Expansion of CD45RA-FOXP3++ regulatory T cells is associated with immune tolerance in patients with combined kidney and bone marrow transplantation.

OBJECTIVES: Simultaneous transplantation of a solid organ and bone marrow from the same donor is a possible means of achieving transplant tolerance. Here, we attempted to identify biomarkers that indicate transplant tolerance for discontinuation of immunosuppressants in combined kidney and bone marrow transplantation (CKBMT). METHODS: Conventional kidney transplant (KT) recipients (n = 20) and CKBMT recipients (n = 6) were included in this study. We examined various immunological parameters by flow cytometry using peripheral blood mononuclear cells (PBMCs), including the frequency and phenotype of regulatory T (Treg) cell subpopulations. We also examined the suppressive activity of the Treg cell population in the setting of mixed lymphocyte reaction (MLR) with or without Treg cell depletion. RESULTS: Among six CKBMT recipients, three successfully discontinued immunosuppressants (tolerant group) and three could not (non-tolerant group). The CD45RA-FOXP3++ Treg cell subpopulation was expanded in CKBMT recipients compared to conventional kidney transplant patients, and this was more obvious in the tolerant group than the non-tolerant group. In addition, high suppressive activity of the Treg cell population was observed in the tolerant group. The ratio of CD45RA-FOXP3++ Treg cells to CD45RA-FOXP3+ cells indicated good discrimination between the tolerant and non-tolerant groups. CONCLUSION: Thus, our findings propose a biomarker that can distinguish CKBMT patients who achieve transplant tolerance and are eligible for discontinuation of immunosuppressants and may provide insight into tolerance mechanisms in CKBMT.

Optimal allogeneic islet dose for transplantation in insulin-dependent diabetic Macaca fascicularis monkeys.

Many groups are working to improve the results of clinical allogeneic islet transplantation in a primate model. However, few studies have focused on the optimal islet dose for achieving normal glycemia without exogenous insulin after transplantation in primate models or on the relationship between rejection and islet amyloid polypeptide (IAPP) expression. We evaluated the dose (10,000, 20,000, and > 25,000 islet equivalents (IEQ)/kg) needed to achieve normal glycemia without exogenous insulin after transplantation using eleven cynomolgus monkeys, and we analyzed the characteristics exhibited in the islets after transplantation. 10,000 IEQ/kg (N = 2) failed to control blood glucose level, despite injection with the highest dose of exogenous insulin, and 20,000 IEQ/kg group (N = 5) achieved unstable control, with a high insulin requirement. However, 25,000 IEQ/kg (N = 4) achieved normal glycemia without exogenous insulin and maintained it for more than 60 days. Immunohistochemistry results from staining islets found in liver biopsies indicated that as the number of transplanted islets decreased, the amount of IAPP accumulation within the islets increased, which accelerated CD3+ T cell infiltration. In conclusion, the optimal transplantation dose for achieving a normal glycemia without exogenous insulin in our cynomolgus monkey model was > 25,000 IEQ/kg, and the accumulation of IAPP early after transplantation, which depends on the transplanted islet dose, can be considered one factor in rejection.

Off-the-Shelf, Immune-Compatible Human Embryonic Stem Cells Generated Via CRISPR-Mediated Genome Editing.

Human embryonic stem cells (hESCs) hold promise in regenerative medicine but allogeneic immune rejections caused by highly polymorphic human leukocyte antigens (HLAs) remain a barrier to their clinical applications. Here, we used a CRISPR/Cas9-mediated HLA-editing strategy to generate a variety of HLA homozygous-like hESC lines from pre-established hESC lines. We edited four pre-established HLA-heterozygous hESC lines and created a mini library of 14 HLA-edited hESC lines in which single HLA-A and HLA-B alleles and both HLA-DR alleles are disrupted. The HLA-edited hESC derivatives elicited both low T cell- and low NK cell-mediated immune responses. Our library would cover about 40% of the Asian-Pacific population. We estimate that HLA-editing of only 19 pre-established hESC lines would give rise to 46 different hESC lines to cover 90% of the Asian-Pacific population. This study offers an opportunity to generate an off-the-shelf HLA-compatible hESC bank, available for immune-compatible cell transplantation, without embryo destruction. Graphical Abstract.

