2.1 Treg subsets in colorectal cancer

At present, increasing evidence suggests that FOXP3⁺ T cells in humans, including suppressive and nonsuppressive subpopulations, have heterogeneous phenotypes and functions.^{4, 10, 49, 54} The effect of abundant Tregs infiltrating tumor tissues such as colorectal cancers (CRCs) is controversial, and some studies have shown that the infiltration of FOXP3⁺ Tregs in tumor tissue is beneficial to the prognosis of patients.^55-58 According to previous research, FOXP3⁺CD4⁺ T cells circulating in human blood can be divided into three main groups based on FOXP3, CD25, and CD45RA: fraction I (Fr. I) CD45RA⁺CD25^low/FOXP3^low naïve Tregs (nTregs), Fr. II CD45RA⁻CD25^high/FOXP3^high effector Tregs (eTregs), and Fr. III CD45RA⁻CD25^low/FOXP3^low cells, the majority of which are not Tregs.⁵⁴ Based on this classification, Saito et al. demonstrated markedly increased numbers of Fr. II in both type A and B CRCs and that Fr. III cells were mainly increased in type B CRCs.⁵⁹ Although Fr. II cells and Fr. III cells are both CD45RA⁻CD45RO⁺ effector-type cells, only Fr. II cells from either type of CRCs tissue showed strong in vitro suppressive activity by highly expressing suppression-related molecules, such as T-cell immunoreceptor with Ig and ITIM domains (TIGIT) and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). Fr. III nonsuppressive FOXP3^low cells were probably derived from non-Tregs following activation with interleukin (IL)-12 and transforming growth factor (TGF)-β. Interestingly, type B CRCs showed significantly better recurrence-free survival than type A CRCs. Thus, Fr. III nonsuppressive FOXP3^low cells could be a biomarker associated with better survival in CRCs. Furthermore, the strategy of increasing local FOXP3^low non-Tregs may effectively inhibit the occurrence and development of CRCs.

A recent study reported that tumor-infiltrating Tregs of CRC express granzyme B (GZMB) immediately after tumor resection, while there are almost no GZMB-expressing Tregs in tumor-associated lymph nodes and circulating lymphocytes.⁶⁰ A number of studies have shown that GZMB-expressing Tregs could induce the apoptosis of effector T cells⁶¹ and were also related to immune homeostasis, mediating tumor immunity,^{62, 63} viral infection, and transplant survival.⁶⁴ Furthermore, the expression of granzymes was highly concentrated in T-cell immunoglobulin- and mucin-domain containing-3 positive (TIM3⁺) Tregs, a subpopulation of Tregs that were enriched in the TME and presented increased suppressive capacity.^65-67 GZMB⁺TIM3⁺ Tregs (Figure 1) were found to present higher cytolytic capacity toward autologous conventional T cells, which depended on GZMB rather than TIM3. Another study reported that CD4⁺CD25^+/highFOXP3⁺ Tregs in peripheral blood mononuclear cells (PBMCs) of patients with CRCs could be divided into two subpopulations, consisting of lymphocyte activation gene 3 negative (LAG3⁻) TIM3⁻ Tregs and LAG3⁺TIM3⁺ Tregs (Figure 1), of which the latter accounted for more than half.⁶⁸ LAG3⁺TIM3⁺ Tregs presented significantly higher expression of TGF-β, IL-10, and CTLA-4 than LAG3⁻TIM3⁻ Tregs, similar to classical Tregs,^{12, 69, 70} which could suppress the expression of MHC-II, CD80/CD86, and TNF-α and increase the expression of IL-10 in macrophages. These findings reported that LAG3⁺TIM3⁺ Tregs could lead to a worse prognosis and survival of CRCs patients.

CCR8 is a chemokine receptor predominantly expressed on Tregs in both mice^{71, 72} and humans.^{30, 73} As a subset of CD4⁺FOXP3⁺ Tregs, CCR8⁺ Tregs have been demonstrated to be a major driver of immunosuppression.^74-76 Villarreal et al. found that tumor-infiltrating CCR8⁺ Tregs are significantly upregulated in CRCs (Figure 1),⁷⁷ similar to previous research in breast⁷⁸ and lung cancer,³⁰ and mAb therapy targeting CCR8 could obviously inhibit tumor growth and improve the prognosis in CRCs mouse models by increasing tumor-specific T cells. Collectively, expressions of GZMB, LAG3 with TIM3, and CCR8 in FOXP3⁺ Tregs all strengthen their immunosuppressive function, and these markers may be potential new targets for immunotherapy of CRCs patients.

In addition, B lymphocyte-induced maturation protein 1(BLIMP-1), encoded by the PR domain zinc finger protein 1 (PRDM1) gene, is a multifunctional transcriptional regulator that has been previously found to be coexpressed with FOXP3 in murine Tregs.⁷⁹ The research has shown that BLIMP-1 expression defines eTregs as a distinct population of mature Tregs that produce IL-10 and display an effector phenotype in mice. To understand the role of these cell populations in the immune regulation of human diseases, a pilot study has demonstrated that CRCs patients with low FOXP3⁺BLIMP-1⁺ eTregs (Figure 1) infiltration in tumors are associated with a high risk of recurrence.⁸⁰ Notably, not only prognostic value, the underlying mechanism of FOXP3⁺BLIMP-1⁺ eTregs in CRCs patients remains to be verified.

Tumor necrosis factor receptor 2 (TNFR2), the receptor of TNF, has been found that is highly expressed in humans and mouse Tregs, especially on eTregs.^81-85 Interestingly, the interaction of TNF with TNFR2 promotes suppressive function and phenotypical stability of Tregs.^{86, 87} Based on this interaction, Nie et al. have reported that blockade of TNFR2 signaling with an antibody could markedly reduce the tumor-infiltrating TNFR2⁺ Tregs (Figure 1) in CRCs, whereas the number of tumor-infiltrating interferon-γ-positive (IFN-γ⁺)CD8⁺ cytotoxic T lymphocytes were significant increased.⁸⁸ Moreover, the combination of TNFR2 blocking and immunotherapy showed a better efficacy, which will be more beneficial for the prognosis of CRCs patients.

