Prolong Islet Allograft Survival Research Articles

Localized cytotoxic T cell–associated antigen 4 and antioxidant islet encapsulation alters macrophage signaling and induces regulatory and anergic T cells to enhance allograft survival

The loss of functional β-cell mass is a hallmark of type 1 diabetes. Islet transplantation represents a promising alternative approach, but immune-mediated graft destruction remains a major challenge. We sought to use islet encapsulation technologies to improve graft survival and function without systemic immunosuppression. We hypothesized islet encapsulation with nanothin coatings consisting of tannic acid (TA), an antioxidant; poly(N-vinylpyrrolidone) (PVPON), a biocompatible polymer; and cytotoxic T cell–associated antigen 4 immunoglobulin (CTLA-4-Ig), an inhibitory immune receptor, will elicit localized immunosuppression to prolong islet allograft function and suppress effector T cell responses. In the absence of systemic immunosuppression, we demonstrated (PVPON/TA/CTLA-4-Ig)-encapsulated NOD.Rag islet grafts maintain function significantly longer than control IgG-containing (PVPON/TA/IgG) and nonencapsulated controls after transplantation into diabetic C57BL/6 mice. This protection coincided with diminished proinflammatory macrophage responses mediated by signal transducer and activator of transcription 1 signaling, decreased proinflammatory T cell effector responses, and CTLA-4-Ig-specific concomitant increases in anergic CD4+ T cells and regulatory T cells. Our results provide evidence that conjugation of CTLA-4-Ig to (PVPON/TA) coatings can suppress T cell activation, enhance regulatory T cell populations, prolong islet allograft survival, and induce localized immunosuppression after transplantation.

HLA-B Matching Prolongs Allograft Survival in Islet Cell Transplantation.

Islet cell transplantation (ITx) is an effective therapeutic approach for selected patients with type 1 diabetes with hypoglycemia unawareness and severe hypoglycemia events. In organ transplantation, human leukocyte antigen (HLA) mismatching between donor and recipient negatively impacts transplant outcomes. We aimed to determine whether HLA matching has an impact on islet allograft survival. Forty-eight patients were followed up after islet transplantation at our institution from 2000 to 2020 in a retrospective cohort. Patients underwent intrahepatic ITx or laparoscopic omental approach. Immunosuppression was dependent upon the protocol. We analyzed HLA data restricted to A, B, and DR loci on allograft survival using survival and subsequent multivariable analyses. Patients were aged 42.8 ± 8.4 years, and 64.3% were female. Diabetes duration was 28.6 ± 11.6 years. Patients matching all three HLA loci presented longer graft survival (P = 0.030). Patients with ≥1 HLA-B matching had longer graft survival compared with zero matching (P = 0.025). The number of HLA-B matching was positively associated with time of graft survival (Spearman's rho = 0.590; P = 0.034). Analyses adjusted for confounders showed that ≥1 matching for HLA-B decreased the risk of allograft failure (P = 0.009). Our data suggest that HLA-B matching between recipients and donors improved islet allograft survival. Matching all three HLA loci (A, B, and DR) was also associated with prolonged islet allograft survival. Prospective studies and a larger sample size are warranted to validate our findings.

Immunoregulatory Sertoli Cell Allografts Engineered to Express Human Insulin Survive Humoral-Mediated Rejection.

An effective treatment and possible cure for type 1 diabetes is transplantation of pancreatic islets. Unfortunately, transplanted islets are rejected by the immune system with humoral-mediated responses being an important part of rejection. Sertoli cells (SC), an immune regulatory cell shown to survive as allografts long-term without immunosuppressants, have the potential to be used as a cell-based gene therapy vehicle to deliver endogenous insulin-a possible alternative to islets. Previously, we transduced a mouse SC line to produce human insulin. After transplantation into diabetic mice, these cells consistently produced low levels of insulin with graft survival of 75% at 50 days post-transplantation. The object of this study was to assess humoral immune regulation by these engineered SC. Both nontransduced and transduced SC survived exposure to human serum with complement in vitro. Analysis of allografts in vivo at 20 and 50 days post-transplantation revealed that despite IgG antibody detection, complement factor deposition was low and grafts survived through 50 days post-transplantation. Furthermore, the transduced SC secreted elevated levels of the complement inhibitor C1q binding protein. Overall, this suggests SC genetically engineered to express insulin maintain their ability to prevent complement-mediated killing. Since inhibiting complement-mediated rejection is important for graft survival, further studies of how SC modifies the immune response could be utilized to advance the use of genetically engineered SC or to prolong islet allograft survival to improve the treatment of diabetes.

