Immunology 2Friday, October 15, 2010, 15:30 - 17:00
Immortalised B cell lines from multiple sclerosis patients produce autoreactive monoclonal antibodies J. Fraussen, K. Vrolix, P. Martinez-Martinez, M. Losen, R. Hupperts, A. Van Diepen, B. Van Wijmeersch, M. De Baets, P. Stinissen, V. Somers (Diepenbeek, BE; Maastricht, Heerlen, NL)
B cells and oligoclonal antibodies are present in the cerebrospinal fluid (CSF) of multiple sclerosis (MS) patients but their target antigens remain unknown. The focus of this study was to produce autoantibodies of MS patients based on B cell immortalization and to further characterize the obtained antibodies based on antigen reactivity and diversity.
Peripheral blood mononuclear cells (PBMC), isolated peripheral immunoglobulin G+ (IgG+) B cells or CSF cells were infected with Epstein-Barr virus (EBV) in the presence of irradiated allogeneic PBMC and B cell stimulating factors to obtain continually dividing B cell lines. B cell immortalization was verified by screening the culture supernatant for antibody production using dot blotting. Clonality of the immortalized B cell lines was verified using a B cell spectratyping procedure. To screen for autoreactivity, binding of the produced antibodies to a human oligodendroglioma (HOG) cell line, PBMC and an epithelial alveolar carcinoma cell line (A549) was analyzed by flow cytometric analysis.
We obtained 288 immortalized B cell lines from 15 MS patients, 29 B cell lines from 8 patients with a non- or other inflammatory neurological disease (NIND/OIND) and 37 B cell lines from 3 patients with clinically isolated syndrome (CIS). Most B cell lines were obtained from the PBMC although 7 B cell lines were isolated from the CSF of 1 MS patient and 2 from 1 NIND patient. B cell spectratyping analysis indicated that more than 82% of the immortalized B cell lines were monoclonal, eliminating the need for cloning. HOG-specific intracellular binding was shown for antibodies from 3 immortalized B cell lines while intracellular binding to both HOG and A549 cells but not to PBMC was demonstrated for 37 other B cell lines, all generated from PBMC of MS patients. In addition, binding to all examined cell types was found in 34 immortalized B cell lines. Further, intracellular binding to HOG cells was confirmed for several of the immortalized B cell lines by immunocytochemistry.
B cell immortalization has proved to be a useful method for the production of antibodies. Autoreactivity of the generated monoclonal antibodies has been demonstrated and is now further examined by detecting antibody binding to healthy and diseased brain tissue from human and rhesus monkey. Moreover, characterization of the obtained B cell lines based on diversity is currently in progress by sequencing the immunoglobulin heavy chain genes.
All listed authors have nothing to disclose.