In situ tetramer staining reveals the presence of islet antigen-reactive CD8+ T cells in pancreatic islets from deceased type 1 diabetes patients.

Type 1 diabetes is a T-cell-mediated disease that is associated with loss of immunological tolerance to selfantigens. The mechanisms involved in maintenance of peripheral tolerance include a specialized subset of regulatory T-cells (Treg) within the CD4 ؉ CD25 ؉ T-cell population, but the function and phenotype of these cells in type 1 diabetes have not been investigated. We hypothesized that a deficiency in the CD4 ؉ CD25 ؉ Treg population or its function could contribute to the lack of self-tolerance evident in patients with type 1 diabetes. We show that although levels of CD4 ؉ CD25 ؉ T-cells are normal in patients with recent-onset adult type 1 diabetes, the ability of the Tregs in this population to suppress T-cell proliferation during in vitro cocultures is markedly reduced compared with control subjects (P ‫؍‬ 0.007). Moreover, in patients with type 1 diabetes, these cocultures display a more proinflammatory phenotype, with increased secretion of interferon-␥ (P ‫؍‬ 0.005) and decreased interleukin-10 production (P ‫؍‬ 0.03). These deficiencies may reflect a disturbance in the balance of the CD4 ؉ CD25 ؉ population, because in patients with type 1 diabetes, a higher proportion of these cells coexpress the early activation marker CD69 (P ‫؍‬ 0.007) and intracellular CTLA-4 (P ‫؍‬ 0.01). These data demonstrate deficiency in function of the CD4 ؉ CD25؉ Treg population that may influence the pathogenesis of type 1 diabetes. Diabetes 54:92-99, 2005

Type 1 diabetes is characterized by T cell-mediated autoimmune destruction of pancreatic ␤ cells. Several studies have suggested an association between Coxsackie enterovirus seroconversion and onset of disease. However, a direct link between ␤ cell viral infection and islet inflammation has not been established. We analyzed pancreatic tissue from six type 1 diabetic and 26 control organ donors. Immunohistochemical, electron microscopy, wholegenome ex vivo nucleotide sequencing, cell culture, and immunological studies demonstrated Coxsackie B4 enterovirus in specimens from three of the six diabetic patients. Infection was specific of ␤ cells, which showed nondestructive islet inflammation mediated mainly by natural killer cells. Islets from enterovirus-positive samples displayed reduced insulin secretion in response to glucose and other secretagogues. In addition, virus extracted from positive islets was able to infect ␤ cells from human islets of nondiabetic donors, causing viral inclusions and signs of pyknosis. None of the control organ donors showed signs of viral infection. These studies provide direct evidence that enterovirus can infect ␤ cells in patients with type 1 diabetes and that infection is associated with inflammation and functional impairment.Coxsackie B4 enterovirus ͉ type 1 diabetes

The final pathway of β cell destruction leading to insulin deficiency, hyperglycemia, and clinical type 1 diabetes is unknown. Here we show that circulating CTLs can kill β cells via recognition of a glucose-regulated epitope. First, we identified 2 naturally processed epitopes from the human preproinsulin signal peptide by elution from HLA-A2 (specifically, the protein encoded by the A*0201 allele) molecules. Processing of these was unconventional, requiring neither the proteasome nor transporter associated with processing (TAP). However, both epitopes were major targets for circulating effector CD8 + T cells from HLA-A2 + patients with type 1 diabetes. Moreover, cloned preproinsulin signal peptide-specific CD8 + T cells killed human β cells in vitro. Critically, at high glucose concentration, β cell presentation of preproinsulin signal epitope increased, as did CTL killing. This study provides direct evidence that autoreactive CTLs are present in the circulation of patients with type 1 diabetes and that they can kill human β cells. These results also identify a mechanism of self-antigen presentation that is under pathophysiological regulation and could expose insulin-producing β cells to increasing cytotoxicity at the later stages of the development of clinical diabetes. Our findings suggest that autoreactive CTLs are important targets for immune-based interventions in type 1 diabetes and argue for early, aggressive insulin therapy to preserve remaining β cells.

