Long-term Islet Allograft Survival Research Articles

Berberine Promotes Induction of Immunological Tolerance to an Allograft via Downregulating Memory CD8+ T-Cells Through Altering the Gut Microbiota.

Emerging evidence has linked the gut microbiota dysbiosis to transplant rejection while memory T-cells pose a threat to long-term transplant survival. However, it's unclear if the gut microbiome alters the formation and function of alloreactive memory T-cells. Here we studied the effects of berberine, a narrow-spectrum antibiotic that is barely absorbed when orally administered, on the gut microbiota, memory T-cells, and allograft survival. In this study, C57BL/6 mice transplanted with islets or a heart from BALB/c mice were treated orally with berberine. Allograft survival was observed, while spleen, and lymph node T-cells from recipient mice were analyzed using a flow cytometer. High-throughput sequencing and qPCR were performed to analyze the gut microbiota. CD8+ T-cells from recipients were cultured with the bacteria to determine potential T-cell memory cross-reactivity to a specific pathogen. We found that berberine suppressed islet allograft rejection, reduced effector CD8+CD44highCD62Llow and central memory CD8+CD44highCD62Lhigh T-cells (TCM), altered the gut microbiota composition and specifically lowered Bacillus cereus abundance. Further, berberine promoted long-term islet allograft survival induced by conventional costimulatory blockade and induced cardiac allograft tolerance as well. Re-colonization of B. cereus upregulated CD8+ TCM cells and reversed long-term islet allograft survival induced by berberine plus the conventional costimulatory blockade. Finally, alloantigen-experienced memory CD8+ T-cells from transplanted recipients rapidly responded to B. cereus in vitro. Thus, berberine prolonged allograft survival by repressing CD8+ TCM through regulating the gut microbiota. We have provided the first evidence that donor-specific memory T-cell generation is linked to a specific microbe and uncovered a novel mechanism underlying the therapeutic effects of berberine. This study may be implicated for suppressing human transplant rejection since berberine is already used in clinic to treat intestinal infections.

Donor apoptotic cell-based therapy for effective inhibition of donor-specific memory T and B cells to promote long-term allograft survival in allosensitized recipients.

Allosensitization constitutes a major barrier in transplantation. Preexisting donor-reactive memory T and B cells and preformed donor-specific antibodies (DSAs) have all been implicated in accelerated allograft rejection in sensitized recipients. Here, we employ a sensitized murine model of islet transplantation to test strategies that promote long-term immunosuppression-free allograft survival. We demonstrate that donor-specific memory T and B cells can be effectively inhibited by peritransplant infusions of donor apoptotic cells in combination with anti-CD40L and rapamycin, and this treatment leads to significant prolongation of islet allograft survival in allosensitized recipients. We further demonstrate that late graft rejection in recipients treated with this regimen is associated with a breakthrough of B cells and their aggressive graft infiltration. Consequently, additional posttransplant B cell depletion effectively prevents late rejection and promotes permanent acceptance of islet allografts. In contrast, persistent low levels of DSAs do not seem to impair graft outcome in these recipients. We propose that B cells contribute to late rejection as antigen-presenting cells for intragraft memory T cell expansion but not to alloantibody production and that a therapeutic strategy combining donor apoptotic cells, anti-CD40L, and rapamycin effectively inhibits proinflammatory B cells and promotes long-term islet allograft survival in such recipients.

Hepatic Stellate Cells Enhance Liver Cancer Progression by Inducing Myeloid-Derived Suppressor Cells through Interleukin-6 Signaling.

The tumor microenvironment, which consists of fibroblasts, smooth muscle cells, endothelial cells, immune cells, epithelial cells, and extracellular matrices, plays a crucial role in tumor progression. Hepatic stellate cells (HSCs), a class of unique liver stromal cells, participate in immunomodulatory activities by inducing the apoptosis of effector T-cells, generation of regulatory T-cells, and development of myeloid-derived suppressor cells (MDSCs) to achieve long-term survival of islet allografts. This study provides in vitro and in vivo evidences that HSCs induce the generation of MDSCs to promote hepatocellular carcinoma (HCC) progression through interleukin (IL)-6 secretion. HSC-induced MDSCs highly expressed inducible nitric oxide synthase (iNOS) and arginase 1 mRNA and presented potent inhibitory T-cell immune responses in the tumor environment. Wild-type HSC-induced MDSCs expressed lower levels of CD40, CD86, and MHC II, and a higher level of B7-H1 surface molecules, as well as increased the production of iNOS and arginase I compared with MDSCs induced by IL-6-deficient HSCs in vitro. A murine-transplanted model of the liver tumor showed that HCCs cotransplanted with HSCs could significantly enhance the tumor area and detect more MDSCs compared with HCCs alone or HCCs cotransplanted with HSCs lacking IL-6. In conclusion, the results indicated that MDSCs are induced mainly by HSCs through IL-6 signaling and produce inhibitory enzymes to reduce T-cell immunity and then promote HCC progression within the tumor microenvironment. Therapies targeting the pathway involved in MDSC production or its immune-modulating pathways can serve as an alternative immunotherapy for HCC.

