Cancer Metastasis Rev DOI 10.1007/s10555-015-9566-0

Regulatory T cells and potential inmmunotherapeutic targets in lung cancer Ding Zhang 1 & Zhihong Chen 1 & Diane C. Wang 1 & Xiangdong Wang 1

# Springer Science+Business Media New York 2015

Abstract Lung cancer and metastasis are two of the most lethal diseases globally and seldom have effective therapies. Immunotherapy is considered as one of the powerful alternatives. Regulatory T cells (Tregs) can suppress the activation of the immune system, maintain immune tolerance to self-antigens, and contribute to immunosuppression of antitumor immunity, which is critical for tumor immune evasion in epithelial malignancies, including lung cancer. The present review gives an overview of the biological functions and regulations of Tregs associated with the development of lung cancer and metastasis and explores the potentials of Treg-oriented therapeutic targets. Subsets and features of Tregs mainly include naturally occurring Tregs (nTregs) (CD4+ nTregs and CD8+ nTregs) and adaptive/induced Tregs (CD4+ iTregs and CD8+ iTregs). Tregs, especially in circulation or regional lymph nodes, play an important role in the progress and metastasis of lung cancer and are considered as therapeutic targets and biomarkers to predict the survival length and recurrence of lung cancer. Increasing understanding of Tregs’ functional mechanisms will lead to a number of clinical trials on the discovery and development of Treg-oriented new therapies. Tregs play important roles in lung cancer and metastasis, and the understanding of Tregs becomes more critical for clinical applications and therapies. Thus, Tregs and associated

Ding Zhang and Zhihong Chen contributed equally to this work. * Xiangdong Wang [email protected] 1

Minhang Hospital, Zhongshan Hospital, Fudan University, Shanghai Institute of Clinical Bioinformatics, Fudan University Center for Clinical Bioinformatics, Shanghai, China

factors can be potential therapeutic targets for lung cancer immunotherapy. Keywords Lung cancer . Regulatory Tcell . Immunotherapy . Metastasis

1 Introduction Lung cancer and metastasis are responsible for the most lethality of diseases [1, 2],with about 15 % overall 5-year survival rates for patients with lung cancer. The immune system protects the host against tumor development, growth, and metastasis [3–5]. Regulatory T cells (Tregs) are a population of T cells suppressing the activation of the immune system and maintaining immune tolerance to self-antigens. Tregs can contribute to immunosuppression of antitumor immunity, allowing the tumor evasion through the immune barrier in epithelial malignancies [6]. Tregs were upregulated or activated in the tumor microenvironment, where increased number of Tregs was correlated with a poor prognosis in epithelial cancers, including lung cancer [7, 8]. The present review gives an overview of the biological functions and regulations of Tregs associated with the development of lung cancer and metastasis and explores the potentials of Treg-oriented therapeutic targets. The present review aims at defining subsets and features of Tregs, potential roles of Tregs from circulation or regional lymph nodes in progress and metastasis of lung cancer, and therapeutic targets or biomarkers to predict the survival length and recurrence of lung cancer. Tregs may suppress the function of immune cells and allow lung cancer escapes probably through cell interaction-dependent or cytokine-mediated suppression, although the exact mechanism of Treg cell-mediated suppression remains unclear.

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2 Subsets and features of Tregs Tregs were initially described as thymus-derived suppressive T cells in the early 1970s [9], to probably interconnect between Bsuppressor cells^ and malignancies [10]. Several phenotypical and functional subsets of distinct Tregs with CD4 and CD8 were described, after CD4+CD25+ T cells were then named as thymus-derived naturally occurring regulatory T cells (nTregs) since 1995 [11]. Tregs are recently divided into naturally occurring Tregs (nTregs) (CD4+ nTregs and CD8+ nTregs) and adaptive/induced Tregs (CD4+ iTregs and CD8+ iTregs) (Fig. 1). 2.1 CD4+ CD25+ nTregs CD4+ CD25+ nTregs are derived from the thymus and represent about 1–2 % of the total peripheral CD4+ T-cell population in human [12], in the control of immune self-tolerance, allograft rejection and allergy, or inhibition of the effector functions during infection and in tumors. CD25 is the αchain of the high-affinity receptor for interleukin-2. The activation of nTregs depends upon IL-2 production and concentration within the microenvironment [13]. Forkhead Box protein 3 (FOXP3) was identified as an important regulator of nTreg development and function [11], while Tregs constitutively express cytotoxic T lymphocyte antigen-4 (CTLA-4) [14] and glucocorticoid-induced tumor necrosis factor receptor family-related gene (GITR) [15]. Few biological functionspecific biomarkers were still discovered to present functional characters of those cells [16]. To discriminate Tregs from conventional (activated) CD4+ T cells, a low expression of CD127 (the α-chain of the IL-7 receptor) and a modulated CD45RB expression were proposed as comarkers together with the expression of FOXP3 and CD25 [17, 18]. Helios, a member of the Ikaros transcription factor family, was recently Fig. 1 Subsets and suppressive mechanisms of Tregs. Tregs are mainly divided into nTregs and iTregs with CD4+ or CD8+ subsets. Both can play the immunosuppressive roles in cell interaction-dependent and/or mediator-dependent manners (by secreting IL-10, IL-35, TGF-β, or other soluble factors). nTregs naturally occurring regulatory T cells, iTregs induced regulatory T cells, Tr1 IL-10-secreting regulatory T cell, Th3 TGF-βsecreting regulatory T cell, Tr35 IL-35-secreting regulatory T cell, IL interleukin, TGF-β transforming growth factor-β

