Islet Allografts Research Articles

MicroRNA-21 silencing prolongs islet allograft survival by inhibiting Th17 cells

It has been reported that microRNA-21 (miR-21) augments Th17 responses and contributes to the pathogenesis of autoimmune diseases. Gene knockout or siRNA-induced knockdown of miR-21 in mice resulted in impaired Th17 differentiation and strong resistance to experimental autoimmune encephalomyelitis (EAE). Recently, we validated the miR-21 target IL-10 mRNA and showed that it exerts a pro-inflammatory role by inhibiting IL-10-expressing regulatory B cell (B10) differentiation. The administration of miR-21 antisense oligonucleotides (antagomiR-21) in vivo potently suppressed the severity of EAE, and the suppressive activity was mediated by an increased number of B10 cells. However, the contribution of the miR-21 pathways involved in transplant rejection remains obscure. In this study, we examined the impact of systemic administration of miR-21 inhibitor on allografts in a pancreatic islet transplantation model. We showed that specific miR-21 silencing in vivo significantly prolonged allograft survival (median survival time (MST) 21 days with antagomiR-21 vs 13 days for the control, p < 0.05), and the change was associated with a decrease in Th17 cells (~3-fold) and an increase in B10 cells (~2.4-fold). Moreover, we found that miR-21-silenced B cells mediate this protective role through pro-inflammatory Th17 responses in an IL-10-dependent fashion. Thus, we have revealed a novel mechanistic pathway that modulates Th17 development and alloimmunity. Targeting miR-21 may represent a valuable therapeutic intervention strategy for preventing transplant rejection.

Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Immunodeficient mice engrafted with human peripheral blood mononuclear cells (PBMCs) support preclinical studies of human pathogens, allograft rejection, and human T-cell function. However, a major limitation of PBMC engraftment is development of acute xenogeneic graft- versus-host disease (GVHD) due to human T-cell recognition of murine major histocompatibility complex (MHC). To address this, we created 2 NOD- scid IL-2 receptor subunit γ ( IL2rg) null (NSG) strains that lack murine MHC class I and II [NSG-β-2-microglobulin ( B2M) null ( IA IE)null and NSG -( Kb Db) null ( IAnull)]. We observed rapid human IgG clearance in NSG- B2Mnull ( IA IE) null mice whereas clearance in NSG -( Kb Db) null ( IAnull) mice and NSG mice was comparable. Injection of human PBMCs into both strains enabled long-term engraftment of human CD4+ and CD8+ T cells without acute GVHD. Engrafted human T-cell function was documented by rejection of human islet allografts. Administration of human IL-2 to NSG -( Kb Db) null ( IAnull) mice via adeno-associated virus vector increased human CD45+ cell engraftment, including an increase in human regulatory T cells. However, high IL-2 levels also induced the development of GVHD. These data document that NSG mice deficient in murine MHC support studies of human immunity in the absence of acute GVHD and enable evaluation of human antibody therapeutics targeting human T cells.-Brehm, M. A., Kenney, L. L., Wiles, M. V., Low, B. E., Tisch, R. M., Burzenski, L., Mueller, C., Greiner, D. L., Shultz, L. D. Lack of acute xenogeneic graft- versus-host disease, but retention of T-cell function following engraftment of human peripheral blood mononuclear cells in NSG mice deficient in MHC class I and II expression.

Mesenchymal stem cells in combination with low-dose rapamycin significantly prolong islet allograft survival through induction of regulatory T cells

We previously demonstrated the protective effect of MSCs in an adaptive transfer mouse model. However, their therapeutic potential in an allogeneic immunocompetent setting mimicking clinical context of islet transplantation remained unknown. The aim of this study was to determine whether MSCs therapy, either by itself, or combined with Rapamycin could benefit the allograft survival of fully MHC-mismatched mouse islet transplant. Combination therapy of MSCs and low-dose Rapamycin significantly prolonged the survival of islet allografts, whereas treatment of MSCs, or Rapamycin alone, had no impact. Interestingly, this protective effect was associated with an induced expansion of regulatory T cells in islet grafts and draining lymph nodes, a skewed T-cell differentiation toward immunotolerance, and a profound suppression of alloreactivity against donor antigen. Our study suggests that a combination therapy of MSCs and low-dose Rapamycin can prolong the survival and preserve the function of islet allograft in the MHC-mismatched mouse model of islet transplantation.

