S. Jahn

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.

Analysis of VH-D-JH gene transcripts in B cells infiltrating the salivary glands and lymph node tissues of patients with Sjögren's syndrome

Objective. In patients with Sjogrens syndrome (SS), B lymphocytes have been found to infiltrate salivary glands, resulting in sialadenitis and keratoconjunctivitis. The disease is frequently associated with benign and neoplastic lymphoproliferation. The present study was undertaken to investigate whether clonal B cell expansion takes place in lymphocytic infiltrations of salivary glands under (auto- [?]) antigen stimulation, by analyzing in more detail the variable part (V H -D-J H ) of the immunoglobulin heavy chain genes expressed in these B cells. Methods. Biopsies of the labial salivary glands and lymph nodes were performed on 2 female patients with SS. The Ig gene rearrangements in these tissues were amplified by reverse transcriptase-polymerase chain reaction using specific primers. Results. A total of 94 V H -D-J H transcripts were cloned and sequenced. Our data suggest a polyclonal origin of the B cell infiltrates. In 92 of the transcripts, V H genes were modified by somatic mutation. Further analysis showed counterselection for replacement mutations within the framework regions, suggesting that those B cells were stimulated and selected for functional expression of a surface Ig. In labial salivary glands from both patients, clonally related B cells became evident. Members of 1 particular clone were found in both the lip and lymph node material. Conclusion. These data provide evidence, on the nucleotide sequence level, that an antigen-triggered clonal B cell expansion takes place in the salivary glands of patients with SS who do not have histologic evidence of developing lymphoma. It may be speculated that those B cell clones expand during disease progression, resulting in lymphomagenesis.

Cutaneous malignant lymphomas.

Abstract Progress in understanding the pathogenesis of cutaneous lymphomas was the focus of a recent meeting ∗ ∗The Clinically Oriented Symposium in Cutaneous Malignant Lymphomas of the European Society of Dermatological Research and the European Organization for Research and Treatment of Cancer was held at Charite, Berlin, Germany, on 24–28 October 1997.. Research in this area should improve diagnosis and treatment of these diseases, and aid the study of other T- and B-cell neoplasias

Purification and immunochemical characterization of a natural human polyreactive monoclonal IgM antibody

In vitro and in vivo experiments to explain the function of natural polyreactive antibodies, usually of the IgM isotype, require large amounts of purified antibodies. We have developed a two-step purification procedure using a human natural polyreactive monoclonal IgM antibody (CB03). This combines hydrophobic interaction chromatography on phenyl-Superose and gel filtration over Superose 12 and readily permits scaling-up to isolate mg to g amounts of antibody. Retention of the CB03 antibody during gel filtration by precipitation and interaction with the gel matrix was overcome by the addition of 10 mM 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate. The yield of purified antibody was 34% and Fab fragments were obtained from the purified CB03 antibody by hot tryptic digestion (yield, 68% of theoretical amount). In an enzyme-linked immunosorbent assay, Fab and complete antibody had similar reaction patterns with different antigens.

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.

Real-time biospecific interaction analysis of a natural human polyreactive monoclonal IgM antibody and its Fab and scFv fragments with several antigens.

Surface plasmon resonance (SPR) was used to investigate the kinetics of interactions between the human monoclonal polyreactive IgM antibody CB03, its Fab as well as its single‐chain variable fragment (scFv) and different antigens. From these experiments apparent binding constants were determined and compared with binding constants obtained by ELISA experiments. In SPR studies with the complete antibody, the polyreactivity of the CB03 antibody as derived from ELISA experiments was confirmed.

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.