The immune system maintains a pool of Regulatory T cells (Tregs), a discrete population of CD4+ lymphocytes that regulates both innate and adaptive immune responses towards self-antigens, virulent agents, cancer, commensal microbiota, and various other allergens (1–5). Tregs play a prominent role in the maintenance of immune tolerance and normal functioning of the immune system or immune homeostasis by eliminating the autoreactive T cells, induction of self-tolerance, and curbing inflammatory processes (6–10).

The suppressive function of a class of T cells was first reported by Gershon and Kondo in the 1970s (11). Gershon’s laboratory discovered that the negative interference exerted by T cells during inflammation was distinct from helper T cells (T_H cells), hypothesizing that T cells not only enhanced but also weakened immune responses by downregulation of some biological functions (11). This suppressor T cell field was interrupted in the mid-1980s when analysis of mouse MHC gene failed to define the I-J DNA region, presumed to encode a molecule or domain of a molecule associated with their suppressive functions (12–14).

Other reasons that diminished interest in Regulatory cells was the inadequacy of specific markers for differentiating Tregs from other T cell populations, uncertainty in the molecular features of suppression, and difficulty in developing antigen-specific suppressor T-cell clones (13, 15). However, in the 1990s, the notion of T cell-mediated suppression resurfaced when a novel subgroup of CD4+ T cells were distinguished that co-expressed the interleukin-2 receptor (IL-2R) alpha-chain (CD25) and its ability to suppress autoimmunity in thymectomized mice (16).

Researchers have investigated the relationship between Tregs and tumors in the tumor microenvironment. Treg participation in anti-tumor immunity was first discovered in 1976 by Fujimoto and Greene (17). In 1999, others showed that in T cell-deficient animals transplanted with CD25+ cell-depleted splenocytes, anti-CD25 Ab depleting CD4+CD25+ Tregs inhibited tumor development (18). Reports have shown that Tregs infiltrate tumors and suppress the function of various immune cells including CD4+ T helper cells, CD8+ cytotoxic T cells, NK cells, and NK T cells (19–21). Thus, Tregs are able to suppress antitumor immunity, enabling tumors to progress faster, and their presence in the tumor microenvironment is directly correlated to a poor prognosis (22).

In humans roughly, 5-10% of the CD4+ T cell population in peripheral blood are comprised of naturally arising Tregs characterized by constitutive expression of CD25 and Forkhead Box 3 (FOXP3) (23–25). Mutations in FOXP3 were discovered to be associated with an autoimmune lymphoproliferative illness in humans termed X-linked autoimmunity-allergic dysregulation syndrome (XLAAD), which was later renamed Immunodysregulation, polyendocrinopathy, enteropathy, and X-linked (IPEX) syndrome (26). IPEX is one of the most well-known Mendelian disorders, characterized by a loss of immunological tolerance caused by a lack of functioning Treg cells (27, 28). As a result of these discoveries, effective Treg suppression is clearly necessary to avoid autoimmune and chronic inflammatory diseases.

Expression of FOXP3 serves as a dominant regulatory pathway in Treg development and function and is vital for Treg cell lineage identity (9, 29). FOXP3 is a member of the forkhead/winged-helix family of transcriptional factors. The gene is located on the X -chromosome and is highly conserved among different species. The FOXP3 protein is a 431 amino acid structure that is encoded by the FOXP3 gene in humans. The protein has four domains: an amino-terminal proline-rich domain mediating transcriptional repression, a central zinc finger, and leucine zipper domains facilitating homo or hereto-dimerization and the C-terminal forkhead domain implicated in nuclear localization and DNA binding activity.

The N-terminal domain has a typical role in the development and function of Tregs (30, 31). The role of foxp3 in modulating immune tolerance was recognized by the discovery of “scurfy” mice exemplified by multi-organ lymphocytic infiltration, associated with mutations in the foxp3 gene (32). In humans, the scurfy phenotype shared molecular and clinical features with IPEX, which was later linked to the human orthologous FOXP3 gene (33).

Tregs appear more resistant to thymic deletion processes and are generated from the thymus during the early stages of fetal development (19). Treg cell activation is antigen-specific, inferring that Treg cells suppressive functions are antigen-dependent. Although self-reactivity of Treg cells has been proposed, extensive TCR repertoire analyses have revealed that self-reactivity is more likely to be the exception instead of the rule (34, 35).

A fraction of CD4+ expressing thymocytes/progenitor cells can differentiate into CD4+ CD25+ FOXP3+ T cells, commonly described as thymus derived Treg (or tTreg), or natural Treg (nTreg). Treg cells can also be generated extrathymically following antigenic stimulation of conventional CD4+ T cells/naive T cells at peripheral sites (lymphoid or non-lymphoid tissues) and therefore are designated as the peripheral (pTreg) Treg cells (in vivo). Upon stimulation with anti-CD3 in the presence of cytokines such as TGF- β and IL-10, induced Tregs (iTreg) cells (in vitro) can be established in cell cultures (36–38). While some investigations have also shown that Helios and neuropilin-1 (Nrp1), a semaphorin III receptor, can be used to identify tTreg and pTreg cells, there are several instances that clearly do not distinguish between thymus and extrathymically derived Tregs (39–41). Even though no one marker distinguishes these three distinct subgroups of Tregs, FOXP3 expression is indispensable for their suppressive function.

