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Background: Adoptive cellular therapy (ACT) with antibody-suppressor CXCR5 + CD8 + T cells (CD8 + T Ab-supp ) inhibits alloantibody production, antibody-mediated rejection (AMR), and prolongs graft survival in multiple transplant mouse models. However, it is not known how conventional immunosuppressive agents impact the efficacy of CD8 + T Ab-supp ACT., Methods: We investigated the efficacy of CD8 + T Ab-supp cell ACT when combined with calcineurin inhibitor (CNi) or mammalian target of rapamycin inhibitor (mTORi) in a murine model of kidney transplant., Results: ACT-mediated decrease in germinal center B cells, posttransplant alloantibody titer, and amelioration of AMR in high alloantibody-producing CCR5 knockout kidney transplant recipients were impaired when ACT was combined with CNi and enhanced when combined with mTORi. CNi (but not mTORi) reduced ACT-mediated in vivo cytotoxicity of IgG + B cells and was associated with increased quantity of germinal center B cells. Neither CNi nor mTORi treatment impacted the expression of cytotoxic effector molecules (FasL, Lamp1, perforin, granzyme B) by CD8 + T Ab-supp after ACT. Concurrent treatment with CNi (but not mTORi) reduced in vivo proliferation of CD8 + T Ab-supp after ACT. The increase in quantity of splenic CD44 + CXCR5 + CD8 + T cells that occurs after ACT was reduced by concurrent treatment with CNi but not by concurrent treatment with mTORi (dose-dependent)., Conclusions: Impaired efficacy of ACT by CNi is attributed to reduced persistence and/or expansion of CD8 + T Ab-supp cells after ACT. In contrast, concurrent immunosuppression with mTORi preserves CD8 + T Ab-supp cells quantity, in vivo proliferation, and in vivo cytotoxic effector function after ACT and enhances suppression of humoral alloimmunity and AMR., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2023 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Liver transplantation (LT) is the treatment of choice for patients with hepatocellular carcinoma (HCC). Recurrence of HCC after LT occurs in 10% to 20% of cases. Preclinical studies to evaluate immune checkpoint inhibitors in conjunction with immunosuppressant treatment in transplant recipients have been lacking. Here, we evaluated the efficacy, safety, and mechanism of programmed cell death-1 (PD1) blockade under tacrolimus treatment in transplant recipients., Methods: We used a murine allogeneic skin transplantation model and murine syngeneic subcutaneous and orthotopic HCC models and measured the tumor volume and the change in tumor-infiltrating lymphocytes under PD1 blockade and tacrolimus treatment., Results: Tacrolimus treatment prolonged allograft survival in the allogeneic transplantation model and enhanced tumor growth in both subcutaneous and orthotopic HCC models. PD1 blockade suppressed tumor growth and lung metastasis in correlation with the number of infiltrating CD8 + T cells. Under tacrolimus treatment, PD1 blockade still resulted in an antitumor effect accompanied by a significant increase in tumor-infiltrating CD8 + T cells, natural killer cells, dendritic cells, and natural killer T cells. Tacrolimus treatment rescued the acceleration of transplant rejection induced by PD1 blockade in the allogeneic transplantation model., Conclusions: Our data suggest that treatment with high-dose tacrolimus in conjunction with PD1 blockade has an antitumor effect and reduces transplant rejection in mouse models of allograft skin transplantation and HCC. Thus, these results suggest that a clinical trial of PD1 inhibitors for HCC in LT merits consideration., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: . Transplant recipients may develop rejection despite having adequate tacrolimus whole blood predose concentrations (C 0 ). The intra-immune cellular concentration is potentially a better target than C 0 . However, little is known regarding intracellular tacrolimus concentration in T-lymphocytes and monocytes. We investigated the tacrolimus concentrations in both cell types and their relation with the expression and activity of FK-binding protein (FKBP)-12 and P-glycoprotein (P-gp)., Methods: . T-lymphocytes and monocytes were isolated from kidney transplant recipients followed by intracellular tacrolimus concentration measurement. FKBP-12 and P-gp were quantified with Western blot, flow cytometry, and the Rhodamine-123 assay. Interleukin-2 and interferon-γ in T-lymphocytes were measured to quantify the effect of tacrolimus., Results: . Tacrolimus concentration in T-lymphocytes was lower than in monocytes (15.3 [8.5-33.4] versus 131.0 [73.5-225.1] pg/million cells; P < 0.001). The activity of P-gp (measured by Rhodamine-123 assay) was higher in T-lymphocytes than in monocytes. Flow cytometry demonstrated a higher expression of P-gp (normalized mean fluorescence intensity 1.5 [1.2-1.7] versus 1.2 [1.1-1.4]; P = 0.012) and a lower expression of FKBP-12 (normalized mean fluorescence intensity 1.3 [1.2-1.7] versus 1.5 [1.4-2.0]; P = 0.011) in T-lymphocytes than monocytes. Western blot confirmed these observations. The addition of verapamil, a P-gp inhibitor, resulted in a 2-fold higher intra-T-cell tacrolimus concentration. This was accompanied by a significantly fewer cytokine-producing cells., Conclusions: . T-lymphocytes have a higher activity of P-gp and lower concentration of the FKBP-12 compared with monocytes. This explains the relatively lower tacrolimus concentration in T-lymphocytes. The addition of verapamil prevents loss of intracellular tacrolimus during the cell isolation process and is required to ensure adequate intracellular concentration measurement., Competing Interests: D.A.H. has received grant support (paid to the Erasmus MC) from Astellas Pharma and Chiesi Pharma and consulting fees from Astellas Pharma, Chiesi Pharma, MedinCell, Novartis Pharma and Sangamo Therapeutics. The other authors declare no conflicts of interest., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Prophylaxis of antibody-mediated rejection (AMR) caused by donor-specific antibodies remains challenging. Given the critical roles of complement activity in antibody-mediated graft injury, we developed a lipid nanoparticle (LNP) formulation of small-interfering RNA against complement C5 (C5 siRNA-LNP) and investigated whether C5 siRNA-LNP could downregulate the complement activity and act as an effective treatment for AMR., Methods: Lewis recipient rats were sensitized by skin grafting from Brown Norway donor rats. Kidney transplantation was performed at 4 wk post-skin grafting.C5 siRNA- or control siRNA-LNP was administered intravenously, and the weekly injections were continued until the study's conclusion. Cyclosporin (CsA) and/or deoxyspergualin (DSG) were used as adjunctive immunosuppressants. Complement activity was evaluated using hemolysis assays. The deposition of C5b9 in the grafts was evaluated using immunohistochemical analysis on day 7 posttransplantation., Results: C5 siRNA-LNP completely suppressed C5 expression and complement activity (hemolytic activity ≤ 20%) 7 d postadministration. C5 siRNA-LNP in combination with CsA and DSG (median survival time: 56.0 d) prolonged graft survival compared with control siRNA-LNP in combination with CsA and DSG (median survival time: 21.0 d; P = 0.0012; log-rank test). Immunohistochemical analysis of the grafts revealed that downregulation of C5 expression was associated with a reduction in C5b9-positive area ( P = 0.0141, Steel-Dwass test)., Conclusions: C5 siRNA-LNP combined with immunosuppressants CsA and DSG downregulated C5 activity and significantly prolonged graft survival compared with control siRNA-LNP with CsA and DSG. Downregulation of C5 expression using C5 siRNA-LNP may be an effective therapeutic approach for AMR., Competing Interests: S.M., C.M., A.T., T.N., Y.K., and T.I. are employees of KAN Research Institute, Inc, Japan; Y.S. is an employee of Eisai, Co, Ltd, Japan; the other authors declare no conflicts of interest. KAN Research Institute, Inc is a subsidiary of Eisai Co, Ltd., (Copyright © 2022 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The current standard immunosuppressive regimens, calcineurin inhibitors, have diabetogenic and anti-vascularization effects on islet grafts. KRP-203, a sphingosine-1-phosphate functional antagonist, exerts its immunomodulatory function through lymphocyte sequestration. However, the effect of this antagonist on islets is unclear. We examined the effect of KRP-203 on the islet function and vascularization and sought a calcineurin-free regimen for islet allotransplantation., Methods: KRP-203 was administered for 14 d to mice, then diabetogenic effect was evaluated by blood glucose levels and a glucose tolerance test. Static glucose stimulation, the breathing index, and insulin/DNA were examined using isolated islets. Islet neovascularization was evaluated using a multiphoton laser scanning microscope. After islet allotransplantation with either KRP-203 alone, sirolimus alone, or both in combination, the graft survival was evaluated by blood glucose levels and immunohistochemical analyses. A mixed lymphocyte reaction was also performed to investigate the immunologic characteristics of KRP-203 and sirolimus., Results: No significant differences in the blood glucose levels or glucose tolerance were observed between the control and KRP-203 groups. Functional assays after islet isolation were also comparable. The multiphoton laser scanning microscope showed no inhibitory effect of KRP-203 on islet neovascularization. Although allogeneic rejection was effectively inhibited by KRP-203 monotherapy (44%), combination therapy prevented rejection in most transplanted mice (83%)., Conclusions: KRP-203 is a desirable immunomodulator for islet transplantation because of the preservation of the endocrine function and lack of interference with islet neovascularization. The combination of KRP-203 with low-dose sirolimus may be promising as a calcineurin-free regimen for islet allotransplantation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

For many individuals with end-stage liver disease, the only treatment option is liver transplantation. However, liver transplant rejection is observed in 24%-80% of transplant patients and lifelong drug regimens that follow the transplant procedure lead to serious side effects. Furthermore, the pool of donor livers available for transplantation is far less than the demand. Well-characterized and physiologically relevant models of liver transplantation are crucial to a deeper understanding of the cellular processes governing the outcomes of liver transplantation and serve as a platform for testing new therapeutic strategies to enhance graft acceptance. Such a model has been found in the rat transplant model, which has an advantageous size for surgical procedures, similar postoperative immunological progression, and high genome match to the human liver. From rat liver transplant studies published in the last 5 years, it is clear that the rat model serves as a strong platform to elucidate transplant immunological mechanisms. Using the model, we have begun to uncover potential players and possible therapeutic targets to restore liver tolerance and preserve host immunocompetence. Here, we present an overview of recent literature for rat liver transplant models, with an aim to highlight the value of the models and to provide future perspectives on how these models could be further characterized to enhance the overall value of rat models to the field of liver transplantation., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 The Author(s). Published by Wolters Kluwer Health, Inc.)

