Regulatory T Cell Metabolism: A Promising Therapeutic Target for Cancer Treatment?

Abstract

Regulatory T (Treg) cells are essential for maintaining immune homeostasis by suppressing excessive immune responses. In the context of cancer, however, Tregs promote immune evasion and tumor progression, particularly through their unique adaptations within the tumor microenvironment (TME). Recent research has emphasized how metabolic characteristics shape Treg activation, migration, and immunosuppressive function, revealing the impact of metabolic pathways on Treg fitness in homeostasis and within the TME. In this review, we first provide an overview of Tregs in cancer immunology, discussing their immunosuppressive roles and properties specific to the TME. We then examine the metabolic requirements for Treg activation and migration under normal conditions, followed by a discussion of how hypoxia, lactate accumulation, nutrient limitation, oxidative stress, and other TME-specific factors alter Treg metabolism and contribute to cancer immune evasion. Finally, we explore therapeutic strategies that target Treg metabolism within the TME, including pharmacological modulation of specific metabolic pathways to diminish Treg-mediated immunosuppression. Thus, we could suggest future directions and clinical implications for Treg-targeted metabolic modulation as a complementary approach in cancer treatment, setting the stage for novel strategies in immunotherapy.

INTRODUCTION

The field of cancer immunology has advanced significantly with the remarkable progress of immunotherapies; however, the tumor microenvironment remains a significant hurdle in cancer treatment, as it regulates tumor progression and directly impacts therapeutic outcomes (1). The TME is a complex, multicellular environment that supports tumor growth and is typically composed of various immune cells, including T lymphocytes, myeloid-derived suppressor cells (MDSCs), tumor-associated macrophages (TAMs), dendritic cells (DCs), NK cells, and stromal components such as cancer-associated fibroblasts, mesenchymal stromal cells, and the extracellular matrix (2). Among these components, immune suppression within the TME remains a major obstacle for effective cancer treatment. Immunosuppressive cells, such as MDSCs, TAMs, and Tregs, are key contributors to immune resistance in the TME (3). Tregs, a specialized subset of CD4⁺ T cells characterized by the expression of the transcription factor Foxp3, are particularly associated with poor tumor prognosis. Elevated levels of immunosuppressive cells, combined with protective niches that shield tumor cells from therapeutic interventions, further exacerbate treatment resistance. In healthy individuals, Tregs are critical for maintaining immune homeostasis and preventing autoimmunity. However, within the TME, Tregs act as potent suppressors of antitumor immunity, representing a significant barrier to effective cancer therapies. Understanding the dual roles of Tregs in immunity, along with their unique adaptations within the TME, is essential for developing innovative cancer treatments that can overcome these challenges.

The role of Tregs in immune regulation

Tregs are indispensable for immune regulation, acting as the guardians of immune tolerance (4,5,6). They mitigate inflammation and immune responses by suppressing the activation and proliferation of effector T cells (Teffs) and modulating antigen-presenting cells (APCs) (7). This suppression is achieved through various mechanisms, including the release of cytokines like IL-10, TGF-β, and IL-35 (6). Additionally, Tregs consume IL-2 via their high expression of the IL-2 receptor (CD25), depriving conventional T cells of this critical survival signal (8,9). By doing so, Tregs dampen excessive immune activation, maintaining self-tolerance and preventing harmful inflammatory responses. Tregs also suppress immune activation by interacting with APCs and modulating their activity. For example, Tregs express CTLA-4, which competes with CD28 on Teffs for binding to the costimulatory molecules CD80 and CD86, thereby reducing DC-mediated T cell activation and promoting an immunosuppressive environment. Furthermore, Tregs can exert contact-dependent suppression by directly inhibiting Teffs and other immune cells. The multifunctional suppressive nature of Tregs is indispensable for immune homeostasis, but it also poses challenges in cancer, where these cells contribute to immune evasion and tumor progression by suppressing antitumor immunity. These functions are essential for maintaining a balanced immune system, yet they also serve as a double-edged sword when co-opted by cancer.

Properties of Tregs in the TME

In the TME, Tregs exhibit unique properties that allow them to adapt and thrive under challenging conditions of hypoxia, nutrient deprivation, and increased immunosuppressive factors such as IL-10 and TGF-β (6,10,11). In addition, these cells are actively recruited to the TME via various chemokines such as CCL22, CCL28, and CCL5, which bind to their respective receptors on Tregs (12,13,14). Moreover, once at the TME, Tregs undergo phenotypic and metabolic reprogramming to enhance their suppressive capacity, making them potent mediators of immunosuppression and contributors to tumor progression. Tumor-associated Tregs exhibit heightened suppressive capabilities, driven by signals such as CTLA-4, PD-1, and metabolic pathways like glycolysis, lipid metabolism, and oxidative phosphorylation (OXPHOS). These adaptations not only enhance their survival but also allow them to dominate the immunosuppressive landscape, contributing to tumor progression and resistance to immunotherapy. By exploring the metabolic underpinnings of Treg function within the TME, we can identify novel opportunities for therapeutic intervention.

In this review, we examine the metabolic regulation of Tregs in both homeostasis and the TME, emphasizing the role of metabolites in facilitating immune evasion. We also explore current strategies for targeting Treg metabolism to inhibit tumor growth and enhance antitumor immune responses. Additionally, we summarize recent advances in the combination of immunotherapy and metabolic inhibitors, highlighting the ways in which the TME influences Treg metabolism. Ultimately, we discuss the therapeutic potential of targeting Tregs and their metabolic pathways as a promising strategy for improving cancer treatment outcomes.

