Liver transplantation (LT) is the ultimate treatment for patients with end-stage liver disease or early hepatocellular carcinoma. In the context of LT, because of the unique immunological characteristics of human liver allograft, 5%-20% of selected LT recipients can achieve operational tolerance. Nonetheless, there remains a risk of rejection in LT patients. Maintaining immune homeostasis is thus crucial for improving clinical outcomes in these patients. In mechanism, several immune cells, including dendritic cells, Kupffer cells, myeloid-derived suppressor cells, hepatic stellate cells, regulatory B cells, and CD4 + regulatory T cells (Treg), contribute to achieving tolerance following LT. In terms of Treg, it plays a role in successfully minimizing immunosuppression or achieving tolerance post-LT while also reducing the risk of rejection. Furthermore, the gut microbiome modulates systemic immune functions along the gut-liver axis. Recent studies have explored changes in the microbiome and its metabolites under various conditions, including post-LT, acute rejection, and tolerance. Certain functional microbiomes and metabolites exhibit immunomodulatory functions, such as the augmentation of Treg, influencing immune homeostasis. Therefore, understanding the mechanisms of tolerance in LT, the role of Treg in tolerance and rejection, as well as their interactions with gut microbiome, is vital for the management of LT patients.

The introduction of calcineurin inhibitors (CNIs) as the primary form of immunosuppression (IS) for liver transplantation (LT) in the late 1970s was a key in increasingly successful outcomes for transplantation over the past 3 decades. Despite the side effects of CNI which directly contribute to the long-term morbidity and mortality post-LT, they will remain the cornerstone of IS in the near future. Efforts to minimize exposure to CNI will require the application of blood and tissue biomarkers that accurately identify the extent of IS and risk of rejection for individual patients.

Vascularized composite allotransplantation is successful in reconstruction of major defects of the upper extremity and face. Both rejection and vascular damage seriously endanger the outcome. The role of adipose-derived stromal cells (ASCs) in suppressing acute rejection of composite allotransplants and their short-term protective effects on vessels remains widely unexplored.

Systemic and local donor-derived ASCs (CD45-CD29+CD90+) versus FK-506 administration was evaluated for reversal of acute rejection and vascular alterations in fully mismatched rat hind-limb transplants.

ASC administration upon grade II rejection significantly delayed but did not suppress progression to grade III rejection (7.6 ± 1.0 days systemic, 7.1 ± 1.1 days local vs. no cell therapy 2.9 ± 1 days; p<0.01, n=38 animals). Pro-inflammatory cytokine blood levels significantly increased in controls from grade II to grade III rejection, whereas ASC significantly lowered the levels for G-CSF, MIP-1α, MIP-3α, IL-1α, IL-1β, IL-18, and Rantes (p<0.05). Local and systemic PKH-26-labeled ASCs homed to the allograft and reversed intragraft vascular alterations in arterioles of rejecting skin and muscle, similarly to FK-506-treated controls (p<0.01).

Although systemic and local ASC therapy reduces progression of acute rejection in vascularized composite allotransplantation, it is not able to revert rejection without additional immunosuppressive therapy. However, graft vasculitis during acute rejection is significantly reduced after cytotherapy.

Heart transplantation remains the gold-standard treatment for end-stage heart failure, yet long-term graft survival is hindered by chronic rejection and the morbidity and mortality caused by lifelong immunosuppression. While advances in medical and device-based therapies have reduced the overall need for transplantation, patients who ultimately require a transplant often present with more advanced disease and comorbidities. Recent advances in tolerance-inducing strategies offer promising avenues to improve allograft acceptance, while minimizing immunosuppressive toxicity. This review explores novel approaches aiming to achieve long-term immunological tolerance, including co-stimulation blockade, mixed chimerism, regulatory T-cell (Treg) therapies, thymic transplantation, and double-organ transplantation. These strategies seek to promote donor-specific unresponsiveness and mitigate chronic rejection. Additionally, expanding the donor pool remains a critical challenge in addressing organ shortages. Innovations such as ABO-incompatible heart transplantation are revolutionizing the field by increasing donor availability and accessibility. In this article, we discuss the mechanistic basis, clinical advancements, and challenges of these approaches, highlighting their potential to transform the future of heart transplantation with emphasis on clinical translation.

The manufacturing process of regulatory T (Treg) cells for clinical application begins with the positive selection of CD25+ cells using superparamagnetic iron oxide nanoparticle (SPION)-conjugated anti-CD25 antibodies (spCD25) and immunomagnetic cell separation technology. Our findings revealed that the interaction of spCD25 with its cell target induced the internalization of the complex spCD25-interleukin-2 receptor. Accumulation of intracellular spCD25 triggered oxidative stress, causing delayed Treg expansion and temporary reduction in suppressor activity. This activation delay hindered the efficient generation of clinically competent cells. During this early phase, Treg cells exhibited elevated mitochondrial superoxide and lipid peroxidation levels, with a concomitant decrease in mitochondrial respiration rates. The results uncovered the increased mitochondrial unfolded protein response. This protective, redox-sensitive activity is inherent in Tregs when contrasted with homologous, spCD25-treated, conventional T cells. Although the temporary effects of spCD25 on clinically competent cells did not impede their use in a safety/feasibility pilot study with kidney transplant recipients, it is reasonable to anticipate a potential reduction in their therapeutic efficacy. The mechanistic understanding of the adverse effects triggered by spCD25 is crucial for improving the manufacturing process of clinically competent Treg cells, a pivotal step in the successful implementation of immune cell therapy in transplantation.

