Regulatory T cells: From Foxp3 to tolerance-inducing therapies

Abstract

The immune system balances self-tolerance and threat defense; regulatory T cells (Tregs) enforce this equilibrium. This review follows their journey from the disputed suppressor-T-cell era to the Foxp3-defined lineage, then integrates how Tregs recalibrate antigen-presenting cells and cytokine networks to sustain immune quiescence and restore homeostasis. These insights shape emerging therapies that expand endogenous Tregs in vivo with low-dose IL-2 or employ ex vivo–engineered cells to re-establish immune balance. We conclude by outlining key challenges—stability, tissue-targeting, scalability—for durable tolerance, and therapeutic applications of recently clarified Treg-mediated tissue-specific homeostasis.

1. INTRODUCTION

The immune system must both eliminate pathogens and cancer (“attack”) and remain unresponsive to self and innocuous antigens (“tolerance”); imbalance of these processes causes autoimmunity, allergy, and transplant rejection (Grover et al., 2021; Shevach, 2000). Regulatory T cells (Tregs), defined by the master transcription factor Foxp3, maintain immune tolerance and are indispensable for homeostasis (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003).

This review retraces the dramatic research trajectory that led to the recognition of Tregs, from the rise and fall of T cell–mediated immunosuppression to the identification of CD25⁺CD4⁺ T cells with suppressive activity and the discovery of Foxp3 as their defining factor. It then examines the diverse mechanisms by which Tregs modulate other immune cells to maintain tolerance and explores how these biological insights have been translated into therapeutic strategies, such as augmenting or engineering Tregs in autoimmune diseases and transplantation. As a complementary perspective, we also briefly address the clinically important but conceptually converse application of depleting tumor-infiltrating Tregs in cancer immunotherapy.

2. HISTORY OF TREG DISCOVERY

2.1. The emergence and repudiation of the suppressor T cell hypothesis

The path from T cell–mediated suppression to Treg-centered therapeutics was not straightforward. The concept of “infectious tolerance” arose in the early 1970s, when murine experiments using ovine red blood cells suggested that thymus-derived cells could transmit immune tolerance to other lymphocytes. (Gershon and Kondo, 1971). This phenomenon implied the existence of “suppressor T cells”—T cells that suppress immune responses. Subsequent work proposed that these cells were CD8⁺ cells acting via the soluble factor, T-suppressor factor, linked to a putative I-J locus within the major histocompatibility complex (MHC) region, but later genetic analyses showed that I-J does not exist, critically undermining the concept (Flood and Louie, 1984; Möller, 1988; Munro, 1983; Okumura et al., 1977; Steinmetz et al., 1982; Taniguchi et al., 1982). The field rapidly waned under the prevailing view that the immune system lacked a dedicated suppressive T cell subtype.

2.2. Identification and acceptance of Tregs

Under these circumstances, Sakaguchi et al. advanced research on immune tolerance using their own approach independent of the T-suppressor factor hypothesis, reporting the existence of a CD4⁺ T cell population with immunosuppressive functions that suppresses autoimmune inflammation (Sakaguchi et al., 1985). Subsequent studies showed that CD4⁺CD5^hi and CD4⁺CD45RB^lo populations markedly suppressed inflammation in murine autoimmune models (Morrissey et al., 1993; Sugihara et al., 1988). Focusing on shared surface features within these subsets, Sakaguchi et al. identified expression of CD25, the IL-2 receptor α chain, and demonstrated that depleting CD25⁺ cells disrupted self-tolerance in vivo (Sakaguchi et al., 1995). Independent groups reproduced these findings (Read et al., 2000; Suri-Payer et al., 1998), establishing a suppressive T cell population distinct from the historical suppressor T cell. Initially termed “immunoregulatory CD4⁺CD25⁺ T cells” (Takahashi et al., 1998; Thornton and Shevach, 1998), this population soon became known as “Tregs” (Sakaguchi, 2000; Shevach, 2000). Using CD25 to enrich Tregs accelerated molecular characterization. Tregs characteristically express cytotoxic T-lymphocyte antigen 4 (CTLA-4), and anti–CTLA-4 antibodies impair their suppressive activity, whereas agonistic signaling through glucocorticoid-induced tumor necrosis factor receptor (GITR) abrogates suppression (Read et al., 2000; Shimizu et al., 2002). Soluble mediators such as TGF-β and IL-10 were also implicated as key components of Treg-mediated suppression (Asseman et al., 1999; Read et al., 2000). Their thymic differentiation has also been reported (Itoh et al., 1999; Jordan et al., 2001).

2.3. Elucidation of the master transcription factor Foxp3

Pivotal genetic discoveries emerged. Brunkow, Ramsdell, and colleagues identified a loss-of-function variant in Foxp3 as the cause of the lethal lymphoproliferative “scurfy” phenotype in mice and sequence variants in FOXP3 as the genetic basis of the multi-organ autoimmune IPEX syndrome in humans (Bennett et al., 2001; Brunkow et al., 2001; Wildin et al., 2001). Building on links between autoimmunity and suppressive T cells, three groups—led by Sakaguchi, Rudensky, and Ramsdell—demonstrated in 2003 that Foxp3 is both necessary and defining for CD4⁺CD25⁺ Tregs. Loss of function eliminates or disables these cells and causes lethal systemic autoimmunity, whereas enforced Foxp3 expression endowed CD4⁺ T cells with the Treg phenotype (CD25, CTLA-4, GITR) and suppressive capacity that ameliorates colitis in the CD4⁺CD45RB^hi T cell transfer model (Fontenot et al., 2003; Hori et al., 2003; Khattri et al., 2003). This established Foxp3 as the master transcription factor programming the Treg lineage, confirming that Foxp3-expressing T cells are Tregs. Clinically, correlations between Treg dynamics and disease activity in autoimmunity and cancer reinforced acceptance of Tregs as pivotal immunoregulatory cells (Bates et al., 2006; Crispin et al., 2003; Curiel et al., 2004; Valencia et al., 2007).