proposed as a potentially specific marker of thymus-derived nTregs [19], although it is still questioned by other studies [20]. Circulating human antigen-reactive CD4+FOXP3+ nTregs or CD4+CD25+FOXP3+ nTregs were identified, isolated, and expanded with minimal loss of functional activity [21]. 2.2 Induced (adaptive) CD4+ regulatory T cells iTregs derived from naive CD4+ T cells in the periphery modulate the immune response to microbial and tissue antigens, different from nTregs which developed in the thymus that play a critical role in regulating self-tolerance and seed peripheral tissues to suppress the activation of effector cells [22, 23]. Subtypes of CD4+ iTregs include IL-10-secreting T regulatory 1 (Tr1) cells, TGF-β-secreting T regulatory (Th3) cells, or CD25+FOXP3+ nTreg-like cells. Tr1 cells produce a high amount of IL-10 and little amounts of FOXP3 and CD25 under the stimulation with immature dendritic cells (DCs) in the presence of IL-10 [24]. Foxp3+ and Foxp3− precursor cells give rise to peripheral Tr1 by a mechanism dependent on TGF-β rather than IL-10 [25]. It indicated that human Tr1 may be an important and clinically feasible target in cancer patients for therapy [26], even though Tr1 Tregs were believed more functionally specialized in the regulation of inflammation [27]. Th3 cells, initially described in the induction of oral tolerance to food antigens [28], may mediate Bbystander suppression^ when encountering the fed autoantigen at the target organ [29]. TGF-β can induce the differentiation of Th3 cells and suppress T-cell responses through the induction of Tregs, rather than direct inhibition of cell proliferation [30]. CD4+CD25+FOXP3+ iTreg-like cells are generated in peripheral lymphoid tissues by the conversion of conventional naive CD4+FoxP3− T cells to FoxP3+ Tregs with polyclonal or antigen-specific activation in the presence of specific immune

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suppressive cytokines [31]. The minimal program for Foxp3+ iTreg development and differentiation requires the stimulation and the appearance of cytokines TGF-β and IL-2 that generated iTregs in mesenteric lymph nodes, during induction of oral tolerance, chronically inflamed tissues, or tumor [31–33]. 2.3 CD8+ nTregs or CD8+ iTregs CD8+ Tregs are divided into naturally occurring and induced CD8+ regulatory T cells, i.e., CD8+ nTregs and CD8+ iTregs, respectively, to mildly suppress T-cell proliferation and interferon-γ production [34], although CD8+ nTregs and CD8+ iTregs express CD25, GITR, CTLA4, and CD103. Several phenotypes of CD8+ iTregs have been identified in peripheral tissue in varied conditions. CD8+ iTregs were also discovered in cancer patients, e.g., ovarian, prostate, or nasopharyngeal carcinoma, and the roles of CD8+ iTregs in cancer are emphasized [35].

