1 Introduction

The tumor microenvironment (TME) constitutes a complex ecosystem that plays a critical role in tumor initiation, progression, invasion, and metastasis (Cai et al., 2024). It comprises a diverse array of cells and extracellular components, including tumor cells, immune cells (such as T cells, B cells, macrophages, dendritic cells, etc.), tumor-associated stromal cells (such as cancer-associated fibroblasts, endothelial cells,etc.), and myeloid-derived suppressor cells (Zahn, 2017). These cellular constituents engage in intricate signaling pathways and intercellular interactions, collectively shaping the distinctive characteristics of the TME. The TME exhibits multiple functional properties; it provides nutrients and releases growth signals that support the proliferation, invasion, and metastasis of tumor cells. Epithelial cells within the TME secrete chemokine ligand 9 (CCL9) and interleukin-23 (IL-23) to promote angiogenesis and establish an immunosuppressive environment (Kortlever et al., 2017). Furthermore, tumor-associated fibroblasts release transforming growth factor-beta (TGF-β) and platelet-derived growth factor (PDGF), which facilitate tumor cell growth and migration (Qu et al., 2019). Conversely, certain immune cells that infiltrate the TME initiate tumor-killing functions; however, the TME provides numerous mechanisms that enable tumor cells to evade immune detection. Tumor cells can secrete inhibitory cytokines, such as interleukin-10 (IL-10) and prostaglandin E2 (PGE2), which suppress the activity of T cells and natural killer (NK) cells, thereby promoting immune evasion (Liu et al., 2022). Additionally, infiltrating Treg cells (Togashi et al., 2019) and myeloid-derived suppressor cells (MDSCs) (Veglia et al., 2021) within the TME predominantly promote immune evasion by suppressing effector immune cells with anti-tumor functions. Furthermore, tumor-associated fibroblasts release vascular endothelial growth factor A (VEGFA), which promotes tumor angiogenesis (Liu X. et al., 2025). Notably, recent studies have identified a context-dependent subset of “fragile” Tregs that exhibit impaired suppressive stability, characterized by reduced FOXP3 expression or Nrp1 deficiency, These unstable Tregs may acquire effector-like properties, including increased production to tumoricidal cytokines such as INFγ, and thereby contribute to anti-tumor immune responses (Overacre-Delgoffe et al., 2017; Overacre-Delgoffe and Vignali, 2018; Liu X. et al., 2024). The complexity of the TME is a significant contributor to drug resistance and metastasis during cancer treatment, thereby impeding the advancement of cancer therapies (Bejarano et al., 2021). The complexity of the TME is a major contributes to drug resistance and metastasis during cancer treatment, thereby limits the progress of cancer therapies.

Regulatory T cells (Tregs), a specialized subset of CD4+ T cells, are integral to the maintenance of immune homeostasis and the suppression of excessive immune responses (Wardell et al., 2025). The Forkhead Box protein P3 (FOXP3) serves as a critical marker for Treg cells and functions as a transcription factor essential for their development and functional maintenance (Josefowicz et al., 2012). FOXP3 is pivotal in mediating the immunosuppressive functions of Tregs, and mutations in the FOXP3 gene are associated with the onset of human Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome, which results in severe autoimmune diseases affecting multiple organs, including the liver, lungs, and skin (Ramsdell and Ziegler, 2014; Ono, 2021). Naturally occurring Tregs (nTregs) are generated in the thymus and inherently possess immunosuppressive capabilities, enabling them to inhibit the activity of both CD4+ and CD8+ T cells. In contrast, inducible Tregs (iTregs) are differentiated from naïve T cells under specific peripheral conditions. These iTregs are predominantly located in peripheral blood and tissues, where they exert suppressive effects on a broad range of immune cells, including CD8+ T cells and NK cells (Li et al., 2020; Savage et al., 2020). Tregs modulate the activation, proliferation, and function of effector T cells through direct cell-to-cell interactions, the secretion of inhibitory cytokines (such as IL-10 and transforming growth factor-bate (TGF-beta)), and the consumption of interleukin-2 (IL-2), thereby preserving immune homeostasis (Nakahashi-Oda et al., 2016; Li et al., 2020). Tregs display a degree of heterogeneity across various tissues, with their function and phenotype adapting to the specific tissue microenvironment. This variability enables Tregs to precisely regulate immune responses under various physiological and pathological conditions (Wyss et al., 2016). Furthermore, Tregs exhibit significant heterogeneity across different tissues, allowing them to adapt to specific microenvironments and precisely regulate immune responses.

