Departments of Cell Biology, Albert Einstein College of Medicine, Bronx, New York, USA.
mBio. 2011 Oct 11;2(5). doi: 10.1128/mBio.00186-11. Print 2011.
Activation-induced cytidine deaminase (AID) mediates the somatic hypermutation (SHM) of immunoglobulin (Ig) variable (V) regions that is required for the generation of antibody diversity and for the affinity maturation of the antibody response against infectious agents and toxic substances. AID preferentially targets WRC (W = A/T, R = A/G) hot spot motifs, particularly WGCW motifs that create overlapping hot spots on both strands. In order to gain a better understanding of the generation of antibody diversity and to create a platform for the in vitro generation of affinity-matured antibodies, we have established a system involving recombinase-mediated cassette exchange (RMCE) to replace the V region and its flanking sequences. This makes it possible to easily manipulate the sequence of the Ig gene within the endogenous heavy chain of the Ramos human Burkitt's lymphoma cell line. Here we show that the newly integrated wild-type (WT) VH regions introduced by RMCE undergo SHM similarly to non-RMCE-modified Ramos cells. Most importantly, we have shown that introducing a cluster of WGCW motifs into the complementary determining region 2 (CDR2) of the human heavy chain V region significantly raised the mutation frequency and number of mutations per sequence compared to WT controls. Thus, we have demonstrated a novel platform in Ramos cells whereby we can easily and quickly manipulate the endogenous human VH region to further explore the regulation and targeting of SHM. This platform will be useful for generating human antibodies with changes in affinity and specificity in vitro.
An effective immune response requires a highly diverse repertoire of affinity-matured antibodies. Activation-induced cytidine deaminase (AID) is required for somatic hypermutation (SHM) of immunoglobulin (Ig) genes. Although a great deal has been learned about the regulation of AID, it remains unclear how it is preferentially targeted to particular motifs, to certain locations within the Ig gene and not to other highly expressed genes in the germinal center B cell. This is an important question because AID is highly mutagenic and is sometimes mistargeted to other highly expressed genes, including proto-oncogenes, leading to B cell lymphomas. Here we describe how we utilize recombinase-mediated cassette exchange (RMCE) to modify the sequence of the endogenous heavy chain locus in the Ramos Burkitt's lymphoma cell line. This platform can be used to explore the regulation and targeting of SHM and to generate human antibodies with changes in affinity and specificity in vitro.
激活诱导的胞嘧啶脱氨酶（AID）介导免疫球蛋白（Ig）可变（V）区的体细胞超突变（SHM），这是产生抗体多样性和针对传染性病原体和有毒物质的抗体反应亲和力成熟所必需的。AID 优先靶向 WRC（W = A/T，R = A/G）热点基序，特别是在两条链上都创建重叠热点的 WGCW 基序。为了更好地理解抗体多样性的产生，并为体外产生亲和力成熟的抗体建立一个平台，我们建立了一个涉及重组酶介导的盒交换（RMCE）的系统来替换 V 区及其侧翼序列。这使得在 Ramos 人伯基特淋巴瘤细胞系的内源性重链内轻松操纵 Ig 基因的序列成为可能。在这里，我们表明，RMCE 引入的新整合的野生型（WT）VH 区与非 RMCE 修饰的 Ramos 细胞相似地经历 SHM。最重要的是，我们已经表明，将 WGCW 基序簇引入人重链 V 区的互补决定区 2（CDR2）中，与 WT 对照相比，显著提高了突变频率和每个序列的突变数量。因此，我们在 Ramos 细胞中展示了一种新的平台，通过该平台，我们可以轻松快速地操纵内源性人 VH 区，以进一步探索 SHM 的调控和靶向。该平台将有助于在体外产生具有亲和力和特异性变化的人抗体。
有效的免疫反应需要高度多样化的亲和力成熟的抗体库。激活诱导的胞嘧啶脱氨酶（AID）是免疫球蛋白（Ig）基因体细胞超突变（SHM）所必需的。尽管已经了解了很多关于 AID 调控的知识，但仍然不清楚它如何优先靶向特定基序，靶向 Ig 基因内的某些位置，而不是生发中心 B 细胞中其他高度表达的基因。这是一个重要的问题，因为 AID 具有高度突变性，有时会错误靶向其他高度表达的基因，包括原癌基因，导致 B 细胞淋巴瘤。在这里，我们描述了如何利用重组酶介导的盒交换（RMCE）修饰 Ramos 伯基特淋巴瘤细胞系内源性重链基因座的序列。该平台可用于探索 SHM 的调控和靶向，并在体外产生具有亲和力和特异性变化的人抗体。