2.4. Discovery of Treg subsets

With Foxp3 providing a lineage anchor, Treg heterogeneity came into focus. Tregs differentiated outside the thymus have also been identified (Apostolou and von Boehmer, 2004; Coombes et al., 2007; Sun et al., 2007). Thymus- and peripheral-derived Tregs are now referred to as thymic Tregs (tTregs) and peripheral Tregs (pTregs), respectively. Collectively, these naturally occurring Tregs in vivo are termed natural Tregs (nTregs), and Tregs induced ex vivo are termed induced Tregs (iTregs; Abbas et al., 2013; Chen et al., 2003; Sakaguchi et al., 2023). Functional partitioning into CCR7⁺CD62L⁺ central Tregs (cTregs) and CCR7⁻CD44⁺ effector Tregs (eTregs) was also described (Smigiel et al., 2014). cTregs reside in T-cell zones of lymphoid organs with IL-2 access, serving as a reservoir; eTregs depend on IRF4–Blimp-1, express high CTLA-4/ICOS/IL-10, and migrate to draining lymph nodes and peripheral tissues to execute suppression (Cretney et al., 2011). Furthermore, tissue-resident Tregs outside lymphoid tissue were discovered, maintaining organ- and context-specific homeostasis (Wang et al., 2020).

3. MECHANISMS OF TREG-MEDIATED IMMUNOSUPPRESSION

Tregs restrain immune responses through contact-dependent suppression of antigen-presenting cells (APC) and soluble-factor–mediated inhibition of conventional T cell (Tconv) responses, including both CD4⁺ and CD8⁺ T cell populations (Fig. 1).

Fig.1.

Schema showing how regulatory T cells (Tregs) suppress CD4⁺ and CD8⁺ conventional T cells (Tconvs)

Tregs (blue) use their high expression of CD25 to consume IL-2 (pink dots), which inhibits Tconv (orange) proliferation. Tregs also interact directly with dendritic cells (DCs; green). Through cytotoxic T-lymphocyte antigen 4 (CTLA-4) and OX40L, Tregs remove CD80/86 and OX40 from the DC surface via trans-endocytosis. Molecules such as LAG-3 and T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) on Tregs bind to MHCII and CD155 on DCs, leading to competitive occupation and inhibitory signaling. Tregs also release inhibitory cytokines. IL-10 (orange dots) from Tregs and tolerogenic DCs itself induces signals to the DC, resulting in decreased production of IL-12, IL-6, CD80/86, and MHCII. Tregs also release TGF-β (dark blue dots) and IL-35 (yellow dots). Tregs use CD39 and CD73 to convert adenosine triphosphate (ATP) to adenosine, further suppressing Tconv T cell receptor (TCR) signaling. These mediators also induce the differentiation of CD4⁺ Tconvs into other regulatory cells, including pTregs (via TGF-β), IL-35 secreting regulatory T cells (iTr35s) (via IL-35), and Type I regulatory cells (Tr1s) (via tryptophan metabolism/IL-10).

*The LAG-3 pathway is depicted as an MHC class II-dependent mechanism that primarily affects CD4⁺ T cell responses.

3.1. Direct interaction with APCs

A canonical mechanism involves membrane-protein–mediated modulation of APCs, particularly dendritic cells (DCs), rendering them tolerogenic DCs. CTLA-4 on Tregs physically deprives CD80/CD86 of DC via trans-endocytosis, diminishing Tconv co-stimulation from CD28-CD80/86; Treg-intrinsic CTLA-4 deficiency or blockade abrogates suppression (Qureshi et al., 2011). Analogous ligand depletion has been reported for OX40–OX40L, where Tregs acquire OX40L from dendritic cells in an OX40-dependent manner, thereby limiting co-stimulatory signaling to autoreactive T cells and promoting their peripheral deletion (Iriki et al., 2021). CTLA-4 engagement also delivers signals, inducing indoleamine 2,3-dioxygenase (IDO) in DCs, which leads to local tryptophan metabolism and the production of kynurenine (Boasso et al., 2005; Grohmann et al., 2002). Tryptophan starvation activates general control nonderepressible 2 in T cells, arresting proliferation, while kynurenines activate the aryl hydrocarbon receptor to promote Treg differentiation from CD4⁺ Tconvs. (Campesato et al., 2020; Mezrich et al., 2010; Munn et al., 2005; Munn et al., 1999).

Lymphocyte activation gene-3 (LAG-3; CD223) binds MHC class II on APC with high affinity, competitively dampening Tconv activation and delivering inhibitory signals that reduce co-stimulation and antigen presentation (Liang et al., 2008; Maruhashi et al., 2022). T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain (TIGIT) on Tregs engage poliovirus receptor (PVR; CD155) on DC, skewing DC cytokines toward IL-10 with concomitant suppression of IL-12 production (Chauvin and Zarour, 2020; Yu et al., 2009). Furthermore, programmed cell death ligand 1 (PD-L1) on APCs promotes Foxp3 induction and Treg differentiation in naive CD4⁺ T cells in the presence of TGF-β via programmed cell death 1 (PD-1) signaling suppression of the AKT–mTOR pathway and increased PTEN (Francisco et al., 2009).

3.2. Suppression via cytokines and soluble mediators

Tregs secrete anti-inflammatory cytokines, notably IL-10 and TGF-β. IL-10 signals through IL-10R–JAK1/TYK2–STAT3 on DC and macrophages to drive an anti-inflammatory program (Riley et al., 1999), reducing production of IL-12/IL-6/TNF and expression of MHC class II and CD80/86, thereby suppressing Th1/Th17 responses (Buelens et al., 1995; de Waal Malefyt et al., 1991; Zhou et al., 2004). Autocrine IL-10 signaling is also essential for Tregs to maintain an immunosuppressive phenotype; Treg-specific IL-10R deletion exacerbates colitis in mice (Chaudhry et al., 2011). TGF-β, via TGFβR–SMAD2/3, suppresses Tconv proliferation and inflammatory cytokines while, together with IL-2, promoting Foxp3 induction and pTreg differentiation (Chen et al., 2003; McKarns et al., 2004; Takimoto et al., 2010).

