Improved Islet Graft Survival Research Articles

Local Dexamethasone Administration Delays Allogeneic Islet Graft Rejection in the Anterior Chamber of the Eye of Non-Human Primates

Pancreatic islet transplantation into the anterior chamber of the eye (ACE) has been shown to improve glycemic control and metabolic parameters of diabetes in both murine and primate models. This novel transplantation site also allows the delivery of therapeutic agents, such as immunosuppressive drugs, locally to prevent islet graft rejection and circumvent unwanted systemic side effects. Local intravitreal administration of micronized dexamethasone implant was performed prior to allogeneic islet transplantation into the ACEs of non-human primates. Two study groups were observed namely allogeneic graft without immunosuppression (n = 4 eyes) and allogeneic graft with local immunosuppression (n = 8 eyes). Survival of islet grafts and dexamethasone concentration in the ACE were assessed in parallel for 24 weeks. Allogeneic islet grafts with local dexamethasone treatment showed significantly better survival than those with no immunosuppression (median survival time- 15 weeks vs 3 weeks, log-rank test p<0.0001). Around 73% of the grafts still survived at week 10 with a single local dexamethasone implant, where the control group showed no graft survival. Dexamethasone treated islet grafts revealed a good functional response to high glucose stimulation despite there was a transient suppression of insulin secretion from week 8 to 12. Our findings show a significant improvement of allografts survival in the ACE with local dexamethasone treatment. These results highlight the feasibility of local administration of pharmacological compounds in the ACE to improve islet graft survival and function. By eliminating the need for systemic immunosuppression, these findings may impact clinical islet transplantation in the treatment of diabetes, and the ACE may serve as a novel therapeutic islet transplantation site with high potential for local pharmacological intervention.

P.159: Investigating the Role of Cathepsin L in Islet Graft Rejection

Type 1 diabetes (T1D) is a T cell-mediated autoimmune disorder in which beta-cell specific destruction leads to life-long exogenous insulin dependence. While islet transplantation can restore euglycemia in these patients, both allogeneic and autoimmune rejection of the islet graft pose challenges limiting graft longevity. CD4+ T cells play a major role in driving T1D autoimmune development and islet graft rejection. Recent discovery of hybrid insulin peptides (HIPs), a fusion of insulin peptides with other beta-cell granule containing peptides, were found to be cognate autoantigens for autoreactive CD4+ T cells in mouse models and in patients with T1D. HIPs are formed within the beta-cell during crinophagy, a process of protein degradation or turnover where insulin granules fuse with lysosomes. By reducing the formation of HIPs, autoimmune-mediated islet graft rejection may be delayed. Cathepsin L (CatL) is a lysosomal endopeptidase enzyme involved in protein degradation and is implicated in the generation of HIPs. Non-Obese Diabetic (NOD) mice lacking CatL (NOD.CatL-/-) exhibited a delay in spontaneous T1D development. Immunophenotyping of islet-infiltrating immune cells from NOD.CatL-/- mice displayed reductions in effector CD4+ T cells and tetramer-specific autoreactive BDC-2.5, BDC-6.9, and insulin T cells compared to NOD mice. We hypothesize that islets deficient in CatL will delay immune-mediated rejection due to the absence of antigenic HIPs following transplantation. CatL-deficient islets may improve islet graft survival due to reduced release of antigenic peptides and passenger CD4+ T cells when transplanted into recipient mice. Isolated islets (250 islets/recipient) from NOD.CatL-/-, NOD.CatL+/-, or NOD.Rag mice, were transplanted under the kidney capsule of streptozotocin-treated and spontaneously diabetic NOD mice and monitored for restoration and maintenance of euglycemia. Both NOD.CatL-/- and NOD.CatL+/- islets restored euglycemia similarly as NOD.Rag islets. Future studies will investigate the impact of CatL-deficiency on long-term graft survival and effects on immune cell infiltration and activation. This work was supported by an NIH/NIDDK R01 award (DK099550), JDRF award SRA-2016-270-S-B, JDRF award 2-SRA-2019-692-S-B, NIH/NIDDK R01 award (DK126456), NIH/NIDDK R01 award (DK127497), NIH/NIGMS (T32GM008111) Cell, Molecular, and Developmental Biology T32 training grant.

