Open Access, Peer-reviewed
pISSN 1598-2629 eISSN 2092-6685
Indexed in PubMed, Scopus and SCIE
    Immune Netw. 2020 Feb;20(1):e4. English.
    Published online Feb 11, 2020.
    https://doi.org/10.4110/in.2020.20.e4
    Copyright © 2020. The Korean Association of Immunologists
    Review

    Regulatory T Cells in Tumor Microenvironment and Approach for Anticancer Immunotherapy

    Jung-Ho Kim,1 Beom Seok Kim,1 and Sang-Kyou Lee1,2
      • 1Research Institute for Precision Immune-Medicine, Good T Cells, Inc., Seoul 03722, Korea.
      • 2Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University, Seoul 03722, Korea.
    • Correspondence to Sang-Kyou Lee. Department of Biotechnology, College of Life Science and Biotechnology, Yonsei University and Research Institute for Precision Immune-Medicine, Good T Cells, Inc., 50 Yonsei-ro, Seodaemun-gu, Seoul 03722, Korea. Email: sjrlee@yonsei.ac.kr
    Received January 19, 2020; Revised January 30, 2020; Accepted February 02, 2020.

    This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (https://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

    Abstract

    Tregs have a role in immunological tolerance and immune homeostasis by suppressing immune reactions, and its therapeutic potential is critical in autoimmune diseases and cancers. There have been multiple studies conducted on Tregs because of their roles in immune suppression and therapeutic potential. In tumor immunity, Tregs can promote the development and progression of tumors by preventing effective anti-tumor immune responses in tumor-bearing hosts. High infiltration of Tregs into tumor tissue results in poor survival in various types of cancer patients. Identifying factors specifically expressed in Tregs that affect the maintenance of stability and function of Tregs is important for understanding cancer pathogenesis and identifying therapeutic targets. Thus, manipulation of Tregs is a promising anticancer strategy, but finding markers for Treg-specific depletion and controlling these cells require fine-tuning and further research. Here, we discuss the role of Tregs in cancer and the development of Treg-targeted therapies to promote cancer immunotherapy.

    Keywords
    T-lymphocytes, regulatory (Treg cells); Tumor microenvironment; Immunotherapy

    INTRODUCTION

    Tregs have been known to function as suppressors of immune responses to self- or foreign-Ags in order to maintain immune homeostasis (1). Tregs are characterized by the expression of a master transcription factor, forkhead box P3 (FOXP3), which is critical for Treg differentiation and function, including secretion of suppressive cytokines and expression of inhibitory surface molecules (1, 2, 3). Severe autoimmune-related diseases leading to scurfy phenotype develop in mice that have the transcription factor FOXP3 gene deleted, and humans with impaired FOXP3 suffer from immune-dysregulation, poly-endocrinopathy, enteropathy, and X-linked syndrome (IPEX), which is characterized by the development of multiple autoimmune disorders (4). Therefore, FOXP3+ Tregs have attracted tremendous interest because of their essential role in maintaining immune tolerance and their therapeutic potential.

    In cancer, a large population of CD4+FOXP3+ T cells infiltrates into several tumor tissues to suppress the effector functions of tumor-specific T cells (5). Therefore, the depletion of Tregs in the tumor microenvironment (TME) leads to anti-tumor effects via the reactivation of effector T (Teff) cells (6). Indeed, in cancer patients, FOXP3+ Tregs migrate into the TME and suppress various types of effector lymphocytes, including CD4+ Th cells and CD8+ CTLs (7, 8).

    Anticancer immunotherapy, especially immune checkpoint inhibitors (ICIs), can reverse the effects of immunosuppression and revitalize dysfunctional or “exhausted” CTLs, enabling them to attack cancer cells (9, 10). mAbs targeting PD-1, PD-L1, and CTLA-4 have exceptional clinical efficacy against various types of cancer (11, 12, 13). However, the efficacy of ICIs proved to be unsatisfactory in most patients, and more effective therapies are required, including combination immunotherapy.

    Here, we discuss the roles Tregs play in cancer and how cancer immunotherapy can be developed by targeting Tregs for immune precision medicine.

    ONTOGENIC CLASSIFICATION AND DEVELOPMENT OF TREGs

    Tregs can be classified into 2 subtypes depending on the site of development (14, 15). Thymus-derived Tregs (tTregs) comprise the immunosuppressive subpopulation that originates from the thymus. tTregs develop by strong interactions between the TCR of CD4/CD8 double-positive or CD4 single-positive thymocytes and self-peptide–MHC complexes in the thymus, resulting in the suppression of autoimmune reactions directed against self-Ags (16, 17). Whereas thymic selection leads to differentiation of self-Ag-specific tTregs, peripheral Tregs (pTregs) induced in peripheral tissues mediate tolerance to innocuous foreign Ags not encountered in the thymus (18). Consequently, pTregs prevent inflammation directed against innocuous Ags, which are expressed by commensal microflora or dietary components. In certain environments, such as a TME, some Teff cells turn into FOXP3+ Tregs in the periphery, which are termed induced Tregs (iTregs). These different subtypes of Tregs share significant similarities, such as their dependence on the activity of the transcription factors FOXP3 and broad complex-tramtrack-bric a brac and Cap'n'collar homology 2 (BACH2); however, some distinguishable features exist (19, 20, 21, 22). tTregs overexpress helios (a member of the Ikaros family of transcription factors) and neurophilin1 (a type 1 transmembrane protein), which are involved in the immunosuppressive activity and dominant Ag recognition, whereas iTregs frequently lack or express less of these proteins(23, 24, 25). On the other hand, an intronic FOXP3 cis-regulatory element, conserved non-coding sequence 1, harboring SMAD3 binding sites, is necessary for pTreg differentiation but is dispensable for tTreg differentiation (26). Additionally, the TCR specificity of tTregs and pTregs is distinct in many ways (18, 27).

    THE SUBTYPE OF TREGs CLASSIFIED BY SUPPRESSIVE FUNCTION

    Tregs were initially defined as CD4+ T cells with high expression of CD25, an α-subunit of IL-2 receptor. However, CD25 is a general marker of T cell activation and not exclusive to Tregs, thus emphasizing the need for additional Treg-specific markers. Although FOXP3 expression is mostly restricted to the Treg population in mice, FOXP3+ T cells in humans possess heterogeneous properties in terms of their phenotype and immunosuppressive functions, despite the high expression level of FOXP3 upon TCR stimulation of Teff cells (28). CD4+CD25+ Tregs expressing low levels of CD127 (the α-chain of the IL-7 receptor) are regarded as functional Tregs with suppressive activities (29, 30). However, TCR stimulation of naïve T cells transiently induces FOXP3 expression along with the downregulation of CD127. Given this fact, CD4+CD25+CD127lo T cells may contain some activated non-Tregs in their population. Therefore, the expression levels of CD45RA, a marker of naïve T cells, have been previously proposed as a complementary marker, as well as CD25 and FOXP3, for alternative classification of Tregs (14, 15, 31). According to this classification, CD4+CD25+FOXP3+ T cells can be categorized into three fractions: naïve Tregs (CD4+CD25loFOXP3loCD45RA+); effector Tregs (eTregs) (CD4+CD25hiFOXP3hiCD45RA−); and non-Tregs (CD4+CD25loFOXP3loCD45RA−) (Figure 1). Naïve Tregs are separated from the thymus but have not yet been stimulated in the periphery, and barely possess any immunosuppressive function. After TCR stimulation, naïve Tregs differentiate into eTregs and thus display highly immunosuppressive activities. However, FOXP3+ non-Tregs are not immunosuppressive but rather are immunostimulatory, providing inflammatory cytokines, such as IFN-γ and IL-17 (31). Therefore, the features of these types of CD4+FOXP3+ T cells are closely connected to human autoimmune and inflammatory diseases. Specifically, eTregs have been referred to as the dominant CD4+FOXP3+ T cell subpopulation in patients with inflammatory diseases (including sarcoidosis), whereas FOXP3+ non-Tregs have been implicated as the predominant subpopulation for those with autoimmune diseases, such as lupus erythematosus (31).

