SKCa activation drives the fate of pluripotent cells toward mesoderm commitment and cardiomyocyte specification, preferentially into nodal-like cardiomyocytes. This provides a novel strategy for the enrichment of cardiomyocytes and in particular, the generation of a specific subtype of cardiomyocytes, pacemaker-like cells, without genetic modification.

OBJECTIVERIP-B7.1 mice expressing the costimulator molecule B7.1 (CD80) on pancreatic β-cells are a well established model to characterize preproinsulin-specific CD8 T-cell responses and experimental autoimmune diabetes (EAD). Different immunization strategies could prime preproinsulin-specific CD8 T-cells in wild-type C57BL/6 (B6) mice, but did not induce diabetes. We tested whether altering the B7-H1 (PD-L1) coinhibition on pancreatic β-cells can reveal a diabetogenic potential of preproinsulin-specific CD8 T-cells.RESEARCH DESIGN AND METHODSDNA-based immunization and adoptive T-cell transfers were used to characterize the induction of preproinsulin-specific CD8 T-cell responses and EAD in RIP-B7.1, B6, B7-H1−/−, PD-1−/− or bone marrow chimeric mice.RESULTSPreproinsulin-specific CD8 T-cells primed in B6 mice revealed their diabetogenic potential after adoptive transfer into congenic RIP-B7.1 hosts. Furthermore, preproinsulin-specific CD8 T-cells primed in anti-B7-H1 antibody-treated B6 mice, or primed in B7-H1−/− or PD-1−/− mice induced EAD. Immunization of bone marrow chimeric mice showed that deficiency of either B7-H.1 in pancreatic β-cells or of PD-1 in autoreactive CD8 T-cells induced EAD.CONCLUSIONSAn imbalance between costimulator (B7.1) and coinhibitor (B7-H1) signals on pancreatic β-cells can trigger pancreatic β-cell-destruction by preproinsulin-specific CD8 T-cells. Hence, regulation of the susceptibility of the β-cells for a preproinsulin-specific CD8 T-cell attack can allow or suppress EAD.

RIP-B7.1 mice express the costimulator molecule B7.1 (CD80) on pancreatic β cells and are a well-established model for studying de novo induction of diabetogenic CD8 T cells. Immunization of RIP-B7.1 mice with preproinsulin (ppins)-encoding plasmid DNA efficiently induces experimental autoimmune diabetes (EAD). EAD is associated with an influx of CD8 T cells specific for the Kb/A12–21 epitope into the pancreatic islets and the subsequent destruction of β cells. In this study, we used this model to investigate how ppins-derived Ags are expressed and processed to prime diabetogenic, Kb/A12–21-specific CD8 T cells. Targeting the Kb/A12–21 epitope, the insulin A chain, or the ppins to the endoplasmic reticulum (ER) (but not to the cytosol and/or nucleus) efficiently elicited Kb/A12–21-specific CD8 T cell responses. The Kb/A12–21 epitope represents the COOH terminus of the ppins molecule and, hence, did not require COOH-terminal processing before binding its restriction element in the ER. However, Kb/A12–21-specific CD8 T cells were also induced by COOH-terminally extended ppins-specific polypeptides expressed in the ER, indicating that the epitope position at the COOH terminus is less important for its diabetogenicity than is targeting the Ag to the ER. The Kb/A12–21 epitope had a low avidity for Kb molecules. When epitopes of unrelated Ags were coprimed at the same site of Ag delivery, “strong” Kb-restricted (but not Db-restricted) CD8 T cell responses led to the suppression of Kb/A12–21-specific CD8 T cell priming and reduced EAD. Thus, direct expression and processing of the “weak” Kb/A12–21 epitope in the ER favor priming of autoreactive CD8 T cells.

Type 1 diabetes mellitus can result from the specific destruction of pancreatic beta cells by autoreactive T cells. As shown here, experimental autoimmune diabetes (EAD) is efficiently induced in RIP-B7.1 mice by preproinsulin (ppins)-encoding DNA vaccines. EAD develops in RIP-B7.1 mice within 3-4 wk after a single immunization with ppinsencoding plasmid DNA. RIP-B7.1 mice develop insulitis, insulin deficiency and hyperglycemia after vaccination with plasmids encoding murine ppins-I or murine ppins-II or human hu-ppins. EAD induction critically depends on CD8 T cells and is independent of CD4 T cells. To be diabetogenic, ppins-specific CD8 T cells had to express IFN-c. Neither expression of perforin nor signaling through the type I IFN receptor is an essential component of this pathogenic CD8 T cell phenotype. Using plasmids encoding truncated ppins variants, we show that EAD is only induced by DNA vaccines encoding the insulin A-chain. Diabetogenic CD8 T cells specifically recognize the K b -restricted A 12-21 epitope of the insulin A-chain. The RIP-B7.1 model hence represents an attractive model for the characterization of cellular and molecular events involved in the CD8 T cell-mediated immune pathogenesis of diabetes.

Aims/hypothesisA strategy to enhance pancreatic islet functional beta cell mass (BCM) while restraining inflammation, through the manipulation of molecular and cellular targets, would provide a means to counteract the deteriorating glycaemic control associated with diabetes mellitus. The aims of the current study were to investigate the therapeutic potential of such a target, the islet-enriched and diabetes-linked transcription factor paired box 4 (PAX4), to restrain experimental autoimmune diabetes (EAD) in the RIP-B7.1 mouse model background and to characterise putative cellular mechanisms associated with preserved BCM.MethodsTwo groups of RIP-B7.1 mice were genetically engineered to: (1) conditionally express either PAX4 (BPTL) or its diabetes-linked mutant variant R129W (mutBPTL) using doxycycline (DOX); and (2) constitutively express luciferase in beta cells through the use of RIP. Mice were treated or not with DOX, and EAD was induced by immunisation with a murine preproinsulin II cDNA expression plasmid. The development of hyperglycaemia was monitored for up to 4 weeks following immunisation and alterations in the BCM were assessed weekly by non-invasive in vivo bioluminescence intensity (BLI). In parallel, BCM, islet cell proliferation and apoptosis were evaluated by immunocytochemistry. Alterations in PAX4- and PAX4R129W-mediated islet gene expression were investigated by microarray profiling. PAX4 preservation of endoplasmic reticulum (ER) homeostasis was assessed using thapsigargin, electron microscopy and intracellular calcium measurements.ResultsPAX4 overexpression blunted EAD, whereas the diabetes-linked mutant variant PAX4R129W did not convey protection. PAX4-expressing islets exhibited reduced insulitis and decreased beta cell apoptosis, correlating with diminished DNA damage and increased islet cell proliferation. Microarray profiling revealed that PAX4 but not PAX4R129W targeted expression of genes implicated in cell cycle and ER homeostasis. Consistent with the latter, islets overexpressing PAX4 were protected against thapsigargin-mediated ER-stress-related apoptosis. Luminal swelling associated with ER stress induced by thapsigargin was rescued in PAX4-overexpressing beta cells, correlating with preserved cytosolic calcium oscillations in response to glucose. In contrast, RNA interference mediated repression of PAX4-sensitised MIN6 cells to thapsigargin cell death.Conclusions/interpretationThe coordinated regulation of distinct cellular pathways particularly related to ER homeostasis by PAX4 not achieved by the mutant variant PAX4R129W alleviates beta cell degeneration and protects against diabetes mellitus. The raw data for the RNA microarray described herein are accessible in the Gene Expression Omnibus database under accession number GSE62846.Electronic supplementary materialThe online version of this article (doi:10.1007/s00125-016-3864-0) contains peer-reviewed but unedited supplementary material, which is available to authorised users.