Receptor tyrosine kinase MerTK suppresses an allogenic type I IFN response to promote transplant tolerance.

Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.

The Fate of Allogeneic Pancreatic Islets following Intraportal Transplantation: Challenges and Solutions.

Pancreatic islet transplantation as a therapeutic option for type 1 diabetes mellitus is gaining widespread attention because this approach can restore physiological insulin secretion, minimize the risk of hypoglycemic unawareness, and reduce the risk of death due to severe hypoglycemia. However, there are many obstacles contributing to the early mass loss of the islets and progressive islet loss in the late stages of clinical islet transplantation, including hypoxia injury, instant blood-mediated inflammatory reactions, inflammatory cytokines, immune rejection, metabolic exhaustion, and immunosuppression-related toxicity that is detrimental to the islet allograft. Here, we discuss the fate of intrahepatic islets infused through the portal vein and propose potential interventions to promote islet allograft survival and improve long-term graft function.

Transcriptomic Analysis Reveals Novel Mechanisms Mediating Islet Dysfunction in the Intrauterine Growth-Restricted Rat

Intrauterine growth restriction (IUGR) increases the risk of type 2 diabetes developing in adulthood. In previous studies that used bilateral uterine artery ligation in a rat model of IUGR, age-associated decline in glucose homeostasis and islet function was revealed. To elucidate mechanisms contributing to IUGR pathogenesis, the islet transcriptome was sequenced from 2-week-old rats, when in vivo glucose tolerance is mildly impaired, and at 10 weeks of age, when rats are hyperglycemic and have reduced β-cell mass. RNA sequencing and functional annotation with Ingenuity Pathway Analysis revealed temporal changes in IUGR islets. For instance, gene expression involving amino acid metabolism was significantly reduced primarily at 2 weeks of age, but ion channel expression, specifically that involved in cell-volume regulation, was more disrupted in adult IUGR islets. Additionally, we observed alterations in the microenvironment of IUGR islets with extracellular matrix genes being significantly increased at 2 weeks of age and significantly decreased at 10 weeks. Specifically, hyaluronan synthase 2 expression and hyaluronan staining were increased in IUGR islets at 2 weeks of age (P < 0.05). Mesenchymal stromal cell-derived factors that have been shown to preserve islet allograft function, such as Anxa1, Cxcl12, and others, also were increased at 2 weeks and decreased in adult islets. Finally, comparisons of differentially expressed genes with those of type 2 diabetic human islets support a role for these pathways in human patients with diabetes. Together, these data point to new mechanisms in the pathogenesis of IUGR-mediated islet dysfunction in type 2 diabetes.

Extending Remission and Reversing New-Onset Type 1 Diabetes by Targeted Ablation of Autoreactive T Cells.

Preserving endogenous insulin production is clinically advantageous and remains a vital unmet challenge in the treatment and reversal of type 1 diabetes. Although broad immunosuppression has had limited success in prolonging the so-called remission period, it comes at the cost of compromising beneficial immunity. Here, we used a novel strategy to specifically deplete the activated diabetogenic T cells that drive pathogenesis while preserving not only endogenous insulin production but also protective immunity. Effector T (Teff) cells, such as diabetogenic T cells, are naturally poised on the edge of apoptosis because of activation-induced DNA damage that stresses the p53 regulation of the cell cycle. We have found that using small molecular inhibitors that further potentiate p53 while inhibiting the G2/M cell cycle checkpoint control drives apoptosis of activated T cells in vivo. When delivered at the onset of disease, these inhibitors significantly reduce diabetogenic Teff cells, prolong remission, preserve functional islets, and protect islet allografts while leaving naive, memory, and regulatory T-cell populations functionally untouched. Thus, the targeted manipulation of p53 and cell cycle checkpoints represents a new therapeutic modality for the preservation of islet β-cells in new-onset type 1 diabetes or after islet transplant.

