Search

Background: Xenogeneic islet transplantation using porcine pancreata has been a promising option for substituting human islet transplantation. Moreover, recent advances in pre-clinical results have put islet xenotransplantation closer to the possibility of clinical application. While preparing for the era of clinical xenotransplantation, developing non-invasive immune monitoring method which could predict the graft fate could benefit the patient. However, there are few reports showing predictive immune parameters associated with the fate of the graft in islet xenotransplantation., Methods: The absolute number and ratio of T-cell subsets have been measured via flow cytometry from the peripheral blood of 16 rhesus monkeys before and after porcine islet xenotransplantation. The correlation between the graft survival and the absolute number or ratio of T cells was retrospectively analyzed., Results: The ratio of CD4 + versus CD8 + T cells was significantly reduced due to CD8 + effector memory cells' increase. Correlation analyses revealed that CD4 + /CD8 + , CD4 + /CD8 + naïve, CD4 + naïve/CD8 + naïve, and CD4 + central memory/CD8 + naïve cell ratios negatively correlated with the duration of graft survival. Conversely, further analyses discovered strong, positive correlation of CD4 + /CD8 + cell ratios within the early graft-rejected monkeys (≤60 days)., Conclusions: This retrospective study demonstrated that CD4 + /CD8 + ratios correlated with graft survival, especially in recipients which rejected the graft in early post-transplantation periods. CD4 + /CD8 + ratios could be used as a surrogate marker to predict the graft fate in pig-to-NHP islet xenotransplantation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Anti-CD154 blockade-based regimens remain unequaled in prolonging graft survival in various organ transplantation models. Several studies have focused on transplantation tolerance with the anti-CD154 blockade, but none of these studies has investigated the mechanisms associated with its use as the sole treatment in animal models, delaying our understanding of anti-CD154 blockade-mediated immune tolerance. The purpose of this study was to investigate the mechanism underlying the anti-CD154 monoclonal antibody (mAb) blockade in inducing immune tolerance using an intrahepatic murine allogeneic islet transplantation model. Allogeneic BALB/c AnHsd (BALB/c) islets were infused into the liver of diabetic C57BL/6 (B6) mice via the cecal vein. Anti-CD154 mAb (MR1) was administered on -1, 0, 1, 3, 5, and 7 d posttransplantation at 0.5 mg per mouse. We showed that short-term MR1 monotherapy could prolong the allogeneic islet grafts to more than 250 d in the murine intrahepatic islet transplantation model. The second islet grafts transplanted under the kidney capsule of the recipients were protected from rejection. We also found that rejection of same-donor skin grafts transplanted to the tolerant mice was modestly delayed. Using a DEREG mouse model, FoxP3+ regulatory T (Treg) cells were shown to play important roles in transplantation tolerance. In mixed lymphocyte reactions, Treg cells from the tolerant mice showed more potency in suppressing BALB/c splenocyte-stimulated Teff cell proliferation than those from naïve mice. In this study, we demonstrated for the first time that a short-term anti-CD154 mAb single treatment could induce FoxP3+ Treg cell-mediated immune tolerance in the intrahepatic murine allogeneic islet transplantation model.

Clinical islet transplantation has recently been a promising treatment option for intractable type 1 diabetes patients. Although early graft loss has been well studied and controlled, the mechanisms of late graft loss largely remains obscure. Since long-term islet graft survival had not been achieved in islet xenotransplantation, it has been impossible to explore the mechanism of late islet graft loss. Fortunately, recent advances where consistent long-term survival (≥6 months) of adult porcine islet grafts was achieved in five independent, diabetic nonhuman primates (NHPs) enabled us to investigate on the late graft loss. Regardless of the conventional immune monitoring methods applied in the post-transplant period, the initiation of late graft loss could rarely be detected before the overt graft loss observed via uncontrolled blood glucose level. Thus, we retrospectively analyzed the gene expression profiles in 2 rhesus monkey recipients using peripheral blood RNA-sequencing (RNA-seq) data to find out the potential cause(s) of late graft loss. Bioinformatic analyses showed that highly relevant immunological pathways were activated in the animal which experienced late graft failure. Further connectivity analyses revealed that the activation of T cell signaling pathways was the most prominent, suggesting that T cell-mediated graft rejection could be the cause of the late-phase islet loss. Indeed, the porcine islets in the biopsied monkey liver samples were heavily infiltrated with CD3 + T cells. Furthermore, hypothesis test using a computational experiment reinforced our conclusion. Taken together, we suggest that bioinformatics analyses with peripheral blood RNA-seq could unveil the cause of insidious late islet graft loss.