Studies have conjointly indicated that tTreg cells can develop into specialized effector Treg subsets, such as tissue resident Treg cells that play a significant role in non-lymphoid organ function (42, 43). Tregs in visceral adipose tissue, muscle Tregs, and skin-resident memory Tregs have been described emphasizing some intriguing biological implications about their functional interactions with local tissues (43–47).

FOXP3 interacts with various transcriptional and chromatin-modifying factors and acts as a “master” regulator of Treg cell development process (9, 48, 49). FOXP3 can modulate the transcriptome of Treg cells by various mechanisms depending on binding partners. FOXP3 forms large protein complexes ranging from 300 KDa to over 12000 KDa encompassing many proteins that have either a direct or indirect interaction within this interactome per se (50, 51).

Binding of AML/Runx1 (Acute myeloid leukemia 1/Runt-related transcription factor 1) to FOXP3 leads to upregulation of Treg-related molecules by repressing IL-2 and IFN-γ levels (52). Runx1 also forms a complex with core-binding factor subunit beta (CBFβ) and precisely adheres to the FOXP3 promoter region’s conserved non-coding sequencing region 2 (CNS2), which is significantly de-methylated in Tregs and is required for FOXP3 expression and Treg cell lineage stability (53). Interaction of FOXP3 with the nuclear factor of activated T cells (NFAT) complements the expression of CD25, cytotoxic T-lymphocyte-associated protein 4 (CTLA4), and glucocorticoid-induced TNF receptor (GITR) while curbing the expression of inflammatory genes IL-2 and IFN-γ, serving as a transcriptional activator of Treg cells (54–56). NFAT is required by both iTregs and pTregs, but not by tTregs. NFAT binds to FOXP3 and suppresses the expression of NFAT-targeted genes. NF-κβ molecules Rels (both RelA and c-Rel) have a role in pTreg formation as well as FOXP3-regulated gene expression and repression (57, 58). RelA deletion, which is Foxp3-specific, causes more severe autoimmune symptoms than c-Rel deletion, indicating that RelA is more essential than c-Rel in Treg function (58).

Additionally, Eos (IKZF4), GATA3, Interferon regulatory factor 4 (IRF4), signal transducer and activator of transcription (STAT3), Retinoic acid receptor-related orphan receptor (RORγT), RORα, YY1, hypoxia-inducible factor 1-alpha (HIF1α), FOXO1, FOXO3, and Satb1 are among the transcription factors that have been reported to interact with FOXP3 to promote Treg identity (50, 59–67). As a result of these numerous descriptions, it has been shown that the presence of these components upstream of FOXP3 orchestrates Treg lineage and makes FOXP3 indispensable for Treg suppressor activity.

The function of FOXP3 can also be regulated by several posttranslational modifications (PTMs) including acetylation, phosphorylation, ubiquitination, and methylation (29). These PTMs govern its DNA binding capability, stability, and protein-protein interactions (transcriptional co-activators, transcriptional repressors, and chromatin remodelers) that modulate Treg suppressive functions. The process of acetylation involves acetylation and deacetylation of specific lysine residues catalyzed by the activities of both Histone acetyltransferases (HATs)/lysine acetyltransferases (KATs) and Histone deacetylases (HDACs). The principal acetylase include Tip60 (KAT5), a member of the MYST family, and p300 (KAT3b) belonging to the p300/CBP family (68, 69). Both Tip60 and p300 act cooperatively to acetylate FOXP3 (70).

p300 interacts with Tip60 to stimulate auto-acetylation and this interaction affects the stability of Tip60. Following this interaction, p300 acetylates the K327 of Tip60, which changes its substrate interaction and acts as a “molecular switch”, permitting Tip60 to change binding partners. Because of this, Tip60 associates with FOXP3, acetylates FOXP3 leading to release of p300. Throughout these events, Tip60 itself acetylates p300, which is indispensable for its HAT activity. Synergistic actions of both Tip60 and p300 contribute to maximal stimulation of FOXP3 repressive transcriptional activity and enhanced stability by preventing its polyubiquitylation-mediated degradation (70, 71).