Epigenetics refers to the mechanisms that regulate gene expression through DNA and histone modifications without altering the DNA sequence, plays a key role in the development and function of Tregs (Ohkura and Sakaguchi, 2020). Among these epigenetic modifications, DNA methylation is an important mechanism. In Tregs, specific hypomethylated regions, such as the Treg-specific demethylated region (TSDR) of the FOXP3 gene, are essential for maintaining stable Foxp3 expression, thereby ensuring the proper function of Tregs (Morikawa et al., 2014). Histone modifications play a critical role in the regulation of epigenetic processes. For instance, histone H3 lysine 4 trimethylation (H3K4me3) is typically enriched near gene promoters and is frequently associated with the initiation of gene transcription (Wang H. et al., 2023). In contrast, H3K27me3 serves as a key repressive post-translational modification that promotes chromatin condensation and inhibits the recruitment of transcription initiation complexes, thereby suppressing gene transcription (Zhang et al., 2020). In Tregs, these histone modification patterns are intricately linked to the expression of Treg-specific genes, influencing both the development and function of Tregs (Beyer and Huehn, 2017). The targeted deletion of the lysine methyltransferase MML1 in Tregs leads to loss of H3K4me3 at transcriptional start sites, consequently impairing Tregs activation, function, and tissue migration (Wang T. et al., 2024). Additionally, non-coding RNAs, including microRNAs (miRNAs) and long non-coding RNAs (lncRNAs), are implicated in the epigenetic regulation of Treg cells. miRNAs modulated gene expression in Treg cells by binding to target mRNAs, thereby inhibiting their translation or promoting their degradation (Shu et al.). Through various modifications, epigenetics regulates the development, function, and stability of Tregs, thereby maintaining immune balance in the body.

In the TME, Treg cell infiltration is often associated with immune evasion of tumors and poor patient prognosis (Dixon et al.). An increase in Treg infiltration within tumor tissue can suppress anti-tumor immune responses through various mechanisms, thereby promoting tumor proliferation and progression. Treg cells can secrete inhibitory cytokines such as IL-10, TGF-β and interleukin-35 (IL-35), which directly suppress the activity of effector T cells and NK cells within the TME, consequently impairing their tumoricidal functions (Vignali et al., 2008; Wang et al., 2018b). Moreover, Tregs can bind to CD80/CD86 on antigen-presenting cells (APCs) via the cell surface protein cytotoxic T-lymphocyte-associated antigen 4 (CLTA4), thereby inhibiting APC function and suppressing T cell activation (Walker, 2013; Tekguc et al., 2021). Additionally, Treg cells contribute to promoting the immunosuppressive microenvironment by modulating other cellular components within in the TME. For example, Tregs can inhibit the secretion of interferon-gamma (INF-γ) by CD8+ T cells, thereby promoting the polarization of macrophages towards the immunosuppressive M2 phenotype (Liu et al., 2019). Additionally, Treg cells can secrete growth factors that promote tumor cell epithelial-to-mesenchymal transition (EMT) and angiogenesis, stimulating the proliferation of fibroblasts and endothelial cells within the TME, thereby enhancing tumor cell proliferation, invasion, and metastasis (Goebel et al., 2015; Li et al., 2020; Ohkura and Sakaguchi, 2020). Finally, Treg cells demonstrate increased metabolic adaptability in the TME, which supports their proliferation and suppressive functions, thereby strengthening the immunosuppressive environment of the TME (Sharma et al., 2024).

Tumor-infiltrating Tregs (TI-Tregs) represent a specialized subset of Treg cells that infiltrate the TME. These TI-Tregs exhibit significant functional and epigenetic distinctions from peripheral Tregs, with epigenetic modifications frequently cited as a pivotal factor underlying these differences (Dixon et al., 2021). Within the TME, TI-Tregs are exposed to various signals that modulate their epigenetic state, ultimately leading to functional changes in these cells. For instance, the DNA methylation patterns of TI-Tregs within the TME are modified, influencing the expression of specific genes (Ohkura and Sakaguchi, 2020). Additionally, chromatin remodeling may enhance the transcription of immunosuppressive genes, thereby enhancing the immunosuppressive capabilities of Tregs. Although Tregs are essential for maintaining immune homeostasis, their presence can impede the efficacy of tumor therapies. A comprehensive understanding of the differences in functional expression, signaling pathways, and surface markers between Tregs in the TME and peripheral Tregs is crucial for the development of targeted strategies aimed at TI-Tregs in cancer treatment. This provides more therapeutic strategies for treating cancer patients.

In this review, we provide a comprehensive overview of the epigenetic characteristics of Treg cells within the TME, including DNA methylation, histone modifications, non-coding RNAs, chromatin remodeling, and lactylation modifications. We further investigate the factors present in the TME that contribute to the epigenetic reprogramming of TI-Treg cells. Furthermore, we review the progress in targeting TI-Treg cells, particularly drugs that specifically target the unique epigenetic features of TI-Tregs. Additionally, based on the unique functional characteristics of Tregs and their significance in tumor treatment, we further discuss the challenges currently faced by strategies targeting TI-Tregs for cancer therapy, and how these issues can be addressed to improve tumor treatment outcomes.

