Liver transplantation (LT) is a critical therapeutic intervention for patients with end-stage liver disease, hepatocellular carcinoma (HCC), and acute liver failure. The evolution of LT from an experimental procedure to a routinely successful treatment has been primarily driven by advances in immunosuppressive therapy. The introduction of calcineurin inhibitors (CNIs) in the 1980s, such as cyclosporine and tacrolimus, was a pivotal development in transplantation, significantly reducing the incidence of acute rejection, increasing graft survival, and solidifying LT as a viable therapeutic option. These immunosuppressive agents have become foundational in transplant medicine, enabling long-term allograft function and improving patient outcomes [1]. Despite these advances, the dependence on long-term immunosuppressive therapy presents a clinical paradox. Although essential for preventing allograft rejection, chronic immunosuppression is associated with substantial adverse effects, including nephrotoxicity, neurotoxicity, new-onset diabetes mellitus, cardiovascular complications, and an increased risk of opportunistic infections and malignancies [2].

The discovery of regulatory T cells (Tregs) in the 1990s marked a paradigm shift in transplant immunology. Unlike conventional immunosuppressive therapies, which nonspecifically dampen immune responses, Tregs offer a targeted immunoregulatory approach that selectively inhibits alloreactive immune responses while preserving overall immune function [3]. Defined by the expression of CD4, CD25, and transcription factor FOXP3, Tregs play a crucial role in maintaining immune homeostasis and preventing autoimmunity by suppressing the activation and proliferation of effector T cells, which could otherwise attack self or transplanted tissues [4]. This unique ability to modulate alloreactivity without compromising systemic immunity has made Tregs a promising target for therapeutic strategies aimed at inducing immune tolerance in transplantation.

In LT, the role of Tregs is particularly significant due to the phenomenon of operational tolerance, wherein a subset of patients achieves long-term graft survival without the need for continuous immunosuppression [5]. Understanding the mechanisms by which Tregs facilitate tolerance has spurred the development of novel therapeutic approaches aimed at enhancing Treg function, with the ultimate objective of reducing or potentially eliminating the requirement for life-long immunosuppression.

Among solid organs, the liver possesses a unique immune environment that is characterized by its capacity to promote immune tolerance; in contrast, other organs rely on rigorous immune surveillance to prevent infection. This immunological privilege is largely due to the liver’s constant exposure to antigens from the gastrointestinal tract, which necessitates a tolerogenic environment to prevent chronic inflammation and immune-mediated tissue damage [6]. Due to these inherent tolerogenic properties, the liver is an optimal candidate for tolerance-based transplantation strategies, with Tregs playing a pivotal role in facilitating this process [7].

Tregs help sustain an immunosuppressive environment by inhibiting the activation and proliferation of effector T cells, which are responsible for mediating graft rejection [8]. Liver transplant recipients with higher levels of circulating Tregs are more likely to achieve operational tolerance, underscoring the pivotal role of Tregs in promoting long-term graft acceptance [9]. Furthermore, Tregs interact with liver-resident cells, including Kupffer cells (KCs), liver sinusoidal endothelial cells (LSECs), and dendritic cells (DCs), to establish a microenvironment that fosters immune tolerance [10].

The liver’s immune microenvironment is also characterized by elevated levels of anti-inflammatory cytokines, such as interleukin (IL)-10 and transforming growth factor (TGF)-β, both of which are essential for Treg differentiation and function. These cytokines, produced by liver-resident immune cells and Tregs, suppress effector T cell responses while promoting the differentiation of naive T cells into Tregs [11]. This dynamic interaction establishes a feedback loop in which Tregs maintain immune tolerance while benefiting from the liver’s intrinsically tolerogenic environment, potentially supporting long-term graft survival with reduced, or even discontinued, immunosuppression.

Recent advances in understanding Treg biology and the interactions of Tregs with immune cells have enabled therapeutic strategies that leverage the liver’s unique immune environment to promote tolerance. Immunosuppressive agents exert varied effects on Tregs; however, comprehensive analyses specifically addressing these impacts in LT remain limited. In this review, we examine these effects and provide an in-depth overview of current findings.

PI3K-Akt-mTOR Pathway

The molecular pathways regulating Treg function, especially the PI3K-Akt-mTOR (phosphatidylinositol 3-kinase-Akt-mammalian target of rapamycin) and MAPK (mitogen-activated protein kinase) signaling pathways, have been studied extensively. These pathways are vital to balancing effector T cell activation with Treg-mediated suppression [12]. In effector T cells, activation of the PI3K-Akt-mTOR pathway drives glycolysis and rapid cell proliferation to support robust immune responses [13]. In contrast, Tregs primarily rely on oxidative phosphorylation and lipid metabolism to sustain their suppressive functions; dysregulation of the PI3K-Akt-mTOR pathway in Tregs can lead to impaired activity and a higher risk of graft rejection [14,15].

