Tetsu Oura

A pilot study of operational tolerance with a regulatory T‐cell‐based cell therapy in living donor liver transplantation

Potent immunosuppressive drugs have significantly improved early patient survival after liver transplantation (LT). However, long‐term results remain unsatisfactory because of adverse events that are largely associated with lifelong immunosuppression. To solve this problem, different strategies have been undertaken to induce operational tolerance, for example, maintenance of normal graft function and histology without immunosuppressive therapy, but have achieved limited success. In this pilot study, we aimed to induce tolerance using a novel regulatory T‐cell‐based cell therapy in living donor LT. Adoptive transfer of an ex vivo‐generated regulatory T‐cell‐enriched cell product was conducted in 10 consecutive adult patients early post‐LT. Cells were generated using a 2‐week coculture of recipient lymphocytes with irradiated donor cells in the presence of anti‐CD80/86 monoclonal antibodies. Immunosuppressive agents were tapered from 6 months, reduced every 3 months, and completely discontinued by 18 months. After the culture, the generated cells displayed cell‐number‐dependent donor‐specific inhibition in the mixed lymphocyte reaction. Infusion of these cells caused no significant adverse events. Currently, all patients are well with normal graft function and histology. Seven patients have completed successful weaning and cessation of immunosuppressive agents. At present, they have been drug free for 16‐33 months; 4 patients have been drug free for more than 24 months. The other 3 recipients with autoimmune liver diseases developed mild rejection during weaning and then resumed conventional low‐dose immunotherapy. Conclusions: A cell therapy using an ex vivo‐generated regulatory T‐cell‐enriched cell product is safe and effective for drug minimization and operational tolerance induction in living donor liver recipients with nonimmunological liver diseases. (Hepatology 2016;64:632‐643)

Long-Term Hepatic Allograft Acceptance Based on CD40 Blockade by ASKP1240 in Nonhuman Primates

Blockade of the CD40–CD154 costimulatory signal is an attractive strategy for immunosuppression and tolerance induction in organ transplantation. Treatment with anti‐CD154 monoclonal antibodies (mAbs) results in potent immunosuppression in nonhuman primates (NHPs). Despite plans for future clinical use, further development of these treatments was halted by complications. As an alternative approach, we have been focusing on the inhibition of the counter receptor, CD40 and have shown that a novel human anti‐CD40 mAb, ASKP1240, markedly prolongs renal allograft survival in NHPs, although allografts eventually underwent chronic allograft nephropathy. On the basis of our previous findings that a CD40–CD154 costimulation blockade induces tolerance to hepatic, but not cardiac, allografts in rodents, we tested here our hypothesis that a blockade of CD40 by ASKP1240 allows acceptance of hepatic allografts in NHPs. A 2‐week ASKP1240 induction treatment prolonged liver allograft survival in NHPs; however, the graft function deteriorated due to chronic rejection. In contrast, a 6‐month ASKP1240 maintenance monotherapy efficiently suppressed both cellular and humoral alloimmune responses and prevented rejection on the hepatic allograft. No serious side effects, including thromboembolic complications, were noted in the ASKP1240‐treated monkeys. We conclude that CD40 blockade by ASKP1240 would be a desirable immunosuppressant for clinical liver transplantation.

A novel NF-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates inflammatory colonic injury in mice

