrocytes was developed applying monoclonal and polyclonal antibodies. The one-step assay is completed within 30 min and enables the detection of 0.3 microgram Cu/Zn SOD per litre. A Cu/Zn SOD concentration of 46 +/- 21.5 micrograms/l and of 1 +/- 0.6 micrograms/mmol creatinine was determined in the serum and the urine, respectively, of healthy individuals. A content of 15 +/- 1.7 ng Cu/Zn SOD was found in 10(6) erythrocytes. Patients with Downs syndrome exhibited a 3.8-fold, a 2-fold and a 1.6-fold higher concentration of Cu/Zn SOD in their serum, urine and erythrocytes.

CD80 AND CD86 COSTIMULATORY MOLECULES ON CIRCULATING T CELLS OF HIV INFECTED INDIVIDUALS

The expression of CD80 and CD86 costimulatory molecules, typical for antigen presenting cells (APC), was measured on circulating T cells of 20 HIV-infected individuals and of 11 HIV-negative healthy controls. The CD80 and CD86 molecules were present on both circulating T subsets of HIV-infected individuals (mean of CD80 expression within CD4+ T cells [CD80/CD4]: 5.0%; and CD86/CD4: 2.6%; CD80/CD8 4.1% and CD86/CD8: 2.7%) and were associated with HLA-DR expression. Some CD80 and CD86 expression was also found in normal controls, and only the expression of CD86 was significantly (P < 0.05) increased on CD4 + and CD8 + T cells of HIV-infected individuals. The expression of CD28 was decreased on T cells of HIV-infected individuals and was negatively correlated to the expression of HLA-DR and CD86 (mean CD28 within CD3+T cells: HIV+ 29.5%, HIV - 67.6%; correlation coefficient, - 0.75 and - 0.71, respectively). The more the disease proceeds, the less CD28 and the more DR and CD86 are found on circulating T cells. This suggests that during HIV infection T cells themselves develop an antigen presenting phenotype by upregulating expression of HLA-DR, CD86 and CD80 molecules.

Establishment of human Ig producing heterohybridomas by fusion of mouse myeloma cells with human lymphocytes derived from peripheral blood, bone marrow, spleen, lymph node, and synovial fluid: Effect of polyclonal prestimulation and cryopreservation

50 fusion experiments were carried out to analyse heterohybridization efficiencies on mouse myeloma cells of the P3 X63 Ag8/653 line with human lymphocytes derived from peripheral blood, bone marrow, lymph node, spleen or synovial fluid. We found higher yields of growing and human Ig-producing hybridoma lines when lymphocytes from spleen or lymph node were fused. Although primary hybridomas could be established from fusions with bone marrow-derived cells, only in nine out of 1616 initially seeded wells was Ig production registered. Four fusions using immune cells from synovial fluid were made without success. Independently of the source of lymphocytes pokeweed mitogen (PWM) prestimulation had no enhancing effect on the percentage of wells with cell growth and this did not alter the IgM:IgG ratio in primary hybridomas (9:1), although cells from all compartments used here (with the exception of bone marrow cells) could be stimulated with PWM to produce both IgG and IgM in cultures. Cryopreserved lymphocytes from different sources could be used for fusions with comparable results registered for the fresh material.

Correlation between the phenotype and the functional capacity of activated T cells in patients with active systemic lupus erythematosus.

Activated T cells in the peripheral blood of patients with systemic lupus erythematosus (SLE) were determined using monoclonal antibodies against activation antigens. Elevated percentages of HLA‐DR+ T cells were found in association with active disease. In contrast, we observed an increase in IL‐2, receptor‐bearing T cells in only six out of 16 patients with active disease. In vitro assays, like spontaneous proliferation, response to IL‐2, production of IL‐2, and immunoglobulin synthesis have shown that the different patterns of activation antigens are related to different functional stages of T‐cell activation. The possible therapeutic consequences are discussed.

The Influence of Haematoporphyrin Derivative and Visible Light on Murine Skin Graft Survival, Epidermal Langerhans Cells and Stimulation of the Allogeneic Mixed Leucocyte Reaction

The influence of combined photochemical treatment with a haematoporphyrin derivative and visible light on antigen‐presenting cells was evaluated. Treatment of murine skin grafts with this procedure prolonged their subsequent survival on allogeneic recipients. The haematoporphyrin derivative and light decreased the ATPase activity of epidermal Langerhans cells in murine skin. When stimulator cells in a human allogeneic mixed leucocyte reaction were treated with the haemaioporphyrin derivative and light, they lost their stimulatory capacity. It is proposed that the haemaioporphyrin derivative and visible lighl interfere, on analogy with ultraviolet radiation, with the function of antigen‐presenting cells.

