Claflin J L, George J, Dell C, Berry J
Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor 48109.
J Immunol. 1989 Nov 1;143(9):3054-63.
The contribution of somatic mutation to the generation of an antibody response was investigated by using the phosphocholine (PC) determinant in the bacterium Proteus morganii as the model Ag. The response to this determinant is restricted to a single VH/VL pair and apparently is derived from only one or two precursors per mouse. In this study we examined hybridoma antibodies from nine individual mice which produced representatives of 12 different clones. We found that all antibodies reactive with the PC Ag of P. morganii contained somatic mutations; the number ranged from 2 to 20. Two clusters of mutations were observed, one in complementarity-determining residue (CDR) 2 and the other in CDR 3 of VH. Examination of a three-dimensional model of M603, an antibody with the same V region composition as the anti-PC antibodies under study, showed that these clusters occupied an area of the binding site which presumably interacts with carrier elements of the PC epitope in P. morganii. A high incidence of recurring mutations were found in both clusters, and one of these was invariant, leading to an Asn for Asp substitution at 95. Ag binding studies with these antibodies and an additional one, which was unmutated except for the invariant substitution at 95, showed that: 1) antibodies having only the 95Asn mutation failed to bind the PC Ag of P. morganii, 2) addition of a second recurring mutation, at 52a (CDR 2), was sufficient to create strong binding to the P. morganii Ag, and 3) accumulation of mutations was directly correlated with increased binding activity for Ag. These results show that somatic mutations play a critical, if not essential, role in generating specificity for this PC Ag, and that Ag, and most likely a carrier element of the epitope, is a primary force in the continued selection and expansion of Ag-reactive B cells.
利用摩根变形杆菌中的磷酸胆碱（PC）决定簇作为模型抗原，研究了体细胞突变对抗体应答产生的贡献。对该决定簇的应答局限于单个VH/VL对，并且显然每只小鼠仅源自一两个前体。在本研究中，我们检测了来自9只单独小鼠的杂交瘤抗体，这些小鼠产生了12个不同克隆的代表抗体。我们发现，所有与摩根变形杆菌PC抗原反应的抗体都含有体细胞突变；突变数量在2到20个之间。观察到两个突变簇，一个在VH的互补决定区（CDR）2，另一个在CDR 3。对M603（一种与所研究的抗PC抗体具有相同V区组成的抗体）的三维模型进行检查发现，这些簇占据了结合位点的一个区域，该区域可能与摩根变形杆菌中PC表位的载体元件相互作用。在两个簇中都发现了高频率的重复突变，其中一个是不变的，导致95位的天冬氨酸被天冬酰胺取代。用这些抗体以及另外一种除95位的不变取代外未发生突变的抗体进行抗原结合研究，结果表明：1）仅具有95位天冬酰胺突变的抗体不能结合摩根变形杆菌的PC抗原；2）在52a（CDR 2）处添加第二个重复突变足以产生与摩根变形杆菌抗原的强结合；3）突变的积累与对抗原结合活性的增加直接相关。这些结果表明，体细胞突变在产生对该PC抗原的特异性方面发挥了关键作用（如果不是必不可少的作用），并且抗原以及最有可能是表位的载体元件是抗原反应性B细胞持续选择和扩增的主要驱动力。
