Transplantation is a life-saving procedure that involves replacing diseased or damaged organs or tissues with healthy ones from a donor. The primary challenge in this process is the recipient's immune system, which recognizes the transplanted tissue as foreign and initiates an immune response against it. To counter this, immunosuppressive therapies are essential to prevent rejection and ensure the survival of the graft. Over the years, various classes of immunosuppressive agents have been developed to minimize graft rejection in recipients, with calcineurin inhibitors (CNIs) such as cyclosporine A (CsA) and tacrolimus being fundamental to treatment protocols [1–3]. CsA, which effectively suppresses the immune response, was approved by the U.S. Food and Drug Administration (FDA) for renal transplantation in 1983. Although these agents are effective in suppressing immune responses, they lack specificity and broadly target the immune system [4]. This lack of specificity can result in significant side effects, including nephrotoxicity, neurotoxicity, and an increased risk of infections and malignancies with prolonged use.

Emerging research has underscored the need for more targeted approaches, particularly those that specifically modulate T cell-mediated immune responses [5,6]. A promising strategy is targeting costimulatory pathways. The development of cytotoxic T lymphocyte antigen-4 (CTLA-4) immunoglobulin (Ig; abatacept) represented a significant breakthrough, as abatacept selectively binds to CD80/86 on antigen-presenting cells (APCs), thereby blocking the essential signals required for T cell activation [7–10]. Nonetheless, the initial form of CTLA-4 Ig exhibited a relatively low binding affinity for CD80/86, which prompted the development of a mutant form known as belatacept [11,12]. Belatacept showed an enhanced affinity for CD80/86 and was approved by the FDA for use in kidney transplantation in 2011. While belatacept marks a significant improvement in transplantation immunotherapy, concerns about its effects on regulatory T (Treg) cells persist. The reduction of Treg cells by belatacept, as reported in various preclinical and clinical studies, raises concerns that its long-term use might compromise graft tolerance [13–15]. Moreover, some studies have indicated that belatacept-resistant T cells, particularly CD28-negative memory T cells, can lead to acute rejection [16]. These findings reflect the complexity of immune regulation in transplantation and underscore the need for therapies that both preserve Treg function and effectively prevent graft rejection.

This review explores the evolution of immunosuppressive therapies in transplantation, with a specific focus on T cell-mediated immune suppression, the critical role of Tregs in maintaining graft tolerance, and the development of more selective therapeutic agents like belatacept. Additionally, this review discusses future strategies to optimize immunosuppression, including potential combination therapies that target both costimulatory pathways and enhance Treg function to improve transplantation outcomes.

CTLA-4 is an essential immune checkpoint molecule that regulates T cell costimulation by competing with CD28 for binding to CD80/86 on APCs [17]. CTLA-4 binds to CD80/86 with a higher affinity than CD28, thereby inhibiting T cell activation by blocking costimulatory signaling through CD28 [18]. This characteristic has positioned CTLA-4 as a key target for developing immunosuppressive therapies to treat diseases characterized by excessive T cell activation, such as autoimmune diseases and transplantation. A notable breakthrough in this field was the development of CTLA-4 Ig (abatacept), a fusion protein combining the extracellular ligand-binding domain of CTLA-4 with the Fc region of human IgG. This design increases molecular stability and prolongs its half-life in circulation [19]. Abatacept effectively moderates the immune system by inhibiting the CD28-mediated signaling pathway in T cells, thereby reducing excessive T cell activity and addressing various immune-mediated diseases. It has been investigated for treating a range of conditions with dysregulated immune responses, including autoimmune, inflammatory, and allergic diseases. In 2005, abatacept received FDA approval for treating rheumatoid arthritis and was later approved for psoriatic arthritis in 2017 [20,21]. More recently, in 2021, it was approved in combination with a CNI and methotrexate for preventing acute graft versus host disease [22]. Despite its success in treating some inflammatory diseases, clinical trials targeting other autoimmune diseases have demonstrated limited effectiveness. For example, in relapse-remitting multiple sclerosis (MS) patients, no significant differences in disease activity were observed between the abatacept and placebo groups (NCT01116427) [23]. Additionally, abatacept proved ineffective in treating moderate-to-severe Crohn disease and ulcerative colitis (NCT00406653) [24]. These findings highlight the limitations of abatacept in treating a broad range of autoimmune diseases.

