Protect Islet Grafts Research Articles

Withaferin A inhibits lymphocyte proliferation, dendritic cell maturation in vitro and prolongs islet allograft survival

The immunosuppressive regimen for clinical allogeneic islet transplantation uses beta cell–toxic compounds such as tacrolimus that cause islet graft loss. Previously we reported that the plant-derived steroidal lactone Withaferin A (WA) can protect islet grafts by inhibiting nuclear factor-kappa B (NF-κB). Since the NF-κB signaling pathway is essential for T-cell activation, we hypothesized that long-term WA administration may also provide an immunosuppressive effect. Treatment of BALB/c donor islets and C57BL/6N recipients with WA alone resulted in 80% islet graft long-term survival vs. 40% in low-dose FK506-treated mice. In vitro, WA significantly blocked mouse and human T-cell proliferation by CD3/CD28 bead stimulation and in mixed lymphocyte reaction assay. Treatment of immature dendritic cells with WA prevented their maturation in response to inflammatory stimuli, as seen by decreased expression of CD83 and human leukocyte antigen–DR isotype. Exosomes released by islets treated with WA contained significantly fewer proinflammatory molecules interleukin-6, interleukin-8, monocyte chemoattractant protein-1, interferon-gamma-induced protein-10, inducible nitric oxide synthase, and cyclooxygenase-2. In conclusion, WA treatment not only reduced inflammation but also prolonged allograft survival, possibly through suppression of dendritic cell maturation and T-cell proliferation. WA has the potential to inhibit both the innate and adaptive immune response to prolong allograft survival.

Applying Immunomodulation to Promote Longevity of Immunoisolated Pancreatic Islet Grafts.

Islet transplantation is a promising therapy for insulin-dependent diabetes, but large-scale application is hampered by the lack of a consistent source of insulin-producing cells and need for lifelong administration of immunosuppressive drugs, which are associated with severe side effects. To avoid chronic immunosuppression, islet grafts can be enveloped in immunoisolating polymeric membranes. These immunoisolating polymeric membranes protect islet grafts from cell-mediated rejection while allowing diffusion of oxygen, nutrients, and insulin. Although clinical trials have shown the safety and feasibility of encapsulated islets to control glucose homeostasis, the strategy does up till now not support long-term graft survival. This partly can be explained by a significant loss of insulin-producing cells in the immediate period after implantation. The loss can be prevented by combining immunoisolation with immunomodulation, such as combined administration of immunomodulating cytokines or coencapsulation of immunomodulating cell types such as regulatory T cells, mesenchymal stem cells, or Sertoli cells. Also, administration of specific antibodies or apoptotic donor leucocytes is considered to create a tolerant microenvironment around immunoisolated grafts. In this review, we describe the outcomes and limitations of these approaches, as well as the recent progress in immunoisolating devices. Impact statement Immunoisolation by enveloping islets in semipermeable membranes allows for successful transplantation of islet grafts in the absence of chronic immunosuppression, but the duration of graft survival is still not permanent. The reasons for long-term final graft failure is not fully understood, but combining immunoisolation with immunomodulation of tissues or host immune system has been proposed to enhance the longevity of grafts. This article reviews the recent progress and challenges of immunoisolation, as well as the benefits and feasibility of combining encapsulation approaches with immunomodulation to promote longevity of encapsulated grafts.

Bone Marrow-Derived Mesenchymal Stem Cells Protect Islet Grafts Against Endoplasmic Reticulum Stress-Induced Apoptosis During the Early Stage After Transplantation.

