Prolonged Graft Survival Time Research Articles

RapaLink-1 outperforms rapamycin in alleviating allogeneic graft rejection by inhibiting the mTORC1-4E-BP1 pathway in mice

Inhibition of mammalian target of rapamycin (mTOR), which is a component of both mTORC1 and mTORC2, leads to clinical benefits for organ transplant recipients. Pathways to inhibit mTOR include strengthening the association of FKBP12-mTOR or competing with ATP at the active site of mTOR, which have been applied to the design of first- and second-generation mTOR inhibitors, respectively. However, the clinical efficacy of these mTOR inhibitors may be limited by side effects, compensatory activation of kinases and attenuation of feedback inhibition of receptor expression. A new generation of mTOR inhibitors possess a core structure similar to rapamycin and covalently link to mTOR kinase inhibitors, resulting in moderate selectivity and potent inhibition of mTORC1. Since the immunosuppressive potential of this class of compounds remains unknown, our goal is to examine the therapeutic efficacy of a third-generation mTOR inhibitor in organ transplantation. In this study, RapaLink-1 outperformed rapamycin in inhibiting T-cell proliferation and significantly prolonged graft survival time. Mechanistically, the ameliorated rejection induced by RapaLink-1 is associated with a reduction in p-4E-BP1 in T cells, resulting in an elevation in Treg cells alongside a decline in Th1 and Th17 cells. For the first time, these studies demonstrate the effectiveness of third-generation mTOR inhibitors in inhibiting allograft rejection, highlighting the potential of this novel class of mTOR inhibitors for further investigation.

Multiple infusions of ex vivo-expanded regulatory T cells promote CD163+ myeloid cells and kidney allograft survival in non-lymphodepleted non-human primates

Clinical verification of adoptively transferred regulatory T cell (Treg) efficacy in transplantation remains challenging. Here, we examined the influence of autologous exvivo-expanded polyclonal Tregs on kidney graft survival in a clinically relevant non-human primate model. Peripheral blood Tregs were isolated and expanded using artificial antigen presenting cells. Immunosuppression was comprised of tapered tacrolimus and CTLA4 immunoglobulin, in five animals each without or with Treg infusions. Escalating Treg doses were administered 6, 10, 13, 16, 20, 23, 27 and 30 days after transplant. Infused Tregs were monitored for Treg signature, anti-apoptotic (Bcl-2) and proliferation (Ki67) marker expression. Treg infusions prolonged median graft survival time significantly from 35 to 70 days. Treg marker (Ki67 and Bcl-2) expression by infused Tregs diminished after their infusion but remained comparable to that of circulating native Tregs. No major changes in circulating donor-reactive T cell responses or total Treg percentages, or in graft-infiltrating T cell subsets were observed with Treg infusion. However, Treg infusion was associated with significant increases in CD163 expression by circulating HLA-DR+ myeloid cells and elevated levels of circulating soluble CD163. Further, graft-infiltrating CD163+ cells were increased with Treg infusion. Thus, multiple Treg infusions were associated with M2-like myeloid cell enhancement that may mediate immunomodulatory, anti-inflammatory and graft reparative effects.

Risk factors and management ofhyperuricemia after renal transplantation.

Hyperuricemia (HUA) is a common complication after renal transplantation. Currently, there is no uniform consensus on factors which increase the risk for and treatment of HUA in renal transplant recipients. The purpose of this review is to summarize current and proposed risk factors and strategies to manage HUA after renal transplantation in order to assist renal function protection and prolong graft survival time.

Mesenchymal stem cell derived exosomes-based immunological signature in a rat model of corneal allograft rejection therapy.

