title: In vitro system for immunoglobulin class switching using BHK cells transfected with murine recombinant CD40 ligand.
abstract: 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 splenic 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 from T to G, when compared with the known sequence from C57BL/6, one of the BALB/c-derived clones was reconstructed to the known CD40L by site-directed mutagenesis. Splenic B cells from BALB/c were induced secretion of Ig isotypes, IgM, IgG1 and IgE when cultured on two types of BHK cells, the transfected 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 cells produced Ig isotypes, IgM, IgG1, IgG2a, IgG2b, IgG3 and IgE. In the similar 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 induced by using these two types of CD40L-expressing BHK cells in vitro.
authors.affiliation: Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
authors.forename: K
authors.initials: K
authors.lastname: Fukushima
authors.forename: Y
authors.initials: Y
authors.lastname: Murata
authors.forename: T
authors.initials: T
authors.lastname: Seto
authors.forename: S
authors.initials: S
authors.lastname: Furusawa
authors.forename: H
authors.initials: H
authors.lastname: Matsuda
mesh_terms: D000818:Animals; D001402:B-Lymphocytes / Q000166:cytology / Q000276:immunology*; D019013:CD40 Antigens / Q000235:genetics*; D018929:Cell Culture Techniques / Q000379:methods; D002478:Cells, Cultured; D006224:Cricetinae; D004797:Enzyme-Linked Immunosorbent Assay; D005260:Female; D017578:Immunoglobulin Class Switching; D007136:Immunoglobulins / Q000096:biosynthesis / Q000235:genetics; D015847:Interleukin-4; D051379:Mice; D008807:Mice, Inbred BALB C; D008810:Mice, Inbred C57BL; D013154:Spleen / Q000276:immunology; D014162:Transfection
chemical_list: D019013:CD40 Antigens; D007136:Immunoglobulins; D015847:Interleukin-4