Early loss of grafted islets is the main obstacle to achieve favorable outcomes of islet transplantation. Mesenchymal stem cells are known to have a protective effect; however, its mechanism remains unclear. We hypothesized that bone marrow-derived mesenchymal stem cells (BMSCs) can protect grafted islets against endoplasmic reticulum stress (ERS)-induced apoptosis. In syngeneic streptozocin-induced diabetic BALB/c mice, islet grafts decreased blood glucose levels; however, the effect was not fully functional from the immediate post-transplant phase. β-Cell apoptosis was proven on days 1 and 3 after transplantation. Ultra-structural evidence of ERS was observed along with increased expressions of marker protein BIP and apoptosis-related protein CHOP. In contrast, BMSC co-transplantation maintained glucose hemostasis, inhibited apoptosis and alleviated ERS. In ex vivo culture, BMSCs improved viability of islets and decreased apoptosis. Increased ERS were observed in cultured islets exposed to hypoxia, but not in the islets cocultured with BMSCs. Furthermore, cocultured BMSCs protected islets against ERS-induced apoptosis as well as improved their insulin secretion, and BMSCs alleviated ERS by improving Myc expression through both stromal cell-derived factor 1 signal and contact effect. In conclusion, BMSCs protected the grafted islets against ERS-induced apoptosis during the early stage after transplantation. This study opens a new arena for ERS-targeted therapy to improve outcomes of islet transplantation. Stem Cells 2018;36:1045-1061.

The Protective Effects of CD39 Overexpression in Multiple Low-Dose Streptozotocin–Induced Diabetes in Mice

Islet allograft survival limits the long-term success of islet transplantation as a potential curative therapy for type 1 diabetes. A number of factors compromise islet survival, including recurrent diabetes. We investigated whether CD39, an ectonucleotidase that promotes the generation of extracellular adenosine, would mitigate diabetes in the T cell–mediated multiple low-dose streptozotocin (MLDS) model. Mice null for CD39 (CD39KO), wild-type mice (WT), and mice overexpressing CD39 (CD39TG) were subjected to MLDS. Adoptive transfer experiments were performed to delineate the efficacy of tissue-restricted overexpression of CD39. The role of adenosine signaling was examined using mutant mice and pharmacological inhibition. The susceptibility to MLDS-induced diabetes was influenced by the level of expression of CD39. CD39KO mice developed diabetes more rapidly and with higher frequency than WT mice. In contrast, CD39TG mice were protected. CD39 overexpression conferred protection through the activation of adenosine 2A receptor and adenosine 2B receptor. Adoptive transfer experiments indicated that tissue-restricted overexpression of CD39 conferred robust protection, suggesting that this may be a useful strategy to protect islet grafts from T cell–mediated injury.

PLG Scaffold Delivered Antigen-Specific Regulatory T Cells Induce Systemic Tolerance in Autoimmune Diabetes

Islet transplantation is a promising treatment for human type 1 diabetes mellitus. Transplantation requires systemic immunosuppression, which has numerous deleterious side effects. Islet antigen-specific regulatory T cells (Tregs) have been shown to protect islet grafts from autoimmune destruction in the nonobese diabetic (NOD) model when co-localized in the kidney capsule. An extra-hepatic transplant site was established by transplanting islet-loaded microporous poly (lactide-co-glycolide) (PLG) scaffolds into abdominal fat. This study examined an autoimmune transplantation model and determined whether co-localized Tregs could protect islet grafts in an extra-hepatic and extra-renal transplant site. Normoglycemia was restored, and co-transplanted Tregs extended graft survival, including several instances of indefinite protection. Transplanted Tregs were replaced by recipient-derived Tregs over time, indicating that islet antigen-specific Tregs induce tolerance to islet grafts through host-derived Tregs. Thus, Tregs provided protection against a diverse repertoire of autoreactive T-cell-receptor specificities mediating diabetes in the NOD model, possibly through a phenomenon previously described as infectious tolerance. Interestingly, the infiltration by Tregs protected a second islet transplant, indicating systemic tolerance to islet antigens. In summary, PLG scaffolds can serve as an alternative delivery system for islet transplantation that allows for the co-localization of immunomodulatory cells within islet grafts and induces long-term graft survival in an autoimmune diabetes model. This method of co-localizing immunomodulatory cells with islets in a clinically translatable transplant site to affect the immune system on a local and systemic level has potential therapeutic implications for human islet transplantation.

