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Islet transplantation is a potential treatment for type 1 diabetes, but the shortage of donor organs limits its routine application. As potential donor animals, we generated transgenic pigs expressing LEA29Y, a high-affinity variant of the T-cell costimulation inhibitor CTLA-4Ig, under the control of the porcine insulin gene promoter. Neonatal islet cell clusters (ICCs) from INSLEA29Y transgenic (LEA-tg) pigs and wild-type controls were transplanted into streptozotocin-induced hyperglycemic NOD-scid IL2Rγ(null) mice. Cloned LEA-tg pigs are healthy and exhibit a strong β-cell-specific transgene expression. LEA-tg ICCs displayed the same potential to normalize glucose homeostasis as wild-type ICCs after transplantation. After adoptive transfer of human peripheral blood mononuclear cells, transplanted LEA-tg ICCs were completely protected from rejection, whereas reoccurrence of hyperglycemia was observed in 80% of mice transplanted with wild-type ICCs. In the current study, we provide the first proof-of-principle report on transgenic pigs with β-cell-specific expression of LEA29Y and their successful application as donors in a xenotransplantation model. This approach may represent a major step toward the development of a novel strategy for pig-to-human islet transplantation without side effects of systemic immunosuppression.

Type 1 diabetes results from selective T-cell-mediated destruction of the insulin-producing beta-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. Here, we used a combinatorial quantum dot major histocompatibility complex multimer technique to simultaneously monitor the presence of HLA-A2 restricted insulin B(10-18), prepro-insulin (PPI)(15-24), islet antigen (IA)-2(797-805), GAD65(114-123), 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. Using this kit, islet autoreactive CD8(+) T-cells recognizing insulin B(10-18), IA-2(797-805), and IGRP(265-273) were shown to be frequently detectable in recent-onset diabetic patients but rarely in healthy control subjects; PPI(15-24) 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. A kit was developed that allows simultaneous detection of CD8(+) T-cells reactive to multiple HLA-A2-restricted beta-cell epitopes requiring limited amounts of blood, without a need for in vitro culture, that is applicable on stored blood samples.

To investigate a new clinically relevant immunoregulatory strategy based on treatment with murine Thymoglobulin mATG Genzyme and CTLA4-Ig in NOD mice to prevent allo- and autoimmune activation using a stringent model of islet transplantation and diabetes reversal. Using allogeneic islet transplantation models as well as NOD mice with recent onset type 1 diabetes, we addressed the therapeutic efficacy and immunomodulatory mechanisms associated with a new immunoregulatory protocol based on prolonged low-dose mATG plus CTLA4-Ig. BALB/c islets transplanted into hyperglycemic NOD mice under prolonged mATG+CTLA4-Ig treatment showed a pronounced delay in allograft rejection compared with untreated mice (mean survival time: 54 vs. 8 days, P < 0.0001). Immunologic analysis of mice receiving transplants revealed a complete abrogation of autoimmune responses and severe downregulation of alloimmunity in response to treatment. The striking effect on autoimmunity was confirmed by 100% diabetes reversal in newly hyperglycemic NOD mice and 100% indefinite survival of syngeneic islet transplantation (NOD.SCID into NOD mice). The capacity to regulate alloimmunity and to abrogate the autoimmune response in NOD mice in different settings confirmed that prolonged mATG+CTLA4-Ig treatment is a clinically relevant strategy to translate to humans with type 1 diabetes.

