The process of human islet isolation triggers a cascade of stressful events in the islets of Langerhans involving activation of apoptosis and necrosis and the production of proinflammatory molecules that negatively influence islet yield and function and may produce detrimental effects after islet transplantation. In this study, we showed that activation of nuclear factor-B (NF-B) and poly(ADP-ribose) polymerase (PARP), two of the major pathways responsible for cellular responses to stress, already occurs in pancreatic cells during the isolation procedure. NF-B؊dependent reactions, such as production and release of interleukin-6 and -8 and macrophage chemoattractant protein 1, were observed days after the isolation procedure in isolated purified islets. Under culture conditions specially designed to mimic isolation stress, islet proinflammatory responses were even more pronounced and correlated with higher islet cell loss and impaired secretory function. Here we present novel evidence that early interventions aimed at reducing oxidative stress of pancreatic cells and islets through the use of the catalytic antioxidant probe AEOL10150 (manganese [III] 5,10,15,20-tetrakis [1,3,-diethyl-2imidazoyl] manganese-porphyrin pentachloride [TDE-2,5-IP]) effectively reduces NF-B binding to DNA, the release of cytokines and chemokines, and PARP activation in islet cells, resulting in higher survival and better insulin release. These findings support the concept that the isolation process predisposes islets to subsequent damage and functional impairment. Blocking oxidative stress can be beneficial in reducing islet vulnerability and can potentially have a significant impact on transplantation outcome.

Successful Ag activation of naive T helper cells requires at least two signals consisting of TCR and CD28 on the T cell interacting with MHC II and CD80/CD86, respectively, on APCs. Recent evidence demonstrates that a third signal consisting of proinflammatory cytokines and reactive oxygen species (ROS) produced by the innate immune response is important in arming the adaptive immune response. In an effort to curtail the generation of an Ag-specific T cell response, we targeted the synthesis of innate immune response signals to generate Ag-specific hyporesponsiveness. We have reported that modulation of redox balance with a catalytic antioxidant effectively inhibited the generation of third signal components from the innate immune response (TNF-α, IL-1β, ROS). In this study, we demonstrate that innate immune-derived signals are necessary for adaptive immune effector function and disruption of these signals with in vivo CA treatment conferred Ag-specific hyporesponsiveness in BALB/c, NOD, DO11.10, and BDC-2.5 mice after immunization. Modulating redox balance led to decreased Ag-specific T cell proliferation and IFN-γ synthesis by diminishing ROS production in the APC, which affected TNF-α levels produced by CD4+ T cells and impairing effector function. These results demonstrate that altering redox status can be effective in T cell-mediated diseases such as autoimmune diabetes to generate Ag-specific immunosuppression because it inhibits the third signal necessary for CD4+ T cells to transition from expansion to effector function.

Type 1 diabetes (T1D) is an autoimmune disease characterized by pancreatic β cell destruction induced by islet reactive T cells that have escaped central tolerance. Many physiological and environmental triggers associated with T1D result in β cell endoplasmic reticulum (ER) stress and dysfunction, increasing the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress induced by environmental and physiological conditions generates abnormally-modified proteins for the T1D autoimmune response. To test this hypothesis we exposed the murine CD4+ diabetogenic BDC2.5 T cell clone to murine islets in which ER stress had been induced chemically (Thapsigargin). The BDC2.5 T cell IFNγ response to these cells was significantly increased compared to non-treated islets. This β cell ER stress increased activity of the calcium (Ca2+)-dependent PTM enzyme tissue transglutaminase 2 (Tgase2), which was necessary for full stress-dependent immunogenicity. Indeed, BDC2.5 T cells responded more strongly to their antigen after its modification by Tgase2. Finally, exposure of non-antigenic murine insulinomas to chemical ER stress in vitro or physiological ER stress in vivo caused increased ER stress and Tgase2 activity, culminating in higher BDC2.5 responses. Thus, β cell ER stress induced by chemical and physiological triggers leads to β cell immunogenicity through Ca2+-dependent PTM. These findings elucidate a mechanism of how β cell proteins are modified and become immunogenic, and reveal a novel opportunity for preventing β cell recognition by autoreactive T cells.

