Islet-Specific Immune Repertoires uncover a Role for Tertiary Lymphoid Organs in T1D

Introduction: Type 1 diabetes (T1D) is an autoimmune disease characterized by the destruction of insulin-producing β-cells in pancreatic islets, leading to insulin deficiency. The immune attack progresses asynchronously across the pancreas, causing variability in the extent of attack experienced by individual islets. Notably, some islets present formation of Tertiary Lymphoid Organs (TLOs) around them. These structures are known to form at sites of chronic inflammation, where they serve as local hubs for B and T cell activation and expansion. Here, we propose to utilize the heterogeneous formation of TLOs to determine the physical site of immune activation. We hypothesize that clonal analysis of somatic hypermutations in B cells across different islets would reveal whether immune activation occurs locally near the targeted islets, or rather in secondary lymphoid organs such as the pancreatic lymph nodes, as commonly thought.

Materials and methods: To test this hypothesis, we developed a novel protocol for sequencing of both the transcriptome and the immune repertoire of single islets. The protocol builds upon existing single-cell transcriptome and immune repertoire sequencing methods, which we adapted to accommodate high-throughput sequencing of single islets.

Results: In a preliminary experiment, we sequenced 96 single islets from a pre-diabetic Non-Obese Diabetic (NOD) mouse. For each islet we successfully identified genes associated with both immune and pancreatic cell types, suggesting that our protocol captures the major cell populations surrounding attacked islets. Immune repertoire analysis revealed clonal expansion of T and B cells, and identified lineages of B cells undergoing hypermutations. Remarkably, rather than a uniform distribution of expanded clones, individual islets exhibited high dominance of specific B cell clonotypes. This supports our hypothesis that B cells are activated and proliferate in the islet’s TLO, rather than exclusively in lymph nodes.

Conclusions: Preliminary results highlight the important role of TLOs during T1Dprogression. Further analysis of these receptor variants, combined with transcriptomic data from the islets could provide deeper insights into the mechanisms triggering the immune attacks on islets and unravel the course of T1D progression. Additionally, these findings may enhance our understanding of other autoimmune diseases with similar asynchronous patterns of immune activation.