BACKGROUND In inflammatory bowel disease (IBD), gut inflammation is associated with the activation of nuclear factor kappa B (NF-κB), a key pro-inflammatory transcription factor. AIM To investigate the therapeutic potential of a novel, specific NF-κB inhibitor, dehydroxymethylepoxyquinomicin (DHMEQ), we examined its effect on IBD using murine experimental colitis models. METHODS The in vitro effect of DHMEQ was evaluated by inflammatory cytokine production and p65 immunostaining using HT-29 and RAW264.7 cells. The in vivo therapeutic effect of DHMEQ was studied in colitis induced by dextran sulphate sodium (DSS) and trinitrobenzenesulphonic acid (TNBS). In these, progression and severity of colitis was mainly assessed by the disease activity index (DAI), histopathology, cellular infiltration, and mRNA expression levels of pro-inflammatory cytokines in the colonic tissues. RESULTS In RAW264.7 cells, DHMEQ significantly inhibited tumour necrosis factor (TNF)-α and interleukin (IL)-6 production induced by LPS in a dose-dependent manner by blocking the nuclear translocation of NF-κB. In addition, DHMEQ inhibited IL-8 production induced by LPS in HT-29 cells. DHMEQ significantly ameliorated DSS colitis as assessed by DAI scores, colonic oedema, and histological scores. Immunohistochemistry revealed that DHMEQ inhibited colonic infiltration of nuclear p65(+) cells, CD4(+) lymphocytes, and F4/80(+) macrophages. mRNA expression levels of the pro-inflammatory cytokines, such as IL-1β, TNF-α, IL-6, IL-12p40, IL-17, and MCP-1 were also suppressed by DHMEQ administration. Furthermore, DHMEQ significantly ameliorated TNBS colitis as assessed by body-weight changes and histological scores. CONCLUSION DHMEQ ameliorated experimental colitis in mice. These results indicate that DHMEQ appears to be an attractive therapeutic agent for IBD.

Does the Permanent Portacaval Shunt for a Small-for-Size Graft in a Living Donor Liver Transplantation Do More Harm Than Good?

In order to obviate a small‐for‐size graft syndrome (SFSGS), a portacaval (PC) shunt had been considered in a case of adult‐to‐adult living donor liver transplantation (AA‐LDLT). In a recent AA‐LDLT case, we adopted the PC shunt to resolve SFSGS; however, graft atrophy was observed in the late period of LDLT, thereby resulting in liver dysfunction. Due to the surgical closure of the PC shunt at 11 months post‐LDLT, the graft regenerated gradually and resulted in the recovery of the liver function. This experience indicates that the portacaval shunt would overcome SFSGS in the early period of LDLT, while it would cause the graft atrophy and the graft dysfunction in the late period of LDLT.

Transient portacaval shunt for a small‐for‐size graft in living donor liver transplantation

Adult-to-adult living donor liver transplantation (AALDLT) is an established treatment option for selected patients with end-stage liver disease. However, its widespread application is limited by the liver volume that can be safely resected from a living donor because a sufficient volume is also required for the recipient. Use of a right lobe graft is widely recommended in AA-LDLT because it can provide sufficient volume to the recipient. However, in comparison with the left lobe graft, the right lobe graft imposes a burden on the donor due to the smaller residual liver volume in the donor. Moreover, the recipient operation with the right lobe graft may be more complicated in consequence of the reconstruction of middle hepatic vein tributaries and plural bile duct. On the other hand, the main problem in using the left lobe graft in AA-LDLT is the small-for-size graft syndrome (SFSGS). The size of the graft required for successful liver transplantation is 30%-40% of the expected liver volume for the recipient or 0.8%-1.0% of the body weight. It is reported that excessive portal venous inflow is a determining factor for injury to endothelial cells and hepatic parenchyma related to SFSGS. A better understanding of the pathophysiology of the small-for-size graft may lead to logical approaches for improving subsequent allograft function. In recent reports, a permanent portacaval (PC) shunt was developed to resolve SFSGS. Partial diversion of the portal flow to the systemic circulation through a PC shunt may be a reasonable approach for attenuating these injuries. However, in this technique, there is every possibility of disturbing appropriate graft regeneration after liver transplantation because a sufficient amount of portal blood does not flow into the graft permanently. In this report, we describe a successful technique for transient PC shunt in small-for-size liver transplantation. A 54-year-old woman with primary biliary cirrhosis underwent LDLT with a left lobe. The actual volume at the back table was 316 ml, which represented 35.8% of the recipient’s estimated standard liver volume. Because the graft volume was small for the recipient, a transient PC shunt was performed between the inferior vena cava and the right branch of the portal vein. To prevent portal hypertension, the PC shunt was performed immediately after total hepatectomy by using a continuous 6-0 Prolene suture. The Endoloop (Ethicon, Somerville, NJ, USA) was passed around the PC shunt beforehand (Fig. 1A). When the graft was implanted, the donor’s hepatic vein was anastomosed to the confluence of the recipient’s middle hepatic vein and left hepatic vein. Subsequently, portal vein anastomosis was performed using a continuous Prolene 6-0 suture. The hepatic vein was then unclamped, and portal reperfusion was initiated. After reperfusion of the portal vein, hepatic artery anastomosis and bile duct reconstruction were performed. After all vascular anastomoses were complete, a 20-French Scale Nelaton tube was inserted into the abdominal cavity. The tip of the tube was located on the PC shunt. The Endoloop was taken out of the abdominal cavity through the tube (Fig. 1B). A hemoclip was fixed on the Endoloop as amarking for the ligation of the Endoloop through a fluoroscope. Another hemoclip was fixed on the opposite side of the Endoloop (Fig. 2). Three-dimensional computed tomography revealed the PC shunt patency 7 days after the operation (Fig. 3). After confirming that the recipient’s clinical condi-