A cell surface ELISA for the screening of monoclonal antibodies to antigens on viable cells in suspension.

To simplify the screening of monoclonal antibodies to different human T cell surface molecules a live cell enzyme-linked immunosorbent assay (cell ELISA) has been established and optimized. The assay was performed in 96-well plates. By using living human T lymphocytes in suspension surface modification by fixation or insolubilization of the cells was avoided. Several parameters influencing sensitivity and specificity were studied. About 150 ng/ml of mouse monoclonal antibodies to cell surface antigens could be detected when using 5 x 10(4) cells per well and a 1/1000 dilution of the anti-mouse IgG-alkaline phosphatase conjugate. This sensitivity permitted the primary screening of cell specific antibodies from hybridoma supernatants. The same detection limit was obtained in flow cytometric analysis. If required, the sensitivity of the cell ELISA could be increased using higher cell numbers and conjugate concentration. When analysing different cell lines with selected antibodies the cell ELISA was found to be as sensitive and specific as the fluorescence assay. The assay was applied to the screening of supernatants from hybridomas developed against human T helper cell clones and the detection of V beta specificities of T cell clones.

Application of a human monoclonal antibody in a rapid competitive anti-HIV ELISA

The ELISA is the established screening technique for the detection of antibodies directed against HIV. The first generation assays, mostly based on the sandwich principle, employed purified virus from cell culture and gave both false-positive and false-negative results. Sandwich-type assays preferentially detect IgG antibodies, require a high serum dilution and are two-step procedures. In order to detect an immune response as early as possible after infection anti-HIV antibodies of the IgM class should also be measured. To this end a competitive ELISA has been developed using a solid phase-adsorbed recombinant HIV envelope protein and an enzyme-labelled human monoclonal antibody. This detects both IgM and IgG antibodies, the results are available within 1 h and a serum predilution is not necessary.

Monoclonal antibodies to p24-core protein of HIV-1 mediate ADCC and inhibit virus spread in vitro.

BACKGROUND Certain antigens of the HIV-1, e.g., gp120-envelop proteins, can be expressed on the membrane of HIV-infected cells. Little is known about the membrane expression of other HIV-antigens and their interaction with specific antibodies. OBJECTIVE To develop murine monoclonal antibodies (mAbs) to the p24-core protein of HIV-1 and to characterise their binding sites and biological activities on HIV-infected T cells. METHODS Monoclonal antibodies were developed from mice hyperimmunised with a recombinant p24-core protein from HIV-1. Two mAbs were epitope-mapped on overlapping peptides and characterised for their reactivity with non-fixed HIV-infected T cells by immunofluorescence staining and flow cytometric analysis. Their biological activities were studied for antibody-dependent cellular cytotoxicity (ADCC) and suppression of viral spread in vitro. RESULTS The epitopes of two selected mAbs were located on the amino terminal region of p24 in the regions 147-152 aa and 178-187 aa, respectively. The antibodies were able to react with living HIV-1 infected cells. The expression of the antigens was time-dependent after the infection of certain cell lines by HIV-1. The mAbs mediated a strong HIV-1-specific ADCC and were able to delay the spread of HIV-1 for about 6 days in cell cultures. CONCLUSIONS Certain epitopes of the p24-core protein of HIV-1 can be expressed on living, HIV-infected T cells and are recognised by specific antibodies. Such antibodies can destroy infected cells by ADCC or delay the virus spread, and therefore, should be considered in immunisation strategies against HIV.

Oligonucleotide fingerprinting as a means to identify and survey long-term cultured B cell hybridomas and T cell lines

Common problems encountered during cell culture are cross-contamination, instability, and inadvertent exchange of cells. Here we report on the application of oligonucleotide fingerprinting as a simple and efficient method to screen hybridomas and T cell lines. Among the fingerprint probes tested, the simple repetitive oligonucleotide (CAC)5/(GTG)5 proved to be most useful for obtaining many fragments specific for each cell line. Because of variable loss of chromosomes, cloned hybridoma cells from one fusion exhibit different fingerprint patterns. Thus, antibody-secreting B cell hybridomas can be distinguished easily even when they originate from the same fusion. Furthermore, we were able to monitor the genomic integrity of myelomas and hybridomas over a period of more than 2 years, thereby highlighting long-term stability. Another application of the method is the control of T cell lines requiring irradiated or mitomycin-treated feeder cells for continuous growth or cloning. There cell lines are always threatened to be overgrown by feeder cells that have escaped from the lethal pretreatment. T cell clones of one individual, known to display differently rearranged T cell receptors, did not show differences in their fingerprint patterns. However, in EBV-transformed cloned B cells, slight differences between clones from the same donor were identified.