Inducing Transient Mixed Chimerism for Allograft Survival Without Maintenance Immunosuppression With Combined Kidney and Bone Marrow Transplantation: Protocol Optimization.

BACKGROUND: Tolerance induction is an important goal in the field of organ transplantation. We have sequentially modified our conditioning regimen for induction of donor-specific tolerance in recipients of major histocompatibility complex-mismatched combined kidney and bone marrow transplantation (CKBMT). METHODS: From December 2011 to May 2017, 8 major histocompatibility complex-mismatched patients received CKBMT. The initial conditioning regimen (protocol 1) consisted of cyclophosphamide (CP), rituximab, rabbit antithymocyte globulin, and thymic irradiation. Tacrolimus and steroids were used for the maintenance of immunosuppression (IS). RESULTS: This regimen was complicated by transient acute kidney injury, which has been the major clinical feature of engraftment syndrome and side effects of CP, although one of 2 subjects successfully discontinued his IS for 14 months. The conditioning regimen was modified by reducing the CP dose and adding fludarabine (protocol 2). The final modification was reducing the fludarabine and rabbit antithymocyte globulin doses (protocol 3). Mixed chimerism, detected by the short tandem repeat method, was achieved transiently in all subjects for 3-20 weeks. Among the 3 subjects treated with protocol 2, IS was successfully discontinued for >35 months in one subject, but the other 2 subjects suffered from severe BK virus-associated nephritis. All 3 subjects treated with protocol 3 tolerated the protocol well and have successfully discontinued IS for >4-41 months. Interestingly, de novo donor-specific antibody was not detected in any subject during all the follow-up periods. CONCLUSIONS: Our clinical trial has shown that long-term renal allograft survival without maintenance IS can be achieved by induction of mixed chimerism following CKBMT.

Comparative study of human and cynomolgus T-cell depletion with rabbit anti-thymocyte globulin (rATG) treatment-for dose adjustment in a non-human primate kidney transplantation model.

Rabbit-antithymocyte globulin (rATG) is commonly used in kidney transplantation (KT) as an induction agent and is also commonly used in non-human primate (NHP) KT models. However, the optimal dose has not been reported. In this study, we evaluated which cumulative dose of rATG was most appropriate for transplantation in NHPs. Cynomolgus monkeys were treated with intravenous 5 mg/kg rATG (Thymoglobulin®, Genzyme Ltd., UK) twice, on days 0 and 2 (a total of 10 mg/kg, n=2), or 4 times, on days 0, 1, 2, and 3 (a total of 20 mg/kg, n=6). In addition, we performed allo-KT in cynomolgus monkeys (n=4) with a cumulative 20 mg/kg dose of rATG with optimized dosing for induction therapy. We further compared immune cells, including naïve, central memory, and effector memory T cells, in reconstituted distributions in human KT patients (n=22). The kinetics of lymphocytes showed a rapid decrease at day 1 that was maintained for 2 weeks in the 20 mg/kg rATG group, while lymphocyte depletion was not maintained for more than 1 week in the 10 mg/kg rATG group. During the early period of rATG treatment in the NHP-KT model, the frequency of total T cells in the 20 mg/kg group showed a pattern of depletion similar with that of KT patients treated with rATG (1.5 mg/kg, 3 days). However, the pattern of reconstituted T cell subpopulations was different, as the number of effector memory cells rebounded in the NHP-KT model. These data indicate that lymphocyte-depletion induced by rATG was influenced by cumulative dose, and that an rATG dose of 20 mg/kg is suitable for induction therapy in renal transplantation in cynomolgus monkeys compared to human KT.

