Kelly P J, Pascual V, Capra J D, Lipsky P E
Department of Internal Medicine, University of Texas Southwestern Medical Center, Dallas 75235.
J Immunol. 1992 Mar 1;148(5):1294-301.
Polyclonal activation of human B cells is achieved by coculture with T cells stimulated by mAb to the CD3 molecular complex. By formal limiting dilution analysis, approximately 60% of human peripheral blood B cells were found to produce Ig in this system. When individual B cells were cultured in microtiter wells with anti-CD3-activated T cells, more than one-third of cultures producing Ig contained multiple Ig H chain isotypes. Similar results were observed when individual IgM-expressing B cells, selected and dispersed by FACS were cultured with anti-CD3-activated T cells. The clonality of the B cells producing multiple Ig isotypes was supported by L chain analysis of the secreted Ig. Of the wells containing more than one H chain isotype, nearly 85% contained only a single L chain type. Clonality was further examined by polymerase chain reaction amplification of cDNA harvested from cultures originally seeded with individual B cells. In general, only a single VH gene family could be amplified from cultures producing more than one Ig isotype. Three separate VH regions were cloned and sequenced. One, a VHIV-mu was nearly identical to a previously described VH gene VH71.4; as second, a VHIV-gamma was very similar to a previously described VH gene segment V-79, whereas a third, a VHIII-gamma differed by 14% in nucleotide sequence from its closest germline counterpart VH3005. These results indicate that anti-CD3-activated T cells not only stimulate the majority of B cells to secrete Ig, but also induce individual B cells to produce multiple Ig H chain isotypes. Additionally, the procedure described provides a reliable method to sample a large proportion of the human peripheral B cell repertoire.
通过与抗CD3分子复合物单克隆抗体刺激的T细胞共培养来实现人B细胞的多克隆激活。通过正式的有限稀释分析，发现在该系统中约60%的人外周血B细胞产生Ig。当单个B细胞在微量滴定孔中与抗CD3激活的T细胞一起培养时，超过三分之一产生Ig的培养物含有多种Ig重链同种型。当通过荧光激活细胞分选术（FACS）选择并分散的单个表达IgM的B细胞与抗CD3激活的T细胞一起培养时，观察到了类似的结果。分泌型Ig的轻链分析支持了产生多种Ig同种型的B细胞的克隆性。在含有不止一种重链同种型的孔中，近85%仅含有单一轻链类型。通过对最初接种单个B细胞的培养物收获的cDNA进行聚合酶链反应扩增，进一步检查克隆性。一般来说，从产生不止一种Ig同种型的培养物中只能扩增出单个VH基因家族。克隆并测序了三个单独的VH区域。一个VHIV-mu与先前描述的VH基因VH71.4几乎相同；第二个VHIV-γ与先前描述的VH基因片段V-79非常相似，而第三个VHIII-γ在核苷酸序列上与其最接近的种系对应物VH3005相差14%。这些结果表明，抗CD3激活的T细胞不仅刺激大多数B细胞分泌Ig, 还诱导单个B细胞产生多种Ig重链同种型。此外，所描述的方法提供了一种可靠的方法来对大部分人外周B细胞库进行取样。