IL-35 has been identified as another Treg-derived suppressive cytokine (Collison et al., 2007). IL-35 consists of EBI3 and p35 (IL-12A) and primarily activates STAT1/STAT4 via receptors formed by IL-12Rβ2 and gp130 on T cells, thereby suppressing Tconv proliferation and reducing IL-17 production (Niedbala et al., 2007; Okada et al., 2017). IL-35 also acts on Tregs themselves, increasing Treg numbers through CCR5-mediated migration and proliferation via AKT–mTOR inhibition. PD-1/TIGIT upregulation stabilizes Tregs’ regulatory function (Shao et al., 2021). IL-35 also induces a distinct Foxp3⁻ T cell subset called IL-35-producing induced regulatory T cells (iTr35s) from naive CD4⁺ T cells. iTr35s exert their immunosuppressive function by producing IL-35 (Collison et al., 2010). IL-35 expands the suppressive milieu beyond T cells by inducing IL-35-producing regulatory B cells, which act suppressively in autoimmunity models (Wang et al., 2014), and by tolerizing DC with lower co-stimulation and enhanced IL-10 production (Haller et al., 2017).

Beyond secreted cytokines, Tregs deprive the environment of IL-2. This function is mediated by IL-2Rα (CD25, p55). IL-2 receptors include the intermediate-affinity receptor, the IL-2Rβ (CD122, p75)/common γ (CD132, p65) complex, and the high-affinity receptor, composed by IL-2Rβ, common γ, and IL-2Rα (Herrmann and Diamantstein, 1988; Waickman et al., 2016). Tconvs and natural killer (NK) cells generally express the CD122/132 complex, while Tregs additionally express CD25 at high levels even under resting conditions (Lokau et al., 2024). Tconvs and NK cells only transiently express CD25 after activation. Consequently, Tregs preferentially capture and consume surrounding IL-2, thereby reducing the proliferation signals of Tconvs (Pandiyan et al., 2007; de la Rosa et al., 2004).

Tregs also co-express ectonucleotidases CD39 and CD73, sequentially converting extracellular adenosine triphosphate (ATP) or adenosine diphosphate to adenosine (Deaglio et al., 2007). Adenosine acting on A2A receptors suppresses T cell receptor (TCR) signaling and cytokine production in Tconvs (Ohta et al., 2006; Zarek et al., 2008) and, in Tregs, enhances TGF-β production and LAG-3 expression as well as Foxp3 (Zarek et al., 2008).

3.3. The spreading range of Treg-mediated immunosuppression

Treg-mediated suppression propagates via direct interaction-induced tolerogenic APC and soluble mediators. These are termed linked- and bystander-suppression, respectively. The former extends to responses against co-presented antigens, including cognate and non-cognate, by the same tolerogenic DC rendered by Tregs. The latter involves cytokine diffusion, affecting neighboring APCs and T cells. Therefore, while the activation of Treg suppressive function is antigen-specific, as it is triggered by TCR signaling, the reach of the suppression is not necessarily antigen-specific (Ohkura and Sakaguchi, 2010; Sakaguchi et al., 2023; Shevach et al., 2001; Vahl et al., 2014).

On the other hand, antigen-specific efficiency in Treg-mediated immunosuppression has also been reported (Mukai et al., 2025; Tang et al., 2004; Wright et al., 2009). A major explanation is that antigen-specific Tregs follow the same homing and chemokine cues as pathogenic cognate antigen-specific Tconvs, leading to their selective accumulation in antigen-bearing tissues and lymph nodes (Selck and Dominguez-Villar, 2021). Additional explanation may come from quantitative biases in peptide–MHC presentation or the formation of peptide–MHC microclusters that favor cognate TCR engagement, even if multiple antigens are presented by single APC (Anderson and Roche, 2015; Bosch et al., 2013; Croft et al., 2013; Muixí et al., 2012).

The concept of infectious tolerance, which originated classically, is now understood as a mechanism whereby Tregs induce an inhibitory phenotype in other T cells (Gravano and Vignali, 2012; Sakaguchi et al., 2023). Specifically, Tregs collaborate with Treg-conditioned tolerogenic DCs to amplify and extend local immunosuppression, including pTreg differentiation by TGF-β and iTr35- and regulatory B cell-induction by IL-35. Furthermore, IL-27 produced by tolerogenic DCs, acting with IL-10, induces the differentiation of Foxp3⁻ IL-10-producing cells, type I regulatory cells (Tr1s; Brockmann et al., 2017; Gregori et al., 2010).

Thus, the mechanism of infectious tolerance should be closely associated with linked/bystander suppression. However, it has been consistently observed in experiments involving antigen-specific or disease-specific experimental settings (Cobbold and Waldmann, 1998; Gershon and Kondo, 1971; Kendal and Waldmann, 2010; Qin et al., 1993). Produced through chimeric antigen receptor (CAR) technology, CAR-Tregs that accumulate to target antigen-bearing tissue induce Treg differentiation in different antigen specific-T cells (Wardell et al., 2025). Whether physiological Tregs in vivo can spread infectious tolerance to T cells recognizing different antigens, via linked- or bystander-suppression, remains unclear.

4. DEVELOPMENT AND CLINICAL APPLICATION OF TREG CELL THERAPY

The discovery of Tregs and Foxp3 catalyzed a therapeutic paradigm harnessing immunosuppression to counteract a Tconv-dominant inflammatory state: Treg cell therapy (Fig. 2). Tregs offer the prospect of fundamental tolerance induction for autoimmune diseases and graft-versus-host disease (GVHD) when standard immunosuppressants fail or cause severe side effects. Treg-mediated suppression offers durable re-programming of immune cells toward homeostasis, potentially bridging acute control and long-term tolerance, and various clinical trials are being conducted. Representative trials reflecting translational trajectory and key quantitative outcomes are summarized in Table 1.