An anti-CD103 antibody-drug conjugate prolongs the survival of pancreatic islet allografts in mice

CD103 mediates T-cell infiltration and organ allograft rejection, and depletion of CD103-expressing cells is a promising therapeutic strategy for allograft intolerance. Recently, we verified that M290-MC-MMAF, an anti-CD103 antibody-drug conjugate, potently eliminates CD103-positive cells in vivo, with high specificity and minimal toxicity. However, the contribution of M290-MC-MMAF to blocking the CD103/E-cadherin pathway involved in transplant rejection remains unclear. Herein, we examined the impact of systemic administration of M290-MC-MMAF on allografts in an islet transplantation model. M290-MC-MMAF treatment maintained the long-term survival of islet allografts (>60 days) compared to mock injection or unconjugated M290 antibody treatment (<18 days). The change was associated with a decrease in CD103+CD8+ effector T cells and an increase in CD4+CD25+ regulatory T cells. CD103+CD8+ effector T-cell transfer or CD4+CD25+ regulatory T-cell depletion resulted in a rapid loss of allografts in long-surviving islet hosts. Moreover, M290-MC-MMAF treatment reduced IL-4, IL-6, and TNF-α expression levels and increased IL-10 expression in the grafts, which presented an immunosuppressive cytokine profile. In conclusion, targeting CD103 with M290-MC-MMAF induced immunosuppression and prolonged the survival of pancreatic islet allografts in mice, indicating the potential clinical value of M290-MC-MMAF in therapeutic interventions for allograft rejection.

Operational immune tolerance towards transplanted allogeneic pancreatic islets in mice and a non-human primate.

Patients with autoimmune type 1 diabetes transplanted with pancreatic islets to their liver experience significant improvement in quality of life through better control of blood sugar and enhanced awareness of hypoglycaemia. However, long-term survival and efficacy of the intrahepatic islet transplant are limited owing to liver-specific complications, such as immediate blood-mediated immune reaction, hypoxia, a highly enzymatic and inflammatory environment and locally elevated levels of drugs including immunosuppressive agents, all of which are injurious to islets. This has spurred a search for new islet transplant sites and for innovative ways to achieve long-term graft survival and efficacy without life-long systemic immunosuppression and its complications. We used our previously established approach of islet transplant in the anterior chamber of the eye in allogeneic recipient mouse models and a baboon model of diabetes, which were treated transiently with anti-CD154/CD40L blocking antibody in the peri-transplant period. Survival of the intraocular islet allografts was assessed by direct visualisation in the eye and metabolic variables (blood glucose and C-peptide measurements). We evaluated longitudinally the cytokine profile in the local microenvironment of the intraocular islet allografts, represented in aqueous humour, under conditions of immune rejection vs tolerance. We also evaluated the recall response in the periphery of the baboon recipient using delayed-type hypersensitivity (DTH) assay, and in mice after repeat transplant in the kidney following initial transplant with allogeneic islets in the eye or kidney. Results in mice showed >300days immunosuppression-free survival of allogeneic islets transplanted in the eye or kidney. Notably, >70% of tolerant mice, initially transplanted in the eye, exhibited >400days of graft survival after re-transplant in the kidney without immunosuppression compared with ~30% in mice that were initially transplanted in the kidney. Cytokine and DTH data provided evidence of T helper 2-driven local and peripheral immune regulatory mechanisms in support of operational immune tolerance towards the islet allografts in both models. We are currently evaluating the safety and efficacy of intraocular islet transplantation in a phase 1 clinical trial. In this study, we demonstrate immunosuppression-free long-term survival of intraocular islet allografts in mice and in a baboon using transient peri-transplant immune intervention. These results highlight the potential for inducing islet transplant immune tolerance through the intraocular route. Therefore, the current findings are conceptually significant and may impact markedly on clinical islet transplantation in the treatment of diabetes.

