Type 1 diabetes studies consistently generate data showing islet beta-cell dysfunction and T cell-mediated anti-beta-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the beta-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single beta-cells. Consistent with immunohistological studies, beta-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These beta-cells also expressed mRNA for Class II and Class II antigen presentation pathway components, but lacked the macrophage marker CD68. Immunohistological study of three independent cohorts of donors with recent-onset type 1 diabetes showed Class II protein and its transcriptional regulator Class II MHC trans-activator protein expressed by a subset of insulin(+)CD68(-) beta-cells, specifically found in islets with lymphocytic infiltrates. beta-Cell surface expression of HLA Class II was detected on a portion of CD45(-)insulin(+) beta-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic beta-cells from donors with type 1 diabetes express Class II molecules on selected cells with other key genes in those pathways and inflammation-associated genes. beta-Cell expression of Class II molecules suggests that beta-cells may interact directly with islet-infiltrating CD4(+) T cells and may play an immunopathogenic role.

Many patients with type 1 diabetes (T1D) have residual beta cells producing small amounts of C-peptide long after disease onset but develop an inadequate glucagon response to hypoglycemia following T1D diagnosis. The features of these residual beta cells and alpha cells in the islet endocrine compartment are largely unknown, due to the difficulty of comprehensive investigation. By studying the T1D pancreas and isolated islets, we show that remnant beta cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D alpha cells was markedly reduced, and these cells had alterations in transcription factors constituting alpha and beta cell identity. In the native pancreas and after placing the T1D islets into a non-autoimmune, normoglycemic in vivo environment, there was no evidence of alpha-to-beta cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

RNA-seq protocols that focus on transcript termini are well suited for applications in which template quantity is limiting. Here we show that, when applied to end-sequencing data, analytical methods designed for global RNA-seq produce computational artifacts. To remedy this, we created the End Sequence Analysis Toolkit (ESAT). As a test, we first compared end-sequencing and bulk RNA-seq using RNA from dendritic cells stimulated with lipopolysaccharide (LPS). As predicted by the telescripting model for transcriptional bursts, ESAT detected an LPS-stimulated shift to shorter 3'-isoforms that was not evident by conventional computational methods. Then, droplet-based microfluidics was used to generate 1000 cDNA libraries, each from an individual pancreatic islet cell. ESAT identified nine distinct cell types, three distinct beta-cell types, and a complex interplay between hormone secretion and vascularization. ESAT, then, offers a much-needed and generally applicable computational pipeline for either bulk or single-cell RNA end-sequencing.