Activated polyreactive B cells are clonally expanded in autoantibody positive and patients with recentonset type 1 diabetes

Autoreactive B cells play an important but ill-defined role in autoimmune type 1 diabetes (T1D). To better understand their contribution to disease, we performed single cell gene expression and BCR-seq on pancreatic islet antigen-reactive (IAR) B cells from the peripheral blood of nondiabetic (ND), autoantibody positive prediabetic (AAB), and recent-onset T1D individuals. We found that the frequency of IAR B cells was increased particularly in AAB, but also in T1D compared to ND donors. Additionally, IAR B cells from AAB and T1D donors exhibited differential gene expression in B cell signaling, pro-inflammatory, infection, and antigen processing and presentation pathways compared to ND donors. Strikingly, both AAB and T1D donors demonstrated a significant increase in particular heavy and light chain V gene usages compared to ND, and these B cells were enriched in islet-reactivity. Shared public clones of IAR B cells were found almost entirely among the AAB and T1D donors. IAR B cells were clonally expanded in the autoimmune donors, particularly the AAB group. Notably, a substantial fraction of IAR B cells in AAB and T1D donors appeared to be polyreactive and was confirmed by production of recombinant monoclonal antibodies. Altogether, these results expand our current understanding of B cells during development of T1D, how autoreactive B cells may become activated, and identify unique BCR repertoire differences that may serve as biomarkers for increased disease risk. These findings could be applied to future therapeutic approaches to prevent or treat T1D, as well as assess response to therapy. This study sought to determine whether differences in the transcriptional phenotype and BCR repertoire of islet antigen reactive B cells exist during development of autoimmunity. We generated uniquely barcoded PE-labeled antigen tetramers for INS, GAD, IA-2, and TET to identify antigen-reactive B cells using scRNA-seq. PBMCs from five ND first-degree relatives, six AAB subjects, and five recent-onset T1D patients were incubated with the various tetramers. Then, PE-binding B cells were sorted and loaded onto the 10X Genomics platform to be analyzed for gene expression and BCR repertoire differences. Recombinant antibodies were generated to confirm antigen-reactivity of some of the identified BCR sequences.

自身反应性B细胞（autoreactive B cells）在自身免疫性1型糖尿病（T1D）中发挥着重要却尚未阐明的作用。为深入阐明其在疾病发生中的贡献，我们对非糖尿病（ND）、自身抗体阳性前驱糖尿病（AAB）及新发T1D个体外周血中的胰岛抗原反应性B细胞（islet antigen-reactive B cells, IAR）开展了单细胞基因表达谱与B细胞受体测序（B cell receptor sequencing, BCR-seq）分析。我们发现，相较于ND受试者，IAR B细胞的频率在AAB群体中尤为升高，在T1D群体中亦显著上调。此外，与ND受试者相比，AAB与T1D群体的IAR B细胞在B细胞信号通路、促炎反应、感染应答及抗原加工提呈通路中呈现出差异基因表达特征。值得注意的是，相较于ND受试者，AAB与T1D群体的B细胞在特定重链与轻链V基因的使用频率上均显著升高，且此类细胞富集胰岛反应性特征。IAR B细胞的公共克隆簇几乎仅存在于AAB与T1D群体中。自身免疫受试者体内的IAR B细胞呈现克隆扩增特征，其中尤以AAB群体最为显著。值得关注的是，AAB与T1D群体中相当比例的IAR B细胞表现出多反应性，这一结论通过重组单克隆抗体的制备得到了验证。综上，本研究结果拓展了当前学界对T1D发生过程中B细胞的认知，阐明了自身反应性B细胞的潜在激活机制，并鉴定出可作为疾病风险升高生物标志物的独特B细胞受体库（BCR repertoire）差异特征。上述发现可应用于未来预防或治疗T1D的干预策略，同时也可用于评估治疗应答情况。本研究旨在探究自身免疫发生过程中，胰岛抗原反应性B细胞的转录表型与B细胞受体库是否存在差异。我们针对胰岛素（INS）、谷氨酸脱羧酶（GAD）、胰岛抗原2（IA-2）及TET制备了带有独特条形码标记的PE标记抗原四聚体，以通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）鉴定抗原反应性B细胞。我们将5名ND一级亲属、6名AAB受试者及5名新发T1D患者的外周血单个核细胞（peripheral blood mononuclear cell, PBMCs）与上述各类四聚体共同孵育。随后，我们分选结合了PE的B细胞，并将其加载至10X Genomics平台，以分析其基因表达谱与B细胞受体库的差异特征。我们还制备了重组抗体，以验证部分鉴定出的BCR序列的抗原反应性。