Type 1 diabetes (T1D) develops when insulin-secreting b-cells, found in the pancreatic islets of Langerhans, are destroyed by infiltrating T cells. How human T cells recognize b-cell-derived antigens remains unclear. Genetic studies have shown that HLA and insulin alleles are the most strongly associated with risk of T1D. These longstanding observations implicate CD4 + T-cell responses against (pro)insulin in the pathogenesis of T1D. To dissect the autoimmune T-cell response against human b-cells, we isolated and characterized 53 CD4 + T-cell clones from within the residual pancreatic islets of a deceased organ donor who had T1D. These 53 clones expressed 47 unique clonotypes, 8 of which encoded proinsulin-specific T-cell receptors. On an individual clone basis, 14 of 53 CD4 + T-cell clones (26%) recognized 6 distinct but overlapping epitopes in the C-peptide of proinsulin. These clones recognized C-peptide epitopes presented by HLA-DQ8 and, notably, HLA-DQ8 transdimers that form in HLA-DQ2/-DQ8 heterozygous individuals. Responses to these epitopes were detected in the peripheral blood mononuclear cells of some people with recent-onset T1D but not in HLAmatched control subjects. Hence, proinsulin-specific, HLA-DQ8, and HLA-DQ8-transdimer-restricted CD4 + T cells are strongly implicated in the autoimmune pathogenesis of human T1D.Type 1 diabetes (T1D) is an autoimmune disease caused by the CD4 + and CD8 + T-cell-mediated destruction of pancreatic insulin-producing b-cells (1). b-Cell destruction leads to primary insulin deficiency, which is treated by exogenous insulin therapy, and currently there is no cure. The pathogenesis of T1D has been well characterized using the NOD mouse model, but the immune basis of T1D in humans is less clear.Genetic association studies have provided powerful insights into the etiology of human T1D (2,3). The HLA class II region has the strongest impact on risk of T1D. Some HLA alleles-DQB1*06:02 for example-dominantly protect against T1D (4). In contrast, of all alleles, HLA-DQ2

Type 1 diabetes (T1D) is an autoimmune disease in which insulin-producing beta cells, found within the islets of Langerhans in the pancreas, are destroyed by islet-infiltrating T cells. Identifying the antigenic targets of beta-cell reactive T cells is critical to gain insight into the pathogenesis of T1D and develop antigen-specific immunotherapies. Several lines of evidence indicate that insulin is an important target of T cells in T1D. Because many human islet-infiltrating CD4+ T cells recognize C-peptide–derived epitopes, we hypothesized that full-length C-peptide (PI33–63), the peptide excised from proinsulin as it is converted to insulin, is a target of CD4+ T cells in people with T1D. CD4+ T cell responses to full-length C-peptide were detected in the blood of: 14 of 23 (>60%) people with recent-onset T1D, 2 of 15 (>13%) people with long-standing T1D, and 1 of 13 (<8%) HLA-matched people without T1D. C-peptide–specific CD4+ T cell clones, isolated from six people with T1D, recognized epitopes from the entire 31 amino acids of C-peptide. Eighty-six percent (19 of 22) of the C-peptide–specific clones were restricted by HLA-DQ8, HLA-DQ2, HLA-DQ8trans, or HLA-DQ2trans, HLA alleles strongly associated with risk of T1D. We also found that full-length C-peptide was a much more potent agonist of some CD4+ T cell clones than an 18mer peptide encompassing the cognate epitope. Collectively, our findings indicate that proinsulin C-peptide is a key target of autoreactive CD4+ T cells in T1D. Hence, full-length C-peptide is a promising candidate for antigen-specific immunotherapy in T1D.

Previous attempts of α-1,3-galactocyltransferase knockout (GalTKO) pig bone marrow (BM) transplantation (Tx) into baboons have demonstrated a loss of macro-chimerism within 24 h in most cases. In order to achieve improved engraftment with persistence of peripheral chimerism, we have developed a new strategy of intra-bone BM (IBBM) Tx. Six baboons received GalTKO BM cells, with one-half of the cells transplanted into the bilateral tibiae directly and the remaining cells injected intravenously (IBBM/BM-Tx) with a conditioning immunosuppressive regimen. In order to assess immune responses induced by the combined IBBM/BM-Tx, three recipients received donor SLA-matched GalTKO kidneys in the peri-operative period of IBBM/BM-Tx (Group 1), and the others received kidneys 2 months after IBBM/BM-Tx (Group 2). Peripheral macro-chimerism was continuously detectable for up to 13 days (mean 7.7 days; range 3–13) post-IBBM/BM-Tx and in three animals, macro-chimerism reappeared at days 10, 14 and 21. Pig CFUs, indicating porcine progenitor cell engraftment, were detected in the host BM in four of six recipients on days 14, 15, 19 and 28. In addition, anti-pig unresponsiveness was observed by in vitro assays. GalTKO/pCMV-kidneys survived for extended periods (47 and 60 days). This strategy may provide a potent adjunct for inducing xenogeneic tolerance through BM-Tx.

