In vitro system for immunoglobulin class switching using BHK cells transfected with murine recombinant CD40 ligand

To develop an in vitro system for mouse immunoglobulin (Ig) class switching , the expression vector of murine CD40 ligand (CD40L) which is expressed on T cells was transfected to BHK cells. By using culture plates coated with the BHK cells expressing the recombinant CD40L, Ig class switching of splen ic B cells was examined. The CD40L mRNA was cloned from splenic T cells of BALB/mice activated with anti-CD3 antibody in vitro. As the No.593 base in the open reading frame sequence of the CD40L from BALB/c spleen differed fr om T to G, when compared with the known sequence from C57BL/6, one of the B ALB/c-derived clones was reconstructed to the known CD40L by site-directed mutagenesis. Splenic B cells from BALB/c were induced secretion of Ig isoty pes, IgM, IgG1 and IgE when cultured on two types of BHK cells, the transfe cted BHK cells with a CD40L clone from BALB/c and the transfected BHK cells with the reconstructed CD40L clone, in the presence of IL-4. However, when splenic B cells from C57BL/6 were cultured on the same systems, the B cell s produced Ig isotypes, IgM, IgG1, IgG2a, IgG2b, IgG3 and IgE. In the simil ar experiments using the transfected BHK cells with a empty vector and the normal BHK cells, none of B cells produced any Ig isotypes other than IgM. These results indicate that Ig class switching of murine B cells can be ind uced by using these two types of CD40L-expressing BHK cells in vitro.