A Simple and Promising Strategy for Pancreatic Islet Preconditioning to Reduce Immunogenicity and Improve Islet Graft Longevity

Introduction An advantage of pancreatic islet transplantation is that the pretransplant in vitro culture period provides an opportunity for cell modification; however, no “islet preconditioning” method has been established yet. We previously reported that the pretreatment of donor islets with an antitumoral antibiotic, mitomycin-C (MMC), and subsequent culture significantly prolonged graft survival in murine renal subcapsular islet transplantations. Here, we introduce an “islet preconditioning” strategy for reducing the immunogenicity of islets and for preventing the activation of proinflammatory events by using MMC in an intraportal islet transplantation model. Methods Freshly isolated BALB/c islets, either untreated or treated for 30 min with 10 μg/mL MMC, were cultured for 20 hours. Subsequently, 600 allogenic islets were transplanted via the portal vein into the liver of C57BL/6N diabetic mice. Recipients did not receive any immunosuppressive treatment. To demonstrate the specific mechanism responsible for prolonging graft survival by islet preconditioning, immunohistochemical studies and inflammatory cytokine assays were conducted. Microarrays were employed to comprehensively analyze the genes expressed in untreated or MMC-treated islets that were subsequently cultured and to identify the biological processes that were most affected by islet preconditioning. Results On islet transplantation, the allograft was rejected by all control mice (n = 11), with a mean survival time (MST) of 13.0 ± 8.3 days. Islet conditioning with MMC significantly prolonged islet allograft survival to an MST of 41.4 ± 7.3 days (n = 14) (P < 0.005). Notably, 42.8% of the recipients accepted islet allografts pretreated with MMC for more than 60 days without any immunosuppression. As shown by immunohistochemical studies, compared with untreated grafts, MMC-treated grafts showed a mild inflammatory cell response and minimal infiltration of immunocompetent cells. In an in vitro study, the expression of IL-6, MCP-1, ICAM-1, and IL-1β was significantly decreased in MMC-treated islets compared with untreated islets. Microarray gene expression analysis identified a significant downregulation of annotated functions associated with the movement of cells and revealed significant downregulation of multiple genes encoding proinflammatory mediators with chemotactic activity. Validation studies also showed that MMC-treated islets secreted lower levels of chemoattractants that reduced the immunogenic potential of islets. Conclusion Islet preconditioning with MMC and subsequent culture can prolong graft survival in intraportal islet transplantations by inhibiting the activation of proinflammatory events and by provoking the suppression of chemoattractants that are responsible for inducing unresponsiveness to islet grafts. This simple and promising islet preconditioning method could be an effective strategy inducing a breakthrough in establishing advanced islet transplantation.

Low Dose IL-2 and Rapamycin Leads to Prolongation of Human Islet Allograft Survival by Inhibition of IFN-gamma+ T cells and Expansion of FOXP3+CD25+ Tregs in a Humanized Islet Transplant Mouse Model

Low Dose IL-2 and Rapamycin Leads to Prolongation of Human Islet Allograft Survival by Inhibition of IFN-gamma+ T cells and Expansion of FOXP3+CD25+ Tregs in a Humanized Islet Transplant Mouse Model

Abstract 5665: NOD-scid IL2rg null (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC

Abstract The study of human immune system-tumor interactions has been limited historically by a paucity of models that fully reproduce human immune system function. Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID mice) has supported studies of human infectious agents, allograft rejection, and human T cell immune function. The Hu-PBL-SCID model has also supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic graft-versus-host disease (GVHD) due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnull with NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(Kb Db)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull (IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(Kb Db)null (IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to engraftment of human T cells that exhibited an approximate 3:1 CD4:CD8 ratio with long term engraftment and without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Administration of human recombinant IL2 using AAV-IL2 to NSG-(Kb Db)null (IAnull) mice resulted in increased human CD45 cell engraftment, including heightened levels of human Tregs. However, high IL2 levels led to the development of acute GVHD. Moreover, NSG-(Kb Db)null (IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II can support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model for evaluation of human antibody therapeutics. Citation Format: Michael A. Brehm, Michael Wiles, Laurie Kenney, Ben Low, Roland M. Tisch, Lisa Burzenski, James G. Keck, Dale L. Greiner, Leonard D. Shultz. NOD-scid IL2rgnull (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5665.