To investigate the incidence and potential predictors of immune tolerance among adult living donor liver transplant (LDLT) recipients.

This case-control study included adult recipients who underwent LDLT between May 2004 and January 2018, with at least a 5-year follow-up after LDLT. We divided the study recipients into two groups: Group 1 (Tolerance Group) included recipients who achieved operational or prope tolerance for at least one year; Group 2 (Control Group) included recipients who did not achieve tolerance. We used logistic regression analysis to study the potential predictors of tolerance after LDLT.

We included 368 recipients, 275 (74.7%) in Group 1 and 93 (25.3%) in Group 2. Operational tolerance occurred in 13/275 (4.7%) recipients and prope tolerance in 262/275 (95.3%) recipients. Age was significantly higher in Group 1. The median time for tolerance among the study recipients was 60 months (36-168). During follow-up, Group 1 showed lower serum levels of bilirubin, liver enzymes, alkaline phosphatase, and gamma-glutamyl transferase. Group 1 had a lower incidence of acute cellular rejection (ACR), recurrent viral hepatitis, and biliary complications. Logistic regression identified preoperative MELD, indication for LDLT, ACR, recurrent viral hepatitis, and biliary complications as significant predictors for allograft tolerance after LDLT.

Allograft tolerance occurred in 74.7% of this cohort. We suggest that the MELD score, indication for LT, ACR, recurrent viral hepatitis, and biliary complications are predictors of allograft tolerance after LDLT.

Pancreatic islet transplantation (PITx) is a promising treatment option for patients with type 1 diabetes mellitus. Previously, we demonstrated that therapy with alloantigen-specific immunomodulatory cells (IMCs) generated ex vivo in the presence of anti-CD80 and CD86 monoclonal antibodies (mAbs), successfully induced tolerance following clinical liver transplantation. To extend IMC therapy to PITx, it is crucial to address the strong inflammatory and innate immune responses that occur immediately after PITx. In this study, we investigated the efficacy of IMCs in modulating macrophage activation and mitigating inflammatory damage of pancreatic islets. IMCs were induced using mouse splenocytes in the presence of anti-mouse anti-CD80 (RM80) and anti-CD86 (GL-1) mAbs. IMCs exerted donor-specific immunosuppressive effects in a mixed lymphocyte reaction. During lipopolysaccharide (LPS) stimulation, the addition of IMCs suppressed conversion to the M1 phenotype and promoted a shift toward the M2 phenotype, particularly under direct cell-cell contact conditions. Nitric oxide production, a hallmark of M1 polarized macrophages, was significantly reduced in LPS-stimulated RAW264 macrophages by IMC treatment. These findings were associated with reduced secretion of pro-inflammatory cytokines, tumoral necrosis factor α, and interleukin-6, and increased interleukin-10 production by macrophages. IMCs effectively prevented macrophage-mediated islet destruction after 12 h of co-culture with LPS-stimulated macrophages and significantly inhibited macrophage migration toward allogeneic islets in vitro. Intraportal co-infusion of IMCs with syngeneic islets in a mouse PITx model resulted in reduced messenger RNA (mRNA) expression of pro-inflammatory cytokines in the recipient liver. Immunohistochemical staining revealed a significantly lower number of F4/80+ macrophages at the transplantation site in IMCs-treated mice. These results demonstrate that IMCs modulate macrophage polarization, promoting a shift toward the M2 phenotype and protecting islets from macrophage-mediated damage. These effects combined with its intrinsic donor antigen-specific immunosuppressive capacity make IMC therapy a promising strategy for improving outcomes after PITx.

Conferring alloantigen-specificity to ex vivo expanded CD4+CD25+FOXP3+ regulatory T cells (Tregs) increases their capacity to counteract effector alloimmune responses following adoptive transfer into transplant recipients. Three strategies are currently undergoing clinical development, which involve the following: (1) expanding Tregs in the presence of donor B cells (donor alloantigen-reactive [DAR] Tregs); (2) culturing Tregs with donor cells in the presence of costimulation blockade (CSB-Tregs); and (3) transducing Tregs with an human leukocyte antigen A2-specific chimeric antigen receptor (CAR-Tregs). Our goal in this study was to assess the relative potency of each of these manufactured Treg products both in vitro and in vivo. When compared with polyclonal Tregs, all 3 manufacturing strategies increased the precursor frequency of alloreactive Tregs, and this was proportional to the overall in vitro immunosuppressive properties of the cell products. Accordingly, CAR-Tregs, which contained the highest frequency of donor-reactive Tregs, exhibited the strongest suppressive effects on a cell-per-cell basis. Similarly, in an in vivo mouse model of graft-vs-host disease, infusion of CAR-Tregs conferred a significantly longer recipient survival than any other Treg product. Our results highlighting the alloantigen-reactivity and associated immunosuppressive properties of different manufactured Treg products have implications for the mechanistic interpretation of currently ongoing clinical trials in transplantation.