    Figure 1
    Classification of human CD4+FOXP3+ T cells. In humans, CD4+FOXP3+ T cells can be classified into three subsets: naïve Tregs (Fr.1), eTregs (Fr.2), and non-Tregs (Fr.3). These three fractions can be distinguished based on the expression of CD45RA, cell surface markers of naive T cells, and the transcription factor FOXP3. Moreover, these subpopulations are functionally different in terms of their suppressive activity. Effector Tregs harbor strong immune suppressive activity, but non-Tregs do not possess immune suppressive activity. In the majority of cancer, eTregs predominantly infiltrate into tumor tissues. In general, the frequency of eTregs in cancer patients is 2~5% in peripheral blood but approximately 10~50% in the tumor tissues. In contrast, naïve Tregs and FOXP3+ non-Tregs are insufficient or absent altogether.

    MECHANISMS OF IMMUNOSUPPRESSION FOR TREGs

    Tregs exert their immunosuppressive function through various modes of action. The first suppressive mechanism is associated with cytokines, and include the expenditure of IL-2 by Tregs with high levels of CD25 expression (32, 33), and suppression by inhibitory cytokines (such as TGF-β, IL-10, and IL-35) (34, 35, 36, 37). Metabolite-related suppressive mechanisms include conversion of ATP into adenosine that can prevent optimal T cell activation (38, 39), as well as the expression of indoleamine 2,3-dioxygenase (IDO) in dendritic cells (DCs), which results in T cell exhaustion by depleting amino acids essential for survival (40). Other important suppressive mechanisms involving immune checkpoint-related pathways include the disruption of Teff cells by the lymphocyte activation gene-3 (LAG-3)-MHC class II interaction, the inducible T-cell costimulator (ICOS)-ICOS ligand (ICOSL)-mediated T cell activation, and the interaction between PD-1/PD-L1 (41). Impairment of Ag-presenting cell (APC) maturation is considered as a crucial mode of action for immune suppression through the binding of CTLA-4 expressed in eTregs, which causes downregulation of CD80/86 expression. Moreover, APCs are directly eliminated by Fas/Fas ligand, perforin, and granzyme B signaling (42). The majority of observations seem to indicate that, CTLA-4-dependent and/or high-affinity IL-2R-dependent suppression of T cell activity is an especially crucial process for immunosuppression by Tregs: mice specifically lacking CTLA-4 in Tregs have impaired Treg-mediated immunosuppression (43); heterozygous CTLA4 mutations have been described in patients with multiple autoimmune symptoms, and are associated with impairments in the immunosuppressive activity of Tregs (44, 45); treating CTLA-4-immunoglobulin fusion protein leads to the conversion of Teff cells into an anergic state (46); high-dose IL-2 neutralizes Treg-mediated suppression of T cell activation and proliferation in vitro (32, 33). Through these mechanisms, Tregs can suppress Ag-specific Teff cells.

    TREG AND CANCER

    Tregs in the TME

    The association between Tregs and tumors in the TME has been studied for decades. The involvement of Tregs in anti-tumor immunity was initially reported in 1999 (47, 48). It is demonstrated that anti-CD25 Ab depleting CD4+CD25+ Tregs retarded tumor growth in T cell-deficient mice transplanted with CD25+ cell-depleted splenocytes. Tregs accumulate at tumor sites and in the peripheral circulation of patients with cancer, and their immunosuppressive function, as well as their number, are increased compared to those found in healthy donors (47, 48). Tregs that have infiltrated into human tumors account for 10%–50% of CD4+ T cells in tumors, which is more abundant relative to the 2%–5% of CD4+ T cells found in the peripheral blood of individuals without cancer. Furthermore, higher levels of tumor-infiltrating Tregs and Treg/Teff cell ratio indicate poor prognosis in patients with various types of cancers, such as non-small cell lung carcinoma (NSCLC), melanoma, and gastric cancer (49, 50). The accumulation of Tregs in tumors are well-studied in the previous reports, elucidating their ability to effectively migrate into tissue sites depending on the expression of multiple chemokine receptors; for example, CXCR5 in Tregs from the lymph node of patients with lung cancer (51). C-C motif chemokine receptor (CCR) 4 with CCL12, CCR4 with CCL17, CCR10 with CCL28, and CXCR4 with CXCL1 have been reported as other chemokine receptors on Tregs with their partner chemokines (51, 52, 53, 54, 55, 56). Treg infiltration into tumor tissues has been extensively investigated in the context of recent “immune-oncology” researches (57, 58). These studies confirmed the conspicuous presence of Tregs among tumor-infiltrating lymphocytes, especially in tumors harboring large immune cell infiltrates (59). Also, based on numerous articles, it is demonstrated that Tregs block antitumor immunity, and thus enhance tumor progression, and their presence in the TME is profoundly linked with unfavorable prognosis, resulting in short OS (60, 61). Notably, Tregs that directly interact with the tumor are more essential for the study of immune evasion by the tumor, because the peripheral Tregs do not always represent immune-tolerant TME (49). Recently, compelling evidence suggests that colorectal cancer (CRC) abundantly infiltrated with the FOXP3hi subset of suppression-competent eTregs lead to poor prognosis, while the presence of pro-inflammatory cytokine-secreting CD4+CD45RAloFOXP3lo T cells (non-Tregs) in tumor tissues is associated with favorable outcomes (50). Therefore, especially in cancer patients with high numbers of tumor-infiltrating Tregs, further analysis needs to be conducted in order to distinguish FOXP3+ non-Tregs from FOXP3hi eTregs in tumors. This will help evaluate the clinical importance of FOXP3+ cells in tumor tissues. In summary, Tregs, particularly in the TME, are a key factor of hindrance in anti-tumor immunity in various types of cancer patients, resulting in the initiation of tumor progression or resistance against cancer immunotherapy (Figure 2).

    Figure 2
    Role of Tregs in immune-evasion of cancer after differentiation from the thymus. Natural Tregs, generated in the thymus, are initially differentiated from the thymocytes by using thymic “positive selection” based on the binding affinity of TCR to the self- peptides-MHC complexes expressed on thymic APCs. The CD4+ T cells which bind to self-peptide-MHC complexes with the highest affinity are removed through apoptosis, and those that cannot bind at all with the complexes will also be removed because of the absence of TCR stimulation. After strong TCR stimulation, these immature precursor cells undergo IL-2-mediated signaling, thus expressing the master transcription factor FOXP3, which orchestrates the differentiation of these cells into Tregs. By contrast, immature T cells with lower affinity for self-peptide–MHC complexes are also positively selected but differentiate into Teff cells. Even though some Teff cells are auto-reactive, Tregs can block the autoimmunity of Teff cells owing to their higher affinity. These immune cells that have departed from the thymus travel through the blood vessels and move wherever they are needed. In the tumor microenvironment, especially, Tregs expressing the chemokine receptors, such as CCR4, CCR5, CCR8, and CCR10, are recruited to and around the tumors by binding to chemokines including CCL1, CCL5, CCL22, and CCL28 that are secreted from various kinds of tumors. Moreover, Tregs constitutively express the IL-2 receptor subunit-α (also known as CD25) that binds to IL-2 with higher affinity, resulting in the depletion of IL-2 from their surroundings. This leads to the reduction of the availability of this cytokine to Teff cells. Tregs also constitutively express CTLA-4, a checkpoint protein suppressing the immune response, which binds to CD80 and CD86 on APC, thereby transmitting suppressive signals to Teff cells. In addition, Tregs secrete cytokines, such as IL-10, IL-35, and TGF-β, which can decrease the activity of APCs and Teff cells and secrete granzymes and perforins that can directly kill these cells. Moreover, abundant adenosine is produced by Tregs via nucleotidase activity of CD39 and CD73, which provides immunosuppressive signals to Teff cells and APCs through the engagement of adenosine A2AR.