Because of reduced antioxidant defenses, beta-cells are especially vulnerable to free radical and inflammatory damage. Commonly used antirejection drugs are excellent at inhibiting the adaptive immune response; however, most are harmful to islets and do not protect well from reactive oxygen species and inflammation resulting from islet isolation and ischemia-reperfusion injury. The aim of this study was to determine whether redox modulation, using the catalytic antioxidant (CA), FBC-007, can improve in vivo islet function post-transplant. The abilities of redox modulation to preserve islet function were analyzed using three models of ischemia-reperfusion injury: 1) streptozotocin (STZ) treatment of human islets, 2) STZ-induced murine model of diabetes, and 3) models of syngeneic, allogeneic, and xenogeneic transplantation. Incubating human islets with catalytic antioxidant during STZ treatment protects from STZ-induced islet damage, and systemic delivery of catalytic antioxidant ablates STZ-induced diabetes in mice. Islets treated with catalytic antioxidant before syngeneic, suboptimal syngeneic, or xenogeneic transplant exhibited superior function compared with untreated controls. Diabetic murine recipients of catalytic antioxidant-treated allogeneic islets exhibited improved glycemic control post-transplant and demonstrated a delay in allograft rejection. Treating recipients systemically with catalytic antioxidant further extended the delay in allograft rejection. Pretreating donor islets with catalytic antioxidant protects from antigen-independent ischemia-reperfusion injury in multiple transplant settings. Treating systemically with catalytic antioxidant protects islets from antigen-independent ischemia-reperfusion injury and hinders the antigen-dependent alloimmune response. These results suggest that the addition of a redox modulation strategy would be a beneficial clinical approach for islet preservation in syngeneic, allogeneic, and xenogeneic transplantation.

For autoimmune conditions like type 1 diabetes to progress, self-reactive CD8⁺ T cells would need to interact with peptide-antigen cross-presented on the surface of antigen-presenting cells in a major histocompatibility complex (MHC) class I-restricted fashion. However, the mechanisms by which autoantigen is cross-presented remain to be identified. In this study, we show cross-presentation of islet-derived autoantigens by B cells. B cells engage self-reactive CD8⁺ T cells in the pancreatic lymph node, driving their proliferative expansion and differentiation into granzyme B⁺interferon-γ⁺lysosomal-associated membrane protein 1⁺ effector cells. B-cell cross-presentation of insulin required proteolytic cleavage and endosomal localization and was sensitive to inhibitors of protein trafficking. Absent B-cell MHC class I, or B-cell receptor restriction to an irrelevant specificity, blunted the expansion of self-reactive CD8⁺ T cells, suggesting B-cell antigen capture and presentation are critical in vivo events for CD8 activation. Indeed, the singular loss of B-cell MHC class I subverted the conversion to clinical diabetes in NOD mice, despite the presence of a pool of activated, and B cell-dependent, interleukin-21-expressing Vβ4⁺CD4⁺ T cells. Thus, B cells govern the transition from clinically silent insulitis to frank diabetes by cross-presenting autoantigen to self-reactive CD8⁺ T cells.

Immunoglobulin-Like Transcript 3-Fc Suppresses T-Cell Responses to Allogeneic Human Islet Transplants in hu-NOD/SCID Mice George Vlad 1 , Vivette D. D'Agati 1 , Qing-Yin Zhang 2 , Zhuoru Liu 2 , Eric K. Ho 1 , Thalachallour Mohanakumar 3 , Mark A. Hardy 2 , Raffaello Cortesini 1 and Nicole Suciu-Foca 1 1 Department of Pathology, College of Physicians and Surgeons of Columbia University, New York, New York 2 Department of Surgery, College of Physicians and Surgeons of Columbia University, New York, New York 3 Department of Surgery, Washington University School of Medicine, St. Louis, Missouri Corresponding author: Dr. Nicole Suciu-Foca, ns20{at}columbia.edu Abstract OBJECTIVE— The aim of our study was to explore the immunomodulatory activity of soluble immunoglobulin (Ig)-like transcript (ILT) 3-Fc in pancreatic islet transplantation and to determine its mechanism of action. RESEARCH DESIGN AND METHODS— NOD/SCID mice in which diabetes was induced by streptozotocin injection were transplanted with human pancreatic islet cells. Mice in which the transplant restored euglycemia were humanized with allogeneic peripheral blood mononuclear cells and treated with ILT3-Fc or control human IgG or left untreated. The blood glucose level was monitored twice a week, and rejection was diagnosed after two consecutive readings >350 mg/dl. Tolerated and rejected grafts were studied histologically and by immunostaining for human T-cells and insulin production. CD4 and CD8 T-cells from the spleen were studied for suppressor activity, expression of cytokines, and CD40L. RESULTS— Although human T-cell engraftment was similar in all groups, ILT3-Fc–treated mice tolerated the islets for the entire period of observation (91 days), whereas control mice rejected the graft within 7 weeks ( P < 0.0001). ILT3-Fc treatment suppressed the expression of cytokines and CD40L and induced the differentiation of human CD8 + T suppressor cells that inhibited Th alloreactivity against graft HLA antigens. T-cells allostimulated in vitro in the presence of ILT3-Fc inhibited CD40L-induced upregulation of CD40 in human pancreatic islet cells. Histochemical studies showed dramatic differences between human pancreatic islets from tolerant, ILT3-Fc–treated mice and control recipients rejecting the grafts. CONCLUSIONS— The data indicated that ILT3-Fc is a potent immunoregulatory agent that suppressed islet allograft rejection in humanized NOD/SCID mice. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 16 April 2008. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted April 11, 2008. Received January 14, 2008. DIABETES