2 The TME drives the epigenetic reprogramming of Tregs2.1 The epigenetic features of Tregs in the TME2.1.1 DNA methylation

DNA methylation is an important epigenetic mechanism that regulates gene expression without altering the underlying DNA sequence, thereby influencing processes such as cell differentiation, development, and disease progression (Jones, 2012; Dai et al., 2021). This process is influenced by variety of upstream signaling pathways, including TGF-β, IL-6, IL-10, which are activated by inflammatory cytokines and other tumor-associated factors in the TME. These signals activate transcription factor like FOXP3 and STAT3, which then initiate specific gene expression changes in Tregs, playing a crucial role in their development and immune-regulatory functions. FOXP3 serves as a pivotal marker for Treg cells, playing a crucial role in their development, differentiation, and function (Fontenot et al., 2003; Hori et al., 2003). The expression of FOXP3 has been observed in various cancers, including pancreatic cancer (Hinz et al., 2007), breast cancer (Liu et al., 2015), and melanoma (Niu et al., 2011), where it exerts different effects (Wang J. et al., 2023; Zhu et al., 2024). Tregs contribute to tumor immune evasion by suppressing the anti-tumor activity of effector T cells through the secretion of inhibitory cytokines, direct interactions with effector cells, and modulation of other cellular components (Vignali et al., 2008; Walker, 2013; Wang et al., 2018b; Liu et al., 2019; Tekguc et al., 2021). Their suppressive function is regulated by epigenetic modifications, particularly DNA methylation (Kumagai et al., 2024). Elevated FOXP3 expression is correlated with its demethylation within the TME (Zhu et al., 2024). In Tregs derived from patients with non-small cell lung cancer, diminished activity of DNA methyltransferases results in the demethylation of eight CpG sites within the FOXP3 promoter. Tumor cells influence the demethylation of the FOXP3 promoter in Tregs, thereby attenuating immune responses and promoting tumor progression (Wang et al., 2011; Zhu et al., 2024). The conserved non-coding sequence 2(CNS2) located within the first intron of the FOXP3 gene encompasses a region rich in CpG dinucleotides, referred to as the Treg-specific demethylation region (TSDR) (Ngalamika et al., 2015), which is crucial for sustaining FOXP3 expression (Baron et al., 2007; Zhuo et al., 2015). In the context of colorectal cancer, STAT5 overexpression in TI-Tregs recruits TET2 to the FOXP3-TSDR, leading to its demethylation and enhancing FOXP3 expression. This interaction between TET2 and FOXP3-TSDR underscores the critical role of epigenetic regulation in Treg function and highlights the complex interplay between different epigenetic layers (Ma et al., 2018). Moreover, increased expression of TET enzymes, coupled with decreased DNA methyltransferase (DNMT) activity, has been shown to result in the hypomethylation of other immune-related genes such as CTLA-4, further enhancing the suppressive function of Tregs in the tumor microenvironment and promoting tumor progression (Sasidharan Nair et al., 2018; Piotrowska et al., 2021). The tumor microenvironment drives demethylation of the FOXP3 and CTLA-4 loci in TI-Tregs. This process is not isolated; rather, it is a coordinated regulation by multiple epigenetic modifications that work in concert with upstream signals and transcriptional networks to fine-tune Treg responses. Further studies are required to elucidate the exact molecular mechanisms and how the TME influences these regulatory networks. Understanding these processes may open new therapeutic avenues for targeting Tregs-mediated immune suppression in cancer therapy.

