Human Antibodies - Volume 3, issue 4

Authors: Jochems, Gijs J. | Klein, Michèl R. | Zeijlemaker, Wim P. | van Lier, René A. W.

Article Type: Research Article

Abstract: Epstein-Barr virus (EBV)–transformed cell lines constitutively secrete lymphotoxin (LT/TNFβ) and not tumor necrosis factor-α (TNFα). To analyze the cellular processes that regulate LT and TNFα secretion by lymphoblastoid cell lines, we studied the role of two signal transduction pathways leading to either protein kinase C (PK-C) or PK-A activation. We demonstrate that PK-C activation, either after cross-linking of surface Ig or by direct activation with phorbolester, leads to increased production of both LT and TNFα, whereas no prominent role for PK-A was found. Interleukin (Il)-4 was found to synergize with PK-C activation in raising levels of secreted LT and TNFα. …Increased levels of LT and TNFα did not correlate with augmented levels of immunoglobulin secreted by the cell lines nor with improved proliferation. These observations demonstrate that EBV B cells respond to B cell activation signals leading to PK-C activation with increased production of both LT and TNFα. It is, however, unlikely that these molecules serve as autostimulatory factors for EBV B cells, but in contrast might play a role in downregulation of biological functions in these cells. Show more

Keywords: LT, lNFα, EBV, B-cell, signal transduction, PK-C

DOI: 10.3233/HAB-1992-3401

Citation: Human Antibodies, vol. 3, no. 4, pp. 162-167, 1992

Authors: Shepherd, A.J. | Boyd, J.E. | Hogg, C.L. | Aw, D. | James, K.

Article Type: Research Article

Abstract: Seven immortalized B cell clones, five of which secreted specific human monoclonal antibodies (MAbs) against hepatitis B, tetanus toxoid, and Rhesus D antigens, were evaluated for their susceptibility to infection by human immunodeficiency virus types 1 and 2 (HIV-1 and HIV-2). Infection was confirmed in three human MAb-producing lines by detection of infectious virus and p24 antigen in culture supernates, by immunofluorescence, and by detection of viral DNA in cells by polymerase chain reaction. The infectable lines were as susceptible to HIV-1 infection as several T cell lines and remained persistently infected for several months, but in contrast to T …cell controls, viral cytopathic effects were not observed. Levels of unintegrated viral DNA in the HB1 B cell line were significantly lower than in the HUT78 T cell line. Cell lines that were susceptible to HIV expressed HLA DR, CD20, and CD21, whereas the uninfectable cell lines did not express any of the markers tested. CD4 was undetectable or present on a small percentage of cells in two of the infectable cell lines. However, infection with HIV-1 was blocked more efficiently in B cells than in T cells by soluble CD4, anti-CD4 MAb, and dextran sulphate. The effect of HIV infection on human MAb secretion was variable, being reduced on a per-cell basis in one line, increased in another, and unchanged in a third. A further 16 Epstein-Barr virus–transformed B cell lines derived from the peripheral blood of a HIV-1 infected individual were susceptible in culture to infection with HIV-1, but with no effect on cell proliferation or total IgG and IgM production. Show more

Keywords: HIV-l, HIV-2, B cell lines, human monoclonal antibody

DOI: 10.3233/HAB-1992-3402

Citation: Human Antibodies, vol. 3, no. 4, pp. 168-176, 1992

Authors: Jonak, Zdenka L. | Henri, Edward | Trulli, Stephen | Jensen, Colleen P. | Muirhead, Katherine A.

Article Type: Research Article

Abstract: Electroporation was used to deliver genomic DNA from a lymphoid tumor to activated/stimulated human peripheral blood lymphocytes to create immortalized lymphoid cell lines. Activation of the recipient lymphocytes was essential for efficient immortalization. A panel of human B cell transfectant clones, each phenotypically representing specific stages of differentiation, resulted from the transfection. Monoclonal antibody production was measured, and the level produced depended on the phenotype of the cells, with the more mature B cell transfectants secreting up to 10 μg/mL of immunoglobulin. The transfectants were stable with respect to their morphological appearance, growth rate, and antibody production. Chromosome analysis indicated …that the transfectants displayed a normal karyotype, devoid of abnormalities. We have shown that electroporation is an effective method of immortalizing human lymphocytes at different stages of differentiation. The transfectants provide a panel of cells that can readily be studied with respect to their phenotypic/karyotypic stability, regulation, and production of immunoglobulin, lymphokines, and growth factors. These data demonstrate the feasibility of generating immortalized human B cells to provide an important resource for the study of B cell differentiation and immortalization. Show more

Keywords: immortalization, novel cell lines, human monoclonal antibodies, in vitro immunization, oncogenic DNA, electroporation

DOI: 10.3233/HAB-1992-3403

Citation: Human Antibodies, vol. 3, no. 4, pp. 177-185, 1992

Authors: Hampe, J. | Nürnberg, P. | Epplen, C. | Jahn, S. | Grunow, R. | Epplen, J. T.

