ANTI-CD40 MONOCLONAL-ANTIBODY INDUCES THE PROLIFERATION OF MURINE B-CELLS AS A B-CELL MITOGEN THROUGH A DISTINCT PATHWAY FROM RECEPTORS FOR ANTIGENS OR LIPOPOLYSACCHARIDE

To study the activation and differentiation of murine B cells, we prepared a hybridoma secreting monoclonal antibody, LB429, which can directly induce the proliferation of murine B cells in vitro. LB429 recognizes a B cell-specific surface molecule of 45 kDa. It recognizes an epitope of murine CD40 produced as a soluble fusion protein with glutathione S-transferase. LB429 stains COS-7 transfectant with murine CD40 cDNA and mature B-cell lines but does not stain pre-B cell lines. Two-color staining demonstrated that the epitope recognized with LB429 appears on the surface of B220(+) cells of spleen and bone marrow. LB429 can induce a strong proliferation of murine B cells from spleen in the absence of initial triggering with anti-IgM antibody or with anti-IgM antibody + IL-4. LB429 induced the cell size enlargement and the cell cycle transition of resting B cells as well as lipopolysaccharide (LPS). LB429 and LPS stimulate B cells synergistically in vitro by accumulating 44.7% of cells in S/G2/M phases of cell cycle. However, stimulation of spleen B cells with LB429 resulted in the increase of sIgM(high+) sIgD(high+) B cells, in contrast LPS showed the proliferation of both sIgM(high+) sIgD(high+) B cells and sIgM(low+) sIgD(high+) B cells. These results suggested that LB429 and LPS cause the proliferation of B cells through different stimulatory pathways. This anti-mouse CD40 antibody (LB429) is a very useful reagent to study the activation and differentiation of B cells in vitro.