2.1.2 Histone modification

The development and function of Tregs depend on the stable expression of Foxp3, and the epigenetic regulatory network of Foxp3 involves various histone modifications. Notably, the enrichment of H3K4me3 at the FOXP3 promoter region serves as a critical hallmark of Treg differentiation. This process is influenced by a network of upstream signaling pathways, including TGF-β, IL-6, and IL-10, which activate key transcription factors such as FOXP3 and STAT3. These transcription factors, in turn, regulate the chromatin landscape of Tregs by promoting or inhibiting the binding of various histone-modifying enzymes, further stabilizing FOXP3 expression. Gene mapping analyses of H3K27me3 modifications in Tregs have demonstrated that excessive activation of Enhancer of zeste homolog 2 (EZH2) can initiate H3K27me3 and promote the differentiation of Treg cells into effector Tregs (Peeters et al., 2024). EZH2 is a key histone methyltransferase that mediates H3K27me3 modifications and is essential for maintaining the suppressive function of Tregs under CD28 stimulation (Peeters et al., 2024). The TME provides hypoxic conditions that further enhance EZH2 activity, leading to increased intracellular levels of H3K27me3, which supports Treg differentiation and stability by inhibiting the expression of pro-inflammatory genes. (Zhuo et al., 2015; Ma et al., 2018). Inhibition of EZH2 results in reduced H3K27me3 levels in TI-Tregs and decreases Foxp3 protein expression, consequently diminishing the stability of TI-Tregs. This demethylation process is not isolated; it involves a cascade of histone-modifying events, including the interaction between EZH2 and transcription factors such as STAT5. This cross-talk between histone modification and transcription factor activity is a key regulatory layer in Treg function. EZH2-deficient TI-Tregs secrete pro-inflammatory cytokines such as TNF-α, IFN-γ, and IL-2, while their capacity to produce IL-10 is impaired. This shift results in a pro-inflammatory phenotype that promotes the recruitment of CD8+ and CD4+ effector T cells to the tumor and enhances their cytotoxic activity (Zhuo et al., 2015; Sasidharan Nair et al., 2018; Piotrowska et al., 2021). Moreover, hypoxia-driven upregulation of EZH2 in the TME facilitates the deposition of H3K27me3 across the genome, leading to the suppression of genes associated with Treg differentiation and function (Goswami et al., 2018). Lysine demethylase 6A (KDM6A) serves as a crucial H3K27me3 demethylase, specifically promoting the demethylation of H3K27me3 (Manna et al., 2015). KDM6A activity is also modulated by upstream cytokine signals, which control its expression in Tregs. Stabilization of KDM6 expression inhibits H3K27me3 demethylation and increases Bcl-2 expression in Tregs, thereby augmenting the anti-apoptotic and suppressive functions of iTregs (Gao et al., 2019). Additionally, the histone demethylase JMJD1 is significantly upregulated in TI-Tregs. As an H3K9me2 demethylase, JMJD1 enhances PD-1 expression while inhibiting AKT and IFN-γ production. Additionally, JMJD1 can directly promote STAT3 demethylation and suppress INF-γ secretion by TI-Tregs (Long et al., 2024). Histone deacetylases (HDACs) and histone acetyltransferases (HATs) function in a reversible manner to regulate the acetylation status of histones H3 and H4 (Dai et al., 2021). In their acetylated state, FOXP3 is stably expressed and exhibits enhanced DNA-binding ability. The knockout of HDAC5 in Tregs reduces the immunosuppressive function of TI-Tregs (Xiao et al., 2016), whereas the knockout of HDAC6 promotes FOXP3 acetylation and strengthens the suppressive function of Tregs (Dahiya et al., 2020). The targeted knockout of HDAC10 in Tregs does not induce autoimmune disease in murine models; however, these cells demonstrated an enhanced suppressive capacity (Dahiya et al., 2020). Conversely, the knockout of HDAC11 results in increased the expression of Foxp3 and TGF-β within Treg cells, thereby enhancing their suppressive function (Huang et al., 2017). The Ep300 gene encodes the adenovirus E1A-associated p300 transcriptional coactivator, which functions as a histone acetyltransferase and modulates transcription through chromatin remodeling (Ghosh et al., 2016; Nicosia et al., 2023). Inhibition of Ep300 leads to a reduction in Foxp3 expression and induces apoptosis in Tregs, consequently impairing their suppressive function and enhancing anti-tumor immunity (Liu et al., 2013). The melanoma antigen family H1 gene (MAGEH1), a member of the type IIMAGE protein family, is expressed across various tissues and operates as an E3 ubiquitin ligase. Comparison of TI-Tregs with peripheral Tregs reveals that TI-Tregs upregulate the expression of MAGEH1 and regulate the ubiquitination status. This process is mediated by upstream signals within the TME, including cytokines and hypoxic factors, and contributes to the survival and suppressive function of Tregs in the tumor microenvironment (Plitas et al., 2016). Histone modifies such as EZH2, JMJD1, HDACs, and p300 modulate Treg stability and effector function through targeted histone methylation and acetylation, thereby enhancing their immunosuppressive activity within the tumor microenvironment and promoting immune evasion.

The distinct epigenetic modifications, including DNA methylation, histone modification, non-coding RNA regulation, chromatin remodeling, and lactylation, contribute to the function divergence between peripheral Tregs and TI-Tregs within the tumor microenvironment.

