Regulatory T cell therapy in solid organ transplantation: clinical applications and laboratory monitoring strategies

Abstract

Regulatory T cells (Tregs) play a fundamental role in maintaining immunological homeostasis and sustaining immune tolerance. In solid organ transplantation, Tregs have emerged as a promising strategy of cellular immunotherapy capable of reducing allograft rejection and mitigating long-term complications. Early-phase clinical studies have established the safety and feasibility of adoptive Treg therapy. Building on these pivotal findings, Treg therapy has created a solid foundation for translational application. However, successful clinical implementation requires a deeper understanding of Treg biology, clarification of their mechanisms of action, and the development of reliable strategies for in vivo monitoring. This review provides a detailed overview of Treg mechanisms, ongoing clinical trials, and methodological approaches for evaluating their phenotype and function through cell-based, protein-based, and gene-based assays. In addition, it highlights key considerations for optimizing therapeutic efficacy and ensuring safety, with the ultimate aim of advancing Treg therapy toward routine clinical use in solid organ transplantation.

HIGHLIGHTS
Regulatory T cells (Tregs) play a central role in immune tolerance after solid organ transplantation. Early clinical trials have demonstrated the safety and potential of Treg therapy. Laboratory assays enable phenotypic and functional monitoring of Tregs. Integrated strategies are needed to optimize Treg therapy and its monitoring.

INTRODUCTION

Regulatory T cells (Tregs), identified primarily by the expression of CD4, CD25, and the transcription factor FOXP3, constitute approximately 1% to 10% of the circulating CD4+ T cell population [1–3]. These cells play a central role in maintaining immune homeostasis and tolerance by suppressing the activation and proliferation of effector T cells, dendritic cells, and other immune components [4]. Their immunosuppressive activity is mediated through several mechanisms, including metabolic disruption, modulation of antigen-presenting cell (APC) function, secretion of immunosuppressive cytokines, and direct cytolysis of target cells [5].

In solid organ transplantation (SOT), conventional immunosuppressive regimens have successfully reduced the incidence of acute rejection. Nevertheless, long-term complications, including opportunistic infections, malignancies, and chronic allograft dysfunction, remain significant challenges [6,7]. In contrast, Treg therapy provides a targeted strategy to promote donor-specific tolerance while minimizing complications and preserving overall immune competence [8]. Preclinical studies employing ex vivo-expanded Tregs or donor antigen-specific Tregs (darTregs) have shown prolonged graft survival with reductions in both rejection and graft-versus-host disease [9–11]. These results support early-phase clinical trials aimed at investigating the immunological effects, safety, and feasibility of Treg therapy in SOT.

Despite these advances, widespread clinical translation of Treg therapy faces major challenges, particularly the absence of standardized and robust assays capable of reliably monitoring phenotypic stability, suppressive function, persistence, and in vivo stability [12,13]. Conventional assays, such as enumeration of total Tregs based on surface markers and viability measurements, provide only limited insight into suppressive capacity or antigen specificity [14,15]. Furthermore, these basic assessments may not reliably correlate with clinical outcomes or immune tolerance status [16,17].

Recent technological developments, including functional suppression assays, cytokine profiling, and epigenetic or single-cell analyses, provide opportunities for more precise immunological monitoring [18,19]. Importantly, these approaches highlight the potential for precision medicine in transplantation, enabling a shift from uniform immunosuppressive regimens to individualized, cell-based therapeutic strategies.

This review offers a comprehensive overview of Treg mechanisms of action, the current progress of clinical trials, and available methodologies for their evaluation. Special attention is given to emerging laboratory technologies that facilitate the monitoring of Treg phenotype, function, and stability using cell-based, protein-based, and gene-based platforms. Moreover, the review discusses the present limitations of Treg therapy and outlines future directions to enhance its safety, efficacy, and applicability in SOT.

