Type 1 diabetes (T1D) is an autoimmune disease characterized by the infiltration of lymphocytes into the insulin-producing β-cells in the pancreas. We have isolated live T cells sorted or grown directly from the isolated, handpicked islets of human donors with T1D. We received ~500 islet equivalent EQ of variable purity (10-90%) from 12 donors with T1D (disease duration 0.42-20 years) and from seven control donors and two donors with type 2 diabetes (T2D). A total of 321 T cell lines and clones were derived from the islets of donors with T1D (3 lines from the 9 control donors). These are 131 CD4+ lines and clones, 47 CD8+ lines and 143 lines that contain both CD4+ and CD8+ T cells. From 50 lines and clones examined to date, we have determined the autoreactivity of 19 and have seen a broad repertoire of T cell autoreactivity in the islets, including characterized targets and post-translationally modified targets. Autoreactivity of CD4+ T cell lines was to three different peptides from glutamic acid decarboxylase 65 (GAD; GAD115-127, GAD274-286, GAD555-567), proinsulin76-90, and to chromogranin A or proinsulin expressed by DR4+DQ8+ B cells transduced with lentivirus containing constructs with the open reading frames corresponding to whole autoantigens. Reactivity to modified peptides included the glucose-regulated protein 78 and islet amyloid polypeptide with arginine to citrulline modifications (GRP78292-305(Arg-Cit297) and IAPP65-84(Arg-Cit 73, 81)), deaminations (IA-2545-562(Gln-Glu 548, 551, 556), and to several insulin hybrid peptides. These autoreactive CD4+ T cell lines and clones secreted only pro-inflammatory cytokines (IFN-γ, TNFα) upon peptide stimulation. For CD8+ T cells from islets, from one donor with T1D, we saw binding of a pool of HLA-A2 pentamers loaded with insulin B10-18, IA-2797-805 and insulin specific glucose-6-phosphatase catalytic subunit related protein, IGRP265-273. These results have implications for the development of successful prevention and reversal therapeutic strategies in T1D.

OBJECTIVE: Glucagon-like peptide-1 induces glucose-dependent insulin secretion and, in rodents, increases proliferation and survival of pancreatic beta cells. To investigate the effects on human beta cells, we used immunodeficient mice transplanted with human islets. The goal was to determine whether lixisenatide, a glucagon-like peptide-1 receptor agonist, improves human islet function and survival in vivo. METHODS: Five independent transplant studies were conducted with human islets from five individual donors. Diabetic human islet-engrafted immunodeficient mice were treated with lixisenatide (50, 150, and 500 microg/kg) or vehicle. Islet function was determined by blood glucose, plasma human insulin/C-peptide, and glucose tolerance tests. Grafts were analyzed for total beta- and alpha-cell number, percent proliferation, and levels of apoptosis. RESULTS: Diabetic mice transplanted with marginal human islet mass and treated with lixisenatide were restored to euglycemia more rapidly than vehicle-treated mice. Glucose tolerance tests, human plasma insulin, and glucose-stimulation indices of lixisenatide-treated mice were significantly improved compared to vehicle-treated mice. The percentages of proliferating or apoptotic beta cells at graft recovery were not different between lixisenatide-treated and vehicle-treated mice. Nevertheless, in one experiment we found a significant twofold to threefold increase in human beta-cell numbers in lixisenatide-treated compared to vehicle-treated mice. CONCLUSION: Diabetic human islet-engrafted immunodeficient mice treated with lixisenatide show improved restoration of normoglycemia, human plasma insulin, and glucose tolerance compared to vehicle-treated mice engrafted with the same donor islets. Because the proliferative capacity of human beta cells is limited, improved beta-cell survival coupled with enhanced beta-cell function following lixisenatide treatment may provide the greatest benefit for diabetic patients with reduced functional islet mass.

PURPOSE OF REVIEW: beta Cells represent one of many cell types in heterogeneous pancreatic islets and play the central role in maintaining glucose homeostasis, such that disrupting beta-cell function leads to diabetes. This review summarizes the methods for isolating and characterizing beta cells, and describes integrated 'omics' approaches used to define the beta cell by its transcriptome and proteome. RECENT FINDINGS: RNA sequencing and mass spectrometry-based protein identification have now identified RNA and protein profiles for mouse and human pancreatic islets and beta cells, and for beta-cell lines. Recent publications have outlined these profiles and, more importantly, have begun to assign the presence or absence of specific genes and regulatory molecules to beta-cell function and dysfunction. Overall, researchers have focused on understanding the pathophysiology of diabetes by connecting genome, transcriptome, proteome, and regulatory RNA profiles with findings from genome-wide association studies. SUMMARY: Studies employing these relatively new techniques promise to identify specific genes or regulatory RNAs with altered expression as beta-cell function begins to deteriorate in the spiral toward the development of diabetes. The ultimate goal is to identify the potential therapeutic targets to prevent beta-cell dysfunction and thereby better treat the individual with diabetes. VIDEO ABSTRACT: http://links.lww.com/COE/A5.

Abstract Pancreatic endocrine cells are co-located into clusters called the islets of Langerhans that are comprised of glucagon producing alpha cells, insulin secreting beta cells, somatostatin generating delta cells, and other cell types. Type 1 diabetes results from an autoimmune process in which autoreactive T cells destroy the insulin producing beta cells, requiring the patient to inject insulin to regulate their blood glucose levels. Thus far, attempts to cure diabetes via islet transplantation have been limited by insufficient donor supply, inconsistent isolated islet quality, continued autoimmunity, alloimmune rejection, and limited beta cell regeneration. Diabetes research has focused on preventing the autoimmune response, promoting stem cell to beta cell differentiation, and defining the factors that influence beta cell proliferation. Islet research, in turn, has been limited to whole islet studies since, isolating the islet cell subtypes has not been possible. Using a method recently developed for mouse islet cells (Pechhold et al. Nat Biotechnol. 2009 Nov; 27(11):1038-42), that uses intracellular hormone staining and flow cytometry, we are able to sort human islets into populations uniquely expressing glucagon, insulin, or somatostatin. Further, we have developed a human gene array to measure candidate gene expression using a quantitative nuclease protection assay (qNPA). This technique uses 50 base oligomers that specifically recognize RNA from each gene of interest, overcoming limitations caused by the harsh conditions required for intracellular staining. We report gene expression analysis for specific hormones and transcription factors expressed in each islet cell population. We are further modifying this technique to study nonhuman primate islets, and investigate the specific proteome and miRNA profiles for individual islet cell populations. The goal of these studies is to characterize the genetic differences between the islet cell populations and understand which factors control beta cell regeneration and proliferation. We have shown that we can purify adult human islets into individual cellular populations. This is the first step in understanding the genetic and environmental components that regulate increased beta cell proliferation and beta cell mass. In the absence of full-length mRNA for RT-PCR or next generation sequencing, the qNPA technique provides candidate gene expression profiles for these cells.