2.1.3 Non-coding RNA

Non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long non-coding RNAs(lncRNAs), play critical regulatory roles in Tregs within the TME. In TI-Tregs, the expression profile of ncRNAs is significantly altered, which profoundly impacts Tregs functionality (Ma et al., 2023). MiRNAs regulate gene expression by binding complementarily to target mRNAs, thereby inhibiting their translation or promoting their degradation (McGeary et al., 2019). In addition to direct gene regulation, miRNAs also influence Treg function through modulation of chromatin accessibility and the expression of key transcription factors. For example, miRNAs can modulate the activity of Treg-specific transcription factors like FOXP3 and STAT3, influencing Treg differentiation and immunosuppressive capacity. Previous studies have demonstrated that miRNAs are essential for maintaining the functional stability of Tregs. For instance, miR-21 and miR-155 have been shown to influence the expression of Foxp3 (Stefani and Slack, 2008; Lu et al., 2009), while miR-142-3p regulates GARP expression in Tregs (Zhou et al., 2013) and can indirectly affect Foxp3 levels by targeting AC9 mRNA (Huang et al., 2009; Jebbawi et al., 2014). Notably, the expression levels of certain miRNAs in TI-Tregs differ from those observed in peripheral Tregs, and these differentially expressed miRNAs participate in regulating Treg proliferation, differentiation, and immunosuppressive activity (Xing et al., 2021). Specifically, miR-155 is upregulated in TI-Tregs, where it enhances immunosuppressive functions by targeting key genes such as ICOSL and suppressing effector T cell activity, thereby facilitating tumor immune evasion (Shu et al., 2017; Chao et al., 2019; Tili et al., 2024). In malignant pleural effusions associated with non-small cell lung cancer, Tregs exhibit downregulation of miR-4772-3p, which leads to increased Helios expression and enhances their immunosuppressive capabilities (Yu W.-Q. et al., 2019). Conversely, miR-142-5p inhibits the expression of the cAMP-hydrolyzing enzyme phosphodiesterase, thereby reducing Tregs metabolism and attenuating their suppressive function (Anandagoda et al., 2019). Additionally, miR-125b-5b modulates the immunosuppressive activity of Tregs by targeting TNFR2; its overexpression results in a reduction of both the proportion and suppressive capacity of TI-Tregs, ultimately promoting anti-tumor immunity (Jiang et al., 2022). In the context of hepatocellular carcinoma (HCC), tumor-derived exosomal circGSE1 promotes the proliferation and accumulation of TI-Tregs within the TME through the miR-324-5p/TGFBR1/Smad3 signaling pathway, thereby contributing to tumor progression. This interaction exemplifies how ncRNAs can indirectly regulate Treg function by influencing the TME’s extracellular vesicle-based communication. (Huang et al., 2022). Furthermore, lncRNAs also regulate gene expression through various mechanisms, including interactions with DNA, RNA, or proteins. In breast cancer, lncRNA SNHG1 upregulates indoleamine 2,3-dioxygenase (IDO) and promotes the expression of Foxp3 and IL-10 by inhibiting miR-448, thereby maintaining the immunosuppressive function of Tregs within the TME (Pei et al., 2018). Similarly, lncRNA SNHG16 enhances the TGF-β1/SMAD5 pathway through miR-16-5p, resulting in elevated CD73 expression in TI-Tregs and strengthening their immunosuppressive activity in the TME (Ni et al., 2020). In HCC, the expression of lncRNA FENDRR is reduced, while miR-423-5p is upregulated, which promotes the proliferation of Tregs within the TME and contributes to immune evasion in liver cancer (Yu Z. et al., 2019). These findings demonstrate how ncRNAs integrate with the epigenetic machinery of Tregs to orchestrate immune suppression within the TME, highlighting their potential as therapeutic targets. TI-Tregs exhibit altered expression patterns of non-coding RNAs, including specific microRNAs and lncRNAs. Thes molecular changes modulate Treg proliferation and suppressive function through the regulation of critical genes and signaling pathways, thereby promoting tumor immune evasion and progression.

2.1.4 Chromatin remodeling

Chromatin remodeling refers to the process by which chromatin structure is altered to regulate gene expression. In TI-Tregs within the TME, chromatin remodeling undergoes substantial modifications that significantly influence their functionality (Dixon et al., 2021; Long et al., 2024). Within the TME, the activity of specific chromatin-remodeling complexes is modified, affecting the gene expression profile and functional state of Tregs. Notably, the SWI/SNF family of nucleosome-remodeling complexes plays a pivotal role in regulating Foxp3 expression, with the BAF complex promoting Foxp3 transcription, and the PBAF complex inhibiting it (Loo et al., 2020). In the context of colorectal cancer, alterations in the composition and activity of the SWI/SNF chromatin-remodeling complex in Tregs altered, leading to the upregulation of genes associated with immunosuppression and enhancing the suppressive function of Tregs (Zhao et al., 2020). These alterations are driven by the TME’s inflammatory cytokines, which activate signaling pathways such as the JAK/STAT pathway, and the PI3K-AKT pathway, which, in turn, modulate the activity of chromatin-remodeling complexes and transcription factors. The interaction between these signaling pathways and chromatin-remodeling factors influences Treg differentiation, function, and stability. Single-cell RNA-seq and ATAC-seq analyses of TI-Tregs and peripheral Tregs from non-small cell lung cancer patients have revealed the upregulation of multiple transcription factors, including BATF, NFAT, SOX4, MEF2, and TBX21. This highlights the importance of chromatin-remodeling complexes and transcription factor networks in maintaining the functionality of Tregs under immune stress conditions within the TME. Targeted knockout of BATF in Tregs has been shown to inhibit the activation, infiltration, and chromatin remodeling of TI-Tregs within the TME, ultimately leading to Treg exhaustion (Itahashi et al., 2022). This highlights the importance of chromatin-remodeling complexes and transcription factor networks in maintaining the functionality of Tregs under immune stress conditions within the TME. TI-Tregs require NFAT and AP-1 to enhance the binding of Foxp3 to DNA and regulate the expression of related target genes. The interaction between Foxp3 and chromatin is modulated by antigens and cytokines within the TME, directly influencing gene expression and the functional stability of TI-Tregs (He et al., 2024). Within the TME, functionally altered SWI/SNF chromatin remodeling complexes cooperate with transcription factors including BATF and Foxp3 to reprogram the gene expression landscape of TI-Treg cells, thereby enhancing their immunosuppressive capacity. This complex interplay between chromatin modifications, transcription factors, and cytokine signaling underscores the dynamic regulation of Treg function within the TME.