REGULATORY T CELLS IN TRANSPLANTATION: MECHANISMS AND CLINICAL TRANSLATION

Tregs play a pivotal role in maintaining immune tolerance through diverse mechanisms, including metabolic disruption, contact-dependent inhibition, cytokine production, and induction of cell death. Building on these fundamental immunoregulatory functions, Treg therapy has emerged as a promising strategy to promote immune tolerance in transplantation. Recent advances in the isolation, expansion, and engineering of Tregs have enabled the development of therapeutic approaches with improved specificity and stability. Ongoing clinical trials are actively assessing the safety, efficacy, and optimal protocols for Treg therapy across different clinical applications.

Action of Regulatory T Cells

Tregs employ multiple distinct immunosuppressive mechanisms to prevent alloimmune responses by suppressing activated effector immune cells (Fig. 1). One key mechanism is the metabolic disruption of essential immune resources [5,20]. Tregs constitutively express high levels of the high-affinity interleukin (IL)-2 receptor alpha chain (CD25), which allows them to efficiently sequester IL-2 [21,22]. This limits IL-2 availability for effector T cells and natural killer cells, ultimately inducing apoptosis of activated effector cells [23,24]. In addition, Tregs stimulate APCs to produce indoleamine 2,3-dioxygenase (IDO) [25]. IDO and other enzymes that catabolize amino acids such as tryptophan and arginine contribute to a metabolic environment unfavorable for effector T cell proliferation [25,26]. A second major mechanism is direct contact-dependent inhibition of APCs [5,20]. Tregs express inhibitory receptors, including cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3), which interact with CD80/86 and major histocompatibility complex II molecules on APCs, respectively [27,28]. These interactions trigger trans-endocytosis, reducing APC costimulatory molecule expression and impairing effector T cell activation [29,30]. A third mechanism involves secretion of immunosuppressive cytokines, such as IL-10, transforming growth factor beta (TGF-β), and IL-35 [31,32]. These cytokines inhibit interferon-γ–producing T helper 1 (Th1) cells and IL-17–producing Th17 cells, while promoting the expansion and stabilization of other regulatory cell subsets [33,34]. A final major mechanism is direct cytotoxicity against effector T cells and APCs through granzyme-dependent apoptotic pathways [35–37]. Other mechanisms, such as tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)/death receptor DR5 interactions and galectin-mediated cytolysis, have also been described [38,39].

Fig. 1.

Mechanisms of immune tolerance mediated by regulatory T cells (Tregs) in solid organ transplantation. Tregs primarily employ four suppressive mechanisms to maintain immune tolerance and prevent allograft rejection. (A) Metabolic disruption: Tregs express high-affinity interleukin (IL)-2 receptors (CD25), enabling them to competitively consume IL-2 and deprive effector T cells (Teffs), leading to their apoptosis. CD39 and CD73 expressed on Tregs convert extracellular ATP into immunosuppressive adenosine, which suppresses effector T cell activity via adenosine receptors. Tregs also transfer cyclic adenosine monophosphate (c-AMP) to Teffs through gap junctions, further impairing their activation. (B) Direct contact–dependent inhibition: Tregs suppress antigen-presenting cells (APCs) by expressing cytotoxic T-lymphocyte associated protein 4 (CTLA-4) and lymphocyte activation gene 3 (LAG-3), which bind CD80/CD86 and major histocompatibility complex (MHC)-II, respectively. These interactions downregulate costimulatory signaling. Additionally, CTLA-4 engagement induces indoleamine 2,3-dioxygenase (IDO) expression in APCs, leading to tryptophan depletion and kynurenine generation, both of which inhibit effector T cell proliferation. (C) Suppressive cytokine production: Tregs secrete immunoregulatory cytokines, including IL-10, transforming growth factor beta (TGF-β), and IL-35, which inhibit activation and proliferation of Teffs and other immune cells. (D) Induction of cell death: Tregs release granzyme A, granzyme B, and perforin, inducing apoptosis in Teffs.

Together, this diverse repertoire of suppressive mechanisms enables Tregs to dynamically regulate immune responses. In the setting of SOT, Tregs act to balance prevention of graft rejection with preservation of protective immunity [40,41].