Specific therapy with modulated DC may restore immunological tolerance, thereby obviating the need for chronic immunosuppression in transplantation or autoimmunity. In this study we compared the tolerizing capacity of dexamethasone (Dex)-and 1a,25-dihydroxyvitamin D3 (VD3)-modulated DC. Treatment of monocytes with either VD3 or Dex resulted in DC with stable, semi-mature phenotypes compared with standard DC, with intermediate levels of co-stimulatory and MHC class II molecules, which remained unaltered after subsequent pro-inflammatory stimulation. IL-12p70 secretion was lost by VD3-and Dex-DC, whereas IL-10 secretion was unaffected. VD3-DC distinctly produced large amounts of TNF-a. Both VD3-and Dex-DC possessed the capacity to convert CD4 T cells into IL-10-secreting Treg potently suppressing the proliferation of responder T cells. However, only Treg induced by VD3-DC exhibited antigen specificity. VD3-, but not Dex-, DC expressed significant high levels of PD-L1 (programmed death-1 ligand), upon activation. Blockade of PD-L1 during priming redirected T cells to produce IFN-c instead of IL-10 and abolished acquisition of regulatory capacity. Our findings demonstrate that both VD3-and Dex-DC possess durable but differential tolerogenic features, acting via different mechanisms. Both are potentially useful to specifically down-regulate unwanted immune responses and induce immune tolerance. These modulated DC appear suitable as adjuvant in antigen-specific clinical vaccination intervention strategies.Key words: Antigen specificity . Autoimmunity . Modulated DC . PD-L1 . Treg IntroductionRestoring immunological tolerance is the ''holy grail'' in the fields of autoimmunity and transplantation. Current applied therapies, which include immunosuppressive drugs, do not target the cause of the disease and, in addition, are associated with considerable non-specific side effects. Therefore, it is desirable to design therapies that specifically target the immunopathogenesis.DC are important modulators of T-cell activity [1]. Depending on the type of pathogen encountered and the profile of co-stimulatory and T-cell polarizing molecules, DC drive the development of either pro-inflammatory Th type 1 (Th1), type 2 (Th2) and type 17 (Th17) cells or protective Treg [2][3][4]. In conjunction with this, human monocyte-derived DC (moDC) can be differentiated into Th1-, Th2-, Th17-or Treg-promoting DC in vitro. Priming of moDC with microbial compounds or tissue-derived factors such as IFN-g, prostaglandin E2 (PGE2) or IL-23 and IL-1 results in enhanced expression of MHC class II and co-stimulatory molecules and drives the development of effector Th1, Th2 and Th17 cells [5][6][7]. It is now clear that certain immunosuppressive drugs and anti-inflammatory agents induce DC with tolerogenic properties [8][9][10][11][12][13][14][15]. For example, DC treated with either dexamethasone (Dex) or the active form of vitamin D, 1a,25-dihydroxyvitamin D 3 (VD3), arrest DC in a semimature state and prevent the up-regulation of co-stimulatory Trea...

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Type 1 diabetes is a T cell-mediated autoimmune disease, and insulin is an important target of the autoimmune response associated with ␤ cell destruction. The mechanism of destruction is still unknown. Here, we provide evidence for CD8 T cell autoreactivity associated with recurrent autoimmunity and loss of ␤ cell function in type 1 diabetic islet transplant recipients. We first identified an insulin B chain peptide (insB10-18) with extraordinary binding affinity to HLA-A2(*0201) that is expressed by the majority of type 1 diabetes patients. We next demonstrated that this peptide is naturally processed by both constitutive and immuno proteasomes and translocated to the endoplasmic reticulum by the peptide transporter TAP1 to allow binding to HLA-A2 in the endoplasmic reticulum and cell surface presentation. Peripheral blood mononuclear cells from a healthy donor were primed in vitro with this peptide, and CD8 T cells were isolated that specifically recognize target cells expressing the insulin B chain peptide. HLA-A2 insB10-18 tetramer staining revealed a strong association between detection of autoreactive CD8 T cells and recurrent autoimmunity after islet transplantation and graft failure in type 1 diabetic patients. We demonstrate that CD8 T cell autoreactivity is associated with ␤ cell destruction in type 1 diabetes in humans.insulin ͉ islet transplantation ͉ cytotoxic T lymphocyte ͉ tetramer

SummaryT cell epitopes represent the molecular code words through which the adaptive immune system communicates. In the context of a T cell-mediated autoimmune disease such as type 1 diabetes, CD4 and CD8 T cell recognition of islet autoantigenic epitopes is a key step in the autoimmune cascade. Epitope recognition takes place during the generation of tolerance, during its loss as the disease process is initiated, and during epitope spreading as islet cell damage is perpetuated. Epitope recognition is also a potentially critical element in therapeutic interventions such as antigen-specific immunotherapy. T cell epitope discovery, therefore, is an important component of type 1 diabetes research, in both human and murine models. With this in mind, in this review we present a comprehensive guide to epitopes that have been identified as T cell targets in autoimmune diabetes. Targets of both CD4 and CD8 T cells are listed for human type 1 diabetes, for humanized [human leucocyte antigen (HLA)-transgenic] mouse models, and for the major spontaneous disease model, the non-obese diabetic (NOD) mouse. Importantly, for each epitope we provide an analysis of the relative stringency with which it has been identified, including whether recognition is spontaneous or induced and whether there is evidence that the epitope is generated from the native protein by natural antigen processing. This analysis provides an important resource for investigating diabetes pathogenesis, for developing antigenspecific therapies, and for developing strategies for T cell monitoring during disease development and therapeutic intervention.