Direct Cytotoxicity

Tregs can induce apoptosis in effector T cells by producing cytotoxic molecules, such as granzyme and perforin [16]. Although granzyme and perforin-mediated cytotoxicity is primarily observed in cytotoxic T cells for the direct elimination of virus-infected or transformed cells, emerging evidence indicates that Tregs also exploit these pathways to suppress immune responses. This cytotoxic mechanism is particularly relevant in specific pathological contexts, such as chronic inflammation or acute rejection episodes following organ transplantation. During these events, autoreactive or alloreactive effector T cells that could potentially exacerbate immune-mediated tissue damage are targeted by Tregs for elimination [17,18].

Suppressive Cytokines

A primary mechanism through which Tregs exert immunosuppressive effects is the secretion of anti-inflammatory cytokines (Fig. 1). IL-10 and TGF-β are key cytokines produced by Tregs, and both play pivotal roles in modulating immune responses [19]. They promote the differentiation of tolerogenic DCs by secreting IL-10 and TGF-β, which inhibit DC maturation and activation [20]. Tolerogenic DCs, in turn, support Treg differentiation while suppressing effector T cell activation.

Figure 1. The role of Tregs in direct and indirect allorecognition pathways in graft rejection. This figure illustrates the role of Tregs in suppressing immune responses and preventing graft damage through direct and indirect allorecognition pathways. In the direct pathway, donor graft cells present alloantigens to recipient CD4⁺ T cells, while in the indirect pathway, recipient APCs present processed donor peptides. Both pathways activate CD8+ T cells and B cells, potentially leading to graft damage. Tregs inhibit T cell activation by depriving effector T cells of IL-2 (via CD25), suppressing dendritic cells through the CTLA-4/CD80 axis, and producing inhibitory cytokines (IL-10, IL-35, and TGF-β). They also modulate B cells via the PD-1/PD-L1 axis and generate adenosine (through CD39/CD73) to protect the graft. MHCII, major histocompatibility complex class II; TCR, T cell receptor; Treg, regulatory T cell; PD-1, programmed cell death protein 1; IL, interleukin; IFN-γ, interferon gamma; ADCC, antibody-dependent cell-mediated cytotoxicity; APC, antigen-presenting cell; TGF, transforming growth factor; DC, dendritic cell; CTLA-4, cytotoxic T-lymphocyte-associated protein 4.

IL-10 suppresses the production of proinflammatory cytokines by effector T cells, DCs, and macrophages, thereby reducing overall inflammation [21]. In addition, IL-10 downregulates the expression of major histocompatibility complex class II molecules and costimulatory molecules, such as CD80 and CD86, on antigen-presenting cells (APCs), thereby limiting their capacity to activate effector T cells [22]. TGF-β plays a dual role in immune regulation. Beyond inhibiting the proliferation and differentiation of effector T cells, TGF-β facilitates the conversion of naive T cells into induced Tregs, a process that is particularly critical in the gut and liver, where Tregs must be continuously replenished to maintain immune homeostasis [23]. TGF-β also modulates the function of APCs, reducing their ability to activate effector T cells while promoting Treg differentiation [24].

Cytotoxic T-Lymphocyte-Associated Protein 4

In addition to cytokine-mediated suppression, Tregs exert immunoregulatory effects through direct cell-cell contact (Fig. 1). A well-characterized mechanism involves the interaction between cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) on Tregs and the costimulatory molecules CD80 and CD86 on APCs [25]. Upon binding to these costimulatory molecules, CTLA-4 prevents APCs from delivering essential activation signals to effector T cells, thereby inhibiting the activation and proliferation of effector T cells [26]. This suppression of costimulation is crucial for maintaining immune tolerance in the liver, where unchecked effector T cell activation can lead to graft rejection [27].

Programmed Cell Death Protein 1

Tregs express programmed cell death protein 1 (PD-1), which interacts with its ligand, PD-L1, on APCs and effector T cells [28]. This PD-1/PD-L1 interaction induces T cell exhaustion, a state characterized by reduced cytokine production and limited proliferative capacity [29]. In the liver, where PD-L1 is abundantly expressed by LSECs and KCs, the PD-1/PD-L1 axis plays a crucial role in maintaining immune quiescence [30,31].