Treg METABOLISM IN HOMEOSTASIS

Naïve T cell activation and Treg generation

Tregs can be categorized based on their site of generation into thymus-derived Tregs (tTregs) and peripheral Tregs (pTregs), each with distinct generation pathways and functions. tTregs originate from developing thymocytes in the thymus during the perinatal period, where they recognize self-ligands to establish central immune tolerance. In contrast, pTregs are generated from developing thymocytes in peripheral tissues to regulate immune responses and maintain homeostasis. In addition to the 2 aforementioned types of Tregs, certain Foxp3⁺CD4⁺ T cells reside in lymphoid follicles, known as follicular Tregs (Tfrs). Tfrs further differentiate from Tregs and dwell in lymphoid follicles to modulate the germinal center reaction. The differentiation of Tregs starts with the activation of naïve T cells whose TCR recognizes self-antigens displayed by MHC molecules (15). T cell activation significantly increases energy demands, leading to marked changes in metabolic pathways—a process known as metabolic reprogramming (16). Following TCR stimulation, the mTORC1 pathway is upregulated, driving the metabolic shift towards glycolysis. Activation of mTORC1 by ASCT2 is essential for the proper naïve T cell activation (17). In addition, upon T cell activation, glutamine uptake is upregulated by costimulatory signals such as CD28, which induces the expression of amino acid transporters (17). Glutamine metabolism is further activated by the ERK/MAPK pathway downstream of TCR/CD28 signaling, highlighting the critical role of glutamine in activated T cells (18). In summary, naïve T cell activation leads to significant metabolic reprogramming, characterized by upregulation of both glycolysis and glutamine metabolism, which are essential for the generation and function of Tregs (Fig. 1).

Figure 1. Treg metabolism in homeostasis. (A) Generation: upon TCR recognition of self-antigens, the ERK pathway is activated, leading to enhanced expression of genes related to Gln metabolism (SNAT, GS, GLS). Gln is taken up via SNAT and converted to Glu by GLS. Glu can be converted back to Gln by GS. This Gln metabolism supports Treg stability. In contrast, after TCR stimulation, CD28 activation triggers the PI3K pathway, which activates mTORC1. This enhances HIF-1α expression, promoting glycolysis and differentiation into Th17. (B) Migration: TCR stimulation and CD28 activation lead to the activation of the PI3K pathway, which subsequently activates mTORC2. mTORC2 activates AKT and inhibits Foxo1, promoting the expression of CCR7 and CD62. Additionally, mTORC2 upregulates GCK, enhancing glycolysis and facilitating cytoskeletal rearrangement, which is critical for T cell migration and activation. (C) Suppressive function: CD28 and PI3K activate mTORC1, which enhances glycolysis. NAD⁺ produced during glycolysis activates SIRT1, resulting in the deacetylation and polyubiquitination of FOXP3, making it more susceptible to degradation. mTORC1 also regulates mitochondrial function and stimulates the mevalonate pathway, which enhances the expression of immunosuppressive molecules (ICOS, CTLA-4). Additionally, FAO promotes acetyl-CoA production, which facilitates the acetylation of FOXP3, contributing to the stabilization of FOXP3 expression and the maintenance of Treg suppressive function. Created with BioRender.com.

SNAT, sodium-coupled neutral amino acid transporter; GS, glutamine synthetase; GLS, glutaminase; Gln, glutamine; Glu, glutamate.

Glycolysis and Treg generation

Glycolysis is a metabolic checkpoint in Treg differentiation. Compared to Th17 cells, Treg generation demands less ATP and exhibits lower levels of glycolysis, allowing the Th17/Treg balance to be regulated by modulating glycolysis levels through various signaling pathways, such as the PI3K-Akt pathway and the mTOR-hypoxia-inducible factor 1 (HIF1) pathway. Among these signaling pathways, mTOR plays a central role in integrating environmental cues and regulating glycolysis levels and Treg differentiation. For pTreg differentiation, mTOR exhibits an inhibitory effect on pTreg generation (19). Research highlights the inhibitory role of mTOR in Treg generation (19,20). Hyperactivation of mTOR by leptin which is cytokine-like hormone can render Tregs less responsive to TCR signals and inducing an anergic state in vitro, impeding their entry into the cell cycle and proliferation (19,21). In contrast, inhibiting mTOR with rapamycin reduces glycolysis levels, promoting Treg generation (22). However, for Tfrs, mTORC1 plays an essential role in the early stage of their differentiation. Research indicates that mTORC1 is more activated in Tfrs than in conventional Tregs. mTORC1 initiates Tfr differentiation through the TCF-1-Bcl-6 axis, thereby increasing the expression of Tfr signaling molecules such as inducible costimulatory (ICOS) and CTLA-4 (23). Downstream of the mTOR pathways, HIF-1α can regulate glycolysis levels and influence Treg differentiation. Additional research shows that HIF-1α can enhance glycolysis by activating glucose transporter (GLUT1, MTC4) and glycolytic enzymes (HK2, GPI, TPI, Eno1, PKM, LDHA) in Th17/Treg differentiation through mTOR downstream pathways, steering T cell precursors towards Th17 differentiation (24). HIF-1α deficiency can reduce glycolysis levels, impair Th17 differentiation, and decrease IL-17 production, while promoting higher Foxp3 induction, which leads to Treg differentiation (24). Collectively, glycolysis levels can be modulated by mTOR pathway, and influence the Th17/Treg balance, shaping the local immune environment (Fig. 1).

Other metabolisms and Treg generation

Tregs exhibit unique metabolic characteristics, relying more on fatty acid oxidation (FAO) than glycolysis for energy. Foxp3 expression requires lipid uptake and oxidation, as shown by research indicating that supplementing media with lipids can counteract ERRα-mediated suppression of Treg differentiation (25,26). However, other studies suggest that activating FAO while inhibiting glycolysis may limit Treg proliferation, although the effect is more pronounced in Teffs. Glutamine metabolism also plays a role in Treg generation. Glutamine is essential for CD4⁺ T cell growth and proliferation following TCR/CD28 signaling but does not affect the initial activation of T cells. The expression of glutamine transporters ASCT2 and SLC7A5, together with TCR/CD28 signaling, activates the mTORC1 pathway, driving the proliferation and differentiation of CD4⁺ T cells (17). Glutamine also steers CD4⁺ T cells toward Th17 differentiation, altering the Th17/Treg balance in tissues. Interestingly, Treg formation is less dependent on glutamine and may even be slightly promoted in a glutamine-free environment (17). Mechanistically, glutamine serves as a precursor in the synthesis of GSH, \\xc9\\x91-KG, and N-glycan branching, influencing ROS stress, tricarboxylic acid (TCA) cycle, fatty acid synthesis, and IL-2 receptor affinity during Th17/Treg differentiation (27). Thus, glutamine not only supports CD4⁺ T cell proliferation but also shapes the fate of T cell intermediates, determining differentiation into either Th17 or Tregs.