OX40, also known as CD134 and TNFRSF4, is a member of the TNFR family which is identified as a negative regulator of FOXP3⁺ Tregs.^89-91 Triggering OX40 with its ligand OX40L on the FOXP3⁺ Tregs abrogates its suppression of T-cell proliferation and effector T cells (Teffs) function.^90-92 Lam JH et al. have found that a high density of tumor-infiltrating CD30⁺OX40⁺ Tregs (Figure 1) in CRCs patients is associated with improved prognosis, which may act as a diagnostic and prognostic biomarker in CRCs.⁹³

2.2 Treg subsets in lung cancer

Lung cancer is known to be the leading cause of cancer-related death worldwide.⁹⁴ There are two subtypes of lung cancer: small-cell lung cancer (SCLC: ∼15%) and non-SCLC (NSCLC: ∼85%).⁹⁵ Evidence from a wide range of sources suggests that high levels of Tregs are associated with metastasis, recurrence, and poor prognosis of lung cancer.^96-100 Koyama et al. have shown that the proportion of CD62L^highCD25⁺CD4⁺ Tregs (Figure 1) is significantly higher in extended-stage disease SCLC, whereas there are more CD62L^lowCD4⁺ T cells in limited-stage SCLC (LD-SCLC).¹⁰⁰ CD62L^lowCD4⁺ T cells that can produce IFN-γ, IL-4, and IL-17 are considered to be immune Teffs. Th17 cells play an essential role in a variety of autoimmune diseases.^101-105 These Teffs deviated to T-helper type 17 (Th17) cells and were driven by IL-23 secreted by monocyte-derived DCs in LD-SCLC. Unfortunately, CD62L^highCD25⁺CD4⁺ Tregs can suppress cytokine production and inhibit the proliferation of Teffs. Therefore, a high Teffs to Tregs ratio could suppress the growth and metastasis of SCLC tumor cells. Based on this study, CD4⁺ T-cell balance may be a helpful biomarker to assess immunologic responses, and increasing tumor-reactive Teffs levels and depleting Tregs may be essential in establishing effective antitumor immunity.

Glycoprotein A repetitions predominant (GARP) has been proven to be highly expressed on the surface of Tregs.¹⁰⁶ Previous studies have shown that GARP⁺ Tregs can secrete TGF-β and possess a strong immunosuppressive function.^107-111 Jin et al. demonstrated that GARP⁺ Tregs (Figure 1) were highly infiltrated in tumor tissues in the early stage of lung cancer and exerted immunosuppressive effects through the GARP-TGF-β pathway to inhibit Teffs.¹¹² Therefore, tumor cells can escape immune surveillance and promote the occurrence and progression of tumors. Further research needs to confirm whether GARP is an important molecule of Tregs and a promising immunotherapy target.

A recent study reported increased numbers of FOXA1⁺ Tregs (Figure 1) in patients with lung cancer.¹⁰⁶.The findings proved that FOXA1⁺ Tregs could inhibit the antitumor immunity of T cells and promote tumor growth by the IFN-β-PI3K-Akt-FOXA1 signaling pathway. Moreover, patients with more FOXA1⁺ Tregs showed more liver metastases and worse treatment responses. FOXA1 hepatocyte nuclear factor 3α is a transcription factor that is associated with the differentiation of embryonic stem cells, bile duct development, and cancer epigenetics.^113-117 According to a previous study, Liu et al. demonstrated that FOXA1⁺ Tregs were induced by IFN-β and possessed a suppressive function in experimental autoimmune encephalomyelitis (EAE) and MS models.¹¹⁸ This subset of Tregs may be a novel target of immunotherapy.

Helios (Ikzf2), a member of the Ikaros transcription factor family, was identified as a specific marker of FOXP3⁺ Tregs^{119, 120} and was shown to be involved in Tregs development and stability.¹²¹ FOXP3⁺Helios⁺ Tregs were present in thymus-derived Tregs (tTregs),¹²² and FOXP3⁺Helios⁻ Tregs were present in peripherally-induced Tregs (pTregs).¹²³ Muto et al. have shown that FOXP3⁺ Helios⁻ Tregs (Figure 1) are apparently higher in NSCLC patients than in healthy controls.¹²⁴ Interestingly, higher percentages of tumor-infiltrated FOXP3⁺ Helios⁻ Tregs were seen in advanced-stage NSCLC with poorer survival. Additionally, Guo et al. also found a subpopulation of Tregs named TNFRSF9⁺ Tregs (Figure 1), which were related to the poor prognosis of NSCLC.¹²⁵ TNFRSF9 is tumor necrosis factor receptor superfamily member 9, also known as CD137 (4-1BB), which is a surface marker that indicates antigen-specific activation.¹²⁶ Moreover, TNFRSF9⁺ Tregs are highly expressed immunosuppressive genes, such as REL¹²⁷ and LAYN,⁴⁷ and may be a major component of functional tumor-infiltrating Tregs in lung cancer. Although studies have shown that the high infiltration of FOXP3⁺Helios⁻ Tregs and TNFRSF9⁺ Tregs in tumors are not conducive to the prognosis of patients, the function and mechanisms of these subtypes of Tregs in tumor-related immune suppression need to be further investigated.

Similar to the previous research, Yan et al. have found that TNFR2⁺ Tregs (Figure 1) could suppress the production of IFN-γ by CD8⁺ T cells through expressed high levels of the CTLA-4.¹²⁸ As result, the high percentage of TNFR2⁺ Tregs in the peripheral blood of lung cancer patients was associated with lymphatic invasion, distant metastasis, more advanced clinical stage, and worse outcomes. Therefore, TNFR2 may prove to be a useful prognostic marker for lung cancer patients and as one of the best potential immune checkpoints on account of its critical role in TME.