3 Tregs in progress and metastasis of lung cancer Several studies demonstrated that tumor-associated T cells from patients with epithelial tumors at early and late stages contained increased proportions of CD4+CD25+ Tregs in the peripheral blood. Those cells secreted immunosuppressive cytokines, e.g., TGF-β, and expressed high levels of CTLA-4 on the cell surface to contribute to Treg-associated immune dysfunction in patients with nonsmall cell lung cancer (NSCLC) [36–40]. CD4+CD25+ Tregs in tumor-infiltrating lymph nodes from patients with NSCLC increased significantly, as compared with tumor-free lymph nodes. The percentage of CD4+CD25+ Tregs in tumor-infiltrating lymph nodes of NSCLC patients was negatively correlated with the amount with CD8+ cells within lymph nodes while positively with the levels of TGF-β1 or IL-10 [41]. CD13+CD4+CD25hi Treg cells as an important subpopulation among CD4+CD25hi Treg cells were identified in the peripheral blood of NSCLC patients [42] and expressed higher levels of Foxp3, CTLA-4, membrane-bound transforming growth factor β1, and programmed death legend-1 (PD-L1). CD13+CD4+CD25hi Treg cells could suppress CD25 expression and proliferation of CD4+CD25− T cells. The percentage of CD13+CD4+CD25hi Tregs among CD4+CD25hi Tregs correlated with pathological stages of NSCLC and tumor burden. The percentage of CD8+CD28− Tregs was also higher in the peripheral blood of patients with lung cancer [39, 43]. Increased Tregs in the circulation were found to be associated with worse overall and relapse-free survivals, tumor FOXP3 expression with better prognosis in NSCLC, tumor-infiltrating Tregs, or high-risks patients [8]. In addition, the reciprocal CD4+ T-cell balance bet w e e n e ff e c t o r C D 6 2 L l o w C D 4 + T e ff e c t o r a n d

CD62LhighCD25+CD4+ Tregs may reflect the stage of small cell lung cancer (SCLC) and predict the survival length and recurrence [44]. Local and systemic immune responses in SCLC patients might be downregulated by the differentiation of functional CD4+CD25+FOXP3+CD127loHeliosTregs, partially depending on IL-15 [45]. Hasegawa et al. [46] analyzed peripheral blood mononuclear cells collected prior to surgery and resected specimens from patients with NSCLC and found that peripheral Tregs (pTregs) were CD4+ and Foxp3+. CD4, CD8, and Foxp3 expressions were observed in central regions as cCD4, cCD8, and cFoxp3 or interstitial regions of the tumor as iCD4, iCD8, and iFoxp3. The higher frequency of pTregs was noticed in patients with pleural, vessel, or lymphatic vessel invasions, or with recurrent cancer. Higher frequencies of cCD4 or cCD8 were detected in patients with T1 cancer. Cancer patients with low levels of cFoxp3/iFoxp3 had higher frequency of pTregs as an independent prognostic factor. It implies that pTregs play an important role in the development of lung cancer recurrence and metastasis. The proportion of Tregs in regional lymph node lymphocytes was higher than in peripheral lymphocytes in NSCLC patients as poor prognostic factor in patients with or without node metastasis, associated with lower 5-year overall survival rates [47]. It was also found that the proportion of Tregs in regional lymph node lymphocytes was higher in patients with lung adenocarcinoma, as compared with lung inflammation or squamous carcinomas [48]. It suggests that Treg target therapy may benefit patients with lung adenocarcinoma. The number of Tregs in tumor stroma was higher than in tumor nest in pathological stage I lung invasive adenocarcinoma. The higher Tregs the stroma had, the poorer the prognosis of patients was. Cancer-associated fibroblasts (CAF) could influence Treg induction probably through TGF-β, vascular endothelial growth factor, or both [49]. It is possible that CAFgenerated immune-regulatory cytokines may induce Tregs in the stroma, forming a tumor-promoting microenvironment in lung adenocarcinoma and leading to poor outcome. In addition, docetaxel-based chemotherapy could reduce nonsuppressive, resting, or activated Treg subsets after 4 cycles of treatment in NSCLC patients, probably through a direct effect [50]. Those lines of evidence suggest that Tregs are involved in the regulation of local antitumor immunity, promotion of cancer progression and metastasis, or patient prognosis.

4 Mechanisms of Treg-targeting immunotherapy for lung cancer Tregs may suppress the function of innate and adaptive immune cells through cell interaction-dependent suppression or cytokine-mediated suppression [51], although the exact mechanism of Treg cell-mediated suppression remains unclear. The

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present review mainly focused on mechanisms of Tregmediated immune suppression and potential Treg-targeting immunotherapy for lung cancer. 4.1 Cell interaction-dependent suppression Tregs can suppress directly immune cells by cell-cell contacts via cell surface molecules such as CTLA-4, programmed death 1 (PD-1)/PD-L1, lymphocyte activation gene (LAG3), GITR, CD39/CD73, CD95–CD95 ligand, granzymes/ perforin, or neuropilin-1 (Nrp-1), as shown in Fig. 2. CTLA4 is homologous to CD28, located on the surface of T cells and expressed highly on Tregs. CTLA-4 binds with higher affinity to B7-1 and B7-2 than CD28 and is more competitive [52]. Such signal of naive T-cell activation in response to tumor cell invasion can be influenced, and Treg-mediated suppression of effector T cells via DCs was reduced by blocking or deleting CTLA-4 [14, 53]. It suggests that targeting CTLA-4 can be an anticancer strategy. Recently, an anti-CTLA-4 agent (Yervoy, ipilimumab) was approved as a new immunotherapy for metastatic melanoma [54]. Tregs increased in metastatic stage and CTLA-4 was overexpressed in the lymphocytes of patients with NSCLC [55]. CTLA-4 and CD28 gene polymorphism was associated with NSCLC [56]. CTLA-4 could induce DCs to express indoleamine 2,3-dioxygenase via interaction between CD80 and CD86, to induce the catabolism of tryptophan into proapoptotic metabolites and suppress effector T cells [57]. It suggests that immunotherapy regimen targeting CTLA-4 of Tregs might benefit lung cancer patients, although