    Molecular and cellular characteristics of Tregs in the tumor

    On the basis of the functional classification of Tregs described above, Tregs in the TME is mostly composed of bona fide Treg (eTreg) cells that overexpress immunosuppressive molecules including CTLA-4 and T cell immunoreceptor with Ig and ITIM domains (TIGIT), which are not expressed much in naïve Tregs (14, 62, 63). Also, transcriptome analysis on human cancer specimens shows that tumor-infiltrating Tregs have high expression levels of Treg-activation surface markers, such as glucocorticoid-induced TNFR-related protein (GITR; also known as TNFRSF18), lymphocyte-activation gene 3 protein (LAG3), T cell immunoglobulin mucin receptor 3 (TIM3; also known as HAVCR2), OX40 (also known as TNFRSF4), and ICOS (64). These phenotypes, distinct from peripheral Tregs, indicate that Tregs in the TME show potent immunosuppressive activities in terms of function and number. One possible mechanism that has been suggested is that proliferating and dying tumor cells produce a large number of self-Ags, which are recognized by Tregs, thereby inducing the activation of Tregs in the TME (65). As part of the mechanism mentioned above, whether Tregs recognize Ags exclusively or share Ags with Th cells remains unclear at this stage (65, 66). Nevertheless, Tregs usually possess a higher binding affinity to TCRs than does Teff cells, resulting in the predominant activation of Tregs in the TME, even in the presence of competition with Teff cells. Furthermore, tumors can harbor some immature dendritic cells, which drive the activation and/or proliferation of Tregs in a TGF-β-dependent manner in animal models. In contrast to the abundant animal studies regarding iTregs, the existence of TGF-β-iTreg cells in humans have not been elucidated clearly; accordingly, investigations on human tumor specimens are crucial to understanding the phenotypes and origins of tumor-infiltrating Tregs.

    Regulation of tumor Ag-specific T cells by Tregs

    Generally, 2 different types of Ags can exist in tumor cells. First, ‘neoantigens’ are non-self-Ags derived from either oncogenic viral proteins or abnormal self- proteins caused by somatic mutations. Second, self-Ags that arise from the aberrant overexpression of endogenous proteins are categorized as ‘shared antigens.’ How CD8+ T cells distinguish each of these 2 types of Ag for anti-tumor immunity remains unclear. Therefore, the different immunosuppressive mechanisms of Tregs against CD8+ T cells specific for shared Ags versus neoantigens need to be resolved through further research. Interestingly, in some animal models, it is suggested that Tregs select for non-self-Ag specific CD8+ T cells harboring high-affinity TCRs by manipulating co-stimulatory signaling (67). In particular, CD8+ T cells targeting self-Ags are more susceptible to Tregs due to the APCs that provide limited co-stimulatory signals (68). By contrast, non-self-specific CD8+ T cells are resistant to suppression by Tregs in humans (68). These results demonstrate that CD8+ T cells specific for neoantigens are more resistant to Treg-mediated immunosuppression, and given this fact, tumors that express shared Ags can serve as more vulnerable targets for cancer immunotherapy.

    TARGETING TREGs FOR CANCER IMMUNOTHERAPY

    Tregs, which express the transcription factor FOXP3, are indispensable for immunological self-tolerance and immune homeostasis. They also disturb tumor immunity and can, therefore, be targeted to elicit an anti-tumor immune response by depleting them or diminishing their suppressive capabilities (69).

    FOXP3, a well-characterized Treg-specific marker and the key phenotype of Tregs to function as suppressive cells, is a transcription factor expressed in the nucleus and is therefore hard to detect for clinical use.

    Therapies targeting Tregs are not likely to be effective against all types of tumors. For example, Treg depletion in animal models led to the regression of tumors from certain cell lines, such as RL-male1 or MethA cells, but did not in other cell lines like AKSL2 or RL-female8 cells (70).

    Thus, the identification of novel and specific biomarkers that distinguish Tregs from other cells in the TME is essential for increasing the possibility of successfully developing effective cancer therapies targeting Tregs.

    Specific surface molecules on Tregs

    Depletion of Tregs or attenuation of their suppressive activity can enhance tumor immunity. Tregs in the TME reveal several cell surface markers, including CD25, CTLA-4, GITR, OX40, ICOS, PD-1, LAG3, TIM3, TIGIT, CCR4, folate receptor (FR) 4 (71) and CD15s (72), and specific mAbs for these cell surface marker can be used to deplete Tregs or hinder their function (Table 1).

    Table 1
    Ab-drug development status of Treg-targeting therapy

    CD25

    Several studies show that the removal of CD25+CD4+Tregs by anti-CD25 mAb or toxin-conjugated anti-IL-2 (Denileukin diftitox) facilitates the activation of Teff cells, which greatly inhibit tumor growth in rodents (47, 48, 73, 74). Treg depletion using an anti-CD25 mAb has been evaluated in clinical trials. When patients with breast cancer were vaccinated with various tumor-associated peptides followed by a treatment with daclizumab—an anti-CD25 mAb—to deplete Tregs, there was robust T cell priming with prolonged stable disease for 6 out of 10 patients and a median progression-free survival of 4.8 months (75). By contrast, another study showed that the administration of daclizumab depleted Teff cells as well as Tregs in patients with melanoma, but neither an antitumor immune response nor Ab production was observed (76). Because activation of Teff cells induces CD25 expression, Treg depletion by targeting CD25 can be accompanied by a deficiency in Teff cells. Thus, anti-CD25 mAb administration may lead to limited efficacy in increasing antitumor T cell responses.

    CTLA-4

    CTLA-4, an immune-checkpoint molecule, is expressed by tumor-infiltrating CD4+ and CD8+ Teff cells and FOXP3+CD4+ Tregs (77). The anti-tumor activity of anti-CTLA-4 mAb was originally thought to be dependent on the reinvigoration of exhausted Teff cells expressing CTLA-4 (78). However, several preclinical studies indicate that the anti-tumor activity of anti-CTLA-4 mAb is instead dependent on the depletion of CTLA-4-expressing Tregs in the TME through Ab-dependent cellular cytotoxicity, thereby increasing the Teff cell to Treg ratio. Consequently, disrupting the function of the Fc portion of the Ab completely abrogated the anti-tumor activity of the anti-CTLA-4 mAb (79, 80, 81, 82). Therefore, further research to address the relative roles of CTLA-4 in Teff cells and Tregs in the TME of various cancers is needed.

    Co-stimulatory molecules (GITR, OX40, and ICOS)

    Co-stimulatory receptors, such as GITR, OX40, and ICOS, highly expressed by Tregs can be candidates for Treg depletion and functional modulation.

    GITR is expressed at a high level by Tregs but at a low level by resting CD4+ and CD8+ T cells, and they play an important role in Treg expansion (83). Activation of GITR signaling through an agonistic anti-GITR mAb inhibits the suppression activity of Tregs and induces Treg-resistant Teff cells (84). The GITR agonists are now being investigated in patients with advanced solid cancer.

    OX40 is constitutively expressed by a subset of Tregs, but is also found on Teff cells (85). Although OX40-agonists are used to stimulate anti-tumor responses of Teff cells, the effect on Tregs in cancer is not well understood. OX40 agonists are being investigated alone or in combination with other immunotherapies in patients with solid cancer or melanoma (86).

    ICOS is important in Treg function and homeostasis (87, 88), and is highly expressed by activated Tregs in tumor-infiltrating lymphocyte (TIL) of gastric cancer patients (89). Agonistic anti-ICOS mAbs, like OX40 and GITR agonists, are expected to have a dual-mode of action involving activation of Ag-specific CD4+ Teff cells and selective depletion of Tregs (90).