Prolonged immunosuppressive therapy in liver transplantation (LT) is associated with significant adverse effects, such as nephrotoxicity, metabolic complications, and heightened risk of infection or malignancy. Regulatory T cells (Tregs) represent a promising target for inducing immune tolerance in LT, with the potential to reduce or eliminate the need for life-long immunosuppression. This review summarizes current knowledge on the roles of Tregs in LT, highlighting their mechanisms and the impact of various immunosuppressive agents on Treg stability and function. The liver's distinct immunological microenvironment, characterized by tolerogenic antigen-presenting cells and high levels of interleukin (IL)-10 and transforming growth factor-β, positions this organ as an ideal setting for Treg-mediated tolerance. We discuss Treg dynamics in LT, their association with rejection risk, and their utility as biomarkers of transplant outcomes. Emerging strategies, including the use of low-dose calcineurin inhibitors with mammalian target of rapamycin inhibitors, adoptive Treg therapy, and low-dose IL-2, aim to enhance Treg function while providing sufficient immunosuppression. Thus, the future of LT involves precision medicine approaches that integrate Treg monitoring with tailored immunosuppressive protocols to optimize long-term outcomes for LT recipients.

Autoimmune diseases, characterized by the immune system's attack on the body's own tissues, affect millions of people worldwide. Current treatments, which primarily rely on broad immunosuppression and symptom management, are often associated with significant adverse effects and necessitate lifelong therapy. This review explores the next generation of therapies for immune-mediated diseases, including chimeric antigen receptor (CAR) T cell and regulatory T cell (Treg)-based approaches, which offer the prospect of targeted, durable disease remission. Notably, we highlight the emergence of CD19-targeted CAR T cell therapies, and their ability to drive sustained remission in B cell-mediated autoimmune diseases, suggesting a possible paradigm shift. Further, we discuss the therapeutic potential of Type 1 and FOXP3+ Treg and CAR-Treg cells, which aim to achieve localized immune modulation by targeting their activity to specific tissues or cell types, thereby minimizing the risk of generalized immunosuppression. By examining the latest advances in this rapidly evolving field, we underscore the potential of these innovative cell therapies to address the unmet need for long-term remission and potential tolerance induction in individuals with autoimmune and immune-mediated diseases.

Liver transplantation (LT) is an established life-saving procedure. The field of LT has changed in the past 10 years from several perspectives, with the expansion of indications, transplantation of patients with acute-on-chronic liver failure, evolution of transplant oncology, the use of donations after cardiac death, new surgical techniques, and prioritisation of recipients on the waiting list. In addition, the advent of organ perfusion machines, the recognition of new forms of rejection, and the attention paid to the transition from paediatric to adult patients, have all improved the management of LT recipients. The purpose of the EASL guidelines presented here is not to cover all aspects of LT but to focus on developments since the previous EASL guidelines published in 2016.

The administration of T cells that have been modified to carry chimeric antigen receptors (CARs) aimed at B cells has been an effective strategy in treating B cell malignancies. This breakthrough has spurred the creation of CAR T cells intended to specifically reduce or alter the faulty immune responses associated with autoimmune disorders. Early positive outcomes from clinical trials involving CAR T cells that target the B cell protein CD19 in patients suffering from autoimmune diseases driven by B cells have been reported. Additional strategies are being developed to broaden the use of CAR T cell therapy and enhance its safety in autoimmune conditions. These include employing chimeric autoantireceptors (CAAR) to specifically eliminate B cells that are reactive to autoantigens, and using regulatory T cells (Tregs) engineered to carry antigen-specific CARs for precise immune modulation. This discussion emphasizes key factors such as choosing the right target cell groups, designing CAR constructs, defining tolerable side effects, and achieving a lasting immune modification, all of which are critical for safely integrating CAR T cell therapy in treating autoimmune diseases.

Infusion of T cells engineered to express chimeric antigen receptors (CARs) that target B cells has proven to be a successful treatment for B cell malignancies. This success inspired the development of CAR T cells to selectively deplete or modulate the aberrant immune responses that underlie autoimmune disease. Promising results are emerging from clinical trials of CAR T cells targeting the B cell protein CD19 in patients with B cell-driven autoimmune diseases. Further approaches are being designed to extend the application and improve safety of CAR T cell therapy in the setting of autoimmunity, including the use of chimeric autoantibody receptors to selectively deplete autoantigen-specific B cells and the use of regulatory T cells engineered to express antigen-specific CARs for targeted immune modulation. Here, we highlight important considerations, such as optimal target cell populations, CAR construct design, acceptable toxicities and potential for lasting immune reset, that will inform the eventual safe adoption of CAR T cell therapy for the treatment of autoimmune diseases.