303-OR: Localized CTLA-4-Ig and Antioxidant Islet Encapsulation Delays Allograft Rejection

Type 1 diabetes is characterized by beta-cell-specific autoimmune destruction, leading to life-long exogenous insulin-dependence. Islet transplantation can restore proper glycemic control, but graft longevity remains low due to immune-mediated rejection. The use of nanothin encapsulation materials consisting of poly (N-vinylpyrrolidone) (PVPON) and tannic acid (TA) , an antioxidant, can delay islet allograft rejection and decrease inflammatory immune responses. However, when (PVPON/TA) -encapsulated C57BL/6 islets were transplanted into streptozocin-treated diabetic NOD mice in the absence of systemic immunosuppression, only 50% of recipients maintained long-term allograft function. Therefore, modification of (PVPON/TA) coatings to include immune inhibitors are needed to prolong islet allograft survival. The adaptability of our (PVPON/TA) layers allow for its conjugation to CTLA-4-Ig, a fusion protein that can block CD28-mediated co-stimulation of T cells. We hypothesize that the addition of CTLA-4-Ig to (PVPON/TA) outer layers, denoted as (PVPON/TA/CTLA-4-Ig) , will extend islet allograft survival by inhibiting proinflammatory effector T cell responses. Transplantation of (PVPON/TA/CTLA-4-Ig) -encapsulated NOD.Rag islets significantly delayed islet allograft rejection in diabetic C57BL/6 mice compared to non- (p<0.0001) and control IgG-containing (PVPON/TA/IgG) -encapsulated islets (p<0.01) . Additionally, (PVPON/TA/CTLA-4-Ig) -encapsulation significantly increased FOXP3+ regulatory and CD73+ FR4+ anergic CD4+ T cell count when compared to both non- and (PVPON/TA/IgG) -encapsulation. Since CTLA-4-Ig can affect the tryptophan catabolism pathway, future studies will investigate the effect of (PVPON/TA/CTLA-4-Ig) -encapsulation on indolamine-2,3-dioxygenase expression in antigen-presenting cells. The success of (PVPON/TA/CTLA-4-Ig) coatings to delay allograft rejection warrant their use in future human translational studies. Disclosure J.Barra: None. K.S.Burnette: None. V.Kozlovskaya: None. E.Kharlampieva: None. H.M.Tse: None. Funding NIH/NIDDK Raward (DK099550) NIH/NIDDK Raward (DK127497) JDRF award (SRA-2016-270-S-B) JDRF award (2-SRA-2019-692-S-B) NIH/NIDDK R56 award (DK126456) NIH/NIDDK Raward (DK126456) NIH/NIGMS (T32GM109780) NIH/NIGMS (T32GM008111)

Comparison of alpha 1- antitrypsin activity and phenotype in type 1 diabetic patients to healthy individuals.

Background and Aims:Alpha 1 antitrypsin (AAT) is an inhibitor of serine protease, which has shown anti-inflammatory reactions in a variety of diseases. It has been thought that that AAT plays a role in prolonging islet allograft survival, preventing the development of type 1 diabetes mellitus (T1DM), and hindering β-cell apoptosis of pancreas. In the current examination, the AAT activity in T1DM and healthy individuals was measured using enzymatic assay.Methods:The present study was conducted on 42 patients with T1DM who referred to the Diabetes Clinic of Rafsanjan, Kerman, Iran, and 42 healthy control individuals who were matched for age, sex and smoking habits. The serum trypsin inhibitory capacity (TIC) was assessed. Plasma samples were analyzed for phenotype, AAT concentration, blood glucose and lipid levels were measured.Results:The activity of plasma AAT and the serum TIC level of patients with T1DM (2.35 ± 0.34 μmol/min/ml) was significantly lower than healthy participants (3.36 ± 0.36 μmol/min/ml). The frequency of phenotype MM in healthy individual was 100%; and in T1DM patients, the prevalence of phenotype MM, MS and MZ was 61.9%, 23.8% and 14.3%, respectively (P < 0.001).Conclusions:It was concluded that that the lack of AAT may be related to the increased risk of T1DM developing.

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

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

IL-10 producing type 2 innate lymphoid cells prolong isletallograft survival.