    Co-inhibitory molecules (TIGIT, LAG3, and TIM3)

    Immune co-inhibitory receptors predominantly expressed by Tregs are also being explored as Treg-targeted immunotherapies. TIGIT marks a population of Tregs with an enhanced suppressive capacity in the TME (91, 92). TIGIT+Tregs have a highly suppressive activity and they express more co-inhibitory molecules, such as LAG3, TIM3, and PD-1 compared to TIGIT-Tregs (91). In contrast, another study showed that TIGIT expression correlated with CD8+ Teff cell exhaustion, and TIGIT blockade increased the production of effector cytokines, such as IFN-γ and TNF-α, by CD8+ Teff cells in a Treg-independent manner (93). Thus, TIGIT blockade may promote anti-tumor immunity through both Treg dependent and independent mechanisms. LAG3 is expressed on TILs, especially on Tregs. Interestingly, CD4+CD25+FOXP3−LAG3+ T cell population from colorectal cancer patients produce immunosuppressive cytokines, such as IL-10 and TGF-β, and show 50% more suppressive activity than FOXP3+ Tregs (94). The humanized LAG3 Ab is under phase I and phase II clinical trials in patients with various solid cancers. TIM3 is expressed on activated T cells and certain subsets of Tregs and binds to several identified ligands (i.e. galectin-9, HMGB1, caecam, phosphatidyl serine) (95, 96). The co-inhibitory function of TIM3 is implicated in tumor evasion and TIM3+ Tregs have an increased suppressive function (97, 98). Co-inhibitory receptors such as LAG3, TIM3, and TIGIT seem to offer an advantage as they are dominantly overexpressed on tumor-infiltrating Tregs. However, broader studies need to be conducted in order to determine their safety and efficacy.

    CCR4

    Chemokine receptors, which allow Tregs to migrate to the TME site, can be a candidate molecule for Treg depletion (99). Tumor-infiltrating macrophages and tumor cells produce the CCL22, which chemoattracts Tregs expressing CCR4 (52, 100, 101). CCR4 is highly expressed by eTregs but not by naive Tregs or most Teff cells, except for some Th2 and Th17 cells in peripheral blood (102). In vitro or in vivo anti-CCR4 mAb treatment selectively depleted eTregs and efficiently induced tumor-specific effector CD4+ and CD8+ T cells (63). Additionally, the administration of an anti-CCR4 mAb (mogamulizumab) on advanced solid cancer patients significantly reduces eTregs in peripheral blood (70). Additional clinical trials are underway with immune checkpoint blockades.

    Treg and Immune checkpoint inhibition

    Immune checkpoint molecules, including CTLA-4 and PD-1, are highly expressed by activated Tregs and Teff cells (49, 77). The role of CTLA-4 in Tregs is mentioned above. The role of the inhibitory receptor PD-1 on Teff cells is well established, but its function in Tregs is less clear. Tregs in the TME show comparable levels of PD-1 expression with that of Teff cells. Because PD-1 signaling in Treg reduces its immunosuppressive activity, PD-1-deficient Tregs might potentiate the activation and immunosuppressive function of Tregs (103). Various studies reported that the anti-PD-1 mAb, nivolumab, reduced the immune-suppressive activity of Tregs (104). However, another research maintains that PD-1 inhibition induced the immune-suppressive activity mediated by Tregs in some cancer patients (105). Therefore, more research is needed to investigate the role of PD-1 in Teff cells and the role of Tregs in the TME.

    Treg modulation factor in the TME

    Cytokines

    The TGF-β and IL-2 signaling pathways are essential to maintain the differentiation and survival of Tregs in the thymus and peripheral tissues. The effect of cancer therapy by IL-2 blockade is still unclear. In particular, hyperactivation of the TGF-β pathway in the TME enhances tumor progression by stimulating angiogenesis and inhibiting innate and adaptive anti-tumor immune responses (106). A type I TGF-β receptor serine/threonine kinase inhibitor (galunisertib) increased the ratio of CD8+ T cells to Tregs in melanoma animal models in a combination treatment with an anti-CTLA-4 mAb (107). In addition, a combination therapy of galunisertib with an anti-PD-1 or anti-PD-L1 mAb is currently underway in clinical trials (108). Thus, the regulation of TGF-β signaling pathways can be a noteworthy candidate for Treg control.

    Targeting intracellular signaling in Tregs

    PI3K signaling pathway, which is crucial for Treg maintenance and function, is a promising target for Treg-directed therapy (109). Inhibitors of PI3K effectively reduced immune suppression by Tregs in mouse models. In particular, selective inactivation of PI3Kδ in Tregs increases the activity of CD8+ T cells, preventing or slowing tumor development, progression, and metastasis (110). Specific ablation of the PI3K-phosphatase and tensin homolog (PTEN)-mTOR pathway in Tregs impairs mitochondrial fitness, upregulates glycolysis, leads to the loss of FOXP3 expression in Tregs, and induces Teff cell activity (111, 112). Combination treatment of pembrolizumab and PI3Kδ inhibitors is currently being explored at an early stage of phase I trial in patients with advanced solid tumors. Also, tyrosine kinase inhibitors, including imatinib and dasatinib, which are known to target specific TCR signaling molecules, have been shown to reduce Treg survival and function through off-target effects (113, 114). In the discontinued clinical trial for dasatinib, Treg reduction was observed and showed favorable clinical outcomes in patients with chronic myeloid leukemia (113).

    CD39 and CD73

    Tregs produce extracellular adenosine by the activity of CD39 and CD73 on their cell surface. Tregs express high levels of CD39 and CD73 and directly inhibit T cell activation via interaction with adenosine A2A receptor (A2AR). Moreover, adenosine increases tolerogenic APCs and enhances the immunosuppressive activity of Tregs (115). Therefore, CD39 and CD73, which are important for adenosine metabolism, can be promising therapeutic targets.

    VEGF signaling

    VEGF receptor (VEGFR) 2 plays an important role in tumor angiogenesis, and this signaling pathway has been shown to increase the infiltration of Tregs into tumors in animal models (116, 117). In addition, blockade of VEGF-VEGFR2 signaling has been reported to inhibit tumor growth by reducing the accumulation of immunosuppressive cells, including Tregs, myeloid-derived suppressor cells, and M2 macrophages in the TME (118). Furthermore, researchers have established that treatment of a humanized anti-VEGFR2 mAb, ramucirumab, led to a decrease in PD-1 expression in CD8+ T cells and a reduction in eTreg infiltration into the TME (49, 119). Thus, targeting VEGFR2 molecules expressed by activated Tregs or blocking the VEGF-VEGFR2 signaling may contribute to cancer therapy through Treg inhibition.

    CONCLUSION

    Tregs serve as a specialized cell lineage that plays an essential role in the immunological tolerance of immune homeostasis through their immune suppressive activity. High levels of Treg infiltration in the TME lead to an undesirable prognosis in patients with various types of cancers. Depleting Tregs and regulating their function in the TME may be potential strategies for cancer therapy. Several Treg-targeted therapies are under investigation, but the lack of specific markers for Tregs has limited their clinical application. Since drugs that selectively deplete Tregs in the TME of cancer patients have not been developed at present, identification of specific targets for disrupting and depleting Tregs is important for the success of cancer immunotherapy. In the future, the development of Treg-targeted therapies based on the TME's comprehensive immune profiling may lead to new therapies and immune precision for individual cancer patients.

    Notes

    Conflict of Interest:The authors declare no potential conflicts of interest.

    Author Contributions:

    • Conceptualization: Lee SK, Kim JH, Kim BS.

    • Investigation: Kim JH, Kim BS.

    • Project administration: Lee SK.

    • Supervision: Lee SK.

    • Writing - original draft: Kim JH, Kim BS.

    • Writing - review & editing: Lee SK, Kim JH, Kim BS.