Mesenchymal stem cells (MSCs) are promising candidates for immunomodulatory therapy that are currently being tested in corneal allograft rejection. In this study, we tested the effects of Mesenchymal stem cells derived exosomes in the corneal allograft rejection model. Mesenchymal stem cells derived exosomes (MSC-exo) were collected and characterized. Wistar-Lewis rat corneal allograft rejection models were established. PKH26 labeled exosomes were used for track experiment. Models were randomly separated into four groups and treated with graded doses of exosomes or same volumn of PBS. Corneal grafts were assessed for rejection degree using slit-lamp biomicroscopy. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4, CD8 and CD25. A comprehensive graft mRNA gene expression array analysis was conducted and checked by real-time polymerase chain reaction (PCR). The nanovesicles obtained were expressing exosome specific protein markers CD9, CD63, CD81. The labeled exosomes could be detected in both cornea and anterior chamber two hours after injection.The 10 μg exosomes subconjunctival injection can effectively prolong graft survival time (MST 16.3 ± 2.5 days). 10 μg exosomes-treated group can inhibit the infiltration of CD4+ and CD25+ T cells. IFN-γ and CXCL11 levels were significantly decreased in grafts obtained from postoperative exosomes-treated rats when compared with controls. MSC-exo can cross biological barrier and play better role directly towards target tissue. MSC-exo can effectively prolong grafts survival time. Th1 signaling pathway was significantly inhibited in the exosomes treated group.

A Cannabinoid 2-Selective Agonist Inhibits Allogeneic Skin Graft Rejection In Vivo.

Previous work from our laboratory showed that a CB2 selective agonist, O-1966, blocked the proliferative response of C57BL/6 mouse spleen cells exposed to spleen cells of C3HeB/FeJ mice in vitro in the mixed lymphocyte reaction (MLR). The MLR is widely accepted as an in vitro correlate of in vivo grant rejection. Mechanisms of the immunosuppression induced by the cannabinoid were explored, and it was shown that O-1966 in this in vitro assay induced CD25+Foxp3+ Treg cells and IL-10, as well as down-regulated mRNA for CD40 and the nuclear form of the transcription factors NF-κB and NFAT in T-cells. The current studies tested the efficacy of O-1966 in prolonging skin grafts in vivo. Full thickness flank skin patches (1-cm2) from C3HeB/FeJ mice were grafted by suturing onto the back of C57BL/6 mice. O-1966 or vehicle was injected intraperitoneally into treated or control groups of animals beginning 1 h pre-op, and then every other day until 14 days post-op. Graft survival was scored based on necrosis and rejection. Treatment with 5 mg/kg of O-1966 prolonged mean graft survival time from 9 to 11 days. Spleens harvested from O-1966 treated mice were significantly smaller than those of vehicle control animals based on weight. Flow cytometry analysis of CD4+ spleen cells showed that O-1966 treated animals had almost a 3-fold increase in CD25+Foxp3+ Treg cells compared to controls. When dissociated spleen cells were placed in culture ex vivo and stimulated with C3HeB/FeJ cells in an MLR, the cells from the O-1966 treated mice were significantly suppressed in their proliferative response to the allogeneic cells. These results support CB2 selective agonists as a new class of compounds to prolong graft survival in transplant patients.

Blockage of P2X7R suppresses Th1/Th17-mediated immune responses and corneal allograft rejection via inhibiting NLRP3 inflammasome activation

P2X7R is a vital modifier of various inflammatory and immune-related diseases. However, the immunomodulatory effects of P2X7R on corneal allograft rejection remains unknown. Here we showed that P2X7R expression was significantly upregulated in corneal grafts of allogeneic transplant mice. Pharmacological blockage of P2X7R remarkably prolonged graft survival time, and reduced inflammatory cell infiltration in corneal grafts, in particular Th1/Th17 cells. Meanwhile, the frequencies of Th1/Th17 cells in draining lymph nodes were significantly decreased in P2X7R blocked allogeneic mice. Further results showed that the effect of P2X7R on promoting Th1/Th17 mediated immune responses in corneal allograft rejection relied heavily on its activation on the NLRP3/caspase-1/IL-1β axis, while P2X7R blockage could mitigate such activation. Nevertheless, the addition of IL-1β in vivo abrogated the protective effect of P2X7R blockage on promoting corneal graft survival. These findings demonstrate that blockage of P2X7R can substantially alleviate corneal allograft rejection and promote grafts survival, highlighting it as a promising target for preventing or treating corneal allograft rejection.

Imaging of Renal Transplant Complications throughout the Life of the Allograft: Comprehensive Multimodality Review.