Acceptance of Murine Islet Allografts without Immunosuppression in the Inguinal Subcutaneous White Adipose Tissue Pretreated with bFGF

Prevention of immune rejection without immunosuppression is a goal for transplant immunology. Previous report demonstrated that rejection of rat islet allografts in sc space of the back between low responder strains was prevented by pretreatment of the site with bFGF (AJT2014). However, it was not in the high responder combination of mice (BALB/c to C57BL/6). Here, we determined whether islet allografts in the inguinal subcutaneous white adipose tissue (ISWAT), a novel site of islet transplantation (tx), pretreated with bFGF are accepted without immunosuppression in mice. An agarose rod containing bFGF was buried in the ISWAT of a C57BL/6 mouse, and 1-3 weeks later 400 BALB/c islets (2 donors) were grafted in the space after the removal of a rod. Recipients were made diabetic by STZ (180mg/kg, iv) at 3 days prior to tx. Eventually, we found that 10μg bFGF/50μl/rod with 2 week interval is the optimal, in which 11/14 recipient mice remained normoglycemic (&amp;lt;200mg/dl) for more than 60 days. Removal of the ISWAT with islets promptly made recipient mice hyperglycemic again. Histologically, intact islets were seen in the ISWAT. In contrast, islet allografts (n=7) in the ISWAT pretreated with vehicle were rejected by 10 days. FACS revealed an expansion of CD8 T cells in the ISWAT rejecting but not accepting islet allografts. In the latter, IL-10+cells including Gr-1+ cells were seen. Re-tx of BALB/c islets (n=3) to the contralateral ISWAT of normoglycemic mice induced rejection of the initial BALB/c grafts and made recipient mice hyperglycemic again, while that of C3H (n=3) did not. These findings indicate that acceptance of murine islet allografts without immunosuppression was achieved when the ISWAT pretreated with bFGF was the site and that the acceptance appeared mediated by local rather than systemic unresponsiveness. Disclosure Y. Nakafusa: None. N. Nitta: None. M. Nakamura: None. Y. Yasunami: None.

Immunosuppressive PLGA TGF-ß1 Microparticles Induce Antigen-Specific T-Cell Tolerance for Enhancing Islet Transplantation Outcomes

Clinical islet transplantation (CIT) is a potential therapy for type 1 diabetes. Allorejection mediated by the host CD4 T cells is a major hurdle for successful CIT, despite potent systemic immunosuppression (SI). To address these issues, we engineered PLGA microparticles (PLGA-MPs) that allow the localized delivery of TGF-β1 for regulatory T cells (TRegs) induction at the transplant site. TGF-β1 is an immunoregulatory agent that promotes the TRegs differentiation from naïve CD4 cells by inducing FoxP3 expression while suppressing inflammatory Th1 and Th2 responses. To date, we have (i) fabricated TGF-β1 loaded PLGA particles by double emulsion (PVA(aq)- CH2Cl2- PVA(aq)) with characterization in size (45±26 μm), TGF-β1 encapsulation efficiency (49.1±0.1%), and release kinetics (durable biphasic release for 14 days); (ii) proved effective TRegs conversion in OVA-OTII antigen-specific murine model (Fig.1); and (iii) shown efficacy for in vivo Treg conversion by MPs in rodent islet allografts, where accepted grafts showed improved insulin production and elevated TRegs. Future studies exploring the competency of PLGA-MPs to provide durable allograft protection in rodent models is on-going. Based on these promising preliminary results, TGF-β1 PLGA-MPs could provide a translational therapeutic strategy to minimize islet graft rejection and the dependency on SI. Disclosure Y. Li: None. A. Frei: Employee; Self; Medline Industries, Inc.. S.D. Barash: None. C.L. Stabler: None.