Treg MIGRATION AND METABOLISM

The migration of Treg through afferent lymphatics involves their interaction with lymphatic endothelium. Tregs display lymphotoxin alpha beta (LTα1β2) on their cell surface, induced by IL-2R activation signal through NF-kB and MAPK signaling pathways. LTα1β2 engages with the lymphatic endothelium, enhancing its permissiveness (28). The migration and recirculation of tTregs from peripheral tissues to lymphoid organs are orchestrated by chemokine signals, such as CCR7/CCL19 or CCL21, which are secreted by the lymphatic endothelium. In contrast, the migration of pTregs is typically under inflammatory conditions and can be activated by a spectrum of inflammatory cytokine (29). CD28 costimulatory signaling initiates Treg migration, whereas CTLA-4 coinhibitory signaling acts to hinder this process (30). Unlike generation, activation, and suppression, Treg migration is a glycolysis-driven process that significantly depends on glucose uptake and metabolic activity. LFA-1, a molecule mediating Treg migration, enhances glucose uptake in Tregs, while CD28 signaling, which initiates migration, further augments glucose uptake (30). Mechanistically, CD28 activates the PI3K-Akt pathway, inducing the expression of glucokinase (GCK), a key enzyme in glycolysis (30), and also activates mTOR, a central regulator of glycolytic metabolism. CD28 can also activate mTOR, central to stimulating the metabolic level of glycolysis. Heightened glycolysis supports Treg migration by supplying ATP via GCK to localized Na⁺/K⁺-ATPase, facilitating cytoskeletal rearrangement critical for cell movement (30). Additionally, mTORC2 activation promotes Treg migration by phosphorylating Akt, which inhibits Foxo1 activity, thereby regulating the expression of migration-related molecules such as CD62L, CCR4, and CD69 (31). mTOR plays a dual role by stimulating Treg migration from the bloodstream to non-lymphoid tissues while inhibiting their recirculation in lymphoid tissues (31). Notably, mTOR-deficient Tregs exhibit reduced migration from the bloodstream to non-lymphoid tissues but enhanced migration to lymphoid tissues, emphasizing the importance of glycolysis and mTOR signaling in Treg migratory behavior (Fig. 1).

Treg SUPPRESSIVE FUNCTION

The immunosuppressive function of Tregs is characterized by their ability to interact with other immune cells and regulate immune responses. Tregs secrete granzyme B and perforin, which induce apoptosis in NK cells, Teff, and other effector immune cells. This suppressive activity is mediated by Foxp3, the master regulatory transcription factor that governs the expression of numerous Treg-specific genes essential for their immunosuppressive function (32). Given that Foxp3 directs the characteristic immunosuppressive functions of Tregs, it can be considered a defining molecular marker of Tregs (Fig. 1).

Glycolysis and Treg suppressive function

Glycolysis plays a dual role in maintaining the Treg function. Some metabolites in glucose metabolism, such as phosphoenolpyruvate (PEP) and mitochondrial ROS, can directly modulate Foxp3 expression, highlighting the connection between glucose metabolism and Treg suppression function (16). Firstly, elevated glycolysis levels are detrimental to Treg stability and function. Autophagy-related molecules, such as Atg7 and Atg5, limit glycolysis by preventing overactivation of the mTORC1-Myc pathway, which is essential for maintaining Treg functional fitness. Deletion of Atg7 or Atg5 has been shown to reduce Treg suppressive capacity (33). Another study demonstrates that glycolysis can modulate the expression of Foxp3 at the post-translational level using histone acetylation (34). The final step of aerobic glycolysis produces NAD+, which activates the deacetylase SIRT1. SIRT1 can mediate the deacetylation of Foxp3, making it more susceptible to polyubiquitination (35). Thus, glycolysis can impair the stability of Treg. Conversely, a moderate level of glycolysis is essential for preserving Treg function. For instance, the glycolytic enzyme enolase-1 induces Foxp3 splice variants that enhance Treg suppressive activity (36). Furthermore, under hypoxic conditions, HIF1-α induces Foxp3 expression, maintaining Treg function (37). Collectively, glycolysis can inhibit Treg function through autophagy and post-translational modification, while also maintaining Treg suppression by post-transcriptional splicing and HIF1 signaling. Despite glycolysis’s influence on Foxp3 expression levels, Foxp3 can also regulate the metabolic level of glycolysis. Foxp3 can mediate metabolic reprogramming of Tregs, such as impairing glycolysis and enhancing OXPHOS. These metabolic features of Tregs make them glucose-dependent, allowing them to perform their immunosuppressive function in low-glucose tissues, such as the colon wall (38).

Fatty acid metabolism and Treg suppressive function

Given that Tregs are less reliant on glucose metabolism, fatty acids are selected as the energy source for Tregs to maintain their suppressive function. Firstly, higher level of FAO is essential for Treg suppressive function. Mechanistically, mTOR can increase IRF4 expression, thereby mediating metabolic reprogramming to enhance OXPHOS and mitochondrial metabolism (39). FAO process can also produce acetyl-CoA, which enhances Foxp3 stability through acetylation, protecting it from ubiquitination and degradation (40). Thus, FAO can maintain Treg function at the transcriptional and post-translational level. Additionally, glycolysis-driven lipid synthesis contributes to Treg functional fitness, and it is regulated by mTORC1. Secondly, glycolysis-driven lipid synthesis contributes to Treg functional fitness, and mTORC1 is a key signaling pathway that regulates fatty acid metabolism and Treg suppressive function. Upon Tregs receiving stimulation from TCR signals and IL-2, mTORC1 is activated. mTORC1 then increases lipogenic metabolism to meet bioenergetic demands (19). Among these lipid synthesis pathways, the mevalonate pathway is notable because it facilitates Tregs proliferation, and it also can upregulate CTLA-4 and ICOS, surface suppressive effector molecules that perform immunosuppressive functions in Tregs (19). Additionally, mTORC1 can also depress mTORC2 to promote the immunosuppressive function (19). Collectively, lipogenic metabolism, regulated by mTORC1, plays a significant role in Treg suppressive function.