Galactose-␣1,3-galactose (␣Gal) epitopes, the synthesis of which requires the enzyme product of ␣1,3-galactosyltransferase (␣1,3GT), are sugar chains on the cell surface of most mammalian species. Notable exceptions are higher primates including Old World monkeys, apes, and humans. The ␣Gal-negative species as well as mice with deletion of the ␣1,3GT gene produce abundant anti␣Gal antibodies. The evolutionary loss of ␣Gal epitopes has been attributed to point mutations in the coding region of the gene. Because no transcripts could be found in the higher primate species with Northern blot analysis, a potential alternative explanation has been loss of upstream regulation of the gene. Here, we have demonstrated that the rhesus promoter is functional. More importantly, a variety of full-length transcripts were detected with sensitive PCR-based methods in the tissues of rhesus monkeys, orangutans, and humans. Five crucial mutations were delineated in the coding region of the human and rhesus and three in the orangutan, any one of which could be responsible for inactivation of the ␣1,3GT gene. Two of the mutations were shared by all three higher primates. These findings, which elucidate the molecular basis for the evolutionary loss of ␣Gal expression, may have implications in medical research.Most mammals express the cell surface carbohydrate epitope galactose-␣1,3-galactose (␣Gal), 1 with notable exceptions that include Old World monkeys, apes, and humans (1). With the loss of the ␣Gal epitopes, the synthesis of which is dependent on enzyme product of the ␣1,3-galactosyltransferase (␣1,3GT) gene, higher primates produce anti-␣Gal antibodies (2) that are responsible for the hyperacute rejection of organs transplanted from ␣Gal-positive donors (3).In 1989, Joziasse et al. (4) reported the sequence of fulllength cDNA clone of the bovine ␣1,3GT gene and demonstrated the presence of this gene in the DNA of human cell lines. Using an 804-bp fragment derived from bovine fulllength cDNA as a probe, they also detected mRNA transcripts in bovine and marmoset (New World monkey) but not in human or African green monkey cell lines (4). The absence of ␣1,3GT mRNA has since been widely viewed as a feature of all Old World monkeys, apes, and humans (5, 6). The molecular basis for the inactivation of the ␣1,3GT gene in the ␣Gal-negative species has been attributed to a mutation(s) localized to a partial sequence of exon 9 (4, 5, 7).Stimulated by the current interest in producing transgenic pigs for clinical use as tissue and organ xenograft donors, the cDNA for the ␣1,3GT gene of the ␣Gal-positive pig was isolated (8), and the coding regions were characterized (9 -12). Further, the full genomic organization of the porcine ␣1,3GT, gene including its upstream regulatory region, was completed (12). A CpG island (i.e. a C connected by a 3Ј-5Ј phosphodiester bond to a G) characteristic of housekeeping genes was observed around exon 1 of the pig ␣1,3GT gene (12). It is of interest that a CpG island was not present in the mouse gene (13-15...

Islet cell autoantigen 69-kDa (ICA69), protein product of the human ICA1 gene, is one target of the immune processes defining the pathogenesis of Type 1 diabetes. We have characterized the genomic structure and functional promoters within the 5-regulatory region of ICA1. 5-RNA ligase-mediated rapid amplification of cDNA ends evaluation of ICA1 transcripts expressed in human islets, testis, heart, and cultured neuroblastoma cells reveals that three 5-untranslated region exons are variably expressed from the ICA1 gene in a tissue-specific manner. Surrounding the transcription initiation sites are motifs characteristic of non-TATA, non-CAAT, GC-rich promoters, including consensus Sp1/GC boxes, an initiator element, cAMP-responsive element-binding protein (CREB) sites, and clusters of other putative transcription factor sites within a genomic CpG island. Luciferase reporter constructs demonstrate that the first two ICA1 exon promoters reciprocally stimulate luciferase expression within islet-(RIN 1046-38 cells) and brain-derived (NMB7) cells in culture; the exon A promoter exhibits greater activity in islet cells, whereas the exon B promoter more efficiently activates transcription in neuronal cells. Mutation of a CREB site within the ICA1 exon B promoter significantly enhances transcriptional activity in both cell lines. Our basic understanding of expression from the functional core promoter elements of ICA1 is an important advance that will not only add to our knowledge of the ICA69 autoantigen but will also facilitate a rational approach to discover the function of ICA69 and to identify relevant ICA1 promoter polymorphisms and their potential associations with disease.

Type 1 diabetes (T1D) is an autoimmune disease in which autoreactive T cells target and destroy islet β cells. The events that break peripheral tolerance in patients genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D cause endoplasmic reticulum (ER) stress, which may increase abnormal protein post-translational modification (PTM). We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells in T1D. Chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased their recognition by diabetogenic BDC2.5 T cells (28-1500 fold) through activation of the PTM enzyme tissue transglutaminase 2 (Tgase; 18 fold). Indeed, reduced Tgase expression decreased stress-induced immunogenicity (58%). Also, physiological conditions in vivo increased ER stress (300 fold) and Tgase activity (44 fold) in primary islets and transplanted insulinomas, increasing BDC2.5 IFNγ responses (22 fold). Thus, ER stress leads to PTM-dependent murine β cell immunogenicity. We are currently translating these findings to a human T1D model. Preliminary data confirm that ER stress in human β cells significantly increased IFNγ secretion by T cells isolated from T1D patients that recognize Tgase-modified β cell antigens (24-360 fold). Thus, Tgase-dependent PTM may also cause immunogenicity in human β cells, demonstrating the relevance of our murine studies to human disease.

Type 1 diabetes (T1D) is an autoimmune disease, largely mediated by autoreactive T cells, in which islet β cells are destroyed. The events that break peripheral tolerance in individuals genetically predisposed to autoimmunity are poorly understood. Many physiological and environmental triggers associated with T1D, such as viral infection, chemicals, reactive oxygen species, and dynamic glucose sensing, result in endoplasmic reticulum (ER) stress. ER stress increases the potential for abnormal post-translational modification (PTM) of proteins. We hypothesized that β cell ER stress generates neo-antigens that activate autoreactive T cells and worsen T1D. Indeed, chemical (Thapsigargin) induction of ER stress in murine islets or insulinomas increased IFNγ secretion from diabetogenic BDC2.5 T cells (28-1500 fold). Chelation of stress-induced calcium (Ca2+) flux reduced this response (27-64%), supporting a role for Ca2+-dependent enzymes such as tissue transglutaminase 2 (Tgase). Tgase activity is higher (18 fold) in β cells under stress, and reduced Tgase expression decreased the BDC2.5 response to β cells under stress (58%). Finally, nonantigenic insulinomas were exposed to normal physiological conditions in vivo. At harvest, these cells had increased ER stress (300 fold), increased Tgase activity (44 fold), and elicited higher BDC2.5 IFNγ responses (22 fold) compared to cultured cells. Thus, β cell ER stress causes formation of neo-antigens that activate autoreactive T cells.