Advances in Pancreatic Islet Transplantation Sites for the Treatment of Diabetes.

Diabetes is a complex disease that affects over 400 million people worldwide. The life-long insulin injections and continuous blood glucose monitoring required in type 1 diabetes (T1D) represent a tremendous clinical and economic burdens that urges the need for a medical solution. Pancreatic islet transplantation holds great promise in the treatment of T1D; however, the difficulty in regulating post-transplantation immune reactions to avoid both allogenic and autoimmune graft rejection represent a bottleneck in the field of islet transplantation. Cell replacement strategies have been performed in hepatic, intramuscular, omentum, and subcutaneous sites, and have been performed in both animal models and human patients. However more optimal transplantation sites and methods of improving islet graft survival are needed to successfully translate these studies to a clinical relevant therapy. In this review, we summarize the current progress in the field as well as methods and sites of islet transplantation, including stem cell-derived functional human islets. We also discuss the contribution of immune cells, vessel formation, extracellular matrix, and nutritional supply on islet graft survival. Developing new transplantation sites with emerging technologies to improve islet graft survival and simplify immune regulation will greatly benefit the future success of islet cell therapy in the treatment of diabetes.

Amniotic Membrane Extract Protects Islets From Serum-Deprivation Induced Impairments and Improves Islet Transplantation Outcome.

Islet culture prior to transplantation is a standard practice in many transplantation centers. Nevertheless, the abundant islet mass loss and function impairment during this serum-deprivation culture period restrain the success of islet transplantation. In the present study, we used a natural biomaterial derived product, amniotic membrane extract (AME), as medium supplementation of islet pretransplant cultivation to investigate its protective effect on islet survival and function and its underlying mechanisms, as well as the engraftment outcome of islets following AME treatment. Results showed that AME supplementation improved islet viability and function, and decreased islet apoptosis and islet loss during serum-deprived culture. This was associated with the increased phosphorylation of PI3K/Akt and MAPK/ERK signaling pathway. Moreover, transplantation of serum-deprivation stressed islets that were pre-treated with AME into diabetic mice revealed better blood glucose control and improved islet graft survival. In conclusion, AME could improve islet survival and function in vivo and in vitro, and was at least partially through increasing phosphorylation of PI3K/Akt and MAPK/ERK signaling pathway.

1681-P: Alpha-1 Antitrypsin Suppresses Macrophage Activation Post Islet Transplantation

Macrophage activation contributes to islet graft loss after transplantation by secretion of proinflammatory cytokines. We showed in previous studies that treatment with alpha-1 antitrypsin (AAT) improved human islet survival after transplantation into the livers of streptozotocin-induced diabetic NOD-SCID mice. To further understand the cellular targets of AAT, we isolated macrophages from control or AAT-treated recipients at day 1 post transplantation (PT) and compared their mRNA profile using RNAseq analysis. 344 genes were changed in AAT-treated recipients compared to controls. Clusters of the inflammatory response, chemokine activity and cytokine and cytokine receptor Gene Otology categories and pathways were significantly affected by AAT-treatment. In vitro, AAT suppressed IFN-γ-induced Raw267.4 cell activation via suppression of STAT1 phosphorylation and iNOS production. This was translated in vitro when human islets were co-transplanted with IFN-γ-treated Raw cells, in the presence or absence of AAT, into diabetic NOD-SCID mice in which macrophages were depleted by liposome. At day 21, all macrophage-depleted diabetic recipients receiving islets alone achieved normoglycemia (n=6). In contrast, only 55.6% (n=9, p=0.001) receiving islets and IFN-γ-treated Raw cells achieved normoglycemia, compared to 90% receiving AAT-treated IFN-γ Raw cells (n=10, p=0.003), suggesting AAT improved islet graft survival via suppressing macrophage activation. The effect of AAT was further confirmed in a major mismatch allogeneic islet transplantation model (Balb/c, H-2b to C57BL/6 H-2d mice) in which macrophage activation contributed to primary nonfunction (PNF) of the transplanted islets. In this model, PNF occurred in 16.7% of AAT-treated recipients (n=6) but in 40% of controls (n=5) when 400 islets were transplanted. In summary, this study further identified that AAT contributed to islet graft survival after transplantation by suppressing macrophage activation. Disclosure W. Gou: None. C. Strange: None. H. Wang: None. Funding National Institutes of Health (DK105183, DK120394, DK118529)