It is generally believed that inflammatory cues can attract noncognate, “bystander” T-cell specificities to sites of inflammation. We have shown that recruitment of naive and in vitro activated autoreactive CD8+ T cells into endogenous islets requires local autoantigen expression. Here, we demonstrate that absence of an autoantigen in syngeneic extrapancreatic islet grafts in diabetic hosts renders the grafts “invisible” to cognate memory (and naive) T cells. We monitored the recruitment of islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP)206–214-reactive CD8+ T cells into IGRP206–214-competent and IGRP206–214-deficient islet grafts in diabetic wild-type or IGRP206–214−/− nonobese diabetic hosts (harboring either naive and memory T cells or only naive IGRP206–214-specific T-cells, respectively). All four host–donor combinations had development of recurrent diabetes within 2 weeks. Wild-type hosts recruited IGRP206–214-specific T cells into IGRP206–214+/+ but not IGRP206–214−/− grafts. In IGRP206–214−/− hosts, there was no recruitment of IGRP206–214-specific T cells, regardless of donor type. Graft-derived IGRP206–214 activated naive IGRP206–214-specific T cells, but graft destruction invariably predated their recruitment. These results indicate that recurrent diabetes is exclusively driven by autoreactive T cells primed during the primary autoimmune response, and demonstrate that local antigen expression is a sine qua non requirement for accumulation of memory T cells into islet grafts. These findings underscore the importance of tackling autoreactive T-cell memory after β-cell replacement therapy.

Type 1 diabetes is an incurable chronic autoimmune disease. Although transplantation of pancreatic islets may serve as a surrogate source of insulin, recipients are subjected to a life of immunosuppression. Interleukin (IL)-21 is necessary for type 1 diabetes in NOD mice. We examined the efficacy of an IL-21-targeted therapy on prevention of diabetes in NOD mice, in combination with syngeneic islet transplantation. In addition, we assessed the role of IL-21 responsiveness in islet allograft rejection in mouse animal models. NOD mice were treated with IL-21R/Fc, an IL-21-neutralizing chimeric protein. This procedure was combined with syngeneic islet transplantation to treat diabetic NOD mice. Survival of allogeneic islet grafts in IL-21R-deficient mice was also assessed. Evidence is provided that IL-21 is continually required by the autoimmune infiltrate, such that insulitis was reduced and reversed and diabetes inhibited by neutralization of IL-21 at a late preclinical stage. Recovery from autoimmune diabetes was achieved by combining neutralization of IL-21 with islet transplantation. Furthermore, IL-21-responsiveness by CD8+ T-cells was sufficient to mediate islet allograft rejection. Neutralization of IL-21 in NOD mice can inhibit diabetes, and when paired with islet transplantation, this therapeutic approach restored normoglycemia. The influence of IL-21 on a graft-mounted immune response was robust, since the absence of IL-21 signaling prevented islet allograft rejection. These findings suggest that therapeutic manipulation of IL-21 may serve as a suitable treatment for patients with type 1 diabetes.