Van Damme et al. and De Simone et al. have discerned that the high frequency of CCR8⁺ tumor-infiltrating Tregs (Figure 1) plays a crucial suppressive function and is correlated with poor prognosis in patients with NSCLC.^{30, 129} The expression of CCR8 on the surface of tumor-infiltrated Tregs may be induced by coregulation of NF-κB and IFN regulatory factor 4 (IRF4).^130-132 Remarkably, combination with anti-CCR8 monotherapy could synergize the efficacy of anti-PD-1 therapy, which provides a beneficial treatment strategy for patients with NSCLC. Moreover, Alvisi et al. have demonstrated that the abundance of IRF4⁺ eTregs (Figure 1) correlated with poor prognosis in patients with NSCLC.¹³³ The findings showed that high expression of IRF4 was present in CCR8⁺ICOS⁺ eTregs, which further contributed to the superior suppressive activity and worse disease-free survival and overall survival of NSCLC patients. Therefore, anti-IRF4 immunotherapy is needed to further research to benefit patients with NSCLC.

One of the ways that Tregs exert their suppression function is by secreting inhibitory cytokines including IL-10, IL-35, and TGF-β.^134-137 Among them, IL-35 appears to play a greater role in inhibitory receptor induction and restriction of central memory T- cell differentiation, while IL-10 plays a greater role in regulating cytokine production and effector function. Sawant et al. found an abundance of IL-10⁺ and IL-35⁺ Tregs (Figure 1) in the TME of patients with NSCLC which cooperatively promoted exhaustion of BLIMP-1-dependent tumor-infiltrating CD8⁺ T cells.¹³⁸ The findings showed that IL-10⁺ and IL-35⁺ Tregs-derived IL-10 and IL-35 collectively induced inhibitory receptors, such as TIM-3 and LAG-3 expression on intratumoral CD8⁺ T cells, which further resulted in T-cell dysfunction and antitumor immunity failure. Therefore, the depletion by IL-10⁺ and IL-35⁺ Tregs may abrogate tumor-immune evasive and resistance to immunotherapy of tumor cells.

2.3 Treg subsets in liver cancer

The liver has been recognized as an immune privilege organ.^{139, 140} Due to its complex immune regulation mechanism, current research is still unclear about its immune regulation. In recent years, there has been an increasing number of studies on the immune microenvironment of the liver at the single-cell level. Zheng et al. used single-cell RNA sequencing to reveal the landscape of infiltrating T cells in liver cancer.⁴⁷ They showed that LAYN was preferentially upregulated in tumor-infiltrated FOXP3⁺Helios⁺ Tregs. LAYN (Layilin), which was first reported in 1998, is a protein-encoding gene located on chromosome11.¹⁴¹ LAYN has been defined as a Treg-specific signature gene¹⁴² that is highly and specifically expressed in tumor-infiltrating Tregs and correlates with poor prognosis in both NSCLC and CRCs patients.³⁰ Similarly, high expression of tumor-infiltrated LAYN⁺FOXP3⁺Helios⁺ Tregs (Figure 1), which possessed suppressive functions, was associated with tumor-infiltrating exhausted CD8⁺ T cells and poor survival in liver cancer. In addition, Sun et al. also found that LAYN⁺ Tregs showed highly expressed LAYN, TNFRSF9, and inducible T-cell costimulator (ICOS, CD278), but the proportion of this cell population in tumor-infiltrating cells was very small, which was different from FOXP3⁺ Tregs.⁴⁸ Hence, further studies are needed to generate mechanistic insights into the function of LAYN⁺ Tregs.

Similar to the Tregs subgroup mentioned above, Kalathil et al. defined GARP⁺/CTLA4⁺ Tregs as a subset of Tregs with immunosuppressive function in advanced liver cancer.¹⁴³ The high frequency of GARP⁺/CTLA4⁺ Tregs (Figure 1) in patients with advanced hepatocellular carcinoma (HCC) may facilitate immune dysregulation, and the expression levels of GARP and CTLA were not associated with the viral infection. However, this finding suggests that depletion of these subpopulations of Tregs may be potential targets of immunotherapy.

In addition, Han et al. identified a new subset of tumor-infiltrating CD4⁺CD69⁺ Tregs (Figure 1) that expressed neither CD25 nor FOXP3, but highly expressed membrane-bound TGF-β1 (mTGF-β1), programmed cell death protein 1 (PD-1, CD279), and CTLA-4.¹⁴⁴ These novel CD4⁺CD69⁺FOXP3⁻ Tregs accounted for the vast majority of tumor-infiltrating Tregs compared with FOXP3⁺ Tregs and could suppress the CD4⁺ T-cell response mainly through mTGF-β1,^{145, 146} which was correlated with tumor progression. The underlying mechanisms of these tumor antigen-induced CD4⁺CD69⁺FOXP3⁻ Tregs in HCC need further investigation for cancer immunotherapy.