2.1.5 Lactylation

Tumor cells undergo metabolic reprogramming, converting pyruvate into lactate, which subsequently accumulates within the TME. This accumulation of lactate acts as a signaling molecule, inducing the lactylation of histone lysine residues and stimulating gene transcription (Zhang D. et al., 2019). Specifically, lactate within the TME facilitates lactylation modifications in Tregs (Gu et al., 2022). Lactate-induced histone modifications are tightly regulated by signaling pathways activated within the TME, including the NF-κB and TGF-β pathways, which modulate transcription factor activity and chromatin accessibility in Tregs. Furthermore, lactate induces histone H3K18 lactylation (H3K18la) within the TME, which enhances NF-κb p65 mediated transcriptional activation, upregulates TNFR2 expression, and accelerates the pathological progression of malignant pleural effusion (MPE) (Xue et al., 2024). This process is not limited to direct histone modifications but is also coordinated with other metabolic signals that modulate Treg differentiation and function. In TI-Tregs, lactate-driven histone lactylation increases the transcription of key genes such as CD39, CD73, and CCR8, all of which contribute to the enhanced immunosuppressive capacity of Tregs within the TME (Sun et al., 2023). The increased levels of lactylation in key proteins within TI-Tregs influence their function and the activity of associated signaling pathways. Additionally, lactate in the TME induces the lactylation of Lys72 in MOESIN, which enhances TGF-β signaling pathways and strengthens the interaction between SMAD3 and MOESIN (Gu et al., 2022), thereby promoting the generation of Tregs in the TME and reinforcing the immunosuppressive nature of the tumor microenvironment (Gu et al., 2022). In patients with colorectal cancer, lactate enhances USP39-mediated RNA splicing, thereby promoting CTLA-4 expression in a Foxp3-dependent manner, which enhances the immunosuppressive function of TI-Tregs (Ding et al., 2024). Additionally, lactylation may influence the metabolic state of Treg cells, allowing them to better adapt to the acidic conditions of the TME. For instance, Tregs can increase the expression of the lactate transporter MCT1 in high-lactate environments to maintain their proliferation and suppressive activity (Watson et al., 2021). In summary, the accumulation of lactate within the TME functions as a signaling molecule that enhances the adaptability and suppressive capacity of Tregs by modifying histones and stimulating the transcription of immunoregulatory genes.

Increased glycolysis, lactate accumulation, and nutrient depletion in the tumor microenvironment result in the accumulation of metabolites such as lactate, 2-HG, and adenosine. These metabolites drive epigenetic changes in TI-Tregs, enhancing their suppressive activity. The modifications promote the stability and survival of TI-Tregs, increasing the expression of FOXP3, IL-10, TGF-beta, CD39, CD73, CTLA-4, and PD-1. This strengthens the immunosuppressive environment, facilitating tumor immune evasion.

2.2 Epigenetic regulation of Tregs mediated by the TME

Various factors within the TME can significantly influence the epigenetic landscape of Tregs, thereby modulating their functional properties and ultimately impacting the antitumor immune response. Epigenetic regulation plays a critical role in controlling Treg cell differentiation and function, involving mechanisms such as DNA methylation, histone acetylation, and chromatin remodeling, which collectively determine the immunosuppressive activity of TI-Tregs (Smiline Girija, 2022). Within the TME, tumor cells secrete metabolites such as TGF-β, lactate, and pyruvate, as well as create hypoxic conditions, all of which can modify the DNA methylation patterns of TI-Tregs (Multhoff and Vaupel, 2021; Xue et al., 2024). Moreover, cytokines such as IL-2, CCL20, IL-17, and IL-6 enhance STAT3 expression in Tregs, thereby facilitating tumor immune evasion (Li and Liu, 2016). Additionally, tumor-associated stromal cells and hypoxia-related signals further modify the epigenetic landscape of TI-Tregs, enhancing their immunosuppressive capacity within the TME (Figure 1) (Macedo-Silva et al., 2019; Sarkar et al., 2022).

