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[Serological analysis of mixed isotype Ab beta Ed alpha class II molecules and preparation of its specific monoclonal
1Department of Immunology, Saga Medical School.
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.
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.