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 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.
Fukushima
K
K
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
Murata
Y
Y
Seto
T
T
Furusawa
S
S
Matsuda
H
H
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
D019013:CD40 Antigens; D007136:Immunoglobulins; D015847:Interleukin-4