Synthetic Immunomodulatory Biomaterials to Improve Islet Graft Survival

Synthetic Immunomodulatory Biomaterials to Improve Islet Graft Survival

Islet cell transplant and the incorporation of Tregs.

T regulatory cells (Tregs) play a central role in maintaining immune homeostasis and peripheral tolerance to foreign antigens in humans. The immune response to alloantigens and recurrence of autoimmunity contribute to pancreatic islet transplant dysfunction, hence the adoptive transfer of Tregs has the potential to significantly improve islet graft survival. In this review, we provide an in-depth analysis of challenges associated with the application of ex-vivo expanded Tregs therapy in pancreatic islet transplant. Tregs administered systemically may poorly migrate to the site of transplantation, which is critical for tolerance induction and graft protection. Intraportal administration of pancreatic tissue exerts some limitations on the ability to cotransplant Tregs at the same site of islet transplantation. In order to maximize therapeutic potential of Tregs, islet transplantation protocols may need additional refinement. Further to this, the Tregs may require cryopreservation in order to make them readily available at the same time as islet transplant. On the basis of current experience and technology, the combination of islet and Treg cotransplantation is feasible and has great potential to improve islet graft survival. The possibility to wean off, or withdraw, traditional immunosuppressive agents and improve patient quality of life makes it an interesting avenue to be pursued.

Alpha-Melanocyte Stimulating Hormone Preserves Islet Graft Survival Through Down-Regulation of Toll-Like Receptors

The induction of Toll-like receptors (TLRs) in β cells is involved in β-cell death and graft rejection after transplantation. This study investigated the ability of alpha-melanocyte stimulating hormone (α-MSH) to protect pancreatic islets and improve graft survival through regulation of TLRs. To test the effect of α-MSH on TLR regulation, we first isolated pancreatic islets from rats pretreated with/without α-MSH and assayed inflammatory cytokines and insulin release, and measured the expression of TLRs. Pancreatic islets were transplanted into the kidney capsule of a diabetes mellitus (DM) mouse with and without prior injection of α-MSH. The blood glucose levels were measured and TLR4 expression in transplanted kidney tissue was assessed. Islet morphology, including size and total mass, was improved in the α-MSH group compared to the control group. The expression of TLRs as well as nitric oxide and monocyte chemoattractant protein 1 production were decreased in islets isolated from α-MSH–treated rats. In DM mice, the normoglycemic ratio was higher in the α-MSH–treated group than in the sham group. Moreover, the high levels of TLR4 expression observed in DM kidney tissue were significantly decreased in islet-transplanted tissue with α-MSH. This study showed that α-MSH protects pancreatic islets from cell death and dysfunction through downregulation of TLRs. In conclusion, α-MSH could contribute to improved islet graft survival and function in pancreatic islet transplantation.

Induction of Protective Genes Leads to Islet Survival and Function

Islet transplantation is the most valid approach to the treatment of type 1 diabetes. However, the function of transplanted islets is often compromised since a large number of β cells undergo apoptosis induced by stress and the immune rejection response elicited by the recipient after transplantation. Conventional treatment for islet transplantation is to administer immunosuppressive drugs to the recipient to suppress the immune rejection response mounted against transplanted islets. Induction of protective genes in the recipient (e.g., heme oxygenase-1 (HO-1), A20/tumor necrosis factor alpha inducible protein3 (tnfaip3), biliverdin reductase (BVR), Bcl2, and others) or administration of one or more of the products of HO-1 to the donor, the islets themselves, and/or the recipient offers an alternative or synergistic approach to improve islet graft survival and function. In this perspective, we summarize studies describing the protective effects of these genes on islet survival and function in rodent allogeneic and xenogeneic transplantation models and the prevention of onset of diabetes, with emphasis on HO-1, A20, and BVR. Such approaches are also appealing to islet autotransplantation in patients with chronic pancreatitis after total pancreatectomy, a procedure that currently only leads to 1/3 of transplanted patients being diabetes-free.