Fig.2.

Schematic overview of approaches to restore immune balance by enhancing regulatory T cells (Tregs).

The schematic illustrates four therapeutic strategies for correcting Tconv-dominant immune imbalance toward restored tolerance (purple box, right).

(1) In vivo Treg augmentation: Endogenous Tregs are selectively expanded using low-dose IL-2, engineered IL-2 variants, with rapamycin.

(2) Ex vivo expanded-nTreg therapy: CD45RA⁺CD127⁻ nTregs are sorted, expanded ex vivo with anti-CD3/CD28, IL-2, and rapamycin, and adoptively transferred.

(3) CAR-Treg therapy: nTregs are engineered with a CAR to confer antigen specificity before expansion and transfer.

(4) Stabilized iTreg therapy: Conventional T cells are converted into stable and functional iTregs (S/F-iTregs) via a two-cycle protocol combining CDK8/19 inhibition, transient CD28 deprivation, retinoic acid, vitamin C, rapamycin, and sustained IL-2, ensuring near-complete Foxp3 CNS2 demethylation prior to transfer.

Abbreviations: Tconv, conventional T cell; Treg, regulatory T cell; nTreg, natural regulatory T cell; iTreg, induced regulatory T cell; CAR, chimeric antigen receptor; IL-2, interleukin-2; RA, retinoic acid; CNS2, conserved non-coding sequence 2

Table 1.

Representative clinical trials of Treg-directed therapies, selected to reflect the translational trajectory.

Therapy Type / Strategy	Target Disease / Condition	Trial Phase	Status	Key Outcomes	Reference	Trial Registration No.
Low-dose IL-2	Chronic GVHD	Phase I/II	Completed	Tregs selectively expanded; clinical response in 52% (phase I) and 61% (phase II) of patients.	Koreth et al. 2011; Koreth et al. 2016	NCT00529035 (Koreth 2011); NCT01366092 (Koreth 2016)
Low-dose IL-2	Hepatitis C virus-associated vasculitis	Phase I/IIa	Completed	Treg recovery; vasculitis improvement in 8/10 patients.	Saadoun et al. 2011	NCT00574652
Low-dose IL-2	Type 1 Diabetes	Phase I/II	Completed	Dose-dependent Treg expansion; no serious adverse events; no significant effect on C-peptide preservation.	Hartemann et al. 2013	NCT01353833
Low-dose IL-2	Systemic Lupus Erythematosus	Phase I/IIa	Completed	Treg frequency selectively increased; SLEDAI reduced in 10/12 patients.	Humrich et al. 2019	DRKS00004858
Low-dose IL-2	Systemic Lupus Erythematosus	Phase II	Completed	Tregs expanded; significant disease activity reduction at week 24.	He et al. 2020	NCT02465580; NCT02932137
Low-dose IL-2	Chronic GVHD	Phase I	Completed	Treg-selective expansion without NK/CD8 activation; 82% PR rate in pediatric cohort.	Whangbo et al. 2019	NCT02318082
Rezpegaldesleukin (NKTR-358)	Atopic Dermatitis, Psoriasis	Phase Ib	Completed	Sustained increases in CD25bright Tregs; 83% EASI improvement at week 12 and exploratory 44.5% PASI improvement at week 12.	Silverberg et al. 2024	NCT04081350 (AD), NCT04119557 (Psoriasis)
Polyclonal nTreg	GVHD	Phase I	Completed	First-in-man; chronic GVHD: MMF withdrawal, major steroid reduction, lung function improvement. (Dose: 1×105 cells/kg) Acute GVHD grade IV: transient stabilization only. (Dose: 3×106 cells/kg total)	Trzonkowski et al. 2009	-
Polyclonal nTreg	Type 1 Diabetes (Pediatric)	Phase I	Completed	Safety confirmed; higher C-peptide levels at 12 months vs matched controls (non-significant). (Dose: 10–20×106 cells/kg)	Marek-Trzonkowska et al. 2012	NKEBN/8/2010
Polyclonal nTreg	Type 1 Diabetes (Adult)	Phase I	Completed	Safety confirmed at doses up to 26×108 cells; Treg persistence demonstrated. (Dose: 0.05–26×108 cells, total)	Bluestone et al. 2015	NCT01210664
Polyclonal nTreg	Type 1 Diabetes (Pediatric)	Phase II	Completed	Primary endpoint (stimulated C-peptide AUC) not met. (Dose: 1×106 cells/kg low-dose or 20×106 cells/kg high-dose)	Bender et al. 2024	NCT02691247
Polyclonal nTreg (Cord blood-derived)	Acute GVHD	Phase I	Completed	Grade II–IV acute GVHD: 43% (Treg) vs 61% (controls). (Dose: up to 10×106 cells/kg)	Brunstein et al. 2011	NCT00602693
Polyclonal nTreg (aAPC expansion)	Acute GVHD	Phase I	Completed	Grade II–IV acute GVHD: 9% (Treg) vs 45% (contemporaneous controls). (Dose: up to 100×106 cells/kg)	Brunstein et al. 2016	NCT00602693
Polyclonal nTreg	Kidney transplantation	Phase I/IIa	Completed	73% of recipients achieved tacrolimus monotherapy vs 0% in reference group. (Dose: 0.5–3×106 cells/kg)	Roemhild et al. 2020	NCT02371434
Donor-antigen reactive nTreg	Kidney transplantation	Phase I/II	Completed	All 3 recipients rejection-free at >6 years on tacrolimus monotherapy. (Dose: 0.86–1.9×104 cells/kg)	Guinan et al. 2023	NCT02129881
HLA-A2-specific CAR-Treg (TX200-TR101)	Kidney transplantation	Phase I/II	Ongoing	–	Schreeb et al. 2022	NCT04817774 (STEADFAST)
HLA-A2-specific CAR-Treg (QEL-001)	Liver transplantation	Phase I	Ongoing	–	–	NCT05234190 (LIBERATE)
iTreg (IL-2, TGF-β, rapamycin, anti-CD3-coated aAPC)	GVHD	Phase I	Completed	Engraftment unimpaired; circulating iTregs detectable for only 14 days; GVHD rates did not differ significantly from controls. (Dose: up to 3×108 cells/kg)	MacMillan et al. 2021	NCT01634217
Anti-tumor immunotherapy
Mogamulizumab (anti-CCR4; Treg depletion)	Relapsed/refractory cutaneous T-cell lymphoma	Phase III	Completed	ORR 28% vs 5% (vorinostat); Treg depletion via CCR4 also depletes effector memory T cells (selectivity limitation).	Kim et al. 2018	NCT01728805 (MAVORIC)
Bempegaldesleukin (CD25-blocked PEG-IL-2) + nivolumab	Advanced melanoma	Phase III	Completed	ORR 27.7% vs 36.0% (nivolumab alone); primary endpoint not met.	Diab et al. 2023	NCT03635983 (PIVOT IO 001)
RG6292 / vopikitug (anti-CD25; Treg depletion)	Solid tumors	Phase I	Completed	≥50% intratumoral Treg reduction achieved; 3 PR (combination arm with atezolizumab).	Belli et al. 2024; Gambardella et al. 2025	NCT04158583, NCT04642365
CHS-114 (anti-CCR8; Treg depletion)	Solid tumors	Phase I	Ongoing	–	Wang et al. 2026	NCT05635643

