Cellular strategies to induce immune tolerance after liver transplantation: Clinical perspectives.

Liver transplantation (LT) has become the most efficient treatment for pediatric and adult end-stage liver disease and the survival time after transplantation is becoming longer due to the development of surgical techniques and perioperative management. However, long-term side-effects of immunosuppressants, like infection, metabolic disorders and malignant tumor are gaining more attention. Immune tolerance is the status in which LT recipients no longer need to take any immunosuppressants, but the liver function and intrahepatic histology maintain normal. The approaches to achieve immune tolerance after transplantation include spontaneous, operational and induced tolerance. The first two means require no specific intervention but withdrawing immunosuppressant gradually during follow-up. No clinical factors or biomarkers so far could accurately predict who are suitable for immunosuppressant withdraw after transplantation. With the understanding to the underlying mechanisms of immune tolerance, many strategies have been developed to induce tolerance in LT recipients. Cellular strategy is one of the most promising methods for immune tolerance induction, including chimerism induced by hematopoietic stem cells and adoptive transfer of regulatory immune cells. The safety and efficacy of various cell products have been evaluated by prospective preclinical and clinical trials, while obstacles still exist before translating into clinical practice. Here, we will summarize the latest perspectives and concerns on the clinical application of cellular strategies in LT recipients.

Reduced Satb1 expression predisposes CD4+ T conventional cells to Treg suppression and promotes transplant survival.

Limiting CD4+ T cell responses is important to prevent solid organ transplant rejection. In a mouse model of costimulation blockade-dependent cardiac allograft tolerance, we previously reported that alloreactive CD4+ conventional T cells (Tconvs) develop dysfunction, losing proliferative capacity. In parallel, induction of transplantation tolerance is dependent on the presence of regulatory T cells (Tregs). Whether susceptibility of CD4+ Tconvs to Treg suppression is modulated during tolerance induction is unknown. We found that alloreactive Tconvs from transplant tolerant mice had augmented sensitivity to Treg suppression when compared with memory T cells from rejector mice and expressed a transcriptional profile distinct from these memory T cells, including down-regulated expression of the transcription factor Special AT-rich sequence-binding protein 1 (Satb1). Mechanistically, Satb1 deficiency in CD4+ T cells limited their expression of CD25 and IL-2, and addition of Tregs, which express higher levels of CD25 than Satb1-deficient Tconvs and successfully competed for IL-2, resulted in greater suppression of Satb1-deficient than wild-type Tconvs in vitro. In vivo, Satb1-deficient Tconvs were more susceptible to Treg suppression, resulting in significantly prolonged skin allograft survival. Overall, our study reveals that transplantation tolerance is associated with Tconvs' susceptibility to Treg suppression, via modulated expression of Tconv-intrinsic Satb1. Targeting Satb1 in the context of Treg-sparing immunosuppressive therapies might be exploited to improve transplant outcomes.

T helper-9 cells and Interleukin-9 in transplantation: The open question.

The role of main TCD4+ lymphocyte subsets including T helper 1 (Th1), Th2, Th17, and T regulatory cells in transplantation has already been described; however, the implication of newly defined lineages such as Th22, Th9, and T follicular helper cells in alloimmune responses remain to be elucidated. In addition to the low number of studies, most evidence about the role of these cells in transplantation has been obtained from experimental studies, which might be insufficient or irrelevant for clinical interpretations. In the present article, we have reviewed the studies that have investigated the role of Th9 and its principal cytokine interleukin-9 (IL-9) in allograft rejection and tolerance induction. However, the findings tend to be controversial since some investigations demonstrate positive effects of Th9 on transplantation outcomes whereas others are suggestive of its detrimental influences. A similar challenge is presented by IL-9 as both advantages and disadvantages of IL-9 expression in allografts have been reported. Moreover, different organs appear to be affected in different ways by Th9 cells and IL-9. Therefore, more research particularly in human patients is required to provide sufficient data for drawing a concrete conclusion about the implication of Th9 and IL-9 in transplantation.