Other metabolisms and Treg suppressive function

Glutamine metabolism influences the stability of Treg. Low levels of glutaminolysis and α-KG production, regulated by mTOR, increase Foxp3 stability through CpG methylation at the Foxp3 locus (31). Thus, low levels of glutamine metabolism can increase the stability of Foxp3 expression and maintain Treg function at the epigenetic level.

Purine catabolism plays a significant role in regulating Treg function, with ATP and NAD⁺ promoting inflammation and adenosine promoting the suppression of immune reactions. Extracellular ATP and NAD⁺ which are released by cell lysis or non-lytic mechanism can activate P2X7 receptor to inhibit Foxp3 expression, thereby impairing Treg suppressive function (41,42). During chronic inflammation, P2X7 signaling can even convert Tregs into Th17 to regulate local immune responses. Extracellular adenosine can bind to the A2A receptor and facilitate generation of Treg and also maintain the suppressive function of Tregs (43). In summary, Treg functional fitness is intricately tied to their metabolic processes, with glycolysis, FAO, and purine catabolism playing distinct yet interconnected roles in maintaining their stability and functionality. These metabolic pathways are essential for modulating Foxp3 expression and, by extension, Treg immunosuppressive capacity.

TUMOR MICROENVIRONMENT AND Treg METABOLIC ADAPTATIONS

Hypoxia

Hypoxia is a hallmark of the TME, complexly linked to cell proliferation, angiogenesis, metabolism, and immune responses (44). The primary regulatory factors of hypoxia are HIFs, which play pivotal roles in modulating Treg metabolism under hypoxic conditions, primarily through the stabilization of HIF-1α (45). HIF-1α is critical for regulating Treg function in hypoxic tumor environments, such as glioblastoma. It enhances glycolysis to promote Treg migration while supporting lipid metabolism to maintain their suppressive function. HIF-1α-deficient Tregs exhibit reduced migration and rely on OXPHOS, leading to diminished immunosuppression and improved tumor control. These Tregs also show enhanced mitochondrial respiration and fatty acid metabolism, indicating a metabolic reprogramming toward lipid utilization for energy production. This metabolic shift is accompanied by signaling changes, including AMPK activation, and mTORC1 suppression (46). Notably, this metabolic adaptation is vital for maintaining their immune-suppressive function, as inhibition of fatty acid metabolism significantly reduces Treg abundance and suppressive markers, including Granzyme B, CD39, CTLA4, and neuropilin-1 (NRP1). Targeting HIF-1α could disrupt Treg-mediated immunosuppression and enhance immune responses in glioblastoma (37,47). In the TME, intratumoral Tregs preferentially utilize lipids over glucose compared to other T cell subsets, a phenomenon reflected by the upregulation of fatty acid transporters, such as CD36 and SLC27A1, on their surface (47). Beyond metabolism, HIF-1α also influences Treg migration. Effective tumor infiltration by Tregs is dependent on glycolysis, suggesting that HIF-1α-driven metabolic pathways contribute to their migration within the TME. Collectively, these findings emphasize that Tregs adapt their metabolic profiles to hypoxic conditions in the TME, favoring lipid metabolism regulated by HIF-1α. This adaptation not only supports their immune-suppressive capacity but also enhances their migratory potential, highlighting HIF-1α as a critical regulator of Treg function within the TME (Fig. 2) (47). The role of HIFs in Treg function is critical for regulating tumor progression, but their specific contributions differ. While HIF-1α destabilizes Tregs and promotes their fragility, leading to enhanced anticancer immunity, HIF-2α plays a contrasting role in maintaining Treg suppressive function and supporting tumor growth. In models where HIF-2α was specifically deleted in Tregs, tumor growth of MC38 colon adenocarcinoma was significantly suppressed, with half of the mice exhibiting complete tumor resistance. Similarly, Treg-specific HIF-2α deletion reduced lung metastases in B16F10 melanoma models, as evidenced by fewer tumor nodules and altered lung morphology. Interestingly, the protective effects of HIF-2α-knockout (KO) Tregs appear to involve a modest upregulation of HIF-1α, which enhances their anticancer properties. These findings highlight the distinct roles of HIF-1α and HIF-2α in Treg function, with HIF-1α promoting Treg fragility and anticancer immunity, while HIF-2α supports their stability and immunosuppressive activity. Targeting HIF-2α in Tregs emerges as a promising therapeutic strategy to suppress tumor growth and metastasis (48).

Figure 2. Treg metabolism in the TME. (A) Hypoxia: under hypoxic conditions in the TME, HIF-1α expression is upregulated. This promotes lipid uptake via CD36, leading to enhanced fatty acid metabolism while reducing glycolysis. Additionally, Tregs exhibit increased immunosuppressive functions, as indicated by the elevated expression of molecules associated with suppression, such as CD39, CTLA-4, NRP1, and Granzyme B. (B) Lactate accumulation: Tregs uptake lactate via MCT1, and lactate is converted to pyruvate by LDH. During pyruvate production, NAD⁺ is converted to NADH and NADH is subsequently regenerated to NAD⁺ in the mitochondria. Mitochondria utilizes pyruvate to generate energy. Also, lactate upregulates PEP, which enhances intracellular Ca²⁺ levels. Elevated Ca²⁺ upregulates NAFT1, leading to PD-1 expression on surface of Tregs. In contrast, FOXP3 downregulates Myc, resulting in reduced glycolysis. (C) Nutrient limitation: Trp depletion activates GCN2, leading to the upregulation of IDO1 and TDO2. These enzymes convert Trp into Kyn, which binds to and activates the AHR. The Kyn-AHR complex enters the nucleus, where it upregulates immune suppressive genes such as FOXP3 and IL-10. Additionally, due to glucose depletion in the TME, Tregs rely on lactate metabolism as described in (B). (D) Oxidative stress: oxidative stress induces Treg apoptosis. Apoptotic Tregs secrete ATP via transporters, which is converted to adenosine by the CD39/CD73 axis. Adenosine suppresses other effector cells, reducing cytokine production (e.g., IFN-γ, TNF-α) and diminishing antitumor immune functions. Created with BioRender.com.