Liver Stromal Cells Contribute to Liver Transplant Tolerance: Involved Cellular and Molecular Mechanisms

Organ transplantation is currently dependent on life-time administration of immunosuppressive drugs to non-specifically suppress immune response, resulting in severe side effects, including infection and cancer. Induction of specific tolerance is an ideal approach, but not generally successful, which however, actually naturally occurs. Thus, ˜1/4 liver allografts in farm pigs are spontaneously accepted, and induce donor specific tolerance. In humans, immunosuppression can be weaned off until completely stopped in ˜1/4 liver transplant patients. The underlying mechanisms are not completely understood. We and others surprisingly found that liver transplant tolerance is dependent on IFN-g stimulation. Mouse liver allograft, otherwise spontaneously accepted, was acutely rejected in IFN-g-/- recipients. In this study, the liver from IFN-gR1-/- mice (B6, H-2b) was transplanted into C3H (H-2k) recipients (n=6). None of them survived >15 days (WT grafts survived >100 days), suggesting that liver transplant tolerance requires intact IFN-g signaling pathway in liver graft. Both flow and histochemical analyses demonstrated markedly increased CD8+ infiltrates in IFN-gR1-/- grafts associated with lower expression of B7-H1 and less infiltrating T cell apoptosis, suggesting a role of B7-H1, an end product of IFN-g signaling in regulating immune response. This was supported by the result that the liver grafts from B7-H1-/- mice were acutely rejected in WT allogeneic recipients, associated with markedly less apoptosis (annexin V) in the graft infiltrating T cells. Liver grafts deficient in IFN-gR1 did not affect much T cell activation (CD44hiCD62Llo), as well as generation of myeloid-derived suppressor cells (MDSC) and Tregs in early phase. However, Tregs were markedly enhanced in long-term survived WT grafts, suggesting that elimination of effectors in early phase is crucial, while the suppressor cells may largely work to maintain tolerance. To identify the cell components in the liver graft responsible for these changes, the graft non-parenchymal cells were isolated at various time points for flow analysis. Almost all graft CD45+ cells were quickly (in one-two days) become recipient origin (H-2k, IFN-gR1+), while CD45- cells remain donor type (H-2b, IFN-gR1-), even in long-term survived grafts, suggesting that liver non-hematopoietic cells may be responsible for these changes. We have studied isolated hepatic stellate cells (HSC) (liver stromal cells, well known for their storing vitamin A and participating in fibrosis) that were exceptionally immunosuppressive. Cotransplantation with HSC effectively protected islet allografts without requirement of immunosuppression. Interestingly, cotransplantation with HSC isolated from IFN-gR1- mice failed to protect islet grafts, associated with markedly more infiltrating CD8+ T cells and less MDSC and Treg cells, indicating that the immune regulatory activity of HSC depends on intact IFN-g signaling pathway. Expression of B7-H1 was upregulated following exposure to IFN-g in a dose dependent manner, suggesting B7-H1 is an end product of IFN-g signaling. HSC isolated from B7-H1-/- mice largely lost their ability to protect islet allgrafts. This was associated with more CD8 T cells graft infiltration. In conclusion, IFN-g/B7-H1 signaling pathway in liver stromal cells play a critical role in establishment of liver transplant tolerance.

No islets left behind: islet autotransplantation for surgery-induced diabetes.

For patients with severe chronic pancreatitis refractory to medical interventions, total pancreatectomy can be considered to relieve the root cause of pain. The goal of a simultaneous islet autotransplant is to prevent or minimize the otherwise inevitable surgical diabetes. Islet autotransplant can successfully preserve some endogenous islet function in the majority of recipients, which mediates protection against brittle diabetes. Most maintain reasonably good glycemic control, while 30 %-40 % successfully discontinue insulin therapy. With islet autotransplants reaching a wider clinical audience, refinements in islet isolation techniques and strategies to protect islet grafts post-transplant may further improve the success of this procedure.