A Review of Cyclophosphamide-Induced Transplantation Tolerance in Mice and Its Relationship With the HLA-Haploidentical Bone Marrow Transplantation/Post-Transplantation Cyclophosphamide Platform.

The bone marrow transplantation (BMT) between haplo-identical combinations (haploBMT) could cause unacceptable bone marrow graft rejection and graft-versus-host disease (GVHD). To cross such barriers, Johns Hopkins platform consisting of haploBMT followed by post-transplantation (PT) cyclophosphamide (Cy) has been used. Although the central mechanism of the Johns Hopkins regimen is Cy-induced tolerance with bone marrow cells (BMC) followed by Cy on days 3 and 4, the mechanisms of Cy-induced tolerance may not be well understood. Here, I review our studies in pursuing skin-tolerance from minor histocompatibility (H) antigen disparity to xenogeneic antigen disparity through fully allogeneic antigen disparity. To overcome fully allogeneic antigen barriers or xenogeneic barriers for skin grafting, pretreatment of the recipients with monoclonal antibodies (mAb) against T cells before cell injection was required. In the cells-followed-by-Cy system providing successful skin tolerance, five mechanisms were identified using the correlation between super-antigens and T-cell receptor (TCR) Vß segments mainly in the H-2-identical murine combinations. Those consist of: 1) clonal destruction of antigen-stimulated-thus-proliferating mature T cells with Cy; 2) peripheral clonal deletion associated with immediate peripheral chimerism; 3) intrathymic clonal deletion associated with intrathymic chimerism; 4) delayed generation of suppressor T (Ts) cells; and 5) delayed generation of clonal anergy. These five mechanisms are insufficient to induce tolerance when the donor-recipient combinations are disparate in MHC antigens plus minor H antigens as is seen in haploBMT. Clonal destruction is incomplete when the antigenic disparity is too strong to establish intrathymic mixed chimerism. Although this incomplete clonal destruction leaves the less-proliferative, antigen-stimulated T cells behind, these cells may confer graft-versus-leukemia (GVL) effects after haploBMT/PTCy.

The self-peptide repertoire plays a critical role in transplant tolerance induction.

While direct allorecognition underpins both solid organ allograft rejection and tolerance induction, the specific molecular targets of most directly alloreactive CD8+ T cells have not been defined. In this study, we used a combination of genetically engineered major histocompatibility complex class I (MHC I) constructs, mice with a hepatocyte-specific mutation in the class I antigen-presentation pathway, and immunopeptidomic analysis to provide definitive evidence for the contribution of the peptide cargo of allogeneic MHC I molecules to transplant tolerance induction. We established a systematic approach for the discovery of directly recognized pMHC epitopes and identified 17 strongly immunogenic H-2Kb-associated peptides recognized by CD8+ T cells from B10.BR (H-2k) mice, 13 of which were also recognized by BALB/c (H-2d) mice. As few as 5 different tetramers used together were able to identify a high proportion of alloreactive T cells within a polyclonal population, suggesting that there are immunodominant allogeneic MHC-peptide complexes that can account for a large component of the alloresponse.

Antibody-mediated rejection of the liver allograft: An update and a clinico-pathological perspective.

Antibody-mediated rejection after liver transplantation is an under-recognised cause of allograft injury. While definitions of acute and chronic antibody-mediated rejection have increased clinical awareness, timely identification and management of antibody-mediated rejection remain difficult because of complexities in diagnosis and histopathology, lack of treatment protocols, and unclear long-term outcomes. While recent cohort studies assessing the importance of donor-specific antibodies have aided in its diagnosis, literature on the treatment of antibody-mediated rejection in liver transplantation remain limited to case reports and small series. Further increasing the awareness and timely recognition of antibody-mediated rejection post-liver transplantation is crucial in order to stimulate future research and the development of protocols for its diagnosis and treatment. This review will summarise recent advances in the clinical diagnosis and treatment of antibody-mediated rejection in liver transplantation, as well as some of the histopathologic features (on liver biopsy tissue) of acute and chronic antibody-mediated rejection.