Tregs also upregulate inhibitory molecules on APCs, such as PD-L1 and ILT3, both of which contribute to the suppression of effector T cells and the induction of immune exhaustion [32]. These interactions between Tregs and APCs establish a feedback loop that reinforces immune tolerance and prevents the activation of effector T cells, protecting the transplanted liver from immune-mediated damage [33].

Indoleamine 2,3-Dioxygenase

A primary mechanism by which Tregs regulate APC function is through the induction of indoleamine 2,3-dioxygenase, an enzyme that depletes tryptophan from the local environment, effectively depriving effector T cells of this essential amino acid and promoting immunosuppression [34,35]. Tregs also promote the differentiation of tolerogenic DCs by secreting IL-10 and TGF-β, which inhibit DC maturation and activation [20]. Tolerogenic DCs, in turn, support Treg differentiation while suppressing effector T cell activation. This mechanism is particularly important in the liver’s tolerogenic environment, where a delicate balance between immune surveillance and immune tolerance is essential for preventing chronic rejection [20,36].

Liver Immunology: A Tolerogenic Microenvironment

The liver’s unique immunological properties make it an ideal organ for studying and promoting immune tolerance. The liver is continually exposed to foreign antigens from the gut, necessitating a high level of immune regulation to prevent chronic inflammation and immune-mediated damage [37]. This specialized immune environment is characterized by the presence of tolerogenic APCs, such as KCs and LSECs, which work in concert with Tregs to maintain immune quiescence [38].

KCs are the resident macrophages of the liver and essential for maintaining immune tolerance. These cells secrete large amounts of IL-10 and TGF-β, which promote the differentiation of Tregs and inhibit the activation of effector T cells [39]. KCs also express low levels of costimulatory molecules, such as CD80 and CD86, further supporting immune tolerance by limiting effector T cell activation [40]. In addition, KCs play a role in the clearance of apoptotic cells and debris from the liver, which is a crucial process for preventing inflammation and preserving tissue homeostasis [41].

LSECs also play a significant role in promoting immune tolerance. LSECs line the liver’s blood vessels and filter blood from the gut, which contains high levels of microbial antigens [42]. LSECs express high levels of PD-L1, which interacts with PD-1 on T cells to inhibit their activation and promote Treg development [43]. Furthermore, LSECs can cross-present antigens to CD8⁺ T cells, leading to their deletion or functional inactivation and thereby contributing to the maintenance of tolerance [44,45].

Changes in Treg Kinetics after Liver Transplantation

The early posttransplant period is marked by significant immunological changes, with Tregs playing a crucial role in shaping long-term graft outcomes. Following LT, Treg populations undergo considerable fluctuations as the immune system responds to ischemia-reperfusion injury, inflammatory signals, and introduction of the allograft [46]. Initially, the number of Tregs in peripheral blood may decline as these cells are recruited to the graft site, where they exert suppressive effects on effector T cells, macrophages, and other immune cells involved in the inflammatory response [47]. This early recruitment of Tregs is essential for establishing a localized immunosuppressive environment that protects the graft from immune-mediated damage and reduces the risk of acute rejection.

Over time, Treg populations stabilize in both the peripheral blood and the graft itself. In patients who develop operational tolerance, which is defined as long-term graft survival without the need for chronic immunosuppressive therapy, Tregs are often found in higher quantities both in the circulation and within the graft compared to patients who experience chronic rejection or require ongoing immunosuppression [48]. These observations highlight the critical role of Tregs not only in preventing acute rejection, but in promoting long-term graft survival and immune tolerance [49]. Furthermore, the dynamics of Treg populations can offer valuable insights into a patient’s risk of rejection, with lower Treg numbers being associated with a greater likelihood of acute rejection episodes [50].

The reconstitution of Treg populations following transplantation is influenced by the type of immunosuppressive therapy administered. CNIs, such as tacrolimus, have been shown to reduce circulating Treg levels, likely due to their inhibitory effects on the production of IL-2, a cytokine essential for Treg survival and function [51]. In contrast, mTOR inhibitors, such as sirolimus and everolimus, support the survival of Tregs and maintain their suppressive function [52]. This differential effect on Tregs has important implications for designing immunosuppressive protocols aimed at promoting tolerance by preserving Treg populations while effectively controlling the risk of rejection.