Background: Belatacept may impair humoral immunity, impacting the effectiveness of SARS-CoV-2 mRNA vaccines in transplant recipients. We investigated immunogenicity after SARS-CoV-2 mRNA vaccines in kidney transplant recipients who are and are not taking belatacept., Methods: Participants were recruited between December 9, 2020, and April 1, 2021. Blood samples were collected after dose 1 and dose 2 (D1, D2) and analyzed using either an anti-SARS-CoV-2 enzyme immunoassay against the S1 domain of the SARS-CoV-2 spike protein or immunoassay against the receptor-binding domain of the SARS-CoV-2 spike protein. Stabilized inverse probability of treatment weights was used to compare immunogenicity, and a weighted logistics regression was used to calculate fold change of positive response., Results: Among the 609 participants studied, 24 (4%) were taking belatacept. After dose 1, 0/24 (0%) belatacept patients had detectable antibodies, compared with 77 of 568 (14%) among the equivalent nonbelatacept population (P = 0.06). After dose 2, 1/19 (5%) belatacept patients had detectable antibodies, compared with 190/381 (50%) among the equivalent nonbelatacept population (P < 0.001). Belatacept use was associated with 16.7-fold lower odds of having a positive post-D2 titer result (P < 0.01)., Conclusions: Additional measures need to be explored to protect kidney transplant recipients taking belatacept. Best safety practices should be continued despite vaccination among this population., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

We discuss what therapeutic regimen might be acceptable/successful in the first clinical trial of genetically engineered pig kidney or heart transplantation. As regimens based on a calcineurin inhibitor or CTLA4-Ig have proved unsuccessful, the regimen we administer to baboons is based on induction therapy with antithymocyte globulin, an anti-CD20 mAb (Rituximab), and cobra venom factor, with maintenance therapy based on blockade of the CD40/CD154 costimulation pathway (with an anti-CD40 mAb), with rapamycin, and a corticosteroid. An anti-inflammatory agent (etanercept) is administered for the first 2 wk, and adjuvant therapy includes prophylaxis against thrombotic complications, anemia, cytomegalovirus, and pneumocystis. Using this regimen, although antibody-mediated rejection certainly can occur, we have documented no definite evidence of an adaptive immune response to the pig xenograft. This regimen could also form the basis for the first clinical trial, except that cobra venom factor will be replaced by a clinically approved agent, for example, a C1-esterase inhibitor. However, none of the agents that block the CD40/CD154 pathway are yet approved for clinical use, and so this hurdle remains to be overcome. The role of anti-inflammatory agents remains unproven. The major difference between this suggested regimen and those used in allotransplantation is the replacement of a calcineurin inhibitor with a costimulation blockade agent, but this does not appear to increase the complications of the regimen., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: The role of regulatory T cells (Treg) in tolerance induction of vascularized composite allotransplantation (VCA) remains unclear. This study was designed to examine characteristics of Treg after VCA and their capacity to rescue allografts from rejection., Methods: Osteomyocutaneous allografts were transplanted from Balb/c to C57BL/6 mice. All mice received costimulatory blockade and a short course of rapamycin. To elucidate the role of Treg for tolerance induction, Treg depletion was performed at postoperative day (POD) 0, 30, or 90. To assess capacity of Treg to rescue allografts from rejection, an injection of 2 × 106 Treg isolated from tolerant mice was applied., Results: Eighty percent of VCA recipient mice using costimulatory blockade and rapamycin regimen developed tolerance. The tolerant recipients had a higher ratio of circulating Treg to effector T cells and elevated interleukin-10 at POD 30. A significantly higher rejection rate was observed when Treg were depleted at POD 30. But Treg depletion at POD 90 had no effect on tolerance. Treg from tolerant recipients showed stronger suppressive potential and the ability to rescue allografts from rejection. Furthermore, transplanted Treg-containing skin grafts from tolerant mice delayed rejection elicited by adoptively transferred effector T cells to Rag2-/- mice., Conclusions: Circulating Treg are crucial for inducing VCA tolerance in the early posttransplant phase, and allograft-residing Treg may maintain tolerance. Treg may, therefore, serve as a potential cellular therapeutic to improve VCA outcomes., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Chronic graft-versus-host disease (GVHD) is a significant cause of morbidity and mortality in transplant patients. We have previously shown that 3 doses of an anti-inducible costimulator (ICOS) mAb transiently ameliorated symptoms and extended survival of dogs affected by chronic GVHD over that of control dogs. The purpose of this study was to specifically correlate changes in T-cell populations in the peripheral blood with anti-ICOS treatment and chronic GVHD progression and regression to reach a better understanding of the mechanism of the disease and prioritize future studies., Methods: Peripheral blood cells from canines transplanted with DLA-mismatched bone marrow and peripheral blood mononuclear cells to generate chronic GVHD were analyzed by flow cytometry using a panel of antibodies specific to helper and cytolytic T cells., Results: Chronic GVHD was specifically associated with an increase in CD4+ICOS+ cells, ICOS+ cells expressing IL-17A, and CD8+ cells generating granzyme B. Treatment with anti-ICOS mAb at onset of chronic GVHD symptoms specifically targeted IL-17A+-expressing cells, transiently relieved symptoms, and lengthened survival but was unable to reduce the percentage of CD8+ T-cells expressing granzyme B., Conclusions: These studies suggested a role for both CD4+ and CD8+ T cells in pathogenesis of chronic GVHD in the canine model. We propose that future studies should focus on further extending survival by developing a treatment that would control both CD4+ and CD8+ T cells., Competing Interests: The authors declare no conflicts of interest., (Copyright © 2021 Wolters Kluwer Health, Inc. All rights reserved.)

Background: Here, we present our initial experience with a prospective protocol of belatacept conversion in patients with chronic active antibody-mediated rejection (caAMR) and a high degree of chronicity at the time of diagnosis., Methods: We converted 19 patients (mean age, 45 ± 12 y) with biopsy-proven caAMR from tacrolimus to belatacept at a median of 44 months post-kidney transplant., Results: At a median of 29 months (interquartile range, 16-46 mo) postconversion, death-censored graft and patient survivals were 89% and 95%, respectively. When compared to a 1:2 propensity-matched control cohort from the INSERM U970 registry maintained on calcineurin inhibitor, the belatacept group had progressive improvement (P = 0.02) in estimated glomerular filtration rate from a mean of 33.9 ± 10 at baseline to 37.8 ± 13 at 6 months and 38.5 ± 12 mL/min/1.73 m2 at 12 months postconversion, as compared to a steady decline noted in the controls (36.2 [baseline] → 33.1 [6 mo] → 32.7 mL/min/1.73 m2 [12 mo] of follow-up). A paired histologic comparison of preconversion and postconversion (performed at median 9.5 mo postconversion) biopsies showed no worsening in microvascular inflammation or chronicity. The paired tissue gene expression analysis showed improved mean total rejection score (0.68 ± 0.26-0.56 ± 0.33; P = 0.02) and a trend toward improved antibody-mediated rejection score (0.64 ± 0.34-0.56 ± 0.39; P = 0.06)., Conclusions: Here, we report that in patients diagnosed with caAMR who were not subjected to intensive salvage immunosuppressive therapies, isolated belatacept conversion alone was associated with stabilization in renal function. These results are bolstered by molecular evidence of improved inflammation., Competing Interests: P.H. has received ownership interest from transcriptome Sciences. G.G. has received honoraria CareDx, Mallinckrodt, Thermo Fisher. The other authors declare no conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Tacrolimus was discovered in 1984 and entered clinical use shortly thereafter, contributing to successful solid organ transplantation across the globe. In this review, we cover development of tacrolimus, its evolving clinical utility, and issues affecting its current usage. Since earliest use of this class of immunosuppressant, concerns for calcineurin-inhibitor toxicity have led to efforts to minimize or eliminate these agents in clinical regimens but with limited success. Current understanding of the role of tacrolimus focuses more on its efficacy in preventing graft rejection and graft loss. As we enter the fourth decade of tacrolimus use, newer studies utilizing novel combinations (as with the mammalian target of rapamycin inhibitor, everolimus, and T-cell costimulation blockade with belatacept) offer potential for enhanced benefits., Competing Interests: The authors declare no funding or conflicts of interest., (Copyright © 2020 Wolters Kluwer Health, Inc. All rights reserved.)