The kidney is the most commonly transplanted solid organ. Advances in surgical techniques, immunosuppression regimens, surveillance imaging, and histopathologic diagnosis of rejection have allowed prolonged graft survival times. However, the demand for kidneys continues to outgrow the available supply, and there are efforts to increase use of donor kidneys with moderate- or high-risk profiles. This highlights the importance of evaluating the renal transplant patient in the context of both donor and recipient risk factors. Radiologists play an integral role within the multidisciplinary team in care of the transplant patient at every stage of the transplant process. In the immediate postoperative period, duplex US is the modality of choice for evaluating the renal allograft. It is useful for establishing a baseline examination for comparison at future surveillance imaging. In the setting of allograft dysfunction, advanced imaging techniques including MRI or contrast-enhanced US may be useful for providing a more specific diagnosis and excluding nonrejection causes of renal dysfunction. When a pathologic diagnosis is deemed necessary to guide therapy, US-guided biopsy is a relatively low-risk, safe procedure. The range of complications of renal transplantation can be organized temporally in relation to the time since surgery and/or according to disease categories, including immunologic (rejection), surgical or iatrogenic, vascular, urinary, infectious, and neoplastic complications. The unique heterotopic location of the renal allograft in the iliac fossa predisposes it to a specific set of complications. As imaging features of infection or malignancy may be nonspecific, awareness of the patient's risk profile and time since transplantation can be used to assign the probability of a certain diagnosis and thus guide more specific diagnostic workup. It is critical to understand variations in vascular anatomy, surgical technique, and independent donor and recipient risk factors to make an accurate diagnosis and initiate appropriate treatment.©RSNA, 2019.

Reducing porcine corneal graft rejection, with an emphasis on porcine endogenous retrovirus transmission safety: a review.

Donor cornea shortage is a primary hurdle in the development of corneal transplantation. Of all species, porcine corneas are the ideal transplantation material for humans. However, the xenoimmune rejection induced by porcine corneal xenotransplantation compromises surgical efficacy. Although the binding of IgM/IgG in human serum to a genetically modified porcine cornea is significantly weaker than that of the wild type (WT), genetically modified porcine corneas do not display a prolonged graft survival time in vivo. Conversely, costimulatory blockade drugs, such as anti-CD40 antibodies, can reduce the xenoimmune response and prolong graft survival time in animal experiments. Moreover, porcine endothelial grafts can survive for more than 6mo with only the subconjunctival injection of a steroid-based immunosuppressants regime; therefore, they show great value for treating corneal endothelial disease. In addition, zoonotic transmission is a primary concern of xenotransplantation. Porcine endogenous retrovirus (PERV) is the most significant virus assessed by ophthalmologists. PERV integrates into the porcine genome and infects human cells in vitro. Fortunately, no evidence from in vivo studies has yet shown that PERV can be transmitted to hosts.

Mouse Model Established by Early Renal Transplantation After Skin Allograft Sensitization Mimics Clinical Antibody-Mediated Rejection.

Antibody-mediated rejection (AMR) is the main barrier to renal graft survival, and mouse renal AMR models are important to study this process. Current mouse models are established by priming the recipient to donor skin for over 7 days before kidney transplantation. The robustness of AMR in these cases is too strong to mimic clinical AMR and it is unclear why altering the priming times ranging from 7 to 91 days fails to reduce the AMR potency in these models. In the present study, we found that the donor-recipient combination and skin graft size were determinants of donor-specific antibody (DSA) development patterns after skin transplantation. DSA-IgG was sustained for over 100 days after skin challenge, accounting for an identical AMR robustness upon different skin priming times over 7 days. However, decreasing the skin priming time within 7 days attenuated the robustness of subsequent renal allograft AMR in C3H to Balb/c mice. Four-day skin priming guaranteed that recipients develop acute renal AMR mixed with a high ratio of graft-infiltrating macrophages, renal grafts survived for a mean of 6.4 ± 2.1 days, characterized by typical AMR histological changes, such as glomerulitis, peritubular capillary (PTC) dilation, and capillaritis, deposition of IgG and C3d in PTCs, but less prevalence of microthrombus, whereas the cellular rejection histological change of tubulitis was absent to mild. With this scheme, we also found that the renal AMR model can be developed using common mouse strains such as C57BL/6 and Balb/c, with mean prolonged renal graft survival times of 14.4 ± 5.0 days. Finally, we proved that donor-matched skin challenge after kidney transplantation did not strongly affect DSA development and kidney graft outcome. These findings may facilitate an understanding and establishment of mouse renal allograft AMR models and promote AMR-associated studies.