Preloading of CD4+CD25+ Tregs to donor islets in vitro prolongs islet allograft survival

Abstract Despite extensive studies on regulatory T cells (Treg) in transplantation (Tx), Treg transfer alone remains ineffective in wild-type (WT) animal recipients, because only a small fraction of systemically administered Tregs can reach an allograft. The objective of this study is to optimize Treg suppression of islet allograft rejection via preloading Tregs to donor islets before Tx. We developed a unique system in which donor islets were activated and incubated with CD4+CD25+ Treg cells, resulting in intraislet recruitment of Tregs before Tx. Donor islets were stimulated by cytokines (IL-1β+ IL-6 + IFNβ) for 24 h and incubated with CD4+CD25+FoxP3+ Tregs for 48 h so that Tregs infiltrated the islets. Islets then were rested in the media for another 24 h before Tx. We found that “Treg-preloaded” islet allografts survived much longer than control islets with long-term islet allograft survival in the hepatic parenchyma in 43% of WT recipient mice (n=7) without any immunosuppressive drug. Importantly, Treg-preloaded islet allografts also survived significantly longer when transplanted in the hepatic parenchyma than in kidney capsule (MST = 62 vs. 39 days, n=7–8, P &amp;lt; 0.05) or the portal vein (MST = 62 vs. 41 days, n=7, P &amp;lt; 0.05). As a control, cytokine-stimulated islets with the similar culture without Treg cells were rejected at the same pace as fresh control islets (14 vs. 16 days, n=7–8, P&amp;gt;0.05). Moreover, BrdU staining demonstrated that graft-infiltrating Thy1.1- T cells of recipients proliferated at a slower pace in islets preloaded with Tregs than in control islets (BrdU+: 21% vs. 43%) one week after Tx. In conclusion, transplanting Treg-preloaded islets in the hepatic parenchyma is a novel approach to prolonging islet allograft survival.

Targeted deletion of Traf2 allows immunosuppression-free islet allograft survival in mice.

Administration of anti-CD40 ligand (CD40L) antibodies has been reported to allow long-term islet allograft survival in non-human primates without the need for exogenous immunosuppression. However, the use of anti-CD40L antibodies was associated with thromboembolic complications. Targeting downstream intracellular components shared between CD40 and other TNF family co-stimulatory molecules could bypass these complications. TNF receptor associated factor 2 (TRAF2) integrates multiple TNF receptor family signalling pathways that are critical for T cell activation and may be a central node of alloimmune responses. T cell-specific Traf2-deficient mice (Traf2TKO) were generated to define the role of TRAF2 in CD4+ T cell effector responses that mediate islet allograft rejection in vivo. In vitro allograft responses were tested using mixed lymphocyte reactions and analysis of IFN-γ and granzyme B effector molecule expression. T cell function was assessed using anti-CD3/CD28-mediated proliferation and T cell polarisation studies. Traf2TKO mice exhibited permanent survival of full MHC-mismatched pancreatic islet allografts without exogenous immunosuppression. Traf2TKO CD4+ T cells exhibited reduced proliferation, activation and acquisition of effector function following T cell receptor stimulation; however, both Traf2TKO CD4+ and CD8+ T cells exhibited impaired alloantigen-mediated proliferation and acquisition of effector function. In polarisation studies, Traf2TKO CD4+ T cells preferentially converted to a T helper (Th)2 phenotype, but exhibited impaired Th17 differentiation. Without TRAF2, thymocytes exhibited dysregulated TNF-mediated induction of c-Jun N-terminal kinase (JNK) and canonical NFκB pathways. Critically, targeting TRAF2 in T cells did not impair the acute phase of CD8-dependent viral immunity. These data highlight a specific requirement for a TRAF2-NFκB and TRAF2-JNK signalling cascade in T cell activation and effector function in rejecting islet allografts. Targeting TRAF2 may be useful as a therapeutic approach for immunosuppression-free islet allograft survival that avoids the thromboembolic complications associated with the use of anti-CD40L antibodies.

Immunosuppression With CD40 Costimulatory Blockade Plus Rapamycin for Simultaneous Islet-Kidney Transplantation in Nonhuman Primates.