Hepatocellular carcinoma (HCC) recurrence appears commonly after liver transplantation (LT), and it severely affected the long-term survival of patients. Previous studies have proved that Rap1A is involved in hepatocarcinogenesis and metastasis, and demonstrated the significant association between KRAS rs712 polymorphism and HCC. However, the relationship between KRAS rs712 polymorphism and HCC recurrence after LT remained unclear. A total of 93 HCC patients who underwent LT from March 2008 to Dec 2015 was analyzed. The genotypes of both donors and recipients had been confirmed as KRAS rs712. The independent risk factors that associated with HCC recurrence were investigated with univariate and multivariate logistic regression analysis. The recurrence-free (RFS) and overall survival (OS) were calculated with Cox regression analysis. The KRAS rs712 genotype frequencies were determined using the Χ2 test and the minor allele frequencies (MAFs) of KRAS rs712 genotypes were calculated by Hardy-Weinberg equilibrium. We found that the recipient KRAS rs712 polymorphism was significantly associated with HCC recurrence after LT. Moreover, the Milan criteria, microvascular invasion and recipient KRAS rs712 genotype were proved to be independent risk factors for HCC recurrence after LT. Patients with donor TG/TT genotypes had a significantly higher RFS and OS than TT genotype. The TNM stage, microvascular invasion, Milan criteria, treatment and recipient KRAS rs712 genotype were independent factors for the RFS of LT patients. Recipient KRAS rs712 polymorphism is associated with HCC recurrence after liver transplantation and plays as a promising bio-predictor of overall survival rate of HCC risks after hepatic transplantation.

Immunosuppression is essential to ensure recipient and graft survivals after liver transplantation (LT). However, our understanding and management of the immune system remain suboptimal. Current immunosuppressive therapy cannot selectively inhibit the graft-specific immune response and entails a significant risk of serious side effects, i.e., among others, de novo cancers, infections, cardiovascular events, renal failure, metabolic syndrome, and late graft fibrosis, with progressive loss of graft function. Pharmacological research, aimed to develop alternative immunosuppressive agents in LT, is behind other solid-organ transplantation subspecialties, and, therefore, the development of new compounds and strategies should get priority in LT. The research trajectories cover mechanisms to induce T-cell exhaustion, to inhibit co-stimulation, to mitigate non-antigen-specific inflammatory response, and, lastly, to minimize the development and action of donor-specific antibodies. Moreover, while cellular modulation techniques are complex, active research is underway to foster the action of T-regulatory cells, to induce tolerogenic dendritic cells, and to promote the function of B-regulatory cells. We herein discuss current lines of research in clinical immunosuppression, particularly focusing on possible applications in the LT setting.

Paediatric heart transplant is an established treatment for end stage heart failure in children, however patients have to commit to lifelong medical surveillance and adhere to daily immunosuppressants to minimise the risk of rejection. Compliance with immunosuppressants can be burdensome with their toxic side effects and need for frequent blood monitoring especially in children. Though the incidence of early rejection episodes has significantly improved overtime, the long-term allograft health and survival is determined by Cardiac Allograft Vasculopathy (CAV) which affects a vast number of post-transplant patients. Once CAV has set in, there is no medical or surgical treatment to reverse it and graft survival is significantly compromised across all age groups. Current treatment strategies include novel immunosuppressant agents and drugs to lower blood lipid levels to address the underlying immunological pathophysiology and to manage traditional cardiac risk factors. Translational researchers are seeking novel immunological approaches that can lead to permanent acceptance of the allograft such as using regulatory T cell (Tregs) immunotherapy. Clinical trials in the setting of graft versus host disease, autoimmunity and kidney and liver transplantation using Tregs have shown the feasibility and safety of this strategy. This review will summarise current knowledge of the latest clinical therapies for CAV and pre-clinical evidence in support of Treg therapy for CAV. We will also discuss the different Treg sources and the considerations of translating this into a feasible immunotherapy in clinical practice in the paediatric population.