Type 2 innate lymphoid cells (ILC2s) are a subset of ILCs with critical roles in immunoregulation. However, the possible role of ILC2s as immunotherapy against allograft rejection remains unclear. Here, we show that IL‐33 significantly prolonged islet allograft survival. IL‐33‐treated mice had elevated numbers of ILC2s and regulatory T cells (Tregs). Depletion of Tregs partially abolished the protective effect of IL‐33 on allograft survival, and additional ILC2 depletion in Treg‐depleted DEREG mice completely abolished the protective effects of IL‐33, indicating that ILC2s play critical roles in IL‐33‐mediated islet graft protection. Two subsets of ILC2s were identified in islet allografts of IL‐33‐treated mice: IL‐10 producing ILC2s (ILC210) and non‐IL‐10 producing ILC2s (non‐ILC 10). Intravenous transfer of ILC210 cells, but not non‐ILC 10, prolonged islet allograft survival in an IL‐10‐dependent manner. Locally transferred ILC210 cells led to long‐term islet graft survival, suggesting that ILC210 cells are required within the allograft for maximal suppressive effect and graft protection. This study has uncovered a major protective role of ILC210 in islet transplantation which could be potentiated as a therapeutic strategy.

The MEK Inhibitor Trametinib Suppresses Major Histocompatibility Antigen-mismatched Rejection Following Pancreatic Islet Transplantation.

Background.Potential adverse effects, such as functional impairment of islets, render conventional immunosuppressive drugs unsuitable for use in islet transplantation. In addition, as a single therapy, they cannot prolong islet allograft survival. Here, we investigated the utility of the mitogen-activated protein kinase inhibitor trametinib and asked whether it ameliorates acute rejection of transplanted islets without the need for conventional immunosuppressants.Methods.Islets from fully major histocompatibility complex-mismatched BALB/c mice were transplanted into streptozotocin-induced diabetic C57BL/6 mice via the portal vein. These mice received trametinib or vehicle (orally) for 28 days. Isolated islets from BALB/c mice were incubated in vitro with different concentrations of trametinib to determine viability and function.Results.Trametinib (0.1 and 0.3 mg/kg) prolonged graft survival significantly (P = 0.0007 and P = 0.005, respectively) when compared with vehicle. Histologic analyses revealed that cellular infiltration of the graft by lymphocytes was inhibited significantly on day 7 (P < 0.05). In addition, trametinib suppressed functional differentiation of naive CD4+ T cells in recipients. Expression of mRNA encoding inflammatory cytokines interleukin (IL)-2, tumor necrosis factor α, and interferon γ in recipients treated with trametinib was also inhibited (P < 0.001, P < 0.05, and P < 0.01, respectively). Trametinib also increased production of IL-4 and IL-10 (P < 0.05 and P = 0.20, respectively). In vitro, islets incubated with different concentrations of trametinib exhibited no harmful effects with respect to viability and function.Conclusions.Trametinib delayed islet graft rejection by inhibiting functional differentiation of naive CD4+ T cells and regulating inflammatory cytokines. Trametinib might be a promising candidate for maintenance immunosuppressive therapy after allogeneic islet transplantation.

Low-Dose Interleukin-2 Combined With Rapamycin Led to an Expansion of CD4+CD25+FOXP3+ Regulatory T Cells and Prolonged Human Islet Allograft Survival in Humanized Mice.

Islet transplantation is an emerging therapy for type 1 diabetes and hypoglycemic unawareness. However, a key challenge for islet transplantation is cellular rejection and the requirement for long-term immunosuppression. In this study, we established a diabetic humanized NOD-scidIL2Rγnull (NSG) mouse model of T-cell-mediated human islet allograft rejection and developed a therapeutic regimen of low-dose recombinant human interleukin-2 (IL-2) combined with low-dose rapamycin to prolong graft survival. NSG mice that had received renal subcapsular human islet allografts and were transfused with 1 × 107 of human spleen mononuclear cells reconstituted human CD45+ cells that were predominantly CD3+ T cells and rejected their grafts with a median survival time of 27 days. IL-2 alone (0.3 × 106 IU/m2 or 1 × 106 IU/m2) or rapamycin alone (0.5-1 mg/kg) for 3 weeks did not prolong survival. However, the combination of rapamycin with IL-2 for 3 weeks significantly prolonged human islet allograft survival. Graft survival was associated with expansion of CD4+CD25+FOXP3+ regulatory T cells (Tregs) and enhanced transforming growth factor-β production by CD4+ T cells. CD8+ T cells showed reduced interferon-γ production and reduced expression of perforin-1. The combination of IL-2 and rapamycin has the potential to inhibit human islet allograft rejection by expanding CD4+FOXP3+ Tregs in vivo and suppressing effector cell function and could be the basis of effective tolerance-based regimens.