Abbreviations: aAPC, artificial antigen-presenting cell; AD, atopic dermatitis; AUC, area under the curve; CAR, chimeric antigen receptor; CCR4, C-C chemokine receptor type 4; CCR8, C-C chemokine receptor type 8; CD, cluster of differentiation; EASI, Eczema Area and Severity Index; GVHD, graft-versus-host disease; HLA, human leukocyte antigen; IL-2, interleukin-2; iTreg, induced regulatory T cell; MMF, mycophenolate mofetil; NK, natural killer; nTreg, naturally occurring regulatory T cell; ORR, overall response rate; PASI, Psoriasis Area and Severity Index; PEG, polyethylene glycol; PR, partial response; SLEDAI, Systemic Lupus Erythematosus Disease Activity Index; TGF-β, transforming growth factor-beta; Treg, regulatory T cell.

4.1. Treg biology as the foundation for therapeutic development

Clinical development has paralleled advances in Treg proliferation, function, and stability—particularly IL-2 dependency, mTOR signaling, and phenotype durability.

Tregs require IL-2 for survival and function even as they deplete environmental IL-2 to restrain effector T cells (de la Rosa et al., 2004; Setoguchi et al., 2005), informing low-dose IL-2 therapy. mTOR signaling orchestrates T cell growth and differentiation. mTORC1 inhibitors, such as rapamycin, suppress effector T cell proliferation while being comparatively permissive/beneficial for Treg expansion and function (Battaglia et al., 2006; Battaglia et al., 2005), reflecting metabolic program differences—glycolysis-biased effectors versus fatty-acid-oxidation-biased Tregs (Michalek et al., 2011). Rapamycin facilitates Foxp3 induction and maintenance, supporting iTreg differentiation (Haxhinasto et al., 2008; Sauer et al., 2008), and has become a mainstay in ex vivo expansion protocols and some clinical regimens (Brunstein et al., 2011).

A central challenge is Treg phenotype stability. Inflammatory milieus rich in IL-6 or IL-1β can destabilize iTregs in particular, leading to loss of Foxp3 and re-differentiation into Th1/Th17 (Bailey-Bucktrout et al., 2013; Zhou et al., 2009). Stability correlates with epigenetic status in the Treg-specific demethylated region (TSDR); for example, Foxp3 CNS2 is stably demethylated in nTregs but often methylated in unstable iTregs (Rossetti et al., 2015).

4.2. In vivo augmentation of Tregs

Early 2010s strategies sought to enhance nTregs without cell manipulation. Low-dose IL-2 (IL-2LD) exploits Tregs’ constitutive high-affinity IL-2 receptor (CD25) to preferentially expand/activate Tregs, avoiding the Tconv/NK activation and toxicity seen with high-dose IL-2; preclinical data were followed by clinical validation (Grinberg-Bleyer et al., 2010; Koreth et al., 2011; Schwartz et al., 2002).

In chronic GVHD, daily subcutaneous IL-2LD was found to selectively expand Tregs and improve symptoms; subsequent trials, including in pediatric practice, corroborate these benefits (Koreth et al., 2016; Koreth et al., 2011). Outside GVHD, Treg recovery with clinical improvement was reported in hepatitis C virus–associated vasculitis, and phase I/II studies in type 1 diabetes confirmed Treg-selective expansion with good tolerability despite being underpowered for clinical efficacy (Hartemann et al., 2013; Saadoun et al., 2011). In systemic lupus erythematosus, an open-label study and a randomized trial demonstrated increased Tregs and reductions in disease activity (He et al., 2020; Humrich et al., 2019), though optimal dosing and long-term safety remain difficult to define in this heterogeneous disease. Across indications, monitoring of pSTAT5, a direct readout of IL-2 receptor signaling that is selectively upregulated in Tregs at low IL-2 doses, is critical for individualized dose titration (Matsuoka et al., 2013). A dose-escalation study demonstrated that the pSTAT5 Treg/non-Treg ratio successfully guided Treg-selective expansion without NK/CD8 activation in adults and children with steroid-refractory chronic GVHD, with clinical responses observed predominantly in the pediatric cohort (Whangbo et al., 2019).