Abatacept has not been approved for use in transplantation. In a study involving allogeneic pancreatic islet transplantation in nonhuman primates, three out of five monkeys treated with abatacept did not show prolonged graft survival [10]. Furthermore, although abatacept binds with similar avidity to both CD80 and CD86, it was found to be less effective at inhibiting CD28 activation in cocultures of CD28-expressing CHO cells with CD86-expressing CHO cells than in those expressing CD80. Additionally, abatacept dissociates from CD86 more rapidly than from CD80 [11]. Collectively, these findings highlight the need to compensate for the relatively weak affinity of CTLA-4 Ig for CD86. To improve the efficacy of abatacept in transplantation, high-throughput screening of 2,300 random mutations in the ligand-binding domain of CTLA-4 identified two specific amino acid substitutions: leucine at position 104 to glutamate and alanine at position 29 to tyrosine (LEA29Y). These mutations significantly improved the binding affinity for both CD80 and CD86, with belatacept exhibiting approximately twice the binding strength to CD80 and nearly four times the affinity for CD86 compared to abatacept [12]. Moreover, belatacept showed a greater suppressive effect on T cell proliferation in vitro and higher therapeutic efficacy in renal transplantation models in vivo than abatacept [11]. Thus, the enhanced binding affinity of belatacept for CD80/86 leads to improved immune regulation, making it particularly promising in transplantation settings where precise control over T cell activation is crucial for preventing graft rejection while minimizing broad immunosuppression. Belatacept has demonstrated superior clinical outcomes in organ transplantation. As a result, the FDA approved belatacept for use in kidney transplantation in 2011, providing a more targeted immunosuppressive option that potentially improves graft survival and reduces toxicity compared to traditional CNIs (Fig. 1) [25].

Figure 1. Historical overview of belatacept development. A historical overview of the understanding of belatacept development from 1991 to 2024 is provided. CTLA-4, cytotoxic T lymphocyte antigen-4; Ig, immunoglobulin; BENEFIT, Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression. Diagram created with BioRender.

Preventing graft rejection mediated by immune responses remains a significant challenge, necessitating the use of immunosuppressive therapies. Several key immunosuppressive drugs have been developed to address this issue. Tacrolimus (FK506), introduced in 1997 for liver transplantation, has become a cornerstone treatment alongside rapamycin (approved in 1999 for transplantation), and mycophenolate mofetil, which was introduced in 1995 for cardiac, renal, and hepatic transplantation [26,27]. The immunosuppressive agents—particularly CNIs, such as tacrolimus and cyclosporine—have significant side effects, most notably nephrotoxicity and neurotoxicity [28]. These side effects have prompted the search for alternative therapies, such as belatacept, a costimulation blocker. Belatacept has shown promise, especially in patients with CNI-induced nephrotoxicity, as it significantly reduces the risk of nephrotoxicity [29,30]. Furthermore, belatacept has been associated with a lower incidence of posttransplant diabetes mellitus in renal transplant patients compared to tacrolimus [31,32]. In the phase III Belatacept Evaluation of Nephroprotection and Efficacy as First-line Immunosuppression (BENEFIT) study, kidney transplant patients treated with belatacept showed a mean calculated glomerular filtration rate that was approximately 21 mL/min/1.73m² higher than those treated with cyclosporine 3 years after treatment (Table 1) [33]. Furthermore, the 7-year long-term BENEFIT study reported that belatacept-treated patients achieved comparable graft survival rates to those on cyclosporine [34]. However, belatacept demonstrated superior renal function at 12 months after treatment, as reflected by the composite renal impairment endpoint, with rates of 54%–55% in the belatacept group versus 78% in the cyclosporine group (NCT00256750). This study further subdivided belatacept-treated patients into a more-intensive (MI) regimen and a less-intensive (LI) regimen. At 84 months posttreatment, both belatacept regimens exhibited improved estimated glomerular filtration rate outcomes compared to cyclosporine, with mean values of 72.1 mL/min/1.73m² for the MI group, 70.4 mL/min/1.73m² for the LI group, and 44.9 mL/min/1.73m² for the cyclosporine group. These findings demonstrate that belatacept induces significantly less nephrotoxicity than CNIs.