Cyclophosphamide and fludarabine monophosphate dose optimization for the non-myeloablative condition in non-human primates to induce transient mixed chimerism via bone marrow transplantation.

Bone marrow preconditioning using cyclophosphamide (CP) is generally used for bone marrow transplantation (BMT). However, because of CP's hepatotoxicity and nephrotoxicity, additional fludarabine (FDR) administration and a reduced dose of CP are used for reduced-intensity preconditioning. Recently, preclinical studies using non-human primates (NHPs) were performed to induce immune tolerance after solid organ transplantation by conducting BMT simultaneously. However, dose optimization of CP and FDR for BMT preconditioning in cynomolgus monkeys has not been conducted. Therefore, the objective of this study was to evaluate the efficacy and tolerability of induction protocols using different doses of CP and FDR. Our results showed that relatively low-dose CP (30 mg/kg×2) combined with additional high-dose FDR (60 mg/m2×4) was associated with sufficient suppression in periphery as well as in bone marrow compared with high-dose CP (60 mg/kg×2) combined with low-dose FDR (30 mg/m2×4) and did not show hepatic or renal toxicity. CD34+ stem cells were also well suppressed with both doses. Therefore, we concluded that the combination of 60 mg/kg of CP with 240 mg/m2 of FDR can be used effectively and safely for non-myeloablative preconditioning for BMT in cynomolgus monkeys.

Polymeric nano-shielded islets with heparin-polyethylene glycol in a non-human primate model.

Intraportal pancreatic islet transplantation incurs huge cell losses during its early stages due to instant blood-mediated inflammatory reactions (IBMIRs), which may also drive regulation of the adaptive immune system. Therefore, a method that evades IBMIR will improve clinical islet transplantation. We used a layer-by-layer approach to shield non-human primate (NHP) islets with polyethylene glycol (nano-shielded islets, NSIs) and polyethylene glycol plus heparin (heparin nano-shielded islets; HNSIs). Islets ranging from 10,000 to 20,000 IEQ/kg body weight were transplanted into 19 cynomolgus monkeys (n = 4, control; n = 5, NSI; and n = 10, HNSI). The mean C-peptide positive graft survival times were 68.5, 64 and 108 days for the control, NSI and HNSI groups, respectively (P = 0.012). HNSI also reduced the factors responsible for IBMIR in vitro. Based on these data, HNSIs in conjunction with clinically established immunosuppressive drug regimens will result in superior outcomes compared to those achieved with the current protocol for clinical islet transplantation.

Therapeutic effects of anti-CD154 antibody in cynomolgus monkeys with advanced rheumatoid arthritis.

Rheumatoid arthritis is one major chronic inflammatory systemic autoimmune disease. The CD154-CD40 interactions play a critical role in the regulation of immune responses and the maintenance of autoimmunity. Therefore, we aimed to determine whether anti-CD154 antibody treatment show positive effects on immunomodulation and clinical improvement of sustained severe rheumatoid arthritis in cynomolgus monkeys. Arthritis was induced using chicken type II collagen (CII) and arthritic monkey were divided into control and anti-CD154 treatment groups based on their concentrations of anti-CII antibodies on week 7 post-immunization. Blood and tissue samples were collected on week 16 post-immunization. Anti-CD154 antibody treatment improved arthritis and movement, and significantly decreased the numbers of proliferating B cells and the serum levels of anti-type II collagen antibody and sCD154 compared with non-treatment group. Further anti-CD154 antibody treatment significantly decreased the percentage of CD4+ cells and the ratio of CD4+ to CD8+ T cells and significantly increased the percentage of CD8+ cells and effector memory CD8+ cells in peripheral blood. We have shown for the first time in a nonhuman primate model of RA that CD154 blockade has beneficial effects. This study might be valuable as preclinical data of CD154 blockade in nonhuman primate models of severe rheumatoid arthritis.