T cell-mediated rejection remains a barrier to the clinical application of islet xenotransplantation. Regulatory T cells (Treg) regulate immune responses by suppressing effector T cells. This study aimed to determine the ability of human Treg to prevent islet xenograft rejection and the mechanism(s) involved. Neonatal porcine islet transplanted NOD-SCID IL2rγ(-/-) mice received human peripheral blood mononuclear cells (PBMC) with in vitro expanded autologous Treg in the absence or presence of anti-human interleukin-10 (IL-10) monoclonal antibody. In addition, human PBMC-reconstituted recipient mice received recombinant human IL-10 (rhIL-10). Adoptive transfer with expanded autologous Treg prevented islet xenograft rejection in human PBMC-reconstituted mice by inhibiting graft infiltration of effector cells and their function. Neutralization of human IL-10 shortened xenograft survival in mice receiving human PBMC and Treg. In addition, rhIL-10 treatment led to prolonged xenograft survival in human PBMC-reconstituted mice. This study demonstrates the ability of human Treg to prevent T-cell effector function and the importance of IL-10 in this response. In vitro Treg expansion was a simple and effective strategy for generating autologous Treg and highlighted a potential adoptive Treg cell therapy to suppress antigraft T-cell responses and reduce the requirement for immunosuppression in islet xenotransplantation.

Dipeptidyl Peptidase IV Inhibition With MK0431 Improves Islet Graft Survival in Diabetic NOD Mice Partially via T-Cell Modulation Su-Jin Kim 1 , Cuilan Nian 1 , Doris J. Doudet 2 and Christopher H.S. McIntosh 1 1 Department of Cellular and Physiological Sciences and the Diabetes Research Group, Life Sciences Institute, University of British Columbia, Vancouver, British Columbia, Canada 2 Department of Medicine, University of British Columbia, Vancouver, British Columbia, Canada Corresponding author: Christopher H.S. McIntosh, mcintoch{at}interchange.ubc.ca Abstract OBJECTIVE— The endopeptidase dipeptidyl peptidase-IV (DPP-IV) has been shown to NH 2 -terminally truncate incretin hormones, glucose-dependent insulinotropic polypeptide, and glucagon-like peptide-1, thus ablating their ability to potentiate glucose-stimulated insulin secretion. Increasing the circulating levels of incretins through administration of DPP-IV inhibitors has therefore been introduced as a therapeutic approach for the treatment of type 2 diabetes. DPP-IV inhibitor treatment has also been shown to preserve islet mass in rodent models of type 1 diabetes. The current study was initiated to define the effects of the DPP-IV inhibitor sitagliptin (MK0431) on transplanted islet survival in nonobese diabetic (NOD) mice, an autoimmune type 1 diabetes model. RESEARCH DESIGN AND METHODS— Effects of MK0431 on islet graft survival in diabetic NOD mice were determined with metabolic studies and micropositron emission tomography imaging, and its underlying molecular mechanisms were assessed. RESULTS— Treatment of NOD mice with MK0431 before and after islet transplantation resulted in prolongation of islet graft survival, whereas treatment after transplantation alone resulted in small beneficial effects compared with nontreated controls. Subsequent studies demonstrated that MK0431 pretreatment resulted in decreased insulitis in diabetic NOD mice and reduced in vitro migration of isolated splenic CD4 + T-cells. Furthermore, in vitro treatment of splenic CD4 + T-cells with DPP-IV resulted in increased migration and activation of protein kinase A (PKA) and Rac1. CONCLUSIONS— Treatment with MK0431 therefore reduced the effect of autoimmunity on graft survival partially by decreasing the homing of CD4 + T-cells into pancreatic β-cells through a pathway involving cAMP/PKA/Rac1 activation. Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 10 December 2008. Readers may use this article as long as the work is properly cited, the use is educational and not for profit, and the work is not altered. See http://creativecommons.org/licenses/by-nc-nd/3.0/ for details. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted December 4, 2008. Received August 12, 2008. DIABETES