Metabolic preconditioning in CD4+ T cells restores inducible immune tolerance in lupus-prone mice.

Autoimmune disease has presented an insurmountable barrier to restoration of durable immune tolerance. Previous studies indicate that chronic therapy with metabolic inhibitors can reduce autoimmune inflammation, but it remains unknown whether acute metabolic modulation enables permanent immune tolerance to be established. In an animal model of lupus, we determined that targeting glucose metabolism with 2-deoxyglucose (2DG) and mitochondrial metabolism with metformin enables endogenous immune tolerance mechanisms to respond to tolerance induction. A 2-week course of 2DG and metformin, when combined with tolerance-inducing therapy anti-CD45RB, prevented renal deposition of autoantibodies for 6 months after initial treatment and restored tolerance induction to allografts in lupus-prone mice. The restoration of durable immune tolerance was linked to changes in T cell surface glycosylation patterns, illustrating a role for glycoregulation in immune tolerance. These findings indicate that metabolic therapy may be applied as a powerful preconditioning to reinvigorate tolerance mechanisms in autoimmune and transplant settings that resist current immune therapies.

Advances in Liver Transplantation: where are we in the pursuit of transplantation tolerance?

Liver transplantation is the ultimate treatment option for end-stage liver disease. Breakthroughs in surgical practice and immunosuppression have seen considerable advancements in survival after transplantation. However, the intricate management of immunosuppressive regimens, balancing desired immunological quiescence while minimizing toxicity has proven challenging. Diminishing improvements in long-term morbidity and mortality have been inextricably linked with the protracted use of these medications. As such, there is now enormous interest to devise protocols that will allow us to minimize or completely withdraw immunosuppressants after transplantation. Immunosuppression withdrawal trials have proved the reality of tolerance following liver transplantation, however, without intervention will only occur after several years at the risk of potential cumulative immunosuppression-related morbidity. Focus has now been directed at accelerating this phenomenon through tolerance-inducing strategies. In this regard, efforts have seen the use of regulatory cell immunotherapy. Here we focus particularly on regulatory T cells, discussing preclinical data that propagated several clinical trials of adoptive cell therapy in liver transplantation. Furthermore, we describe efforts to further optimize the specificity and survival of regulatory cell therapy guided by concurrent immunomonitoring studies and the development of novel technologies including chimeric antigen receptors and co-administration of low-dose IL-2.

ACTH treatment promotes murine cardiac allograft acceptance.

Heart transplantation is the optimal therapy for patients with end-stage heart disease, but its long-term outcome remains inadequate. Recent studies have highlighted the importance of the melanocortin receptors (MCRs) in inflammation, but how MCRs regulate the balance between alloreactive T cells and Tregs, and whether they impact chronic heart transplant rejection, is unknown. Here, we found that Tregs express MC2R, and MC2R expression was highest among all MCRs by Tregs. Our data indicate that adrenocorticotropic hormone (ACTH), the sole ligand for MC2R, promoted the formation of Tregs by increasing the expression of IL-2Rα (CD25) in CD4+ T cells and activation of STAT5 in CD4+CD25+ T cells. ACTH treatment also improved the survival of heart allografts and increased the formation of Tregs in CD28KO mice. ACTH treatment synergized with the tolerogenic effect of CTLA-4-Ig, resulting in long-term survival of heart allografts and an increase in intragraft Tregs. ACTH administration also demonstrated higher prolongation of heart allograft survival in transgenic mouse recipients with both complete KO and conditional KO of PI3Kγ in T cells. Finally, ACTH treatment reduced chronic rejection markedly. These data demonstrate that ACTH treatment improved heart transplant outcomes, and this effect correlated with an increase in Tregs.