Fig. 2 Contact-dependent suppression and potential targets of Tregs for lung cancer immunotherapy. a CTLA-4 binds to B7.1/2 costimulatory molecules on DCs with CD28 on T cells; b LAG-3 binds MHC class II molecules expressed on DCs with high affinity; c agonist anti-GITR antibodies could overcome self-tolerance and reverse Treg suppression; d the CD39 ectoenzyme produces AMP from ATP or ADP, which is subsequently converted into extracellular adenosine by the CD73 ectoenzyme. Adenosine binding to ADORA2A on activated T effector cells or NKs generates immunosuppressive effect; e exogenous CD95 results in a systemic decrease of Tregs through AICD; f CD4+CD25+

the overexpression of CTLA-4 in radically resected NSCLC indicates a reduced death rate [58]. The blocking of CTLA-4 permitted antitumor T cells to acquire effector function and improved the survival rate, evidenced by randomized phase III studies in patients with metastatic cancer [59–61]. Long-term follow-up from a clinical trial showed that a 5-year survival rate was approximately 15 % of treated patients after enrollment [60]. CTLA-4 blockade is moving forward in lung cancer because the immune checkpoint molecule likely evolved to protect self-tissues from autoimmunity. A phase II study in NSCLC on the activity of ipilimumab plus paclitaxel or carboplatin showed that phased ipilimumab plus paclitaxel or carboplatin improved immune-related progression-free survival and progression-free survival [62]. Abscopal responses of NSCLC patients led to a posttreatment increase in tumorinfiltrating cytotoxic lymphocytes, tumor regression, or normalization of tumor markers 1 year after treatment with concurrent radiotherapy and ipilimumab [63]. Another phase II study in extensive disease SCLC showed that phased ipilimumab rather than concurrent ipilimumab improved immune-related progression-free survival while not progression-free survival [64], although results from some phase II trials of ipilimumab in advanced NSCLC and SCLC are encouraging and phase III trials are still ongoing. PD-1, one of the immune checkpoint molecules, is expressed on activated and exhausted peripheral T cells in lung cancer and on tumor cells, and PD-1/PD-L1 interaction provided a negative signal for antigen-induced T-cell activation [65–67]. PD-1 expressed on tumor-infiltrating T cells

Tregs and CD4 + CD25 − T cells regulate death/growth arrest by differentially utilizing the granzyme–perforin pathway. LL-37 regulates CD4+CD25+FoxP3+ Tregs through apoptosis; g Nrp-1 expresses on Tregs and promotes interactions with immature DCs. DCs dendritic cells, CTLA-4 cytotoxic T lymphocyte antigen-4, LAG-3 lymphocyte activation gene 3, GITR glucocorticoid-induced tumor necrosis factor receptor, ADORA2A adenosine A2A receptor, AICD activation-induced cell death, LL-37 a human cathelicidin, Nrp-1 neuropilin-1, LAP latent form of TGF-β

