[Serological analysis of mixed isotype Ab beta Ed alpha class II molecules and preparation of its specific monoclonal antibody].

The existence of mixed isotype Ab beta Ed alpha class II molecules on Ed alpha gene-introduced C57BL/6 transgenic (B6Ed alpha) mouse spleen cells was demonstrated by a flow cytometric analysis. Immunofluorescence staining of B 6 Ed alpha transgenic spleen cells by biotin-conjugated anti-Ab beta monoclonal antibody (mAb) was not completely blocked by unconjugated anti-Ab alpha mAb. Remaining fluorescence was completely blocked by unconjugated anti-Ed alpha mAb but not by anti-Eb beta mAb. Similar results were obtained in immunofluorescence staining with biotin-conjugated anti-Ed alpha mAb which was blocked by unconjugated anti-Eb beta mAb. We have also made an mAb (SEA40) that specifically recognizes Ab beta Ed alpha molecule. SEA40 reacts with B6Ed alpha and (BALB/c x B6Ed alpha) F1 spleen cells. The specificity of SEA40 was confirmed by experiments which showed that unconjugated anti-Ab beta mAb and anti-Ed alpha mAb both blocked the immunofluorescence staining of B6Ed alpha spleen cells with biotin-conjugated SEA40. Using this (SEA40) mAb, we showed that normal H-2d X H-2b) F1 spleen B cells, which do not normally express Ab beta Ed alpha mixed isotype molecules, were induced to express Ab beta Ed alpha molecules upon stimulation by interleukin-4, immunization with Complete Freund's Adjuvant (CFA) or injection with parental spleen cells (GVHD mice). These results suggest that normal murine B cells have a potential to express new class II specificities when activated by immunological stimuli and provide important implications for further complexity of class II-mediated immune responses including MHC restriction. A possible involvement of mixed isotype class II molecules in the association of disease susceptibility to MHC haplotypes and the induction of autoimmunity is discussed.