Inhibition of NLRC5 regulates cytokine expression in CD4+ T helper lymphocytes and prolongs murine islet and skin allograft survival.

The purpose of this study was to study the effect of inhibiting NLRC5 expression and function on CD4 + T cells, and islet and skin transplantation in mice. A murine skin graft model and islet cell transplantation model were established, and the expression of NLRC5 was compared in rejection and immune tolerance groups. Mice spleen-derived CD4 + T cells were cultured, purified, and enriched in vitro, and transfected with the shRNA lentiviral vector NLRC5-RNAi-GFP. Changes in cytokine secretion were detected to understand changes in immunological function. Murine islet and skin transplantation models were injected with CD4 + T cells transfected with the lentivirus, and the survival time of the grafts and the levels of IFN-γ and IL-10 were compared between groups. The expression of NLRC5 mRNA in islet and skin grafts was significantly increased. In vitro experiments showed that the expression of IL-4 and IL-10 was up-regulated in CD4 + T cells, and T cells differentiation turned to Th2 after inhibition of NLRC5. In vivo experiments showed that inhibition of NLRC5 prolonged islet and skin graft survival. Pathological examination showed that the rejection of transplanted skin and islets in the NLRC5-RNAi group was mild, and there was a correlation between high expression of NLRC5 and rejection of mouse islet and skin grafts. In summary, inhibition of NLRC5 can prolong islet and skin graft survival induce transplant immune tolerance through induction of the secretion of Th2 cytokines by CD4 + T cells.

Clinical and Basic Research Progress on Treg-Induced Immune Tolerance in Liver Transplantation.

Rejection after organ transplantation is a cause of graft failure. Effectively reducing rejection and inducing tolerance is a challenge in the field of transplantation immunology. The liver, as an immunologically privileged organ, has high rates of spontaneous and operational tolerance after transplantation, allowing it to maintain its normal function for long periods. Although modern immunosuppression regimens have serious toxicity and side effects, it is very risky to discontinue immunosuppression regimens blindly. A more effective treatment to induce immune tolerance is the most sought-after goal in transplant medicine. Tregs have been shown to play a pivotal role in the regulation of immune balance, and infusion of Tregs can also effectively prevent rejection and cure autoimmune diseases without significant side effects. Given the immune characteristics of the liver, the correct use of Tregs can more effectively induce the occurrence of operational tolerance for liver transplants than for other organ transplants. This review mainly summarizes the latest research advances regarding the characteristics of the hepatic immune microenvironment, operational tolerance, Treg generation in vitro, and the application of Tregs in liver transplantation. It is hoped that this review will provide a deeper understanding of Tregs as the most effective treatment to induce and maintain operational tolerance after liver transplantation.

Influence of the microbiome on solid organ transplant survival.

The microbiome is an environmental factor in intricate symbiotic relationship with its hosts' immune system, potentially shaping anticancer immunity, autoimmunity, and transplant responses. The focus of this review is to discuss recent findings tying the microbiota to transplant outcomes and alloimmunity. The microbiota changes dynamically following transplantation, but whether these changes affect transplant outcomes can be difficult to parse out. New data reveal effects of the microbiota locally, as well as systemically, depending on the mucosal/epithelial surface colonized, the specific commensal communities present and the nature of microbial-derived molecules produced. These complex interactions result in the microbiota potentially impacting transplantation at different levels, including modulation of donor and/or recipient cells, alterations in the priming and/or effector phases of the alloimmune response, availability or metabolism of immunosuppressive drugs, transplant fate or post-transplant complications.

Cell therapy in transplantation: A comprehensive review of the current applications of cell therapy in transplant patients with the focus on Tregs, CAR Tregs, and Mesenchymal stem cells.