Correlation Between Treg Frequency and Incidence of Acute Rejection in Liver Transplantation

The relationship between Treg frequency and the incidence of acute rejection in LT has been studied extensively. Numerous studies have consistently shown that lower Treg frequencies in the early posttransplant period are associated with a heightened risk of acute rejection, whereas higher Treg frequencies correlate with improved graft survival and the development of operational tolerance [53]. This correlation suggests that Treg levels could serve as a valuable biomarker for predicting transplant outcomes and personalizing immunosuppressive therapies to meet each patient’s specific needs.

A previous study monitored Treg frequencies in LT recipients during the first month posttransplantation. The researchers observed that patients who experienced acute rejection had significantly lower Treg counts than those who did not develop rejection [54]. Another study demonstrated that patients with higher Treg levels were more likely to achieve operational tolerance, underscoring the potential of Tregs as a predictive biomarker of long-term transplant outcomes [55]. This association between Treg levels and rejection risk highlights the therapeutic potential of targeting Tregs to reduce rejection incidence and promote tolerance, ultimately reducing the need for life-long immunosuppression.

Recent advances in understanding Treg biology have promoted the exploration of various strategies aimed at enhancing Treg function or expanding Treg populations to prevent rejection and promote tolerance. These strategies include cytokine therapies, such as low-dose IL-2, Treg expansion protocols, and adoptive Treg transfer. Early results from clinical trials investigating these approaches have shown promise in reducing rejection rates and promoting long-term graft survival [56,57]. However, further research is needed to optimize these therapies and establish their long-term efficacy and safety in LT recipients.

Calcineurin Inhibitors: Tacrolimus and Cyclosporine

CNIs, such as tacrolimus and cyclosporine, have long been the cornerstone of immunosuppressive therapy in LT. These agents work by inhibiting the calcineurin pathway, which is essential for the activation of nuclear factor of activated T cells (NFAT), a key transcription factor involved in the production of IL-2 and other cytokines that drive T cell activation and proliferation [58]. By blocking NFAT activation, CNIs suppress the immune response, effectively preventing acute rejection of the transplanted liver [59].

Though effective in reducing rejection rates, CNIs have been shown to exert negative effects on Tregs. Similar to conventional effector T cells, Tregs depend on IL-2 for their survival and function. Consequently, the inhibition of IL-2 production by CNIs can lead to a reduction in Treg levels and impair their suppressive function [60]. This reduction in Tregs may contribute to the increased risk of chronic rejection and other long-term complications observed in patients on CNI-based regimens [61]. These findings have prompted efforts to develop alternative immunosuppressive strategies that preserve Treg function while still providing adequate immunosuppression to prevent rejection.

A promising approach involves the use of low-dose CNIs in combination with other immunosuppressive agents, such as mTOR inhibitors or mycophenolate mofetil (MMF). These combination therapies aim to reduce the overall immunosuppressive burden while preserving Treg populations and promoting immune tolerance [62]. Clinical trials have shown that patients receiving low-dose CNIs in combination with mTOR inhibitors have higher levels of circulating Tregs and more favorable long-term graft outcomes than those on CNI monotherapy [63].

Our group recently examined the effects of tacrolimus on Tregs in LT [64]. The findings suggested that tacrolimus-based immunosuppression significantly reduces Treg counts, particularly in patients who experience acute rejection (Fig. 2). Notably, the study also demonstrated an inverse correlation between serum tacrolimus levels and Treg levels in patients with acute rejection, suggesting that these patients may be more susceptible to tacrolimus-induced apoptosis of Tregs. These findings underscore the importance of carefully selecting immunosuppressive regimens that effectively prevent rejection while preserving Treg function.

Figure 2. Effects of immunosuppressive regimens on Treg dynamics post-LT. This figure illustrates the impact of various immunosuppressive agents on Tregs following LT. Glucocorticoids initially increase Treg levels at low doses but decrease them at high doses. High doses of tacrolimus reduce Tregs, whereas mycophenolate mofetil has a neutral effect on Tregs. Basiliximab, administered on days 0 and 4, has a transient Treg depletion effect. Everolimus, which may be introduced around day 30, increases Treg levels. Early reduction of Tregs by day 7 is associated with a higher risk of graft rejection, as indicated by flow cytometry data for FOXP3⁺CD25⁺ Tregs. Treg, regulatory T cell; IV, intravenous; PO, oral; LT, liver transplantation.

Mammalian Target of Rapamycin Inhibitors: Sirolimus and Everolimus

The mTOR inhibitors, including sirolimus and everolimus, have emerged as valuable alternatives to CNIs in LT due to their unique ability to selectively inhibit the proliferation of effector T cells while preserving Treg populations. mTOR inhibitors block the mTOR pathway, which regulates cell growth, metabolism, and proliferation [65]. Unlike CNIs, which broadly suppress immune responses, mTOR inhibitors have selective actions, effectively controlling the immune response without significantly impairing Treg function [66]. This selective immunosuppressive effect makes mTOR inhibitors attractive candidates for promoting tolerance in LT [67].

