The surface receptor OX40 is expressed by both effector T cells and Tregs. Despite its role as a potent costimulatory molecule for activated effector T cells, OX40 plays a critical role in regulating FOXP3 Tregs. According to Vue et al., activation of OX40 on mature FOXP3 Tregs completely disables their ability to inhibit effector T cell proliferation and cytokine production. Interestingly, OX40 signaling on Tregs does not appear to affect their survival and proliferation but suppresses FOXP3 expression (51). OX40 consistently impedes the capacity of TGFB to elicit FOXP3 expression in activated effector T cells. Consequently, OX40 functions as a potent negative regulator of both inherent FOXP3 Tregs and Tregs that are produced by activated effector T cells. This illustrates that OX40 governs a pivotal checkpoint in Treg homeostasis (51). The co-stimulation of OX40 has emerged as a promising method to combat the suppressive effects of Tregs (52). OX40 agonists have been observed to augment antitumor immunity in multiple tumor models and are currently being investigated as a new cancer immunotherapy target in clinical trials. The primary OX40 agonist antibodies employed in clinical research are MEDI0562, MEDI6469, INBRX-106, PF04518600, and SL279252 (53). Another novel approach for improving antitumor immunity involves the in situ reprogramming of tumor cells into “artificial” antigen-presenting cells (APCs) that express OX40L using nanoparticles that are loaded with an OX40L plasmid. This method has demonstrated the capacity to induce T cell proliferation in vitro, while also eliciting potent antitumor immune responses in vivo without any notable toxicity (54). While targeted therapy for OX40 has exhibited remarkable outcomes in mice with tumors, preliminary clinical evidence suggests that its efficacy in humans is moderate when utilized independently. Nevertheless, when employed with immunotherapies that target inhibitory receptors such as anti-PD-1 and anti-PD-L1, OX40 costimulation demonstrates potential as a promising strategy (55).

The secondary stimulus for Treg function is activated concurrently with TCR stimulation and facilitated by the costimulatory receptor CD28. The protein CD28 from the immunoglobulin superfamily contains a single “V-like” extracellular domain that positively regulates Treg cell response. It is the most typical and best- studied costimulatory transmembrane protein (56). CD28 is of critical importance in the development of Treg cells in the thymus, as well as in the survival, proliferation, suppressor function, and homeostasis of Treg cells in the periphery (57, 58). However, it is still unknown how CD28 regulates Treg development and homeostasis. In addition, CD28 plays a crucial role in preventing T cell anergy by providing a co-stimulatory signal. Without this signal, T cells may either remain inactive or develop tolerance to antigens. Therefore, CD28 is identified as an essential second signal for T cell activation. In the nonobese diabetic (NOD) mouse model, wherein CD28 or its ligands (CD80 [B7-1] and CD86 [B7-2]) have been knocked out, the number of natural Tregs (nTregs) decreased significantly, resulting in accelerated autoimmunity in mice (58). The development of CD28 blockers could potentially offer a more targeted therapy for cancer. Drugs like abatacept (CTLA4-Ig) and belatacept (LEA29Y) affect the CD28 signaling pathway by acting as blocking agents for CD28/CD80-CD86 interactions. Abatacept, for example, inhibits CD28 from binding to CD80-CD86 on the APC surface, thereby hindering CD28-mediated costimulatory signaling that is essential for Treg cell activation and reducing the resulting downstream inflammation response (59). These medications are employed in clinical settings to manage autoimmune diseases like psoriasis and rheumatoid arthritis (RA), wherein genetic abnormalities or modified posttranslational variations of the CTLA-4 gene or its promoter are detected. However, more research is needed to determine how strong the immunosuppressive effects of these drugs are in cancer settings. Inhibition of downstream components of the CD28 signaling pathway is another potential target for modulating Treg cell activation. Vang et al. demonstrated that the effective development of Tregs requires the P187YAP motif in the cytoplasmic tail of CD28 (60). The CBM complex (CARD11/CARMA1-BCL10-MALT1) is activated by the LCK-PRKC (protein kinase C) pathway, which is linked to CD28 through this motif (61). This complex then activates the JNK2 complex and the IKK complex, which, in turn, triggers the activation of the classical NF-κB pathway (60, 61). REL is one of the NF-κB family members required for the development of progenitor Tregs or the development of mature Tregs. A xanthine derivative called PTXF (pentoxifylline) has specific and dose-dependent effects on REL and exhibits competitive and nonselective phosphodiesterase inhibitory activity (62). Ghosh et al. demonstrated that in a mouse model of melanoma inhibition of C-Rel by PTXF, similar to genetic deletion of C-Rel, reduced Tregs and improved antitumor response (63). Inhibition of MALT1 in tumor-bearing mice with a drug induces Treg cells to secrete the immunostimulatory cytokine IFNγ only in tumor tissue, resulting in stunted tumor growth. This antitumor effect is due to macrophage activation and upregulation of MHC class I on tumor cells (64). EZH2 (enhancer of zeste 2 polycomb repressive complex 2 subunit) is expressed in Treg cells via CD28 signaling (1). Once activated, FOXP3 and EZH2 can develop a complex crucial for preserving the identity of nTregs. Suppression of EZH2 in Tregs (using Foxp3-cre ezh2^fl/fl mice) and employing CPI-1205 (a pharmacological inhibitor of EZH2) resulted in functional modifications in Tregs and improved cytotoxic activity of effector T cells (65).

