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

BackgroundIslet cell transplantation can cure type 1 diabetes (T1D), but only a minority of recipients remains insulin–independent in the following years. We tested the hypothesis that allograft rejection and recurrent autoimmunity contribute to this progressive loss of islet allograft function.Methodology/Principal FindingsTwenty-one T1D patients received cultured islet cell grafts prepared from multiple donors and transplanted under anti-thymocyte globulin (ATG) induction and tacrolimus plus mycophenolate mofetil (MMF) maintenance immunosuppression. Immunity against auto- and alloantigens was measured before and during one year after transplantation. Cellular auto- and alloreactivity was assessed by lymphocyte stimulation tests against autoantigens and cytotoxic T lymphocyte precursor assays, respectively. Humoral reactivity was measured by auto- and alloantibodies. Clinical outcome parameters - including time until insulin independence, insulin independence at one year, and C-peptide levels over one year- remained blinded until their correlation with immunological parameters. All patients showed significant improvement of metabolic control and 13 out of 21 became insulin-independent. Multivariate analyses showed that presence of cellular autoimmunity before and after transplantation is associated with delayed insulin-independence (p = 0.001 and p = 0.01, respectively) and lower circulating C-peptide levels during the first year after transplantation (p = 0.002 and p = 0.02, respectively). Seven out of eight patients without pre-existent T-cell autoreactivity became insulin-independent, versus none of the four patients reactive to both islet autoantigens GAD and IA-2 before transplantation. Autoantibody levels and cellular alloreactivity had no significant association with outcome.Conclusions/SignificanceIn this cohort study, cellular islet-specific autoimmunity associates with clinical outcome of islet cell transplantation under ATG-tacrolimus-MMF immunosuppression. Tailored immunotherapy targeting cellular islet autoreactivity may be required. Monitoring cellular immune reactivity can be useful to identify factors influencing graft survival and to assess efficacy of immunosuppression.Trial RegistrationClinicaltrials.gov NCT00623610

Islet transplantation is associated with a high rate of early graft failure caused by early immune attack and poor functionality of islets. Apoptosis of islet cells appears soon after islet isolation and primarily involves the ␤-cell. The purpose of this study was to determine the effect of ligation to extracellular matrix (ECM) proteins on survival of the islets of Langerhans following islet isolation. Islets that had been cultured for 24 h on collagen type I showed an islet survival of 59.7 ؎ 8.7%, while islets that had been cultured on collagen type IV and laminin showed an islet survival of 88.6 ؎ 10.3 and 94.3 ؎ 5.6%, respectively. Islets that had been pretreated with anti-␤1 antibodies and argenin-glycin-aspartic acid (RGD) peptides showed a decrease in the level of apoptosis by a factor of 2.5 and 3.1, respectively, and an increase of phospho-Akt Ser 473 activity by a factor of 3.1 and 2.9, respectively, compared with untreated islets. When detached from their natural ECM surrounding in the pancreas, islet cells undergo apoptosis, unless islets are cultured on collagen IV or laminin or treated with anti-␤1 integrin antibodies or RGD peptides to mimic ECM ligation. These results indicate that inhibition of anoikis may offer opportunities to improve function and viability of islet cells. Diabetes 55:312-317, 2006 T ransplantation of islets of Langerhans has the potential to become a widely applicable treatment for type 1 diabetes. Unfortunately, islet transplantation is associated with a high rate of early graft failure (1-4), which is caused by early immune attack, poorly functioning islets that were presumably damaged during isolation, or both. Apoptosis of islet cells appears soon after islet isolation and primarily involves the ␤-cell (5).During islet isolation by collagenase treatment, during purification, and during pretransplanting culture, the microenvironment of the islet is destroyed. Extracellular matrix (ECM) is an important component of the islet microenvironment that prevents cellular stress, which could impair ␤-cell function and survival (6,7). The islet basement membrane is composed of the ECM proteins laminin, fibronectin, and collagen types I, III, IV, and V (8,9). Cell-matrix interactions are necessary because they provide signals for proliferation, differentation, and migration (10). These interactions are mediated by integrins, which are a diverse class of ␣␤ heterodimeric transmembrane receptors that form ligands for the ECM (10 -12).Cells disconnected from their ECM can undergo apoptosis by an integrin-mediated death signal called anoikis (6,13,14). The pancreatic cells express the integrins ␣3␤1, ␣5␤1, and ␣v␤3 (7,15), which are also known to bind argenin-glycin-aspartic acid (RGD) sequences on ECM molecules. Cell attachment mediated by the ␣5␤1 and the ␣v␤3 integrin promotes cell survival by upregulation of Bcl-2 (16,17). Integrins are believed to mediate survival by activating a specific signaling pathway. One of its ligands is integrin-linked kinase (ILK), which can phosphorylate...