Clinical studies have shown that patients treated with mTOR inhibitors, such as sirolimus or everolimus, maintain higher levels of circulating Tregs than those treated with CNIs [68]. This preservation of Tregs is particularly critical in LT, as Tregs play an essential role in maintaining immune tolerance and preventing rejection. In addition to their favorable impact on Tregs, mTOR inhibitors have been associated with lower rates of chronic rejection and improved long-term graft survival, supporting their use in tolerance-promoting immunosuppressive protocols [69].

The dual benefits of mTOR inhibitors (i.e., preservation of Tregs and reduced recurrence of HCC) make them particularly advantageous for LT recipients with a history of HCC. mTOR inhibitors have demonstrated antitumor properties, inhibiting cancer cell proliferation and reducing the risk of posttransplant tumor recurrence [70]. This antitumor activity is especially valuable for patients at higher risk of HCC recurrence, allowing for effective immunosuppression while simultaneously addressing oncological concerns [71,72].

Despite these advantages of mTOR inhibitors, several challenges remain. Common adverse effects include hyperlipidemia, delayed wound healing, and proteinuria, which can limit their use in certain patient populations [73]. In addition, optimizing the dose of mTOR inhibitors, especially in combination with CNIs or other immunosuppressive agents, is an active area of research aimed at balancing side effect management with immunosuppressive efficacy. As research continues to refine the use of mTOR inhibitors in LT, these agents are likely to play an increasingly prominent role in immunosuppressive regimens.

Corticosteroids

Corticosteroids, such as prednisone and methylprednisolone, have long been a cornerstone of immunosuppressive therapy in solid organ transplantation, including LT. These agents exert their immunosuppressive effects by inhibiting the production of proinflammatory cytokines, reducing APC activation, and suppressing effector T cell activity [74]. Corticosteroids are typically administered in the early posttransplant period to prevent acute rejection and control inflammation [75].

Corticosteroids have complex and dose-dependent effects on Tregs. At low doses, corticosteroids have been shown to promote Treg expansion and function, likely by promoting IL-2 signaling and suppressing proinflammatory cytokines that inhibit Treg activity [76]. This effect makes corticosteroids a valuable tool for fostering immune tolerance in LT, especially in the early posttransplant period when the risk of acute rejection is highest. However, high-dose corticosteroid therapy can have detrimental effects on Tregs by inducing apoptosis and disrupting the balance between effector T cells and Tregs [77]. Long-term corticosteroid use is also associated with various adverse effects, including hyperglycemia, hypertension, osteoporosis, and an increased risk of infection [78]. Therefore, many transplant centers have adopted corticosteroid-sparing protocols that aim to minimize or discontinue corticosteroid use as soon as possible posttransplant, particularly in patients with stable graft function who do not require ongoing immunosuppression [79].

Corticosteroid-sparing regimens often combine low-dose corticosteroids with other immunosuppressive agents, such as CNIs, mTOR inhibitors, or MMF. These combination therapies enable effective immunosuppression while mitigating the side effects associated with prolonged corticosteroid use [80]. The development of corticosteroid-sparing protocols represents important progress in the management of LT recipients, offering a more personalized approach to immunosuppression that considers each patient’s specific needs and risks.

Anti-CD25 Antibodies: Basiliximab and Daclizumab

Anti-CD25 antibodies, such as basiliximab and daclizumab, have been widely used as part of induction therapy in LT. These agents target the IL-2 receptor alpha chain (CD25) expressed on T cells, including Tregs. By blocking the IL-2 receptor, anti-CD25 antibodies inhibit the activation and proliferation of T cells, thereby reducing the risk of acute rejection [81]. This approach is especially beneficial for high-risk patients who require more aggressive immunosuppression to prevent early rejection episodes [82].

However, as Tregs also express CD25, anti-CD25 antibodies may inadvertently deplete Treg populations, particularly when combined with CNIs, which further suppress IL-2 production [83]. This potential for Treg depletion raises concerns about the long-term impact of anti-CD25 therapy on immune tolerance. Although these agents effectively prevent early rejection, their use may hinder the immune system’s ability to achieve long-term tolerance, potentially increasing the risk of chronic rejection or other complications.