The effects of local administration of mesenchymal stem cells on rat corneal allograft rejection

BackgroundMesenchymal stem cells (MSCs) have been reported to promote long-term cellular and organ transplant acceptance due to their immunotherapeutic characteristics. Previous work from our lab using a rat allograft model has shown that systemic infusion of MSCs inhibited corneal allograft rejection and prolonged graft survival. Here, we further investigated the effects of local MSCs administration in the same animal model.MethodsDonor-derived MSCs were isolated and cultured while corneal grafts obtained from Wistar rats were transplanted into Lewis rat hosts. Hosts were then randomly separated into four groups and treated with previously cultured MSCs at different times and doses. Graft survival was clinically assessed using slit-lamp biomicroscopy and the median survival time (MST) was calculated. Grafts were examined histologically using hematoxylin-eosin (H-E) staining and immunohistochemically using antibodies against CD4. A comprehensive graft analysis of IL-2, IL-4, IL-10, and IFN-γ expression was also conducted using both real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA).ResultsPostoperative MSCs injection prolonged graft survival time when compared with controls (MST 9.8 ± 1.2 days). Injection twice of MSCs (MST 12.6 ± 1.4 days) was more effective than a single injection (MST 10.8 ± 1.3 days). MSCs-treated groups also showed suppression of inflammatory cell as well as CD4 + T cell infiltration in the allograft region. IL-4 and IL-10 levels were significantly increased in grafts obtained from postoperative twice MSCs-treated rats when compared with controls. There were no significant differences in IL-2 or IFN-γ expression across groups.ConclusionsSubconjunctival injection of MSCs in rats was effective in prolonging corneal allograft survival. This effect was mediated by inhibition of inflammatory and immune responses, indicating an anti-inflammatory shift in the balance of T helper (Th)1 to T helper(Th) 2.

Effects of AS2541019, a novel selective PI3Kδ inhibitor, on antibody production and hamster to rat xenotransplantation

B cell-mediated antibodies play a critical role in protecting the body from infections; however, excessive antibody production is involved in the pathogenesis of autoimmune diseases and transplanted organ rejection. Regulation of antibody production is therefore crucial for overcoming these complications. Phosphatidylinositol-3-kinase p110δ (PI3Kδ), a member of the family of PI3K lipid kinases, is a key mediator of B cell activation and proliferation, with a small molecule PI3Kδ inhibitor having been approved for the treatment of B cell lymphoma. However, the effect of PI3Kδ inhibitors on B cell-mediated antibody production has not been clearly elucidated. In this study, we investigated the effect of the selective PI3Kδ inhibitor, AS2541019, on B cell immunity and antibody production. Our results show that AS2541019 effectively prevented B cell activation and proliferation in vitro, and that oral administration of AS2541019 resulted in significant inhibition of both T-dependent and T-independent de novo antibody production in peripheral blood. Further, in a hamster to rat concordant xenotransplant model, AS2541019 significantly prolonged graft survival time by inhibiting xenoreactive antibody production. Therefore, our study demonstrates that the selective PI3Kδ inhibitor AS2541019 inhibits antibody production through potent inhibitory effects on B cell activation, and can protect against organ dysfunction.

Mesenchymal stem cells increase skin graft survival time and up-regulate PD-L1 expression in splenocytes of mice

Recently, mesenchymal stem cells (MSCs) have gained considerable interests as hopeful therapeutic cells in transplantation due to their immunoregulatory functions. But exact mechanisms underlying MSCs immunoregulatory function is not fully understood. Herein, in addition to investigate the ability of MSCs to prolong graft survival time, the effects of them on the expression of PD-L1 and IDO immunomodulatory molecules in splenocytes of skin graft recipient mice was clarified. To achieve this goal, full-thickness skins were transplanted from C57BL/6 to BALB/c mice. MSCs were isolated from bone marrow of BALB/c mice and injected to the recipient mice. Skin graft survival was monitored daily to determine graft rejection time. On days 2, 5 and 10 post skin transplantation, serum cytokine levels and expression of PD-L1 and IDO mRNA and protein in the splenocytes of recipient mice were evaluated. The results showed that administration of MSCs prolonged skin graft survival time from 11 to 14 days. On days 2 and 5 post transplantation, splenocytes PD-L1 expression and IL-10 serum level in MSCs treated mice were higher than those in the controls, while IL-2 and IFN-γ levels were lower. Rejection in MSCs treated mice was accompanied by an increase in IL-2 and IFN-γ, and decrease in PD-L1 expression and IL-10 level. No difference in the expression of IDO between MSCs treated mice and controls was observed. In conclusion, we found that one of the mechanisms underlying MSCs immunomodulatory function could be up-regulating PD-L1 expression.