Our findings indicate that embedding of hepatocytes within their normal liver ECM surroundings maintains their survival. When detached from their natural surrounding hepatocytes enter into apoptosis, unless treated with anti-beta1-integrin antibodies or RGD peptides. This knowledge will allow improvement of hepatocyte transplantation efficiency.

Islet cell transplantation can cure type 1 diabetes, but allograft rejection and recurrent autoimmunity may contribute to decreasing insulin independence over time. In this study we report the association of allograft-specific proliferative and cytokine profiles with clinical outcome.Peripheral blood mononuclear cells were obtained of 20 islet recipients. Cytokine values in mixed lymphocyte cultures (MLC) were determined using stimulator cells with graft-specific HLA class II. Qualitative and quantitative cytokine profiles were determined before and after islet transplantation, blinded from clinical outcome. Cytotoxic T Lymphocyte precursor (CTLp) assays were performed to determine HLA class I alloreactivity.Allograft-specific cytokine profiles were skewed toward a Th2 or regulatory (Treg) phenotype after transplantation in insulin-independent, but not in insulin-requiring recipients. IFNc /IL10 ratio and MLC proliferation decreased after transplantation in insulinindependent recipients (p = 0.006 and p = 0.01, respectively). Production of the Treg cytokine IL10 inversely correlated with proliferation in alloreactive MLC (p = 0.008) and CTLp (p = 0.005). Production of IL10 combined with low-MLC reactivity associated significantly with insulin independence.The significant correlation between allograft-specific cytokine profiles and clinical outcome may reflect the induction of immune regulation in successfully transplanted recipients. Islet donor-specific IL10 production correlates with low alloreactivity and superior islet function.

Aims/hypothesis. Large quantities of pure viable donor islets are necessary for clinical transplantation. At present, low yields and low viability of pancreatic islets after transplantation necessitate the use of multiple donors for a single recipient. In this study an improved method for obtaining large quantities of pure viable islets of Langerhans for transplantation was developed in the rat. Methods. Islets of Langerhans were isolated from Albino Oxford rats. The donor pancreata were perfused in situ with iron oxide, which resulted in entrapment of iron particles in the capillaries of the islets. Subsequently, the islets were isolated by magnetic retraction. Islets obtained with this method were compared with islets obtained by density gradient-isolated islets with respect to yields, purity, and insulin production capacity. Islets isolated with the magnetic retraction method were transplanted under the renal capsule of streptozotocin-induced diabetic recipients. Blood-glucose levels in the recipients were monitored for 2 months after transplantation.Results. This method yielded more pure and viable islets than the conventional protocol. No contamination of exocrine tissue was observed after isolation. Furthermore, the islets isolated by magnetic retraction stained strongly positive for insulin during the entire observation period in vitro, and produced high amounts of insulin upon a challenge with glucose. The islets that were obtained by this new protocol were suitable for safe and effective transplantation. Conclusions/interpretation. We have shown that both the quantity and quality of islets obtained with this method were sufficient to induce insulin independence in a diabetic recipient using islets from only one donor. [Diabetologia (2004) 47:55-61]

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Type 1 diabetes (T1D) is a multifactorial disease characterized by the infiltration and subsequent destruction of the pancreatic insulin-producing beta cells by autoreactive T cells. CD8(+) T cells play an essential role in this beta cell destruction. However, little is known about the target antigens of CD8(+) T cells in human T1D patients. The aim of this study was to assess whether an epitope derived from the islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP), IGRP(265-273,) which has recently been identified as a target in non-obese diabetic (NOD) mice and is fully homologous to the human epitope, is a target of human diabetogenic CD8(+) T cells. We isolated a human CD8 T cell clone against this epitope, which confirms that this IGRP epitope is shared across species.