Anti-Inflammatory Thalidomide Improves Islet Grafts Survival and Functions in a Xenogenic Environment

Thalidomide possesses both anti-inflammatory and anti-angiogenic properties. This study investigates its potential application in islet transplantation with a xenogenic transplantation model. Transplantation was performed using C57Bl/6 mice and NMRI nu/nu mice as recipients of porcine islets. Moreover, islet graft vasculature and inflammation were investigated to identify the mechanisms of thalidomide action. In the immunocompetent environment of C57Bl/6 mice, a fast graft rejection was observed. The group treated with thalidomide 200 mg/kg BW per day achieved and maintained euglycemia in the complete observation period for 42 days. The treated mice had more functional islet graft mass with less leukocyte infiltration. The pro-inflammatory TNF-α and VEGF content in islet grafted kidneys was significantly lowered by the treatment. By comparison, thalidomide was not effective in improving graft survival in immunocompromised nude mice. It strongly inhibited the VEGF and TNF-α-induced endothelial proliferation of isolated pig islets in a dose dependent manner. The magnitude of thalidomide's inhibitory effect was nearly identical to the effect of VEGF- receptor 2 inhibitor SU416 and anti-TNF-receptor 1 neutralizing antibody, and was reversed by sphingosine-1-phosphate. In conclusion, the anti-inflammatory effect of thalidomide improved islet graft survival and function in a transplantation model with a maximum immune barrier.

Marginal mass islet transplantation with autologous mesenchymal stem cells promotes long-term islet allograft survival and sustained normoglycemia

Allogeneic islet transplantation is an option to treat diabetes however there are obstacles that are limiting its clinical use. We have examined whether mesenchymal stem cells (MSC) improve islet graft survival and whether such therapy allows for better graft acceptance with reduced requirement for immunosuppression. In vitro-expanded syngeneic bone marrow-derived MSC were co-transplanted with islets into omental pouch in a rat model of streptozotocin-induced diabetes. Marginal mass syngeneic islet transplantation into the omentum with MSC promoted sustained normoglycemia. Interestingly, allogeneic islets +MSC, but not islets alone, with short-term use of immunosuppression enhanced long-term islet graft survival, insulin expression in the grafts and induced normal serum insulin levels and normoglycemia. T cells from recipients transplanted with allogeneic islets +MSC produced low levels of IFN-γ and TNF-α upon ex-vivo activation, and this transplantation protocol promoted the generation of IL-10-secreting CD4 + T cells. These data encourage further preclinical and eventually, clinical MSC-based islet transplantation to improve the outcome of allogeneic islet transplantation in the treatment of diabetes.

Tacrolimus and Sirolimus Cause Insulin Resistance in Normal Sprague Dawley Rats

Tacrolimus-sirolimus immunosuppression has improved islet graft survival but may affect islet function. We studied the effects of tacrolimus, sirolimus, or both in normal adult male Sprague Dawley rats. Glucose and insulin response to oral glucose load and pancreas pathology were evaluated after two weeks of daily tacrolimus (1-8 mg/kg/day), sirolimus (0.08-8 mg/kg/day), or low-dose sirolimus (0.08 mg/kg/day) plus tacrolimus (1 mg/kg/day) treatment compared to controls. Tacrolimus and sirolimus each caused dose-dependent hyperglycemia with hyperinsulinemia in response to oral glucose compared to controls, suggesting insulin resistance. At the highest doses of sirolimus, fasting insulin concentrations were high and did not increase with oral glucose suggesting loss of first phase insulin release. The combination of low doses of tacrolimus and sirolimus, at concentrations used in clinical transplantation, resulted in hyperglycemia without hyperinsulinemia after oral glucose administration. The combination of tacrolimus and sirolimus decreased islet size, and increased islet apoptosis more than either medication alone, or controls. In summary, short-term therapy with either tacrolimus or sirolimus causes insulin resistance in normal rats. Combination tacrolimus-sirolimus causes greater islet changes suggesting early islet failure.