Lactate accumulation

Tumors consume large amounts of energy to support their growth and survival. To achieve this, they take up excessive glucose and perform glycolysis, favoring this pathway regardless of oxygen availability—a phenomenon known as the Warburg effect. Glycolysis primarily produces lactate, leading to its accumulation in the TME (49). This high concentration of lactate weakens the function of most immune cells but enhances the suppressive capacity of Tregs. Tregs thrive in high-lactate conditions due to mechanisms closely tied to Foxp3 expression. Foxp3 suppresses glycolysis by downregulating MYC, a transcription factor critical for activating glycolysis in T cells and promotes OXPHOS instead. By binding to the MYC promoter, Foxp3 reduces MYC expression, leading to a metabolic shift in Tregs from glycolysis to OXPHOS (49,50). Moreover, Foxp3⁺ Tregs oxidize lactate into pyruvate instead of producing lactate, enabling them to adapt to and resist the suppressive effects of high lactate concentrations—a process dependent on LDH. Additionally, Tregs exhibit resistance to NAD⁺ depletion through NAD⁺ regeneration via OXPHOS (38,51). In some cases of gastric cancer, G-protein-coupled receptor 81 which is natural receptor of lactate, is highly expressed and Treg infiltration is increased in tumor by the upregulation of the chemokine, remarkably CX3CL1. The infiltrated Tregs exhibit enhanced immunosuppressive function (52). Furthermore, lactate in TME can also promote PD-1 expression on Tregs (53). Tregs found in glycolytic tumor tissues, which highly express LDHA and MYC, display increased PD-1 expression. PD-1^hi Tregs show significantly higher expression of MCT1, a lactate transporter, and CD147 which is closely associated with MCT1. Elevated lactate level enhanced PD-1 expression and enhanced suppressive functions in Tregs. The mechanism involves lactate uptake through MCT1, regulated by Foxp3 which increases PEP, intracellular Ca²⁺ levels, and intranuclear NFAT1 expression in Tregs. Notably, PD-1^hi Tregs show increased immune suppressive function after anti-PD-1 monoclonal antibody treatment. Thus, PD-1^hi Tregs can be predictive marker for immune checkpoint blockade (ICB) therapy and targeting tumor LDHA or Tregs MCT1 may provide a solution to overcome treatment resistance (Fig. 2) (54). In summary, Tregs maintain their suppressive function in high-lactate conditions while enhancing PD-1 expression, making PD-1^hi Tregs a predictive marker for ICB therapy. Targeting tumor LDHA or Treg MCT1 presents a potential strategy to overcome resistance to such treatments.

Nutrient limitation

Nutrient limitation is a common feature of the TME due to dysfunctional angiogenic signaling and the physical compression of blood and lymphatic vessels within the tumor. This leads to reduced delivery of essential nutrients such as glucose and amino acids. Additionally, tumors consume a substantial amount of nutrients to sustain their rapid growth and metastasis (55). As a result, cancer cells and immune cells must compete for these limited resources. Immune cells, such as CD8⁺ T cells, that fail to acquire sufficient nutrients exhibit reduced antitumor functionality (56). Tregs, however, can avoid nutrient competition in the TME by altering their metabolism. Instead of relying on glucose, Tregs utilize alternative carbon sources like lactate (51). Cancer cells typically uptake glucose and produce lactate through glycolysis, which accumulates in the TME. Tregs use the lactate transporter MCT1 to absorb lactate and integrate it into their metabolism. This metabolic adaptation allows Tregs to maintain their suppressive function and achieve metabolic stability. From a therapeutic perspective, this reliance on lactate results in poor responses to ICB therapies, such as anti-PD-L1 (51). Enhancing the response to immune checkpoint therapies may involve targeting Treg metabolism. For instance, forcing Tregs to rely on glucose instead of lactate could reduce their stability and suppressive function, thereby improving therapy outcomes. Amino acid limitations in the TME also impact Treg function. For example, tryptophan (Trp) depletion activates the GCN2 kinase pathway. Activated GCN2 increases the expression of enzymes such as indoleamine 2,3-dioxygenase 1 (IDO1) and Trp 2,3-dioxygenase 2, which convert Trp into kynurenine (Kyn). Kyn, in turn, activates the aryl hydrocarbon receptor (AHR), enhancing the immunosuppressive function of Tregs by upregulated FOXP3 and IL-10 expression (Fig. 2) (57). Overall, Tregs can adapt to nutrient limitations, such as glucose and amino acid scarcity, in the TME by reprogramming their metabolism. This metabolic flexibility enables them to sustain their suppressive features, highlighting the potential for therapeutic strategies that target Treg metabolic pathways to improve cancer immunotherapy.

Oxidative stress

Oxidative stress is mediated by elevated levels of ROS, which are highly reactive oxygen-containing molecules, including superoxide, peroxides, and non-radical hydrogen peroxide (58). ROS are primarily generated by the electron transport chain and OXPHOS during aerobic respiration in mitochondria. While ROS play diverse roles in cell survival and function, maintaining their equilibrium is crucial. Under hypoxic conditions, however, cancer cells shift their metabolism from OXPHOS to glycolysis for energy production, leading to increased ROS generation (59). ROS also significantly impact immune cells, particularly Tregs, by altering their properties and enhancing their suppressive function. Oxidative stress can activate the PI3K/AKT pathway, which is closely related to Tregs immune suppressive functions by stabilizing FOXP3 expression (20,60,61). Also, oxidative stress can induce apoptosis in Tregs, and apoptotic Tregs release ATP, which is subsequently converted into adenosine via the CD39-CD73 axis. This adenosine suppresses the production of cytokines such as TNF-α and IFN-γ, resulting in diminished antitumor activity and reduced efficacy of PD-1 therapy (62). Additionally, in immune checkpoint therapy non-responders, the knockdown of complex I to inhibit oxidative stress has been shown to increase Treg populations among tumor-infiltrating lymphocytes (TILs) (Fig. 2). This suggests that regulating Treg infiltration into tumors is a critical factor for enhancing therapeutic efficacy (63).