Table 1. Clinical trials using belatacept in transplantation

NCT number	Title	Status	Conditions	Interventions	Outcomes	Phase
Overview of clinical trials on belatacept
NCT02103855	Switch from calcineurin inhibitor to belatacept in pancreas transplant recipients	Completed (2016)	Nephrotoxicity	Belatacept	• Higher rejection incidence (belatacept, 8.4%; CNI, 3.6%) • Prevent worsening renal and pancreatic graft function	Phase IV
NCT00402168	A study of BMS-224818 (belatacept) in patients who have undergone a kidney transplant and are currently on stable cyclosporine or tacrolimus regimen with or without corticosteroids	Completed (2013)	Kidney transplantation	Belatacept Cyclosporine A Tacrolimus	• Comparable acute rejection rate • More nonserious mucocutaneous fungal infection in belatacept group • Higher cGFR	Phase II
NCT00578448	Belatacept pharmacokinetic trial in renal transplantation	Completed (2012)	Kidney transplantation	Belatacept	• 4/12 acute rejection	Phase II
NCT02327403	Belatacept conversion in proteinuric kidney transplant recipients	Completed (2020)	Proteinuria	Belatacept	• Reduced proteinuria • More stable allograft function	Phase II
NCT01820572	A study in maintenance kidney transplant recipients following conversion to Nulojix (belatacept)-based	Completed (2019)	Kidney transplantation	Belatacept Tacrolimus Cyclosporine	• Similar rate of death/graft loss • Improved renal function with lower incidence of dnDSA	Phase III
NCT00455013	A phase II study of belatacept (BMS-224818) with a steroid-free regimen in subjects undergoing kidney transplantation	Completed (2012)	Disorders related to kidney transplantation	Thymoglobulin Belatacept Sirolimus Tacrolimus MMF	• Improved renal function (cGFR) that persisted for 12 months • Lower rate of rejection rate in belatacept	Phase II
NCT03388008	Belatacept pilot study in lung transplantation immunosuppression in lung transplantation	Completed (2022)	Lung transplant rejection, antibody-mediated rejection	Belatacept Tacrolimus ATG MMF Methylprednisolone Prednisone	• No differences in the incidence of DSA, acute cellular rejection and antibody-mediated rejection	Phase II
NCT00035555	Study comparing the safety and efficacy of belatacept with that of cyclosporine in patients with a transplanted kidney	Completed (2012)	Graft rejection, kidney transplantation	Belatacept Cyclosporine MMF Corticosteroids	• No differences in acute rejection ratio compared with CNI	Phase II
NCT00114777	Study of belatacept in subjects who are undergoing a renal transplant	Completed (2014)	Kidney transplantation	Cyclosporin A Belatacept: less-intensive regimen Belatacept: more-intensive regimen	• Higher cGFR in belatacept • Similar rate of biopsy-proven acute rejection • Comparable patients and graft survival	Phase III
NCT00256750	Belatacept evaluation of nephroprotection and efficacy as first-line immunosuppression (BENEFIT)	Completed (2015)	Kidney transplantation	Cyclosporine Belatacept less-intensive Belatacept more-intensive	• Improved eGFR index than cyclosporine • Reduced the risk of death or graft loss in belatacept	Phase III
NCT00565773	Belatacept post depletional repopulation to facilitate tolerance	Completed (2017)	Kidney transplantation	Belatacept Sirolimus Alemtuzumab	• No costimulation-resistant rejection (costimulation blockade resistant rejection)	Phase II
Current ongoing clinical trials utilizing belatacept
NCT04786067	Use of DNA testing to help transition kidney transplant recipients to belatacept-only immunosuppression	Recruiting	Kidney transplant immunosuppression	Belatacept	-	Phase IV
NCT05345717	Novel desensitization kidney transplantation	Recruiting	Kidney transplantation, end stage renal disease	Belatacept	-	Phase I Phase II
NCT04180085	Pilot Study: interest of belatacept as a non-nephrotoxic immunosuppressive treatment in cardiac transplant patients at risk of chronic renal failure (BELACOEUR)	Recruiting	Heart transplant failure	Belatacept	-	Phase II
NCT02310867	Immunomodulation to optimize vascularized composite allograft integration for limb loss therapy	Recruiting	Immunosuppression	Belatacept	-	Phase II
NCT04477629	Belatacept in de novo heart transplantation	Recruiting	Heart transplantation	Belatacept Tacrolimus MMF Corticosteroid	-	Phase II
NCT05017545	Carfilzomib and belatacept for desensitization	Recruiting	Highly sensitized prospective kidney transplant recipients	Carfilzomib Belatacept Bone marrow aspiration	-	Phase I Phase II
NCT04827979	Daratumumab and belatacept for desensitization	Recruiting	Highly sensitized prospective kidney transplant recipients	Daratumumab Belatacept Bone marrow aspiration	-	Phase I Phase II
NCT05562869	Belatacept as a replacement for CNIs 3 to 12 months post-transplantation in patients with early graft dysfunction	Recruiting	Chronic kidney failure	Belatacept	-	Phase III
NCT05669001	A study of TCD601 in de novo renal transplant recipients	Recruiting	Kidney transplantation	TCD601 Belatacept ATG Tacrolimus Methiopropamine Corticosteroids	-	Phase II
NCT04877288	A study to evaluate the benefits and risks of conversion of existing adolescent kidney transplant recipients aged 12 to <18 years to a belatacept-based immunosuppressive regimen as compared to continuation of a calcineurin inhibitor-based regimen, and their adherence to immunosuppressive medications	Recruiting	Renal allograft recipients	Belatacept Tacrolimus Cyclosporine A MMF Enteric-coated mycophenolate sodium Corticosteroids	-	Phase III
NCT06055608	Advancing transplantation outcomes in children	Recruiting	Kidney transplantation	Sirolimus Belatacept MMF Tacrolimus (group 1) ATG Tacrolimus (group 2)	-	Phase II
NCT05975450	Subcutaneous abatacept in renal transplant recipients	Recruiting	Kidney transplant recipients	Abatacept 125 mg/mL syringe	-	Phase I