Pedroza-Gonzalez et al. have shown that the expression of glucocorticoid-induced TNFR (GITR) and ICOS are upregulated in activated tumor-infiltrating Tregs in patients with primary or metastatic liver cancer.¹⁴⁷ GITR, also known as TNFRSF18, is a member of the TNF/nerve growth factor receptor family,^148-150 and it is mainly expressed on CD4⁺CD25⁺ Tregs¹⁵¹ (Figure 1). The findings demonstrated that GITR ligation could abrogate the tumor-infiltrating Tregs-mediated suppression of effector T cells through treatment with soluble GITR ligand, which was consistent with previous research reports.^151-153 Moreover, Tu et al. reported that ICOS⁺FOXP3⁺ Tregs (Figure 1) were significantly increased in the tumor tissues of patients with HCC.¹⁵⁴ ICOS⁺ Tregs could produce a mass of IL-10 and TGF-β1,¹⁵⁵ and the levels of tumor-infiltrating ICOS⁺ Tregs were higher than those in tumor adjacent tissues in several cancers.^156-158 Likewise, higher ICOS⁺ Tregs levels and ICOS⁺ Tregs/CD4⁺ T-cell ratios indicated a worse prognosis in HCC. In addition, ICOS⁺ Tregs were concentrated in high chemokine (C-C motif) ligand (CCL20)-expressed areas. CCL20, the ligand targeting CCR6, has been demonstrated to be highly expressed in triggering receptors expressed on myeloid cell-1 (TREM-1)-positive tumor-associated macrophages (TAMs)¹⁵⁹ in liver cancer. The findings also found that CCR6⁺ Tregs (Figure 1) accounted for the majority of intratumoral Tregs. Therefore, TREM-1⁺ TAMs could recruit CCR6⁺ Tregs through producing CCL20, which induced CD8⁺ T-cell exhaustion and was associated with poor prognosis in patients with HCC. In conclusion, CCL20 may be a potential target for immunotherapy.

A recent study has identified that CCR4⁺ Tregs (Figure 1) are the predominant type of Tregs in HBV⁺HCC.¹⁶⁰ CCR4 is mainly expressed on Tregs and other T-helper cells, which can mediate Tregs trafficking into the TME by interacting with its ligands, CCL22 and CCL17.¹⁶¹ The findings showed that the high frequency of CCR4⁺ Tregs exhibited potently immunosuppressive stem-like specificity by upregulating transcription factor 1 (TCF1), PD-1, and CTLA-4 levels and secreting more IL-10 and IL-35. Moreover, targeting CCR4⁺ Tregs could alleviate sorafenib resistance and might improve the curative effect of immune checkpoint blockade (ICB) for patients with HBV⁺HCC.

What is noteworthy is that low glucose, or rather high-lactic acid (LA), and hypoxic environments may impaired the survival and functions of effector T cells and are not conducive to antitumor immunotherapy.^162-164 Kumagai et al. have reported that enhanced PD-1 expression on eTregs (Figure 1) was observed in low-glucose TME of HCC on account of eTregs actively absorbed LA through monocarboxylate transporter 1 (MCT1) and promoted nuclear factor of activated T cells-1 (NFAT1) translocation into the nucleus.¹⁶⁵ The deletion of MCT1, a transporter of LA,^{166, 167} plays a pivotal role for tumor-infiltrating Tregs to uptake lactate to inhibit tumor growth and enhance immunotherapy response.¹⁶⁸ In addition, NFAT1 positively regulates the expression of PD-1 on T cells.^169-172 Therefore, molecular-targeted immunotherapy against LA provides a novel insight for the clinic.

2.4 Treg subsets in gastric cancer

In recent years, increasing attention has been given to the application of ICBs, such as anti-PD-1 monoclonal antibodies (mAbs), in tumor immunity.^{173, 174} Some previous studies have shown that a fraction of Tregs expresses PD-1.^175-178 However, the effects of anti-PD-1 mAbs on PD-1⁺ Tregs in the tumor environment are currently poorly understood. Kamada et al. found that tumor-infiltrating eTregs highly express PD-1 and that the proliferation and immunosuppressive activity of PD-1⁺ eTregs (Figure 1) could be reinforced by anti-PD-1 mAb therapy in patients with gastric cancer (GC).¹⁷⁹ As a result, the patients treated with anti-PD-1 mAb aggravated the progression of the disease, which was consistent with the results of previous studies.^{180, 181} Therefore, the pros and cons of anti-PD-1 mAb therapy and its promotion of PD-1⁺ eTregs suppressive function still need sufficient clinical data to verify.

Several studies have confirmed the limited efficacy of ICBs in the treatment of tumors,^182-187 and establishing more effective therapies is necessary. Vascular endothelial growth factor (VEGF) is secreted by a variety of tumors and accumulates abundantly in the TME, which mainly acts as an immunosuppressive factor.¹⁸⁸ VEGF acts on the vascular endothelium through specific receptors, and VEGF receptor 2 (VEGFR2) is the most important signaling receptor.^{189, 190} In addition, VEGFR2 is selectively expressed by FOXP3^high CD4⁺ Tregs.¹⁹¹ Tada et al. revealed that VEGFR2⁺FOXP3⁺ eTregs (Figure 1) highly expressed Ki67 in GC tumor tissues.¹⁹² Ramucirumab (RAM), an anti-VEGFR2 mAb,^193-195 could reduce the frequency of VEGFR2⁺FOXP3⁺ eTregs. Furthermore, PD-1 ligand (PD-L1) expression and CD8⁺ T-cell infiltration in tumor-infiltrating lymphocytes were enhanced after treatment with RAM, which was beneficial to the killing of tumor cells. It can be seen that inhibiting the expression of VEGFR2 in Tregs may be a potentially beneficial treatment option.

T-cell chemotaxis homing mediated by chemokine receptors plays a critical role in immune homeostasis.¹⁹⁶ The chemokine receptor CCR7 has been proven to guide T-cell chemotactic homing to tumor tissues.¹⁹⁷ Furthermore, Mao et al. found that tumor-infiltrating Tregs in GC expressed little CCR7 (Figure 1).¹⁹⁸ Most tumor-infiltrating Tregs exhibited a CD45RA⁻CCR7⁻ phenotype, which was induced by tumor-derived TNF-α and could inhibit the secretion of IFN-γ and proliferation of effector CD8⁺ T cells. The high proportion of these subsets of Tregs in the tumor tissue was associated with tumor progression and poor prognosis. In the future, reversing the expression of CCR7 in Tregs may prevent tumor progression.