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were correlated with the prognosis of patients with NSCLC, which could be influenced by PD-1 blockade on a single agent [68–70]. Antibodies against PD-1 have recently entered phase III clinical trials, where the rate of adverse events at grades 3 and 4 was lower in PD-1 blockade than in CTLA-4 blockade [71]. The PD-1/PD-ligand pathway may act more peripherally than the CTLA-4/B7-1 pathway which may play a role in lymph nodes. Clinical studies on nivolumab against PD-1 and ipilimumab showed that the combination may provide a feasible regimen as the first-line treatment for chemotherapynaive patients with NSCLC [72]. LAG-3 (CD223), a CD4 homologue, can bind major histocompatibility complex (MHC) class II molecules with high affinity, for maximal suppressive activity of nTregs and iTregs [73]. The depletion of LAG-3+CD4+ T cells enhanced tumor-specific CD8 + T-cell reactivity, and LAG-3–Ig fusion protein mediated tumor control and regression [74, 75]. Based on the targeting potential of LAG-3–Ig, a new drug (IMP321, Immutep, Paris) was developed and tested in clinical trials. IMP321 is well tolerated and associated with an enhanced TH1 CD4+ T cell and CD8+ T cell [76, 77]. The combination of IMP321 with taxane-based chemotherapy in women with breast cancer demonstrated a promisingly objective response rate [78]. A recent study found an enhanced suppressive capacity of LAG-3+CD4+CD25+ T cells, as compared with LAG-3− T cells from tumor sites of patients [79]. It indicates that LAG-3 could be a promising target in lung cancer immunotherapy, although further studies are still needed to clarify the role of LAG-3 in lung cancer. GITR is considered as a target for cancer immunotherapy on the basis of the evidence that agonist anti-GITR antibodies could overcome self-tolerance and reverse Treg suppression [80]. Therapeutic effects of anti-GITR antibodies were primarily demonstrated in melanoma [81], colon tumors [82], lymphoma [83], or vaccine-treated-tumors [84]. The combination of DTA-1 with adoptive T-cell therapy or CTLA-4 blockade could synergistically enhance tumor regression of fibrosarcoma and colon tumors [85, 86]. The addition of DCs expressing anti-CTLA-4 to the original glucocorticoid-induced tumor necrosis factor receptor ligand (GITRL) and TYRP-2 expressing DC vaccine further enhanced the efficacy of the approach [87]. The agonist anti-GITR antibodies in tumor immunotherapy could make T effectors resistant to Treg suppression [88], indicating that GITR may have different effects on intratumor and peripheral Tregs [89]. GITR-induced changes in nTreg phenotype and function may depend upon phosphorylation of c-Jun N-terminal kinase (JNK) [90]. It indicates that the regulation of JNK phosphorylation plays a central role in GITR-induced changes in Tregs with therapeutic implications and that JNK inhibitors can act as another regulator of GITR-mediated lung cancer immunotherapy.

The CD39 ectoenzyme produces AMP from ATP or ADP, which is subsequently converted into extracellular adenosine by the CD73 ectoenzyme [91]. Humans can express low levels of adenosine deaminase, while T effector cells are enriched in adenosine deaminase with low levels of CD39 and CD73. It may influence Tregs to produce and sustain high concentrations of extracellular adenosine [92]. Adenosine binding to adenosine A2A receptor (ADORA2A) on activated T effector cells or natural killer (NK) cells generates an immunosuppressive effect through the coordinated expression of CD39/CD73 as one of the mechanisms of Treg-mediated immunosuppression [93]. The ectonucleotidases CD39/CD73 and ADORA2A appear as possible targets for novel treatments in cancer [94]. In addition, exosomes from other cancer cells could express CD39/CD73 to suppress T cells through adenosine production [95]. It is needed to furthermore clarify the expression of CD39 and/or CD73 on exosomes from lung cancer tissues and the axis of CD39/CD73 and ADORA2A as a potential target for inhibition of Treg-mediated immunosuppression and lung cancer immunotherapy. CD95 (APO-1, Fas) is a member of the death receptor family and a subfamily of the tumor necrosis factor receptor superfamily. Activation-induced cell death is involved in the removal of activated T cells and Ag-presenting B cells depending, at least partially, on death receptor CD95 and the ligand CD95L [96, 97]. Human Tregs in lung cancer could express Fas and FasL [98] and low levels of CD95L upon stimulation, inducing the resistance to activation-induced cell death in Tregs. The exogenous CD95 stimulation by administration of an agonistic anti-CD95 antibody reduced systemic numbers of Tregs and Foxp3+GFP+ Treg numbers in pooled lymph nodes. Chen et al. [99] engineered cancer cells with CD95L to kill Tregs within the tumor, leading to a decrease in tumor mass. CD95L-expressing conventional CD4+ T in the tumor can reduce the number of Tregs [100]. Tregs were depleted from the inflamed tissue site by an unknown cell type expressing CD95L [101]. It implies that Tregs are susceptible to homeostatic controls by exogenous CD95 stimulation, which provides a new potential target for Treg-targeting lung cancer immunotherapy. The perforin/granzyme pathway is a central component of antitumor and antiviral effector immune responses mediated by cytotoxic T lymphocytes and NK cells. Granzyme B and perforin were also involved in the suppression of nodal metastasis of lung cancer cells [102]. Tregs suppressed the ability of NK cells and cytotoxic T lymphocytes to clear tumor cells via granzymes A/B/perforin within Tregs [103]. Nonregulatory responder CD4+ T cells could resist Treg suppression by production of granzyme B [104]. A recent study suggests that lung cancer cells utilize the increased PI-9 (granzyme B inhibitor) expression to protect from granzyme Bmediated cytotoxicity as an immune evasion mechanism of lung cancer [105]. Czystowska et al. [106] further

