Myelin protein P0-specific IgM producing monoclonal B cell lines were established from polyneuropathy patients with monoclonal gammopathy of undetermined significance (MGUS)

Abstract

Monoclonal expansion of B cells and plasma cells, producing antibodies against 'self' molecules, can be found not only in different autoimmune diseases, such as peripheral neuropathy (PN), but also in malignancies, such as Waldenström's macroglobulinaemia and B-type of chronic lymphocytic leukaemia (B-CLL), as well as in precancerous conditions including monoclonal gammopathy of undetermined significance (MGUS). About 50% of patients with PN-MGUS have serum antibodies against peripheral nerve myelin, but the specific role of these antibodies remains uncertain. The aims of the study were to establish, and characterize, myelin-specific B cell clones from peripheral blood of patients with PN-MGUS, by selection of cells bearing specific membrane Ig-receptors for myelin protein P0, using beads coated with P0. P0-coated magnetic beads were used for selection of cells, which subsequently were transformed by Epstein--Barr virus. The specificity of secreted antibodies was tested by ELISA. Two of the clones producing anti-P0 antibodies were selected and expanded. The magnetic selection procedure was repeated and new clones established. The cells were CD5+ positive, although the expression declined in vitro over time. The anti-P0 antibodies were of IgM-lambda type. The antibodies belonged to the VH3 gene family with presence of somatic mutations. The IgM reacted with P0 and myelin-associated glycoprotein (MAG), and showed no evidence for polyreactivity, in contrast to other IgM CD5+ clones included in the study as controls. The expanded clones expressed CD80 and HLA-DR, which is compatible with properties of antigen-presenting cells. The immunomagnetic selection technique was successfully used for isolation of antimyelin protein P0-specific clones. The cell lines may provide useful tools in studies of monoclonal gammopathies, leukaemia, and autoimmune diseases, including aspects of antigen-presentation by these cells followed by T cell activation.

Fig. 1

Direct immunofluorescence performed on TJ1 B cell line using FITC-anti-kappa, FITC-anti-lambda, and isotype control mouse IgG-FITC.

Fig. 2

TJ1 cells were analysed by two-colour immunofluorescence. Anti-CD19-PE MoAb conjugate and protein P₀-FITC conjugate was used for staining the cells. (a) Gating for lymphocytes. (b) CD19 (FL2-H) intensity on y-axis plotted against P₀-FITC intensity on x-axis. (c) CD19⁺ and P₀-FITC positive cells. (d) Isotype control used for setting background gating for CD19.

Fig. 3

Flow cytometry analysis of TJ2 cells. CD19, CD80 and HLA-DR specific MoAbs were used for indirect immunofluorescence staining. 10 000 cells were counted.

Fig. 4

Nucleotide sequence of TJ IgM compared with V_H3-15 germ line.