Activation of NFκB dependent apoptotic pathway in pancreatic islet cells by hypoxia

Insulin producing β-cells are exposed to hypoxic stress in the early period after islet transplantation. Although it has been suggested that hypoxia leads to islet loss, the underlying mechanisms are unclear. In this study, the early transcriptional profiles of b cell response to hypoxia were determined by using the Affymetrix Murine Genome Array U74Av2 GeneChip (about 12,422 genes). In addition to the upregulation of genes associated with glycolysis, genes related to apoptosis and stress response were also upregulated. The downregulated genes on hypoxia are classified as transcription and inflammatory response. These findings were confirmed by TUNEL assay and real-time reverse transcription-PCR for mRNA of genes related to apoptosis. On the other hand, we have found that the transcription factor NFκB was activated by hypoxia in islet cells. The affected genes involved in the activated NFκB pathway were mainly categorized as apoptosis-related genes by quantitative real-time PCR array (84 genes). Our comprehensive analysis of transcriptional changes of islets by hypoxia may assist the development of strategies that protect islet grafts from early loss.

Overexpression of thioredoxin in islets transduced by a lentiviral vector prolongs graft survival in autoimmune diabetic NOD mice

Pancreatic islet transplantation is considered an appropriate treatment to achieve insulin independence in type I diabetic patients. However, islet isolation and transplantation-induced oxidative stress and autoimmune-mediated destruction are still the major obstacles to the long-term survival of graft islets in this potential therapy. To protect islet grafts from inflammatory damage and prolong their survival, we transduced islets with an antioxidative gene thioredoxin (TRX) using a lentiviral vector before transplantation. We hypothesized that the overexpression of TRX in islets would prolong islet graft survival when transplanted into diabetic non-obese diabetic (NOD) mice.MethodsIslets were isolated from NOD mice and transduced with lentivirus carrying TRX (Lt-TRX) or enhanced green fluorescence protein (Lt-eGFP), respectively. Transduced islets were transplanted under the left kidney capsule of female diabetic NOD mice, and blood glucose concentration was monitored daily after transplantation. The histology of the islet graft was assessed at the end of the study. The protective effect of TRX on islets was investigated.ResultsThe lentiviral vector effectively transduced islets without altering the glucose-stimulating insulin-secretory function of islets. Overexpression of TRX in islets reduced hydrogen peroxide-induced cytotoxicity in vitro. After transplantation into diabetic NOD mice, euglycemia was maintained for significantly longer in Lt-TRX-transduced islets than in Lt-eGFP-transduced islets; the mean graft survival was 18 vs. 6.5 days (n = 9 and 10, respectively, p < 0.05).ConclusionWe successfully transduced the TRX gene into islets and demonstrated that these genetically modified grafts are resistant to inflammatory insult and survived longer in diabetic recipients. Our results further support the concept that the reactive oxygen species (ROS) scavenger and antiapoptotic functions of TRX are critical to islet survival after transplantation.

TGF-β-induced TCR-Tg/NOD regulatory T cells suppress both autoimmune diabetes transfer and islet graft destruction in an antigen dependent manner (B75)

Abstract Regulatory T cells (Tregs) are understood to fall into two categories: natural Tregs and induced Tregs. Induced Tregs can be produced by activating CD4 T cells in the presence of TGF-β. Such induced Tregs are of great promise for immunotherapy, but it has been unclear if they require stimulation by antigen in order to suppress in vivo. We have investigated the limits of induced Tregs in the NOD mouse model of type I diabetes. We produced TGF-β-induced Tregs from the 6.9 TCR-Tg/NOD mouse that are specific for islet autoantigen. In some experiments we used a congenic mouse, the NOD.C6, that specifically lacks the antigen for the 6.9 TCR-Tg T cells but not for other islet-specific T cells. These Tregs express Foxp3, and produce much less IFN-γ than non-Tregs. We have shown that induced Tregs can prevent transfer of diabetes by effector T cells in NOD mice, but fail to protect in NOD.C6 mice that lack the Treg antigen. These Tregs decrease inflammatory cytokine production and macrophage infiltration in the pancreas. In human patients with diabetes, induced Tregs might be used to protect islet grafts from immune destruction, and we have demonstrated that our induced Tregs can prevent islet graft destruction in NOD mice. In addition, we have grafted islets from the NOD.C6 mouse. By using these antigen-free islets, we have conclusively demonstrated that induced Tregs require activation by their antigen in the islet graft in order to protect.