Exendin-4 treatment improves metabolic control after rat islet transplantation to athymic mice with streptozotocin-induced diabetes

Early islet graft survival is crucial in determining the outcome after clinical islet transplantation. Exendin-4 has anti-apoptotic and beta cell proliferative properties, which could improve islet graft survival and function. The aim of these studies was to evaluate the effect of exendin-4 on graft function after islet transplantation. Rat islets were transplanted under the kidney capsule of diabetic athymic mice. First, we performed a dose-finding study and found that 30 islets just failed to cure diabetic mice. In the following two studies, we transplanted 30 islets and treated the mice that had received these islets with exendin-4 i.p. (100 ng/mouse) once daily for 1 week. Blood glucose levels and body weights were used as evaluation criteria. In the short-term study evaluation was done at day 8. This study was followed by a long-term study that was evaluated at 4 weeks. In this study, islets were precultured with exendin-4 (0.1 nmol/l) in addition to the treatment given to mouse-recipients of transplanted islets. The cured mice underwent an intraperitoneal glucose tolerance test (IPGTT). In the short-term study, 63% of exendin-4-treated mice achieved graft function compared with 21% of untreated mice (p = 0.033). In the long-term study, 88% of treated mice had functioning grafts compared with 22% of controls (p = 0.015). Cured mice showed a normal response in the IPGTT, comparable to that of healthy mice. Exendin-4-treated mice gained significantly more weight than their untreated counterparts. Islet preculture and a short course of therapy with exendin-4 improves metabolic control after rat islet transplantation in athymic mice. The beneficial effect lasts beyond the treatment period.

1,25-dihydroxyvitamin D3 protects human pancreatic islets against cytokine-induced apoptosis via down-regulation of the fas receptor

Beta cell loss occurs at the onset of type 1 diabetes and after islet graft. It results from the dysfunction and destruction of beta cells mainly achieved by apoptosis. One of the mediators believed to be involved in beta cell apoptosis is Fas, a transmembrane cell surface receptor transducing an apoptotic death signal and contributing to the pathogenesis of several autoimmune diseases. Fas expression is particularly induced in beta cells by inflammatory cytokines secreted by islet-infiltrating mononuclear cells and makes cells susceptible to apoptosis by interaction with Fas-ligand expressing cells. We have previously demonstrated that 1,25(OH)2D3, the active metabolite of vitamin D, known to exhibit immunomodulatory properties and prevent the development of type 1 diabetes in NOD mice, is efficient against apoptosis induced by cytokines in human pancreatic islets in vitro. The effects were mainly mediated by the inactivation of NF-kappa-B. In this study we demonstrated that 1,25(OH)2D3 was also able to counteract cytokine-induced Fas expression in human islets both at the mRNA and protein levels. These results were reinforced by our microarray analysis highlighting the beneficial effects of 1,25(OH)2D3 on death signals induced by Fas activation. Our results provides additional evidence that 1,25(OH)2D3 may be an interesting tool to help prevent the onset of type 1 diabetes and improve islet graft survival.