Over the last 50 years, liver transplantation has evolved into a procedure routinely performed in many countries worldwide. Those able to access this therapy frequently experience a miraculous risk-benefit ratio, particularly if they face the imminently life-threatening disease. Over the decades, the success of liver transplantation, with dramatic improvements in early posttransplant survival, has aggressively driven demand. However, despite the emergence of living donors to augment deceased donors as a source of organs, supply has lagged far behind demand. As a result, rationing has been an unfortunate focus in recent decades. Recent shifts in the epidemiology of liver disease combined with transformative innovations in liver preservation suggest that the underlying premise of organ shortage may erode in the foreseeable future. The focus will sharpen on improving equitable access while mitigating constraints related to workforce training, infrastructure for organ recovery and rehabilitation, and their associated costs. Research efforts in liver preservation will undoubtedly blossom with the aim of optimizing both the timing and conditions of transplantation. Coupled with advances in genetic engineering, regenerative biology, and cellular therapies, the portfolio of innovation, both broad and deep, offers the promise that, in the future, liver transplantation will not only be broadly available to those in need but also represent a highly durable life-saving therapy.

Regulatory T-cells (Tregs) play a key role in immune homeostasis after organ transplantation. However, the role of CD4+ T cell subsets in early acute rejection is still not well understood. Therefore, our aim was to determine changes in CD4+ T-cell subsets in living donor liver transplantation (LDLT).

LDLT patients were assessed for T-cell subsets, Tregs frequencies and their functionality by flow-cytometry at peri- and post-transplant in the span of 1 year.

33 patients were followed up and 11 (33%) patients have developed early acute cellular rejection (ACR). At peri-transplant time point, MFI of Foxp3+ Tregs was significantly increased compared to HC (P = 0.04). However, CD4+CD25+Foxp3+/CD127- Tregs numbers and IL-10, IL-17 and TGF-β secreting functional Tregs were significantly decreased at 3 months compared to peri-transplant (P = 0.003). But in patients with rejection, CD4+CD25+FOXP3+ and CD4+CD25+CD127- Tregs were significantly decreased at day 3 compared to no rejection group (P = 0.048). Patients with rejection also showed significantly decreased numbers of IL-17 and TGF-β secreting CD4+CD25+FOXP3+ Tregs at peri-transplant time (P = 0.04, P = 0.03) compared to no rejection. Further, rejection group showed decreased terminally differentiated effector memory (TEMRA) at peri-transplant and day 7 (P = 0.048 and P = 0.01). Additionally, CD4+ central memory (CM) was decreased at peri-transplant (P = 0.05), 1 month (P = 0.04), and 3 to 6 month (P = 0.02).

Tregs frequencies were significantly decreased in peri-TX in rejection patients. Further, decreased frequencies of CD4+ TEMRA and CD4+ CM at day 7 and 1 month were associated with rejection.

Posttransplantation complications pose a major challenge to the long-term survival and quality of life of organ transplant recipients. These complications encompass immune-mediated complications, infectious complications, metabolic complications, and malignancies, with each type influenced by various risk factors and pathological mechanisms. The molecular mechanisms underlying posttransplantation complications involve a complex interplay of immunological, metabolic, and oncogenic processes, including innate and adaptive immune activation, immunosuppressant side effects, and viral reactivation. Here, we provide a comprehensive overview of the clinical features, risk factors, and molecular mechanisms of major posttransplantation complications. We systematically summarize the current understanding of the immunological basis of allograft rejection and graft-versus-host disease, the metabolic dysregulation associated with immunosuppressive agents, and the role of oncogenic viruses in posttransplantation malignancies. Furthermore, we discuss potential prevention and intervention strategies based on these mechanistic insights, highlighting the importance of optimizing immunosuppressive regimens, enhancing infection prophylaxis, and implementing targeted therapies. We also emphasize the need for future research to develop individualized complication control strategies under the guidance of precision medicine, ultimately improving the prognosis and quality of life of transplant recipients.

Solid organ transplantation is hindered by immune-mediated chronic graft dysfunction and the side effects of immunosuppressive therapy. Regulatory T cells (Tregs) are crucial for modulating immune responses post-transplantation; however, the transfer of polyspecific Tregs alone is insufficient to induce allotolerance in rodent models.

To enhance the efficacy of adoptive Treg therapy, we investigated different immune interventions in the recipients. By utilizing an immunogenic skin transplant model and existing transplantation medicine reagents, we facilitated the clinical translation of our findings. Specifically, antigen-specific Tregs were used.

Our study demonstrated that combining the available induction therapies with drug-induced T-cell proliferation due to lymphopenia effectively increased the Treg/T effector ratios. This results in significant Treg accumulation within the graft, leading to long-term tolerance after the transfer of antigen-specific Tregs. Importantly, all the animals achieved operational tolerance, which boosted the presence of adoptively transferred Tregs within the graft.

This protocol offers a means to establish tolerance by utilizing antigen-specific Tregs. These results have promising implications for future trials involving adoptive Treg therapy in organ transplantation.