Despite these clinical advances, IL-2LD carries a narrow therapeutic window and requires frequent subcutaneous injections (Boyman and Sprent, 2012; Koreth et al., 2011; Matsuoka et al., 2013). To address these limitations, engineered IL-2 agonists with enhanced Treg selectivity and optimized half-life have entered clinical testing. PEGylation strategies bias binding away from intermediate-affinity IL-2 receptors and toward high-affinity receptors (Ptacin et al., 2024). Rezpegaldesleukin, a Treg-targeted IL-2 agonist, showed sustained increases in CD25^bright Tregs and clinical signals of efficacy in both atopic dermatitis and psoriasis in phase Ib studies, with responses sustained beyond 36 weeks after treatment discontinuation in both conditions (Silverberg et al., 2024).

Nevertheless, antigen specificity and tissue targeting remain unaddressable by IL-2-based approaches alone, providing a key rationale for the strategies discussed in subsequent sections.

4.3. Ex vivo–expanded nTreg therapy

Adoptive transfer of ex vivo–expanded Tregs has been pursued since the Treg discovery era. Early approaches isolated CD4⁺CD25⁺ nTregs from peripheral blood and polyclonally expanded them using anti-CD3/CD28 beads with IL-2. Foundational methods were established in the early 2000s, including large-scale expansion and the insight that CD45RA⁺ naïve Tregs yield more homogeneous, stable products; adding CD127^low improved purity and reproducibility (Hoffmann et al., 2006; Liu et al., 2006). Rapamycin has frequently been incorporated to preferentially expand and stabilize Tregs (Battaglia et al., 2006; Battaglia et al., 2005).

Clinically, Trzonkowski et al. first reported Treg infusion in steroid-refractory GVHD, demonstrating durable clinical benefit at 1×10⁵ cells/kg in chronic GVHD but only transient stabilization at higher doses in grade IV acute GVHD (Trzonkowski et al., 2009). Building on this safety signal, phase I trials in pediatric and adult type 1 diabetes showed safety at doses in the 10⁶–10⁷ cells/kg range, with preliminary signals of C-peptide preservation in the pediatric cohort (Bluestone et al., 2015; Marek-Trzonkowska et al., 2012). A subsequent randomized phase II trial in pediatric type 1 diabetes with 1–20×10⁶ cells/kg, however, did not meet its primary endpoint, indicating a need for optimized indications and dosing (Bender et al., 2024).

In umbilical cord blood transplantation, Brunstein et al. reported that cord blood–derived nTregs expanded with rapamycin reduced acute GVHD incidence at doses up to 10×10⁶ cells/kg (Brunstein et al., 2011); a subsequent trial using artificial APC (K562-derived KT64/86) enabled 10-fold higher doses, up to 100×10⁶ cells/kg, with further reduction in GVHD incidence (Brunstein et al., 2016).

In kidney transplantation, polyclonal nTreg therapy demonstrated safety and feasibility at doses of 0.5–3×10⁶ cells/kg, with 73% of recipients achieving tacrolimus monotherapy versus none in the reference group (Roemhild et al., 2020). To improve potency through antigen specificity, donor-antigen reactive Tregs expanded via the native TCR have been tested at ~10⁴ cells/kg, with all three recipients rejection-free on tacrolimus monotherapy beyond six years post-transplant (Guinan et al., 2023). However, reliance on the endogenous TCR repertoire limits targeting precision and scalability, motivating the development of synthetic CAR-based constructs (Fritsche et al., 2020).

4.4. CAR-Treg therapy

To enhance targeting, CAR-Tregs recognize specific antigens independently of the native TCR. Proof-of-concept was shown using trinitrophenol-specific and carcinoembryonic antigen-specific CAR-Tregs in colitis models (Blat et al., 2014; Elinav et al., 2009; Elinav et al., 2008). These findings demonstrated that CAR-directed homing amplifies local suppression, a principle then applied to transplantation, where donor-human leukocyte antigen (HLA)–specific targeting offered a clinically tractable entry point.

MacDonald et al. first demonstrated that HLA-A2–specific CAR-Tregs showed preferential interaction with HLA-A2⁺ targets and outperformed polyclonal Tregs in suppressing allogeneic T cell responses in vitro (MacDonald et al., 2016); Boardman et al. then showed that such CAR-Tregs preferentially transmigrated toward HLA-A2⁺ tissues and more effectively attenuated alloimmune-mediated skin graft injury than polyclonal Tregs in immunodeficient mice (Boardman et al., 2017). To bridge these findings toward clinical use, Dawson et al. systematically compared four CAR constructs, identified a fully humanized design as optimal, and confirmed preferential CAR-Treg accumulation at graft sites by in vivo imaging, collectively establishing the CAR design criteria required for first-in-human trials (Dawson et al., 2019). A phase I/II trial of HLA-A2–specific CAR-Tregs in kidney transplantation is underway (Schreeb et al., 2022). Building on the same HLA-A2–targeting concept, two independent CAR-Treg approaches are also advancing in parallel: Noyan et al. demonstrated that a distinct HLA-A2-CAR construct similarly preserved Foxp3 expression and TSDR demethylation while completely preventing allograft rejection in humanized mice without immunosuppression (Noyan et al., 2017), and a separately designed HLA-A2–specific CAR-Treg product (QEL-001) is under investigation in liver transplant recipients.

4.5. CAR-Tregs and infectious tolerance

Beyond enhancing antigen-specificity, Levings et al. are exploring advanced strategies using the target-homing capabilities of CAR-Tregs.

In an HLA-haploidentical cardiac transplant model, using donor hearts expressing HLA-A2 as the CAR target alongside two additional trackable antigens (2W and OVA), combining A2.CAR-Tregs with an otherwise subtherapeutic dose of anti-CD154 prolonged graft survival in a subset of recipients. In these acceptors, endogenous Tregs specific for the non-CAR donor antigen 2W expanded, and donor-specific CD8⁺ T cell responses against OVA were simultaneously suppressed, together demonstrating that CAR-Tregs induced infectious tolerance across multiple antigen specificities beyond the directly targeted HLA-A2. However, the mechanism underlying this infectious tolerance remains incompletely understood, and whether it reflects cytokine spreading or direct CAR-Treg engagement of recipient APCs presenting acquired donor HLA-A2 has yet to be determined (Durgam et al., 2025).