The lack of a reliable immunosuppressive regimen that effectively suppresses both renal and islet allograft rejection without islet toxicity hampers a wider clinical application of simultaneous islet-kidney transplantation (SIK). Seven MHC-mismatched SIKs were performed in diabetic cynomolgus monkeys. Two recipients received rabbit antithymocyte globulin (ATG) induction followed by daily tacrolimus and rapamycin (ATG/Tac/Rapa), and five recipients were treated with anti-CD40 monoclonal antibody (mAb) and rapamycin (aCD40/Rapa). Anti-inflammatory therapy, including anti-interleukin-6 receptor mAb and anti-tumor necrosis factor-α mAb, was given in both groups. The ATG/Tac/Rapa recipients failed to achieve long-term islet allograft survival (19 and 26 days) due to poor islet engraftment and cytomegalovirus pneumonia. In contrast, the aCD40/Rapa regimen provided long-term islet and kidney allograft survival (90, 94, >120, >120, and >120 days), with only one recipient developing evidence of allograft rejection. The aCD40/Rapa regimen was also tested in four kidney-alone transplant recipients. All four recipients achieved long-term renal allograft survival (100% at day 120), which was superior to renal allograft survival (62.9% at day 120) with triple immunosuppressive regimen (tacrolimus, mycophenolate mofetil, and steroids). The combination of anti-CD40 mAb and rapamycin is an effective and nontoxic immunosuppressive regimen that uses only clinically available agents for kidney and islet recipients.

Immunization with donor splenocytes in testis of recipient mice promotes induction of long-term survival of islet allografts transplanted in a conventional site (TRAN1P.920)

Abstract Immune responses are subdued in immune privileged sites, which provide a unique opportunity to prolong cellular allograft survival. We and others have previously shown that inducing tolerance of islet allografts transplanted in murine testes is easier than in conventional sites. However, it is not practical to transplant islets in human testes. Here we found that immunization of B6 recipients with irradiated Balb/C splenocytes through testicular injection, 7 and 14 days before islet transplantation under the kidney capsule, promotes long-term allograft survival induced by low doses of anti-CD40L (MR1) or CTLA4-Ig (0.1mg/day, days 0,2,4,6), with 75% islet allografts achieving long-term survival (&amp;gt;120 days). Same Ab treatment without the immunization failed to do so. Immunization without MR1 or CTLA4-Ig did not prolong allograft survival. We also found that islet transplantation under renal capsule of previously immunized recipients generates much less CD44+CD8+ memory T cells but induces more CD4+CD25+ Tregs than under renal capsule of unimmunized recipients. These Tregs exhibited donor-specific suppression of alloimmune responses in vitro. These findings reveal new mechanisms of immune privilege and may provide novel approaches to inducing long-term allograft survival or transplant tolerance in both immunologically privileged and unprivileged sites.

Enhancement of islet engraftment and achievement of long-term islet allograft survival by Toll-like receptor 4 blockade.

Toll-like receptors are key players in sterile inflammation phenomena and can link the innate and adaptive immune systems by enhancing graft immunogenicity. They are also considered mediators of types 1 and 2 diabetes development. The aim of the present study was to assess the role of Toll-like receptor-4 (TLR4) in mediating the inflammatory and immune responses to pancreatic islets, thereby promoting inflammatory destruction and immune rejection of islet grafts. Experiments were conducted in murine and human in vitro systems and in vivo murine islet transplant models, using species-specific anti-TLR4 monoclonal antibodies. In vitro, mixed lymphocyte-islet reaction experiments were performed to assess T-cell activation and proliferation. In vivo, both a syngeneic (B6-to-B6) marginal mass islet transplant model to assess the impact of TLR4 blockade on islet engraftment and an allogeneic (DBA1-to-B6) model were used. In vitro TLR4 blockade decreased lipopolysaccharide-mediated β-cell apoptosis and T-cell activation and proliferation against allogeneic islets. In vivo, TLR4 blockade resulted in significantly better syngeneic marginal mass islet engraftment and in indefinite allogeneic islet graft survival. Tolerance was not observed because donor-specific skin graft rechallenge in nonrejecting animals resulted in rejection of both skin and islets, but without accelerated rejection as compared to naive animals. Taken together, our data indicate that TLR4 blockade leads to a significant improvement of syngeneic islet engraftment and of allogeneic islet graft survival. A mechanism of graft accommodation with concurrent inhibition of donor-specific immune memory is likely to be involved.