CTLA4 is a related inhibitor of the costimulatory T-cell molecule CD28. While CD28 promotes T-cell activation, CTLA4 serves as an immune checkpoint and downregulated T-cell responses (66). Similar to CD28, CTLA4 binds to CD80 and CD86 on APCs, but with higher affinity and avidity than CD28, allowing it to outcompete CD28 in terms of ligands. Activation of CD28 induces expression of CTLA4 as a negative feedback mechanism in effector T cells and inhibits activation of effector T cells, whereas expression of CTLA4 in TI-Tregs, most likely due to their highly activated state, enhances their immunosuppressive activity in the TME (67). Monoclonal antibodies directed toward CTLA-4 have been found to provide an immunotherapeutic effect against cancer (CITE), however, they are also associated with inducing severe immunotherapy-related adverse events (irAE). Despite this, the targeting of CTLA-4 remains a valuable approach for cancer immunotherapy owing to its remarkable long-term benefits provided the irAEs could be effectively managed (68). Further research is warranted to design safer reagents targeting CTLA-4. Several therapeutic molecules are currently being developed to selectively knock down or disrupt CTLA4 function in TI-Tregs. Drugs that block CTLA4 are called ICIs (immune checkpoint inhibitors) (66, 69). These drugs include ipilimumab (Yervoy) and tremelimumab. Inhibition of CTLA4 by ipilimumab and tremelimumab allows for CD28-mediated positive signaling and activation of cytotoxic T-cell responses (69). Ipilimumab was the first antibody approved by the FDA. It is a fully human anti-CTLA4 (IgG1) monoclonal antibody that has been shown to improve the long-term survival of melanoma patients (70).

GITR (TNF receptor superfamily member 18) is categorized under the TNFR superfamily, and its expression is more common in Treg cells as compared to conventional CD4 and CD8 T cells. GITR can interact with its corresponding ligand, GITRL, which is expressed in APCs and endothelial cells (71, 72). The activation of GITR on Treg cells leads to instability and reduction of suppressive function, whereas activation of GITR in conventional T cells results in a co-stimulatory signal that enhances T effector cell viability, cytokine production, and effector function (72). Studies have provided evidence that the resistance to immunotherapy in glioblastoma mice models can be effectively reduced through the targeted activation of Treg cells by GITR. In a study conducted by Amoozgar et al., it was demonstrated that the activation of GITR in Treg cells by an agonistic antibody (αGITR) led to the transformation of immunosuppressive Treg cells in the TME into immunostimulatory Th1-like CD4 T cells, enhancing the immune response against the tumor (73). ΑGITR promotes the differentiation of CD4 Treg cells into CD4 effector T cells, reduced the ability of Treg cells to suppress effector T cells, and induced potent antitumor effector cells in GBM (73). Other potent GITR mAbs currently in ongoing clinical trials include MEDI1873 (NCT02583165) (74), AMG 228 (NCT02437916) (75), BMS-986156 (NCT02598960) (76), and GWN323 (NCT02740270) (77). A Phase 1 clinical trial administered the anti-GITR antibody TRX518 to patients with cancer that cannot be cured. The study established that TRX518 is safe to use as monotherapy and has immune effects (78). However, although Tregs were reduced and the ratios of T effector cells to Tregs increased, there were no notable clinical responses. Similarly, in mice with advanced tumors, GITR agonist alone could not activate effector T cells because of persistent exhaustion (78, 79). Overcoming the resistance of advanced tumors to anti-GITR monotherapy may be achieved through T-cell reinvigoration with other immune checkpoint inhibitors, such as PD-1 blockade. These findings have prompted the investigation of TRX518 in combination with the PD-1 pathway blockade in patients with advanced refractory tumors (NCT02628574) (78).

