Carachterization of B cell stimulatory factor capable of inducing autoantibody production and its receptor

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1) Culture fluid supernatant of murine B151K12 T-cell hybridoma grown in protein-free RPMI1640 medium was concentrated by 20-50% ammonium sulfate precipitation and applied onto DEAE-Sephadex A-50 column. The active fraction in the ionexchangechromatography was further purified with GlcNAc copolymer (chitin) column by taking advantage of the specific binding ability of B151-TRF2 to GlcNAc residue(s). B151-TRF2 activity in eluate fraction was purified approximately 2,000-fold. In consistent with the results of functional analysis, binding experiments utilizing purified B151-TRF2 fraction labelled with ^<125>I revealed that sugar moiety is not detected on B151-TRF2 molecule and that B151-TRF2 binds to B cells through interaction with their surface terminal GlcNAc residue(s). 2) B cell subpopulation identified by Jl monoclonal antibody (mAb) specific for terminal GlcNAc residue(s) was shown to be involved in the B151-TRF2 response. Furthermore, Western blotting analysis of SDS-solublized m … More embrane fraction with ^<125>I-Jl mAb revealed four bands with M.W of approximately 25K, 30K, 30K, 40K and 60K. 3) Among B cell hybridomas established by fusing P3Ul myeloma with spleen cells from rats hyperimmunized with purified B151-TRF2, we selected out a clone 5-6-9 which produces mAb capable of specifically inhibiting B151-TRF2 activity. Culture plate (24W) coated with purified 5-6-9 mAb was able to absorb B151-TRF2 activity. 4) Immunogenetic study on autoantibody production by B151-TRF2 demonstrated that in vitro anti-Bromelain treated mouse red blood cells (BrMRBC) antibody response of unprimed murine B cells induced by polyclonal B cells activation B151-TRF2 is controlled by I-region gene within H-2 complex. Interestingly, self-Ia recognitionspecificity expressed on b cells was closely associated with in vivo generation of BrMRBC-specific B cell clone. This finding has important implication to the problems of H-2-linked Ir-gene control of B cell response, especially generation of antibody repertoire. Less