SummaryType 1 diabetes (T1D) develops when there are insufficient insulinproducing beta cells to maintain glucose homeostasis. The prevailing view has been that T1D is caused by immune-mediated destruction of the pancreatic beta cells. However, several recent papers have challenged the long-standing paradigm describing T1D as a tissue-specific autoimmune disease. These authors have highlighted the gaps in our knowledge and understanding of the aetiology of T1D in humans. Here we review the evidence and argue the case for the autoimmune basis of human T1D. In particular, recent analysis of human islet-infiltrating T cells brings important new evidence to this question. Further data in support of the autoimmune basis of T1D from many fields, including genetics, experimental therapies and immunology, is discussed. Finally, we highlight some of the persistent questions relating to the pathogenesis of human type 1 diabetes that remain to be answered.

The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035T>C and 4452T>C, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452T>C transition in tRNA(Met) represented a polymorphism; however, 9035T>C conversion in the ATP6 gene was responsible for a defective F(0)-ATPase. Accordingly, mutant cybrids had a reduced oligomycin-sensitive ATP hydrolyzing activity. They had less than half of the steady-state content of ATP and nearly an 8-fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with additional insults, i.e., glucose deprivation or tertiary-butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ(10) and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035T>C mutation and could be responsible for the development of clinical symptoms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease.

We have previously reported that transplantation (Tx) of prevascularized donor islets as composite Islet-Kidneys (IK) reversed diabetic hyperglycemia in both miniature swine and baboons. In order to enhance this strategy's potential clinical applicability, we have now combined this approach with hematopoietic stem cells (HSC) Tx in an attempt to induce tolerance in non-human primates. IKs were prepared by isolating islets from 70% partial pancreatectomies and injecting them beneath the autologous renal capsule of five rhesus monkey donors at least 3 months before allogeneic IKTx. HSCTx was performed following mobilization and leukapheresis of the donors, and conditioning of the recipients with total body irradiation, T-cell depletion and cyclosporine. One IK was harvested for histologic analysis and four were transplanted into diabetic recipients. IKTx was performed either 20–22 (n=3) or 208 (n=1) days after HSCTx. All animals accepted IKs without rejection. All recipients required >20 U/day of insulin before IKTx to maintain less than 200mg/dl, whereas after IKTx 3 animals required minimal doses of insulin (1–3 U/day) and one animal was insulin-free. These results constitute a proof-of-principle that this IK tolerance strategy may provide a cure for both end-stage renal disease and diabetes without the need for immunosuppression.

Diphtheria toxin (DT)-based anti-CD3 immunotoxins have clinical relevance in numerous applications including autoimmune disease therapies and organ transplantation tolerance protocols. Pre-existing anti-DT antibodies acquired either by vaccination against diphtheria toxin or infections with C. diphtheriae may interfere or inhibit the function of these anti-CD3 immunotoxins. Previously, a full-length anti-rhesus monkey CD3 immunotoxin, FN18-CRM9, was shown to be less effective at depleting circulating T cells in animals with pre-existing anti-DT antibody titers than in animals without antibodies, and subsequent doses were ineffective. In this study, the T cell depletion function of a truncated DT based recombinant anti-monkey CD3 immunotoxin, A-dmDT390-scfbDb (C207), as part of a reduced intensity conditioning regimen prior to hematopoietic cell transplantation, was compared between two groups of monkeys: those with and without pre-existing anti-diphtheria titers. T cell depletion was comparable in both groups of monkeys, and therefore appeared to be unaffected by the presence of moderate levels of pre-existing anti-diphtheria antibodies.

Loss of chimerism is an undesirable outcome of allogeneic hematopoietic cell transplantation (HCT) after reduced-intensity conditioning. Understanding the nature of cellular and humoral immune responses to HCT after graft loss could lead to improved retransplantation strategies. We investigated the immunologic responses after graft loss in miniature swine recipients of haploidentical HCT that received reduced-intensity conditioning. After the loss of peripheral blood chimerism, antidonor cellular responses were present without detectable antidonor antibody. Reexposure to donor hematopoietic cells after graft loss induced a sensitized antidonor cellular response. No induced antidonor antibody response could be detected despite evidence of cellular sensitization to donor cells. In contrast, unconditioned animals exposed repeatedly to similar doses of haploidentical donor cells developed antidonor antibody responses. These results could have important implications for the design of treatment strategies to overcome antidonor responses in HCT and improve the outcome of retransplantation after graft loss.