Qu et al. have also confirmed that TNFR2⁺ Tregs (Figure 1) preferentially accumulate in TME of GC, which express high levels of CTLA-4 and CCR6 and possess strong suppressive activity by activating the TNF-α/TNFR2 signaling pathway.¹⁹⁹ In addition, the higher level of TNFR2⁺ Treg infiltration was correlated to a poorer prognosis in GC patients. Based on the results, targeting TNFR2⁺ Tregs may be an immunotherapeutic strategy for GC.

2.5 Treg subsets in other cancers

Many different subtypes of Tregs have also been found in other types of tumors. CCR8 has been identified to be upregulated in intratumoral Tregs in a variety of tumors.^{30, 78} A recent study on breast cancer showed that CCR8⁺ Tregs (Figure 2), a subset of peripheral blood Fr. II, could be recruited to human breast tumors through the CCL1-CCR8 axis.²⁰⁰ Intratumoral CCR8⁺ Tregs highly expressed surface-activated markers such as CTLA-4, CD39, and PD-1. Activated CCR8⁺ Tregs have the potential for immunosuppression and are significantly related to the clinical outcome of patients with breast cancer.

Neuropilin-1 (Nrp1), a cell surface glycoprotein of the semaphorin III receptor, was reported to be an exclusive surface marker of Tregs and is associated with suppressive activity.^{201, 202} Indeed, the expression of Nrp1 on human Tregs has always been controversial, with some suggesting that peripheral human Tregs do not express Nrp1 while others suggest that Nrp1 is expressed on a subset of CD8⁺ TIL in human NSCLC.^{203, 204} In many subsequent studies, Battaglia et al. have shown that depletion of Nrp1⁺ Tregs (Figure 2) in tumor-draining lymph nodes is directly related to a favorable response to chemoradiotherapy in cervical cancer.²⁰⁵ Overacre-Delgoffe et al. have found that a high proportion of human Tregs expressed Nrp1 in melanoma and neck squamous cell carcinoma (HNSCC) and peripheral blood lymphocyte (PBL) Tregs from these cancer patients also possessed a clear population of Nrp1⁺ Tregs in contrast to healthy donor PBL Tregs.²⁰⁶ Interestingly, Nrp1 expression in intratumoral Tregs appeared to correlate with poor prognosis in both melanoma and HNSCC. The findings also suggested that targeting Nrp1⁺ intratumoral Tregs with an Nrp1 mAb may be therapeutic. Furthermore, Bell et al. have also found that OX40 (Figure 2) is particularly expressed on the surface of Tregs of HNSCC patients.²⁰⁷ The anti-OX40 clinical trial is expected to be conducted in patients with advanced cancers and yield good clinical benefits.

Not only that, but several studies have also found that OX40 (Figure 2) is predominantly expressed on the tumor-infiltrated FOXP3⁺ Tregs of cutaneous squamous cell carcinoma²⁰⁸ and peripheral FOXP3⁺ Tregs of patients with breast cancer.²⁰⁹ The application of anti-OX40 could promote tumor-infiltrated CD4⁺ T-cell proliferation and reduce tumor metastasis,²⁰⁸ which provides a therapeutic target for tumors.

Govindaraj et al. have revealed that TNFR2⁺ Tregs (Figure 2) are presented at high levels within the ascites of ovarian cancer, which are more suppressive compared to peripheral blood TNFR2⁺ Tregs.²¹⁰ TNFR2⁺ Tregs mainly expressed high levels of CD39, CD73, TGF-β, and GARP in the cell surface to increase their suppressive capacity, which further inhibited the production of IFN-γ by Teffs. Moreover, Zhang et al. demonstrated that TNFR2⁺ Tregs (Figure 2) infiltrated both peripheral and tumors in patients with cervical intraepithelial neoplasia and cervical cancer, and the proportion of TNFR2⁺ Tregs was found to be associated with the clinical stages of cervical cancer.²¹¹ Therefore, TNFR2⁺ Tregs may be a potential immunotherapy target to improve survival for patients with ovarian cancer and cervical cancer.

Th17 cells have considerable plasticity in autoimmune diseases and other chronic inflammatory diseases and mainly produce IL-17.^212-215 Th17 cells and Tregs are indispensable in maintaining immune homeostasis, and Th17-Treg imbalance is related to inflammatory immunosuppression in cancer. Downs-Canner et al. revealed that Th17 cells presented a novel source of tumor-induced FOXP3⁺ Tregs in tumor-bearing mouse models.²¹⁶ It is generally known that TGF-β is essential in producing pTregs, iTregs, and some TH17 cells,^{217, 218} and the above findings have demonstrated that Th17 (IL-17A⁺FOXP3⁻) cells progressively transdifferentiate into IL-17A⁺FOXP3⁺ and ex-Th17 IL-17A⁻FOXP3⁺ T cells promoted by TGF-β during tumor progression, which are subpopulations of suppressive Tregs (Figure 2). In contrast, when FOXP3⁺ Tregs are exposed to IL-6 with or without IL-1β and IL-23, FOXP3 becomes downregulated in favor of expressing Th17 genes including IL-17, IL-22, IL-23-receptor (IL-23R), and retinoid-related orphan receptor-γt (RORγt).²¹⁹ Based on this report, inhibiting the conversion of Th17 cells into Tregs may serve as a valuable targeting strategy in tumor immunotherapy.

