OBJECTIVEType 1 diabetes results from selective T-cell–mediated destruction of the insulin-producing β-cells in the pancreas. In this process, islet epitope–specific CD8+ T-cells play a pivotal role. Thus, monitoring of multiple islet–specific CD8+ T-cells may prove to be valuable for measuring disease activity, progression, and intervention. Yet, conventional detection techniques (ELISPOT and HLA tetramers) require many cells and are relatively insensitive.RESEARCH DESIGN AND METHODSHere, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B10–18, prepro-insulin (PPI)15–24, islet antigen (IA)-2797–805, GAD65114–123, islet-specific glucose-6-phosphatase catalytic subunit–related protein (IGRP)265–273, and prepro islet amyloid polypeptide (ppIAPP)5–13–specific CD8+ T-cells in recent-onset diabetic patients, their siblings, healthy control subjects, and islet cell transplantation recipients.RESULTSUsing this kit, islet autoreactive CD8+ T-cells recognizing insulin B10–18, IA-2797–805, and IGRP265–273 were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI15–24 proved to be the most sensitive epitope. Applying the “Diab-Q-kit” to samples of islet cell transplantation recipients allowed detection of changes of autoreactive T-cell frequencies against multiple islet cell–derived epitopes that were associated with disease activity and correlated with clinical outcome.CONCLUSIONSA kit was developed that allows simultaneous detection of CD8+ T-cells reactive to multiple HLA-A2–restricted β-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

OBJECTIVEThe metabolic outcome of islet cell transplants in type 1 diabetic patients is variable. This retrospective analysis examines whether differences in recipient characteristics at the time of transplantation are correlated with inadequate graft function.RESEARCH DESIGN AND METHODSThirty nonuremic C-peptide–negative type 1 diabetic patients had received an intraportal islet cell graft of comparable size under an ATG-tacrolimus–mycophenolate mofetil regimen. Baseline patient characteristics were compared with outcome parameters during the first 6 posttransplant months (i.e., plasma C-peptide, glycemic variability, and gain of insulin independence). Correlations in univariate analysis were further examined in a multivariate model.RESULTSPatients that did not become insulin independent exhibited significantly higher counts of B-cells as well as a T-cell autoreactivity against insulinoma-associated protein 2 (IA2) and/or GAD. In one of them, a liver biopsy during posttransplant year 2 showed B-cell accumulations near insulin-positive β-cell aggregates. Higher baseline total lymphocytes and T-cell autoreactivity were also correlated with lower plasma C-peptide levels and higher glycemic variability.CONCLUSIONSHigher total and B-cell counts and presence of T-cell autoreactivity at baseline are independently associated with lower graft function in type 1 diabetic patients receiving intraportal islet cells under ATG-tacrolimus–mycophenolate mofetil therapy. Prospective studies are needed to assess whether control of these characteristics can help increase the function of islet cell grafts during the first year posttransplantation.

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.

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CD4+ CD25 bright+ FoxP3 + T cells are potent regulators of T-cell reactivity, but their possible involvement in donor-specific nonresponsiveness after clinical kidney transplantation remains to be elucidated. We assessed the proliferative donor-reactivity in 33 kidney allograft recipients who were maintained on a combination of proliferation inhibitors (mycophenolate mofetil ( Altogether, we conclude that in long-term immunosuppressed kidney allograft patients functional regulatory CD4 + CD25 bright+ T cells circulate but that these cells mediate donor non reactivity only in a subset of patients.

Aims/hypothesis Simultaneous kidney-pancreas transplantation is an established treatment for patients with type 1 diabetes and end-stage renal failure, even though restored beta cell function may become affected by recurrent islet autoimmunity or graft rejection. We characterised infiltrating lymphocytes isolated from a pancreatic graft with normal endocrine function in a kidney-pancreas recipient with type 1 diabetes. Methods The pancreas graft was removed due to recurrent graft pancreatitis of unknown cause. Pancreas-infiltrating lymphocytes and peripheral blood mononuclear cells (PBMC) were isolated and characterised phenotypically and functionally.Results Compared with PBMC, pancreas-infiltrating lymphocytes exhibited a distinct activation/memory phenotype and T cell receptor profile that were indicative of selective infiltration of the pancreas. Islet autoreactive CD8 + T cells could be detected in the pancreas and were increased in frequency compared with PBMC. Additionally, an augmentation of CD8 + CD28 − regulatory T cells was observed in the pancreas; these induced expression of the inhibitory receptor immunoglobulin-like transcript-3 on antigen-presenting cells in a donor HLA class I-specific manner. Conclusions/interpretation These data demonstrate the simultaneous presence of regulatory and effector T cells in the pancreas allograft of a recipient with type 1 diabetes. They also indicate that circulating islet autoreactive T cells may reflect immunological processes in pancreatic tissue, even though their frequency in the periphery may lead to underestimation of their presence in the pancreas. Additional specificities were also present in the pancreas that were undetectable in the circulation.Keywords Effector T cells . Pancreas transplantation . Regulatory T cells . Type 1 diabetesAbbreviations APC antigen presenting cell CCR7 chemokine (C-C motif) receptor 7 CDR3 complementarity-determining region 3 IMDM Iscove's Modified Dulbecco's Medium IGRP islet-specific glucose-6-phosphatase catalytic subunit-related protein ILT immunoglobulin-like transcript mAbs monoclonal antibodies MACS magnetic-activated cell-sorting Diabetologia (2009) 52:494-503

These observations suggest that, after clinical heart transplantation, FOXP3+ T cells do not prevent acute rejection, but rather are a response to antidonor effector T-cell activity.

Summary CD4+ CD25bright+ FoxP3+ regulatory T cells (Tregs) may control donor‐specific allogeneic responses in kidney transplant recipients. Recent evidence demonstrated that three phenotypical Treg‐subsets, naive (CCR7+CD45RO−), central‐memory (CCR7+CD45RO+) and effector‐memory (CCR7−CD45RO+), are essential for the development and function of antigen‐specific suppression in the lymphoid and peripheral tissues. Also, it has been appreciated that Tregs are affected by immunosuppressive agents. In clinical practice, however, the effect of a single drug remains to be determined. Therefore, we analyzed the effect of several immunosuppressive agents on the number, phenotype and function of peripheral Tregs from 46 stable kidney transplant recipients. These patients were converted to monotherapy with tacrolimus (n = 15), rapamycin (n = 17) or mycophenolate mofetil (n = 14). Blood was obtained at inclusion and 6 months thereafter. The number of Tregs increased significantly in patients on monotherapy with rapamycin (P < 0.001), which was caused by increased numbers of Tregs with a central‐memory and an effector‐memory phenotype (both P < 0.05). At 6 months after conversion, however, the suppressive function of Tregs did not significantly change in co‐cultures stimulated with donor‐Ag. Therefore, monotherapy with rapamycin allows the signals that are needed to increase the number of functional Tregs with a memory phenotype, thereby enhancing the potential capacity to regulate donor‐specific responses in the lymphoid and the peripheral tissues.