Understanding distinct gene expression patterns of normal adult and developing fetal human pancreatic α- and β-cells is crucial for developing stem cell therapies, islet regeneration strategies, and therapies designed to increase β-cell function in patients with diabetes (type 1 or 2). Toward that end, we have developed methods to highly purify α-, β-, and δ-cells from human fetal and adult pancreata by intracellular staining for the cell-specific hormone content, sorting the subpopulations by flow cytometry, and, using next-generation RNA sequencing, we report the detailed transcriptomes of fetal and adult α- and β-cells. We observed that human islet composition was not influenced by age, sex, or BMI, and transcripts for inflammatory gene products were noted in fetal β-cells. In addition, within highly purified adult glucagon-expressing α-cells, we observed surprisingly high insulin mRNA expression, but not insulin protein expression. This transcriptome analysis from highly purified islet α- and β-cell subsets from fetal and adult pancreata offers clear implications for strategies that seek to increase insulin expression in type 1 and type 2 diabetes.

A major therapeutic goal for type 1 diabetes (T1D) is to induce autoantigen-specific tolerance of T cells. This could suppress autoimmunity in those at risk for the development of T1D, as well as in those with established disease who receive islet replacement or regeneration therapy. Because functional studies of human autoreactive T cell responses have been limited largely to peripheral blood–derived T cells1–3, it is unclear how representative the peripheral T cell repertoire is of T cells infiltrating the islets. Our knowledge of the insulitic T cell repertoire is derived from histological and immunohistochemical analyses of insulitis4–8, the identification of autoreactive CD8+ T cells in situ, in islets of human leukocyte antigen (HLA)-A2+ donors9 and isolation and identification of DQ8 and DQ2–DQ8 heterodimer–restricted, proinsulin-reactive CD4+ T cells grown from islets of a single donor with T1D10. Here we present an analysis of 50 of a total of 236 CD4+ and CD8+ T cell lines grown from individual handpicked islets or clones directly sorted from handpicked, dispersed islets from nine donors with T1D. Seventeen of these T cell lines and clones reacted to a broad range of studied native islet antigens and to post-translationally modified peptides. These studies demonstrate the existence of a variety of islet-infiltrating, islet-autoantigen reactive T cells in individuals with T1D, and these data have implications for the design of successful immunotherapies.

Many patients with type 1 diabetes (T1D) have residual β 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 β cells and α 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 β cells appeared to maintain several aspects of regulated insulin secretion. However, the function of T1D α cells was markedly reduced, and these cells had alterations in transcription factors constituting α and β 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 α-to-β cell conversion. These results suggest an explanation for the disordered T1D counterregulatory glucagon response to hypoglycemia.

Type 1 diabetes studies consistently generate data showing islet b-cell dysfunction and T cell-mediated anti-b-cell-specific autoimmunity. To explore the pathogenesis, we interrogated the b-cell transcriptomes from donors with and without type 1 diabetes using both bulk-sorted and single b-cells. Consistent with immunohistological studies, b-cells from donors with type 1 diabetes displayed increased Class I transcripts and associated mRNA species. These b-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 2 b-cells, specifically found in islets with lymphocytic infiltrates. b-Cell surface expression of HLA Class II was detected on a portion of CD45 2 insulin + b-cells from donors with type 1 diabetes by immunofluorescence and flow cytometry. Our data demonstrate that pancreatic b-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. b-Cell expression of Class II molecules suggests that b-cells may interact directly with islet-infiltrating CD4 + T cells and may play an immunopathogenic role. The immune system plays a critical role in human type 1 diabetes pathogenesis. Varying proportions of T-cell subsets (CD8 + and CD4 +) and B cells infiltrate the pancreatic islets (1) and target b-cells by recognizing type 1 diabetes-associated autoantigens (2,3). The immunological mechanisms recruiting these cells to the islets had remained incompletely understood because, until recently, islets from donors with type 1 diabetes were not available for study. Antigen presentation to T cells is mediated by antigen-presenting cells (APCs) via two classes of HLA molecules: HLA Class I, recognized by CD8 +-expressing T cells (Class I is present on nearly all nucleated cells), and