Salvia miltiorrhiza inhibits allospecific memory CD8+ T cell generation and allograft rejection in mice

Abstract It’s well known that memory T cells pose a threat to long-term allograft survival. The objective of this study is to suppress generation of donor-specific memory T cells via short-term treatment with a natural and non-cytotoxic herb. Previous studies have demonstrated that Chinese herb Salvia miltiorrhiza (Danshen) regulates immune responses. In this study, B6 mice were transplanted with BALB/c islets and treated with Danshen and/or low doses of rapamycin (0.25 mg/kg/day). To generate allospecific memory T cells, Thy1.1+ CD8+CD44low T cells were sorted by FACSAria and injected into B6 recipients one day before transplantation. Graft-infiltrating T cell proliferation was determined by BrdU staining while the memory cells were enumerated via FACS. We found that Danshen alone significantly extended islet allograft survival (MST = 28 vs. 13 days, n=9, P&amp;lt;0.05). Interestingly, administration of both Danshen and low doses of rapamycin further prolonged islet allograft survival (MST = 83 versus 28 days), with 44% of recipients achieving long-term islet graft survival (&amp;gt;100 days). Moreover, Danshen suppressed generation of memory CD8+ T cells compared to control two months after transplantation (Thy1.1+CD8+CD44high cell numbers: 1.3±0.2 vs. 2.5±0.3, ×104/LN, P&amp;lt;0.05 and 4.3±0.6 vs. 8.5±0.9, ×105/spleen, P&amp;lt;0.05) while Danshen plus rapamycin further reduced CD8+ memory T cells compared to Danshen alone (0.7±0.1 vs. 1.3±0.2/LN, P&amp;lt;0.05 and 2.6±0.4 vs. 4.3±0.6,/spleen, P&amp;lt;0.05). Finally, Danshen alone inhibited graft-infiltrating T cell proliferation at one week (52±4 vs. 35±3 %, P&amp;lt;0.05). Thus, Danshen inhibited donor-specific memory CD8+ T cell generation and promoted induction of long-term murine allograft survival.

B Cell derived IL-4 promotes IL-10 secreting TIM-1+ regulatory B cell expansion and regulates Th2 T cell responses

Abstract TIM-1+ B cells are enriched for IL-10+ B regulatory cells (Bregs). However, TIM-1+ B cells are also enriched for IL-4+ B effector 2 cells (Be2). Tolerogenic α-TIM-1 (RMT1-10) prolongs islet allograft survival (GS) by expanding (IL-10+) Bregs. Interestingly, IL-4KO and IL-4Rα-KO mice are deficient in Bregs and are not responsive to αTIM-1. To establish the importance of IL-4R signaling, and the source of IL-4 for Breg induction, we made tamoxifen (TAM) inducible BALB/c hCD20Cre.ERT2+/− X IL-4Rαfl/fl (B-IL-4RαKO) and hCD20Cre.ERT2+/− X IL-4/13fl/fl mice (B-IL-4KO) to respectively delete IL-4Rα or IL-4/IL-13 in B cells. After TAM, αTIM-1 treatment of alloimmunized B-IL-4RαKO mice had ↓ TIM-1+ B cells (50%), Be2 (50%) and Bregs (40%) vs. IL-4Rαfl/fl littermate controls and a concomittant 33%↑ in IFNγ+ and 33% ↓ in IL-10+ CD4 T cells, and a 20%↑ in IFNγ+ CD8 T cells vs. controls. Finally, α–TIM-1 led to &amp;gt;100d GS in 75% of control mice, but failed to improve GS in B-IL-4RαKO mice (MST 20d, p=0.03). Similarly, TAM treated B-IL-4KO mice had a 3x ↓ in Be2 and Bregs vs. IL-4fl/fl controls, and a 6x drop in IL-4+ CD4 T cells. Importantly, B-IL-4KO mice rejected islets more rapidly (MST 20d) than controls (MST 31d, p=0.04), and α–TIM-1 failed to improve B-IL-4KO GS (MST 17d) vs. controls (MST &amp;gt;100d). In contrast, in Th2 dominant OVA-induced allergic airway disease (AAD), B-IL-4KO mice had 3x decrease in total lymphocyte infiltrate, 5x decrease CD4 T cells and 8x decrease eosinophils in BAL fluid vs. controls. CD4+ T cells from whole lung tissue produced 2x less IL-4 and 3x less IL-5 vs. controls. Hence, IL-4 production by B cells is important for both Breg induction and Th2 polarization and absence of B cell IL-4/IL-13 promotes allograft rejection yet reduces AAD.