Regulatory T Cell Therapy: Evolving Strategies in Transplantation

Because of their central role in regulating alloimmune responses, Tregs have emerged as promising therapeutic candidates for donor-specific immunomodulation in SOT [41]. To translate this potential into clinical application, multiple strategies have been developed to expand Tregs and enhance their antigen specificity. These strategies can be broadly categorized into three types: polyclonal Tregs, darTregs, and chimeric antigen receptor (CAR)-Tregs (Table 1).

Table 1.

Comparative overview of Treg-based immunotherapies in transplantation

Feature	Polyclonal Tregs	darTregs	CAR-Tregs
Mechanism of action	• Ex vivo-expanded Tregs that react to a wide range of antigens	• Selected and expanded Tregs that specifically recognize donor-alloantigens via native TCRs	• Genetically engineered Tregs expressing CARs for enhanced antigen specificity
Antigen specificity	• Low • Broad and nonspecific	• High • Targeted to donor antigen	• Very high • Engineered to recognize a defined antigen (e.g., HLA-A2, tissue-specific antigens)
Persistence (in vivo)	• Limited • Typically detectable for days to weeks after infusion; may not persist long-term	• Intermediate • Shown to be detectable in tissues for a limited period, may wane in weeks	• Variable • Shown to persist in animal models for weeks; human data is limited but may offer improved persistence
Clinical trial outcomes	• Shown to be safe in early-phase trials (e.g., kidney, liver transplant). • Some success in reducing immunosuppression	• Early trials (e.g., the ONE Study) suggest potential in promoting donor-specific tolerance • Ongoing studies underway	• Preclinical models show strong efficacy in suppressing rejection • Human trials are in early stages
Advantages	• Easier to manufacture; extensively studied; broadly applicable	• Greater efficacy with fewer cells; targeted suppression	• Highly specific; potential for durable and localized tolerance
Limitations	• Nonspecific; may require repeated dosing; risk of off-target effects	• Technically challenging; requires donor-recipient matching and ex vivo expansion	• Engineering complexity; unknown long-term safety and efficacy; regulatory hurdles

Treg, regulatory T cell; darTreg, donor-specific regulatory T cell; CAR, chimeric antigen receptor; TCR, T cell receptor; HLA, human leukocyte antigen.

Polyclonal Tregs can be expanded either ex vivo or in vivo without specific antigen stimulation [42]. Ex vivo expansion typically employs nonspecific stimuli, such as anti-CD3/CD28 antibodies with IL-2, under controlled laboratory conditions [43–45]. Polyclonal Tregs exhibit broad antigen reactivity and are relatively simple to manufacture; however, their lack of antigen specificity may necessitate infusion of large cell numbers to achieve therapeutic efficacy [46]. In contrast, in vivo expansion strategies rely on administration of low-dose IL-2 to selectively promote endogenous Treg proliferation in recipients [47–49]. In this context, the Transplantation Without Overimmunosuppression (TWO) study evaluated autologous polyclonal Treg therapy in living donor kidney transplantation. This trial showed that polyclonal Treg therapy enabled successful minimization of immunosuppression with low-dose tacrolimus monotherapy, leading to fewer complications and stable long-term graft function [50].

To improve specificity, darTregs are induced by coculturing ex vivo-expanded Tregs with donor-derived alloantigens or donor cells [51]. Clinical protocols for darTreg expansion have employed CD40L-stimulated B cells or low-dose IL-2 combined with rapamycin [52,53]. These approaches demonstrated enhanced suppressive potency and antigen specificity in humanized mouse models. The ONE Study consortium further evaluated the safety and feasibility of darTreg administration in kidney transplantation [54]. By selectively recognizing and suppressing donor-specific immune responses, darTregs provide the strongest clinical evidence supporting targeted immunotherapy [55]. However, both polyclonal and darTreg therapies face limitations in yield, as Tregs represent only 3% to 17% of the final cell product. Thus, optimal dosing requirements and the degree of antigen specificity needed for therapeutic success remain unresolved [56,57].