Moreover, in a diabetic model of allogeneic HLA-A2⁺ islet transplantation into immunodeficient mice co-injected with islet antigen-specific (BDC2.5) effector T cells, HLA-A2-CAR-Tregs induced pTreg differentiation from BDC2.5 T cells. Crucially, these newly induced BDC2.5 Tregs persisted and kept suppressing diabetes even after CAR-Treg depletion using diphtheria toxin, demonstrating that CAR-Treg-induced infectious tolerance is a viable therapeutic strategy (Wardell et al., 2025). These studies provide preclinical evidence that A2.CAR-Tregs can induce infectious tolerance across distinct transplant contexts, and confirming this phenomenon in humans and harnessing it efficiently in clinical protocols are key next steps.

4.6. iTreg therapy

To overcome nTreg scarcity and genetic modification complexity, strategies to generate iTregs from naïve CD4⁺ T cells ex vivo have long been pursued. Foxp3⁺ iTregs were induced by TCR stimulation with TGF-β + IL-2 (Chen et al., 2003), but early iTregs often showed Foxp3 instability and permitted conversion to inflammatory phenotypes under stress (Zhou et al., 2009). Then, TSDR (Foxp3 CNS2) demethylation subsequently emerged as the key to lineage stability (Ohkura et al., 2012), distinguishing epigenetically stable nTregs from unstable iTregs and establishing the benchmark for subsequent optimization efforts. Retinoic acid enhances iTreg induction efficiency (Benson et al., 2007), mTOR inhibition with rapamycin facilitates Foxp3 induction/maintenance (Chen et al., 2021; Haxhinasto et al., 2008; Lu et al., 2014), and sustained IL-2 signaling proved essential for Foxp3 stability (Chen et al., 2011).

The clinical potential of these interventions was directly tested in a phase I trial in which donor-derived iTregs generated using IL-2, rapamycin, TGF-β, and artificial APCs were administered at doses up to 3×10⁸ cells/kg for GVHD prevention following HLA-identical sibling peripheral blood stem cell transplantation. Infusions were well tolerated and engraftment was not impaired, but circulating iTregs were detectable for only 14 days, and GVHD rates did not significantly differ from contemporary controls (MacMillan et al., 2021). Notably, no TSDR demethylation was reported in the infused products, consistent with the limited persistence and lack of efficacy.

Subsequent preclinical work identified additional interventions targeting this epigenetic barrier. Vitamin C promotes TET-mediated CNS2 demethylation (Kasahara et al., 2017; Sasidharan Nair et al., 2016), CDK8/19 inhibition facilitates Foxp3 induction from antigen-experienced T cells (Akamatsu et al., 2019), and transient CD28 deprivation during early induction promotes hypomethylation across Treg signature loci (Mikami et al., 2020). In 2025, building on these insights, Sakaguchi and Mikami’s group reported a two-cycle protocol centered on CDK8/19 inhibition, transient CD28 deprivation, and sustained IL-2, retinoic acid, and vitamin C. The resulting iTregs, namely stable and functional iTregs (S/F-iTregs), achieved extensive TSDR demethylation and showed transcriptomic/epigenomic profiles closely resembling those of nTregs. Crucially, S/F-iTregs maintained Foxp3 under strongly inflammatory conditions (IL-6/IL-1β) and conferred potent therapeutic efficacy in murine colitis and arthritis models (Mikami et al., 2025).

Mukai et al. further converted pathogenic Dsg3-specific CD4⁺ T cells into S/F-iTregs using a similar protocol, demonstrating antigen-specific immunosuppression in autoimmune models. Specifically, in a T cell-mediated dermatitis model, the antigen-specific S/F-iTregs retained their Foxp3 phenotype and persisted in greater numbers in skin-draining lymph nodes compared to non-specific S/F-iTregs. They also suppressed anti-Dsg3 autoantibodies and clinical scores in a pemphigus model. Furthermore, analogous S/F-iTregs were generated from pemphigus patient T cells, providing initial evidence for the feasibility of antigen- and disease-specific Treg therapy (Mukai et al., 2025). Together, S/F-iTregs and their antigen-specific derivatives represent a mechanistically well-defined iTreg platform that supports renewed efforts toward clinical translation, in which confirming durable lineage stability under chronic inflammation in humans will be a key milestone.

4.7. Treg-targeted cancer immunotherapy

In contrast, in cancer immunotherapy Tregs represent a therapeutic problem rather than a therapeutic product, as noted in the Introduction, and the goal is to selectively deplete intratumoral Tregs (TI-Tregs) while preserving systemic immunity. Mogamulizumab, targeting CCR4 highly expressed on Tregs, demonstrated clinical activity in cutaneous T cell lymphoma, but its expression on effector T cells limits tumor selectivity (Kim et al., 2018). As an alternative strategy, bempegaldesleukin, a PEGylated IL-2 conjugate whose CD25-binding site is sterically blocked, was designed to preferentially signal through CD122/CD132 on CD8⁺ T cells and NK cells while sparing Treg expansion. Combined with nivolumab, however, it failed to improve outcomes over monotherapy in a phase III trial of advanced melanoma (Diab et al., 2023). Direct Treg depletion via RG6292 (vopikitug), an anti-CD25 antibody engineered to exploit the higher CD25 density on Tregs, achieved sustained peripheral Treg depletion in phase I studies but only limited objective responses, suggesting incomplete intratumoral elimination as the underlying limitation (Gambardella et al., 2025).

These results have directed attention toward CCR8, which is highly and selectively expressed on intratumoral Tregs across multiple solid tumor types with minimal expression on peripheral Tregs or effector T cells (De Simone et al., 2016; Plitas et al., 2016). Anti-CCR8 antibody CHS-114 selectively eliminates CCR8⁺ intratumoral Tregs with antitumor activity in preclinical models, and phase I evaluation is ongoing (Wang et al., 2026).