Others

In the TME, genes associated with lipid metabolism significantly influence the function and survival of Tregs. Intratumoral Tregs exhibit elevated expression of lipid metabolism-related genes and demonstrate enhanced fatty acid absorption and storage compared to circulating Tregs (64). Among these, sterol regulatory element binding protein (SREBP) and fatty acid synthase (FASN) play crucial roles in maintaining the metabolic balance of Tregs, particularly in regulating their immunosuppressive functions. In SREBP cleavage-associating protein-deficient mice, where SREBP-related gene expression in Tregs is inhibited, the proportion of Tregs increased; however, a significant proportion of these Tregs began producing IFN-γ. This indicates an abundance of less stable Tregs, characterized by reduced expression of the immune suppressive receptor PD-1, which in turn diminishes their suppressive function. As a result, Teff activity improved, leading to enhanced anti-tumor responses. Similarly, Tregs deficient in FASN showed reduced immune suppressive capacity, which contributed to decreased tumor growth (65). The CD36 gene also plays a key role in Treg metabolism. In CD36 KO mice, intratumoral Tregs displayed impaired fatty acid absorption and storage, accompanied by reduced tumor growth and increased expression of anti-tumor cytokines such as IFN-γ and TNF. CD36 deficient mice showed decreased Treg accumulation in tumors and increased apoptosis of Tregs. CD36 further influences mitochondrial function through PPAR-β signaling, creating an amplification loop that enhances CD36 expression and mitochondrial fitness. In CD36 KO mice, mitochondrial membrane potential and cristae density were reduced, leading to impaired OXPHOS and a metabolic shift toward glycolysis. Additionally, a lower NAD/NADH ratio created harsher conditions for Tregs to survive in the high-lactate environment of the TME (66). Another gene, FABP5, also plays a critical role in Treg survival and suppressive function. Inhibition of FABP5 impairs mitochondrial metabolism, exposing mitochondrial DNA to the cytoplasm and activating the cGAS-STING pathway, which induces type 1 IFN signaling. This inhibition reduces mitochondrial mass, OXPHOS, and the production of specific phospholipids in the mitochondrial inner membrane. Paradoxically, the activation of type 1 IFN signaling by FABP5 inhibition increases the production of IL-10, an immunosuppressive cytokine, along with elevated expression of Treg markers such as CD25 and ICOS. These changes enhance Treg survival and immunosuppressive function (67). Therefore, targeting genes related to the lipid metabolism of Tregs, such as SREBP, FASN, CD36, and FABP5, presents a promising strategy to improve the outcomes of cancer immunotherapy by disrupting Treg-mediated immune suppression in the TME.

THERAPEUTIC POTENTIAL: STRATEGIES TARGETING METABOLISM FOR THERAPY

Metabolic inhibitors: targeting Treg function

The unique metabolic dependencies of Tregs within the TME have emerged as promising therapeutic targets in cancer immunology. To sustain their survival and suppressive functions under nutrient-deprived, hypoxic conditions, Tregs rely on distinct metabolic pathways, including OXPHOS, FAO, and glycolysis. Therapeutically targeting these pathways offers a strategy to selectively disrupt Treg activity while preserving or even enhancing the function of effector immune cells.

Glucose metabolism plays a pivotal role in maintaining Treg suppressive functions, making it a critical target for disrupting tumor immune evasion. Similar to tumor cells, Tregs exhibit increased glucose consumption, promoting cellular senescence and suppressing Teff activity (68). Exploiting this metabolic vulnerability, TLR8 signaling has been shown to selectively inhibit glucose uptake and glycolysis in Tregs, reversing their suppressive functions and enhancing antitumor immunity (69). Glycolysis inhibitors such as 2-deoxy-D-glucose (2-DG) and galloflavin further suppress glycolysis-related gene and protein expression in Tregs, impairing their ability to suppress Teff proliferation, particularly in ovarian cancer models. These findings highlight glycolysis as a promising therapeutic target to enhance the efficacy of cancer immunotherapy (70).

Another example is CPI-613 (devimistat), which inhibits pyruvate dehydrogenase, a key enzyme for the transition from glycolysis to the TCA cycle. By disrupting bioenergetic and biosynthetic pathways, CPI-613 significantly reduces Treg populations and metabolic adaptations in lymph node-derived regulatory cells in immunosuppressive lymph nodes. This inhibition enhances immune responses against human xenografted lung cancer cells in preclinical models (71).

The pharmacological inhibition of MCT1 using AR-C155858 also demonstrates potential by reducing PD-1 expression on Tregs, thereby suppressing their proliferation and immune suppressive functions. This approach has shown efficacy in reducing colon carcinoma growth (54). Similarly, targeting LDHA, which catalyzes the conversion of pyruvate to lactic acid and sustains the immunosuppressive TME, using agents like GSK2837808A and GNE-140, significantly reduces lactic acid production in tumors (54,72). ML-05, a novel lactate production inhibitor, disrupts ATP production and induces ROS generation, leading to G1 phase cell cycle arrest. In a mouse model of B16F10 melanoma, ML-05 administration significantly reduced lactate levels, suppressed tumor growth, and enhanced antitumor immunity by activating Th1 cells and GMZB⁺CD8⁺ T cells within the TME (73).

IDO1, which regulates Trp metabolism, plays a crucial role in modulating the immune microenvironment. IDO1-mediated Trp depletion and the accumulation of Kyn suppress Teff function while hyperactivating Tregs, thereby promoting tumor immune evasion (74,75). Inhibitors of IDO1 disrupt this pathway, limiting Treg-mediated immune suppression and reactivating antitumor immunity (57).

Targeting lipid metabolism has also shown promise. Drugs like TOFA and C75 have limited efficacy as monotherapies for tumor control (76,77), but targeting CD36 with antibodies effectively reduces Treg suppression and inhibits tumor growth (66). Additionally, metabolic inhibitors targeting FAO, such as etomoxir, selectively reduce Treg-mediated immune suppression, thereby enhancing antitumor immune responses (76).

As these therapeutic approaches evolve, ongoing research must focus on refining the specificity and safety of metabolic inhibitors to minimize systemic immune dysregulation while maximizing therapeutic efficacy. The continued advancement of these strategies holds significant promises for overcoming immunotherapy resistance and reshaping the TME to favor anti-tumor immunity.