CNI, calcineurin inhibitor; cGFR, calculated glomerular filtration rate; DSA, donor-specific antibody; MMF, mycophenolate mofetil; ATG; anti-thymocyte globulin; eGFR, estimated glomerular filtration rate.

However, while belatacept offers advantages such as improved renal function and reduced nephrotoxicity, it is also associated with a higher incidence of acute rejection and poorer allograft survival compared to CNIs. Specifically, acute rejection rates were reported as 24% in the MI belatacept group and 17% in the LI group, versus 10% in the cyclosporine group in a phase III study [34]. Similarly, a phase II study of kidney transplant patients treated with belatacept every 8 weeks for 10 years showed a significantly higher rate of biopsy-proven acute rejection compared to cyclosporine: 37.0% in the belatacept group, and 25.8% in the cyclosporine group [35]. These findings suggest that relying solely on a limited set of indicators, such as nephrotoxicity and kidney function, is insufficient to fully assess the clinical efficacy of belatacept, highlighting the need to identify additional indicators related to the rejection mechanism. Moreover, despite observed clinical improvements, there are still limitations, underscoring the need for the development of new therapeutic strategies such as combination therapy to address these challenges. One reason for this increased risk of acute rejection is believed to be the persistence of effector and memory T cells, particularly CD28-negative T cells, which are antigen-experienced and highly differentiated [36]. Clinical reports have shown that CD28-negative CD4 and CD8 T cells accumulate in rejected renal grafts, and these T cells, which have an effector/memory phenotype, produce high levels of granzyme B and interferon (IFN)-γ [37]. Moreover, it has been reported that renal transplant patients who experience acute graft rejection have a higher frequency of memory Th17 cells, which express high levels of CTLA-4 and interleukin (IL)-17 and are CD45RA-negative, compared to stable patients.

Belatacept is also associated with a risk of adverse effects related to immunosuppression, such as an increased rate of infections. Studies have shown that kidney transplant patients who switched from tacrolimus to belatacept experienced a higher infection rate than those who remained on tacrolimus (42% with belatacept vs. 29.9% with tacrolimus) [38]. In addition to the elevated infection risk, treatment with belatacept has been linked to reduced vaccine efficacy. For example, total serum IgG and rHA-specific IgG levels after trivalent inactivated vaccination were significantly lower in renal transplant patients treated with belatacept compared to those receiving tacrolimus [39]. Notably, organ transplant recipients on belatacept therapy exhibited a complete absence of humoral response, indicating a substantially diminished degree of immune protection [40]. Taken together, belatacept may not fully prevent the transplant rejection in some patients and still have some side effects.

