Tertiary lymphoid organs in islets at the early stages of autoimmune diabetes, are local hubs for autoantibody maturation

Introduction: The debut of treatments which delay the onset of T1D, highlights the importance of its pre-symptomatic detection. Currently, early diagnosis relies on serum detection of autoantibodies which target up to five known autoantigens. Improving the accuracy of T1D diagnosis requires better understanding of the processes that lead to beta cell ablation, and expansion of the repertoire of known autoantibody targets.

Results: scRNA-seq on islets of NOD mice identified more than 40 different cell types in the insulitic niche during the pre-symptomatic phase of autoimmune diabetes. Compared to non-attacked islets, insulitic islets are enriched for cells characteristic of tertiary lymphoid organs (TLO) - transient structures that form in sites of severe inflammation and serve as local lymph nodes. Although T1D-associated TLOs have been reported in both mouse and human islets, their functional role is poorly understood. We noticed that attacked islets are highly enriched for both active and naïve B cells, and that a subpopulation of the former express Aicda – an inducer of somatic hypermutations (SHM) during the antibody maturation process. Accordingly, single cell T and B cell receptor sequencing uncovered lineage trajectories which follow SHM accumulation within islet-associated B-cell clones. Altogether, this shows that islet-TLOs serve as local hubs that recruit naïve lymphocytes and activate them in situ; and that final antibody maturation occurs in the insulitic niche surrounding islets, and is not confined to “canonical” lymph nodes. Subsequently, identifying key antigens which drive the autoimmune process should focus on the antibody repertoire that forms in attacked islets. To undertake this task, we sequenced more than 6,000 B-cell receptors (BCRs) from islets of pre-symptomatic NOD mice, and generated equivalent antibodies to 8 BCRs of activated clones, in vitro. Surprisingly, 6 of the tested antibodies bind beta-cell associated proteins, most of which are localized to the cells’ membrane.

Conclusions: Our findings show that islet-TLOs “train” naïve B-cells and drive their maturation. Utilizing this, we interrogated the antibody repertoire of attacked islets, and discovered novel autoantibodies that target beta cell associated antigens. Altogether, these findings are expected to advance the development of accurate tools for early diagnosis of T1D.