EFFECTS OF RECOMBINANT SOLUBLE CD40 LIGAND IN HEMATOPOIESIS

Abstract

CD40 is a 50KD membrane glycoprotein expressed by cells such as B lymphocytes, macrophages, monocytes, dendritic cells, epithelial cells and some carcinomas (Banchereau et al., 1994). This molecule was shown by in vitro studies to be involved in B cell activation, differentiation, proliferation, and several other important activities such as isotype switching, generation of B cells within germinal centers. The in vivo functional significance of CD40 stimulation in normal mice is not known. Furthermore, the effect of CD40 stimulation on hematopoietic progenitor cells has not been determined. To study this effect, normal BALB/c mice were treated intraperitoneally with 100µg, 10µg, and 1µg of soluble recombinant murine CD40 ligand(srmCD4OL) every other day for three days to measure increases in cellularity. Cellularity of the bone marrow and spleen increased approximately 61% when compared to mice that did not receive srmCD4OL. A 62% increase was seen in granulocytic precursors.. The splenic response of mice treated with the bacterial B cell mitogen lipopolysaccharide(LPS) increased 22% when compared to mice that received only glycerol. To study the effect further, in vitro experiments were performed with murine and human bone marrow cells using growth promoting cytokines such as GM-CSF, IL-3, SCF, IL-11, and M-CSF in the presence and absence of srm/srhCD4OL. Hematopoietic progenitor formation was increased between 28- 70%. Ligand alone had no effect which suggests that the ligand functions in a costimulatory manner. To examine if the effects of the ligand were direct, murine stem cells were separated from a whole population of murine bone marrow cells, and human hematopoietic progenitor cells were separated from a whole population of human cord blood or bone marrow cells, and these cells were stimulated with srm/srhCD4OL using cytokines. The increase in cellularity was approximately 40% in BMC treated with cytokines and srhCD4OL when compared to BMC treated with cytokines alone. These results demonstrate that srm/srhCD4OL could be used to stimulate proliferation and differentiation of hematopoietic progenitors, suggesting that CD40 stimulation by its ligand could be used clinically to accelerate hematopoiesis.