CAR-Tregs represent the most advanced approach, employing genetic engineering to confer precise donor specificity [58]. These cells are engineered to express synthetic receptors that recognize donor human leukocyte antigen (HLA) molecules on graft tissues [59,60]. CAR-Tregs offer distinct advantages, including uniform antigen specificity, lower therapeutic cell requirements, improved scalability, and preservation of natural T cell receptor (TCR)-mediated reactivity.

Collectively, these evolving strategies signify a paradigm shift from broad immunosuppression to targeted immune regulation. Progress in Treg therapy underscores the urgent need for standardized laboratory monitoring methods to evaluate Treg stability, function, and in vivo persistence, which are key requirements for safe and effective clinical translation (Table 2).

Table 2.

Clinical trials of Treg therapy in solid organ transplantation on clincialtrials.gov

Organ	Study ID	Acronym	Cell typea)	Phase	Cell dose	Location
Kidney	NCT06552169	RETIRE	Polyclonal Tregs	II	-	Taiwan, U.S.
Kidney	NCT02091232	ONE Study	Belatacept-conditioned Tregs	I	1.0 × 106	U.S.
Kidney	NCT03867617	-	Polyclonal with donor bone marrow	I, II	1.0 × 107	Austria
Kidney	NCT02129881	OneTreg1	Polyclonal Tregs	I, II	1-10 × 106/kg	U.K.
Kidney	NCT06777719	Eight Treg	Polyclonal Tregs	I	2.096 × 106 CD8+ Tregs/kg	France
Kidney	NCT02371434	ONEnTreg13	Polyclonal Tregs	I, II	0.5, 1.0, and 2.5-3 × 106/kg	Germany
Kidney	NCT01446484	-	Polyclonal Tregs	I, II	2.0 × 108	Russia
Kidney	NCT04817774	STEADFAST	CAR-Tregs	I, II	104 to 109 cells/kg	Belgium, Netherlands, U.K.
Kidney	NCT02244801	DART	Donor-alloantigen-reactive Tregs	I	3.0 and 9.0 × 108	U.S.
Kidney	NCT02145325	TRACT	Polyclonal Tregs	I	0.5, 1.0, and 5.0 × 109	U.S.
Kidney	NCT02088931	TASKp	Polyclonal Tregs	I	3.2 × 108	U.S.
Kidney	NCT02711826	TASK	Polyclonal Tregs	-	550±450 × 106	U.S.
Liver	NCT02474199	ARTEMIS	Donor-alloantigen-reactive Tregs	I, II	4 × 108	U.S.
Liver	NCT02188719	deLTa	Donor-alloantigen-reactive Tregs	I	50, 200, 800 × 106	U.S.
Liver	NCT02166177	ThRIL	Polyclonal Tregs	I, II	0.5-1 and 3-4.5 × 106/kg	U.K.
Liver	NCT03654040	LITTMUS-UCSF	Polyclonal Tregs	I, II	90 to 500 × 108	U.S.
Heart	NCT04924491	THYTECH	Polyclonal Treg	I, II	1.0 × 107	Spain

Treg, regulatory T cell; CAR, chimeric antigen receptor.

^a)The source of all cell types was autologous expanded Tregs.

Clinical Trials of Regulatory T Cell Therapy

Early in vitro investigations of Treg therapy in SOT were essential for promoting efficient translation toward clinical implementation. Optimized manufacturing protocols can now generate up to 2 × 10⁹ highly purified Tregs (>90% CD4⁺CD25^highCD127^lowFOXP3⁺) within a 2-week culture period [61]. In vitro studies relevant to clinical translation have shown that darTregs exhibit superior suppressive capacity compared with polyclonal Tregs, enabling effective immunoregulation at lower cell doses [62–64]. In vivo studies in immunocompetent mice further demonstrated that HLA-A2–targeted CAR-Tregs preferentially accumulate within HLA-A2⁺ allografts, highlighting their antigen-specific homing ability [65]. Boardman et al. [66] further suggested that such selective migration significantly enhances the protective efficacy of CAR-Tregs and is supported by both CAR- and TCR-mediated reactivity.