Organ transplantation is a practical treatment for patients with end-stage organ failure. Despite the advances in short-term graft survival, long-term graft survival remains the main challenge considering the increased mortality and morbidity associated with chronic rejection and the toxicity of immunosuppressive drugs. Since a novel therapeutic strategy to induce allograft tolerance seems urgent, focusing on developing novel and safe approaches to prolong graft survival is one of the main goals of transplant investigators. Researchers in the field of organ transplantation are interested in suppressing or optimizing the immune responses by focusing on immune cells including mesenchymal stem cells (MSCs), polyclonal regulatory Tcells (Tregs), and antigen-specific Tregs engineered with chimeric antigen receptors (CAR Tregs). We review the mechanistic pathways, phenotypic and functional characteristics of these cells, and their promising application in organ transplantation.

Regulatory T cells and transplantation tolerance: Emerging from the darkness?

The field of tissue transplantation has revolutionized the treatment of patients with failing organs. Its success, thus far, has depended on combinations of immunosuppressive drugs that damp host immunity, while also imposing numerous unwanted side-effects. There is a longstanding recognition that better treatment outcomes, will come from replacing these drugs, fully or in part, by taking advantage of tractable physiological mechanisms of self-tolerance. The past 50 years have seen many advances in the field of self-tolerance, but perhaps, the most tractable of these has been the more recent discovery of a subset T-cells (Treg) whose role is to regulate or damp immunity. This article is intended to first provide the reader with some historical background to explain why we have been slow to identify these cells, despite numerous clues to their existence, and also to indicate how little we know about how they achieve their regulatory function in averting transplant rejection. However, as is often the case in immunology, the therapeutic needs often dictate that our advances move to translation even before detailed explanations of the science are available. The final part of the article will briefly summarize how Treg are being harnessed as agents to interface with or perhaps, replace current drug combinations.

The mTOR Deficiency in Monocytic Myeloid-Derived Suppressor Cells Protects Mouse Cardiac Allografts by Inducing Allograft Tolerance.

Background: Myeloid-derived suppressor cells (MDSCs) can prevent allograft rejection and induce immune tolerance in transplantation models. Previous studies have demonstrated that inhibition of mTOR signaling can enhance the MDSC protective effect in heart transplantation (HTx) by promoting MDSC expansion. In addition, mTOR inhibition is related to autophagy. The present study investigated the protective mechanism of mTOR-deficient monocytic MDSCs (M-MDSCs) in mouse HTx. Methods: Myeloid-specific mTOR conditional knockout mice were generated to obtain mTOR-/- M-MDSCs. The proliferation and immunosuppressive function of mTOR-/- M-MDSCs were determined by flow cytometry and T cell proliferation assays. The mTOR-/- M-MDSC intracellular autophagy levels were determined using western blotting and electron microscopy. RNAseq analysis was performed for wild-type (WT) and mTOR-/- M-MDSCs. Allogeneic HTx mouse model was established and treated with WT or mTOR-/- M-MDSCs. Enzyme-linked immunosorbent assay, flow cytometry, and immunohistochemistry assays were performed to determine WT and mTOR-/- M-MDSC-induced immune tolerance. Results: The mTOR deficiency promoted M-MDSC differentiation and enhanced intracellular autophagy levels in vivo and in vitro. mTOR deficiency also enhanced the immunosuppressive function of M-MDSCs. In addition, infusing with WT and mTOR-/- M-MDSCs prolonged cardiac allograft survival and established immune tolerance in recipient mice by inhibiting T cell activation and inducing regulatory T cells. Conclusion: mTOR deficiency enhances the immunosuppressive function of M-MDSCs and prolongs mouse cardiac allograft survival.

The Unique Immunomodulatory Properties of MSC-Derived Exosomes in Organ Transplantation.