Use of CTLA4Ig for Induction of Mixed Chimerism and Renal Allograft Tolerance in Nonhuman Primates

We have previously reported successful induction of renal allograft tolerance via a mixed chimerism approach in nonhuman primates. In those studies, we found that costimulatory blockade with anti‐CD154 mAb was an effective adjunctive therapy for induction of renal allograft tolerance. However, since anti‐CD154 mAb is not clinically available, we have evaluated CTLA4Ig as an alternative agent for effecting costimulation blockade in this treatment protocol. Two CTLA4Igs, abatacept and belatacept, were substituted for anti‐CD154 mAb in the conditioning regimen (low dose total body irradiation, thymic irradiation, anti‐thymocyte globulin and a 1‐month posttransplant course of cyclosporine [CyA]). Three recipients treated with the abatacept regimen failed to develop comparable lymphoid chimerism to that achieved with anti‐CD154 mAb treatment and these recipients rejected their kidney allografts early. With the belatacept regimen, four of five recipients developed chimerism and three of these achieved long‐term renal allograft survival (>861, >796 and >378 days) without maintenance immunosuppression. Neither chimerism nor long‐term allograft survival were achieved in two recipients treated with the belatacept regimen but with a lower, subtherapeutic dose of CyA. This study indicates that CD28/B7 blockade with belatacept can provide a clinically applicable alternative to anti‐CD154 mAb for promoting chimerism and renal allograft tolerance.

Chimerism-based tolerance in organ transplantation: preclinical and clinical studies

Induction of allograft tolerance has been considered the ultimate goal in organ transplantation. Although numerous protocols to induce allograft tolerance have been reported in mice, a chimerism‐based approach through donor haematopoietic stem cell transplantation has been the only approach to date that induced allograft tolerance reproducibly following kidney transplantation in man. Renal allograft tolerance has been achieved by induction of either transient mixed chimerism or persistent full donor chimerism. Although the risk of rejection may be low in tolerance achieved via durable full donor chimerism, the development of graft‐versus‐host disease (GVHD) has limited the wider clinical application of this approach. In contrast, tolerance induced by transient mixed chimerism has not been associated with GVHD, but the risk of allograft rejection is more difficult to predict after the disappearance of haematopoietic chimerism. Current efforts are directed towards the development of more clinically feasible and reliable approaches to induce more durable mixed chimerism in order to widen the clinical applicability of these treatment regimens.