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demonstrated that human CD4 + CD25 + Tregs and CD4+CD25− T cells reciprocally regulate death/growth arrest by differentially utilizing the granzyme–perforin pathway depending on IL-2 concentrations. It seems that the roles of the granzyme–perforin pathway in lung cancer immunity are complicated, probably depending on the microenvironment. LL-37, a human cathelicidin, can kill stimulated and nonstimulated CD4+CD25+FoxP3+ Tregs through apoptosis, rather than CD4+CD25− T cells [107]. LL-37-induced apoptosis in Tregs depends upon granzymes A and B from cytolytic granules. Administration of LL-37 within the tumor could influence the adaptive antitumor immune response by killing Tregs and, thus, inhibiting the suppressor activity. The role of LL-37 in lung cancer immunotherapy needs to be furthermore clarified. Nrp-1 expressed on the surface of CD4+CD25+ Tregs independent of the activation can be downregulated in naive CD4+CD25− T cells after the stimulation [108]. Nrp-1 is expressed by most Tregs rather than naive T helper cells and promotes prolonged interactions with immature DCs [109]. Nrp-1 is also a high-affinity receptor for latent and active TGF-β1, activates the latent form, and is relevant to Treg activity in tumor biology [110]. Nrp1+ Tregs showed to be more efficient to induce the suppression, and Nrp1+ and Nrp1− Tregs decreased in tumor-draining lymph nodes of patients with cervical cancer following preoperative chemoradiotherapy [111]. Nrp1+ Treg elimination may facilitate the generation of T cells to mediate the destruction of the neoplastic cells after cytotoxic therapy. Recently, Nrp-1 was identified as a valid antiangiogenic target in multimalignancy, including lung cancer, and as a potential direct antitumor target

Fig. 3 Role of TGF-β in lung cancer pathogenesis and progression. TGF-β within lung cancer microenvironment upregulates B7H1 and GITRL expression in DCs and de novo generation and expansion of Tregs. TGF-β induces epithelial-to-mesenchymal-like transition (EMT), associated with early NSCLC progression, while the process can be inhibited by BMP7 and MiR-23a. TGF-β induces expression of FOXP3 in lung cancer cells. TGF-β induces lung cancer cells to death

of immunotherapy and antiangiogenic therapy for lung cancer [112].

4.2 Soluble factor-mediated suppression of Tregs Major immunosuppressive cytokines or factors of Tregs include TGF-β, IL-10, IL-35, galectin-1, or prostaglandin E2 (PGE2), as explained in Fig. 3. TGF-β can regulate the biological function of CD4+ T cells and demethylate FOXP3 promoter in CD4+CD25− T cells to maintain lymphocyte homeostasis and increase FOXP3 expression [113]. TGF-β within the lung cancer microenvironment could upregulate PD-L1 and GITRL expression in DC and Treg generation [114]. TGF-β plays a role in tumorigenesis and highly expresses in the lungs at risk for lung cancer [115, 116]. The expression of FOXP3 within tumor cells is associated with tumor progression and metastasis, as a poor prognostic factor [117]. Treatment of TGF-β2 and DNA methyltransferase inhibitor could upregulate FOXP3 expression in lung cancer cells [118]. It is possible to have TGF-β as a target for lung cancer immunotherapy, although the role of TGF-β signaling in tumorigenesis needs to be further clarified [118]. The epithelial-to-mesenchymal-like transition associated with early progression of NSCLC can be induced by TGF-β [119]. However, the loss of TGF-β-induced tumor suppressor function due to genetic and epigenetic alterations of TβRII may play a critical role in lung carcinogenesis [120]. The downregulation of TβRII in lung cancer depended on histone deacetylase and can be restored by the inhibitors in lung cancer patients [121]. It is necessary to balance TGF-β-induced tumor suppression and pathogenesis when TGF-β in the

by binding to TβRII on lung cancer cells. TGF-β transforming growth factor-β, Tregs regulatory T cells, DCs dendritic cells, CTLs cytotoxic T lymphocytes, GITRL glucocorticoid-induced tumor necrosis factor receptor ligand, EMT epithelial-to-mesenchymal-like transition, NSCLC nonsmall cell lung cancer, BMP7 bone morphogenetic protein 7, MiR23 microRNA 23a, TβRII TGF-β type II receptor