Methods for suppressing the host immune system over the long term and improving transplantation tolerance remain a primary issue in organ transplantation. Cell therapy is an emerging therapeutic strategy for immunomodulation after transplantation. Mesenchymal stem cells (MSCs) are adult multipotent stem cells with wide differentiation potential and immunosuppressive properties, which are mostly used in regenerative medicine and immunomodulation. In addition, emerging research suggests that MSC-derived exosomes have the same therapeutic effects as MSCs in many diseases, while avoiding many of the risks associated with cell transplantation. Their unique immunomodulatory properties are particularly important in the immune system-overactive graft environment. In this paper, we review the effects of MSC-derived exosomes in the immune regulation mechanism after organ transplantation and graft-versus-host disease (GvHD) from various perspectives, including immunosuppression, influencing factors, anti-inflammatory properties, mediation of tissue repair and regeneration, and the induction of immune tolerance. At present, the great potential of MSC-derived exosomes in immunotherapy has attracted a great deal of attention. Furthermore, we discuss the latest insights on MSC-derived exosomes in organ transplantation and GvHD, especially its commercial production concepts, which aim to provide new strategies for improving the prognosis of organ transplantation patients.

Adoptive Transfer of Regulatory Immune Cells in Organ Transplantation.

Chronic graft rejection remains a significant barrier to solid organ transplantation as a treatment for end-organ failure. Patients receiving organ transplants typically require systemic immunosuppression in the form of pharmacological immunosuppressants for the duration of their lives, leaving these patients vulnerable to opportunistic infections, malignancies, and other use-restricting side-effects. In recent years, a substantial amount of research has focused on the use of cell-based therapies for the induction of graft tolerance. Inducing or adoptively transferring regulatory cell types, including regulatory T cells, myeloid-derived suppressor cells, and IL-10 secreting B cells, has the potential to produce graft-specific tolerance in transplant recipients. Significant progress has been made in the optimization of these cell-based therapeutic strategies as our understanding of their underlying mechanisms increases and new immunoengineering technologies become more widely available. Still, many questions remain to be answered regarding optimal cell types to use, appropriate dosage and timing, and adjuvant therapies. In this review, we summarize what is known about the cellular mechanisms that underly the current cell-based therapies being developed for the prevention of allograft rejection, the different strategies being explored to optimize these therapies, and all of the completed and ongoing clinical trials involving these therapies.

Inducing immune tolerance with dendritic cell-targeting nanomedicines.

Induced tolerogenic dendritic cells are a powerful immunotherapy for autoimmune disease that have shown promise in laboratory models of disease and early clinical trials. In contrast to conventional immunosuppressive treatments, tolerogenic immunotherapy leverages the cells and function of the immune system to quell the autoreactive lymphocytes responsible for damage and disease. The principle techniques of isolating and reprogramming dendritic cells (DCs), central to this approach, are well characterized. However, the broader application of this technology is limited by its high cost and bespoke nature. Nanomedicine offers an alternative route by performing this reprogramming process in situ. Here, we review the challenges and opportunities in using nanoparticles as a delivery mechanism to target DCs and induce immunomodulation, emphasizing their versatility. We then highlight their potential to solve critical problems in organ transplantation and increasingly prevalent autoimmune disorders such as type 1 diabetes mellitus and multiple sclerosis, where new immunotherapy approaches have begun to show promise.

Profiling the liver graft.

PURPOSE OF REVIEW: Achieving operational tolerance remains a priority in liver transplantation. Although several biomarkers of tolerance and rejection have been identified, few have been reproducible and validated across centers, and therefore have yet to reach clinical practice. Here we summarize findings from prior seminal studies and review current developments in profiling the liver allograft. RECENT FINDINGS: Substantial efforts and progress have been made in the recent years towards the discovery of reliable biomarkers that can predict and guide successful immunosuppression withdrawal. Recent studies have also investigated the transcriptomic signatures underlying not only acute rejection but also subclinical inflammation and chronic allograft injury. SUMMARY: As new genomic and sequencing technologies continue to develop, clinical trials are underway to validate biomarkers of tolerance, as well as better understand the mechanisms of both acute and subclinical rejection, with the goal of maximizing allograft survival. Altogether, this will hopefully enable the implementation of immunosuppression withdrawal protocols into clinical practice and make operational tolerance reliably attainable in the near future.