Treg cells play a crucial role in preventing graft rejection and have therapeutic applications in allogeneic transplantation through various mechanisms. FOXP3, a key transcription factor in Treg cells, enables them to maintain their suppressive characteristics, including CD25, CTLA-4, IL-10, and transforming growth factor (TGF)-β. Treg cells express IL-2Rα (CD25) at high levels compared to conventional T cells, which depletes IL-2 from the environment, thereby suppressing the activation of conventional T cells [41]. A study involving 24 renal transplant patients demonstrated an increase in CD4⁺CD25^high Treg cells in the peripheral blood 5 days posttransplant, with this increase sustained for up to 24 months. Notably, Treg cells were more abundant in donor-specific hyporesponders than in nondonor-specific hyporesponders at 24 months posttransplantation, suggesting that Treg cells are essential for regulating alloreactive responses [42]. In a DBA/2 (H-2d) to C57BL/6 (H-2b) kidney allograft model, disruption of Foxp3 gene expression using diphtheria toxin led to allograft rejection, underscoring the critical role of FOXP3 in maintaining allograft tolerance [43]. Additionally, Foxp3 mRNA levels are elevated in tolerant kidneys, serving as a diagnostic marker for graft rejection [44–46]. In islet allograft models, Treg cells infiltrate transplanted tissues and lymph nodes through CCR2, CCR5, and CCR7-dependent mechanisms, exhibiting a suppressive, tolerogenic phenotype while secreting TGF-β and IL-10 [47]. Similarly, in the DBA/2 (H-2d) to C57BL/6 (H-2b) kidney allograft model, infiltrating Treg cells showed increased expression of IL-10 and TGF-β expression in the kidney, promoting long-term survival (up to 100 days) [43]. Blockade of IL-10 or TGF-β with a neutralizing antibody abrogated allograft rejection, suggesting that IL-10 and TGF-β secreting Treg cells contribute to maintaining tolerance in transplant settings [48,49]. In heart allografts from Act.2W-OVA+ BALB/c × C57BL/6 F1 donors to C57BL/6 recipients, donor-specific 2W:I-Ab Treg cells highly express CD73 [50]. CD73 and CD39 are ectonucleotidases that convert environmental ATP to ADP and AMP, which are further converted to adenosine [51]. Adenosine impairs T cell activation and metabolic activity. These findings suggest that Treg cells, with suppressive properties including FOXP3, CD25, IL-10, TGF-β, and CD73, infiltrate transplanted tissues, enhancing immune tolerance posttransplantation.

It is well established that alloreactive Treg cells are more effective in preventing graft rejection than polyclonal Treg cells. For instance, in anti-bm12 (ABM) T cell receptor (TCR) transgenic mice, where all CD4 T cells recognize major histocompatibility complex (MHC) class II I-Abm12, bm12 heart allografts were accepted due to the presence of ABM Treg cells, despite a high frequency of alloreactive T cells [52]. ABM Treg cells significantly suppressed the proliferation of ABM TCR transgenic CD4⁺CD25^– responder T cells compared to wild-type polyclonal Treg cells. Similarly, alloreactive human Treg cells, induced by CD40L-activated allogeneic B cells, demonstrated greater suppression of responder T cell activation compared to polyclonal human Treg cells, both in vitro and in humanized skin transplant mouse models [53]. These results demonstrate that alloreactive Treg cells serve as key regulators of T cell activation and are critical for preventing graft rejection.

CD28 costimulatory signaling is crucial for both the induction and maintenance of FOXP3. In a heart transplantation model involving an MHC class II mismatch, Treg populations in the spleen were found to be reduced in CD28 knockout and CD80/86 knockout mice compared to C57BL/6 wild-type mice [54]. This reduction in Treg cells was linked to increased acute allograft rejection in the knockout mice, suggesting that the absence of CD28 and CD80/86 signaling intensifies immune responses and hastens the rejection of transplanted organs [55]. Consistent with these observations, hCTLA-4 Ig not only diminished the population and function of splenic FOXP3+ CD4 Treg cells but also decreased the levels of Helios+ FOXP3+ thymic Treg cells. In the realm of autoimmune diseases, MS patients treated with abatacept exhibited a reduction in Treg populations within peripheral blood mononuclear cells after 28 weeks of treatment. Importantly, there was a notable decrease in CD45RO+ memory Treg cells, indicating that abatacept may particularly affect the memory Treg subset in MS patients during extended treatment periods [56]. The mechanisms underlying CD28 signaling in Treg cells are not completely understood; however, some studies suggest that CD28 signaling enhances the nuclear localization of RelA/NF-κB, which then binds to the FOXP3 promoter region and stimulates FOXP3 expression (Fig. 2) [57]. Additionally, the PI3K-AKT-mTOR pathway is reported to boost Foxp3 expression through chromatin remodeling shortly after CD28 stimulation [58]. These findings imply that belatacept may inhibit these downstream CD28 signaling pathways, thereby reducing Foxp3 expression essential for maintaining the suppressive properties of Treg cells. While belatacept presents several advantages over traditional CNIs, including reduced nephrotoxicity and a lower incidence of posttransplant diabetes mellitus, its associated higher rates of acute rejection, coupled with the reduction in Treg levels and the presence of uncontrolled effector/memory T cells, present significant challenges. Addressing these issues, particularly through strategies that preserve or enhance Treg function, is crucial for improving outcomes in organ transplantation.