Metabolic supplements: reprogramming the TME

In addition to metabolic inhibitors, metabolic supplements offer an alternative strategy to reprogram the TME and modulate Treg function. By altering nutrient availability and supporting effector immune cell metabolism, these supplements aim to counteract the suppressive influence of Tregs and enhance antitumor immune responses. For instance, deficiencies in key amino acids, such as Trp and glutamine, within the TME are known to increase Treg populations while impairing Teff proliferation and function (78). Modifying nutrient availability, such as replenishing amino acid levels, has been shown to suppress Treg function while restoring Teff activity (79). Glutamine supplementation has been explored as a strategy to counteract Treg dominance and enhance Teff responses (80). Similarly, pyruvate supplementation boosts glycolytic flux in Teffs, promoting their activation and proliferation while indirectly reducing Treg reliance on alternative fuel sources like lactate (81,82). However, both strategies are heavily dependent on the environmental niche, making it challenging to precisely target Treg metabolism within the TME.

Additionally, Tregs depend on the transferrin receptor CD71 for iron uptake, which is critical for their proliferation and metabolic fitness, particularly within the TME. A deficiency in CD71 impairs Treg expansion and tissue-specific adaptation, disrupting systemic iron homeostasis and gut microbiota composition. These findings highlight the close relationship between iron metabolism and Treg functionality, providing potential therapeutic strategies to modulate Treg activity in cancer and immune-related disorders (83).

Overall, these findings suggest that metabolic supplements represent a complementary approach to immunotherapy, offering a means to recalibrate the TME and improve immune responses against cancer.

Combination therapy: overcoming immunotherapy resistance

Targeting Treg metabolism through pharmacological approaches offers a promising strategy to combat tumor-induced immunosuppression. However, these approaches face limitations in effectively enhancing antitumor immunity. Despite the success of cancer immunotherapies, such as ICB, resistance remains a significant challenge. Recent studies suggest that combining Treg metabolism-targeting strategies with ICBs or other cancer treatments can synergistically overcome resistance and improve therapeutic outcomes. Here, we describe the current research progress on Treg metabolic targeting combined with other anti-tumor therapeutic agents, as summarized in Table 1. In highly glycolytic tumors, the active immunosuppressive mechanism of lactate via a metabolic checkpoint specific to Tregs, which enhances PD-1 expression and suppressive activity, contributes to the limited efficacy of PD-1 blockade therapy, suggesting a potential for developing molecular-targeted therapies against lactate in cancer immunotherapy (54). Inhibiting glycolysis in Tregs has been shown to reduce their suppressive function, enabling ICBs to more effectively promote antitumor immunity (84). Additionally, blocking IDO, a key enzyme in Trp metabolism, disrupts Treg activity and has demonstrated synergy with anti-PD-1 therapy in preclinical models (85). Although IDO1 inhibition alone has shown limited direct anticancer efficacy in most patients, combining IDO1 inhibitors with ICB, such as anti-PD-1 or anti-PD-L1 therapies, has demonstrated promising potential (85). Navoximod (GDC-0919), an investigational IDO1 inhibitor, has been shown in preclinical models to enhance intratumoral CD8⁺ T cell activation and suppress tumor growth when combined with the PD-L1 inhibitor atezolizumab. Early-phase clinical studies have confirmed the tolerability of navoximod and suggested that its combination with immune checkpoint inhibitors could augment anti-tumor immune responses by reversing Treg-mediated immunosuppression in the TME (86). This highlights the potential of targeting IDO1 in combination therapies to enhance the efficacy of cancer immunotherapy.

Table 1. Current research progress on Treg metabolic targeting combined with other anti-tumor therapeutic agents.

Metabolic pathway	Target pathway/Inhibitors	Combination therapeutic agents	Influence Treg and tumor growth	Ref.
Glucose metabolism	AR-C155858 (MCT-1/2 inhibitor)	Anti-PD-1	Reduces PD-1 expression, suppresses Treg proliferation, and inhibits tumor growth	(54,96)
	2-DG	Anti-PD-L1	Inhibit Treg accumulation and tumor growth	(97)
FAO	BAY-876	AptCTLA-4 and aptPD-L1	Reduces Treg-mediated suppression and enhances Teff activation, Inhibit tumor growth	(84)
Trp metabolism	Navoximod GDC-0919	Anti-PD-L1	Inhibits the differentiation and function of Tregs	(86)
	BGB-5777	Anti-PD-1 (with WBRT)	Reduces Treg accumulation and synergizes with PD-1 blockade	(85)
	Epacadostat	Anti-PD-1	Reduces Treg accumulation and enhances T cell-mediated antitumor response	(98,99)
Lipid metabolism	Anti-CD36 antibody	Anti-PD-1 antibody	Inhibit Treg accumulation, suppressive function and tumor growth	(66)
	SERBP gene targets	Anti-PD-1 antibody	Inhibit Treg accumulation and survival and tumor growth	(65)
Pyruvate metabolism	CPI-613 (PDH inhibitor)	Anti-PD-L1 antibody	Reduces Treg populations and enhances antitumor immune responses (observed in lysosomal acid lipase KO mice)	(71)
OXPHOS	Metformin	Anti-PD-1 antibody	Reduces Treg populations and enhances antitumor immune responses	(88)

WBRT, whole brain radiotherapy; PDH, pyruvate dehydrogenase.

Furthermore, inhibiting lipid metabolism presents promising opportunities for combination treatments with ICB therapy. For instance, combining anti-CD36 treatment with anti-PD-1 therapy has been shown to reduce tumor growth by inhibiting Treg accumulation and suppressive function (66). Additionally, targeting the SREBP pathway, which regulates de novo lipid synthesis, demonstrates synergistic effects when combined with anti-PD-1 therapy (65). FASN inhibitors also hold potential for combination treatments, though their synergistic effects with ICB therapy have yet to be established. Preclinical studies have demonstrated that FASN inhibitors, such as cerulenin and orlistat, enhance the efficacy of bestatin, an antitumor drug. By upregulating the peptide transporter PEPT1, FASN inhibitors facilitate increased cellular uptake of bestatin, leading to heightened antitumor activity. Combined treatments significantly suppress tumor growth, enhance apoptotic markers, and outperform single-agent therapies in xenograft models, including HT1080 and C26 tumors (87). These findings highlight the potential of targeting tumor metabolism with FASN inhibitors to amplify the effects of metabolically influenced cancer therapies.