Immunological Challenges Facing Translation of Alginate Encapsulated Porcine Islet Xenotransplantation to Human Clinical Trials.

Transplantation of alginate-encapsulated islets has the potential to treat patients suffering from type I diabetes, a condition characterized by an autoimmune attack against insulin-secreting beta cells. However, there are multiple immunological challenges associated with this procedure, all of which must be adequately addressed prior to translation from trials in small animal and nonhuman primate models to human clinical trials. Principal threats to graft viability include immune-mediated destruction triggered by immunogenic alginate impurities, unfavorable polymer composition and surface characteristics, and release of membrane-permeable antigens, as well as damage associated molecular patterns (DAMPs) by the encapsulated islets themselves. The lack of standardization of significant parameters of bioencapsulation device design and manufacture (i.e., purification protocols, surface-modification grafting techniques, alginate composition modifications) between labs is yet another obstacle that must be overcome before a clinically effective and applicable protocol for encapsulating islets can be implemented. Nonetheless, substantial progress is being made, as is evident from prolonged graft survival times and improved protection from immune-mediated graft destruction reported by various research groups, but also with regard to discoveries of specific pathways involved in explaining observed outcomes. Progress in the latter is essential for a comprehensive understanding of the mechanisms responsible for the varying levels of immunogenicity of certain alginate devices. Successful translation of encapsulated islet transplantation from in vitro and animal model testing to human clinical trials hinges on application of this knowledge of the pathways and interactions which comprise immune-mediated rejection. Thus, this review not only focuses on the different factors contributing to provocation of the immune reaction by encapsulated islets, but also on the defining characteristics of the response itself.

Prolongation of kidney allograft survival regulated by indoleamine 2, 3-dioxygenase in immature dendritic cells generated from recipient type bone marrow progenitors.

Immature dendritic cells (iDCs) are bone marrow-derived professional antigen-presenting cells, exhibit very low levels of the co-stimulatory molecules CD80 (B7-1), CD86 (B7-2), and CD40 and major histocompatibility complex (MHC) class II and play a critical role in triggering antigen-specific immunotolerance. The enzyme indoleamine 2, 3-dioxygenase (IDO) is a cytosolic tryptophan catabolism rate-limiting step enzyme. IDO secreted by DCs shows an association with the suppression of T-cell responses and promotion of tolerance. In this study, BN rat recipients were pre-injected with donor renal alloantigen-treated recipient iDCs before kidney transplantation. The renal allograft exhibited a lighter renal rejection response, prolonged graft survival time, and an increasing content of CD4+CD25+Foxp3+ regulatory T cells (Tregs). Additionally, up-regulated secretion of Th2 cytokines were found in recipient sera post-transplantation. Transfection of si-IDO1 RNA into renal-antigen-treated recipient iDCs reversed these changes, which suggested that IDO channel signaling may be involved in iDC-induced allograft immunotolerance. These results suggested that iDC-induced and IDO-mediated allograft immunotolerance might be a potentially feasible tactic to prolong allograft survival, in addition to immunosuppressive drugs.