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microenvironments can be targeted by immunotherapy for lung cancer. IL-10 is commonly regarded as an anti-inflammatory and immunosuppressive cytokine, allowing tumor immune escape (Fig. 4). It was evidenced that human lung cancer cells could induce IL-10 production from T cells and increase the capacity of T-cell-derived IL-10, associated with clinical prognosis of patients with NSCLC [122, 123]. Polymorphisms of IL-10 genes were associated with the occurrence of NSCLC [124]. NSCLC origin soluble mediators may play an immunoregulatory role through induction of IL-10 production [125]. On the other hand, IL-10 might also possess anticancer properties due to the immunostimulating and antiangiogenic functions [126] and is associated with worse prognosis in NSCLC

[127]. The survival advantage of patients with stage I NSCLC may be related to the antitumor activity of the CD8+/IL-10+ cell phenotype [128]. IL-35 is identified as a novel immunosuppressive/antiinflammatory cytokine, of which two subunits, IL-12A and Epstein-Barr virus induced 3, are produced by Tregs [129, 130]. IL-35 could suppress T-cell proliferation and convert naive T cells into IL-35-producing induced regulatory T cells to mediate the suppression (Fig. 5) [131]. IL-35 is signaled through a unique heterodimer of receptor chains IL-12Rβ2 and gp130 or homodimers of each or both chains, dependent upon the activation of transcription factors STAT1 and STAT4, to bound to distinct sites in promoters of genes encoding p35 and Epstein-Barr virus induced 3 [132]. IL35-producing induced regulatory T cells can be induced in an IL-35- and/or IL-10-dependent manner within the tumor microenvironment to contribute to Treg-mediated tumor progression [131]. Galectin-1 (Gal-1) is a homodimer of noncovalently associated 14 kDa subunits with two carbohydrate recognition domains to enable cell adhesion and cross-linking of several glycoproteins with branched or repeating Galβ1-4GlcNAc sequences on T cells [133]. Gal-1 overexpressed on Tregs and blockade of Gal-1 binding reduced the inhibitory effects of CD4+CD25+ T cells [134]. The primary roles of Gal-1 in cancer progression and metastasis are attributed to suppress Tcell immune responses, promote tumor angiogenesis, and increase tumor cell adhesion and invasion, including lung cancer [135]. Gal-1 might locally contribute to the apoptotic elimination of infiltrating effector T cells and favor tumor progression [136]. Blockage of Gal-1 expression stimulated the

Fig. 5 Role of IL-35 in a tumor microenvironment and IL-35 signaling pathway. IL-35 has two subunits, IL-12A and Epstein-Barr virus induced 3 (EBi3). IL-35 suppresses T-cell proliferation and converts naive T cells and Tconv cells into IL-35-producing induced regulatory T cells (iTr35 cells). The generation of iTr35 cells within cancer can be induced in an IL-35- or IL-10-dependent manner. IL-35 signaled through IL-12Rβ2

and gp130 required for IL-35 expression and conversion into iTr35 cells. The signaling through IL-35 receptor depends on transcription factors STAT1 and STAT4. iTr35s IL-35-producing regulatory T cells, Tconv conventional T cell, EBi3 Epstein-Barr virus induced 3, B16 B16 melanoma cells, MC38 colorectal adenocarcinoma cells, IL-35 interleukin-35, IL-35R IL-35 receptor, IL-2A interleukin-2A

Fig. 4 Role of IL-10 in lung cancer. Lung cancer cells can produce and also induce T cell to produce IL-10, partially depending on PGE2. IL-10 can impair antitumor immunity, due to defects in T-cell and DC function. IL-10 might possess anticancer properties on the basis of immunostimulating and antiangiogenic functions. Tr1 IL-10-secreting T regulatory, DCs dendritic cells

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Fig. 6 Role of galectin-1 in Tregs and lung cancer microenvironment and signaling pathway of galectin-1 (Gal-1)-induced T-cell apoptosis. Gal-1 overexpresses on Tregs, upregulates upon the activation, and overexpresses on cancer cells. Primary roles of Gal-1 in cancer progression and metastasis are through inducing T-cell apoptosis. Signaling pathways of Gal-1-induced T-cell apoptosis are delineated.

Tregs regulatory T cells, DCs dendritic cells, Gal-1 galectin-1, Txnip thioredoxin-interacting protein, ASK1 apoptosis signal-regulating kinase 1, MAPK mitogen-activated protein kinase, JNK c-Jun amino-terminal kinase, AP-1 activator protein-1, ER endoplasmic reticulum, TRE TPA responsive element, Bcl-2 B-cell lymphoma-2, ZAP70 zeta-chainassociated protein kinase

generation of a tumor-specific T-cell-mediated response and tumor rejection [137] and reduced lung metastasis with increased CD4+ and CD8+ T cells and cancer cell adherence [138]. Gal-1 could induce the expression of IL-10 in monocytes and monocyte-derived DCs [139]. It suggests that the Gal-1/IL-10 functional axis may be crucial in lung cancermediated immune suppression. It is evidenced that the JNK/ c-Jun/AP-1 pathway with other T-cell apoptosis pathways plays a key role for T-cell death regulation in response to Gal-1 stimulation [140], as shown in Fig. 6.