Background: During intraportal pancreatic islet transplantation (PITx), early inflammatory reactions cause an immediate loss of more than half of the transplanted graft and potentiate subsequent allograft rejection. Previous findings suggest that cibinetide, a selective innate repair receptor agonist, exerts islet protective and antiinflammatory properties and improved transplant efficacy in syngeneic mouse PITx model. In a stepwise approach toward a clinical application, we have here investigated the short- and long-term effects of cibinetide in an allogeneic mouse PITx model., Methods: Streptozotocin-induced diabetic C57BL/6N (H-2) mice were transplanted with 320 (marginal) or 450 (standard) islets from BALB/c (H-2) mice via the portal vein. Recipients were treated perioperative and thereafter daily during 14 d with cibinetide (120 µg/kg), with or without tacrolimus injection (0.4 mg/kg/d) during days 4-14 after transplantation. Graft function was assessed using nonfasting glucose measurements. Relative gene expressions of proinflammatory cytokines and proinsulin of the graft-bearing liver were assessed by quantitative polymerase chain reaction. Cibinetide's effects on dendritic cell maturation were investigated in vitro., Results: Cibinetide ameliorated the local inflammatory responses in the liver and improved glycemic control immediately after allogeneic PITx and significantly delayed the onset of allograft loss. Combination treatment with cibinetide and low-dose tacrolimus significantly improved long-term graft survival following allogeneic PITx. In vitro experiments indicated that cibinetide lowered bone-marrow-derived-immature-dendritic cell maturation and subsequently reduced allogeneic T-cell response., Conclusions: Cibinetide reduced the initial transplantation-related severe inflammation and delayed the subsequent alloreactivity. Cibinetide, in combination with low-dose tacrolimus, could significantly improve long-term graft survival in allogeneic PITx.

Background: Despite the benefits of ex vivo lung perfusion (EVLP) such as lung reconditioning, preservation, and evaluation before transplantation, deleterious effects, including activation of proinflammatory cascades and alteration of metabolic profiles have been reported. Although patient outcomes have been favorable, further studies addressing optimal conditions are warranted. In this study, we investigated the role of the immunosuppressant drug cyclosporine A (CyA) in preserving mitochondrial function and subsequently preventing proinflammatory changes in lung grafts during EVLP., Methods: Using rat heart-lung blocks after 1-hour cold preservation, an acellular normothermic EVLP system was established for 4 hours. CyA was added into perfusate at a final concentration of 1 μM. The evaluation included lung graft function, lung compliance, and pulmonary vascular resistance as well as biochemical marker measurement in the perfusate at multiple time points. After EVLP, single orthotopic lung transplantation was performed, and the grafts were assessed 2 hours after reperfusion., Results: Lung grafts on EVLP with CyA exhibited significantly better functional and physiological parameters as compared with those without CyA treatment. CyA administration attenuated proinflammatory changes and prohibited glucose consumption during EVLP through mitigating mitochondrial dysfunction in lung grafts. CyA-preconditioned lungs showed better posttransplant lung early graft function and less inflammatory events compared with control., Conclusions: During EVLP, CyA administration can have a preconditioning effect through both its anti-inflammatory and mitochondrial protective properties, leading to improved lung graft preservation, which may result in enhanced graft quality after transplantation.

Background: Antibody-dependent cell-mediated cytotoxicity (ADCC) is an important pathway responsible for antibody-mediated rejection (AMR). Imlifidase (IdeS) cleaves human IgG into F(ab')2 and Fc fragments, potentially inhibiting ADCC. Here we examined the effect of IdeS on allo-antibody-mediated NK cell activation (Allo-CFC) and ADCC in vitro., Methods: For Allo-CFC, normal whole blood was incubated with third-party peripheral blood mononuclear cells (PBMCs) pretreated with anti-HLA antibody positive (HS) or negative (NC) sera to measure IFNγ+ NK cell%. For ADCC, normal PBMCs were incubated with Farage B (FB) cells with HS or NC sera to measure 7-AAD+ lysed FB cell%. To assess the effect of IdeS on these assays, serum-treated PBMCs (Allo-CFC-1) and serum used for PBMC pretreatment (Allo-CFC-2) in Allo-CFC, and serum used for ADCC were preincubated with IdeS. Sera from IdeS-treated patients were also tested for Allo-CFC (Allo-CFC-3)., Results: IFNγ+ NK cell% were significantly elevated in HS versus NC sera in Allo-CFC-1 (10 ± 3% versus 2 ± 1%, P = 0.001), Allo-CFC-2 (20 ± 10% versus 4 ± 2%, P = 0.01) and 7AAD+ FB cell% (11 ± 3% versus 4 ± 2%, P = 0.02) in ADCC. These were significantly reduced by IdeS treatment. Patient sera with significantly reduced anti-HLA antibody levels at 1 day postimlifidase lost the capacity to activate NK cells in Allo-CFC-3, but those at 1-3 months postimlifidase regained the capacity., Conclusions: IdeS inhibited NK cell activation and ADCC in vitro and in treated patients. These results and reported inhibition of complement activating anti-HLA antibodies by IdeS suggest its possible role in treatment of AMR.

Medication nonadherence (MNA) after solid organ transplantation is highly prevalent and associated with (late) (sub)clinical acute rejection, graft dysfunction and graft loss, development of donor-specific anti-HLA antibodies, and antibody-mediated rejection. MNA is predominantly unintentional and originates from barriers to adherence that are often multifactorial and complex. Tools to establish an early diagnosis of MNA include incorporation of MNA as a vital sign in daily clinical practice, self-reporting using validated questionnaires, calculating intrapatient variability in drug exposure and applying electronic monitoring, and recent audio and video technologies such as in home telemonitoring. MNA is a modifiable risk factor after organ transplantation, and treatment is most effective if a multimodal approach is used. Management of MNA comprises education (cognitive) and counseling (behavioral) that require the involvement of a trained multidisciplinary team [ideally physician, nurse (specialist), social worker, transplant coordinator, psychologist, clinical pharmacist], electronic reminder and support systems (eg, Medication Event Monitoring System, smartphone), and different types of novel mobile health applications as well as simplification of the medication dosing regimen. Future studies that evaluate novel therapeutic approaches for MNA should assure the use of reliable MNA measures, focus on multimodal individualized therapy for enriched nonadherent target populations (eg, adolescents), and incorporate clinically relevant endpoints. Costs, time, and personnel investments should be taken into account when assessing scalability and cost-effectiveness of novel therapeutic strategies. This review provides suggestions how different types of transplant centers can set up a dedicated MNA program according to available resources to define and achieve realistic clinical goals in managing MNA.

Background: Renal allograft rejection is more frequent under belatacept-based, compared with tacrolimus-based, immunosuppression. We studied kidney transplant recipients experiencing rejection under belatacept-based early corticosteroid withdrawal following T-cell-depleting induction in a recent randomized trial (Belatacept-based Early Steroid Withdrawal Trial, clinicaltrials.gov NCT01729494) to determine mechanisms of rejection and treatment., Methods: Peripheral mononuclear cells, serum creatinine levels, and renal biopsies were collected from 8 patients undergoing belatacept-refractory rejection (BRR). We used flow cytometry, histology, and immunofluorescence to characterize CD8 effector memory T cell (TEM) populations in the periphery and graft before and after mammalian target of rapamycin (mTOR) inhibition., Results: Here, we found that patients with BRR did not respond to standard antirejection therapy and had a substantial increase in alloreactive CD8 T cells with a CD28/DR/CD38/CD45RO TEM. These cells had increased activation of the mTOR pathway, as assessed by phosphorylated ribosomal protein S6 expression. Notably, everolimus (an mTOR inhibitor) treatment of patients with BRR halted the in vivo proliferation of TEM cells and their ex vivo alloreactivity and resulted in their significant reduction in the peripheral blood. The frequency of circulating FoxP3 regulatory T cells was not altered. Importantly, everolimus led to rapid resolution of rejection as confirmed by histology., Conclusions: Thus, while prior work has shown that concomitant belatacept + mTOR inhibitor therapy is effective for maintenance immunosuppression, our preliminary data suggest that everolimus may provide an available means for effecting "rescue" therapy for rejections occurring under belatacept that are refractory to traditional antirejection therapy with corticosteroids and polyclonal antilymphocyte globulin.

T-cell immunity undergoes a complex and continuous remodeling with aging. Understanding those dynamics is essential in refining immunosuppression. Aging is linked to phenotypic and metabolic changes in T-cell immunity, many resulting into impaired function and compromised effectiveness. Those changes may impact clinical immunosuppression with evidences suggesting age-specific efficacies of some (CNI and mammalian target of rapamycin inhibitors) but not necessarily all immunosuppressants. Metabolic changes of T cells with aging have only recently been appreciated and may provide novel ways of immunosuppression. Here, we provide an update on changes of T-cell immunity in aging.