As mentioned in the previous description, TIGIT is an immunosuppressive-related immunoreceptor expressed on eTregs (Fr. II), which can facilitate Tregs development.^220-222 DNAM-1 (DNAX accessory molecule-1, CD226),²²³ a competitive costimulatory counter-receptor of TIGIT,^{220, 224, 225} enhances T-cell activation,^{223, 226} while TIGIT can inhibit T-cell responses through T cell-intrinsic inhibitory functions.²²⁷ Fourcade et al. found that tumor-infiltrating Tregs express high-level TIGIT and low-level CD226 in melanoma.²²⁸ TIGIT⁺ Tregs (Figure 2) showed suppression and stable characteristics as well as a large amount of enrichment in tumors. The research indicated that a high proportion of TIGIT⁺ Tregs and a high TIGIT/CD226 ratio in Tregs in the tumor immune microenvironment were associated with poor clinical prognosis. Recently, Wu et al. have found that the frequency of TIGIT⁺ Tregs was significantly increased in patients with bladder cancer by single-cell sequencing.²²⁹ Interestingly, the TIGIT⁺ Tregs also highly expressed IL-32 which further promoted the migration and invasion of tumor cells. As a result, targeting TIGIT to suppress the metastasis of tumor may be a novel insight for patients with bladder cancer. In addition, Yang et al. have demonstrated that abundantly tumor-infiltrating TIGIT⁺ Tregs show enhanced suppressive capacity by inhibiting the activation and proliferation of CD8⁺ T cells in patients with follicular lymphoma.²³⁰ The findings also identified several other types of TIGIT⁺ T cell subsets, including exhausted TIGIT⁺ non-Tregs/follicular helper T cells (Tfhs) and effector TIGIT⁺ T cells. In conclusion, increased numbers of TIGIT⁺ T cells were correlated with inferior outcomes and poor survival and the blockade of TIGIT signaling may have therapeutic potential for patients with follicular lymphoma. Moreover, another study reported that the expression of TIGIT (Figure 2) elevated in the PBMCs and tumor tissues of patients with HNSCC.²³¹ TIGIT could activate its ligand CD155,²³² and the activation of TIGIT/CD155 signaling was associated with the pathologic grade and lymph node metastasis of HNSCC. Consistent with previous studies, targeting TIGIT/CD155 signaling could reduce the secretion of TGF-β and may be a potential therapeutic strategy for HNSCC patients.

3.1 Treg subsets in systemic lupus erythematosus

SLE, a chronic autoimmune systemic disease with extremely varied clinical manifestations and complex pathogenesis, has been characterized by the breakdown of immunological tolerance and antinuclear antibody production.²³³ Some studies have shown that the suppressive function of Tregs is weakened in patients with active SLE, but the specific mechanisms remain unknown.^{234, 235} It was recently reported that IL-21, a proinflammatory cytokine in SLE, could suppress FOXP3⁺ Tregs differentiation and suppressive activity by inhibiting CTLA-4 and GATA-3 expression.²³⁶ GATA-3 is a transcription factor that mediates the expression of FOXP3²³⁷ and is essential for Tregs development and function.²³⁸ CTLA-4 is associated with Tregs activation. Interestingly, in contrast to IL-21, IL-2 and TGF-β promoted the expression of CTLA-4⁺ Tregs and GATA-3⁺ Tregs in SLE (Figure 3). Therefore, inhibiting the production of IL-21 from the source can restore the suppressive function of CTLA-4⁺ Tregs and GATA-3⁺ Tregs, which may be beneficial to the treatment of SLE patients.

Follicular regulatory T cells (Tfrs), one of the subsets of Tregs that specifically regulate the function of Tfhs through expressing CXCR5 and FOXP3, mainly suppress the activation of Tfhs in germinal centers to maintain immune homeostasis.^239-241 The dysregulation of Tfrs may eventually contribute to the progression of autoimmune diseases.^242-244 Liu et al. have demonstrated an elevated level of CD4⁺CXCR5⁺FOXP3⁺ Tfrs (Figure 3) in peripheral blood of SLE patients.²⁴⁴ The high proportion of Tfrs could suppress Tfhs function and the frequency of Tfrs was positively associated with autoantibodies (AAbs) and clinical severity of SLE patients, which might be a response to the enhanced humoral immunity and provided novel insight during SLE pathogenesis. In subsequent studies, researchers have found that IL-2 plays a pivotal role in maintaining the suppression function of Tfrs.^245-247 When serum IL-2 levels were reduced in patients with SLE, the proportion of CD4⁺CXCR5⁺FOXP3^–PD-1^high Tfhs was increased, whereas the proportion of CD4⁺CXCR5⁺CD45RA^–FOXP3^high Tfrs was decreased.²⁴⁵ However, stimulation with an exogenous low dose of IL-2 could restore the function of Tfrs and converse Tfhs to Tfrs by regulation of FOXP3 and BCL6 through histone modification. Similarly, high expression of PD-1 was explored on Tfrs (Figure 3) with dysfunction of suppressing Tfhs proliferation and activation in patients with SLE due to IL-2 deficiency, which could also be rescued by low-dose IL-2 treatment.²⁴⁶ As result, IL-2 treatment may provide potential therapeutic benefits for SLE.

A recent study has identified that P-selectin glycoprotein ligand-1 (PSGL-1), an adhesion molecule that can be expressed by T cells,^{248, 249} is highly expressed on Tfrs (Figure 3) in patients with SLE.²⁵⁰ PSGL-1 interaction with selectin impaired the suppression function of Tfrs and contributed to the pathogenesis of SLE via inhibition of the TGF-β pathway and reduced expression of FOXP3. Moreover, the circulating P-selectin derived from platelets was activated and correlated with disease severity in the patients with SLE. Blocking P-selectin may improve inflammation and as a potential therapeutic target for SLE.