IMPACT OF MICROGRAVITY CULTURE ON SURVIVAL OF ALLOGENEIC MOUSE PANCREATIC ISLET TRANSPLANTS

P684 Aims: Diabetes mellitus (Type 1) remains a major cause of morbidity and mortality despite significant advances in medical management. Clinical pancreatic islet allotransplantation is a low-risk alternative to whole pancreas transplantation, offering great promise in treating patients. Although an Edmonton therapeutic protocol has dramatically improved the survival of islet allografts, the majority of patients eventually develop impaired graft function and chronic rejection. Therefore, the ultimate goal—to radically improve islet graft survival—is to induce transplantation tolerance. Methods/Results: We have developed a unique mouse model of tolerance to pancreatic islet allografts [C57BL10 (H2b) to C3H (H2k)] by culturing islets in microgravity conditions using rotating bioreactors. Islets cultured in bioreactors for 7 days displayed decreased immunogenicity (lack of dendritic cells), resulting in long-term allograft survival (>100 days; n = 18) with superior islet morphology and function. Indeed, transmission electron microscopy showed gradual loss of dendritic cells and scanning electron microscopy revealed development of multiple nutritional channels in islets cultured in the bioreactor. Similar results were obtained in C57BL10 to Balb/c (H2d) donor/recipient combination. However, although a 7-day culture produced long-term survival (>100 days; n = 4; p = 0.001), a limited 3-day culture shortened the survival to a mean survival time (MST) of 21.6 ± 5.8 days (n = 5; p = 0.026); fresh allogeneic islets survived 13.8 ± 2.7 days (n = 5). Interestingly, the results were different in Stat6 or Stat4 knockout (KO) mice deficient for Th2 and Th1 cells, respectively. Diabetic Stat6 KO mice rejected fresh islet allografts at 12.1 ± 1.6 days (n = 8) and Stat4 KO mice at 13.3 ± 2.4 days. Islets cultured for 3 days in the bioreactor achieved long-term survivals in all Stat4 KO recipients (>100 days; n = 7; p = 0.001). In contrast, Stat6 KO recipients transplanted with 3-day bioreactor islets produced a mixed response with some long-term survivals (>100 days; n = 4; p=0.001) and some rejectors (22.0 ± 1.7 days; n = 3; p = 0.01. Conclusions: Microgravity condition decreases immunogenicity of allogeneic islets. Furthermore, bioreactor-cultured islets induce stable long-term allograft survival by induction of IL-4/Stat6-dependent Th2reg cells.

The effect of simultaneous CD154 and LFA-1 blockade on the survival of allogeneic islet grafts in nonobese diabetic mice.

The rate of success in clinical transplantation of islets of Langerhans has dramatically improved with perspectives of wide-scale applicability for patients with type 1 diabetes. One drawback is the need for lifelong immunosuppression, which is associated with significant side effects. Immunomodulatory strategies devoid of side effects and with tolerogenic potential, such as co-stimulatory blockade, would be a great improvement if successful. In this study, the authors have explored the effect of simultaneous blockade of CD40/CD154 and intercellular adhesion molecule (ICAM)/lymphocyte function-associated antigen (LFA)-1 interactions. Spontaneously diabetic nonobese diabetic (NOD) mice underwent transplantation with allogeneic (C57BL/6) islets and were treated with anti-CD154 monoclonal antibody (mAb) (500 microg, three doses), anti-LFA-1 mAb (100 microg, three doses), or a combination of both in the early peritransplant period. In another set of experiments, LFA-1 engagement was impaired by transplanting islets isolated from ICAM-1-knockout (KO) mice. Untreated animals rejected their grafts within 10 days. LFA-1 blockade alone did not result in improved islet graft survival, whereas CD154 blockade alone increased graft survival to 18 days. Simultaneous blockade of both pathways led to significantly improved islet graft survival to 30 days (ICAM-1-KO islets plus anti-CD154), 35 days (anti-LFA-1 plus anti-CD154), and 44 days (ICAM-1-KO islets plus anti-LFA-1 plus anti-CD154). These data suggest that a synergistic effect for prolonged graft survival can be obtained by simultaneously targeting CD154 and LFA-1 in the challenging model of islet allotransplantation in NOD mice. The observation of similar results with anti-LFA-1 mAb and with ICAM-1-KO grafts suggests a key role of direct antigen presentation for the activation of LFA-1-driven signaling.