Long-Term Function of Islet Allografts Transplanted on the Omentum Using a Biological Scaffold

The liver is the preferred site for islet transplantation (ITx) but not ideal due to limitations affecting engraftment. We evaluated the safety and efficacy of ITx in the omentum using a resorbable biological scaffold in 3 subjects with type 1 diabetes and negative C-peptide. Islets were combined with autologous plasma and thrombin to generate a biologic scaffold and layered laparoscopically on the omentum. Induction was with anti-thymocyte globulin and etanercept. Maintenance was with mycophenolate sodium and tacrolimus. Demographics, islet dose and metabolic data are shown in the Table.Subject #Age (years)Duration T1DM (years)BMI (Kg/m2)IEQ†/KgMMTT‡ Stimulated Glucose (mg/dl)/C-peptide (ng/ml)Insulin dose U/Kg/day (Units/day)HbA1c%6 months12 months24 monthsPre-ITxLatestPre-ITxLatest1432621.511,386181/3.32277/1.79317/0.490.62 (33)0.14 (7)6.85.32321625.39,635372/0.88374/0.65332/0.52*0.45 (31)0.45 (30)5.75.6346422412,648277/2.47--0.45 (20)0.2 (11)6.36.3* 18 months Post-ITx † IEQ= islet equivalents ‡ MMTT = mixed meal tolerance test Subject 1 was insulin independent for 15 months and maintains stable glycemic control. Subject 2 had marginal graft function with persistence of severe hypoglycemia (SH) and then underwent intrahepatic ITx resulting in insulin independence. Subject 3 had a 55% reduction in insulin dose and maintains excellent glycemic control. Our initial experience demonstrates feasibility and safety of ITx on the omentum. Graft function persisted throughout follow-up (6-24 months) resulting in improved glycemic control and absence of SH (subjects 1 and 3). Results suggest a significant loss of islets early post-ITx followed by gradual functional decline similar to intrahepatic ITx. Strategies to improve oxygen delivery and neo-vascularization and minimize immunosuppression are needed to improve long-term outcomes at this site. Disclosure D. Baidal: None. C. Ricordi: None. D.M. Berman: Stock/Shareholder; Self; Pfizer Inc. A. Pileggi: Stock/Shareholder; Self; Converge Biotech, Inc.. Stock/Shareholder; Spouse/Partner; Converge Biotech, Inc. A.M. Alvarez Gil: None. N. Padilla: None. G. Ciancio: None. E. Linetsky: None. R. Alejandro: None.

Persistence of Allosensitization after Islet Allograft Failure

Allosensitization (PRA+) has been reported after discontinuation of immunosuppression (IS) following graft failure (GF) in islet transplant (ITx) recipients. Persistence of PRA+ after GF is unknown. Thirteen ITx alone (IA) and 5 ITx subjects who also received donor CD34+ hematopoietic stem cells (IBM) and developed GF enrolled in the study “Allosensitization After Islet Transplantation.” Panel reactive antibodies (PRA) were measured yearly while having graft function, within 1 year post-GF and yearly after study enrollment. Two subjects who developed GF but remained on mycophenolate mofetil (MMF) for 2 years and 18 subjects with persistent graft function (15 IA and 3 islet after kidney [IAK]) were also evaluated. 80% IBM subjects developed high PRA+ (&amp;gt;89%) (Figure 1a) while 70% IA subjects were PRA+ at enrollment (0.8-6 years post-GF) and 42%-55% were PRA+ throughout follow-up. PRA results were variable (Figure 1b). The 2 subjects taking MMF post-GF and 1 subject who required IVIG pre-GF (for parvovirus infection) were not PRA+. Four subjects with persistent graft function by 10 years became PRA+ (2 IA and 2 IAK; Figure 1c). After stopping IS, 70% IA subjects became PRA+ with persistence of PRA (although variable) for at least 10 years post-GF. PRA+ was higher in IBM compared to IA subjects. While receiving IS, PRA+ was minimal. Persistence of PRA+ remains a concern due to potentially prolonged wait list time for subsequent organ transplantation if needed. Disclosure P. Rios: None. D. Baidal: None. N. Padilla: None. A.M. Alvarez Gil: None. S. Madiraju: None. J. Ambut: None. A.M. Mantero: None. S. Messinger Cayetano: None. C. Ricordi: None. R. Alejandro: None.