Targeting OXPHOS using metformin in combination with anti-PD-1 therapy enhances antitumor immunity by normalizing tumor vasculature and improving the TME. This combination reduces the Treg population, stimulates the proliferation of CD8⁺ TILs, and induces high levels of IFN-γ, which play crucial roles in mediating these effects (88).

The combination of metabolic modulation and other cancer therapies offers a promising strategy to overcome immunotherapy resistance. By disrupting the suppressive influence of Tregs, these approaches enhance the efficacy of conventional and emerging cancer treatments. Future research should focus on optimizing the timing, dosing, and combination of these therapies to minimize toxicity while maximizing clinical benefits. Additionally, identifying predictive biomarkers for patient response will be critical to successfully implementing these strategies in personalized cancer treatments.

CHALLENGES AND RISKS OF TARGETING Treg METABOLISM

While targeting Treg metabolism holds great promise for enhancing antitumor immunity, several challenges and risks must be carefully considered before clinical application. The primary concerns include off-target effects, immune-related adverse events (irAEs), and the difficulty of selectively targeting tumor-infiltrating Tregs. Many metabolic pathways exploited by Tregs—such as glycolysis, FAO, and OXPHOS—are also essential for Teff activation and proliferation. Consequently, their inhibition may dampen antitumor immune responses. For instance, the glycolysis inhibitor 2-DG not only suppresses Treg function but also reduces Teff proliferation, thereby limiting the overall immune response (24,89,90). Since Tregs play a central role in maintaining immune tolerance, disrupting their metabolism may lead to autoimmune-like toxicities and chronic inflammation. Clinical observations from ICB therapies, such as anti-PD-1 or anti-CTLA-4, have shown that reducing Treg function can increase the risk of irAEs, including colitis, dermatitis, and endocrinopathies (91). Likewise, metabolic inhibitors targeting Tregs could disrupt immune homeostasis and trigger systemic inflammatory responses. For example, inhibiting FABP with BMS309403, has been associated with reduced Treg proliferation and Foxp3 expression inducing autoimmunity (92). To minimize these risks, understanding the unique features of intratumoral Tregs is crucial, as it may allow for more precise targeting while minimizing side effects. Intratumoral Tregs exhibit highly upregulated lipid metabolism, making it a promising target for tumor growth regulation with minimal off-target effects or risk of autoimmunity (65,66). Additionally, intratumoral Tregs rely on Foxo1 inactivation for migration and immune suppression, and selectively targeting the Foxo signaling pathway can deplete tumor-associated Tregs while preserving systemic immune tolerance, thereby enhancing CD8⁺ T cell responses and inhibiting tumor growth (93). Inducing Treg fragility represents another potential strategy for selectively targeting intratumoral Tregs. For instance, targeting Nrp1 selectively disrupts intratumoral Treg stability without affecting peripheral tolerance, promoting Treg fragility, enhancing antitumor immunity, and improving responsiveness to anti-PD-1 therapy (94). Therefore, a deeper understanding of the metabolic and functional distinctions of intratumoral Tregs is essential for identifying the most effective and safe therapeutic targets.

CONCLUDING REMARKS

Recent advancements in understanding Treg metabolism have led to strategies such as metabolic inhibitors, nutrient modulation, and dual-action therapies that simultaneously enhance Teffs. However, translating these findings into clinical practice requires addressing key challenges, including specificity, toxicity, and resistance mechanisms. A major question is how to achieve Treg-specific targeting within the TME while sparing pTreg that maintain immune tolerance—potential strategies include targeting metabolic signaling pathways or molecules, leveraging tumor-specific Treg markers, CAR-Tregs, bispecific antibodies, and lipid nanoparticle-mediated in vivo targeted delivery to enhance precision and reduce off-target effects. Additionally, the dosage of metabolic inhibitors can differentially affect Treg function. For instance, etomoxir, an inhibitor of CPT1, a key enzyme involved in FAO, selectively impacts Treg metabolism at lower doses; however, at higher doses, it may exert off-target effects beyond CPT1 inhibition (95). Furthermore, Treg plasticity within the TME and its impact on therapy remain poorly understood. Employing single-cell transcriptomics, metabolic flux analysis, and fate-mapping models could provide deeper insights into how metabolic reprogramming alters Treg stability and function. Another critical direction is integrating metabolic therapies with existing immunotherapies, such as immune checkpoint inhibitors, to enhance antitumor immune responses while mitigating immune suppression. Investigating combination therapies that include metabolic inhibitors, ICBs, and cytokine modulation (e.g., IL-2 variants or TGF-β inhibitors) in preclinical tumor models could help optimize therapeutic synergy. As our understanding of Treg biology continues to expand, refining these strategies through targeted experimental approaches will be crucial for developing effective and durable cancer immunotherapies.

Abbreviations

2-DG

2-deoxy-D-glucose

AHR

aryl hydrocarbon receptor

APC

antigen-presenting cell

DC

dendritic cell

FAO

fatty acid oxidation

FASN

fatty acid synthase

GCK

glucokinase

HIF

hypoxia-inducible factor

ICB

immune checkpoint blockade

ICOS

inducible costimulatory

IDO

indoleamine 2,3-dioxygenase

irAE

immune-related adverse event

KO

knockout

Kyn

kynurenine

MDSC

myeloid-derived suppressor cell

NRP1

neuropilin-1

OXPHOS

oxidative phosphorylation

PEP

phosphoenolpyruvate

pTreg

peripheral regulatory T cell

SREBP

sterol regulatory element binding protein

TAM

tumor-associated macrophage

TCA

tricarboxylic acid

Teff

effector T cell

Tfr

follicular regulatory T cell

TIL

tumor-infiltrating lymphocyte

TME

tumor microenvironment

Trp

tryptophan

tTreg

thymus-derived regulatory T cell