Chronic Antibody-Mediated Rejection in Nonhuman Primate Renal Allografts: Validation of Human Histological and Molecular Phenotypes

Molecular testing represents a promising adjunct for the diagnosis of antibody‐mediated rejection (AMR). Here, we apply a novel gene expression platform in sequential formalin‐fixed paraffin‐embedded samples from nonhuman primate (NHP) renal transplants. We analyzed 34 previously described gene transcripts related to AMR in humans in 197 archival NHP samples, including 102 from recipients that developed chronic AMR, 80 from recipients without AMR, and 15 normal native nephrectomies. Three endothelial genes (VWF, DARC, and CAV1), derived from 10‐fold cross‐validation receiver operating characteristic curve analysis, demonstrated excellent discrimination between AMR and non‐AMR samples (area under the curve = 0.92). This three‐gene set correlated with classic features of AMR, including glomerulitis, capillaritis, glomerulopathy, C4d deposition, and DSAs (r = 0.39–0.63, p < 0.001). Principal component analysis confirmed the association between three‐gene set expression and AMR and highlighted the ambiguity of v lesions and ptc lesions between AMR and T cell–mediated rejection (TCMR). Elevated three‐gene set expression corresponded with the development of immunopathological evidence of rejection and often preceded it. Many recipients demonstrated mixed AMR and TCMR, suggesting that this represents the natural pattern of rejection. These data provide NHP animal model validation of recent updates to the Banff classification including the assessment of molecular markers for diagnosing AMR.

Kidney Versus Islet Allograft Survival After Induction of Mixed Chimerism With Combined Donor Bone Marrow Transplantation.

We have previously reported successful induction of transient mixed chimerism and long-term acceptance of renal allografts in MHC mismatched nonhuman primates. In this study, we attempted to extend this tolerance induction approach to islet allografts. A total of eight recipients underwent MHC mismatched combined islet and bone marrow (BM) transplantation after induction of diabetes by streptozotocin. Three recipients were treated after a nonmyeloablative conditioning regimen that included low-dose total body and thymic irradiation, horse Atgam (ATG), six doses of anti-CD154 monoclonal antibody (mAb), and a 1-month course of cyclosporine (CyA) (Islet A). In Islet B, anti-CD8 mAb was administered in place of CyA. In Islet C, two recipients were treated with Islet B, but without ATG. The results were compared with previously reported results of eight cynomolgus monkeys that received combined kidney and BM transplantation (Kidney A) following the same conditioning regimen used in Islet A. The majority of kidney/BM recipients achieved long-term renal allograft survival after induction of transient chimerism. However, prolonged islet survival was not achieved in similarly conditioned islet/BM recipients (Islet A), despite induction of comparable levels of chimerism. In order to rule out islet allograft loss due to CyA toxicity, three recipients were treated with anti-CD8 mAb in place of CyA. Although these recipients developed significantly superior mixed chimerism and more prolonged islet allograft survival (61, 103, and 113 days), islet function was lost soon after the disappearance of chimerism. In Islet C recipients, neither prolonged chimerism nor islet survival was observed (30 and 40 days). Significant improvement of mixed chimerism induction and islet allograft survival were achieved with a CyA-free regimen that included anti-CD8 mAb. However, unlike the kidney allograft, islet allograft tolerance was not induced with transient chimerism. Induction of more durable mixed chimerism may be necessary for induction of islet allograft tolerance.

Pilot Study Evaluating Regulatory T Cell-Promoting Immunosuppression and Nonimmunogenic Donor Antigen Delivery in a Nonhuman Primate Islet Allotransplantation Model

The full potential of islet transplantation will only be realized through the development of tolerogenic regimens that obviate the need for maintenance immunosuppression. Here, we report an immunotherapy regimen that combines 1‐ethyl‐3‐(3′‐dimethylaminopropyl)‐carbodiimide (ECDI)‐treated donor lymphoid cell infusion (ECDI‐DLI) with thymoglobulin, anti‐interleukin‐6 receptor antibody and rapamycin to achieve prolonged allogeneic islet graft survival in a nonhuman primate (NHP) model. Prolonged graft survival is associated with Treg expansion, donor‐specific T cell hyporesponsiveness and a transient absence of donor‐specific alloantibody production during the period of graft survival. This regimen shows promise for clinical translation.