Prevention of autoimmune diabetes and islet allograft rejection by beta cell expression of XIAP: Insight into possible mechanisms of local immunomodulation.

Overexpression of the X-linked inhibitor of apoptosis (XIAP) prevents islet allograft rejection. We constructed an adeno-associated virus expressing XIAP driven by the rat insulin promoter (dsAAV8-RIP-XIAP) for long-term beta-cell gene expression in vivo. Pancreatic delivery of dsAAV8-RIP-XIAP prevented autoimmune diabetes in 70% of non-obese diabetic (NOD) mice, associated with decreased insulitis. Islets from Balb/c mice transduced with dsAAV8-RIP-XIAP were protected following transplantation into streptozotocin (STZ)-diabetic Bl/6 recipients, associated with decreased graft infiltration. Interestingly, dsAAV8-RIP-XIAP transduction induced expression of lactate dehydrogenase (LDHA) and monocarboxylate transporter 1 (MCT1), two genes normally suppressed in beta cells and involved in production and release of lactate, a metabolite known to suppress local immune responses. Transduction of Balb/c islets with AAV8-RIP-LDHA-MCT1 tended to prolong allograft survival following transplant into STZ-diabetic Bl/6 recipients. These findings suggest that XIAP has therapeutic potential in autoimmune diabetes and raise the possibility that local lactate production may play a role in XIAP-mediated immunomodulation.

Local immunomodulation Fas ligand-engineered biomaterials achieves allogeneic islet graft acceptance.

Islet transplantation is a promising therapy for type 1 diabetes. However, chronic immunosuppression to control rejection of allogeneic islets induces morbidities and impairs islet function. T-effector cells are responsible for islet allograft rejection and express Fas death receptor following activation, becoming sensitive to Fas-mediated apoptosis. Here, we report that localized immunomodulation using microgels presenting an apoptotic form of Fas ligand (SA-FasL) results in prolonged survival of allogeneic islet grafts in diabetic mice. A short course of rapamycin treatment boosted the immunomodulatory efficacy of SA-FasL-microgels, resulting in acceptance and function of allografts over 200 days. Survivors generated normal systemic responses to donor antigens, implying immune privilege of the graft, and had increased CD4+CD25+FoxP3+ T-regulatory cells in the graft and draining lymph nodes. Deletion of T-regulatory cells resulted in acute rejection of established islet allografts. This localized immunomodulatory biomaterial-enabled approach may provide an alternative to chronic immunosuppression for clinical islet transplantation.

A Novel Subcutaneous Site of Islet Transplantation Superior to the Liver

Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved. We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice. In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice. Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Novel Consistently Ex-vivo Expanded CD8+CD25+FoxP3+Treg Cells Prolongs the Allogeneic Islet Survival