Despite these concerns, anti-CD25 antibodies remain an essential component of immunosuppressive regimens, particularly in high-risk patients or those receiving induction therapy [84]. Further research is needed to determine the optimal use of these agents in combination with other immunosuppressive drugs to preserve Treg function while effectively preventing rejection [85]. In addition, newer therapies targeting other immune system components, such as costimulatory blockade or immune checkpoint inhibitors, are being investigated as potential alternatives to anti-CD25 antibodies, aiming to increase immune tolerance while minimizing the risk of rejection [86].

Mycophenolate Mofetil

Mycophenolic acid, administered as either MMF or mycophenolate sodium, is a widely used antiproliferative agent in transplantation. It functions by inhibiting inosine monophosphate dehydrogenase, a key enzyme in the de novo synthesis of purine nucleotides, which is critical for lymphocyte function. However, inconsistent findings have been reported regarding MMF's impact on Tregs in vitro. Some studies suggest that MMF facilitates the differentiation of Tregs from conventional T cells [87,88], while others indicate that MMF exhibits dose-dependent negative effects on Tregs, impairing their viability and proliferative capacity [89]. The specific effects of MMF, as well as azathioprine, on Tregs in transplant recipients remain inconclusive, but MMF might have a neutral effect.

Emerging Immunosuppressive Agents: Janus Kinase Inhibitors

Janus kinase (JAK) inhibitors represent a novel class of immunosuppressive agents that have shown considerable promise in transplantation due to their ability to modulate immune responses while preserving Treg function. JAK inhibitors target the JAK-STAT (signal transducer and activator of transcription) signaling pathway, which plays a key role in activating several proinflammatory cytokines, including IL-6 and interferon gamma [90]. By inhibiting this pathway, JAK inhibitors reduce the activation of effector T cells and promote a more tolerogenic immune environment, thereby decreasing the likelihood of graft rejection [91].

Recent studies have shown that JAK inhibitors can increase the number of circulating Tregs in transplant recipients, potentially improving long-term graft outcomes by promoting immune tolerance [92]. In addition to their immunosuppressive effects, JAK inhibitors have demonstrated efficacy in reducing the incidence of graft-versus-host disease (GVHD) in patients undergoing hematopoietic stem cell transplantation (HSCT), further highlighting their potential in solid organ transplantation [93].

Although JAK inhibitors have not yet been tested clinically in LT recipients, their capacity to preserve Treg function while preventing rejection makes them an attractive option for future immunosuppressive protocols. Ongoing clinical trials will help establish the safety and efficacy of JAK inhibitors in solid organ transplant recipients and their potential role in promoting long-term immune tolerance [94]. However, as with any emerging therapy, the long-term effects of JAK inhibitors on the immune system must be carefully considered, including their impact on Tregs and other immune cell populations.

Low-Dose Calcineurin Inhibitors with Mammalian Target of Rapamycin Inhibitors

The preservation of Tregs in LT may be a critical component of evolving immunosuppressive strategies aimed at promoting long-term immune tolerance. Given the essential role of Tregs in suppressing alloreactive immune responses, recent therapeutic approaches have focused on maintaining or improving Treg function while achieving adequate immunosuppression to prevent rejection. One promising strategy involves combining low-dose CNIs with mTOR inhibitors, such as sirolimus or everolimus. This combination can help preserve Treg populations and sustain their suppressive function [69], potentially reducing rejection rates and minimizing the adverse effects commonly associated with high-dose CNIs, such as nephrotoxicity and neurotoxicity [95]. Furthermore, the reduced risk of HCC recurrence makes this combination therapy particularly advantageous for patients with a history of HCC [71]. The optimal dosing of CNIs and mTOR inhibitors in combination therapy is still under investigation, as mTOR inhibitors are associated with side effects, including hyperlipidemia, delayed wound healing, and proteinuria, which can limit their use in certain patient populations [73]. Additional research is required to determine the best strategies for balancing efficacy and safety.

Adoptive Treg Cell Therapy

Treg cell therapy has emerged as one of the most promising developments in the field of transplantation. This approach involves isolating, expanding ex vivo, and reinfusing autologous or allogeneic Tregs into transplant recipients, with the goal of suppressing alloreactive immune responses and promoting long-term graft survival. Building on the success of Treg cell therapy in treating autoimmune diseases and preventing GVHD in HSCT, there is increasing interest in applying this approach to solid organ transplantation, particularly LT [96].

