June Goldman

Comparative biochemical and genetic characterization of clonally related human B-cell lines secreting pathogenic anti-Pr2 cold agglutinins

To study the biology of cold autoimmune hemolytic anemia, Epstein-Barr virus (EBV)-transformed B-cell clones were established from a patient with splenic lymphoma associated with immune hemolysis due to an anti-Pr2 cold autoantibody. Studies were performed comparing the cold autoantibody present in culture supernatants of these cell lines to the pathogenic cold autoantibodies present in the patients plasma. Cytogenetic studies of splenic lymphocytes demonstrated an abnormal karyotype (51XX, +3, +9, +12, +13, +18). After EBV transformation, eight clones secreting IgM, kappa anti-Pr were isolated; each clone had the same abnormal karyotype as above. DNA isolated from the clones and spleen was analyzed by Southern blot hybridization with JH, C mu, and C kappa probes; identical gene rearrangements were seen in each case. Anti-Pr antibodies, isolated from culture supernatant and serum were compared by isoelectric focusing (IEF) and demonstrated similar banding patterns. Distinctive binding patterns, however, were observed in 2/8 clones, suggesting structural differences. Adsorption studies with red blood cells further showed that the observed IEF banding patterns were solely due to anti-Pr cold autoantibody. With a thin-layer chromatography method, the biochemical determinants recognized by the cold autoantibodies were defined as glycolipids containing Neu Ac alpha 2-3Gal beta 1-4Glc sequences. The data demonstrate that the autoantibodies of the EBV-transformed B-cell lines were similar to the pathogenic monoclonal serum autoantibody in both structure and specificity. These clonal cell lines may thus serve to further study the biology of human B-cell lymphomas with defined autoantibody specificity.

Production of human warm-reacting red cell monoclonal autoantibodies by Epstein-Barr virus transformation

Monoclonal antibody technology has been used in both murine and human systems to produce a variety of antibodies that react with the human red cell (RBC). RBC monoclonal autoantibodies have been obtained from animal models of autoimmune hemolytic anemia (AIHA), but to date no warm‐reactive monoclonal autoantibodies have been generated from human B cells. Using the Epstein‐Barr virus (EBV) transformation method, clones of RBC autoantibodies were generated from two patients with AIHA. These antibodies reacted preferentially at 37° C, agglutinated or bound to a variety of different RBC phenotypes, and were IgM in nature. The serologic reactivity of one clone showed a relative specificity to e+ RBCs that was similar to that seen in the patients serum. These results are the first to demonstrate that warm‐reactive RBC autoantibodies can be obtained from patients with AIHA using the technique of EBV transformation, and they further substantiate the existence of warm‐reactive IgM RBC autoantibodies in the spectrum of warm AIHA.

Anti-idiotypic antibodies specific for a pathologic anti-Pr2 cold agglutinin

The heterogeneity of human red cell (RBC) autoantibodies may be assessed by using anti‐idiotypic antibodies. In this study, mouse monoclonal anti‐idiotypic antibodies were produced against a pathologic RBC autoantibody with anti‐Pr2 specificity. Epstein‐Barr virus‐transformed B‐cell clones were established from a patient who had splenic lymphoma and associated immune hemolysis due to an anti‐Pr2 cold autoantibody. Two of the eight clones producing this autoantibody were used to immunize mice for the establishment of hybridomas, and four monoclonal anti‐idiotypic antibodies were isolated (2 IgG1 kappa and 2 IgM kappa). By the use of these anti‐idiotypic antibodies, strong cross reactivity was seen on enzyme‐linked immunosorbent assay with other anti‐Pr2‐producing clones from the same patient, but no cross‐reactivity was seen with RBC autoantibodies from other individuals having anti‐Pr or different specificities. Each of the anti‐idiotypic antibodies inhibited hemagglutination (HA) by the patients anti‐Pr2 but failed to inhibit HA by antisera of a different RBC specificity. Cross‐competition experiments indicated that all of the anti‐idiotypic antibodies may recognize the same or a closely related idiotope on the anti‐Pr2 autoantibody. These studies suggested that the four anti‐idiotypic antibodies are directed against the same (or closely related) idiotypic determinant(s), unique to this patients anti‐Pr2 and located at or near the antigen‐binding site. These anti‐idiotypic antibodies may be useful tools for the study of this autoimmune response or for the development of immune therapeutic agents. Additional panels of anti‐idiotypic antibodies will be necessary to elucidate further the idiotypic diversity of RBC autoantibodies occurring both in healthy individuals and in patients with immune hemolysis.

[23] Production of carbohydrate-specific human monoclonal antibodies in vitro

Publisher Summary This chapter focuses on the production of carbohydrate–specific human monoclonal antibodies in vitro . The method described in this chapter is the current approach used in many laboratories to isolate autoreactive B-cells, with specificity for carbohydrate determinants on human red blood cells. The approaches that have been successfully employed for this purpose are quite diverse with respect to the initial seeding density of the lymphocytes, the activation of lymphocytes, cloning of cells under limiting dilution conditions, the presence of T lymphocytes, and the use of hybridomas. This diversity in methods is probably related to ultimate purposes for which the Epstein-Barr virus (EBV)-transformed B-cell clones have be used. For example, with regard to the initial seeding density of lymphocytes, it is important to evaluate the probable frequency of the desired (auto-) antigen-specific B cells in the source of these cells (peripheral blood, lymph node, spleen, and bone marrow).