Background: Plasmapheresis in combination with immunoglobulin and rituximab is often used to induce accommodation in ABO-incompatible (ABOi) living-donor transplantation; however, this regimen cannot be applied to cases of ABOi deceased-donor transplantation. Here, we investigated whether an anti-complement component 5 (C5) antibody-based regimen can induce accommodation in ABOi heart transplantation., Methods: Both IgM and IgG anti-blood type A antibodies were induced in wild-type mice by sensitization using human blood type A antigen. Heterotopic ABOi heart transplantation was performed from human blood type A-transgenic C57BL/6J mice to sensitized wild-type DBA/2 mice., Results: Either anti-C5 antibody or conventional triple immunosuppressants (corticosteroid, tacrolimus, mycophenolate mofetil) alone did not induce accommodation in majority of ABOi heart allografts, whereas their combination induced accommodation in more than 70% of cases despite the presence of anti-A antibodies. The combination therapy markedly suppressed the infiltration of T cells and macrophages into ABOi allografts, despite mild deposition of IgG and C4d. T-cell activation and differentiation into Th1, Th2, and Th17 cells were suppressed along with CD49dCD4 T and follicular helper T cells in the combination treatment group. CD24 B cells, including both CD24CD23 marginal zone B cells and CD24CD23 T2-marginal zone B cells, were increased in the accommodation group., Conclusions: C5 inhibitor-based immunosuppression induced accommodation in murine ABOi heart transplantation, presenting a promising strategy for ABOi deceased-donor transplantation.

Background: Experimental and preclinical evidence suggest that adoptive transfer of regulatory T (Treg) cells could be an appropriate therapeutic strategy to induce tolerance and improve graft survival in transplanted patients. The University of Kentucky Transplant Service Line is developing a novel phase I/II clinical trial with ex vivo expanded autologous Treg cells as an adoptive cellular therapy in renal transplant recipients who are using everolimus (EVR)-based immunosuppressive regimen., Methods: The aim of this study was to determine the mechanisms of action and efficacy of EVR for the development of functionally competent Treg cell-based adoptive immunotherapy in transplantation to integrate a common EVR-based regimen in vivo (in the patient) and ex vivo (in the expansion of autologous Treg cells). CD25 Treg cells were selected from leukapheresis product with a GMP-compliant cell separation system and placed in 5-day (short) or 21-day (long) culture with EVR or rapamycin (RAPA). Multi-parametric flow cytometry analyses were used to monitor the expansion rates, phenotype, autophagic flux, and suppressor function of the cells. phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin signaling pathway profiles of treated cells were analyzed by Western blot and cell bioenergetic parameters by extracellular flux analysis., Results: EVR-treated cells showed temporary slower growth, lower metabolic rates, and reduced phosphorylation of protein kinase B compared with RAPA-treated cells. In spite of these differences, the expansion rates, phenotype, and suppressor function of long-term Treg cells in culture with EVR were similar to those with RAPA., Conclusions: Our results support the feasibility of EVR to expand functionally competent Treg cells for their clinical use.

Background: Systemic hypertension is prevalent in heart transplant recipients and has been partially attributed to treatment with calcineurin inhibitors (CNIs). SCandinavian HEart transplant De-novo stUdy with earLy calcineurin inhibitors avoidancE trial was the first randomized trial to study early withdrawal of CNIs in de novo heart transplant recipients, comparing an everolimus-based immunosuppressive regimen with conventional CNI-based treatment. As a prespecified secondary endpoint, blood pressure was repeatedly compared across treatment arms., Methods: The The SCandinavian HEart transplant De-novo stUdy with earLy calcineurin inhibitors avoidancE trial was a prospective, multicenter, randomized, controlled, parallel-group, open-label trial in de novo adult heart transplant recipients, undertaken at transplant centers in Scandinavia. Blood pressure was assessed with 24-hour ambulatory blood pressure monitoring up to 3 years after heart transplantation (HTx) in 83 patients., Results: Overall, systolic blood pressure fell with time, from 138 ± 15 mm Hg 2 weeks after HTx to 134 ± 11 mm Hg after 12 months and 132 ± 14 mm Hg after 36 months (P = 0.003). Diastolic blood pressure did not change over time. After 12 months, there was a numerically larger fall in systolic blood pressure in the everolimus arm (between-group difference 8 mm Hg; P = 0.053), and after 36 months, there was a significant between group difference of 13 mm Hg (P = 0.02) in favor of everolimus., Conclusions: In this first, randomized trial with early CNI avoidance in de novo HTx recipients, we observed a modest fall in systolic blood pressure over the first 1 to 3 years after transplantation. The fall in systolic blood pressure was more pronounced in patients allocated to everolimus.

Background: We recently reported anti-CD40 monoclonal antibody and rapamycin (aCD40/rapa) to be a reliable, nontoxic, immunosuppressive regimen for combined islet and kidney transplantation (CIKTx) in nonhuman primates. In the current study, we attempted to induce allograft tolerance through the mixed chimerism approach using a conditioning regimen with aCD40 and belatacept (Bela)., Methods: Five CIKTx or kidney transplant alone recipients were treated with aCD40/rapa for 4 months. All recipients then received a conditioning regimen including horse antithymocyte globulin and aCD40/Bela. The results were compared with previous reports of recipients treated with Bela-based regimens., Results: All 3 CIKTx recipients developed mixed chimerism, which was significantly superior to that observed in the previous Bela-based studies. Nevertheless, all CIKTx recipients in this study lost their islet and renal allografts as a result of cellular and humoral rejection on days 140, 89, and 84. The 2 kidney transplant-alone recipients were treated with the same conditioning regimen and suffered rejection on days 127 and 116, despite the development of excellent chimerism. B lymphocyte reconstitution dominated by memory phenotypes was associated with early development of donor-specific antibodies in 4 of 5 recipients. In vitro assays showed no donor-specific regulatory T cell expansion, which has been consistently observed in tolerant recipients with our mixed chimerism approach., Conclusions: Despite displaying excellent immunosuppressive efficacy, costimulatory blockade with anti-CD40 monoclonal antibody (2C10R4) may inhibit the induction of renal or islet allograft tolerance via a mixed chimerism approach.

Background: Brachial plexus injuries are devastating. Current reconstructive treatments achieve limited partial functionality. Vascularized brachial plexus allotransplantation could offer the best nerve graft fulfilling the like-with-like principle. In this experimental study, we assessed the feasibility of rat brachial plexus allotransplantation and analyzed its functional outcomes., Methods: A free vascularized brachial plexus with a chimeric compound skin paddle flap based on the subclavian vessels was transplanted from a Brown Norway rat to a Lewis rat. This study has 2 parts. Protocol I aimed to develop the vascularized brachial plexus allotransplantation (VBP-allo) model. Four groups are compared: no reconstruction, VBP-allo with and without cyclosporine A immunosuppression, VBP autotransplantation (VBP-auto). Protocol II compared the recovery of the biceps muscle and forearm flexors when using all 5, 2 (C5 + C6) or 1 (isolated C6) spinal nerve as the donor nerves. The assessment was performed on week 16 and included muscle weight, functionality (grooming tests, muscle strength), electrophysiology and histomorphology of the targeted muscles., Results: Protocol I showed, the VBP-allo with cyclosporine A immunosuppression was electrophysiologically and functionally comparable to VBP-auto and significantly superior to negative controls and absent immunosuppression. In protocol II, all groups had a comparable functional recovery in the biceps muscle. Only with 5 donor nerves did the forearm show good results compared with only 1 or 2 donor nerves., Conclusions: This study demonstrated a useful vascularized complete brachial plexus allotransplantation rodent model with successful forelimb function restoration under immunosuppression. Only the allotransplantation including all 5 roots as donor nerves achieved a forearm recovery.

Background: CXC motif chemokine receptor 4 (CXCR4) blockade is pursued as an alternative to mesenchymal stem cell treatment in transplantation based on our previous report that burixafor, through CXCR4 antagonism, mobilizes immunomodulatory mesenchymal stem cells. Here, we explored the efficacy of combining mycophenolate mofetil (MMF)-based immunosuppressants with repetitive burixafor administration., Methods: Swine heterotopic cardiac allograft recipients received MMF and corticosteroids (control, n = 10) combined with burixafor as a 2-dose (burixafor2D, n = 7) or 2-dose plus booster injections (burixafor2D + B, n = 5) regimen. The efficacy endpoints were graft survival, freedom from first acute rejection, and the severity of intimal hyperplasia. Each specimen was sacrificed either at its first graft arrest or after 150 days., Results: After 150 days, all specimens in the control group had died, but 28.5% of the burixafor2D group survived, and 60% of the burixafor2D + B group survived (P = 0.0088). Although the control group demonstrated acute rejection at a median of 33.5 days, the burixafor2D + B group survived without acute rejection for a median of 136 days (P = 0.0209). Burixafor administration significantly attenuated the incidence rate of acute rejection (P = 0.002) and the severity of intimal hyperplasia (P = 0.0097) at end point relative to the controls. These findings were associated with reduced cell infiltrates in the allografts, and modulation of C-reactive protein profiles in the circulation., Conclusions: The augmentation of conventional MMF plus corticosteroids with a CXCR4 antagonist is potentially effective in improving outcomes after heart transplantation in minipigs. Future studies are warranted into optimizing the therapeutic regimens for humans.

