Fukushima K, Murata Y, Seto T, Furusawa S, Matsuda H
Tsukuba Research Laboratories, Eisai Co., Ltd., Ibaraki, Japan.
J Vet Med Sci. 1999 Jun;61(6):615-9. doi: 10.1292/jvms.61.615.
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.
为了开发一种用于小鼠免疫球蛋白（Ig）类别转换的体外系统，将在T细胞上表达的小鼠CD40配体（CD40L）的表达载体转染到BHK细胞中。通过使用涂有表达重组CD40L的BHK细胞的培养板，检测脾B细胞的Ig类别转换。CD40L mRNA从体外被抗CD3抗体激活的BALB/c小鼠的脾T细胞中克隆得到。由于与C57BL/6的已知序列相比，BALB/c脾CD40L开放阅读框序列中的第593个碱基从T变为G，因此通过定点诱变将一个BALB/c来源的克隆重建为已知的CD40L。在IL-4存在的情况下，当BALB/c的脾B细胞在两种类型的BHK细胞上培养时，即转染了来自BALB/c的CD40L克隆的BHK细胞和转染了重建的CD40L克隆的BHK细胞，诱导分泌了Ig同种型IgM、IgG1和IgE。然而，当C57BL/6的脾B细胞在相同系统上培养时，B细胞产生了Ig同种型IgM、IgG1、IgG2a、IgG2b、IgG3和IgE。在使用转染了空载体的BHK细胞和正常BHK细胞的类似实验中，除了IgM之外，没有B细胞产生任何Ig同种型。这些结果表明，使用这两种表达CD40L的BHK细胞可以在体外诱导小鼠B细胞的Ig类别转换。