The IL2RA (interleukin-2 receptor subunit alpha)/CD25 is expressed on activated CD4+ and CD8+ T cells, as well as on Tregs, in a constitutive manner. It is noteworthy that Tregs exhibit a greater upregulation of IL2RA expression when compared to CD4+ and CD8+ T cells. This observation implies that targeting IL2RA could be a promising strategy for cancer therapy, as supported by prior research (80). The development, control, proliferation, and maintenance of Tregs depend on IL2. The binding of IL2 to IL2R leads to phosphorylation, dimerization and translocation of STAT5 (signal transducer and activator of transcription 5) to the nucleus, where they direct the transcription of target genes required for the Treg immunosuppressive phenotype, largely through direct regulation of FOXP3 expression (81). CD25 also interferes with the optimal functioning of effector T cells by depriving surrounding effector T cells of IL2. IL2 is necessary for the differentiation and fate of effector T cells after immune activation (82). Therefore, an efficient strategy to reprogram Tregs would entail the creation of a recombinant IL-2 that demonstrates heightened selectivity toward the IL-2 receptor on natural killer (NK) and naïve CD8 T-cells, while abstaining from binding to the IL-2 receptor on Tregs. The first approach targeting IL2RA was the use of denileukin diptitox, a fusion protein of IL2 and diphtheria toxin (83). More recently, the recombinant anti-IL2RA immunotoxins and daclizumab have been evaluated for their ability to block IL2 binding to IL2RA in Tregs (83). However, the mechanism of action of these drugs remains to be elucidated. Proleukin (recombinant human interleukin-2) has been identified as an effective therapy for metastatic melanoma and renal cell carcinoma (RCC) (84–86). However, the need for the high-dose regime of Proleukin because of its short half-life, and the potential to expand Tregs, has raised concerns about the use of this therapy. MDNA11 is a new IL-2R agonist designed to address the limitations associated with IL-2 therapy (86, 87). This has been made possible by implementing two crucial modifications: first, the selectivity of the receptors has been altered in favor of anti-cancer immune cells, which has led to a significant improvement in therapeutic effectiveness, and second, the immunotherapeutic has been fused with albumin to prolong its half-life, eliminating the need for a high dosage of administration (87). MDNA11 exhibits an increased attraction to CD122 (IL-2 receptor on NK and naïve CD8 T cells), but does not bind to CD25 on Treg cells. This leads to a restriction of Treg stimulation, while simultaneously prompting an amplified activation of NK and naïve CD8 T cells when compared to Proleukin. In in vivo models, MDNA11 effectively restricted tumor growth when used alone, and when combined with inhibitors of immune checkpoints, it resulted in tumor eradication with a once-weekly dosing schedule (87).

Glucose is needed for effector T cells to function and kill cancer cells. Similarly, cancer cells use glucose and compete with T cells for it, effectively reducing the amount of available glucose and, consequently, reducing the anti-cancer response (89). However, Tregs are less dependent on glucose uptake due to their master transcriptional regulator FOXP3, which shifts their metabolism from aerobic glycolysis to mitochondrial pathways, allowing Tregs to thrive in TME (90). In addition, in human FOXP3⁺ Tregs, FOXP3 inhibits AKT phosphorylation, which in turn prevents Glut1 (the main glucose transporter of Tregs) from being expressed on the cell surface. The most direct method for metabolic reprogramming of Tregs is the PI3K-AKT–MTOR pathway, which is essential in the control of various metabolic pathways, including glucose metabolism (91, 92). An important signal that appears to abolish the Treg suppressive phenotype is the activation of the PI3K → AKT → MTOR axis. In effector T cells, this axis is crucial for inducing proliferation and achieving effector function. However, in Tregs, this pathway can have a profound negative effect on suppressive activity (91). Low glucose levels in the TME promote Treg cell suppressive activity, as downstream activation of the PI3K → AKT → MTOR signaling pathway renders Tregs unstable. Thus, pharmacological intervention to enable AKT activation increases glucose uptake and glycolysis via the upregulation of Glut1 and subsequent destabilization of Tregs. PTEN (phosphatase and tensin homolog) is the main negative regulator of PI3K. PTEN catalyzes the dephosphorylation of the inositol ring 3′-phosphate in phosphatidylinositol-3-phosphate)PtdIns3P(and reverses the PtdIns3K reaction (93). Mice that carried a particular PTEN Treg deletion exhibited a substantial decrease in tumor growth and even experienced rapid regression of tumors (94). In addition, PTEN deficiency in Tregs increases FOXP3 instability, which is likely the result of increased glycolytic metabolism and decreased oxidative phosphorylation (OXPHOS) (95). The effective operation of Tregs depends on the OXPHOS pathway. Studies have shown that deleting key regulators of OXPHOS, namely Peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α) or NAD-dependent deacetylase sirtuin-3 can disrupt Treg-dependent suppressive function and impair their survival (96). Histone deacetylase-9 (Hdac9) inhibits myocyte enhancer factor 2 (MEF2), which induces the expression of genes essential to OXPHOS. Interestingly, deleting Hdac9 has been found to increase Treg suppressive function (96). PTEN inhibitor drugs are under active investigation for their immunostimulatory impacts (94). VHL (von Hippel–Lindau tumor suppressor) could be an effective target for the activation of the PI3K → AKT → MTOR pathway. Lee et al. demonstrated that Treg cells deficient in VHL exhibit elevated expression of HIF1-α (hypoxia inducible factor 1 subunit alpha). The family of prolyl hydroxylase domain-containing proteins, namely PHD1, PHD2, and PHD3, play a crucial role in driving Treg programming in metastatic niches. This is achieved through the catalyzation of post-translational hydroxylation of HIF1α, which leads to its degradation (97). The targeted deletion of the HIF1-α E3 ubiquitin ligase VHL in Tregs results in elevated levels of HIF1-α (98). This increase in HIF1-α level impairs Treg stability by upregulating the transcription of glycolytic enzymes and directly binding to FOXP3 and inducing its degradation (98). Moreover, HIF1-α directly binds to the Ifng promoter in Tregs, ultimately leading to their conversion into Th1-like cells and the impairment of their suppressive function (99). Basu et al. demonstrated that SC79, a small molecular activator of AKT, is able to overcome the effects of FOXP3-mediated AKT repression, leading to significant upregulation of Glut1 in both primary Tregs and conventional T cells (92).

The elevation in lipid oxidation implies that this metabolic route may serve as a crucial source of energy for Treg cells, which is in line with the recent findings that these cells can maintain their viability even under conditions of glucose deprivation or exposure to 2-deoxyglucose, a glycolysis inhibitor (100). However, a comprehensive understanding of how lipids regulate the development and function of Tregs is still lacking. The two most highly activated processes in activated Tregs obtained from human liver cancer are glycolysis and lipid biosynthesis. The utilization of various metabolic pathways may serve as an immune escape mechanism that grants a selective advantage to Tregs in the TME (101, 102). As a result, disrupting lipid metabolism could potentially serve as a therapeutic target for cancer therapy. Several studies have revealed that the genes responsible for lipid metabolism, including CD36, a fatty acid(FA) transporter, were found to be upregulated in TI-Treg cells, playing a crucial role in their ability to suppress antitumor CD8+ T cell responses. Notably, mice with Treg cell-specific loss of CD36 were found to exhibit a reduced count of Treg cells in their tumors, but higher numbers of TI-CD8 + T cells that produced IFN-γ and TNF (tumor necrosis factor) (103). In mice with tumors deficient in Cd36, administration of monoclonal antibodies to PD-1 was found to limit tumor progression and increase survival compared with wild-type mice. This suggests that the antitumor effect of CD36 blockade can be enhanced by reinvigorating exhausted T cells using ICIs. These results indicate that targeting CD36 in Treg cells has the potential to reprogram the TME to immunostimulatory conditions, which may therapeutically complement the effect of PD-1 blockade to combat T cell exhaustion (104). Lim et al. showed that inhibition of FAS (Fatty acid synthase) and SREBF (sterol regulatory element binding transcription factor)-dependent metabolic signaling in Treg cells induces potent antitumor immune responses without autoimmune toxicity (105). In an in vitro model of Treg proliferation, treatment with 5-tetradecyloxy-2-furoic acid (TOFA), an ACC (acetyl-CoA carboxylase) inhibitor, −a crucial enzyme in the FAS cascade - significantly reduced Treg proliferation in a dose-dependent manner. TOFA treatment specifically abolished FA accumulation in proliferating cells, demonstrating the importance of FAS in both Treg expansion and lipid pool formation (102). Signaling between CD70 and CD27 was identified as the only significant contact interaction between CD4+ naïve T cells, Tregs, and nasopharyngeal carcinoma (NPC) cells in the TME (106). This interaction strengthens a lipid signaling network in Treg cells that involves mitochondrial integrity, cholesterol homeostasis, and FA metabolism. Knockout of CD70 has been shown to inhibit Treg-mediated suppression, restoring CD8+ T cell immunity. CD70 blockade may act synergistically with anti-PD -1 treatment to reinvigorate T cell immunity against NPC. Anti-CD70+ anti-PD-1 therapy has been studied in preclinical animal models and showed enhanced tumor-killing efficacy (106).