After translational studies, initial clinical trials confirmed the safety and feasibility of both expanded polyclonal and darTregs [42,46,56,67]. In 2016, Todo et al. [56] reported the first clinical application of darTregs in living donor liver transplantation, showing that adoptive transfer of ex vivo-expanded darTregs was feasible, well tolerated, and permitted complete withdrawal of immunosuppressants in selected recipients. Similarly, the TRACT trial (NCT02145325) in living donor kidney transplant recipients demonstrated that autologous polyclonal Treg infusion was safe, well tolerated, and associated with increased circulating FOXP3⁺ Tregs, with no rejection episodes or serious infections [68]. These results indicate that Treg therapy has the potential to reduce immunosuppressant requirements, support long-term graft stability, and preserve protective immune responses, thereby laying the foundation for broader clinical use in SOT.

Such encouraging results have been made possible by advances in good manufacturing practice (GMP)-compliant Treg production. For example, at the University of California, San Francisco GMP facility, Tregs were manufactured through a standardized process [45,52]. CD4⁺CD25⁺CD127^low cells were isolated by flow cytometry following sequential magnetic selection and then subjected to a 3-week ex vivo expansion with CD3/CD28 antibodies, IL-2, and sirolimus. This approach yielded up to 2 × 10⁹ cells with >90% FOXP3⁺ purity [52,69,70].

Despite these technical advances, several clinical challenges remain. Key issues include maintaining Treg stability, defining optimal dosing and infusion schedules, and clarifying the influence of immunosuppressive regimens on Treg persistence and function. With respect to stability, the Brunstein trial showed that umbilical cord blood-derived Tregs peaked at day 2 but became undetectable in circulation beyond 14 days. In contrast, the Trzonkowski trial reported a progressive decline in FOXP3⁺ cells, from 90% to 70% to 40%, at days 75, 82, and 93, respectively, despite three infusions [71,72]. These findings highlight the need to optimize culture duration and stimulation protocols to preserve FOXP3 expression and functional integrity of Treg products. Regarding dosing and infusion schedules, trials have applied a broad range, from 0.43 × 10⁶/kg to 5 × 10⁹ total cells, at different posttransplant time points (e.g., days +10, +13, or +60). These choices are typically influenced by patient immunological risk and individual trial design [45,52,56,68,73]. Furthermore, the choice of immunosuppressive regimen is critical. Rapamycin, for instance, supports Treg survival and expansion more effectively than calcineurin inhibitors, while lymphodepletion strategies such as alemtuzumab may enhance Treg engraftment but require careful coordination to avoid unintended depletion [74,75].

For successful clinical translation, harmonized and standardized protocols are essential to optimize outcomes [15]. The ONE Study consortium demonstrated effective multicenter coordination, showing that a single infusion of autologous Tregs in kidney transplant recipients was safe, maintained stable graft function, and reduced the incidence of viral infections [54,76]. In parallel, validated biomarkers are needed for reliable monitoring of therapeutic efficacy. Advances in high-resolution technologies are expected to deepen understanding of Treg heterogeneity and mechanisms of action [77]. Ultimately, the development of robust, scalable, and antigen-specific Treg products will be critical for realizing the full therapeutic potential of Tregs in transplantation.

MONITORING OF REGULATORY T CELLS

The successful application of Treg therapy in SOT requires accurate enumeration and functional assessment of Tregs. Comprehensive monitoring before, during, and after therapeutic infusion depends on highly sensitive and specific qualitative and quantitative assays. Multiple analytical platforms have been developed, including cell-based, protein-based, and gene-based assays. Each provides distinct insights into Treg phenotype and functional activity, including suppression of proliferation, modulation of activation markers, and regulation of cytokine production (Fig. 2).

Fig. 2.