Efficient elimination of CD103-expressing cells by anti-CD103 antibody drug conjugates in immunocompetent mice.

CD103 plays an important role in the destruction of islet allografts, and previous studies found that a CD103 immunotoxin (M290-Saporin, or M290-SAP) promoted the long-term survival of pancreatic islet allografts. However, systemic toxicity to the host and the bystander effects of M290-SAP obscure the underlying mechanisms of action and restrict its clinical applications. To overcome these shortcomings, anti-CD103 M290 was conjugated to different cytotoxic agents through cleavable or uncleavable linkages to form three distinct antibody-drug conjugates (ADCs): M290-MC-vc-PAB-MMAE, M290-MC-MMAF, and M290-MCC-DM1. The drug-to-antibody ratio (DAR) and the purity of the ADCs were determined by HIC-HPLC and SEC-HPLC, respectively. The binding characteristics, internalization and cytotoxicity of M290 and the corresponding ADCs were evaluated in vitro. The cell depletion efficacies of the various M290-ADCs against CD103-positive cells were then evaluated in vivo. The M290-ADCs maintained the initial binding affinity for the CD103-positive cell surface antigen and then quickly internalized the CD103-positive cell. Surprisingly, all M290-ADCs potently depleted CD103-positive cells in vivo, with high specificity and reduced toxicity. Our findings show that M290-ADCs have potent and selective depletion effects on CD103-expressing cells in immunocompetent mice. These data indicate that M290-ADCs could potentially serve as a therapeutic intervention to block the CD103/E-cadherin pathway.

B-Cell Depletion Improves Islet Allograft Survival with Anti-CD45RB

A short course of anti-CD45RB leads to long-term islet allograft survival and donor-specific tolerance in approximately half of immunocompetent mice. We have previously demonstrated that anti-CD45RB antibody-mediated tolerance requires B-cells for cardiac allograft survival. We therefore asked whether B-cells were also required for anti-CD45RB antibody-mediated survival of islets. Unexpectedly, we found that nearly 100% of islet allografts survive long term in B-cell-deficient mice. Similarly, B-cell depletion by anti-CD22/cal augmented anti-CD45RB-mediated tolerance when administered pretransplant, although it had no effect on tolerance induction when administered posttransplant. Our results demonstrate that the role of B-cells in promoting tolerance with anti-CD45RB is graft specific, promoting tolerance in cardiac grafts but resisting tolerance in islet transplantation. These findings may help elucidate the varied action of B-cells in promoting tolerance versus rejection.

Selective Depletion of Alloreactive T Cells Leads to Long-Term Islet Allograft Survival across a Major Histocompatibility Complex Mismatch in Diabetic Mice

Islet cell transplantation as a therapy for type 1 diabetes has been limited by progressive graft loss. Significant immunosuppression including T-cell ablation has been used in an attempt to limit islet rejection. Here, we show that CD3(+) lymphocytes depleted of alloreactive T cells selected from a mixed lymphocyte reaction (MLR), where responder BALB/c splenocytes stained with carboxyfluorescein succinimidyl ester (CFSE) were stimulated with irradiated C57BL/6 splenocytes for 5 days, infused into diabetic immunodeficient mice are capable of restoring a broad T-cell repertoire and specifically do not reject islet transplants from the strain (C57BL/6) used in the original depletion. These mice demonstrate reconstitution with CD4(+) and CD8(+) T cells, the capacity to reject third-party grafts (CBA), and restoration of interferon-γ (IFN-γ) responses to third-party alloantigens. Over time, both forkhead box P3-positive (Foxp3(+)) T regulatory cells (Tregs) and γδ T cells expand, suggesting a role for peripheral tolerance, in addition to the initial depletion of alloreactive T cells, in long-term islet graft survival. Our results suggest that immune restoration with CD3(+) lymphocytes where alloreactive T cells are removed can restore cognate immunity without islet allograft loss and recurrence of diabetes.

Novel sphingosine-1-phosphate receptor modulator KRP203 combined with locally delivered regulatory T cells induces permanent acceptance of pancreatic islet allografts.