In an earlier study from our laboratory, we first showed that Tip60, HDA7, and HDAC9 were associated with FOXP3 in a dynamic ensemble forming a chromatin remodeling complex where Tip60 constituted as a vital subunit in the repression complex. We found that FOXP3 exerted higher repression in the presence of over-expressed Tip60 levels when compared to its HAT deficient mutant forms. Further, the knockdown of Tip60 eased transcriptional repression (69). In subsequent studies from our laboratory, we showed that the conditional knockout of Tip60, but not p300 in vivo, leads to scurfy like an autoimmune disease by significantly lowering the Treg cell population in spleen and lymph nodes (70). Further, naïve CD4+ T cells transduced with foxp3 and Tip60 mutants (Q377/G380E and K327Q) exhibited lowered Treg suppressive function compared to foxp3 and wild type (WT) Tip60.

In disease processes such as IPEX, one of the most common mutations (A384T) occurring in the C-terminal forkhead domain disrupts the Tip60-FOXP3 interaction affecting Treg functions. Our laboratory has identified small allosteric modifiers (SGF003 and B7A) that are able to target Tip60. We employed a cavity-induced allosteric modification (CIAM) approach that can help stabilize Tip60-FOXP3 interaction and can restore Treg suppressive capacity (71, 72). These observations identify some of the important roles of Tip60 in Treg biology [ Figure 1 , adapted from (70, 71)].

Methylation of FOXP3 occurs on arginine residues by the activity of Protein methyl transferases (PRMTs) exerts a pivotal role in Treg function. PRMT5 is the core type II protein arginine methyltransferases found abundant in all eukaryotic species (73, 74). In recent studies, it was demonstrated that the cholesterol biosynthesis metabolic gene expression program that induces RORγt agonistic activity and promotes Th17 differentiation and experimental autoimmune encephalomyelitis is dependent on “PRMT5 expression” in newly activated T cells (75). Additionally, recruitment of STAT3 by PRMT surmounted the inhibition of Th17 differentiation mediated by STAT5 activation induced by IL-2. As a result, PRMT influenced Th17 differentiation by controlling the reciprocal recruitment of STAT3 and STAT5. Identifying PRMT members that act as a potential target for reducing RORγt-dependent production of pathogenic Th17 cells may be relevant to therapeutics that may relieve Th17-mediated autoimmunity (76).

Conditional deletion of PRMT5 in Treg cells alters their quantity and function, resulting in a scurfy phenotype, according to recent data from our laboratory. We found that in Foxp3+ cells, silencing PRMT5 resulted in reduced suppressive activity, which affected the relevance of PRMT5 in Treg function maintenance. Inhibiting PRMT5 affected anti-tumor immunity by limiting the infiltration of Treg cells into tumor sites. In a syngeneic mouse model of breast cancer (erbB2/neu), we reported that DS-437, a PRMT5 inhibitor, dramatically improved anti-HER2/neu therapy in rodents (77). Taken together, these findings point to the critical functions of PRMT5 and PRMT1 in regulating FOXP3 function by regulating Treg cell activity and function.

Tregs have the potential to alter immune function in cells of both the innate and adaptive immune systems. Treg cells suppress a myriad of immune cells, notably B cells, CD4+ T cells, CD8+ cytotoxic T cells, NK cells, NKT cells, macrophages, dendritic cells, neutrophils, and T cells (1, 78). Tregs use a variety of immunosuppressive methods to dampen immune responses ( Figure 2 ). These include (a) immunosuppressive cytokines such as TGF-β, IL-10, and IL-35 (b) Metabolic perturbations involving CD25 (IL-2 receptor alpha) dependent cytokine deprivation facilitated apoptosis, immunosuppressive adenosine by ectoenzymes CD39 and CD73 and c-AMP mediated inhibition, (c) granzyme and perforin mediated cytolysis (d) Interaction with antigen-presenting cells (APC) such as dendritic cells (DCs) to modulate their maturation and function (CTLA4 and LAG3) and (e) extracellular vesicles (EVs) generated from Tregs (8, 9, 24, 78–83).

Distinct processes may function in different situations, with one taking precedence in one process and the other in the another. Alternatively, multiple suppressive mechanisms act in concert and may do so synergistically, such that limiting of any one of these pathways not enough to compromise suppression activity significantly (84).

In the immune system, Treg cells perform both positive and negative roles. Immune suppression mechanisms to limit autoimmunity, transplantation and maintain immune homeostasis are positive effects, whereas circumvention of antitumor immunity is a negative effect. Tregs immunosuppressive activity in tumors environments represents a significant barrier to efficient anti-tumor immunity. As a result, research into the roles and activities of Tregs is needed to fully understand their potential as immunotherapeutic targets and to develop new tumor immunotherapy methods.

Treg biology is complex. Understanding the molecules and detailed structural and functional domains of regulatory proteins as well as signaling pathways in target cells affected by Treg may be needed for potential therapeutic interventions of effective immune response.

All authors contributed to data analysis, drafting or revising the article, gave final approval of the version to be published, agreed to the submitted journal, and agree to be accountable for all aspects of the work.

This work was supported by grants from the National Institutes of Health (R01CA219034 and R21AI35359) and Breast Cancer Research Foundation (BCRF-17-061) to MG.

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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