Epigenetic differences between peripheral Tregs and TI-Tregs. This figure was drawn using Figdraw (https://www.figdraw.com).

2.2.1 Regulation of Treg epigenetics by metabolic metabolites within the TME

Metabolic reprogramming of tumor cells within the TME leads to nutrient depletion and the accumulation of immunosuppressive metabolic byproducts. These metabolites have the capacity to modify epigenetic programs and signaling networks, thereby affecting the differentiation, proliferation, and activation of immune effector cells, ultimately modulating the function of Tregs (Li Y. et al., 2021). The metabolite 2-hydroxyglutarate (2-HG), frequently produced by a metabolite commonly produced by IDH mutations in gliomas and acute myeloid leukemia, can alter genome-wide histone and DNA methylation (Xu et al., 2011). The generation of 2-HG directly reduces methylation at the FOXP3 locus, enhancing Foxp3 expression (Xu et al., 2017; Li Y. et al., 2021). In addition, TME-derived metabolites can indirectly regulate Treg epigenetics by influencing their metabolic pathways. Dysregulated glycolysis in tumor cells increases lactate production in the TME (Barbieri et al., 2023), which is closely associated with increased acidity (Brand et al., 2016; Feng et al., 2022). Acidic conditions in the TME disrupt one-carbon metabolism in Tregs, leading to altered intracellular metabolite levels and consequently affecting epigenetic modifications (Mani et al., 2024). Under acidic conditions within the TME, the metabolic reprogramming of nTregs results in alterations to the one-carbon folate pathway, leading to decreased levels of S-adenosylmethionine (SAM), folate, and glutathione. This metabolic adaptation enhances the immunosuppressive activity of nTregs in a sustained manner, thereby promoting tumor immune evasion (Mani et al., 2024). Moreover, the accumulation of lactate in the TME induces histone lactylation in Tregs, which affects the activity of histone-modifying enzymes and alters histone marks (Qu et al., 2023). Lactate functions not only as a metabolic substrate for Tregs but also supports their suppressive function. Tregs modulate their metabolic and functional states in response to nutrient availability. Targeting lactate metabolism directly or mitigating TME acidity can reduce Treg-mediated suppression of cytotoxic immune cells, thereby enhancing antitumor immunity (Watson et al., 2021). Furthermore, hypoxic conditions within the TME upregulate the expression of CD39 and CD73 through hypoxia-inducible factor 1(HIF-1) and TGF-β signaling pathways, resulting in increased ATP hydrolysis and elevated adenosine levels (Antonioli et al., 2013). Adenosine interacts with A2A receptors, influencing intracellular signaling pathways and altering the epigenetic landscape of Tregs, thereby enhancing their immunosuppressive functions (Zhu et al., 2023). TI-Tregs depend on a metabolic network comprising glycolysis, fatty acid synthesis, and fatty acid oxidation to endure and proliferate under challenging conditions (Pacella et al., 2018). These cells acquire and utilize extracellular free fatty acids to fulfill their metabolic requirements, thus supporting their survival and suppressive capabilities through lipid metabolism (Berod et al., 2014; Gerriets et al., 2015; Field et al., 2020). Fatty acid-binding proteins (FABPs), which are integral to lipid uptake and intracellular transport, play a crucial role in this process (Furuhashi and Hotamisligil, 2008). TI-Tregs preserve mitochondrial integrity and regulatory function through elevated expression of FABPs (Rolph et al., 2006). The inhibition of FABP5 in Tregs leads to the release of mitochondrial DNA release and activates the cGAS-STING-dependent type I IFN signaling, resulting in increased IL-10 production and enhanced immunosuppressive activity (Field et al., 2020). Metabolic byproducts and conditions in the TME-including 2-HG, lactate-derived acidity, hypoxia, and adenosine drive epigenetic reprogramming and metabolic adaptation in Tregs via DNA methylation, histone lactylation, disruption of one-carbon metabolism, and FABP dependent lipid metabolism, ultimately bolstering their immunosuppressive function and enabling tumor immune evasion (Figure 2).

Metabolic reprogramming of tumor-infiltrating Treg cells. This figure was drawn using Figdraw (https://www.figdraw.com).