KRP203, a structural FTY720 analogue, has 5-fold greater selectivity for binding to sphingosine-1-phosphate receptor (S1PR) 1 (S1PR(1)) versus S1PR3 and 100-fold greater selectivity over S1PR(2) and S1PR(5). Although the immunoregulatory effects of FTY720 have been tested in clinical and experimental research, the therapeutic efficacy of KRP203 in allograft models remains elusive. In this study, we investigated the potential of KRP203 alone and in combination with intragraft injection of CD4(+)CD25(+)FoxP3(+) regulatory T cells (Tregs) to induce islet allograft tolerance. BALB/c (H-2(d)) mice received transplants of fresh C57BL/10 (H-2(b)) islet allografts under the kidney capsule and were treated for 7 days with 0.3, 1.0, or 3.0 mg/kg KRP203 alone or in combination with intragraft-infused Tregs. Untreated BALB/c mice acutely rejected C57BL/10 islet allografts at a mean survival time of 13.8 ± 2.7 days (n=5). A 7-day dosing of 0.3 or 1.0 mg/kg KRP203 produced long-term islet allograft survival (9200 days) in one of five and two of seven recipients, respectively. A 3 mg/kg KRP203 dose resulted in islet graft survival for more than 200 days in 5 of 12 recipients. Whereas recipients that received 500 allogeneic islets admixed with 5 x 10(5) - 7 x 10(5) Tregssurvived 83.6 ± 67.2 days, addition of transient 3 mg/kg KRP203 therapy induced prolonged drug-free graft survival (9200 days) in all recipients. A brief treatment with KRP203 significantly prolonged islet allograft survival, whereas additional intragraft delivery of Tregs induced tolerogenic effects selective to islet alloantigens.

Roles of Toll-Like Receptors in Allogeneic Islet Transplantation

Toll-like receptors (TLRs) are involved in the rejection of solid organ allografts. However, the roles of TLRs in islets are still controversial. We investigated the roles of TLRs in donor islets together with those in recipients in allogeneic islet transplantation. To assess the roles of TLRs in either donor islets or recipients, allogeneic islet transplantation was performed using myeloid differentiation factor 88 (MyD88)-knockout (KO), TLR4-KO, or Toll/interleukin-1 receptor domain-containing adaptor-inducing interferon-β (TRIF)-KO mice. Both polyriboinosinic polyribocytidylic acid and lipopolysaccharide (LPS) stimulation induced the mRNA expression of regulated and normal T cell expressed and secreted, interferon-γ-inducible protein-10, monocyte chemotactic protein-1, interleukin-8, and inducible nitric oxide synthase in murine islets, whereas the induction was attenuated in TRIF-KO, interferon-β promoter stimulator-1-KO, and TLR4-KO mice. When islets from MyD88-KO, TLR4-KO, or TRIF-KO C57BL/6 mice were transplanted to BALB/c recipients, graft survival was not better than that of wild-type (WT) islets. However, the survival of the MyD88-KO islet allograft was significantly prolonged when combined with anti-CD40L. In parallel, LPS stimulation in donor islets interfered with anti-CD40L blockade-mediated long-term survival of islet allografts in TLR4-KO recipients. LPS stimulation increased the perigraft infiltration of both T cells and macrophages. Then again, when islets from WT BALB/c mice were transplanted to MyD88-KO, TRIF-KO, or WT C57BL/6 mice, there was no difference in graft survival, although some of the MyD88-KO recipients obtained long-term graft survival. However, anti-CD40L prolonged graft survival significantly in MyD88-KO recipients. The absence of MyD88 in either donors or recipients decreased the perigraft infiltration of inflammatory cells when combined with anti-CD40L. TLRs in both donor islets and recipients are involved in islet allograft rejection.

Short-Term Treatment of VCAM-1-Targeting Antibody Induces Immunological Tolerance Against Allogeneic Islet Grafts