Tolerance is the Holy Grail of solid organ transplantation (SOT) and remains its primary challenge since its inception. In this topic, the seminal contributions of Thomas Starzl at Pittsburgh University outlined foundational principles of graft acceptance and tolerance, with chimerism emerging as a pivotal factor. Immunologically, intestinal transplantation (ITx) poses a unique hurdle due to the inherent characteristics and functions of the small bowel, resulting in increased immunogenicity. This necessitates heavy immunosuppression (IS) while IS drugs side effects cause significant morbidity. In addition, current IS therapies fall short of inducing clinical tolerance and their discontinuation has been proven unattainable in most cases. This underscores the unfulfilled need for immunological modulation to safely reduce IS-related burdens. To address this challenge, the Leuven Immunomodulatory Protocol (LIP), introduced in 2000, incorporates various pro-tolerogenic interventions in both the donor to the recipient, with the aim of facilitating graft acceptance and improving outcome. This review seeks to provide an overview of the current understanding of tolerance in ITx and outline recent advances in this domain.

Adeno-associated virus (AAV) gene therapy is making rapid strides owing to its wide range of therapeutic applications. However, development of serious immune responses to the capsid antigen or the therapeutic transgene product hinders its full clinical impact. Immune suppressive (IS) drug treatments have been used in various clinical trials to prevent the deleterious effects of cytotoxic T cells to the viral vector or transgene, although there is no consensus on the best treatment regimen, dosage, or schedule. Regulatory T cells (Tregs) are crucial for maintaining tolerance against self or nonself antigens. Of importance, Tregs also play an important role in dampening immune responses to AAV gene therapy, including tolerance induction to the transgene product. Approaches to harness the tolerogenic effect of Tregs include the use of selective IS drugs that expand existing Tregs, and skew activated conventional T cells into antigen-specific peripherally induced Tregs. In addition, Tregs can be expanded ex vivo and delivered as cellular therapy. Furthermore, receptor engineering can be used to increase the potency and specificity of Tregs allowing for suppression at lower doses and reducing the risk of disrupting protective immunity. Because immune-mediated toxicities to AAV vectors are a concern in the clinic, strategies that can enhance or preserve Treg function should be considered to improve both the safety and efficacy of AAV gene therapy.

Induced Tregs (iTregs) have great promise in adoptive immunotherapy for treatment of autoimmune diseases. This report investigates the impacts of substrate stiffness on human Treg induction, providing a powerful yet simple approach to improving production of these cells. Conventional CD4+ human T cells were activated on materials of different elastic modulus and cultured under suppressive conditions. Enhanced Treg induction was observed on softer materials as early as 3 days following activation and persisted for multiple weeks. Substrate stiffness also affected epigenetic modification of Treg specific genes and Treg suppressive capacity. Tregs induced on substrates of an optimal stiffness balance quantity and suppressive quality.

Foxp3+ T regulatory (Treg) cells prevent allograft rejection and graft-versus-host disease. Although polyclonal Tregs have been used both in animal models and in humans, the fine specificity of their suppressive function is poorly defined. We have generated mouse recipient-derived alloantigen-specific Tregs in vitro and explored the fine specificity of their suppressive function and their mechanism of action in vitro and in vivo. In vitro, when alloantigen and peptide Ag were both presented on the same dendritic cell, both responses were suppressed by iTregs specific either for the alloantigen or for the peptide Ag. In vivo, iTreg suppression was limited to the cognate Ag, and no bystander suppression was observed when both allo-antigen and peptide Ag were present on the same dendritic cell. In vitro, alloantigen-specific Tregs captured cognate MHC but failed to capture noncognate MHC. Our results demonstrate that a polyclonal population of iTregs generated from naive T cells can mediate highly specific function in vivo and support the view that Treg therapy, even with unselected polyclonal populations, is likely to be target antigen-specific and that bystander responses to self-antigens or to infectious agents are unlikely.

Regulatory T cells (Tregs) are essential for maintaining the immune balance in normal and pathological conditions. In autoimmune diseases and transplantation, they restrain the loss of self-tolerance and promote engraftment, whereas in cancer, an increase in Treg numbers is mostly associated with tumor growth and poor prognosis. Numerous markers and their combinations have been used to identify Treg subsets, demonstrating the phenotypic diversity of Tregs. The complexity of Treg identification can be hampered by the unstable expression of some markers, the decrease in the expression of a specific marker over time or the emergence of a new marker. It remains unclear whether such phenotypic shifts are due to new conditions or whether the observed changes are due to initially different populations. In the first case, cellular plasticity is observed, whereas in the second, cellular heterogeneity is observed. The difference between these terms in relation to Tregs is rather blurred. Considering the promising perspectives of Tregs in regenerative cell-based therapy, the existing confusing data on Treg phenotypes require further investigation and analysis. In our review, we introduce criteria that allow us to distinguish between the heterogeneity and plasticity of Tregs normally and pathologically, taking a closer look at their diversity and drawing the line between two terms.

