Characterization of an Immunosuppressive Anti-CD40 Ligand Monoclonal Antibody

Abstract

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 (VH) and light chain variable region (VL) genes were cloned using PCR. The cloned VH gene coded for 123 amino acid residues and belonged to the subgroup III(D). The VL 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.

SCID mouse models to study human cancer pathogenesis and approaches to therapy: potential, limitations, and future directions

“…The HuPBL/SCID mouse Model #2 h as been effectively utilized to study the role of co-stimulatory molecules CD28, CD80, CD86, CD40 and CD154 in the development of human immune responses against allo-and xeno-antigens (55,56,(152)(153)(154). On the other hand, very few studies have been done in Model #2 to investigate the role of co-stimulatory molecules in tumor immunity.…”

Impact of the CD40-CD40L Dyad in Alzheimers Disease

“…Studies originally identified CD40 on B lymphocytes, where it was shown to mediate T cell-dependent B cell activation and differentiation. It is found on most immune and some non-immune cells including macrophages, dendritic cells, endothelial cells, smooth muscle cells, and B cells, as well as astrocytes and microglia [5,10–15]. Membrane-bound CD40 and its cognate CD40 ligand (CD40L–also known as CD154), a trimeric 33-kDa type II membrane glycoprotein, play a critical role in microglial phenotypic transformation from a ramified (resting), to an activated macrophage morphology [16] and enhance surface expression of inflammatory markers [17,18].…”

Soluble CD40 ligand in dementia

“…Landmark studies originally identified CD40 on B lymphocytes, where it was shown to mediate T-cell-dependent B-cell activation and differentiation. It is found on most immune and some nonimmune cells, including macrophages, dendritic cells, endothelial cells, smooth muscle cells and B cells, as well as astrocytes and microglia (9)(10)(11)(12)(13)(14)(15). CD40-L, also known as CD154, is a trimeric 33-kDa type II membrane glycoprotein that is predominantly expressed by activated T and B cells, astrocytes and platelets (13,16,17).…”