The metabolism of amino acids provides support for the synthesis of proteins and nucleotides crucial for the rapid proliferation of Treg cells. Nevertheless, the signaling of amino acids in Treg cells and their significance in vivo are presently obscure. During the proliferation and activation of Treg cells, a number of major amino acid transporters are expressed, including LAT1, LAT4, CAT-1, SLC3A2, and SLC7A11 (107, 108). For example, the amino acid transporter SLC3A2 is required for maintaining the proliferative state of Treg cells and their suppressive activity by transporting branched-chain amino acids (BCAAs), including leucine, isoleucine, and valine (109, 110). Knockdown of Slc3a2 in mice results in impaired in vivo replication and reduced numbers of Treg cells (110). In addition to BCAAs, arginine, glutamine, serine, tryptophan, glutathione, and glutamate modulate Treg production and function. Tryptophan catabolism is mediated primarily by the rate-limiting enzyme IDO, and there is increasing evidence that IDO-induced metabolites of tryptophan, including kynurenine and 3-hydroxyanthranilic acid (3-HAA), promote the proliferation of Tregs (111). Recently, a correlation between the IDO signaling pathway and Treg biology has been showed in several stages. The first link is attributed to the ability of IDO -expressing dendritic cells (DCs) to drive the differentiation of naïve CD4 (+) T cells toward a Treg phenotype. The second link is the ability of IDO-expressing DCs to activate Tregs and significantly enhance target cell suppression. Finally, the third link is attributed to the ability of IDO to deter the inflammation-induced transformation of Tregs into T helper-like cells. These findings emphasize IDO’s potential as an appealing target for therapeutic intervention, given its involvement in stabilizing and strengthening the suppressive phenotype and deterring the reprogramming of Tregs into T helper-like cells (112). Numerous preclinical and clinical trials have been carried out to examine the effectiveness of IDO1 inhibitors, with the most studied ones being NLG-8189, Epacadostat (INCB024360), NLG-919 (GDC-0919), and Linrodostat (113). The signaling of MTORC1 (mechanistic target of rapamycin complex 1) kinase plays a crucial role in the Treg cell responses to amino acids, specifically arginine, and leucine (108). The activation of MTORC1 is amino acid-dependent and required the activation of GTPases such as RRAG (RRAGA or RRAGB) and RHEB (RHEB1 or RHEB2). The knockdown of these GTPases causes a decline in Treg cell accumulation and suppressive function and is associated with the emergence of deadly autoimmunity (108). The identification of RRAG and RHEB as downstream targets of amino acid signaling implies the potential therapeutic target of these proteins to regulate Treg cell responses in autoimmune diseases and cancer.

Autophagy and lysosomal activities hold critical significance in governing the metabolic efficacy of Treg cells and their cell lineage stability in the TME. Suppression of ATG7 (autophagy-related 7) or ATG5 (autophagy-related 5) in Treg cells results in elevated apoptosis rates and reduced lineage stability (114). Furthermore, it should be noted that the suppressive impact of autophagy on MTORC1 is a contributing factor to the survival and stability of Treg cells. In Treg cells that are deficient in autophagy, the expression of MTORC1 is dysregulated, which in turn promotes the expression of c-Myc and glycolysis metabolic pathway (115). The stability impairment of autophagy-deficient Treg cells is partially restored by pharmacologically inhibiting MTORC1, MYC, or glycolytic activities (114). Conversely, lysosomal knockout of Traf3ip3/T3jam (TRAF3 interacting protein 3) in mice impairs Treg cell suppressive functions and loss of signature gene Foxp3 expression, leading to the development of inflammatory diseases, stronger antitumor T cell responses, and a significant reduction in tumor size (116). TRAF3IP3 actively suppresses MTORC1 signaling at the lysosome and limits glycolytic metabolism, thereby preserving Treg cell identity and function (116). The Foxp3CreAtg7fl/fl mouse model was used to illustrate the effects of Treg-restricted autophagy deficiency on MC38 colon adenocarcinoma cells that were inoculated (117). Tumor growth was significantly reduced and the tumor site exhibited a remarkable increase in TI -CD8+ cells, increased IFN-γ expression in effector CD4+ and CD8+ T cells, and a remarkable decrease in Tregs (117). These results demonstrate the critical role that autophagy plays in Treg-mediated suppression of anti-tumor immune responses. Figure 1 illustrates immune signaling pathways involved in the regulation of Treg cell metabolism and adaptation to non-lymphoid tissues.