Pascual V, Liu Y J, Magalski A, de Bouteiller O, Banchereau J, Capra J D
Department of Microbilogy, University of Texas Southwestern Medical Center at Dallas.
J Exp Med. 1994 Jul 1;180(1):329-39. doi: 10.1084/jem.180.1.329.
Using a series of phenotypic markers that include immunoglobulin (Ig)D, IgM, IgG, CD23, CD44, Bcl-2, CD38, CD10, CD77, and Ki67, human tonsillar B cells were separated into five fractions representing different stages of B cell differentiation that included sIgD+ (Bm1 and Bm2), germinal center (Bm3 and Bm4), and memory (Bm5) B cells. To establish whether the initiation of somatic mutation correlated with this phenotypic characterization, we performed polymerase chain reaction and subsequent sequence analysis of the Ig heavy chain variable region genes from each of the B cell subsets. We studied the genes from the smallest VH families (VH4, VH5, and VH6) in order to facilitate the mutational analysis. In agreement with previous reports, we found that the somatic mutation machinery is activated only after B cells reach the germinal center and become centroblasts (Bm3). Whereas 47 independently rearranged IgM transcripts from the Bm1 and Bm2 subsets were nearly germline encoded, 57 Bm3-, and Bm4-, and Bm5-derived IgM transcripts had accumulated an average of 5.7 point mutations within the VH gene segment. gamma transcripts corresponding to the same VH gene families were isolated from subsets Bm3, Bm4, and Bm5, and had accumulated an average of 9.5 somatic mutations. We conclude that the molecular events underlying the process of somatic mutation takes place during the transition from IgD+, CD23+ B cells (Bm2) to the IgD-, CD23-, germinal center centroblast (Bm3). Furthermore, the analysis of Ig variable region transcripts from the different subpopulations confirms that the pathway of B cell differentiation from virgin B cell throughout the germinal center up to the memory compartment can be traced with phenotypic markers. The availability of these subpopulations should permit the identification of the functional molecules relevant to each stage of B cell differentiation.
利用一系列表型标志物，包括免疫球蛋白（Ig）D、IgM、IgG、CD23、CD44、Bcl-2、CD38、CD10、CD77和Ki67，将人扁桃体B细胞分离为五个代表B细胞分化不同阶段的组分，包括表面IgD+（Bm1和Bm2）、生发中心（Bm3和Bm4）和记忆（Bm5）B细胞。为了确定体细胞突变的起始是否与这种表型特征相关，我们对每个B细胞亚群的Ig重链可变区基因进行了聚合酶链反应及后续序列分析。我们研究了最小的VH家族（VH4、VH5和VH6）的基因，以便于进行突变分析。与先前的报道一致，我们发现体细胞突变机制仅在B细胞到达生发中心并成为中心母细胞（Bm3）后才被激活。来自Bm1和Bm2亚群的47个独立重排的IgM转录本几乎是种系编码的，而来自Bm3、Bm4和Bm5的57个IgM转录本在VH基因区段内平均积累了5.7个点突变。从Bm3、Bm4和Bm5亚群中分离出对应于相同VH基因家族的γ转录本，其平均积累了9.5个体细胞突变。我们得出结论，体细胞突变过程的分子事件发生在从IgD+、CD23+B细胞（Bm2）向IgD-、CD23-生发中心中心母细胞（Bm3）的转变过程中。此外，对不同亚群的Ig可变区转录本的分析证实，从初始B细胞到生发中心直至记忆区室的B细胞分化途径可以用表型标志物进行追踪。这些亚群的可得性应有助于鉴定与B细胞分化各阶段相关的功能分子。