Figure 2. CD28 signaling in FOXP3 expression and the effects of belatacept. CD28 costimulatory signaling is essential for the induction and maintenance of FOXP3 by activating the PI3K-AKT-mTOR and RelA/NFκB signaling pathways. RelA binds to the FOXP3 promoter region to induce FOXP3 expression, whereas the PI3K pathway regulates chromatin remodeling to enhance FOXP3 expression. FOXP3 is the key transcription factor in Treg cells, promoting the expression of suppressive functional markers such as CD25, CTLA-4, IL-10, TGF-β, CD39 and CD73, which collectively suppress alloreactive conventional T cells. However, belatacept blocks this CD28 signaling pathway by inhibiting CD28 binding to CD80/86 on APCs, thereby reducing FOXP3 expression. APC, antigen-presenting cell; MHC, major histocompatibility complex; Treg, regulatory T; TCR, T cell receptor; mTOR, mammalian target of rapamycin; CTLA-4, cytotoxic T lymphocyte antigen-4; IL, interleukin; TGF, transforming growth factor. Diagram created with BioRender.

Transplant tolerance mediated by Treg cells has been explored in various organs, including the kidney, heart, liver, skin, and islets. Each organ presents unique immunological challenges, such as variability in antigen presentation, immune privilege status, and inflammatory environments. For example, islet transplantation often involves autoimmune components, whereas kidney and heart transplants primarily encounter alloimmune responses. Despite these organ-specific nuances, therapeutic strategies aimed at enhancing Treg function—such as Treg cell transfer, endogenous induction, and advanced genetic engineering—are widely applicable across different organ transplantation models. Consequently, this review organizes Treg-based therapeutic strategies and synthesizes key findings from studies across various organs to provide comprehensive insights.

Alloreactive Regulatory T Cell Transfer Therapy

Based on the role of Treg cells in transplantation, several studies have investigated alloreactive Treg cell therapy in mouse models. In a preclinical study, alloreactive H2-Kb-specific Treg cells expanded in vitro were transferred into CBA mice (H2K) that had received skin grafts from CBK (H2K + Kb) mice [59]. These Treg cells migrated to the graft-draining lymph nodes and selectively inhibited the rejection of CBK skin grafts. In vitro-expanded allogenic Treg cells have demonstrated a greater suppressive capacity compared to freshly isolated polyclonal Treg cells. The prevention of graft rejection by alloreactive Treg cell transfer has also been confirmed in models of islet, heart, and bone marrow transplantation in mice [60–62]. To assess the clinical potential of Treg cell therapy, human donor-specific CD4⁺ CD25⁺CD127^low Treg cells were expanded ex vivo using donor B cells in the presence of IL-2, TGF-β, and sirolimus [63]. These Treg cells exhibited stronger suppressive function compared to polyclonal Treg cells and maintained demethylation of the CNS2 region of the Foxp3 gene, indicating sustained stability. Treg cell therapy has been tested in phase 1/2A trials in kidney transplantation as part of the ONE study [64]. In this study, ex vivo-expanded donor-antigen-reactive Treg cell therapy was found to be feasible and safe, with Treg-specific demethylation maintained at 60 weeks, suggesting stability of Treg cells and the potential for long-term regulation in the transplant environment [65]. A phase 2b clinical trial is currently ongoing to assess the transfer of autologous ex vivo-expanded Treg cells in kidney transplantation [66]. Although these advantages of Treg cell therapy exist, it still has some limitations, including a reduction in Treg cell numbers and functionality in inflammatory environments after transfer [67].

Chimeric Antigen Receptor Regulatory T Cells

To address these limitations by specifically targeting alloreactive Treg cells, chimeric antigen receptor (CAR) Treg cells have been developed. A CAR specific to HLA-A2, featuring a CD3z-CD28 chain in its cytoplasmic domain, demonstrated a stronger suppressive function than polyclonal Treg cells in vitro [68]. Moreover, these human CAR Treg cells more effectively regulated alloimmune responses in a human HLA-A2⁺ skin xenograft mouse model compared to polyclonal Treg cells. In another study, using a single MHC mismatch model where a heart from a B6.HLA-A2 donor was transplanted into a C57BL/6 recipient, HLA-A2-specific CAR Treg cells extended graft survival compared to polyclonal Treg cells, demonstrating the efficacy of alloantigen-specific CAR Treg therapy [69]. A phase I/IIa study of CAR Treg cell therapy in kidney transplantation is currently underway to assess their tolerability and safety (NCT05234190). However, in a multiple MHC mismatch model using hearts from BALB/c × B6.HLA-A2 F1 donors, CAR Treg cells extended survival by only 3 days, which was further prolonged to 100 days when combined with rapamycin. These findings highlight the limitations of CAR Treg therapy in multiple MHC mismatch settings and suggest the need for appropriate combination treatments tailored to the context [69].