Ex vivo and systemic transfer of adenovirus-mediated CTLA4Ig gene combined with a short course of FK506 therapy prolongs islet graft survival

Adenovirus-mediated CTLA4Ig gene transfer has been reported to enhance graft survival in several rodent transplantation models. In this study, we investigated the efficacy of ex vivo and systemic transfer of the CTLA4Ig gene by adenoviral vectors in pancreatic islet allo-transplantation. Islet grafts from BN rats were transplanted to chemically induced diabetic LEW rats. First, ex vivo CTLA4Ig gene transfer into isolated islets was performed prior to transplantation. Survival of transduced grafts under the kidney capsule was slightly prolonged (8.6±1.3 days) compared with survival of untransduced grafts (6.7±1.2 days); when combined with a short course of FK506, graft survival was further extended (32.6±10.7 days vs. 13.7±1.0 days with FK506 alone). Secondly, systemic gene transfer was accomplished by intravenous administration immediately after the transplantation procedure. In these animals, islet grafts under the kidney capsule survived longer (15.2±3.3 days) than in controls (6.7±1.2 days), and when FK506 was administered perioperatively, all the islet grafts survived for more than 100 days. In systemically transduced recipients, the survival of islet grafts transplanted into the liver was not significantly different from that of the grafts placed under the kidney capsule. In order to examine organ-specific immunogenicity, heterotopic BN cardiac grafts were transplanted to LEW rats intra-abdominally, with the virus transferred systemically as in the islet model. In contrast to the islet grafts, all the cardiac grafts were accepted for longer than 100 days, even without FK506 therapy. Finally, the LEW recipients with long-surviving islet or cardiac grafts were re-transplanted with islet grafts from the same donor strain (BN) on day 100. The second islet grafts survived longer than 100 days in half of the cardiac recipients, but consistently failed in the islet recipients. We conclude that in this transplant model, CTLA4Ig gene transfer and FK506 treatment synergistically improved islet graft survival, systemic transfer of the gene was more effective than ex vivo transfer to the islets, and donor-specific tolerance could not be achieved for islet transplantation but was achieved for cardiac transplantation.

Prolonged xenograft survival of islets infected with small doses of adenovirus expressing CTLA4Ig.

Systemic administration of the inhibitor of costimulation, CTLA4Ig, has been shown to prolong islet graft survival. The purpose of this study was to compare local and systemic expression of murine CTLA4Ig in transplants of rat islets into mice. Murine CTLA4Ig was made by joining two polymerase chain reaction products, the extracellular portion of CTLA4 and the Fc portion of IgG2a. Recombinant adenovirus expressing CTLA4Ig (AdCTLA4Ig) was generated using the strategy of Cre-lox recombination. Isolated rat islets infected with AdCTLA4Ig at multiplicities of infection (MOIs) ranging from 0.1 to 10 were transplanted into streptozocin diabetic male B6AF1 mice. Control islets were mock infected or infected with AdLacZ or AdsIg, a recombinant adenovirus expressing only the Fc portion of IgG2a. Also, AdCTLA4Ig and control viruses were injected intramuscularly into mouse transplant recipients at the time of islet transplantation to provide CTLA4Ig systemically. Control islets transplanted into diabetic mice were rejected in 13-17 days. Islets infected with AdCTLA4Ig had dose-dependent prolongation of graft survival. Prolonged survival was even found with very low MOIs of 0.1 and 0.5, with survivals of 24+/-4.2 and 25+/-2.2 days, respectively. Survival with an MOI of 10 was 39+/-8.7 days. With intramuscular injection, no prolongation was found at the lowest relative MOIs of 0.2 and 1, but there was dose-dependent prolongation of graft survival with larger doses. At the highest relative MOI of 400, survival was prolonged to 58+/-10 days. Rat islets infected with AdCTLA4Ig transplanted into mice had prolonged graft survival. Prolonged survival with MOIs as low as 0.1 and 0.5 indicates that only a minority of islet cells need to express CTLA4Ig to exert an effect. Moreover, the results suggest that the improved islet graft survival is due to a local influence of CTLA4Ig.