Background: Anti-CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4 + regulatory T cells (T reg s) in xeno-immune tolerance. However, the characteristics of T reg s and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined., Method: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti-CD154 mAb on 0, 1, 3, 5, and 7 days post-transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme-linked immunosorbent spot (ELISPOT) assay, and mixed-lymphocyte reaction., Results: Short-term anti-CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4 + Foxp3 + T reg s in the peri-graft site, but not in the spleen and kidney-draining lymph node of the recipient mice. Depletion of T reg s evoked graft rejection, and adoptive transfer of T reg s from anti-CD154 mAb-treated recipients provided protection to the graft from rejection. These T reg s showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen-specific., Conclusions: In this study, we showed that anti-CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine-specific CD4 + Foxp3 + T reg s in the peri-graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation., (© 2019 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Recent progress in xenotransplantation of porcine islets to non-human primates (NHPs) gives hope for human clinical trials in the near future. Thus, implementation of an appropriate monitoring method to detect the development of detrimental porcine antigen-specific cellular immune responses is necessary. The enzyme-linked immunospot (ELISpot) assay has been widely used to monitor antigen-specific alloreactive T-cell responses in humans; however, the utility of porcine islet-specific ELISpot assay has not yet been thoroughly evaluated for pig-to-NHPs intraportal islet xenotransplantation., Methods: The optimal ELISpot assay conditions, including the number of responder and stimulator cells and the provision of costimulation, were determined. Then, ELISpot assays were conducted on serial stocks of peripheral blood mononuclear cell (PBMC) samples previously isolated from NHP recipients transplanted with porcine islets. Either splenocytes from donor pigs or pancreatic islets from third-party pigs were used for antigen stimulation. At the same time, the ratio of CD4(+) /CD8(+) T cells and the percentage of CD4(+) FoxP3(+) T cells in the peripheral blood were evaluated. Finally, liver biopsy samples were evaluated to assess the immunopathology of the grafts., Results: The optimal conditions for the ELISpot assay were defined as 2.5 × 10(5) responder cells incubated with 5.0 × 10(5) stimulator cells in 96-well, flat-bottom plates without further costimulation. Using donor splenocytes as stimulators, a serial interferon-gamma (IFN-γ) ELISpot assay with PBMCs from the monkeys with prolonged porcine islet grafts (>180 days) demonstrated that the number of donor antigen-specific IFN-γ-producing cells significantly increased upon overt graft rejection. However, use of third-party porcine islets as stimulators did not reflect graft rejection, suggesting that the use of donor-specific PBMCs, and not tissue (porcine islet)-specific cells, as stimulators could better serve the purpose of this assay in adult porcine islet transplantation. IFN-γ spot number was neither influenced by the peripheral blood CD4(+) /CD8(+) T-cell ratio nor the percentage of CD4(+) FoxP3(+) T cells. Finally, in cases of overt graft rejection, the number of IFN-γ spots and the graft-infiltrating T cells in biopsied liver samples increased simultaneously., Conclusion: Use of PBMCs in a porcine antigen-specific IFN-γ ELISpot assay is a reliable method for monitoring T-cell-mediated rejection in pig-to-NHP islet xenotransplantation., (© 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Pancreatic islet transplantation is a promising treatment option for Type 1 diabetes, but organ supply shortage limits its wide adoption. Pig islets are the most promising alternative source and many important measures such as donor animal selection, pig islet production release criteria, preclinical data and zoonosis surveillance prior to human clinical trials have been put forward as a consensus through the efforts of the International Xenotransplantation Association. To bring pig islet transplantation to clinical reality, the development of clinically applicable immunosuppression regimens and methods to minimize immunosuppression to reduce side effects should be established. This review encompasses immune rejection mechanisms in islet xenotransplantation, immunosuppression regimens that have enabled long-term graft survival in pig-to-nonhuman primate experiments and strategies for minimizing immunosuppression in islet xenotransplantation. By thoroughly examining the drugs that are currently available and in development and their individual targets within the immune response, the best strategy for enabling clinical trials of pig islets for Type 1 diabetes will be proposed.