The concept of Treg cell therapy is based on the ability of Tregs to specifically suppress immune responses against the transplanted organ without broadly suppressing the entire immune system. This targeted suppression offers the potential to reduce, or even eliminate, the need for chronic immunosuppressive therapy, especially in patients who achieve operational tolerance. Several early-phase clinical trials have demonstrated that Treg cell therapy is both safe and effective, and that it increases circulating Treg levels, improves immune regulation, and reduces rejection risk [57].

One major challenge in implementing Treg cell therapy in LT is the difficulty of expanding sufficient numbers of functional Tregs for therapeutic use. Tregs constitute only a small fraction of the total T cell population in peripheral blood. Their expansion in vitro requires specialized culture conditions, including growth factors essential for Treg survival and proliferation, such as IL-2 [97]. Advances in cell culture methods, such as optimized growth protocols, have made it increasingly feasible to generate large quantities of Tregs for therapeutic use [98].

Another critical challenge is ensuring the stability and suppressive function of Tregs after infusion into patients, as demonstrated in the recent ARTEMIS clinical trial [99]. Tregs must maintain their regulatory phenotype and resist conversion into proinflammatory effector T cells, a phenomenon known as Treg instability. Stability is especially important in the inflammatory environment of the transplanted liver, where Tregs may be exposed to proinflammatory cytokines that could impair their regulatory function [100]. Strategies to promote Treg stability, such as genetic modification or the use of pharmacological agents that support Treg function, are currently under investigation in preclinical and clinical studies [101]. Another area of focus is the identification of biomarkers to determine which patients are most likely to benefit from Treg cell therapy and to optimize the timing of Treg infusion in relation to the transplant [102]. The successful implementation of Treg cell therapy in LT could not only reduce the need for life-long immunosuppression, but also pave the way for its application in other solid organ transplants and autoimmune diseases.

Despite these challenges, the potential benefits of Treg cell therapy are substantial. This approach offers a targeted, personalized treatment that promotes immune tolerance and minimizes the need for chronic immunosuppression. As research progresses, Treg cell therapy could transform the LT field, providing a new paradigm for achieving long-term graft survival with minimal immunosuppression.

Expanding Tregs Through Low-Dose Interleukin-2 Therapy

Low-dose IL-2 therapy is one of the most promising strategies for selectively expanding Tregs in LT. IL-2 is a cytokine that plays a crucial role in the survival, proliferation, and function of Tregs. High doses of IL-2 can stimulate the proliferation of effector T cells, whereas low doses have been shown to selectively expand Tregs without activating effector T cells, providing a novel therapeutic approach for promoting immune tolerance and reducing the need for long-term immunosuppression [103]. In the context of LT, low-dose IL-2 therapy could be particularly beneficial for patients at high risk of rejection or those transitioning to reduced immunosuppressive regimens.

The selective expansion of Tregs with low-dose IL-2 therapy is thought to be due to the differential expression of CD25 on Tregs compared to effector T cells. Tregs express high levels of CD25, enabling them to efficiently capture and respond to low doses of IL-2. In contrast, effector T cells express lower levels of CD25 and are less responsive to low-dose IL-2 therapy. This selective Treg expansion offers a unique opportunity to modulate the immune response in favor of tolerance without broadly suppressing the entire immune system [104]. A recent clinical trial demonstrated that low-dose IL-2 therapy successfully expanded circulating Tregs, but this expansion did not result in liver trafficking of these cells or the induction of liver allograft tolerance [105], highlighting the need to complement this strategy. Combining IL-2 targeting strategies, such as IL-2 receptor engineering, with adoptive Treg cell therapy may represent a promising approach for enhancing therapeutic efficacy.

Treg-Based Biomarkers for Predicting Transplant Outcomes

The identification of reliable biomarkers to predict transplant outcomes and assess the need for adjustments in immunosuppression has been a major focus of research in LT, and Tregs have emerged as one of the most promising candidates. Early studies showed that higher levels of circulating Tregs after transplantation are associated with better graft outcomes and a reduced risk of both acute and chronic rejection [106]. A study conducted by our group highlighted the potential of Treg monitoring in the early posttransplant period as a prognostic tool in LT [64]. Specifically, peripheral blood Treg frequency 7 days posttransplant could serve as an independent predictor of acute rejection, with an optimal cutoff of 4.7%. This Treg reduction and its association with acute rejection might be attributed to the reduction of the activated Treg subset, which highly expresses FOXP3 but lacks CD45RA expression. This population was also reduced along with the total Treg population, in contrast to other Treg subsets. Patients with Treg levels below this threshold had a significantly higher risk of rejection, emphasizing the utility of early Treg monitoring in identifying high-risk patients who may benefit from tailored immunosuppressive strategies. This reduced Treg frequency also correlated with early serum tacrolimus levels, suggesting a potential link between immunosuppressive dosing and Treg dynamics. The ability to predict rejection risk based on Treg levels could enable clinicians to personalize immunosuppression regimens, potentially reducing rejection incidence while minimizing the side effects of over-immunosuppression.