CD4+CD25hiCD127lo/-FOXP3+ regulatory T cells (Tregs) play a key role in preventing autoimmunity. In autoimmune type 1 diabetes (T1D), adoptive transfer of autologous polyclonal Tregs has been shown to be safe in adults in phase 1 clinical trials. We explored factors contributing to efficacy of autologous polyclonal expanded Tregs (expTregs) in a randomized phase 2 multi-center, double-blind, clinical trial (Sanford/Lisata Therapeutics T-Rex phase 2 trial, ClinicalTrials.gov NCT02691247). One hundred ten treated children and adolescents with new-onset T1D were randomized 1:1:1 to high-dose (20 × 106 cells/kilogram) or low-dose (1 × 106 cells/kilogram) treatments or to matching placebo. Cytometry as well as bulk and single-cell RNA sequencing were performed on selected expTregs and peripheral blood samples from participants. The single doses of expTregs were safe but did not prevent decline in residual β cell function over 1 year compared to placebo (P = 0.94 low dose, P = 0.21 high dose), regardless of age or baseline C-peptide. ExpTregs were highly activated and suppressive in vitro. A transient increase of activated memory Tregs was detectable 1 week after infusion in the high-dose cohort, suggesting effective transfer of expTregs. However, the in vitro fold expansion of expTregs varied across participants, even when accounting for age, and lower fold expansion and its associated gene signature were linked with better C-peptide preservation regardless of Treg dose. These results suggest that a single dose of polyclonal expTregs does not alter progression in T1D; instead, Treg quality may be an important factor.

Donor-specific tolerance remains a goal in transplantation because it could improve graft survival and reduce morbidity. Cotransplantation of donor hematopoietic cells to achieve chimerism is a promising approach for tolerance induction, which was successfully tested in clinical trials. However, current protocols are associated with side effects related to the myelosuppressive recipient conditioning, which makes it difficult to introduce them as standard therapy. More recently, adoptive cell therapy with polyclonal or donor-specific regulatory T cells (Treg) proved safe and feasible in several transplant trials, but it is unclear whether it can induce tolerance on its own. The combination of both approaches-Treg therapy and hematopoietic cell transplantation-leads to chimerism and tolerance without myelosuppressive treatment in murine models. Treg therapy promotes engraftment of allogeneic hematopoietic cells, reducing conditioning requirements and enhancing regulatory mechanisms maintaining tolerance. This review discusses possible modes of action of transferred Treg in experimental chimerism models and describes translational efforts investigating the potent synergy of Treg and chimerism.

Balancing the immune response after solid organ transplantation (SOT) and vascularized composite allotransplantation (VCA) remains an ongoing clinical challenge. While immunosuppressants can effectively reduce acute rejection rates following transplant surgery, some patients still experience recurrent acute rejection episodes, which in turn may progress to chronic rejection. Furthermore, these immunosuppressive regimens are associated with an increased risk of malignancies and metabolic disorders. Despite significant advancements in the field, these IS related side effects persist as clinical hurdles, emphasizing the need for innovative therapeutic strategies to improve transplant survival and longevity. Cellular therapy, a novel therapeutic approach, has emerged as a potential pathway to promote immune tolerance while minimizing systemic side-effects of standard IS regiments. Various cell types, including chimeric antigen receptor T cells (CAR-T), mesenchymal stromal cells (MSCs), regulatory myeloid cells (RMCs) and regulatory T cells (Tregs), offer unique immunomodulatory properties that may help achieve improved outcomes in transplant patients. This review aims to elucidate the role of cellular therapies, particularly MSCs, T cells, Tregs, RMCs, macrophages, and dendritic cells in SOT and VCA. We explore the immunological features of each cell type, their capacity for immune regulation, and the prospective advantages and obstacles linked to their application in transplant patients. An in-depth outline of the current state of the technology may help SOT and VCA providers refine their perioperative treatment strategies while laying the foundation for further trials that investigate cellular therapeutics in transplantation surgery.

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.

We demonstrated that an agonistic anti-B and T lymphocyte attenuator antibody (3C10) prolonged cardiac survival by inducing regulatory T cells (Treg). However, the mechanisms of immune tolerance in the recipients remained unclear. In this study, we investigated the graft-protective and intercellular immunomodulatory effects of adoptive transfer (AT) of 3C10-induced Tregs in a murine cardiac allograft transplant model.

Thirty days after transplantation of a C57BL/6 heart into the primary 3C10-treated CBA recipients, splenic CD4+CD25+ cells from these recipients (3C10/AT group) or naïve CBA mice (no-treatment group) were adoptively transferred into secondary CBA recipients with a C57BL/6 heart. To confirm the requirement for 3C10-induced Tregs, we administered an anti-interleukin-2 receptor alpha antibody (PC-61) to secondary CBA recipients. Additionally, histologic and fluorescent staining, cell proliferation analysis, flow cytometry, and donor-specific antibody (DSA) measurements were performed.

3C10/AT-treated CBA recipients resulted in significantly prolonged allograft survival (median survival time [MST], >50 days). Allografts displayed prolonged function with preservation of vessel structure by maintaining high numbers of splenic CD4+CD25+Foxp3+ Treg and intramyocardial CD4+Foxp3+ cells. DSA levels were suppressed in 3C10/AT-treated CBA recipients. Moreover, PC-61 administration resulted in a shorter MSTs of cardiac allograft survivals, a detrimental increase in DSA production, and enhanced expression of programmed cell death (PD)-1.