De novo donor-specific antibody (DSA) formation is a major problem in transplantation, and associated with long-term graft decline and loss as well as sensitization, limiting future transplant options. Forming high-affinity, long-lived antibody responses involves a process called the germinal center (GC) reaction, and requires interaction between several cell types, including GC B cells, T follicular helper (Tfh) and T follicular regulatory (Tfr) cells. T follicular regulatory cells are an essential component of the GC reaction, limiting its size and reducing nonspecific or self-reactive responses.An imbalance between helper function and regulatory function can lead to excessive antibody production. High proportions of Tfh cells have been associated with DSA formation in transplantation; therefore, Tfr cells are likely to play an important role in limiting DSA production. Understanding the signals that govern Tfr cell development and the balance between helper and regulatory function within the GC is key to understanding how these cells might be manipulated to reduce the risk of DSA development.This review discusses the development and function of Tfr cells and their relevance to transplantation. In particular how current and future immunosuppressive strategies might allow us to skew the ratio between Tfr and Tfh cells to increase or decrease the risk of de novo DSA formation.

Chronic rejection is the Achilles heel of modern lung transplantation, characterized by a slow, progressive decline in allograft function. Clinically, this manifests as obstructive disease, restrictive disease, or a mixture of the 2 depending on the underlying pathology. The 2 major phenotypes of chronic rejection include bronchiolitis obliterans syndrome and restrictive allograft syndrome. The last decade of research has revealed that each of these phenotypes has a unique underlying pathophysiology which may require a distinct treatment regimen for optimal control. Insights into the intricate alloimmune pathways contributing to chronic rejection have been gained from both large and small animal models, suggesting directions for future research. In this review, we explore the pathological hallmarks of chronic rejection, recent insights gained from both clinical and basic science research, and the current state of animal models of chronic lung rejection.

Background: Islet transplantation is an attractive treatment for patients with insulin-dependent diabetes mellitus, and currently, the liver is the favored transplantation site. However, an alternative site is desirable because of the low efficiency of hepatic transplantation, requiring 2 to 3 donors for a single recipient, and because the transplanted islets cannot be accessed or retrieved., Methods: We developed a novel procedure of islet transplantation to the inguinal subcutaneous white adipose tissue (ISWAT) of mice and described functional and morphological characteristics of transplanted syngeneic islets. Also, it was determined whether islet allograft rejection in the ISWAT can be prevented by immunosuppressive agents. Furthermore, it was examined whether human islets function when grafted in this particular site of immune-deficient mice., Results: In this site, transplanted islets are engrafted as clusters and function to reverse streptozotocin-induced diabetes in mice. Importantly, transplanted islets can be visualized by computed tomography and are easily retrievable, and allograft rejection is preventable by blockade of costimulatory signals. Of much importance, the efficiency of islet transplantation in this site is superior to the liver, in which hyperglycemia of diabetic recipient mice is ameliorated after transplantation of 200 syngeneic islets (the islet number yielded from 1 mouse pancreas) to the ISWAT but not to the liver. Furthermore, human islets transplanted in this particular site function to reverse diabetes in immune-deficient mice., Conclusions: Thus, the ISWAT is superior to the liver as the site of islet transplantation, which may lead to improved outcome of clinical islet transplantation.

Background: There exists a need for a minimally invasive method of islet transplantation without immunosuppressive drugs for the treatment of type 1 diabetes., Methods: In diabetic August Copenhagen Irish rats, an agarose rod containing the cyclic oligopeptide SEK-1005 (agarose-SEK rod) was implanted at 2 dorsal subcutaneous sites. Then these rods were removed, and 1500 islets of Langerhans isolated from Fischer 344 rats were transplanted into each of the pockets., Results: Ten days after implantation of agarose-SEK rods, vascularized pockets were present. Nonfasting blood glucose levels confirmed long-term survival of the allogeneic islet grafts, without immunosuppressive therapy, in 8 of 10 recipients. Flow cytometry and gene expression analyses were performed to investigate the mechanisms underlying graft acceptance. Agarose-SEK rod implantation led to the formation of granulomatous tissue containing regulatory T cells that suppressed immune reactions against the allogeneic islet grafts., Conclusions: These results indicate that the use of an agarose-SEK rod to prevascularize a subcutaneous site may be a useful method for achieving successful allogeneic islet transplantation without immunosuppression.

Background: The identification of pathway(s) playing a pivotal role in peritransplant detrimental inflammatory events represents the crucial step toward a better management and outcome of pancreatic islet transplanted patients. Recently, we selected the CXCR1/2 inhibition as a relevant strategy in enhancing pancreatic islet survival after transplantation., Methods: Here, the most clinically used anti-inflammatory compounds (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with a CXCR1/2 inhibitor were evaluated in their ability to improve engraftment or delay graft rejection. To rule out bias related to transplantation site, we used well-established preclinical syngeneic (250 C57BL/6 equivalent islets in C57BL/6) and allogeneic (400 Balb/c equivalent islets in C57BL6) intrahepatic islet transplantation platforms., Results: In mice, we confirmed that targeting the CXCR1/2 pathway is crucial in preserving islet function and improving engraftment. In the allogeneic setting, CXCR1/2 inhibitor alone could reduce the overall recruitment of transplant-induced leukocytes and significantly prolong the time to graft rejection both as a single agent and in combination with immunosuppression. No other anti-inflammatory compounds tested (IL1-receptor antagonist, steroids, and TNF-α inhibitor) alone or in combination with CXCR1/2 inhibitor improve islet engraftment and significantly delay graft rejection in the presence of MMF + FK-506 immunosuppressive treatment., Conclusions: These findings indicate that only the CXCR1/2-mediated axis plays a crucial role in controlling the islet damage and should be a target for intervention to improve the efficiency of islet transplantation.

Background: Although the elderly represents a rapidly growing population among transplant recipients, age-specific aspects have not been considered sufficiently in clinical trials. Moreover, age-specific effects of immunosuppressive therapies remain poorly understood., Methods: Here, we assessed the impact of rapamycin on alloimmune responses in old recipients using a fully major histocompatibility complex-mismatched murine transplantation model., Results: Old untreated recipients displayed a prolonged skin graft survival compared to their young counterparts, an observation that confirmed data of our previous experiments. Rapamycin led to a significant prolongation of graft survival in both young and old recipients. However, graft survival was age-dependent and extended in old versus young recipients (19 days vs 12 days, P = 0.004). This age-specific effect was not linked to changes in frequencies or subset composition of either cluster of differentiation (CD)8 or CD4 T cells. Moreover, antiproliferative effects of rapamycin on CD8 and CD4 T cells as assessed by in vivo bromdesoxyuridine incorporation were comparable and age-independent. In contrast, the systemic production of IL-10 was markedly elevated in old recipients treated with rapamycin. In parallel to this shift in cytokine balance, IFN-γ/IL-10 double-positive regulatory type 1 cells emerged during T helper type 1 differentiation of old T helper cells in presence of rapamycin. Similarly, CD4IFN-γIL-10 cells expanded among Foxp3-negative cells after in vivo treatment of old recipients with rapamycin., Conclusions: Our results highlight novel aspects of age-dependent immunosuppressive effects of rapamycin, with relevance for age-specific immunosuppressive regimens.

Background: Ischemia-reperfusion injury after liver transplantation (LT) impairs graft function and affects prognosis of recipients. Isoglycyrrhizinate magnesium (Iso) is a hepatoprotective drug usually used after liver injury. In this study, we intended to explore whether Iso alone have protective effect after ischemia-reperfusion injury in a rat model of liver transplantation. We also aimed to study whether Iso could enhance the hepatoprotective effect of FK506 (tacrolimus) and underlying mechanism., Methods: Rats after LT were treated with different concentration of FK506 with or without, Iso or lower-dose FK506 plus Iso. Alanine transaminase, aspartate transaminase, and albumin level were measured after 48 hours, 72 hours, and 7 days. A cell ischemic/reperfusion model was established to further study the mechanism of hepatoprotective effect of FK506 and Iso., Results: Iso treatment alone had no effect on liver grafts after LT, but lower-dose FK506 + Iso was better for maintenance of liver function than lower-dose FK506 alone at 48 hours, 72 hours, and 7 days after LT. In terms of mechanism, FK506 induced autophagy which resulted in significantly reduced apoptosis and maintained proliferative potential. However, autophagy induced by FK506 also lead to high-mobility group box (HMGB) 1 release from nuclei, resulting in hepatocyte injury through triggering of p38 phosphorylation and chemokine release. Iso effectively inhibited the release of HMGB1 and downstream inflammatory cytokines., Conclusions: Iso could inhibit release of HMGB1 by FK506 and enhance the hepatoprotective effect of FK506 in rat LT. Combining Iso with FK506 would be promising for the patients after LT.