2.2.2 The influence of cytokine networks on the epigenetic regulation of Tregs

The cytokine network within the TME plays a crucial role in regulating the epigenetic state of Tregs. Cytokines present in the TME, including interleukins and TGF-β, interact with receptors on Tregs to initiate intracellular signaling cascades that influence the activity of epigenetic enzymes (Yang et al., 2023). Various cytokines affect epigenetic modifications at critical Treg loci through multiple signaling pathways, thereby modulating Treg differentiation, function, and stability (Li and Liu, 2016). In the context of hepatocellular carcinoma, the CCL20–IL-17–IL-6 cytokine signaling axis is instrumental in regulating Treg cell activity. Similarly, in lung cancer, there is a significant increase in CCL20 mRNA expression, which is associated with upregulation of CCR6 on Tregs and elevated levels of STAT3 within these cells. These modifications in the cytokine network promote immune suppression, thereby promoting tumor metastasis (Li and Liu, 2016). Additionally, cytokines can modulate Treg function by influencing the epigenetic modifications of the FOXP3 gene. IL-2 shapes the epigenetic landscape of thymus-derived Tregs by regulating the localization of SATB1, which in turn controls genome-wide chromatin accessibility and Treg functionality (Chorro et al., 2018). TGF-β modulates FOXP3 methylation and the differentiation of Tregs through UHRF1. TGF-β induces Uhrf1 phosphorylation and nuclear exclusion, leading to its proteasome dependent degradation and promoting Treg differentiation (Sun et al., 2019). Furthermore, TGF-β activates Smad signaling and can enhance the expression of DNA methyltransferases (DNMTs), leading to locus-specific modifications in DNA methylation that influence the expression of Treg-relevant genes (Ohkura and Sakaguchi, 2020). Tumor-derived cytokines, notably interleukins and TGF-β, remodel the epigenetic architecture of Treg through signal dependent control of FOXP3 expression and chromatin remodeling, This reprogramming locks in Treg differentiation and stability, constraining antitumor immunity and advancing disease progression.

2.2.3 Tumor-associated stromal cells induce epigenetic reprogramming of TI-Tregs

Tumor-associated stromal cells, including cancer-associated fibroblasts (CAFs) (Liao et al., 2019; Micalet et al., 2024) and tumor-associated macrophages (TAMs) (Zhang R. et al., 2019), constitute critical elements of the TME and significantly contribute to the regulation of the epigenetic landscape of TI-Tregs through cellular interactions (Yang et al., 2023). Within the TME, CAFs secrete a variety of cytokines and chemokines, such as CCL22, CCL18, and CXCL12, which not only recruit Tregs to tumor sites but also impact their epigenetic reprogramming (Wang et al., 2017; Sarkar et al., 2022). CCL22 interacts with the CCR4 receptor on Tregs, initiating intracellular signaling pathways that modify histone modifications and enhance the expression of genes linked to immunosuppressive functions. In patients with breast cancer, FOXP3 and HAT1 in Tregs modify the acetylation status of the CCR4 promoter, thereby enhancing CCR4 expression. These CCR4+ Tregs are subsequently attracted to the tumor tissue by CCL22 and CCL17 secreted within the TME (Sarkar et al., 2022). Additionally, macrophages within TME are activated and polarized towards the immunosuppressive M2 phenotype (Wang S. et al., 2024). Cytokines and metabolites released by these cells attract Tregs to the tumor microenvironment and contribute to their epigenetic remodeling (Wang et al., 2017; DeNardo and Ruffell, 2019; Wang S. et al., 2024). Arginase-2 secreted by M2 macrophages induces alterations in the metabolic state of Tregs within the tumor, leading to modifications in metabolite production and influencing the activity of epigenetic regulatory enzymes. Consequently, the altered metabolic state affects the activity of epigenetic regulatory enzymes, reshaping DNA methylation and histone modification patterns within Treg cells and ultimately enhancing the immunosuppressive capacity of TI-Tregs (Brüne et al., 2015). Tumor-associated stromal cells, including CAF and TAM, actively remodel the epigenetic landscape of TI-Tregs through secreted factors and cellular interactions, such as CCL22-CCR4 signaling and metabolic modulation via arginase-2-which enhance the expression of immunosuppressive genes and reshape DNA methylation and histone modification patterns, ultimately strengthening Treg-mediated immune suppression within the TME.

2.2.4 The role of hypoxic condition in the TME in regulating the epigenetic reprogramming of Tregs

Hypoxia is a prevalent characteristic of numerous solid tumors (Godet et al., 2019) and contributes to tumor immune evasion and progression through the induction of epigenetic modifications in Tregs (Macedo-Silva et al., 2019). Hypoxia-inducible factors (HIFs) are activated under hypoxic conditions and play a crucial role in modulating the epigenetic landscape of Tregs, thereby enhancing their immunosuppressive function (Kao et al., 2022; Wu et al., 2022). In the context of esophageal cancer, the formation of hypoxic regions and the activation of HIF-1α are linked to resistance to radiotherapy. Furthermore, hypoxia influences various epigenetic mechanisms, including antigen presentation, cellular stress responses, DNA methylation, and histone methylation, which collectively affect Tregs function within the TME (Macedo-Silva et al., 2019). In addition to direct effects, hypoxia modulates the secretion of tumor-derived exosomes, thereby indirectly regulating the epigenetic state of Tregs. In hepatocellular carcinoma, hypoxia decreases the secretion of miR-101, resulting in macrophage activation and inflammation, which subsequently reshape the epigenetic profile and immunosuppressive