3.2 Treg subsets in MS

In 1992, MS was described as an autoimmune disease caused by pathogenic T cells specific for myelin-Ags in the central nervous system.²⁵¹ At present, research on the function of Tregs in MS patients is still controversial.^{252, 253} In addition to Th1 cells, studies have reported that IL-17 is highly expressed in MS patients, so it could be speculated that Th17 cells play a key role in mediating autoimmune inflammation.^{104, 254} Moreover, it has been reported that human FOXP3⁺ Tregs can secrete IL-17.²⁵⁵ Subsequently, however, Fletcher et al. demonstrated that FOXP3⁺CD39⁺ Tregs (Figure 3) could suppress the production of IL-17 by Th17 cells, while FOXP3⁺CD39⁻ Tregs could produce IL-17 in MS patients.²⁵⁶ Unfortunately, the proportion of FOXP3⁺CD39⁺ Tregs was reduced, and their function was impaired. CD39 is an ectonucleotidase that can hydrolyze ATP into AMP, and the hydrolysis of ATP is considered to be an important mechanism of immunoregulation.²⁵⁷ As a result, injection of FOXP3⁺CD39⁺ Tregs and exclusion of FOXP3⁺CD39⁻ Tregs may be a potential immunotherapy strategy for MS.

In the previous description, we mentioned that FOXA1⁺ Tregs (Figure 3) were found in lung cancer patients, which were reported earlier in EAE and MS models.¹¹⁸ The findings showed that FOXA1 could bind to PD-L1, which was essential for FOXA1⁺ Tregs to kill activated T cells. More importantly, in addition to the IFN-α/β receptor signaling inherent in T cells, the development of FOXA1⁺ Tregs could also be induced by IFN-β. Therefore, clinical treatment with IFN-β to induce more suppressive FOXA1⁺ Tregs may generate favorable outcomes for MS patients.

Similarly, Dominguez-Villar et al. found that IFN-γ⁺FOXP3⁺ Tregs (Figure 3) were significantly more abundant than IFN-γ⁻FOXP3⁺ Tregs in MS.²⁵⁸ IL-12, a proinflammatory cytokine that is highly expressed in individuals with MS,²⁵⁹ could stimulate the secretion of IFN-γ by Tregs. The findings indicated that the suppressive activity of IFN-γ⁺FOXP3⁺ Tregs was inhibited through the secretion of IFN-γ stimulated by IL-12. Prospective studies may focus on the frequency of IFN-γ⁺FOXP3⁺ Tregs at risk for developing MS.

Recently, Haque et al. have evaluated the frequencies of circulating CXCR5⁺PD-1⁺ Tfhs and CXCR5⁺PD-1⁺ FOXP3⁺CD25⁺ Tfrs (Figure 3) in patients with MS.²⁶⁰ It was proven that the frequency of circulating CXCR5⁺PD-1⁺ Tfhs was increased in MS patients, whereas the frequency of circulating CXCR5⁺PD-1⁺ FOXP3⁺CD25⁺ Tfrs was significantly decreased. Consistent with previous research, a high proportion of circulating CXCR5⁺PD-1⁺ Tfhs secreted abundance of IL-21,²⁶¹ which contributed to the production of pathogenic autoantibody.^262-264 Correspondingly, a lower frequency of circulating Tfrs was correlated to the reduction of IL-10 which might increase the severity of MS.^265-267 The findings suggested that improved IL-10 secretion by circulating Tfrs may serve as a potential therapeutic target for patients with MS.

3.3 Treg subsets in rheumatoid arthritis

Rheumatoid arthritis (RA), one of the common types of chronic and pharmacologically complex systemic autoimmune disease observed in the elderly population,^{242, 268-270} is characterized by the presence of AAbs such as rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibody.^{271, 272} Liu et al. have found that the frequency of circulating CD4⁺CXCR5⁺FOXP3⁺ Tfrs (Figure 3) and the ratio of Tfrs/Tfhs were significantly increased in patients with stable RA than in patients with active RA or healthy people.²⁴² The circulating CD4⁺CXCR5⁺FOXP3⁺ Tfrs with enhanced suppressive function could alleviate autoimmunity in RA patients by reducing IgG and IgM levels, and the proportion of Tfrs was negatively correlated with the disease severity, which provided a novel insight into RA pathogenesis.

Santinon et al. have also demonstrated that the suppressive TNFR2⁺ Tregs (Figure 3) play a key role in patients with RA.²⁷³ They confirmed that TNF treatment could promote the proliferation of TNFR2⁺ Tregs and ameliorate inflammation by activation of TNF–TNFR2 signaling^{274, 275} and enhance the expression of CD25. Targeting TNFR2 signaling may be a potential therapeutic strategy in RA.

Given that TIM3⁺FOXP3⁺ Tregs possess potent suppression of proinflammatory responses, Sun et al. have examined the frequency and function of TIM3⁺FOXP3⁺ Tregs (Figure 3) in patients with RA.²⁷⁶ They demonstrated the proportion of TIM3⁺FOXP3⁺ Tregs, which could potently suppress IFN-γ and TNF-α inflammation from Teffs by producing high IL-10 and TGF-β, was significantly decreased in patients with RA. As result, the frequency of TIM3⁺FOXP3⁺ Tregs was negatively associated with the pathogenesis of RA, which might be an immunotherapeutic target for patients with RA.

3.4 Treg subsets in type 1 diabetes

T1D, a chronic autoimmune disorder, is characterized by insulin deficiency and resultant hyperglycaemia.^277-279 The complex interactions between the pancreatic β-cell and innate and adaptive immune systems lead to the progression of T1D.²⁸⁰ In humans, Tfrs are identified that can inhibit the production of AAbs and restore glucose tolerance.^{239, 281, 282} Vecchione et al. have reported that the frequency of CXCR5⁻FOXP3⁺ Tregs was higher in AAb⁺ patients than in AAb⁻ patients, which may be caused by active autoimmunity and metabolic dysfunction in AAb⁺ patients.²⁷⁹ Furthermore, they also validated that CXCR5⁺FOXP3⁺ Tfrs (Figure 3), which were needed to maintain peripheral tolerance by regulating diabetogenic Tfhs and B cells, were reduced in spleen and pancreatic lymph node of patients with T1D. Therefore, Tfrs ACT may be a potential therapeutic modality to treat T1D.