AT of 3C10-induced Tregs may be a promising graft-protective strategy to prolong allograft survival and suppress DSA production, driven by the promotion of splenic and graft-infiltrating Tregs and collaboration with PD-1+ T cells and Treg.

CD4+ CD25+ FOXP3+ T regulatory cells (Tregs) are a subset of the immunomodulatory cell population that can inhibit both innate and adaptive immunity by various regulatory mechanisms. In hepatic microenvironment, proliferation, plasticity, migration, and function of Tregs are interrelated to the remaining immune cells and their secreted cytokines and chemokines. In normal conditions, Tregs protect the liver from inflammatory and auto-immune responses, while disruption of this crosstalk between Tregs and other immune cells may result in the progression of chronic liver diseases and the development of hepatic malignancy. In this review, we analyze the deviance of this protective nature of Tregs in response to chronic inflammation and its involvement in inducing liver fibrosis, cirrhosis, and hepatocellular carcinoma. We will also provide a detailed emphasis on the relevance of Tregs as an effective immunotherapeutic option for autoimmune diseases, liver transplantation, and chronic liver diseases including liver cancer.

Immunotherapies have revolutionized the landscape of cancer treatment. Regulatory T cells (Tregs), as crucial components of the tumor immune environment, has great therapeutic potential. However, nonspecific inhibition of Tregs in therapies may not lead to enhanced antitumor responses, but could also trigger autoimmune reactions in patients, resulting in intolerable treatment side effects. Hence, the precision targeting and inhibition of tumor-infiltrating Tregs is of paramount importance. In this overview, we summarize the characteristics and subpopulations of Tregs within tumor microenvironment and their inhibitory mechanisms in antitumor responses. Furthermore, we discuss the current major strategies targeting regulatory T cells, weighing their advantages and limitations, and summarize representative clinical trials targeting Tregs in cancer treatment. We believe that developing therapies that specifically target and suppress tumor-infiltrating Tregs holds great promise for advancing immune-based therapies.

Long-term organ transplant survival remains suboptimal, and life-long immunosuppression predisposes transplant recipients to an increased risk of infection, malignancy, and kidney toxicity. Promoting the regulatory arm of the immune system by expanding Tregs may allow immunosuppression minimization and improve long-term graft outcomes. While low-dose IL-2 treatment can expand Tregs, it has a short half-life and off-target expansion of NK and effector T cells, limiting its clinical applicability. Here, we designed a humanized mutein IL-2 with high Treg selectivity and a prolonged half-life due to the fusion of an Fc domain, which we termed mIL-2. We showed selective and sustainable Treg expansion by mIL-2 in 2 murine models of skin transplantation. This expansion led to donor-specific tolerance through robust increases in polyclonal and antigen-specific Tregs, along with enhanced Treg-suppressive function. We also showed that Treg expansion by mIL-2 could overcome the failure of calcineurin inhibitors or costimulation blockade to prolong the survival of major-mismatched skin grafts. Validating its translational potential, mIL-2 induced a selective and sustainable in vivo Treg expansion in cynomolgus monkeys and showed selectivity for human Tregs in vitro and in a humanized mouse model. This work demonstrated that mIL-2 can enhance immune regulation and promote long-term allograft survival, potentially minimizing immunosuppression.

Regulatory T cells (Tregs) are lymphocytes that play a central role in peripheral immune tolerance. Tregs are promising targets for the prevention and suppression of autoimmune diseases, allergies, and graft-versus-host disease, and treatments aimed at regulating their functions are being developed. In this study, we created a new modality consisting of a protein molecule that suppressed excessive immune responses by effectively and preferentially expanding Tregs. Recent studies reported that tumor necrosis factor receptor type 2 (TNFR2) expressed on Tregs is involved in the proliferation and activation of Tregs. Therefore, we created a functional immunocytokine, named TNFR2-ICK-Ig, consisting of a fusion protein of an anti-TNFR2 single-chain Fv (scFv) and a TNFR2 agonist TNF-α mutant protein, as a new modality that strongly enhances TNFR2 signaling. The formation of agonist-receptor multimerization (TNFR2 cluster) is effective for the induction of a strong TNFR2 signal, similar to the TNFR2 signaling mechanism exhibited by membrane-bound TNF. TNFR2-ICK-Ig improved the TNFR2 signaling activity and promoted TNFR2 cluster formation compared to a TNFR2 agonist TNF-α mutant protein that did not have an immunocytokine structure. Furthermore, the Treg expansion efficiency was enhanced. TNFR2-ICK-Ig promotes its effects via scFv, which crosslinks receptors whereas the agonists transmit stimulatory signals. Therefore, this novel molecule expands Tregs via strong TNFR2 signaling by the formation of TNFR2 clustering.

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