Background: Little is known about how new-generation adenosine triphosphate-competitive mechanistic target of rapamycin (mTOR) kinase inhibitors affect immunity and allograft rejection., Methods: mTOR complex (C) 1 and 2 signaling in dendritic cells and T cells was analyzed by Western blotting, whereas immune cell populations in normal and heart allograft recipient mice were analyzed by flow cytometry. Alloreactive T cell proliferation was quantified in mixed leukocyte reaction; intracellular cytokine production and serum antidonor IgG levels were determined by flow analysis and immunofluorescence staining used to detect IgG in allografts., Results: The novel target of rapamycin kinase inhibitor AZD2014 impaired dendritic cell differentiation and T cell proliferation in vitro and depressed immune cells and allospecific T cell responses in vivo. A 9-day course of AZD2014 (10 mg/kg, intraperitoneally, twice daily) or rapamycin (RAPA) (1 mg/kg, intraperitoneally, daily) prolonged median heart allograft survival time significantly (25 days for AZD2014, 100 days for RAPA, 9.5 days for control). Like RAPA, AZD2014 suppressed graft mononuclear cell infiltration, increased regulatory T cell to effector memory T cell ratios and reduced T follicular helper and B cells 7 days posttransplant. By 21 days (10 days after drug withdrawal), however, T follicular helper and B cells and donor-specific IgG1 and IgG2c antibody titers were significantly lower in RAPA-treated compared with AZD2014-treated mice. Elevated regulatory T cell to effector memory T cell ratios were maintained after RAPA, but not AZD2014 withdrawal., Conclusions: Immunomodulatory effects of AZD2014, unlike those of RAPA, were not sustained after drug withdrawal, possibly reflecting distinct pharmacokinetics or/and inhibitory effects of AZD2014 on mTORC2.

Background: Serum (extracellular) histone levels are increased in inflammatory states and in the presence of coagulation dysfunction, for example, trauma, chemical/ischemic injury, infection. There is increasing evidence of a systemic inflammatory response associated with the presence of a pig xenograft in a nonhuman primate. We evaluated extracellular histone levels in baboons with various pig xenografts., Methods: We measured serum histones in baboons with pig heterotopic heart (n = 8), life-supporting kidney (n = 5), orthotopic liver (n = 4), and artery patch (n = 9) grafts by enzyme-linked immunosorbent assay. C-reactive protein (CRP), free triiodothyronine (fT3), serum amyloid A (SAA), and platelet counts were also measured, all of which may provide an indication of an inflammatory state. We investigated the effect of histones on platelet aggregation and on cytotoxicity of pig cells in vitro., Results: Serum histones increased when baboons developed consumptive coagulopathy (eg, thrombocytopenia) or infection. CRP levels tended to be higher and fT3 levels lower when consumptive coagulopathy developed. Measurement of SAA correlated fairly well with CRP and indicated the state of inflammation. Treatment of the recipient with tocilizumab reduced the level of serum histones, CRP, and SAA, and increased the level of fT3 and platelet counts. In vitro, histone-induced platelet aggregation and endothelial cell apoptosis were both significantly reduced by the NF-κB pathway inhibitor, parthenolide., Conclusions: These noninvasive assays may be useful for monitoring the health status of nonhuman primate recipients of pig organ grafts and may help in management after xenotransplantation. Tocilizumab and NF-κB inhibitors might prove valuable in reducing the inflammatory response to a pig xenograft.

Background: Specific blockade of T cell costimulation pathway is a promising immunomodulatory approach being developed to replace our current clinical immunosuppression therapies. The goal of this study is to compare results associated with 3 monoclonal antibodies directed against the CD40/CD154 T cell costimulation pathway., Methods: Cynomolgus monkey heterotopic cardiac allograft recipients were treated with either IDEC-131 (humanized αCD154, n = 9), 5C8H1 (mouse-human chimeric αCD154, n = 5), or 2C10R4 (mouse-rhesus chimeric αCD40, n = 6) monotherapy using a consistent, comparable dosing regimen for 3 months after transplant., Results: Relative to the previously reported IDEC-131-treated allografts, median survival time (35 ± 31 days) was significantly prolonged in both 5C8H1-treated (142 ± 26, P < 0.002) and 2C10R4-treated (124 ± 37, P < 0.020) allografts. IDEC-131-treated grafts had higher cardiac allograft vasculopathy severity scores during treatment relative to either 5C8H1 (P = 0.008) or 2C10R4 (P = 0.0002). Both 5C8H1 (5 of 5 animals, P = 0.02) and 2C10R4 (6/6, P = 0.007), but not IDEC-131 (2/9), completely attenuated IgM antidonor alloantibody (alloAb) production during treatment; 5C8H1 (5/5) more consistently attenuated IgG alloAb production compared to 2C10R4 (4/6) and IDEC-131 (0/9). All evaluable explanted grafts experienced antibody-mediated rejection. Only 2C10R4-treated animals exhibited a modest, transient drop in CD20 lymphocytes from baseline at day 14 after transplant (-457 ± 152 cells/μL) compared with 5C8H1-treated animals (16 ± 25, P = 0.037), and the resurgent B cells were primarily of a naive phenotype., Conclusions: In this model, CD154/CD40 axis blockade using IDEC-131 is an inferior immunomodulatory treatment than 5C8H1 or 2C10R4, which have similar efficacy to prolong graft survival and to delay cardiac allograft vasculopathy development and antidonor alloAb production during treatment.

Background: Although islet xenotransplantation is a promising therapy for type 1 diabetes, its clinical application has been hampered by cellular rejection and the requirement for high levels of immunosuppression. The aim of this study was to determine the role of Foxp3 regulatory T (Treg) cells in costimulation blockade-induced dominant tolerance to porcine neonatal islet cell cluster (NICC) xenografts in mice., Methods: Porcine-NICC were transplanted under the renal capsule of BALB/c or C57BL/6 recipients and given a single dose of CTLA4-Fc at the time of transplant and 4doses of anti-CD154 mAb to day 6. Depletion of Foxp3Treg cell was performed in DEpletion of REGulatory T cells mice at day 80 posttransplantation. Foxp3Treg cell from spleens of treated BALB/c mice (tolerant Treg cell), and splenocytes were cotransferred into islet transplanted nonobese diabetic background with severe combined immunodeficiency mice to assess suppressive function., Results: In treated mice, increased numbers of Foxp3Treg cell were identified in the porcine-NICC xenografts, draining lymph node, and spleen. Porcine-NICC xenografts from treated mice expressed elevated levels of TGF-β, IL-10 and IFN-γ. Porcine-NICC xenograft tolerance was abrogated after depletion of Foxp3Treg cell. Tolerant Treg cell produced high levels of IL-10 and had diverse T cell receptor Vβ repertoires with an oligoclonal expansion in CDR3 of T cell receptor Vβ14. These tolerant Treg cells had the capacity to transfer dominant tolerance and specifically exhibited more potent regulatory function to porcine-NICC xenografts that naive Treg cell., Conclusions: This study demonstrated that short-term costimulation blockade-induced dominant tolerance and that Foxp3Treg cell played an essential role in its maintenance. Foxp3Treg cells were activated and had more potent regulatory function in vivo than naive Treg cells.

Background: Antibody-mediated rejection (AMR) of most solid organs is characterized by evidence of complement activation and/or intragraft macrophages (C4d + and CD68+ biopsies). We previously demonstrated that crosslinking of HLA I by antibodies triggered endothelial activation and monocyte adhesion. We hypothesized that activation of the classical complement pathway at the endothelial cell surface by HLA antibodies would enhance monocyte adhesion through soluble split product generation, in parallel with direct endothelial activation downstream of HLA signaling., Methods: Primary human aortic endothelial cells (HAEC) were stimulated with HLA class I antibodies in the presence of intact human serum complement. C3a and C5a generation, endothelial P-selectin expression, and adhesion of human primary and immortalized monocytes (Mono Mac 6) were measured. Alternatively, HAEC or monocytes were directly stimulated with purified C3a or C5a. Classical complement activation was inhibited by pretreatment of complement with an anti-C1s antibody (TNT003)., Results: Treatment of HAEC with HLA antibody and human complement increased the formation of C3a and C5a. Monocyte recruitment by human HLA antibodies was enhanced in the presence of intact human serum complement or purified C3a or C5a. Specific inhibition of the classical complement pathway using TNT003 or C1q-depleted serum significantly reduced adhesion of monocytes in the presence of human complement., Conclusions: Despite persistent endothelial viability in the presence of HLA antibodies and complement, upstream complement anaphylatoxin production exacerbates endothelial exocytosis and leukocyte recruitment. Upstream inhibition of classical complement may be therapeutic to dampen mononuclear cell recruitment and endothelial activation characteristic of microvascular inflammation during AMR.

Background: Recipients of vascularized composite allografts require aggressive and lifelong immunosuppression, and because the surgery is usually performed in nonlife-threatening situations, the development of strategies to minimize immunosuppression is especially pertinent for this procedure. We investigated how complement affects acute graft injury, alloimmunity, and immunosuppressive therapy., Methods: Vascularized composite allografts were transplanted from Balb/C to C57BL/6 mice that were complement deficient (C3 or double C3a Receptor (R)/C5aR), or treated with a targeted complement inhibitor (CR2-Crry). Allografts were analyzed for acute inflammation and injury, subacute T cell response, and survival in the absence and presence of cyclosporine A (CsA) therapy., Results: Allografts in C3-deficient or CR2-Crry-treated recipients were protected from skin and muscle ischemia-reperfusion injury (IRI). C3aR/C5aR-deficient recipients were more modestly protected. IgM and C3d colocalized within allografts from wild type and C3aR/C5aR-deficient recipients indicating IgM-mediated complement activation, and C3d deposition was almost absent in allografts from C3-deficient and CR2-Crry-treated recipients. Inflammatory cell infiltration and P-selectin expression was also significantly reduced in C3-deficient and CR2-Crry-treated recipients. Acute treatment with CR2-Crry or with 3 mg/kg per day CsA modestly, but significantly increased median allograft survival from 5.8 to 7.4 and 7.2 days, respectively. However, combined acute CR2-Crry treatment and CsA therapy increased mean graft survival to 17.2 days. Protection was associated with significantly reduced T cell infiltration of allografts and Tc1 cells in recipient spleens., Conclusions: Complement-mediated IRI augments graft allogenicity, and appropriate complement inhibition ameliorates IRI, decreases alloimmune priming and allows more immune-sparing CsA dosing.

