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. Overall design: 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.