Article Type: Research Article

Abstract: 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. Show more

Keywords: hybridoma, DNA fingerprinting, cell culture, feeder cells, EBV transformation

DOI: 10.3233/HAB-1992-3404

Citation: Human Antibodies, vol. 3, no. 4, pp. 186-190, 1992

Authors: Looney, James E. | Knight, David M. | Arevalo-Moore, Maria | Trinh, Han | Pak, Koon-Yan | Dalesandro, Margaret R. | Rieber, E. Peter | Riethmuller, Gert | Daddona, Peter E. | Ghrayeb, John

Article Type: Research Article

Abstract: The use of murine anti-CD4 monoclonal antibodies (MAbs) has shown considerable promise for the treatment of allograft rejection and rheumatoid arthritis. We have constructed mouse-human anti-CD4 antibodies with the goal of increasing their clinical potential by decreasing immunogenicity and improving effector functions. The chimeric antibodies were constructed by cloning the heavy and light chain variable regions of M-T412, a murine antibody raised against the human CD4 antigen, and joining them to the human G1, G4, or kappa constant regions in mammalian expression vectors. After transfection into mouse myeloma cells, stable cell lines were isolated that secrete up to 140 μg/ml …chimeric antibody in static culture. The chimeric antibodies were equivalent to the murine antibody in their binding characteristics and relative affinities. However, the chimeric M-T412 MAbs have enhanced activity when compared to the murine G2a MAb in mediating antibody-dependent cell-mediated cytotoxicity using human CD4+ target and effector cells. Show more

Keywords: chimeric monoclonal antibody, M-T412, CD4

DOI: 10.3233/HAB-1992-3405

Citation: Human Antibodies, vol. 3, no. 4, pp. 191-200, 1992

Authors: Neil, G. A. | Sammons, D. W.

Article Type: Research Article

Abstract: Human hybridomas with specificity for recombinant hepatitis B surface antigen (HBSAg) were produced by adoptive transfer of human peripheral blood mononuclear cells prepared from HBSAg-immune donors to CB.17 mice bearing the severe combined immunodeficiency (SCID) phenotype. A total of ten SCID-Hu mice were immunized with recombinant HBSAg. Eight SCID-Hu mice found to have human HBSAg antibody in their serum were sacrificed, and single-cell suspensions were made from their spleens. The SCID-Hu spleen cells were electrofused to the mouse-human fusion partner H7. HBSAg-specific hybridomas were recovered from all fusions. This method may provide the means for the production of other human …hybridomas and may, In some cases, circumvent the need for in vitro immunization or Epstein-Barr virus transformation of human B lymphocytes. Show more

Keywords: SCID-Hu mice, human hybridomas, hepatitis B

DOI: 10.3233/HAB-1992-3406

Citation: Human Antibodies, vol. 3, no. 4, pp. 201-205, 1992

Authors: Torres, Anthony R. | Healey, Mark C. | Johnston, Alice V. | McKnight, Michael E.

Article Type: Research Article

Abstract: An agamma calf serum (ACS) was compared to fetal bovine serum (FBS) and calf serum supplemented growth media for both murine and human fusion partners and derived hybridoma cells. The variables analyzed were cloning efficiencies, growth characteristics, and MAb production levels. Cultures were established in each serum source, and supernatants were kept for analysis. For cloning efficiencies, the results indicate that although there is no clear advantage in the performance of FBS-supplemented medium, ACS-supplemented RPMI 1640 medium can be used to clone hybridomas and the fusion partners. When analyzed for MAb production in the various sera, the hybridoma cell lines …used in this study appeared to produce up to twice as much immunoglobulin when grown in ACS as when grown in supplemented medium. IgG- and IgM-secreting hybridoma cultures should be checked independently for antibody production. Six out of eight murine and human hybridoma cell lines produced higher levels of MAbs when grown in ACS. Show more

Keywords: monoclonals, hybridomas, antibodies, serum, serum-free, cell-culture

DOI: 10.3233/HAB-1992-3407

Citation: Human Antibodies, vol. 3, no. 4, pp. 206-211, 1992

Article Type: Other

DOI: 10.3233/HAB-1992-3408

Citation: Human Antibodies, vol. 3, no. 4, pp. 212-213, 1992

Article Type: Correction

DOI: 10.3233/HAB-1992-3409

Citation: Human Antibodies, vol. 3, no. 4, pp. 214-214, 1992