PGE2 was found to promote the development of Tregs [141], of which cyclooxygenase (COX)-2-dependent production contributes to TGF-β-induced Treg function in human NSCLC [142, 143]. It seems that TGF-β and PGE2 signaling may be important in therapeutic interventions in lung cancer. Figure 7 demonstrates that COX2 and PGE2 are essential for the EP2- and EP4-dependent induction of Tregs in cancer, interaction of DCs with Tregs, or the suppressive activity of Tregs [144]. A recent study demonstrated that COX2 -1195G/ A polymorphism could influence the infiltration of Tregs into

Fig. 7 Role of PGE2 in Treg-mediated antitumor immunosuppression and lung cancer microenvironment. PGE 2 are small molecule derivatives of arachidonic acid (AA), produced by cyclooxygenases (COX; COX 1 /COX 2 ) and PG synthases. It promotes Treg differentiation from naive T cell and expansion of re-existing Tregs. PGE2 can mediate the suppressive activity of Tregs. PGE2 selectively

suppresses effector functions of the Th1-, CTL-, and NK cell-mediated type 1 immunity and supports activation of DCs. PGE2 contributes cell proliferation and migration of lung cancer. Tregs regulatory T cells, DCs dendritic cells, AA arachidonic acid, COX2 cyclooxygenase-2, PG synthases prostaglandin synthases, PGE2 prostaglandin E2, TGF-β transforming growth factor-β

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NSCLC which was reflected by COX2 SNP factor as a prognostic factor [145]. PGE2, in an autocrine or paracrine fashion, modulates transcriptional repressors of E-cadherin and regulates COX2-dependent E-cadherin expression in NSCLC [146, 147]. It suggests that the blocking of PGE2 production or activity by genetic or pharmacological interventions may benefit patients with NSCLC. The possible link between tumor COX2 overexpression and elevated Erk-mediated cancer cell proliferation and migration may contribute to EGFR inhibitor resistance in NSCLC [148]. Increased expression of PGE2 in the lung tumor microenvironment may initiate a mitogen-signaling cascade composed of EP4, βArrestin1, and c-Src, to mediate lung cancer cell migration [149]. Selective targeting of EP4 with a ligand antagonist may provide an efficient approach to better manage patients with advanced lung cancer. Changes in urine PGE-M, a major PGE2 metabolite, reflect the levels of systemic and intratumoral PGE2. A phase II clinical trial demonstrated that the more than 70 % decline in PGE-M was associated with an apparent survival benefit in NSCLC patients [150].

Authors’ contributions DZ contributed to the collection of data, discussion, writing, and review of figures, and ZHC, DCW, and XDW contributed to the revision, structure design, manuscript preparation, and discussion. Conflict of interest The authors declare no conflict of interest.

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5 Conclusion The present article gives an overview of the biological functions and regulations of Tregs associated with the development of lung cancer and metastasis and explores the potentials of Treg-oriented therapeutic targets. Subsets and features of Tregs mainly include naturally occurring Tregs (CD4+ nTregs and CD8+ nTregs) and adaptive/induced Tregs (CD4+ iTregs and CD8+ iTregs). Tregs, especially in circulation or regional lymph nodes, play an important role in the progress and metastasis of lung cancer and are considered as therapeutic targets and biomarkers to predict the survival length and recurrence of lung cancer. Tregs may suppress the function of immune cells and allow lung cancer escapes probably through cell interaction-dependent or cytokine-mediated suppression, although the exact mechanism of Treg cell-mediated suppression remains unclear. Increasing understanding of Tregs’ functional mechanisms will lead to a number of clinical trials on the discovery and development of Treg-oriented new therapies. Tregs as one of the therapeutic target candidates for lung cancer need global forces and databases to integrate the genes, proteins, receptors, signal pathways, and functions with clinical informatics and phenotypes together.

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14. Acknowledgments The work was supported by Zhongshan Distinguished Professor Grant (XDW), The National Nature Science Foundation of China (91230204, 81270099, 81320108001, 81270131, 81300010), The Shanghai Committee of Science and Technology (12JC1402200, 12431900207, 11410708600, 14431905100), Operation Funding of Shanghai Institute of Clinical Bioinformatics, and Ministry of Education, Academic Special Science and Research Foundation for PhD Education (20130071110043).

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