Claflin J L, Berry J, Flaherty D, Dunnick W
J Immunol. 1987 May 1;138(9):3060-8.
The variable region sequences of light and heavy chains (VL and VH) were determined for 11 hybridoma antibodies produced in response to the PC moiety on Proteus morganii. These hybridomas were derived from two separate fusions, one obtained from mice early in a secondary response and the other from late in a secondary response. All of these antibodies possessed a cross-reactive idiotype found on anti-PC antibodies in the M603 family, and exhibited preferential specificity for PC in the context of P. morganii. We found that all of the antibodies were derived from a single VH/VL pair. VH was encoded by V1, DFL16.1 and JH1, and VL was encoded by a consensus VK8 gene and JK5. Antibodies differed from each other by somatic point mutations that occurred at a high rate. The mutations in VL were approximately one-third as abundant as those in VH and were randomly distributed throughout the molecule. Mutations in VH were concentrated in CDR 2 and 3 and had a replacement to silent ratio that was three to six times greater than predicted from random accumulation. Based on the sequence data, a single genealogic tree with multiple branches could accommodate all the hybrids from a fusion. We concluded that in both examples the anti-PC response arose by somatic mutation and stepwise selection from a single precursor. Antigen binding studies with these 11 hybridomas and a 12th that had no mutations revealed that the acquisition of preferential specificity for antigen was dependent on somatic mutation of germline genes. Additional binding studies demonstrated that continued selection during clonal expansion was probably antigen driven. An unexpected finding was five independently selected antibodies from one fusion that had identically mutated VH and VL sequences. We suggest that the hypermutation mechanism is not a continuously active process during clonal expansion and that it is regulated, probably during the mid to late phase of the primary response.
测定了11种针对摩根氏变形杆菌上PC部分产生的杂交瘤抗体的轻链和重链可变区序列（VL和VH）。这些杂交瘤来源于两次独立的融合，一次是在二次应答早期从小鼠获得，另一次是在二次应答后期获得。所有这些抗体都具有在M603家族抗PC抗体上发现的交叉反应独特型，并且在摩根氏变形杆菌的背景下对PC表现出优先特异性。我们发现所有抗体都源自单个VH/VL对。VH由V1、DFL16.1和JH1编码，VL由一个共有VK8基因和JK5编码。抗体之间因高发生率的体细胞点突变而不同。VL中的突变数量约为VH中的三分之一，且随机分布于整个分子。VH中的突变集中在互补决定区2和3，其替换与沉默的比例比随机积累预测的大三到六倍。基于序列数据，一个具有多个分支的单一家系树可以容纳来自一次融合的所有杂交体。我们得出结论，在这两个例子中，抗PC应答都是通过体细胞突变和从单个前体的逐步选择产生的。对这11种杂交瘤和第12种无突变杂交瘤进行的抗原结合研究表明，对抗原优先特异性的获得取决于种系基因的体细胞突变。额外的结合研究表明，克隆扩增过程中的持续选择可能是由抗原驱动的。一个意外发现是来自一次融合的5种独立选择的抗体具有相同突变的VH和VL序列。我们认为，高突变机制在克隆扩增过程中不是一个持续活跃的过程，并且它可能在初次应答的中期到后期受到调控。