Purpose Of Review: Porcine islet xenotransplantation is a promising alternative to overcome the shortage of organ donors. For the successful application of islet xenotransplantation, robust immune/inflammatory responses against porcine islets should be thoroughly controlled. Over the last few decades, there have been numerous attempts to surmount xenogeneic immune barriers. In this review, we summarize the current progress in immunomodulatory therapy for the clinical application of porcine islet xenotransplantation.Recent Findings: Long-term graft survival of porcine islets was achieved by using anti-CD154 Ab-based regimens in a preclinical non-human primate (NHP) model. However, owing to a serious complication of thromboembolism in clinical trials, the development of an anti-CD154 Ab-sparing immunosuppressant procedure is required. The efficacy of new immunosuppressive practices that employ anti-CD40 Abs or other immunosuppressive reagents has been tested in a NHP model to realize their utility in porcine islet xenotransplantation. The recent progress in the development of immunomodulatory approaches, including the immunosuppressive regimen, which enables long-term graft survival in a pig-to-non-human primate islet xenotransplantation model, with their potential clinical applicability was reviewed. [ABSTRACT FROM AUTHOR]

Background: Anti‐CD154 monoclonal antibody (mAb) treatment has been known to have potential to induce immune tolerance in organ transplantation. Several studies have suggested the involvement of CD4+ regulatory T cells (Tregs) in xeno‐immune tolerance. However, the characteristics of Tregs and the mechanisms of their regulatory functions in islet xenotransplantation have not been clearly defined. Method: Adult porcine islet cells were isolated and purified, and were transplanted under the kidney capsule of diabetic C57BL/6J mice with the administration of 0.5 mg/mouse of anti‐CD154 mAb on 0, 1, 3, 5, and 7 days post‐transplantation (DPT). The graft survival was monitored by blood glucose level. The islet graft and recipients' cells were analyzed by immunohistochemistry (IHC), flow cytometry, enzyme‐linked immunosorbent spot (ELISPOT) assay, and mixed‐lymphocyte reaction. Results: Short‐term anti‐CD154 mAb monotherapy enabled the porcine islet graft to survive indefinitely in diabetic mice (n = 18). Immunohistochemical staining showed significantly higher ratio of CD4+Foxp3+ Tregs in the peri‐graft site, but not in the spleen and kidney‐draining lymph node of the recipient mice. Depletion of Tregs evoked graft rejection, and adoptive transfer of Tregs from anti‐CD154 mAb‐treated recipients provided protection to the graft from rejection. These Tregs showed more potent suppressive capacity than those from the untreated control and were found to be porcine antigen‐specific. Conclusions: In this study, we showed that anti‐CD154 mAb monotherapy resulted in indefinite porcine islet graft survival in mice. The porcine‐specific CD4+Foxp3+Tregs in the peri‐graft site played the critical role in the protection of islet graft from rejection, which suggests a prospective immunosuppressive strategy for islet xenotransplantation. [ABSTRACT FROM AUTHOR]