4.8. Safety considerations and lineage stability

Polyclonal Treg preparations carry inherent risks of bystander immunosuppression and impaired cancer immune surveillance; antigen-specific platforms such as CAR-Tregs aim to mitigate these (Fritsche et al., 2020; Raffin et al., 2020). Lineage instability represents a further hazard: ex-Foxp3 cells converted under inflammatory conditions can re-differentiate into pathogenic Th17 effectors, making near-complete TSDR demethylation an ongoing in vivo safety requirement rather than a one-time manufacturing checkpoint (Bailey-Bucktrout et al., 2013; Komatsu et al., 2014). To navigate these risks, the ONE Study demonstrated that infectious complications in seven parallel regulatory cell therapy trials in kidney transplantation did not exceed those in standard-of-care controls (Guinan et al., 2023; Roemhild et al., 2020; Sawitzki et al., 2020). A multiparametric monitoring framework combining a pSTAT5 Treg/non-Treg ratio for IL-2LD dose calibration with FOXP3 TSDR demethylation and inflammatory cytokine profiling for lineage surveillance provides a practical framework for broader clinical translation (Rossetti et al., 2015; Whangbo et al., 2019).

5. TISSUE-RESIDENT TREGS AND THERAPEUTIC PERSPECTIVES

Beyond immunoregulatory functions, tissue-resident Tregs outside lymphoid organs offer new therapeutic possibilities. In visceral adipose tissue, PPARγ-dependent Tregs regulate metabolic homeostasis and inflammation (Cipolletta et al., 2012). In skeletal muscle, Tregs accumulate post-injury, aiding the transition from inflammation to regeneration (Burzyn et al., 2013). Even in the central nervous system, Tregs infiltrate during steady-state and inflammation, resolving neuroinflammation and promoting tissue repair (Dombrowski et al., 2017; Liesz et al., 2009).

Rosenblum et al. elucidated the unique functions of skin Tregs. Skin Tregs localize near hair follicles and possess a “non-classical” function: promoting hair follicle stem cell differentiation and proliferation via the Notch ligand Jagged 1 to facilitate regeneration (Ali et al., 2017). They stably express Cxcr4 via glucocorticoid receptor signaling and are recruited to hair follicles by Cxcl12 from follicular epithelial cells (Cohen et al., 2025). Skin Tregs also express epidermal growth factor receptor, suppressing inflammation and promoting re-epithelialization during early wound healing (Nosbaum et al., 2016). Additionally, skin Tregs, which preferentially express high levels of GATA3, maintain local homeostasis, preventing fibroblast activation and skin fibrosis by suppressing local Th2 responses (Kalekar et al., 2019).

These findings enable organ- and disease-specific Treg therapies beyond immunosuppression. Strategies include restoring dysfunctional local Tregs or actively recruiting Tregs by targeting localization mechanisms such as the Cxcr4-Cxcl12 axis. Regenerative applications are also anticipated, such as administering Tregs or mimics during skeletal muscle or skin injury to promote repair. Tissue Treg biology offers potential for new therapies toward homeostatic reconstitution and tissue regeneration, with broad applications across autoimmunity, transplantation, metabolic disorders, fibrosis, and tissue degeneration.

6. CONCLUSIONS AND FUTURE DIRECTIONS

This review summarizes the historical ascent of Tregs to the forefront of immunology, their intricate suppressive mechanisms, and their translation to clinical arenas. Once questioned, the concept of T cells with suppressive function gained legitimacy through Sakaguchi’s demonstration of Tregs and the discovery of their master gene Foxp3 by Brunkow and Ramsdell, establishing these cells as a centerpiece of immune tolerance. Tregs deploy multilayered mechanisms to safeguard immune and tissue homeostasis.

The 2025 Nobel Prize awarded to Sakaguchi, Brunkow, and Ramsdell for their findings regarding peripheral immune tolerance symbolized the profound impact of Treg biology on medicine. Therapeutic exploitation of Tregs’ suppressive capacity is now advancing across multiple modalities—from IL-2LD to specificity endowed by CAR-Tregs to iTreg-stabilization strategies including the recently described S/F-iTregs. In parallel, tissue-resident Tregs have extended this paradigm to local control of metabolism, fibrosis, and tissue repair, suggesting prospects for regenerative Treg therapies.

Looking ahead, key challenges include ensuring lineage stability at clinical scale, efficiently inducing and delivering tissue/antigen-specific Tregs, and maintaining durable tolerance. Continued progress in Treg research is expected to support the development of more durable tolerance-inducing therapies in autoimmunity and transplantation.

Abbreviations:

APC

antigen-presenting cell

ATP

adenosine triphosphate

CAR

chimeric antigen receptor

cTreg

central Treg

CTLA-4

cytotoxic T-lymphocyte antigen 4

DC

dendritic cell

eTreg

effector Treg

GITR

glucocorticoid-induced tumor necrosis factor receptor

GVHD

graft-versus-host disease

HLA

human leukocyte antigen

IDO

indoleamine 2,3-dioxygenase

iTr35

IL-35-producing induced regulatory T cell

IL-2LD

low-dose IL-2

IPEX

immune dysregulation, polyendocrinopathy, enteropathy, X-linked

iTreg

induced Treg

LAG-3

lymphocyte activation gene-3

MHC

major histocompatibility complex

NK

natural killer

nTreg

natural Treg

PD-1

programmed cell death 1

PD-L1

programmed cell death ligand 1

pTreg

peripheral Treg

PVR

poliovirus receptor

S/F-iTreg

stable and functional iTreg

TCR

T cell receptor

Tconv

conventional T cell

TIGIT

T-cell immunoglobulin and immunoreceptor tyrosine-based inhibitory domain

Treg

regulatory T cell

Tr1

Type I regulatory cell

TSDR

Treg-specific demethylated region

tTreg

thymic Treg

TI-Treg

tumor-infiltrating Treg