Preliminary data indicates that stromal cells within the vicinity of islets acquire properties that can organize ectopic lymph node like structures (known as tertiary lymphoid structures or TLS)in NOD mice. These properties include differentiating of stromal cells into specific cell types that can secrete chemokines to organize infiltration of T and B cells and secrete growth factors to organize endothelial cells into venules through which these lymphocytes traffic. These ectopic lymph nodes correlate with disease progression and mice that don’t develop disease do not show an evidence of these TLS. In this application, we utilize Co–Detection–by–InDEXing (CODEX), a multiplexed single–cell imaging technology that incorporates DNA barcoded antibodies to visualize 100 + cellular markers at the single–cell level to map islet stromal cells and TLS in nPOD samples to map tissue architecture and characterize stromal–immune interactions in T1D. As TLS have been recently been shown to be a feature of T1D pancreas in humans, we would like to assess whether the stromal cells in nPOD donors acquire TLS organizing properties. We also assess the subtypes of stromal cells with lymphoid organizing properties and expression of cell type specific markers of stromal cell subtypes involved in TLS organization. We have built our CODEX panel to include markers of lymphoid organizing stromal cells that will enable us to identify the sub–types of cells. The CODEX panel includes markers for T cells, B cells and endothelial structures called high endothelial venules (HEVs).We will also be able to assess the activation state of lymphocytes. Finally we haver markers for endocrine cells. We envision that such analysis of T1D samples will allow us to establish whether stromal cells are strategically positioned in the islet to provide a scaffold and orchestrate TLS formation by modulating immune cell maturation, migration and activation.