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.

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.

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.

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.

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.

Immunosuppressive effect of arsenic trioxide on islet xenotransplantation prolongs xenograft survival in mice

The role of arsenic trioxide (As2O3) in inhibiting immune rejection and prolonging islet allograft survival has been identified in islet allotransplantation. This study aims to explore the role of As2O3 in islet xenotransplantation and the action mechanism. The streptozotocin (STZ) was used in C57BL/6 mice to induce the type 1 diabetes mellitus (T1DM) for xenotransplantation models establishment. Donor islets were isolated by digesting. The flow cytometry (FCM) was used to analyze lymphocyte types. The blood sugar level was detected by using intraperitoneal glucose tolerance test (IPGTT). The serum level of cytokines was determined by the enzyme-linked immunosorbent assay (ELIZA). The cell proliferation was measured by MTT assay. The mRNA levels were quantified with qRT-PCR. As2O3 prolonged the survival of the recipient mice but had no influence on body weight. As2O3 protected the function of xenograft in insulin secretion and suppressed immune rejection of recipient. As2O3 inhibited proliferation of T lymphocyte and increased the proportion of Foxp3+ regulatory T cells in recipient mice. As2O3 inhibited activation and promoted clonal anergy of T lymphocyte. As2O3 decreased total number of B cells and reduced partial antibody levels in recipient mice. As2O3 and leflunomide showed a synergistic effect in suppressing islet xenotransplant rejection. As2O3 prolongs islet xenograft survival by inhibiting cellular immune response, and increasing Foxp3+ regulatory T cells, while decreasing partial antibody levels in serum.

C-Jun N-terminal kinase 1 defective CD4+CD25+FoxP3+ cells prolong islet allograft survival in diabetic mice

CD4+CD25+FoxP3+ cells (Tregs) inhibit inflammatory immune responses to allografts. Here, we found that co-transplantation of allogeneic pancreatic islets with Tregs that are defective in c-Jun N-terminal kinase 1 (JNK1) signaling prolongs islet allograft survival in the liver parenchyma of chemically induced diabetic mice (CDM). Adoptively transferred JNK1−/− but not wild-type (WT) Tregs survive longer in the liver parenchyma of CDM. JNK1−/− Tregs are resistant to apoptosis and express anti-apoptotic molecules. JNK1−/− Tregs express higher levels of lymphocyte activation gene-3 molecule (LAG-3) on their surface and produce higher amounts of the anti-inflammatory cytokine interleukin (IL)-10 compared with WT Tregs. JNK1−/− Tregs inhibit liver alloimmune responses more efficiently than WT Tregs. JNK1−/− but not WT Tregs are able to inhibit IL-17 and IL-21 production through enhanced LAG-3 expression and IL-10 production. Our study identifies a novel role of JNK1 signaling in Tregs that enhances islet allograft survival in the liver parenchyma of CDM.

Dendritic Cells Transduced with Single Immunoglobulin IL-1-Related Receptor Exhibit Immature Properties and Prolong Islet Allograft Survival.

Members of toll-like receptor-interleukin 1 receptor signaling [TLR/IL-1R (TIR)] superfamily mediate maturation of dendritic cells (DCs) and launch immune response in transplanted organs. In this study, we hypothesized that TIR8, also known as single immunoglobulin IL-1-related receptor (SIGIRR) molecule, refrain DCs from maturation and induce immune tolerance of transplanted organ. DCs were transduced with the recombinant adenovirus Ad5F35 to highly express SIGIRR (DC-SIGIRR), then injected to murine recipient before islet transplantation. It revealed that DCs transduced with SIGIRR had low expression of major histocompatibility and costimulatory molecules along with strong phagocytic ability in vitro assay. The data demonstrated that recipients treated with DC-SIGIRR had satisfying islet allograft function and long survival times, with an increase of Treg and reduction of Th17 in both spleen and draining lymph nodes in vivo. Therefore, genetic modification of SIGIRR inhibits DC activation and maturation, affects differentiation of T cell subsets, protects allograft biological function, and prolongs graft survival.

Stealing Tregs’ Identity (by Deleting NF-κB)

Stealing Tregs’ Identity (by Deleting NF-κB)