    Abbreviations

    A2AR A2A receptor
    APC Ag-presenting cell
    BACH2 broad complex-tramtrack-bric a brac and Cap'n'collar homology 2
    CCR C-C motif chemokine receptor
    CNS1 conserved non-coding sequence 1
    CRC colorectal cancer
    DC dendritic cell
    eTreg effector Treg
    FOXP3 Forkhead box p 3
    FR folate receptor
    GITR glucocorticoid-induced TNFR-related protein
    ICI immune checkpoint inhibitor
    ICOS inducible T-cell costimulatory
    IDO indoleamine 2,3-dioxygenase
    IPEX immune-dysregulation, poly-endocrinopathy, enteropathy, and X-linked syndrome
    iTreg induced Treg
    LAG3 lymphocyte-activation gene 3 protein
    LAG-3 lymphocyte activation gene-3
    PTEN phosphatase and tensin homolog
    pTreg Peripheral Treg
    Teff effector T
    TIGIT T cell immunoreceptor with Ig and ITIM domains
    TIL tumor-infiltrating lymphocyte
    TIM3 T cell immunoglobulin mucin receptor 3
    TME tumor microenvironment
    tTreg thymus-derived Treg
    VEGFR VEGF receptor

    ACKNOWLEDGEMENTS

    This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government, Ministry of Science and ICT (MSIT) (NRF-2017R1A2A1A17069807); Global Research Laboratory (GRL) Program through the NRF funded by the MSIT (NRF-2016K1A1A2912755).