Further research is needed to standardize the use of Tregs as a biomarker given the variability in Treg levels across different patient populations and the influence of factors, such as immunosuppressive regimens, infection, and individual patient characteristics. Technological advances, such as flow cytometry, mass cytometry, and single-cell RNA sequencing, have made it increasingly feasible to accurately quantify Treg populations and assess their functional capacity in the context of LT.

The heterogeneity of Tregs has led researchers to explore the potential of Treg subset analysis to obtain more precise insights into a patient’s immune status. Tregs are not a homogeneous population; they can be classified into different subpopulations based on activation state, cytokine profile, tissue-homing properties, and expression of suppressive molecules, such as CTLA-4, PD-1, and inducible T cell costimulator (ICOS) [107]. These subsets vary in their capacity to suppress immune responses, making it important to identify which subpopulations are most relevant to promoting tolerance in LT. For example, activated Tregs, characterized by high expression of CTLA-4 and PD-1, may be more effective at suppressing effector T cell responses than resting Tregs [108]. Identifying and monitoring these activated subsets could provide a more accurate reflection of the immune environment and help guide immunosuppressive therapy.

Tregs have fundamentally reshaped the field of LT by providing a pathway toward immune tolerance, thereby reducing the need for chronic, broad-spectrum immunosuppression. Although traditional immunosuppressive therapies effectively prevent acute rejection, their nonspecific mechanisms often lead to long-term complications, including an increased risk of infection, malignancy, and drug-induced toxicity. Emerging therapies that focus on preserving or enhancing Treg function, such as mTOR inhibitors, low-dose IL-2 therapy, and Treg-based cell therapies, are poised to shift the paradigm of transplantation management toward a more targeted and personalized approach.

Continued advances in Treg-based therapies are likely in LT, with an increasing emphasis on developing precision medicine strategies that integrate Treg monitoring with individualized immunosuppressive protocols. The use of Tregs as biomarkers for predicting transplant outcomes is promising for reducing the immunosuppressive burden in patients who achieve operational tolerance while still ensuring adequate protection against rejection. Advances in genomics, proteomics, and metabolomics, along with the integration of artificial intelligence and machine learning, will undoubtedly play a pivotal role in refining these approaches and unlocking new avenues for personalized care.

Furthermore, novel gene-editing technologies, such as CRISPR-Cas9, have the potential to create “super-Tregs” with enhanced stability and suppressive function, potentially paving the way for new strategies for the prevention of graft rejection [109]. These engineered Tregs could be designed to resist proinflammatory signals, maintain their regulatory phenotype, and offer more durable immune tolerance. As gene-editing techniques evolve, they may become a central component of next-generation immunosuppressive therapies, transforming the future of solid organ transplantation. Chimeric antigen receptor (CAR) Tregs offer a targeted approach to preventing graft rejection by specifically recognizing donor antigens, such as mismatched human leukocyte antigen molecules [110]. Compared to polyclonal Tregs, CAR Tregs exhibit superior suppressive capacity, enhanced migration, and improved retention within grafts, potentially reducing the reliance on broad-spectrum immunosuppression. Advances in gene-editing technologies may further improve the stability and functionality of CAR Tregs, paving the way for their application in achieving long-term immune tolerance in LT.

In conclusion, Tregs represent a powerful tool for promoting immune tolerance in LT. By harnessing the suppressive capabilities of Tregs and incorporating them into advanced immunosuppressive protocols, the field of LT can progress toward achieving long-term graft survival with minimal complications. Continued investment in Treg biology, biomarker development, and innovative therapeutic strategies will be essential to advancing the field and improving outcomes for LT recipients.

Conflict of Interest

Su-Hyung Park is an editorial board member of the journal but was not involved in the peer reviewer selection, evaluation, or decision process of this article. No other potential conflict of interest relevant to this article was reported.

Funding/Support

This study was supported by a Research Fund of Seoul St. Mary’s Hospital, The Catholic University of Korea (to JWH) and by a grant from the National Research Foundation of Korea (NRF) funded by the Korean government (MSIT) (NRF-2022R1A2C3007292 to SHP).

Author Contributions

All the work was done by Ji Won Han and Su-Hyung Park. All authors read and approved the final manuscript.