Background: MicroRNAs (miRNAs) are small noncoding RNA molecules that regulate the posttranscriptional expression of target genes and are important regulators in immune responses. Previous studies demonstrated that the miRNA, miR-182 was significantly increased during allograft rejection. Further, the transcription factor Forkhead box (FOX) protein 1, (FOXO1) was shown to be a target of miR-182. The aim of this study is to further examine the role of miR-182 in alloimmune responses., Methods: Transplantation of BALB/c cardiac allografts was performed in C57BL/6, miR-182, B6.129S-H2 (MHC II and CD4 T cell-deficient) and B6.129S2-Tap1 (MHC I and CD8 T cell-deficient) mice, with or without CTLA-4Ig administration. T cell phenotype, FOXO1 protein levels and graft infiltrating lymphocytes were determined in C57BL/6 or miR-182 mice by flow cytometric analysis, Western blot, and immunohistochemistry, respectively., Results: We now show that T cells, mainly CD4 are the main cellular source of miR-182 during allograft rejection. In the absence of miR-182, CTLA-4Ig treatment significantly increased allograft survival (31.5 days C57BL/6 vs 60 days miR-182; P < 0.01). Further, CTLA4-Ig treatment inhibits miR-182 expression, increases FOXO1 levels, and reduces the percentage of CD4CD44 T cells after transplantation. Fewer T cells infiltrate the cardiac allografts, and memory T cells are significantly decreased in allograft recipients deficient in miR-182 with CTLA4-Ig treatment (P < 0.01)., Conclusions: Our findings suggest that miR-182 contributes to the T-cell responses to alloantigen especially under costimulation blockade. Therapeutics that target specific miRNAs may prove beneficial in transplantation.

Background: Adoptive transfer of forkhead box protein (FOX)3 regulatory T (Treg) cells offers a promising strategy to reduce damage to an allograft by the recipient's immune system. Identification of cell surface markers sufficient to purify Treg cells expanded ex vivo to remove cellular contaminants requires optimization. Furthermore, the expanded Treg must be able to survive, expand, and suppress in allograft recipients exposed to immunosuppressants, such as tacrolimus (TAC). Reduced CD127 expression enhances identification of Treg in the human CD4CD25 population. CD45RA expression identifies naive CD4CD25 Treg with an enhanced stability of Treg phenotype., Methods: We combine an analysis of CD45RA, CD25, and CD127 expression to identify subpopulations of CD4CD127CD25 cells. Regulatory T cells were sorted according to expression of CD25 and CD45RA and expanded in the presence of a physiological relevant concentration of TAC. Regulatory T cell-specific demethylation region (TSDR) demethylation, FOXP3 expression, and suppression were analyzed., Results: CD4CD127CD25CD45RA Treg cells had a stable TSDR demethylated FOXP3 phenotype after expansion whereas CD4CD127CD25CD45RA Treg cell lost the TSDR demethylated phenotype. CD45RA Treg had a greater capacity to suppress after expansion with TAC., Conclusions: Although CD45RA Treg retained a greater suppressive capacity when expanded with TAC, the marked loss of the TSDR demethylated status highlights the potential for loss of stability of these cells in transplant recipients treated with TAC based immunosuppression. We show that a population of CD4CD127CD45RA Regulatory T cell may offer the best compromise between susceptibility to loss of suppression when exposed to TAC and maintenance of a TSDR demethylated phenotype following in vitro expansion., Competing Interests: The authors declare no conflicts of interest.

Background: Most strategies for antirejection and tolerance induction in clinical transplantation have focused on modifying adaptive immunity, it is unclear whether pharmacological suppressing the innate immune system can promote transplant tolerance., Methods: We inhibited innate immunity by using our self-generated inhibitor of myeloid differentiation factor 88 (MyD88), TJ-M2010-5, and investigated its therapeutic effects and mechanisms in cardiac and skin transplant models., Results: TJ-M2010-5 directly and indirectly interacted with the Toll/IL-1R domain of MyD88, inhibiting MyD88 homodimerization. In vitro, TJ-M2010-5 inhibited maturation of dendritic cells, which suppressed nuclear translocation of NF-κB and T cell activation. In vivo, short-term (10 days) monotherapy of TJ-M2010-5 resulted in long time survival of 50% of the cardiac allografts, and longer-term (14 days) combination treatment of TJ-M2010-5 with CD154 mAb resulted in survival of 29% of skin allografts, which outperformed far more than CsA did and stimulated the proliferation of CD4CD25FoxP3 Regulatory T cells in recipient mice., Conclusions: Pharmacological inhibition of MyD88 signaling by this novel inhibitor TJ-M2010-5 shows a powerful anti-rejection effect, which may have therapeutic potential in clinical transplantation. The inhibition of both innate and adaptive immunity may be necessary for tolerance induction in nonsolid organs.

Background: Immunoglobulin-cytokine fusion molecules have been shown to be the new generation of immunomodulating agents in transplantation tolerance induction. In the present study, we tested whether immunoregulatory cytokine fusion proteins of IL-10/Fc, TGF-β/Fc, or IL-2/Fc would enhance allogeneic bone marrow cell (BMC) engraftment and promote tolerance induction., Methods: B6 (H2) mice were conditioned with anti-CD154 (MR1) and rapamycin (Rapa) plus 100 cGy total body irradiation (MR1/Rapa/100 cGy) and transplanted with allogeneic B10.D2 (H2) BMC. Recipients were treated with lytic IL-2/Fc, nonlytic IL-2/Fc, TGF-β/Fc, or IL-10/Fc fusion proteins to promote chimerism to induce tolerance., Results: Donor chimerism was achieved in 20% of recipients conditioned with MR1/Rapa/100 cGy. The addition of TGF-β/Fc (5- or 10-day treatment) or nonlytic IL-2/Fc (10-day treatment) fusion proteins to the conditioning resulted in engraftment in nearly 100% of recipients. In contrast, lytic IL-2/Fc or IL-10/Fc had no effect. The combination of nonlytic IL-2/Fc and TGF-β/Fc had a synergistic effect to promote engraftment and resulted in significantly higher donor chimerism compared with recipients conditioned with TGF-β/MR1/Rapa/100 cGy. Engraftment was durable in the majority of chimeras and increased over time. The chimeras accepted donor skin grafts and promptly rejected third-party skin grafts. Moreover, specific T cell receptor-Vβ5.½ and TCR-Vβ11 clonal deletion was detected in host T cells in chimeras, suggesting central tolerance to donor alloantigens., Conclusions: Allogeneic BMC engraftment is enhanced with TGF-β/Fc fusion protein treatment. TGF-β/Fc and nonlytic IL-2/Fc exert a synergistic effect in promotion of alloengraftment and donor-specific transplant tolerance, significantly decreasing the minimum total body irradiation dose required.

Background: Anti-CD154 monotherapy is associated with antidonor allo-antibody (Ab) elaboration, cardiac allograft vasculopathy (CAV), and allograft failure in preclinical primate cell and organ transplant models. In the context of calcineurin inhibitors (CNI), these pathogenic phenomena are delayed by preemptive "induction" B cell depletion., Methods: αCD154 (IDEC-131)-treated cynomolgus monkey heart allograft recipients were given peritransplant rituximab (αCD20) alone or with rabbit antihuman thymocyte globulin., Results: Relative to previously reported reference groups, αCD20 significantly prolonged survival, delayed Ab detection, and attenuated CAV within 3 months in αCD154-treated recipients (αCD154 + αCD20 graft median survival time > 90 days, n = 7, vs 28 days for αCD154 alone (IDEC-131), n = 21; P = 0.05). Addition of rabbit antihuman thymocyte globulin to αCD154 (n = 6) or αCD154 + αCD20 (n = 10) improved graft protection from graft rejection and failure during treatment but was associated with significant morbidity in 8 of 16 recipients (6 infections, 2 drug-related complications). In αCD20-treated animals, detection of antidonor Ab and relatively severe CAV were anticipated by appearance of CD20 cells (>1% of lymphocytes) in peripheral blood and were associated with low αCD154 trough levels (below 100 μg/mL)., Conclusions: These observations support the hypothesis that efficient preemptive "induction" CD20 B cell depletion consistently modulates pathogenic alloimmunity and attenuates CAV in this translational model, extending our prior findings with calcineurin inhibitors to the context of CD154 blockade., Competing Interests: The authors declare no conflicts of interest.