Abstract In addition to CD4+ regulatory T cells(Tregs), adoptive transfer of CD8+CD25+FoxP3+T cells is emerging as an alternative adjunctive therapy to diminish current reliance on lifelong, nonspecific immunosuppression after transplantation. Here we evaluated the possible therapeutic application of consistently ex-vivo expanded mouse CD8+CD25+FoxP3+T cells to prevent the rejection of allogeneic islets. Around 80% of mouse CD8+CD25+T and CD4+CD25+T cells expressed FoxP3+T cells in spleen. Higher level of FoxP3 was expressed in CD8+Tregs compared to CD4+Tregs. Ex-vivo enriched CD8+CD25+Treg cells were successfully and consistently expanded to 600–1,000 times from the initial culture for 12 days with optimized culture conditions. High concentration of IL-2 (&amp;gt;500units) and TGF-beta together with aCD3-and aCD28 stimulation were essential to the massive ex-vivo expansion. Expanded Tregs showed 99% of FoxP3 expression and expressed high level of CD27, CD47 and Lag-3 in their surface. Very high amount of Granzyme B and minor amount of IFN-r and IL-17 were xpressed. Expanded CD8+Tregs successfully inhibits the proliferation and activation of CD4+T cells in vitro. In the allogenic islet transplantation model (Balb.Cà B6), Single injection of ex-vivo expanded CD8+Tregs prolonged islet allograft survival more than 100 days after the reconstitution with CD90.2+T cells in immunodeficient B6 rag2(−/−) mice by inhibiting antigen-specific IFN-r producing cell generation. In this study, we proved that ex-vivo expanded CD8+Treg retain regulatory activity and can protect islet allograft rejection by single administration. These implies that ex-vivo expanded CD8+Tregs has clinical relevance of an adjunctive cellular therapy.

Extending remission and reversing new onset type 1 diabetes by targeted ablation of autoreactive T cells

Abstract Preserving endogenous insulin production is clinically advantageous and remains a vital unmet challenge in the treatment and reversal of type 1 diabetes (T1D). While broad immunosuppression has had limited success in prolonging the so-called remission period, it comes at the cost of compromising beneficial immunity. Here, we employed an alternate strategy to specifically deplete the activated diabetogenic T cells that drive pathogenesis while preserving not only endogenous insulin production but also protective immunity. Effector T cells, such as diabetogenic T cells, are naturally poised on the edge of apoptosis because of activation-induced DNA damage that stresses the p53 regulation of cell cycle. We have found that the combined use of small molecular inhibitors that further potentiates p53 while inhibiting G2/M cell cycle checkpoint control drives apoptosis of activated T cells in vivo. When delivered at the onset of T1D these inhibitors eliminate diabetogenic effector T cells, prolong remission, preserve functional islets, and protect islet allografts while leaving naïve, memory, and regulatory T cell populations functionally untouched. Thus, the targeted manipulation of p53 and cell cycle checkpoints represents a new therapeutic modality for the preservation of islet beta cells in new onset type 1 diabetes or after islet transplant.

NOD-scid IL2rg null (NSG) mice deficient in murine MHC Class I and Class II expression support engraftment of functional human T cells in the absence of acute xenogeneic GVHD following injection of PBMC

Abstract Humanized mice are emerging as an exciting platform to study human immuno-oncology and provide preclinical tools to test cutting edge immunotherapies. An effective and simplistic approach to engraft immunodeficient mice with functional human T cells is the injection of human peripheral blood mononuclear cells (PBMC). This PBMC engraftment model (termed Hu-PBL-SCID) has supported studies of human immune system-tumor interactions following injection of human PBMC into immunodeficient mice implanted with patient-derived (PDX) tumors. However, a major limitation of this model is the development of acute xenogeneic GVHD due to human T cell recognition of murine MHC class I and class II. To address this limitation, we created two strains of NSG mice that lack murine MHC antigens, one by crossing NSG-B2Mnullwith NSG-(IA IEnull) mice, the second by knocking out the MHC class II IAb gene in NSG-(KbDb)null mice using TALEN technology. We observed that human IgG clearance was rapid in NSG-B2Mnull(IA IEnull) mice that lack functional FcRn activity whereas clearance in NSG-(KbDb)null(IAnull) mice was comparable to that observed in NSG mice. Injection of human PBMC into both strains led to long term engraftment of human T cells without development of acute GVHD. The engrafted human T cells were functional as documented by their ability to reject human islet allografts. Moreover, NSG-(KbDb)null(IAnull) mice injected with a PDX tumor and PBMC from an allogeneic donor supported significant tumor growth in the absence of acute GVHD. These data document that NSG mice deficient in murine MHC class I and class II support studies of human immune system-tumor interactions in the absence of acute GVHD and provide a model to evaluate human antibody therapeutics.