The interaction between CD40 ligand (CD40L) and its counter-receptor CD40 is critically important in T and B-cell costimulation and generation of the humoral immune response. But several questions still remain unsolved, particularly in the human in vivo system. To clarify the precise function of CD40L and develop an immunosuppressive agent, we have generated a murine monoclonal antibody (MAb), 2B2 specific for human CD40L. The specificity of this MAb for human CD40L was verified by enzyme-linked immunoadsorbent assay (ELISA) and flow cytometry. MAb 2B2 immunoprecipitated proteins of molecular weight 35 and 28 kD on human peripheral blood lymphocytes (PBLs) stimulated with phorbol 12-myristate-13-acetate (PMA) plus ionomycin. Then we have studied the biological effect of MAb 2B2 in severe combined immunodeficiency (SCID) mice reconstituted with human PBLs. The data showed that this MAb strongly suppressed human IgG production of human B cells transplanted in SCID mice, indicating that this MAb 2B2 could be used to regulate unwanted immune responses associated with autoimmune disease. Then we analyzed the sequence of MAb 2B2. The 2B2 heavy chain variable region (V(H)) and light chain variable region (V(L)) genes were cloned using PCR. The cloned V(H) gene coded for 123 amino acid residues and belonged to the subgroup III(D). The V(L) gene coded for 126 amino acid and belonged to the subgroup V. Collectively, these results will be used to develop an immunosuppressive chimeric or humanized anti-CD40L antibody.
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