Endogenous Induction of Regulatory T Cells

Another approach has been developed that involves either expanding or inducing Treg cells endogenously within the body. Low-dose IL-2 has been tested in various autoimmune diseases, including type 1 diabetes, systemic lupus erythematosus, and psoriatic arthritis. These studies have demonstrated an increase in Treg cell numbers and effective control of inflammation [70]. However, IL-2 also affects conventional T cells and NK cells, which limits its specificity to Treg cells. A recent study has developed mutated IL-2 conjugates with a human Fc domain that selectively binds to CD25 and increases stability [71]. This humanized IL-2 mutein selectively increased Treg cells in humanized mouse models and cynomolgus monkeys, promoting immune tolerance in a minor-mismatch (B6.mOVA to B6) skin transplantation model. Rapamycin (sirolimus) inhibits DNA synthesis and arrests the cell cycle by binding to mammalian target of rapamycin. In a syngeneic liver transplant model, rapamycin increased CD4⁺FOXP3⁺ Treg cells, whereas cyclosporine treatment reduced their numbers [72].

Consequently, some studies have explored tolerance induction by combining rapamycin with IL-2. In a model of MHC-mismatched hindlimb transplantation, local administration of microparticles releasing TGF-β1, IL-2, and rapamycin promoted Treg cell differentiation and reduced Th1 cells, leading to long-term prevention of graft rejection for approximately 300 days [73]. In a human renal subcapsular islet allograft model in humanized NSG mice, neither IL-2 nor rapamycin alone could prevent rejection. However, their combination increased survival rates by expanding Treg cells, suggesting potential for application in human transplant patients [74]. Additionally, a case study on low-dose IL-2 treatment in skin transplantation patients showed increased CD4⁺CD127^-CD25⁺ Treg cells for 48 weeks, with enhanced expression of suppressive molecules such as TIM3 and LAG3 [75].

The cytoplasmic domain of CTLA-4 can transduce inhibitory signals to T cells, even without ligand interaction, by recruiting SHP-2 and PP2A [76]. dNP2-ctCTLA-4 is a conjugate of the cytoplasmic domain of CTLA-4 and a dNP2, a cell-penetrating peptide [77–79]. This conjugate has been shown to regulate the immune response in a human skin graft model in humanized SCID/beige mice by controlling effector T cell infiltration and cytokine production in grafted tissues [80]. Furthermore, dNP2-ctCTLA-4 has been shown to induce Treg differentiation, highlighting its potential to promote transplant tolerance through Treg cells [81,82]. These findings suggest that while the cytoplas- mic domain of CTLA-4 regulates transplant models by modulating T cell signaling, the extracellular domain, as exemplified by CTLA-4 Ig, manages transplant models by inhibiting costimulatory signals.

Limitations of Regulatory T Therapy

Although several studies have highlighted the importance of maintaining immune tolerance with Treg cells, Treg cell therapy alone may not fully prevent rejection, necessitating the use of additional immunosuppressants [60,62,83]. For example, in an islet allograft model, the transfer of donor-specific Treg cells did not prevent rejection, resulting in survival rates similar to those of the group that did not receive Treg cells [62]. However, when 80% of donor-reactive T cells were eliminated through donor-specific transfusion and cyclophosphamide treatment, the transfer of donor-specific Treg cells significantly improved survival rates. Effector T cells heavily infiltrate graft tissues, yet transferred Treg cells, which depend on IL-2 for growth and survival, may compete for IL-2 with conventional effector T cells under certain conditions. Therefore, strategies to selectively increase endogenous Treg cells in vivo have been investigated. In a minor-mismatch model, mutated IL-2 effectively enhanced survival rates in skin transplants, but it failed to regulate immune responses in a major-mismatch model (BALB/c to B6 skin transplantation) [71]. These findings indicate that increasing Treg cells alone is not sufficient to control the activation and pathogenicity of effector T cells. Indeed, combining IL-2 with immunosuppressants such as tacrolimus significantly reduces rejection compared to IL-2 treatment alone [71]. Thus, while targeting Treg cells in transplantation holds promise, combination therapies involving immunosuppressants and Treg therapy may be a more effective strategy.

