Prak E L, Trounstine M, Huszar D, Weigert M
Department of Molecular Biology, Princeton University, New Jersey 08544.
J Exp Med. 1994 Nov 1;180(5):1805-15. doi: 10.1084/jem.180.5.1805.
The genetic organization of the kappa and lambda light chain loci permits multiple, successive rearrangement attempts at each allele. Multiple rearrangements allow autoreactive B cells to escape clonal deletion by editing their surface receptors. Editing may also facilitate efficient B cell production by salvaging cells with nonproductive light chain (L chain) rearrangements. To study receptor editing of kappa L chains, we have characterized B cells from mice hemizygous for the targeted inactivation of kappa (JCkD/wt) which have an anti-DNA heavy chain transgene, 3H9. Hybridomas from JCkD/wt mice exhibited an increased frequency of rearrangements to downstream Jk segments (such as Jk5) compared with most surveys from normal mice, consistent with receptor editing by sequential kappa locus rearrangements in JCkD/wt. We observed an even higher frequency of rearrangements to Jk5 in 3H9 JCkD/wt animals compared with nontransgenic JCkD/wt, consistent with editing of autoreactive kappa in 3H9 JCkD/wt. We also recovered a large number of 3H9 JCkD/wt lines with Vk12/13-Jk5 rearrangements and could demonstrate by PCR and Southern analysis that up to three quarters of these lines underwent multiple kappa rearrangements. To investigate editing at the lambda locus, we used homozygous kappa-deficient animals (JCkD/JCkD and 3H9 JCkD/JCkD). The frequencies of V lambda 1 and V lambda 2 rearrangements among splenic hybridomas in 3H9 JCkD/JCkD were reduced by 75% whereas V lambda X was increased 5-10-fold, compared with nontransgenic JCkD/JCkD animals. This indicates that V lambda 1 and V lambda 2 are negatively regulated in 3H9 JCkD/JCkD, consistent with earlier studies that showed that the 3H9 heavy chain, in combination with lambda 1 binds DNA. As successive lambda rearrangements to V lambda X do not inactivate V lambda 1, the consequence of lambda editing in 3H9 JCkD/JCkD would be failed allelic exclusion at lambda. However, analysis of 18 3H9 JCkD/JCkD hybridomas with V lambda 1 and V lambda X DNA rearrangements revealed that most of these lines do not have productive lambda 1 rearrangements. In sum, both kappa and lambda loci undergo editing to recover from nonproductive rearrangement, but only kappa locus editing appears to play a substantial role in rescuing autoreactive B cells from deletion.
κ和λ轻链基因座的遗传组织允许每个等位基因进行多次连续的重排尝试。多次重排使自身反应性B细胞能够通过编辑其表面受体来逃避克隆清除。编辑还可以通过挽救具有无功能轻链（L链）重排的细胞来促进高效的B细胞产生。为了研究κ轻链的受体编辑，我们对κ基因靶向失活的半合子小鼠（JCkD/wt）的B细胞进行了表征，这些小鼠带有抗DNA重链转基因3H9。与来自正常小鼠的大多数研究相比，JCkD/wt小鼠的杂交瘤显示重排至下游Jκ片段（如Jκ5）的频率增加，这与JCkD/wt中κ基因座的顺序重排导致的受体编辑一致。我们观察到，与非转基因JCkD/wt相比，3H9 JCkD/wt动物中重排至Jκ5的频率更高，这与3H9 JCkD/wt中自身反应性κ的编辑一致。我们还获得了大量具有Vk12/13-Jκ5重排的3H9 JCkD/wt细胞系，并通过PCR和Southern分析证明，这些细胞系中多达四分之三经历了多次κ重排。为了研究λ基因座的编辑，我们使用了纯合κ缺陷动物（JCkD/JCkD和3H9 JCkD/JCkD）。与非转基因JCkD/JCkD动物相比，3H9 JCkD/JCkD脾杂交瘤中Vλ1和Vλ2重排的频率降低了75%，而VλX增加了5至10倍。这表明在3H9 JCkD/JCkD中Vλ1和Vλ2受到负调控，这与早期研究一致，该研究表明3H9重链与λ1结合可结合DNA。由于连续向VλX的λ重排不会使Vλ1失活，3H9 JCkD/JCkD中λ编辑的结果将是λ基因座的等位基因排斥失败。然而，对18个具有Vλ1和VλX DNA重排的3H9 JCkD/JCkD杂交瘤的分析表明，这些细胞系中的大多数没有有功能的λ1重排。总之，κ和λ基因座都经历编辑以从不产生功能的重排中恢复，但只有κ基因座编辑似乎在拯救自身反应性B细胞免于清除方面发挥了重要作用。