    References

      1. Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol 2012;30:531–564.
      1. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol 2003;4:330–336.
      1. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science 2003;299:1057–1061.
      1. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, Kelly TE, Saulsbury FT, Chance PF, Ochs HD. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet 2001;27:20–21.
      1. Motz GT, Coukos G. Deciphering and reversing tumor immune suppression. Immunity 2013;39:61–73.
      1. Baecher-Allan C, Brown JA, Freeman GJ, Hafler DA. CD4+CD25high regulatory cells in human peripheral blood. J Immunol 2001;167:1245–1253.
      1. Spranger S, Spaapen RM, Zha Y, Williams J, Meng Y, Ha TT, Gajewski TF. Up-regulation of PD-L1, IDO, and Tregs in the melanoma tumor microenvironment is driven by CD8+ T cells. Sci Transl Med 2013;5:200ra116
      1. Williams JB, Horton BL, Zheng Y, Duan Y, Powell JD, Gajewski TF. The EGR2 targets LAG-3 and 4-1BB describe and regulate dysfunctional antigen-specific CD8+ T cells in the tumor microenvironment. J Exp Med 2017;214:381–400.
      1. Pardoll DM. The blockade of immune checkpoints in cancer immunotherapy. Nat Rev Cancer 2012;12:252–264.
      1. Zou W, Wolchok JD, Chen L. PD-L1 (B7-H1) and PD-1 pathway blockade for cancer therapy: mechanisms, response biomarkers, and combinations. Sci Transl Med 2016;8:328rv4
      1. Brahmer JR, Tykodi SS, Chow LQ, Hwu WJ, Topalian SL, Hwu P, Drake CG, Camacho LH, Kauh J, Odunsi K, et al. Safety and activity of anti-PD-L1 antibody in patients with advanced cancer. N Engl J Med 2012;366:2455–2465.
      1. Hodi FS, O'Day SJ, McDermott DF, Weber RW, Sosman JA, Haanen JB, Gonzalez R, Robert C, Schadendorf D, Hassel JC, et al. Improved survival with ipilimumab in patients with metastatic melanoma. N Engl J Med 2010;363:711–723.
      1. Topalian SL, Hodi FS, Brahmer JR, Gettinger SN, Smith DC, McDermott DF, Powderly JD, Carvajal RD, Sosman JA, Atkins MB, et al. Safety, activity, and immune correlates of anti-PD-1 antibody in cancer. N Engl J Med 2012;366:2443–2454.
      1. Togashi Y, Nishikawa H. Regulatory T cells: molecular and cellular basis for immunoregulation. Curr Top Microbiol Immunol 2017;410:3–27.
      1. Sakaguchi S, Miyara M, Costantino CM, Hafler DA. FOXP3+ regulatory T cells in the human immune system. Nat Rev Immunol 2010;10:490–500.
      1. Hsieh CS, Zheng Y, Liang Y, Fontenot JD, Rudensky AY. An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat Immunol 2006;7:401–410.
      1. Wong J, Obst R, Correia-Neves M, Losyev G, Mathis D, Benoist C. Adaptation of TCR repertoires to self-peptides in regulatory and nonregulatory CD4+ T cells. J Immunol 2007;178:7032–7041.
      1. Yadav M, Stephan S, Bluestone JA. Peripherally induced Tregs - role in immune homeostasis and autoimmunity. Front Immunol 2013;4:232.
      1. Ziegler SF. FOXP3: of mice and men. Annu Rev Immunol 2006;24:209–226.
      1. Roychoudhuri R, Hirahara K, Mousavi K, Clever D, Klebanoff CA, Bonelli M, Sciumè G, Zare H, Vahedi G, Dema B, et al. BACH2 represses effector programs to stabilize Treg-mediated immune homeostasis. Nature 2013;498:506–510.
      1. Igarashi K, Kurosaki T, Roychoudhuri R. BACH transcription factors in innate and adaptive immunity. Nat Rev Immunol 2017;17:437–450.
      1. Kim EH, Gasper DJ, Lee SH, Plisch EH, Svaren J, Suresh M. Bach2 regulates homeostasis of Foxp3+ regulatory T cells and protects against fatal lung disease in mice. J Immunol 2014;192:985–995.
      1. Overacre-Delgoffe AE, Chikina M, Dadey RE, Yano H, Brunazzi EA, Shayan G, Horne W, Moskovitz JM, Kolls JK, Sander C, et al. Interferon-γ drives Treg fragility to promote anti-tumor immunity. Cell 2017;169:1130–1141.e11.
      1. Sarris M, Andersen KG, Randow F, Mayr L, Betz AG. Neuropilin-1 expression on regulatory T cells enhances their interactions with dendritic cells during antigen recognition. Immunity 2008;28:402–413.
      1. Getnet D, Grosso JF, Goldberg MV, Harris TJ, Yen HR, Bruno TC, Durham NM, Hipkiss EL, Pyle KJ, Wada S. A role for the transcription factor Helios in human CD4+CD25+ regulatory T cells. Mol Immunol 2010;47:1595–1600.
      1. Zheng Y, Josefowicz S, Chaudhry A, Peng XP, Forbush K, Rudensky AY. Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate. Nature 2010;463:808–812.
      1. Lathrop SK, Santacruz NA, Pham D, Luo J, Hsieh CS. Antigen-specific peripheral shaping of the natural regulatory T cell population. J Exp Med 2008;205:3105–3117.
      1. Tran DQ, Ramsey H, Shevach EM. Induction of FOXP3 expression in naive human CD4+FOXP3 T cells by T-cell receptor stimulation is transforming growth factor-beta dependent but does not confer a regulatory phenotype. Blood 2007;110:2983–2990.
      1. Liu W, Putnam AL, Xu-Yu Z, Szot GL, Lee MR, Zhu S, Gottlieb PA, Kapranov P, Gingeras TR, Fazekas de St Groth B, et al. CD127 expression inversely correlates with FoxP3 and suppressive function of human CD4+ T reg cells. J Exp Med 2006;203:1701–1711.
      1. Seddiki N, Santner-Nanan B, Martinson J, Zaunders J, Sasson S, Landay A, Solomon M, Selby W, Alexander SI, Nanan R, et al. Expression of interleukin (IL)-2 and IL-7 receptors discriminates between human regulatory and activated T cells. J Exp Med 2006;203:1693–1700.
      1. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, Parizot C, Taflin C, Heike T, Valeyre D, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity 2009;30:899–911.
      1. Thornton AM, Shevach EM. CD4+CD25+ immunoregulatory T cells suppress polyclonal T cell activation in vitro by inhibiting interleukin 2 production. J Exp Med 1998;188:287–296.
      1. Takahashi T, Kuniyasu Y, Toda M, Sakaguchi N, Itoh M, Iwata M, Shimizu J, Sakaguchi S. Immunologic self-tolerance maintained by CD25+CD4+ naturally anergic and suppressive T cells: induction of autoimmune disease by breaking their anergic/suppressive state. Int Immunol 1998;10:1969–1980.
      1. Steinbrink K, Wölfl M, Jonuleit H, Knop J, Enk AH. Induction of tolerance by IL-10-treated dendritic cells. J Immunol 1997;159:4772–4780.
      1. Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, Cross R, Sehy D, Blumberg RS, Vignali DA. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature 2007;450:566–569.
      1. Turnis ME, Sawant DV, Szymczak-Workman AL, Andrews LP, Delgoffe GM, Yano H, Beres AJ, Vogel P, Workman CJ, Vignali DA. Interleukin-35 limits anti-tumor immunity. Immunity 2016;44:316–329.
      1. Jarnicki AG, Lysaght J, Todryk S, Mills KH. Suppression of antitumor immunity by IL-10 and TGF-beta-producing T cells infiltrating the growing tumor: influence of tumor environment on the induction of CD4+ and CD8+ regulatory T cells. J Immunol 2006;177:896–904.
      1. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, Chen JF, Enjyoji K, Linden J, Oukka M, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med 2007;204:1257–1265.
      1. Wilson JM, Ross WG, Agbai ON, Frazier R, Figler RA, Rieger J, Linden J, Ernst PB. The A2B adenosine receptor impairs the maturation and immunogenicity of dendritic cells. J Immunol 2009;182:4616–4623.
      1. Uyttenhove C, Pilotte L, Théate I, Stroobant V, Colau D, Parmentier N, Boon T, Van den Eynde BJ. Evidence for a tumoral immune resistance mechanism based on tryptophan degradation by indoleamine 2,3-dioxygenase. Nat Med 2003;9:1269–1274.
      1. Saleh R, Elkord E. Treg-mediated acquired resistance to immune checkpoint inhibitors. Cancer Lett 2019;457:168–179.
      1. Burchell JT, Strickland DH, Stumbles PA. The role of dendritic cells and regulatory T cells in the regulation of allergic asthma. Pharmacol Ther 2010;125:1–10.
      1. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, Nomura T, Sakaguchi S. CTLA-4 control over Foxp3+ regulatory T cell function. Science 2008;322:271–275.
      1. Schubert D, Bode C, Kenefeck R, Hou TZ, Wing JB, Kennedy A, Bulashevska A, Petersen BS, Schäffer AA, Grüning BA, et al. Autosomal dominant immune dysregulation syndrome in humans with CTLA4 mutations. Nat Med 2014;20:1410–1416.
      1. Kuehn HS, Ouyang W, Lo B, Deenick EK, Niemela JE, Avery DT, Schickel JN, Tran DQ, Stoddard J, Zhang Y, et al. Immune dysregulation in human subjects with heterozygous germline mutations in CTLA4. Science 2014;345:1623–1627.