Analytical approaches used to assess regulatory T cell (Treg) phenotype and function. (A) Cell-based assays include flow cytometry for phenotypic characterization, [³H]-thymidine incorporation, and carboxyfluorescein succinimidyl ester (CFSE) dilution assays for evaluating proliferative suppression, and ATP-based assays for cytotoxic activity. (B) Protein-based assays such as mass spectrometry provide insights into metabolic activity, while enzyme-linked immunosorbent assays (ELISA) and intracellular cytokine staining by flow cytometry are used to quantify cytokine production. (C) Gene-based assays include reverse transcription polymerase chain reaction (RT-PCR) for RNA expression analysis, methylation assays to detect epigenetic modifications (e.g., FOXP3 Treg-specific demethylated region), and single-cell RNA sequencing (scRNA-seq) for high-resolution transcriptional profiling. Together, these complementary methods enable comprehensive monitoring of Treg identity and function in both research and clinical contexts.

Cell-Based Phenotypic and Functional Assays

Flow cytometry remains the most widely used cell-based assay for evaluating Treg phenotypes. Phenotypically, Tregs are defined by expression of CD4, CD25, and the transcription factor FOXP3, along with reduced CD127 expression [14]. To address phenotypic heterogeneity, standardized flow cytometry panels incorporating CD3, CD4, CD25, CD127, and FOXP3 have been developed [78]. Additional markers such as Ki67, CD45RA, CTLA-4, and Helios are often included to improve both phenotypic resolution and functional characterization [14,79–82]. The adoption of prealiquoted dried antibody panels and TruCOUNT tubes has enhanced reproducibility and reduced variability across platforms [83,84]. Despite these advances, the absence of fully standardized protocols—particularly regarding gating strategies and interassay consistency—remains a major limitation for broader clinical application.

The suppressive function of Tregs is commonly assessed with in vitro proliferation-based assays, such as [³H]-thymidine incorporation and carboxyfluorescein succinimidyl ester (CFSE) dye dilution [85,86]. In the [³H]-thymidine assay, suppression is measured by quantifying radioactive thymidine uptake in CD4⁺CD25^– conventional T cells (Tconv) stimulated with anti-CD3 antibodies [87]. This approach is highly sensitive, enabling detection of suppression at low Treg-to-Tconv ratios and providing quantitative proliferation data [85]. However, it underestimates Treg numbers because it cannot distinguish proliferating Tregs from responder T cells within cocultures, and it is further limited by the use of radioactive isotopes and strict timing requirements [88].

The CFSE dye dilution assay allows simultaneous analysis of Tregs and responder T cells, enabling calculation of proliferation indices and suppression rates by multiparameter flow cytometry [89,90]. Nonetheless, this technique is hindered by technical challenges, including substantial cell loss during CFSE staining and reduced signal intensity caused by dye dilution during successive divisions. Importantly, proliferation-based assays such as [³H]-thymidine and CFSE dilution do not assess the ability of Tregs to suppress antigen-specific T cell responses [88].

Protein-Based Phenotypic and Functional Assays

Tregs exert immunosuppressive effects through metabolic disruption and secretion of inhibitory cytokines [29,31,32]. Based on these mechanisms, metabolic profiling and cytokine assays can serve as functional readouts. Metabolic activity is often assessed by measuring CD39/CD73-mediated adenosine production, using techniques such as flow cytometry, immunostaining, mass spectrometry, and western blotting [91]. However, the immunosuppressive effects of Treg-derived adenosine may be offset by effector T cells expressing the CD26-adenosine deaminase complex, which catalyzes adenosine degradation to inosine [92]. Cytokine assays quantify suppressive cytokines such as TGF-β, IL-10, and IL-35 using enzyme-linked immunosorbent assay (ELISA) or flow cytometry [93,94]. ELISA remains the gold standard for cytokine quantification, offering high sensitivity for single or multiplex detection [95]. However, ELISA is limited by difficulties in producing reliable standard curves across wide dynamic ranges and by risks of cross-reactivity, both of which affect accuracy and reproducibility [96]. Its greatest limitation is the inability to identify the specific cell types responsible for cytokine production [97]. In contrast, flow cytometry enables cytokine detection at the single-cell level within heterogeneous Treg populations [98]. Multiparametric approaches, including intracellular staining, permit simultaneous assessment of multiple cytokines, thereby generating a more comprehensive functional profile.

