Higher Order Restrictions Of The Immunoglobulin Repertoire In CLL Stereotyped Subsets #2 And #169

Stereotyped subset #2 (IGHV3-21/IGLV3-21) is the largest subset in CLL (~3% of all patients). Assignment to subset #2 is clinically relevant since these patients experience an aggressive disease irrespective of the somatic hypermutation (SHM) status of the clonotypic immunoglobulin heavy variable (IGHV) gene. Low-throughput evidence suggests that stereotyped subset #169, a minor CLL subset (~0.21% of all CLL), resembles subset #2 at the immunogenetic level. In specific: (i) the clonotypic heavy chains (HC) of subset #169 are encoded by the IGHV3-48 gene, closely related to the IGHV3-21 gene; (ii) both subsets carry VH CDR3 of 9-amino acids (aa) with a conserved aspartic acid (D) at VH CDR3 position 3; and, (iii) both subsets bear light chains (LC) encoded by the IGLV3-21 gene with restricted VL CDR3. Here we assessed more thoroughly the ontogenetic relationship of CLL subsets #2 and #169 as evidenced in their respective immunogenetic signatures. Using next-generation sequencing methodologies, intraclonal diversification due to ongoing SHM was identified in both heavy and light chain genes of both subsets, albeit more intense in the latter. Furthermore, we identified the presence of common light chain clonotypes which also displayed shared SHMs, with some remarkable examples of virtually ubiquitous SHMs in all analyzed cases at either clonal or subclonal level, strongly suggestive of common antigenic drive. This phenomenon was apparent in the heavy chain as well, where, despite using different, albeit phylogenetically related genes, identical SHMs were detected between CLL subsets #2 and #169. The present high-throughput immunogenetic evidence cements the immunogenetic relatedness of CLL stereotyped subsets #2 and #169, further highlighting the role of antigen selection throughout their natural history.

定型亚组2（IGHV3-21/IGLV3-21）是慢性淋巴细胞白血病（CLL，Chronic Lymphocytic Leukemia）中规模最大的亚组，约占所有患者的3%。将患者归为该亚组具有临床意义，因为无论克隆型免疫球蛋白重链可变区（IGHV，immunoglobulin heavy variable）基因的体细胞超突变（SHM，somatic hypermutation）状态如何，此类患者的疾病均呈侵袭性进展。低通量测序证据显示，作为慢性淋巴细胞白血病小众亚组（约占所有CLL病例的0.21%）的定型亚组169，在免疫遗传学层面与亚组2高度相似。具体而言：(i) 亚组169的克隆型重链（HC，heavy chain）由IGHV3-48基因编码，该基因与IGHV3-21基因亲缘关系密切；(ii) 两个亚组均携带长度为9个氨基酸（aa，amino acid）的VH CDR3，且在VH CDR3的第3位均保有保守的天冬氨酸（D，aspartic acid）；(iii) 两个亚组的轻链（LC，light chain）均由IGLV3-21基因编码，且VL CDR3序列受限。本研究基于两个亚组各自的免疫遗传学特征，对慢性淋巴细胞白血病亚组2与169的个体发育关联展开了更为深入的评估。通过下一代测序（NGS，next-generation sequencing）技术，我们在两个亚组的重链与轻链基因中均检测到了由持续体细胞超突变驱动的克隆内多样性，且后者的克隆内多样性程度更高。此外，我们还发现了共享体细胞超突变模式的共有轻链克隆型，部分案例中几乎所有受检病例（无论处于克隆还是亚克隆层面）均存在普遍存在的体细胞超突变，这强烈提示二者存在共同的抗原驱动机制。重链层面也观察到了这一现象：尽管两个亚组使用的IGHV基因存在差异，但二者在系统发育上亲缘关系密切，且在慢性淋巴细胞白血病亚组2与169中检测到了完全一致的体细胞超突变模式。本研究获得的高通量免疫遗传学证据进一步证实了慢性淋巴细胞白血病定型亚组2与169的免疫遗传学关联性，并进一步凸显了抗原选择在二者自然病程中的关键作用。