University of Oklahoma Health Sciences Center, Oklahoma Medical Research Foundation, and Department of Veterans Affairs Medical Center, Oklahoma City, Oklahoma.
Oklahoma Medical Research Foundation, Oklahoma City.
Arthritis Rheumatol. 2018 Jul;70(7):1102-1113. doi: 10.1002/art.40458. Epub 2018 May 11.
To better understand the role of B cells, the potential mechanisms responsible for their aberrant activation, and the production of autoantibodies in the pathogenesis of Sjögren's syndrome (SS), this study explored patterns of selection pressure and sites of N-glycosylation acquired by somatic mutation (acN-glyc) in the IgG variable (V) regions of antibody-secreting cells (ASCs) isolated from the minor salivary glands of patients with SS and non-SS control patients with sicca symptoms.
A novel method to produce and characterize recombinant monoclonal antibodies (mAb) from single cell-sorted ASC infiltrates was applied to concurrently probe expressed genes (all heavy- and light-chain isotypes as well as any other gene of interest not related to immunoglobulin) in the labial salivary glands of patients with SS and non-SS controls. V regions were amplified by reverse transcription-polymerase chain reaction, sequenced, and analyzed for the incidence of N-glycosylation and selection pressure. For specificity testing, the amplified regions were expressed as either the native mAb or mutant mAb lacking the acN-glyc motif. Protein modeling was used to demonstrate how even an acN-glyc site outside of the complementarity-determining region could participate in, or inhibit, antigen binding.
V-region sequence analyses revealed clonal expansions and evidence of secondary light-chain editing and allelic inclusion, of which neither of the latter two have previously been reported in patients with SS. Increased frequencies of acN-glyc were found in the sequences from patients with SS, and these acN-glyc regions were associated with an increased number of replacement mutations and lowered selection pressure. A clonal set of polyreactive mAb with differential framework region 1 acN-glyc motifs was also identified, and removal of the acN-glyc could nearly abolish binding to autoantigens.
These findings support the notion of an alternative mechanism for the selection and proliferation of some autoreactive B cells, involving V-region N-glycosylation, in patients with SS.
为了更好地理解 B 细胞在干燥综合征 (SS) 发病机制中的作用、导致其异常激活的潜在机制以及自身抗体的产生，本研究探讨了从 SS 患者和有干燥症状的非 SS 对照患者的小唾液腺分离的分泌抗体细胞 (ASC) 的 IgG 可变 (V) 区中体细胞突变获得的选择压力模式和 N-糖基化位点 (acN-glyc)。
应用一种从单细胞分选 ASC 浸润物中产生和鉴定重组单克隆抗体 (mAb) 的新方法，同时探测 SS 患者和非 SS 对照患者的唇腺中表达的基因 (所有重链和轻链同型以及任何与免疫球蛋白无关的感兴趣基因)。通过逆转录-聚合酶链反应扩增 V 区，测序并分析 N-糖基化和选择压力的发生率。为了特异性测试，扩增区域被表达为天然 mAb 或缺乏 acN-glyc 基序的突变 mAb。蛋白质建模用于证明即使是互补决定区之外的 acN-glyc 位点也可以参与或抑制抗原结合。
V 区序列分析显示克隆扩增和证据表明次要轻链编辑和等位基因包含，其中后两者以前在 SS 患者中均未报道过。在 SS 患者的序列中发现 acN-glyc 的频率增加，并且这些 acN-glyc 区域与更多的替换突变和降低的选择压力相关。还鉴定了具有差异框架 1 acN-glyc 基序的多反应性 mAb 的克隆集，并且去除 acN-glyc 几乎可以完全消除对自身抗原的结合。
这些发现支持了在 SS 患者中存在一种替代机制来选择和增殖某些自身反应性 B 细胞的观点，涉及 V 区 N-糖基化。