Background: Blocking adhesion domain of VCAM-1 prevents trafficking of immune cells into the inflammatory sites, and thereby prolongs longterm survival of allogeneic islet grafts in mice. In this study, we developed novel antibody targeting the sixth non-adhesive immunoglobulin-like domain of VCAM-1 and evaluated its efficacy with allogeneic-islet transplantation model. Methods: Chimeric rabbit/human VCAM-1 antibody (anti-VCAM-1-D6) targeting Ig-like domain six of VCAM-1 was generated using phage display technology. Balb/c islets were transplanted underneath of the kidney capsule of diabetic C57BL/6 mouse with the transient anti-VCAM-1-D6 (100ug/day on day -1, 0,1,2,3,4,5,6,7 after transplantation) treatment. Antigen-specific tolerance induction was confirmed by second-party transplantation of Balb/c islets or tail skin. Results: Short-term treatment of anti-VCAM-1-D6 antibody led to the long-term survival of islet allografts in 5 out of 5 mice. Donor-specific-IL-2, IFN-r, Granzyme B- secreting cell population significantly decreased in VACM-1-D6 treated-mouse in ELISPOT analysis. In the grafts, peri-islet infiltrates of leukocytes were observed with intact insulin-positive beta cells in long-term survived mice. Importantly, secondary-islet allograft, but not secondary-skin graft, was survived without evident graft rejection for more than 80 days, meaning tissue-specific tolerance was induced by the short-term treatment of anti-VCAM-1 antibody. Conclusion: Developing VCAM-1 targeting antibody may be a promising therapeutic modality in inducing antigen-specific tolerance in allogeneic islet transplantation.

Use of Modified ET-Kyoto Solution (ET-Kyoto/Neutrophil Elastase Inhibitor) Significantly Improves Islet Yield and Survival

Introduction: Islet transplantation is a minimal invasive approach for β-cell replacement. However, a sufficient number of islets derived from two or more donor pancreas are usually required to achieve insulin independence. Thus, there is a need for novel strategies that increase islet yield, maintain high islet quality, and protect transplanted islet grafts. Indeed, the islet isolation procedure itself can lead to tissue destruction and activation of cellular and non-cellular components of the pancreas, including resident neutrophils, macrophages and T cells, which probably play an important role in impairment of islet survival. In the present study, we focused on the role of neutrophils, in particular neutrophil elastase (NE), against both islets during islet isolation and survival, because NE, released from activated neutrophils can directly cause tissue injury, and may also amplify inflammatory responses in acute organ injury. Objectives: The objectives of in this study were to determine whether the addition of NE inhibitor [sivelestat (ONO-5046)] to the islet isolation solution improves islet yield and viability. We also investigated the cytoprotective effects of sivelestat in islet recipients. Materials and methods:Islet isolation: Fresh islets were isolated from male C57BL/6 mice using either UW, UW containing 20 μM of sivelestat (ie: S-UW), ET-Kyoto or ET-Kyoto containing 20 μM of sivelestat (ie: S-Kyoto), respectively. Islet viability assay: To determine the amelioration of islet viability, TMRE assay was performed. Glucose-Stimulated Insulin Release: To determine the changes in the endocrinological potency of isolated islets, static glucose challenge was performed. Measurement of neutrophil elastase activity: To determine whether neutrophils are activated and release neutrophil elastase during pancreas digestion, neutrophil elastase activity was measured by the incubation with N-methoxysuccinyl-Ala-Ala-Pro-Val-p-nitroanilide as the substrate. Islet transplant experiments: The recipient 10-12-week-old male Balbc/A mice were rendered diabetic by a injection of streptozotocin. 500 of freshly isolated islets were transplanted under the kidney capsule. After transplantation, non-fasting blood glucose level was monitored. Moreover, to assess the beneficial effects of monotherapy with intraperitoneal sivelestat, sivelestat was administered at 100 mg/kg/day/for one day before transplantation and every day until 14 days after transplantation. To determine the effects of sivelestat administration on glucose tolerance, IPGTT was conducted at one week post-transplantation. Results: Data are summarized in the Table.[Tanemura et al. result table]Conclusion: We succeeded in isolating large numbers of islets using a new preservation solution, S-Kyoto solution, and in significant prolongation of islet graft survival in recipient mice treated with sivelestat. Therefore, treatment with NE inhibitors is potentially suitable for better harvest of transplantable islets and long-term islet allograft survival, allowing successful management of diabetes with islets from a single donor.