Background: Blockade of CD28-mediated T cell costimulation by a modified cytotoxic T lymphocyte-associated antigen 4 (CTLA4-Ig), belatacept, is a clinically effective immunosuppressive therapy for the prevention of renal allograft rejection. Use of belatacept-based calcineurin inhibitor-free immunosuppression, however, has demonstrated an increased frequency of cellular rejection episodes and immunosuppression-related safety issues relative to conventional regimens. Furthermore, belatacept typically requires infusion for its administration chronically, which may present an inconvenience to patients. To address these issues, a novel CTLA4-Ig variant, ASP2409, with improved CD86 binding selectivity and affinity relative to belatacept was created using DNA shuffling directed evolution methods., Methods: We evaluated the immunosuppressive effect of ASP2409 on in vitro alloimmune T cell responses, in vivo tetanus toxoid (TTx)-induced immunological responses and renal transplantation in cynomolgus monkeys., Results: ASP2409 had 6.1-fold higher and 2.1-fold lower binding affinity to monkey CD86 and CD80 relative to belatacept, respectively. ASP2409 was 18-fold more potent in suppressing in vitro alloimmune T cell responses relative to belatacept. In a cynomolgus monkey TTx immunization model, ASP2409 inhibited anti-TTx immune responses at a 10-fold lower dose level than belatacept. In a cynomolgus monkey renal transplantation model, subcutaneous injection of 1 mg/kg ASP2409 prevented allograft rejection through complete CD86 and partial CD80 receptor occupancies and dramatically prolonged renal allograft survival in combination with tacrolimus or mycophenolate mofetil/methylprednisolone., Conclusions: These results support the potential of ASP2409 as an improved CTLA4-Ig for maintenance immunosuppression in organ transplantation.

Immunosuppression strategies that selectively inhibit effector T cells while preserving and even enhancing CD4FOXP3 regulatory T cells (Treg) permit immune self-regulation and may allow minimization of immunosuppression and associated toxicities. Many immunosuppressive drugs were developed before the identity and function of Treg were appreciated. A good understanding of the interactions between Treg and immunosuppressive agents will be valuable to the effective design of more tolerable immunosuppression regimens. This review will discuss preclinical and clinical evidence regarding the influence of current and emerging immunosuppressive drugs on Treg homeostasis, stability, and function as a guideline for the selection and development of Treg-friendly immunosuppressive regimens.

Background: De novo alloantibodies (donor-specific antibody) contribute to antibody-mediated rejection and poor long-term graft survival. Because the development of donor-specific antibody is associated with early graft loss of cell transplants and reduced long-term survival of solid organ transplants, we hypothesized that conventional immunosuppressives, calcineurin inhibitors (CNi), and mammalian target of rapamycin inhibitors (mTORi), may not be as effective for suppression of humoral alloimmunity as for cell-mediated immunity., Methods: Wild-type or CD8-depleted mice were transplanted with allogeneic hepatocytes. Recipients were treated with mTORi and/or CNi and serially monitored for alloantibody and graft survival. The direct effect of mTORi and CNi on alloprimed B cell function was investigated in Rag1 mice adoptively transferred with alloprimed IgG1 B cells. The efficacy of mTORi and/or CNi to suppress CD8-mediated cytotoxicity of IgG1 B cells was evaluated in in vitro and in vivo cytotoxicity assays., Results: Mammalian target of rapamycin inhibitors, but not CNi, reduced alloantibody production in transplant recipients, directly suppressed alloantibody production by alloprimed IgG1 B cells and delayed graft rejection in both low and high alloantibody producers. Combination treatment with mTORi and CNi resulted in loss of the inhibitory effect observed for mTORi monotherapy in part due to CNi suppression of CD8 T cells which downregulate alloantibody production (CD8 TAb-supp cells)., Conclusions: Our data support that mTORi is a potent inhibitor of humoral immunity through suppression of alloprimed B cells and preservation of CD8 TAb-supp cells. In contrast, alloantibody is readily detected in CNi-treated recipients because CNi does not suppress alloprimed B cells and interferes with downregulatory CD8 TAb-supp cells., Competing Interests: The authors of this manuscript have no conflicts of interest to disclose.

Background: Current transplant immunosuppression regimens have numerous limitations. Recent evidence suggests histone deacetylase inhibitors (HDACis) may represent a class of drug with immunosuppressive properties. This study compares cyclosporin A (CyA) with the pan-HDACi suberoylanilide hydroxamic acid (SAHA) and a novel HDAC6-specific inhibitor (KA1010) in models of alloreactivity., Methods: Proliferation and mixed lymphocyte reaction (MLR)-based assays were used to determine the immunosuppressive effect of compounds, and a murine model of allogeneic skin transplantation was adopted to assess the in vivo effects of HDAC6 inhibition., Results: KA1010 displayed superior inhibitory effects on the activation of peripheral mononuclear cells using in vitro models of transplantation. In a 1-way MLR, KA1010 (5 μΜ) reduced parent cell proliferation from 92% to 64% (P = 0.001). A 2-way MLR, adopting IFN-γ production as a marker of alloresponse, resulting in up to 91% reduction. Dose-response curves revealed dose-dependent profiles with greater potency of HDACis over CyA (IC50 values of 82.0 nM and 13.4 nM for KA1010 and SAHA).Mice treated with KA1010 displayed no significant features of skin allograft rejection upon histological analysis at 70 days and graft survival of 80% in subjects treated with 160 mg/kg. Immunological assessment, revealed a significant increase in CD4CD25forkhead box P3 regulatory T cells (from 18% to 25%, P = 0.0002) and a corresponding reduction in CD4 T cells (from 58% to 42%, P = 0.0009)., Conclusions: HDAC6 may represent an optimal target for future immunosuppressant therapeutics with a particular role in transplantation. In this article, we have demonstrated a superior immunosuppressive effect of KA1010 over both CyA and SAHA, in the models of allotransplantation adopted.

Background: Immune responses are somewhat suppressed in immune privileged sites, including the testes, which provide a preexisting opportunity to prolong allograft survival. Previous studies have shown that intratesticular islet allografts enjoy extended survival even without any immunosuppression. However, it is unknown if testicular immune privilege can be exploited to prolong the survival of a solid allograft, including the skin, because it is impractical to implant a solid tissue in human testes., Methods: To immunize recipient mice, splenocytes from BALB/c mice were injected into the testis of C57BL/6 recipients 1 week before skin transplantation. CD8 + CD122+ and CD4 + FoxP3+ regulatory T [Treg] cells were quantified by fluorescence-activated cell sorting., Results: Although donor-antigen inoculation alone did not delay skin allograft rejection, it significantly extended the allograft survival when combined with CD40/CD40L or B7/CD28 costimulatory blockade and further induced long-term skin allograft acceptance when both costimulatory pathways were blocked. Similarly, donor-antigen inoculation suppressed alloreactive T cell proliferation in draining lymph nodes of skin recipients in the presence of the same costimulatory blockade. Interestingly, donor-antigen inoculation via intratesticular injection increased CD8 + CD122+, but not CD4 + FoxP3+, Treg numbers after transplantation. However, both CD8 + CD122+ and CD4 + CD25+ Treg cells induced by donor-antigen inoculation and the costimulatory blockade were more potent in suppression than that induced without the inoculation. Depletion of CD8+ or CD25+ T cells largely abrogated long-term skin allograft survival induced by donor-antigen inoculation and the costimulatory blockade., Conclusions: Intratesticular inoculation with donor antigens promotes long-term skin allograft survival induced by conventional costimulatory blockade via the induction of both CD8 + CD122+ and CD4 + CD25+ Treg cells.

Background: Immune rejection continues to threaten all tissue transplants. Here we sought to determine whether platelet (P)- and endothelial (E)-selectin mediate T cell recruitment in corneal transplantation and whether their blockade can reduce T cell graft infiltration and improve long-term corneal allograft survival., Methods: In a murine model of allogeneic corneal transplantation, we used PCR and immunohistochemistry to investigate expression of P- and E-selectin in rejected versus accepted allografts and lymph node flow cytometry to assess expression of selectin ligands by effector T cells. Using P- and E-selectin neutralizing antibodies, we evaluated the effect of blockade on CD4 T cell recruitment, as well as the effect of anti-E-selectin on long-term allograft survival., Results: The P- (93.3-fold, P < 0.05) and E-selectin (17.1-fold, P < 0.005) are upregulated in rejected versus accepted allogeneic transplants. Type 1 T helper cells from hosts with accepted and rejected grafts express high levels of P-selectin glycoprotein ligand 1 and glycosylated CD43. In vivo blockade of P (0.47 ± 0.03, P < 0.05) and E selectin (0.49 ± 0.1, P < 0.05) reduced the number of recruited T cells compared with IgG control (0.98 ± 0.1). Anti-E-selectin reduced the number of mature antigen-presenting cells trafficking to lymphoid tissue compared with control (6.96 ± 0.9 vs 12.67 ± 0.5, P < 0.05). Anti-E-selectin treatment delayed graft rejection and increased survival compared with control, although this difference did not reach statistical significance., Conclusions: In a model of corneal transplantation, P- and E-selectin mediate T cell recruitment to the graft, E-selectin mediates APC trafficking to lymphoid tissue, and blockade of E-selectin has a modest effect on improving long-term graft survival.