CD25 siRNA induces Treg/Th1 cytokine expression in rat corneal transplantation models

Corneal graft rejection is the major reason for transplant failure. CD25 plays an important role in the induction of corneal graft rejection by regulating CD4+ T cell function. Furthermore, CD25-mediated signaling is closely associated with the expression of Treg cytokines (IL-10, TGF-β) and Th1 cytokines (IFN-γ, IL-1β, and TNF-α). In the current study, corneal transplantation was performed on Wistar rats as donors and Sprague-Dawley rats as recipients. The survival curves indicated that CD25 siRNA treatment significantly prolonged graft survival time (mean survival time [MST], 14.8 ± 0.7 days) as compared with controls (MST, 7.6 ± 0.7 days; n = 12, p < 0.01). HE staining showed that CD25 siRNA alleviated inflammatory cell infiltration. At days 3, 7, 14, and 21, the mRNA and protein expression of CD25 in the CD25 siRNA groups were less than those of the control group, although the most significant decrease of CD25 protein was at day 3. The expression of IL-10 and TGF-β in the CD25 siRNA group increased, while IFN-γ, IL-1β, and TNF-α expression decreased, as well as no significant changes in Foxp3 expression were observed at day 14 post-operation. In conclusion, CD25 siRNA gene therapy played a protective role in corneal graft rejection via up-regulation of Treg cytokine expression and down-regulation of Th1 cytokine expression.

The Effect of Traditional Chinese Formula Danchaiheji on the Differentiation of Regulatory Dendritic Cells.

Recently, regulatory dendritic cells (DCregs), a newly described dendritic cell subset with potent immunomodulatory function, have attracted increased attention for their utility in treating immune response-related diseases, such as graft-versus-host disease, hypersensitivity, and autoimmune diseases. Danchaiheji (DCHJ) is a traditional Chinese formula that has been used for many years in the clinic. However, whether DCHJ can program dendritic cells towards a regulatory phenotype and the underlying mechanism behind this process remain unknown. Herein, we investigate the effects of traditional Chinese DCHJ on DCregs differentiation and a mouse model of skin transplantation. The current study demonstrates that DCHJ can induce dendritic cells to differentiate into DCregs, which are represented by high CD11b and low CD86 and HLA-DR expression as well as the secretion of IL-10 and TGF-β. In addition, DCHJ inhibited DC migration and T cell proliferation, which correlated with increased IDO expression. Furthermore, DCHJ significantly prolonged skin graft survival time in a mouse model of skin transplantation without any liver or kidney toxicity. The traditional Chinese formula DCHJ has the potential to be a potent immunosuppressive agent with high efficiency and nontoxicity.

Effects of CD25siRNA gene transfer on high-risk rat corneal graft rejection.

Corneal graft rejection is the major cause of corneal failures. Previous studies have shown that the CD25 monoclonal antibody can inhibit corneal graft rejection during the acute phase of rejection in rat models. In the current study, we evaluated the safety and efficacy of the topical Entranster™ vector in rat corneal applications and further investigated the effects of CD25siRNA gene transfer on high-risk rat corneal graft rejection. Fluorescence detection, clinical assessment, hematoxylin and eosin (HE), terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL) and CD11b assays were used to evaluate the safety and efficacy of CD25siRNA gene transfer in normal SD rat corneas. Orthotopic corneal transplants were performed in alkali burned SD rats. Corneal recipients were divided into four groups that received different treatments. Clinical assessment, western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), immunohistochemistry, HE and transmission electron microscopy (TEM) were performed on all grafts. Low toxicity, no immunogenicity and high transfection efficiency were observed in rat corneas treated with the Entranster™ vector. Reduced endothelial cell apoptosis, inflammatory cell infiltration and graft neovascularisation were observed in the CD25siRNA treatment groups. The graft survival curves showed that CD25siRNA treatment significantly prolonged graft survival time, with better graft transparency and less graft oedema. Lower CD25 and higher IL-10 expression were detected in the CD25siRNA treatment groups during the study period, and a higher FOXP3 level was found in the CD25siRNA group than in the CD25siRNA-twice group on days 14 and 21 post-operation. The Entranster™ vector is an effective vector for corneal gene therapy. CD25 siRNA gene transfer inhibits corneal graft rejection via upregulation of anti-inflammatory molecule expression.

The roles of IL-2 and IL-10 enhance anti-CD45RBmAb immune inhibition in allograft skin.