      1. Perez VL, Van Parijs L, Biuckians A, Zheng XX, Strom TB, Abbas AK. Induction of peripheral T cell tolerance in vivo requires CTLA-4 engagement. Immunity 1997;6:411–417.
      1. Onizuka S, Tawara I, Shimizu J, Sakaguchi S, Fujita T, Nakayama E. Tumor rejection by in vivo administration of anti-CD25 (interleukin-2 receptor alpha) monoclonal antibody. Cancer Res 1999;59:3128–3133.
      1. Shimizu J, Yamazaki S, Sakaguchi S. Induction of tumor immunity by removing CD25+CD4+ T cells: a common basis between tumor immunity and autoimmunity. J Immunol 1999;163:5211–5218.
      1. Tada Y, Togashi Y, Kotani D, Kuwata T, Sato E, Kawazoe A, Doi T, Wada H, Nishikawa H, Shitara K. Targeting VEGFR2 with Ramucirumab strongly impacts effector/activated regulatory T cells and CD8+ T cells in the tumor microenvironment. J Immunother Cancer 2018;6:106.
      1. Saito T, Nishikawa H, Wada H, Nagano Y, Sugiyama D, Atarashi K, Maeda Y, Hamaguchi M, Ohkura N, Sato E, et al. Two FOXP3+CD4+ T cell subpopulations distinctly control the prognosis of colorectal cancers. Nat Med 2016;22:679–684.
      1. Akimova T, Zhang T, Negorev D, Singhal S, Stadanlick J, Rao A, Annunziata M, Levine MH, Beier UH, Diamond JM, et al. Human lung tumor FOXP3+ Tregs upregulate four “Treg-locking” transcription factors. JCI Insight 2017;2:94075.
      1. Curiel TJ, Coukos G, Zou L, Alvarez X, Cheng P, Mottram P, Evdemon-Hogan M, Conejo-Garcia JR, Zhang L, Burow M, et al. Specific recruitment of regulatory T cells in ovarian carcinoma fosters immune privilege and predicts reduced survival. Nat Med 2004;10:942–949.
      1. Takeuchi Y, Nishikawa H. Roles of regulatory T cells in cancer immunity. Int Immunol 2016;28:401–409.
      1. Gobert M, Treilleux I, Bendriss-Vermare N, Bachelot T, Goddard-Leon S, Arfi V, Biota C, Doffin AC, Durand I, Olive D, et al. Regulatory T cells recruited through CCL22/CCR4 are selectively activated in lymphoid infiltrates surrounding primary breast tumors and lead to an adverse clinical outcome. Cancer Res 2009;69:2000–2009.
      1. Tan MC, Goedegebuure PS, Belt BA, Flaherty B, Sankpal N, Gillanders WE, Eberlein TJ, Hsieh CS, Linehan DC. Disruption of CCR5-dependent homing of regulatory T cells inhibits tumor growth in a murine model of pancreatic cancer. J Immunol 2009;182:1746–1755.
      1. Kryczek I, Wei S, Zhu G, Myers L, Mottram P, Cheng P, Chen L, Coukos G, Zou W. Relationship between B7-H4, regulatory T cells, and patient outcome in human ovarian carcinoma. Cancer Res 2007;67:8900–8905.
      1. Ladoire S, Martin F, Ghiringhelli F. Prognostic role of FOXP3+ regulatory T cells infiltrating human carcinomas: the paradox of colorectal cancer. Cancer Immunol Immunother 2011;60:909–918.
      1. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, Platell C, Iacopetta B. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol 2009;27:186–192.
      1. Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay NH, Mosseri V, Laccourreye O, Bruneval P, Fridman WH, et al. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin Cancer Res 2006;12:465–472.
      1. Petersen RP, Campa MJ, Sperlazza J, Conlon D, Joshi MB, Harpole DH Jr, Patz EF Jr. Tumor infiltrating Foxp3+ regulatory T-cells are associated with recurrence in pathologic stage I NSCLC patients. Cancer 2006;107:2866–2872.
      1. Marshall EA, Ng KW, Kung SH, Conway EM, Martinez VD, Halvorsen EC, Rowbotham DA, Vucic EA, Plumb AW, Becker-Santos DD, et al. Emerging roles of T helper 17 and regulatory T cells in lung cancer progression and metastasis. Mol Cancer 2016;15:67.
      1. Fridman WH, Pagès F, Sautès-Fridman C, Galon J. The immune contexture in human tumours: impact on clinical outcome. Nat Rev Cancer 2012;12:298–306.
      1. Sugiyama D, Nishikawa H, Maeda Y, Nishioka M, Tanemura A, Katayama I, Ezoe S, Kanakura Y, Sato E, Fukumori Y, et al. Anti-CCR4 mAb selectively depletes effector-type FoxP3+CD4+ regulatory T cells, evoking antitumor immune responses in humans. Proc Natl Acad Sci U S A 2013;110:17945–17950.
      1. De Simone M, Arrigoni A, Rossetti G, Gruarin P, Ranzani V, Politano C, Bonnal RJ, Provasi E, Sarnicola ML, Panzeri I, et al. Transcriptional landscape of human tissue lymphocytes unveils uniqueness of tumor-infiltrating T regulatory cells. Immunity 2016;45:1135–1147.
      1. Nishikawa H, Kato T, Tawara I, Saito K, Ikeda H, Kuribayashi K, Allen PM, Schreiber RD, Sakaguchi S, Old LJ, et al. Definition of target antigens for naturally occurring CD4+ CD25+ regulatory T cells. J Exp Med 2005;201:681–686.
      1. Nishikawa H, Kato T, Tawara I, Ikeda H, Kuribayashi K, Allen PM, Schreiber RD, Old LJ, Shiku H. IFN-gamma controls the generation/activation of CD4+ CD25+ regulatory T cells in antitumor immune response. J Immunol 2005;175:4433–4440.
      1. Pace L, Tempez A, Arnold-Schrauf C, Lemaitre F, Bousso P, Fetler L, Sparwasser T, Amigorena S. Regulatory T cells increase the avidity of primary CD8+ T cell responses and promote memory. Science 2012;338:532–536.
      1. Maeda Y, Nishikawa H, Sugiyama D, Ha D, Hamaguchi M, Saito T, Nishioka M, Wing JB, Adeegbe D, Katayama I, et al. Detection of self-reactive CD8+ T cells with an anergic phenotype in healthy individuals. Science 2014;346:1536–1540.
      1. Nishikawa H, Sakaguchi S. Regulatory T cells in cancer immunotherapy. Curr Opin Immunol 2014;27:1–7.
      1. Kurose K, Ohue Y, Wada H, Iida S, Ishida T, Kojima T, Doi T, Suzuki S, Isobe M, Funakoshi T, et al. Phase Ia study of FoxP3+ CD4 Treg depletion by infusion of a humanized anti-CCR4 antibody, KW-0761, in cancer patients. Clin Cancer Res 2015;21:4327–4336.
      1. Jia Z, Zhao R, Tian Y, Huang Z, Tian Z, Shen Z, Wang Q, Wang J, Fu X, Wu Y, et al. A novel splice variant of FR4 predominantly expressed in CD4+CD25+ regulatory T cells. Immunol Invest 2009;38:718–729.
      1. Miyara M, Chader D, Sage E, Sugiyama D, Nishikawa H, Bouvry D, Claër L, Hingorani R, Balderas R, Rohrer J, et al. Sialyl Lewis x (CD15s) identifies highly differentiated and most suppressive FOXP3high regulatory T cells in humans. Proc Natl Acad Sci U S A 2015;112:7225–7230.
      1. Foss F. Clinical experience with denileukin diftitox (ONTAK). Semin Oncol 2006;33:S11–S16.
      1. Steitz J, Brück J, Lenz J, Knop J, Tüting T. Depletion of CD25+ CD4+ T cells and treatment with tyrosinase-related protein 2-transduced dendritic cells enhance the interferon alpha-induced, CD8+ T-cell-dependent immune defense of B16 melanoma. Cancer Res 2001;61:8643–8646.
      1. Rech AJ, Mick R, Martin S, Recio A, Aqui NA, Powell DJ Jr, Colligon TA, Trosko JA, Leinbach LI, Pletcher CH, et al. CD25 blockade depletes and selectively reprograms regulatory T cells in concert with immunotherapy in cancer patients. Sci Transl Med 2012;4:134ra62
      1. Jacobs JF, Punt CJ, Lesterhuis WJ, Sutmuller RP, Brouwer HM, Scharenborg NM, Klasen IS, Hilbrands LB, Figdor CG, de Vries IJ, et al. Dendritic cell vaccination in combination with anti-CD25 monoclonal antibody treatment: a phase I/II study in metastatic melanoma patients. Clin Cancer Res 2010;16:5067–5078.
      1. Romano E, Kusio-Kobialka M, Foukas PG, Baumgaertner P, Meyer C, Ballabeni P, Michielin O, Weide B, Romero P, Speiser DE. Ipilimumab-dependent cell-mediated cytotoxicity of regulatory T cells ex vivo by nonclassical monocytes in melanoma patients. Proc Natl Acad Sci U S A 2015;112:6140–6145.
      1. Ribas A. Tumor immunotherapy directed at PD-1. N Engl J Med 2012;366:2517–2519.
      1. Arce Vargas F, Furness AJ, Litchfield K, Joshi K, Rosenthal R, Ghorani E, Solomon I, Lesko MH, Ruef N, Roddie C, et al. Fc effector function contributes to the activity of human anti-CTLA-4 antibodies. Cancer Cell 2018;33:649–663.e4.
      1. Bulliard Y, Jolicoeur R, Windman M, Rue SM, Ettenberg S, Knee DA, Wilson NS, Dranoff G, Brogdon JL. Activating Fc γ receptors contribute to the antitumor activities of immunoregulatory receptor-targeting antibodies. J Exp Med 2013;210:1685–1693.
      1. Selby MJ, Engelhardt JJ, Quigley M, Henning KA, Chen T, Srinivasan M, Korman AJ. Anti-CTLA-4 antibodies of IgG2a isotype enhance antitumor activity through reduction of intratumoral regulatory T cells. Cancer Immunol Res 2013;1:32–42.
      1. Simpson TR, Li F, Montalvo-Ortiz W, Sepulveda MA, Bergerhoff K, Arce F, Roddie C, Henry JY, Yagita H, Wolchok JD, et al. Fc-dependent depletion of tumor-infiltrating regulatory T cells co-defines the efficacy of anti-CTLA-4 therapy against melanoma. J Exp Med 2013;210:1695–1710.