The TIM family comprises TIM1, TIM3, and TIM4, and there is little known about the expression of TIM1 and TIM-4 on Tregs. Guo et al. have evaluated the frequencies of circulating TIM1⁺ Tregs and TMI4⁺ Tregs (Figure 3) in patients with TID.²⁸³ Their study revealed that TIM1⁺ Tregs and TMI4⁺ Tregs were significantly decreased in PBMCs of patients with T1D as compared with healthy people. Inhibition of TIM1 could reduce FOXP3 expression and inhibit Tregs development,²⁸⁴ while TIM4 regulated the activation of naive T cells and proliferation of activated T cells.^{285, 286} However, the specific functions and potential mechanisms of TIM1⁺ Tregs and TMI4⁺ Tregs in patients with T1D may need to be further explored.

4.1 Treg subsets in

Although the current improvement in surgical techniques and the application of immunosuppression have improved survival after organ transplantation, immune-mediated injury is still a critical cause of allograft failure.^{287, 288} Notch signaling plays a significant role in immune cell fate determination and differentiation.²⁸⁹ Notch-1, a Notch receptor of the Notch family, is pivotal for regulating fate decisions in the T-cell lineage.²⁹⁰ More recently, Magee et al. found that Notch-1 was highly expressed in FOXP3⁺ Tregs and that the proportion of Notch-1⁺ Tregs (Figure 4) was significantly higher in patients with a kidney transplant than in healthy controls.²⁹¹ Furthermore, the results showed that inhibition of Notch-1 could reduce the frequency and function of effector T cells, whereas the survival, proliferation, and suppressive function of Tregs were enhanced. In agreement with a previous study,²⁹² treatment targeting Notch-1⁺ Tregs may improve allograft tolerance and prolong the survival of solid organ transplantation in humans.

In addition, Hoerning et al. confirmed that the expression of CXCR3 on human CD4⁺FOXP3⁺ Tregs was associated with better renal transplant function (Figure 4).²⁹³ It has been reported that CXCR3 promotes Tregs recruitment and Teffs interactions, which limits autoimmune-mediated tissue damage in EAE.²⁹⁴ The CXCR3⁺FOXP3⁺ Tregs also highly expressed GARP, indicating that this subset of Tregs possessed a strong immunosuppressive function. Interestingly, treatment with tacrolimus did not affect the expression of CXCR3 on the surface of Tregs. CXCR3⁺FOXP3⁺ Tregs showed the capacity to translocate to the site of inflammation and could control renal allograft rejection.

4.2 Treg subsets in graft-versus-host disease

Acute GVHD is a very common complication in hematopoietic stem cells transplantation (HSCT).²⁹⁵ Trzonkowski et al. first described a method for the adoptive transfer of CD4⁺CD25⁺CD127⁻ Tregs expanded in vitro for the treatment of patients with acute and chronic GVHD, which could improve their condition.²⁹⁶ Subsequently, several HSCT clinical phase I and phase II studies were carried out in human leukocyte antigen (HLA)–haploidentical^{297, 298} or umbilical cord blood²⁹⁹ transplantation setting and using HLA-matched donors,³⁰⁰ which further confirmed the stability of the immunosuppressive function of Tregs expanded in vitro and the feasibility of preventing GVHD. In addition, some more detailed induction strategies and subtypes of Tregs have been proved to have stronger mechanisms of suppressive function. Among them, two clinical studies have demonstrated that low-dose IL-2 can better induce the expansion of functional Tregs and ameliorate the manifestations of chronic GVHD (cGVHD).^{301, 302} Notably, Asano et al. have demonstrated that low doses of IL-2 enhance PD-1 expression of central memory Tregs in patients with cGVHD, suggesting that the PD-1 pathway is a critical homeostasis and tolerance regulator for Tregs.¹⁷⁸ Ulbar et al. have used an automated, clinical-grade protocol to expand Tregs in vitro.³⁰³ The expression levels of IL-10, GZMB, and IL-35 of these expanded Tregs were significantly increased, and could protect mice from GVHD. Among them, the subtype of TIM3⁺ Tregs (Figure 4) exhibited a highly immunosuppressive function. We hope these findings may facilitate the development of therapeutic strategies that promote immune tolerance and reverse symptoms of GVHD in humans.

IL-33 and its receptor suppression of tumorigenicity-2 (ST2) have been reported to be associated with Tregs immunobiology, and ST2⁺FOXP3⁺ Tregs (Figure 4) could be augmented following administration of IL-33.^304-306 Consequently, Matta et al. demonstrated that IL-33 mediated the expansion of ST2⁺FOXP3⁺ Tregs via activation of p38 MAPK signaling after HSCT, which could protect against acute GVHD.³⁰⁷ In xenogeneic GVHD (xGVHD), Wang et al. have demonstrated that latency-associated peptide (LAP) can be used as a unique cell-surface marker to distinguish bona fide Tregs from activated FOXP3⁺ and FOXP3⁻ non-Tregs, which is beneficial to improve the purity of Tregs expanded in vitro.³⁰⁸ The subset of LAP⁺FOXP3⁺ Tregs (Figure 4) possessed a fully suppressive function and adoptive transfer of LAP⁺ Tregs could delay the development of xGVHD. It may be important to testify their safety and stability in the future.

The bone marrow niche is important for the potential of HSCs to maintain multifunctionality, such as quiescent status, multidirectional differentiation, and self-renewal, in which the adenosine signaling pathway plays a pivotal role.^{309, 310} It was worth noting that CD150⁺ Tregs (Figure 4) could secrete adenosine to maintain the quiescent status of HSCs, which activated the AMPK pathway to promote energy metabolism to inhibit the GVHD and intestinal cell apoptosis secondary to HSCT. Their results provided potential feasibility of inducing the expression of CD150⁺ Tregs as the therapeutic strategy to prevent GVHD for patients after HSCT.