S.T. Kiessig

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.

Immune Restoration in Children after Partial Splenectomy

Splenectomy (SE) is recognized to be a therapeutical approach in treating children with severe autoimmune diseases (chronic idiopathic thrombocytopenia; hemolytic anemia) or hypersplenism because of portal hypertension. Nevertheless, removal of a main immune organ results in elevated infection risk for these patients. Partial splenectomy (PSE) was developed as a therapeutical compromise to retain immunologically active spleen tissue. Here, we document the analysis of immune parameters obtained from children after both partial and total splenectomy, which have been followed up for a period of more than 6 years: (i) Lymphocytes from both groups of patients failed to produce IgG in response to pokeweed mitogen in vitro. This was observed in 11/20 splenectomized patients even 10 years after operation, whereas in PSE patients a restoration of this parameter after 1-2 years was seen. (ii) In patients after PSE, but not in splenectomized persons, an elevated number of HLA-class II positive cells had been detected suggesting a different situation of immune regulation following this operation. However, in parallel with an improvement of B cell in vitro activity this parameter was found to achieve normal values. Our findings indicate that partial splenectomy may be a therapeutical alternative, if the therapeutic goal can be achieved by this procedure.

Human hybridomas derived from CDS+ B lymphocytes of patients with chronic lymphocytic leukemia (B-CLL) produce multi-specific natural IgM (kappa) antibodies

Great numbers of CD5+ B lymphocytes were detected in the peripheral blood of patients with B‐CLL. To study the antibody repertoire of this immune cell subpopulation on a monoclonal level, we fused the lymphocytes derived from five different donors to a highly efficient HAT‐sensitive heteromye‐loma line (CB‐F7). A fusion frequency of up to 10‐5 allowed us to analyse hundreds of initial hybridoma lines per fusion. In all culture supernatants in three out of five fusions IgM lambda antibodies were detected, in two experiments only IgM kappa was measured, suggesting monoclonality of the primary hybridoma cell lines. The later fusions resulted in hybridomas producing multi‐specific antibodies against both an autoantigen and an infectious agent: (i) dsDNA/influenza virus haemagglutinin; (ii) dsDNA/class V outer membrane protein type C from Neisseria meningitidis. However, no antibodies of the described specificity were detected in blood sera of patients, indicating a ‘switch‐on’ of the immunoglobulin secretion capacity of malignant B cells during fusion to a myeloma partner. We discuss the results as further evidence for the natural multi‐reactive antibody repertoire of CD5+ B cells.

Human monoclonal IgM antibodies from foetal B-cell hybridomas directed against a surface antigen on human tumour cells

In order to assess the existence of B lymphocytes capable of producing anti-tumour antibodies in non-tumour-bearing individuals, human lymphocytes derived from foetuses and adults were fused with the heteromyeloma cell line CB-F7. By indirect immunofluorescence, 29 out of 4,472 IgM-producing hybridomas (from 8 foetuses and 8 adults) were shown to produce antibodies which bind to colon carcinoma lines Colo205 and SW620, Raji lymphoma cells and small cell carcinoma of the lung. In vitro growth of tumour cells recognized by these antibodies was inhibited. The antibodies also mediated complement-dependent cytotoxicity. All antibodies tested recognized a cell surface molecule of 55 kDa. Southern blot hybridization analysis of hybridoma DNA with a human JH probe showed that the hybridomas were derived from clonally unrelated B cells. These results demonstrate that human foetal and adult B cells from non-tumour-bearing individuals are able to produce IgM antibodies recognizing defined cell surface molecules expressed on some tumour cells.

A myelopeptide from unstimulated bone marrow cells with immunomodulatory actiivity in lymphocyte cultures from healthy donors and patients with hypogammaglobulinemia and active systemic lupus erythematosus

A myelopeptide (SAP) was derived from culture supernatants of unstimulated animal bone marrow. SAP consists of a group of peptides with a molecular weight of about 2000 D, having a broad variety of biological activities. Testing immunoregulatory properties of the purified factor Petrov, Mickhailova & Zacharova [(1971). Immunoglobin synthesis in syngenic cells of different lymphoid tissues. J. Immun., 106 1086-1089] found enhanced antibody production in mice (SAP-stimulator of antibody production). We show here that the substance could induce expression of activation markers on human lymphocytes (4F2, HLA-class II antigens, thermostable SE rosette formation) and potentiate their appearance in combination with mitogens (PWM, PHA, Con A). Although SAP was not mitogenic for itself, it enhanced lectin-induced 3H-thymidine incorporation and T-cell-dependent B-cell differentiation in a dose-dependent manner. The factor was able to reconstitute disturbed PWM-driven Ig synthesis in lymphocyte cultures derived from two patients with hypogammaglobulinemia and a healthy non-responder to PWM. On the other side, SAP potentiated the inhibitory activity of PWM on elevated spontaneous IgG secretion in cultures derived from patients with active SLE. Findings of this study indicate immunomodulatory capacity of SAP on human peripheral blood lymphocytes possible via T-cell activation. The results suggest a potential therapeutic application of SAP in patients with disturbances in the T-dependent B-cell differentiation.

SIMULTANEOUS CULTIVATION OF TWO HYBRIDOMA CELL LINES IN A HOLLOW FIBER BIOREACTOR

ABSTRACT A human monoclonal IgM-antibody and a murine monoclonal IgG3 antibody, were produced simultaneous in a simple hollow fiber bioreactor (CELL-PHARM 1) over 32 days. Therefore two hollow fiber cartridges were introduced parallel into the oxygenated perfusion circuit. Each of the cell lines was inocculated in the extra-capillary space of one of the cartridges. The possibility of simultaneous independent production of the two antibodies could be shown. Not antibody molecules nor their fragments could be detected within the common perfusion circuit over the whole fermentation procedure. Differences of concentration of glucose and some amino acids within the two cartridges were found.

APPEARANCE OF NONSPECIFIC AMOUNTS OF MONOCLONAL ANTIBODY DURING FERMENTATION CAUSED BY DECREASED CELL VIABILITY

A monoclonal anti-HIV-1 antibody (ab) from a murine hybridoma cell line was produced by repeated batch fermentation to compare the product quality in respect of its antigen (ag) binding specificity during cultivation. Cells are cultivated in a 1.21 stirred suspension reactor in a repeated batch mode. Cultivation was finished after cell viability dropped below 30%. Absolute amounts of specific reactive ab were detected by ag nonspecific and ag specific ELISA-techniques, respectively. It could be shown that the concentration of released specific non-reactive abs correlated with the decreased cell viability. Simultaneous immunoblots showed that this release could not be explained by the appearance of cytoplasmatic unlinked heavy and light immunoglobulin chains only.