      1. van Olffen RW, Koning N, van Gisbergen KP, Wensveen FM, Hoek RM, Boon L, Hamann J, van Lier RA, Nolte MA. GITR triggering induces expansion of both effector and regulatory CD4+ T cells in vivo . J Immunol 2009;182:7490–7500.
      1. Nishikawa H, Kato T, Hirayama M, Orito Y, Sato E, Harada N, Gnjatic S, Old LJ, Shiku H. Regulatory T cell-resistant CD8+ T cells induced by glucocorticoid-induced tumor necrosis factor receptor signaling. Cancer Res 2008;68:5948–5954.
      1. Buchan SL, Rogel A, Al-Shamkhani A. The immunobiology of CD27 and OX40 and their potential as targets for cancer immunotherapy. Blood 2018;131:39–48.
      1. Curti BD, Kovacsovics-Bankowski M, Morris N, Walker E, Chisholm L, Floyd K, Walker J, Gonzalez I, Meeuwsen T, Fox BA, et al. OX40 is a potent immune-stimulating target in late-stage cancer patients. Cancer Res 2013;73:7189–7198.
      1. Herman AE, Freeman GJ, Mathis D, Benoist C. CD4+CD25+ T regulatory cells dependent on ICOS promote regulation of effector cells in the prediabetic lesion. J Exp Med 2004;199:1479–1489.
      1. Burmeister Y, Lischke T, Dahler AC, Mages HW, Lam KP, Coyle AJ, Kroczek RA, Hutloff A. ICOS controls the pool size of effector-memory and regulatory T cells. J Immunol 2008;180:774–782.
      1. Nagase H, Takeoka T, Urakawa S, Morimoto-Okazawa A, Kawashima A, Iwahori K, Takiguchi S, Nishikawa H, Sato E, Sakaguchi S, et al. ICOS+ Foxp3+ TILs in gastric cancer are prognostic markers and effector regulatory T cells associated with Helicobacter pylori. Int J Cancer 2017;140:686–695.
      1. Burris HA, Callahan MK, Tolcher AW, Kummar S, Falchook GS, Pachynski RK, Tykodi SS, Gibney GT, Seiwert TY, Gainor JF, et al. Phase 1 safety of ICOS agonist antibody JTX-2011 alone and with nivolumab (nivo) in advanced solid tumors; predicted vs observed pharmacokinetics (PK) in ICONIC. J Clin Oncol 2017;35:3033.
      1. Kurtulus S, Sakuishi K, Ngiow SF, Joller N, Tan DJ, Teng MW, Smyth MJ, Kuchroo VK, Anderson AC. TIGIT predominantly regulates the immune response via regulatory T cells. J Clin Invest 2015;125:4053–4062.
      1. Joller N, Lozano E, Burkett PR, Patel B, Xiao S, Zhu C, Xia J, Tan TG, Sefik E, Yajnik V, et al. Treg cells expressing the coinhibitory molecule TIGIT selectively inhibit proinflammatory Th1 and Th17 cell responses. Immunity 2014;40:569–581.
      1. Johnston RJ, Comps-Agrar L, Hackney J, Yu X, Huseni M, Yang Y, Park S, Javinal V, Chiu H, Irving B, et al. The immunoreceptor TIGIT regulates antitumor and antiviral CD8+ T cell effector function. Cancer Cell 2014;26:923–937.
      1. Scurr M, Ladell K, Besneux M, Christian A, Hockey T, Smart K, Bridgeman H, Hargest R, Phillips S, Davies M, et al. Highly prevalent colorectal cancer-infiltrating LAP+ Foxp3− T cells exhibit more potent immunosuppressive activity than Foxp3+ regulatory T cells. Mucosal Immunol 2014;7:428–439.
      1. Anderson AC, Joller N, Kuchroo VK. Lag-3, Tim-3, and TIGIT: co-inhibitory receptors with specialized functions in immune regulation. Immunity 2016;44:989–1004.
      1. Zhu C, Anderson AC, Schubart A, Xiong H, Imitola J, Khoury SJ, Zheng XX, Strom TB, Kuchroo VK. The Tim-3 ligand galectin-9 negatively regulates T helper type 1 immunity. Nat Immunol 2005;6:1245–1252.
      1. Das M, Zhu C, Kuchroo VK. Tim-3 and its role in regulating anti-tumor immunity. Immunol Rev 2017;276:97–111.
      1. Sakuishi K, Ngiow SF, Sullivan JM, Teng MW, Kuchroo VK, Smyth MJ, Anderson AC. TIM3+FOXP3+ regulatory T cells are tissue-specific promoters of T-cell dysfunction in cancer. OncoImmunology 2013;2:e23849
      1. Campbell DJ, Koch MA. Phenotypical and functional specialization of FOXP3+ regulatory T cells. Nat Rev Immunol 2011;11:119–130.
      1. Ishida T, Ueda R. CCR4 as a novel molecular target for immunotherapy of cancer. Cancer Sci 2006;97:1139–1146.
      1. Nishikawa H, Sakaguchi S. Regulatory T cells in tumor immunity. Int J Cancer 2010;127:759–767.
      1. Kurose K, Ohue Y, Oka M. Anti-CCR4 mAb and regulatory T cells. Gan To Kagaku Ryoho 2013;40:1150–1155.
      1. Zhang B, Chikuma S, Hori S, Fagarasan S, Honjo T. Nonoverlapping roles of PD-1 and FoxP3 in maintaining immune tolerance in a novel autoimmune pancreatitis mouse model. Proc Natl Acad Sci U S A 2016;113:8490–8495.
      1. Gianchecchi E, Fierabracci A. Inhibitory receptors and pathways of lymphocytes: the role of PD-1 in Treg development and their involvement in autoimmunity onset and cancer progression. Front Immunol 2018;9:2374.
      1. Kamada T, Togashi Y, Tay C, Ha D, Sasaki A, Nakamura Y, Sato E, Fukuoka S, Tada Y, Tanaka A, et al. PD-1+ regulatory T cells amplified by PD-1 blockade promote hyperprogression of cancer. Proc Natl Acad Sci U S A 2019;116:9999–10008.
      1. Colak S, Ten Dijke P. Targeting TGF-β signaling in cancer. Trends Cancer 2017;3:56–71.
      1. Holmgaard RB, Schaer DA, Li Y, Castaneda SP, Murphy MY, Xu X, Inigo I, Dobkin J, Manro JR, Iversen PW, et al. Targeting the TGFβ pathway with galunisertib, a TGFβRI small molecule inhibitor, promotes anti-tumor immunity leading to durable, complete responses, as monotherapy and in combination with checkpoint blockade. J Immunother Cancer 2018;6:47.
      1. Strauss J, Heery CR, Schlom J, Madan RA, Cao L, Kang Z, Lamping E, Marté JL, Donahue RN, Grenga I, et al. Phase I trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGFβ, in advanced solid tumors. Clin Cancer Res 2018;24:1287–1295.
      1. Ahmad S, Abu-Eid R, Shrimali R, Webb M, Verma V, Doroodchi A, Berrong Z, Samara R, Rodriguez PC, Mkrtichyan M, et al. Differential PI3Kδ signaling in CD4+ T-cell subsets enables selective targeting of T regulatory cells to enhance cancer immunotherapy. Cancer Res 2017;77:1892–1904.
      1. Ali K, Soond DR, Pineiro R, Hagemann T, Pearce W, Lim EL, Bouabe H, Scudamore CL, Hancox T, Maecker H, et al. Inactivation of PI(3)K p110δ breaks regulatory T-cell-mediated immune tolerance to cancer. Nature 2014;510:407–411.
      1. Huynh A, DuPage M, Priyadharshini B, Sage PT, Quiros J, Borges CM, Townamchai N, Gerriets VA, Rathmell JC, Sharpe AH, et al. Control of PI(3) kinase in Treg cells maintains homeostasis and lineage stability. Nat Immunol 2015;16:188–196.
      1. Shrestha S, Yang K, Guy C, Vogel P, Neale G, Chi H. Treg cells require the phosphatase PTEN to restrain TH1 and TFH cell responses. Nat Immunol 2015;16:178–187.
      1. Imagawa J, Tanaka H, Okada M, Nakamae H, Hino M, Murai K, Ishida Y, Kumagai T, Sato S, Ohashi K, et al. Discontinuation of dasatinib in patients with chronic myeloid leukaemia who have maintained deep molecular response for longer than 1 year (DADI trial): a multicentre phase 2 trial. Lancet Haematol 2015;2:e528–e535.
      1. Vahl JC, Drees C, Heger K, Heink S, Fischer JC, Nedjic J, Ohkura N, Morikawa H, Poeck H, Schallenberg S, et al. Continuous T cell receptor signals maintain a functional regulatory T cell pool. Immunity 2014;41:722–736.
      1. Ohta A, Sitkovsky M. Extracellular adenosine-mediated modulation of regulatory T cells. Front Immunol 2014;5:304.
      1. Terme M, Pernot S, Marcheteau E, Sandoval F, Benhamouda N, Colussi O, Dubreuil O, Carpentier AF, Tartour E, Taieb J. VEGFA-VEGFR pathway blockade inhibits tumor-induced regulatory T-cell proliferation in colorectal cancer. Cancer Res 2013;73:539–549.
      1. Zhu P, Hu C, Hui K, Jiang X. The role and significance of VEGFR2+ regulatory T cells in tumor immunity. Onco Targets Ther 2017;10:4315–4319.
      1. Roland CL, Lynn KD, Toombs JE, Dineen SP, Udugamasooriya DG, Brekken RA. Cytokine levels correlate with immune cell infiltration after anti-VEGF therapy in preclinical mouse models of breast cancer. PLoS One 2009;4:e7669
      1. Voron T, Colussi O, Marcheteau E, Pernot S, Nizard M, Pointet AL, Latreche S, Bergaya S, Benhamouda N, Tanchot C, et al. VEGF-A modulates expression of inhibitory checkpoints on CD8+ T cells in tumors. J Exp Med 2015;212:139–148.
    Cited by
    Crossref 74
    Google Scholar 
    PubMed Central 42
    Web of Science 62
    MeSH Terms
    Autoimmune Diseases
    Homeostasis
    Humans
    Immunity
    Immunotherapy*
    T-Lymphocytes, Regulatory*
    Tumor Microenvironment*
    Metrics
    Share
    Links to
    Figures
    slide
    slide

    1 / 2

    Tables
    slide

    1 / 1

    ORCID IDs
    Funding Information
    PERMALINK