As a new type of immune tolerance inducer, anti-CD45RB monoclonal antibodies (anti-CD45RBmAb) can prolong the graft survival time of animal organs or cell transplantation as well as induce stable immune tolerance. Both interleukin (IL)-2 and IL-10 have important roles in the induction and maintenance of immunological tolerance. However, whether these cytokines combined with anti-CD45RBmAb can promote immune tolerance is poorly understood. Therefore, we investigated the effect of IL-2 and IL-10 in vitro and in vivo on the tolerance induction by anti-CD45RBmAb. The changes of Treg and Th17 cells and Th1/Th2 cytokines in anti-CD45RBmAb induced prolongation of skin allograft survival in mice. The finding of a role for IL-2 is novel, of interest, IL-2 promoted anti-CD45RBmAb-induced CD4(+) T cell differentiation into Treg and Th2 cells and suppressed Th17 and Th1 cells. IL-2 enhanced the induction of immune tolerance by anti-CD45RBmAb and significantly prolonged skin graft survival time in vivo. In contrast, this effect should be demonstrated experimentally by neutralizing IL-2 and inhibition of the effect of anti-CD45RBmAb, and neutralizing IL-10 showed no effect for anti-CD45RBmAb-induced tolerance. These data reveal that IL-2 significantly enhances anti-CD45RBmAb-induced immune tolerance via up-regulated T regulatory (Treg) cells and the balance of Th1/Th2 shifts. Conversely, IL-10 showed no effect on anti-CD45RBmAb-induced tolerance.

ASKP1240, a Fully Human Anti-CD40 Monoclonal Antibody, Prolongs Pancreatic Islet Allograft Survival in Nonhuman Primates

A strategy for inhibiting CD40 has been considered as an alternative approach for immunosuppression because of undesirable effects of anti-CD154 monoclonal antibodies (mAbs). Previously, we demonstrated that ASKP1240, which is a fully human anti-CD40 mAb, significantly prolonged kidney and liver allograft survival in cynomolgus monkeys without causing thromboembolic complications. Herein, we evaluated the effect of ASKP1240 on pancreatic islet transplantation (PITx) in cynomolgus monkeys. Diabetes was induced by total pancreatectomy, and islet allografts were transplanted into the liver. Following PITx (8201–12 438 IEQ/kg), blood glucose levels normalized promptly in all animals. Control islet allografts were rejected within 9 days (n = 3), whereas ASKP1240 (10 mg/kg) given on postoperative days 0, 4, 7, 11 and 14 (induction treatment, n = 5) significantly prolonged graft survival time (GST) to >15, >23, 210, 250 and >608 days, respectively. When ASKP1240 (5 mg/kg) was administered weekly thereafter up to post-PITx 6 months (maintenance treatment, n = 4), GST was markedly prolonged to >96, >115, 523 and >607 days. During the ASKP1240 treatment period, both anti-donor cellular responses and development of anti-donor antibodies were abolished, and no serious adverse events were noted. ASKP1240 appears to be a promising candidate for immunosuppression in clinical PITx.

Suppression of NF‐kappaB p65 expression attenuates delayed xenograft rejection

Delayed xenograft rejection (DXR) involves type II vascular endothelial cell (VEC) activation including upregulation of pro-inflammatory genes, which contributes to infiltration into the graft and a complex process of cytokine production. Approaches to prevent DXR have shown limited success. In this study, we modified heart donors using siRNA in an attempt to attenuate DXR and to improve xenograft survival in the mouse-to-rat heterotopic heart transplant model. siRNA technology was used to inhibit NF-kappaB p65 gene expression in vivo in mice. After the donor was transfected with siRNA, the effects of NF-kappaB siRNA on DXR and expression of NF-kappaB and pro-inflammatory genes were evaluated in a concordant mouse-to-rat cardiac xenograft model. Treatment of NF-kappaB siRNA prolonged median heart graft survival time in the recipient rats from 1.7days in a PBS control group to 5.4days in the NF-kappaB siRNA-treated group (P<0.05). Compared with normal mouse hearts, the NF-kappaB p65 mRNA relative levels following siRNA injection in the donors decreased significantly (approximately 70% reduction) in grafts harvested 12h after transplantation. The mRNA levels of VCAM-1, ICAM-1, and interleukin-1 displayed a similar reduction. Histological evaluation using light and electron microscopy showed that damage of endothelial cells after NF-kappaB siRNA treament occured at a later time. Transfection of NF-kappaB p65 siRNA in donor animals can delay the emergence of DXR. This treatment may be used as part of strategies to minimize the complex and multi-faceted rejection responses in vascularized xenografts.