Gene Combination Transfer to Block Autoimmune Damage in Transplanted Islets of Langerhans

Islet transplantation therapy would be applicable to a wider range of diabetic patients if donor islet acceptance and protection were possible without systemic immunosuppression of the recipient. To this aim, gene transfer to isolated donor islets ex vivo is one method that has shown promise. This study examines the combined effect of selected immunomodulatory and anti-inflammatory genes known to extend the functional viability of pancreatic islet grafts in an autoimmune system. These genes, indoleamine 2,3-dioxygenase (IDO), manganese superoxide dismutase (MnSOD), and interleukin (IL)-1 receptor antagonist protein (IRAP), were transferred to isolated NOD donor islets ex vivo then transplanted to NODscid recipients and evaluated in vivo after diabetogenic T-cell challenge. The length of time the recipient remained euglycemic was used to measure the ability of the transgenes to protect the graft from autoimmune destruction. Although the results of these cotransfections gave little evidence of a synergistic relationship, they were useful to show that gene combinations can be used to more efficiently protect islet grafts from diabetogenic T cells.

The testicular-derived Sertoli cell: cellular immunoscience to enable transplantation.

There is a renewed enthusiasm for the potential of cellular transplantation as a therapy for numerous clinical disorders. The revived interest is largely due to the unprecedented success of the "Edmonton protocol," which produced a 100% cure rate for type I diabetics following the transplantation of human islet allografts together with a modified immunosuppressive regimen. While these data provide a clear and unequivocal demonstration that transplantation is a viable treatment strategy, the shortage of suitable donor tissue together with the debilitating consequences of lifelong immunosuppression necessitate a concerted effort to develop novel means to enable transplantation on a widespread basis. This review outlines the use of Sertoli cells to provide local immunoprotection to cografted discordant cells, including those from xenogeneic sources. Sertoli cells are normally found in the testes where one of their functions is to provide local immunologic protection to developing germ cells. Isolated Sertoli cells 1) engraft and self-protect when transplanted into allogeneic and xenogeneic environments, 2) protect cografted allogeneic and xenogeneic cells from immune destruction, 3) protect islet grafts to reverse diabetes in animal models, 4) enable survival and function of cografted foreign dopaminergic neurons in rodent models of Parkinson's disease (PD), and 5) promote regeneration of damaged striatal dopaminergic circuitry in those same PD models. These benefits are discussed in the context of several potential underlying biological mechanisms. While the majority of work to date has focused on Sertoli cells to facilitate transplantation for diabetes and PD, the generalized ability of these unique cells to potently suppress the local immune environment opens additional clinical possibilities.