Biologic and immunomodulatory properties of mesenchymal stromal cells derived from human pancreatic islets

Mesenchymal stromal cells (MSC) have now been shown to reside in numerous tissues throughout the body, including the pancreas. Ex vivo culture-expanded MSC derived from many tissues display important interactions with different types of immune cells in vitro and potentially play a significant role in tissue homeostasis in vivo. In this study, we investigated the biologic and immunomodulatory properties of human pancreatic islet-derived MSC. We culture-expanded MSC from cadaveric human pancreatic islets and characterized them using flow cytometry, differentiation assays and nuclear magnetic resonance-based metabolomics. We also investigated the immunologic properties of pancreatic islet-derived MSC compared with bone marrow (BM) MSC. Pancreatic islet and BM-derived MSC expressed the same cell-surface markers by flow cytometry, and both could differentiate into bone, fat and cartilage. Metabolomics analysis of MSC from BM and pancreatic islets also showed a similar set of metabolic markers but quantitative polymerase chain reactions showed that pancreatic islet MSC expressed more interleukin(IL)-1b, IL-6, STAT3 and FGF9 compared with BM MSC, and less IL-10. However, similar to BM MSC, pancreatic islet MSC were able to suppress proliferation of allogeneic T lymphocytes stimulated with anti-CD3 and anti-CD28 antibodies. Our in vitro analysis shows pancreatic islet-derived MSC have phenotypic, biologic and immunomodulatory characteristics similar, but not identical, to BM-derived MSC. We propose that pancreatic islet-derived MSC could potentially play an important role in improving the outcome of pancreatic islet transplantation by promoting engraftment and creating a favorable immune environment for long-term survival of islet allografts.

CTLA4Ig Prevents Alloantibody Formation Following Nonhuman Primate Islet Transplantation Using the CD40-Specific Antibody 3A8

Islet transplantation to treat type 1 diabetes has been limited in part by toxicities of current immunosuppression and recipient humoral sensitization. Blockade of the CD28/CD80/86 and CD40/CD154 pathways has shown promise to remedy both these limitations, but translation has been hampered by difficulties in translating CD154-directed therapies. Prior CD40-directed regimens have led to prolonged islet survival, but fail to prevent humoral allosensitization. We therefore evaluated the addition of CTLA4Ig to a CD40 blockade-based regimen in nonhuman primate (NHP) alloislet transplantation. Diabetic rhesus macaques were transplanted allogeneic islets using the CD40-specific antibody 3A8, basiliximab induction, and sirolimus with or without CTLA4Ig maintenance therapy. Allograft survival was determined by fasting blood glucose levels and flow cytometric techniques were used to test for donor-specific antibody (DSA) formation. CTLA4Ig plus 3A8, basiliximab and sirolimus was well tolerated and induced long-term islet allograft survival. The addition of CTLA4Ig prevented DSA formation, but did not facilitate withdrawal of the 3A8-based regimen. Thus, CTLA4Ig combines with a CD40-specific regimen to prevent DSA formation in NHPs, and offers a potentially translatable calcineurin inhibitor-free protocol inclusive of a single investigational agent for use in clinical islet transplantation without relying upon CD154 blockade.

Murine Islet Allograft Tolerance Upon Blockade of the B-Lymphocyte Stimulator, BLyS/BAFF

Immunologic rejection is a major barrier to successful long-term outcomes in clinical transplantation. The importance of B lymphocytes-and their secretory products, alloantibodies-in the pathogenesis of allograft rejection is accepted. Furthermore, it is now clear that the dominant regulator of peripheral B-cell homeostasis and tolerance is the B-lymphocyte stimulator (BLyS), also referred to as the B-cell activating factor (BAFF). Recently, a novel class of clinical immunotherapeutic agents specific for BLyS, and its family of cytokines, has emerged for the treatment of B-cell-mediated diseases. In this study, we demonstrate the potential utility of BLyS-directed immunotherapy in preventing allograft rejection using a murine islet transplantation model. A transient period of mature peripheral B-cell depletion was induced by means of in vivo BLyS neutralization using a murine analog of the monoclonal antibody, Benlysta. Subsequently, fully major histocompatibility complex-mismatched islets were transplanted into naïve diabetic mice followed by a short course of rapamycin. After BLyS neutralization, indefinite islet allograft survival was achieved. Induction therapy with rapamycin was necessary, but not sufficient, for the achievement of this long-term graft survival. The tolerant state was associated with (1) abrogation of the donor-specific antibody response, (2) transient preponderance of immature/transitional B cells in all lymphoid organs, (3) impaired CD4 T-cell activation during the period of B-cell depletion, and (4) presence of a "regulatory" cytokine milieu. In vivo BLyS neutralization effectively induces humoral tolerance and promotes long-term islet allograft survival in mice. Therefore, B-lymphocyte-directed immunotherapy targeting the homeostatic regulator, BLyS, may be effective in promoting transplantation tolerance.