Gene-Based Molecular Assays

The suppressive functions of Tregs are largely determined by the expression of key genes, notably FOXP3 and CTLA-4 [99,100]. Transcriptional profiling of these and other Treg-associated genes can be performed using reverse transcription polymerase chain reaction or RNA sequencing [101,102]. Chauhan et al. [103] demonstrated that Tregs with high FOXP3 expression more effectively suppressed effector T cell proliferation and produced increased IL-10 and TGF-β. In contrast, Zheng et al. [104] reported that while both FOXP3 and CTLA-4 are upregulated upon activation, suppressive function correlates more strongly with CTLA-4 than with FOXP3. Importantly, some studies have shown that activated non-Tregs can transiently express FOXP3 without acquiring suppressive activity [105]. Thus, FOXP3 alone is insufficient to distinguish bona fide Tregs from activated non-Tregs. Epigenetic modifications, such as DNA methylation patterns in the FOXP3 Treg-specific demethylated region (TSDR), provide a more reliable means of distinguishing Tregs from other T cell subsets [106–109]. These differences can be quantified by sequencing-based assays, which calculate a demethylation index by comparing the number of demethylated TSDR copies with the total amplified TSDR copies [110,111].

More recently, single-cell RNA sequencing (scRNA-seq) has emerged as a powerful tool for tracking immune cell dynamics in tolerance and rejection after Treg therapy in SOT [112,113]. This technology enables high-resolution profiling of infused Tregs and recipient immune cells, identifying transcriptional changes, activation states, and lineage heterogeneity. Clinical application of scRNA-seq is expected to provide deeper mechanistic insights into graft-host immune interactions, elucidate therapeutic effects, and facilitate discovery of novel biomarkers for monitoring Treg therapy.

LIMITATIONS OF REGULATORY T CELL THERAPY

The clinical translation of Treg therapy in SOT faces several major challenges. First, the phenotypic and functional heterogeneity of Treg populations complicates standardization of therapeutic products and undermines consistency in suppressive function [114]. Orozco et al. [13] emphasized that variable Treg subsets and unstable FOXP3 expression impair phenotypic stability and functional capacity. Similarly, Raffin et al. [12] highlighted that Treg instability and plasticity under inflammatory conditions raise concerns regarding both safety and efficacy, as unstable Tregs may convert into pathogenic effector T cells. Another challenge is the potential for resistance to Treg-mediated suppression. In autoimmune diseases, specific effector T cell subsets have been shown to evade Treg regulation, contributing to disease progression [115]. This observation suggests that resistance to Treg-mediated suppression may also occur within the inflammatory environment of SOT and should be carefully considered when applying Treg therapy clinically. A further limitation is the absence of validated biomarkers for in vivo persistence, migration, and functional activity. Without reliable biomarkers, monitoring infused Tregs and optimizing therapeutic protocols remain difficult, hindering assessment of efficacy [116]. To address these challenges, harmonized multiomic biomarker approaches are needed. These include FOXP3 TSDR demethylation to evaluate stability, single-cell transcriptomic and epigenomic profiling for subset characterization, and TCR repertoire analysis for lineage tracking. Such integrated strategies can precisely identify stable and suppressive Treg subsets while enabling real-time monitoring after infusion. Next-generation monitoring will therefore be indispensable for developing robust, safe, and clinically effective Treg therapies in SOT.

CONCLUSION

As Treg therapy progresses toward broader clinical application in SOT, reliable monitoring of Treg phenotype and function has become increasingly crucial. Given the inherent cellular heterogeneity, potential lineage instability, and dynamic functional states of Tregs, conventional monitoring approaches are insufficient for assessing therapeutic efficacy in vivo. Accurate assessment tools, including cell-based, protein-based, and gene-based assays, are essential not only to evaluate efficacy and ensure safety but also to guide individualized immunomodulatory strategies. In the absence of standardized monitoring frameworks, interpretation of clinical outcomes and refinement of therapeutic protocols remain limited. Therefore, the development and implementation of comprehensive, sensitive, and clinically validated monitoring strategies must be prioritized. Such efforts will be critical to fully realize the therapeutic potential of Treg therapy in transplantation.