Advances in islet transplantation and the UK islet transplant consortium

AbstractIslet transplantation has surpassed whole pancreas transplantation as a cure for diabetes mellitus since the success of the Edmonton group. It is a safer procedure and requires minimal surgical intervention, yet offers excellent results in terms of achieving insulin independence. It also offers a more physiological insulin profile than current insulin replacement therapy. The UK Islet Transplant Consortium, founded by Diabetes UK, is in the process of establishing a programme of clinical islet transplantation in the UK.The immunosuppression required to protect islet grafts remains a threat to the long‐term health of transplant recipients and research into the possibility of circumventing the need for immunosuppression continues. The main areas under investigation include immunoisolation of islet grafts, HLA class II mismatching between donor and recipient, destruction of immune‐reactive dendritic cells in culture, in vitro pre‐treatment of islets to reduce their immunogenicity and genetic modification of islets to produce localised immunosuppression and prevent the release of MHC molecules.Because of the shortage of donors, researchers are looking toward alternative sources of glucose‐responsive insulin‐secreting tissue. Xenotransplantation would offer an attractive solution if the risk of transferring animal endogenous retroviruses can be eliminated. The expansion of mature beta‐cells, beta‐cell progenitors in pancreatic ducts or embryonic stem cells into viable tissue for transplant is also being researched in the hope of finding a solution for the tissue shortage issue. Encouraging advances have been made in many of these areas, creating a sense of anticipation and excitement for the future. Copyright © 2003 John Wiley &amp; Sons, Ltd.

Immunotherapy with nondepleting anti-CD4 monoclonal antibodies but not CD28 antagonists protects islet graft in spontaneously diabetic nod mice from autoimmune destruction and allogeneic and xenogeneic graft rejection.

T-cell activation and the subsequent induction of effector functions require not only the recognition of antigen peptides bound to MHC molecules by T-cell receptor (TCR) for antigen but also a costimulatory signal provided by antigen presenting cells. CD4 T-cell activation and function require the CD4 molecule as a coreceptor of TCR. The CD28/B7 pathway is a major costimulatory signal for T-cell activation and differentiation. The effect of targeting CD4 by nondepleting anti-CD4 monoclonal antibodies (mAbs) versus blocking CD28/B7 by CTLA4Ig, anti-CD80 mAbs, and anti-CD86 mAbs on the prevention of recurrence of autoimmune diabetes after MHC-matched nonobese diabetes-resistant (NOR) islet transplantation in nonobese diabetic (NOD) mice were compared. Whether nondepleting anti-CD4 mAbs prolong allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice were studied. Furthermore, the effect of nondepleting anti-CD4 mAbs combined with CTLA4Ig on allogeneic islet graft survival in NOD mice was investigated. Recurrence of autoimmune diabetes can be prevented by nondepleting anti-CD4 mAbs. Blocking the CD28/B7 costimulatory pathway by CTLA4Ig or by anti-CD80 mAbs and anti-CD86 mAbs cannot prevent recurrence of autoimmune diabetes after islet transplantation. Short-term treatment with nondepleting anti-CD4 mAbs significantly prolongs allogeneic islet graft survival and xenogeneic pig islet graft survival in diabetic NOD mice. But nondepleting anti-CD4 mAbs combined with CTLA4Ig decreased allogeneic islet graft survival. Nondepleting anti-CD4 mAbs but not CD28 antagonists protect islet grafts in diabetic NOD mice from autoimmune destruction and allogeneic and xenogeneic graft rejection. The efficacy of nondepleting anti-CD4 mAbs is compromised when it combines with CTLA4Ig.

Islet transplantation

Since the first appreciation of the role of the pancreas in diabetes, investigators have tried to replace the function of the insulin-producing β cells. Because type I diabetes is caused by autoimmune destruction of these β cells, transplanting islets is attractive, although somewhat elusive. Rodent islet transplants were performed in the 1970s, but the application to humans required many advances that spanned a decade. Even after the methods for the automated large-scale isolation of the number and quality of cells required for human investigation were available, it was not until 1990 that the first successful human islet allografts were reported. Human islet autografts were increasingly successful during this period, giving hope for successes with allografts. Long-term results of euglycemia with islet autografts and allografts have now established the feasibility of islet transplantation in humans. Although there are many clear advantages to cellular transplants compared with whole-organ transplants, the success rate is currently unacceptably low because of rejection. Recent developments in the field of organ acceptance and tolerance and the successful use of barrier devices in large animals and recently in humans to protect islet grafts provide real hope that more widespread application of islet transplantation in healthy patients may be possible.