A strategy that combines blocking coinhibitory signals to inhibit T cell activation with enhancing the Treg population or their suppressive function offers a promising approach to preventing allograft rejection. Increasing immune tolerance can counteract the reduction of Treg cells caused by belatacept, and blocking coinhibitory signaling can address the limitations of Treg therapy. In this context, several studies have explored combinatory approaches involving belatacept alongside Treg-enhancing drugs. Concomitant treatment of belatacept with the IL-2/anti-IL-2 complex, which selectively increases Treg cells, has been shown to improve survival rates compared to belatacept alone by restoring Treg cells that are reduced by belatacept in a cardiac allograft model [84]. However, the adoptive transfer of CD4⁺CD25⁺ Treg cells expanded ex vivo with belatacept treatment did not improve rejection rates compared to the belatacept-only group. These results suggest that transferred Treg cells might not expand sufficiently in vivo due to competition for IL-2 with endogenous Treg cells. A phase II study involving 20 kidney transplant patients treated with belatacept and sirolimus, and followed up for 24 months (NCT00565773) found that CD4 and CD8 T cells were significantly reduced posttreatment compared to pretreatment, while CD25^+-FOXP3⁺ Treg cells and regulatory B cells increased, leading to successful prevention of rejection in all patients [85]. An ongoing phase II study is evaluating the combination of rapamycin and belatacept treatment in kidney transplantation (NCT06055608). Additionally, IFN-β treatment demonstrated synergistic effects when used with CTLA-4 Ig compared to CTLA-4 Ig alone in a BALB/c (H-2d) to C57BL/6 (H-2b) heart allograft model [86]. IFN-β increases Treg cells through the pSTAT1 signaling pathway and P300-dependent FOXP3 acetylation, both of which are involved in FOXP3 expression and stability. Furthermore, the JAK 1/3 inhibitor tofacitinib, when administered with CTLA-4 Ig in a heart allograft model, promoted long-term survival compared to CTLA-4 Ig alone [87]. Tofacitinib treatment showed a correlation with an increase in Treg cells in heart tissue, indicating that inhibiting effector T cells, such as Th17 cells, can improve the therapeutic efficacy of belatacept. A phase I/II study has been conducted to evaluate the effects of belatacept and tocilizumab, another Th17 cell blocker, on Treg modulation in kidney transplant patients (NCT04066114). Three patients experienced rejection, and some developed urinary tract infections and acute kidney injury. However, the effects on Treg cells have not yet been reported.

The role of Treg cells in maintaining immune tolerance is well established, particularly in the context of transplantation. Although belatacept therapies show significant promise in modulating immune responses, their effectiveness is limited by challenges such as the reduction of Treg cells and resistance from certain effector/memory T cells, especially those that are CD28-negative. These limitations highlight the complexity of achieving long-term graft survival without compromising immune tolerance. Recent developments in combination therapies, such as using belatacept alongside agents that promote Treg expansion like IL-2 or rapamycin, present promising avenues for improving transplant outcomes. However, these therapies are still in the early stages, and clinical trials are necessary to fully assess their efficacy in transplantation. The future of transplantation may lie in combining therapies that effectively balance immune cell inhibition with the enhancement of Treg cells (Fig. 3). Addressing the limitations of current belatacept regimens by augmenting Treg cells represents a crucial next step in advancing transplant tolerance. By refining these strategies, it may become possible to reduce graft rejection rates, improve long-term outcomes, and minimize the need for lifelong immunosuppression in transplant recipients.

Figure 3. Perspectives on therapeutic strategies for transplantation. Belatacept binds to CD80/86 on APCs, inhibiting the activation of alloreactive effector T cells by blocking CD28 signaling. However, it also inhibits CD28 signaling in Treg cells, impairing their differentiation and maintenance, and it cannot regulate CD28-negative T cells. Strategies that increase Treg cells, such as low-dose IL-2, can restore Treg cells inhibited by belatacept and effectively inhibit not only alloreactive effector T cells but also CD28-negative effector T cells. Combining belatacept with drugs that enhance Treg cells can effectively prevent graft rejection and maintain immune tolerance. Treg, regulatory T; TCR, T cell receptor; MHC, major histocompatibility complex; APC, antigen-presenting cell; IL, interleukin; IFN, interferon; CTLA-4, cytotoxic T lymphocyte antigen-4. Diagram created with BioRender.

Conflict of Interest

No potential conflict of interest relevant to this article was reported.

Funding/Support

This research received funding through grants awarded to JMC from the Korea Drug Development Fund (No. RS-2023-00217324) and to GRK from the Sejong Science Fellow (No. RS-2023-00210407) of the National Research Foundation, sponsored by the Korean government.

Author Contributions

Conceptualization: all authors. Data curation: GRK, KHN. Writing–original draft: all authors. Writing–review & editing: all authors. All authors read and approved the final manuscript.