Convergences and Divergences of Thymus- and Peripherally Derived Regulatory T Cells in Cancer

The expansion of regulatory T cells (T_reg) is a common event characterizing the vast majority of human and experimental tumors and it is now well established that T_reg represent a crucial hurdle for a successful immunotherapy. T_reg are currently classified, according to their origin, into thymus-derived T_reg (tT_reg) or peripherally induced T_reg (pT_reg) cells. Controversy exists over the prevalent mechanism accounting for T_reg expansion in tumors, since both tT_reg proliferation and de novo pT_reg differentiation may occur. Since tT_reg and pT_reg are believed as preferentially self-specific or broadly directed to non-self and tumor-specific antigens, respectively, the balance between tT_reg and pT_reg accumulation may impact on the repertoire of antigen specificities recognized by T_reg in tumors. The prevalence of tT_reg or pT_reg may also affect the outcome of immunotherapies based on tumor-antigen vaccination or T_reg depletion. The mechanisms dictating pT_reg induction or tT_reg expansion/stability are a matter of intense investigation and the most recent results depict a complex landscape. Indeed, selected T_reg subsets may display peculiar characteristics in terms of stability, suppressive function, and cytokine production, depending on microenvironmental signals. These features may be differentially distributed between pT_reg and tT_reg and may significantly affect the possibility of manipulating T_reg in cancer therapy. We propose here that innovative immunotherapeutic strategies may be directed at diverting unstable/uncommitted T_reg, mostly enriched in the pT_reg pool, into tumor-specific effectors, while preserving systemic immune tolerance ensured by self-specific tT_reg.

T_reg Suppress Pro-Tumoral Inflammation or Anti-Tumor Response

Introduction

Tumor onset is a very complex process, in which cells of both innate and adaptive immune system play crucial roles in inhibiting or fostering tumor development. The awareness that the immune system could act as an extrinsic tumor suppressor or as a tumor-sculpting player resulted in the cancer immunoediting theory, which described the interaction between tumor and host as consisting of three different phases: elimination, equilibrium, and escape (1). During the last phase of this process, transformed cells acquire the ability to subvert the control exerted by immune cells thus originating the clinically evident pathology. The escape is due to different mechanisms, including reduced immunogenicity (low expression level of MHC class I and loss of antigen expression), acquired resistance to the cytotoxic functions of immune cells, and accumulation in the tumor microenvironment of immunosuppressive mediators, like regulatory T cells (T_reg) (1). The first marker to be identified as distinguishing T_reg from the other CD4⁺ T lymphocytes was CD25 (2) and depletion of CD25-positive cells unveiled anti-tumor immunity in experimental models (3). Few years later, the transcription factor Forkhead Box P3 (Foxp3) was indicated as the master regulator of T_reg (4, 5). In support of the crucial roles played by Foxp3 in T_reg fate determination and immune homeostasis, Foxp3 mutations have been recognized as responsible for human Immune Dysfunction, Polyendocrinopathy, Enteropathy, X-linked (IPEX) syndrome (6, 7), and for the phenotype of scurfy mutant mice (8), both characterized by fatal autoimmune lymphoproliferation linked to severe defects in T_reg development/functions. However, very recent data have demonstrated that the complete development of the T_reg lineage requires the concomitant, Foxp3-independent, establishment of a T_reg-specific pattern of DNA hypomethylation (9).

According to recently proposed recommendations (10), T_reg are classified into two principal subsets based on their developmental origin: thymus-derived T_reg (tT_reg) develop in the thymus, while peripherally induced T_reg (pT_reg) develop in vivo in the periphery from conventional T cells (T_conv), through a process called “conversion” (11). T_reg can also be induced in vitro (and are called iT_reg) under TGF-β and/or retinoic acid exposure, but their complete commitment into fully differentiated T_reg is still under debate (12). In physiological conditions, the pool of T_reg, encompassing both tT_reg and pT_reg, which represents about the 5–10% of the circulating CD4⁺ T lymphocytes, assures peripheral self-tolerance and prevents exacerbated immune responses (7, 8). A huge amount of data now demonstrates that tumor onset and progression perturb T_reg homeostasis and lead to increased T_reg/T_conv and T_reg/CD8 ratios both in peripheral blood and in the tumor microenvironment (13). The accumulation of T_reg at tumor sites may be due to the concomitant or the preferential occurrence of distinct events, such as the recruitment of T_reg from periphery, the proliferation of pre-existing T_reg in the tumor microenvironment, and the de novo conversion of tumor-infiltrating CD4⁺ lymphocytes (TIL) into pT_reg (14, 15). Despite controversy existing over the dominant suppression mechanism, and despite the incomplete understanding of the biological meaning of T_reg accumulation in cancer, it is well accepted that T_reg are crucial players in tumor biology and that the modulation of their function is an indispensable requisite for the development of successful anti-tumor immune-therapies.

Mechanisms of T_reg Suppression in Tumors

It was recently demonstrated that T_reg infiltrating different tissues have a specific gene signature (16), thus T_reg may use peculiar suppression mechanisms in response to microenvironmental stimuli. This specialization may represent the basis for designing immune interventions targeting specific T_reg functions in a given tissue while sparing systemic immune homeostasis. Even though a tumor T_reg-specific gene signature has not been delineated yet, some mechanisms have been described to contribute to T_reg suppression in tumors, which can be clustered in three main types: surface molecules, enzymatic activities, and cytokines (Figure 1).

FIGURE 1

Figure 1. T_reg suppressive mechanisms in tumor microenvironment. T_reg use different strategies to inhibit target cells within the tumor mass. Three types of T_reg-related molecules can mediate these suppressive mechanisms: (1) surface molecules (upper panel); (2) enzymes (middle panel), and (3) cytokines (lower panel). (1) Among the surface molecules expressed by T_reg, CTLA-4, LAG-3, Nrp-1, and RANKL have a well-demonstrated role in promoting tumor progression, mainly modulating DC activation and function. In particular CTLA-4 and LAG-3, binding to CD80/CD86 (B7-1/2), and MHCII respectively, significantly impair DC capacity to activate T_conv. In addition CTLA-4 promotes IDO expression and the production of the pro-apoptotic metabolite kynurenine. Nrp-1 instead stabilizes T_reg-DC contact, allowing T_reg to adequately suppress DC. Although the course of action of RANKL is not yet well defined, its expression is associated to tumor metastatization. (2) The two ecto-enzymes CD39 and CD73 generate from ATP pericellular adenosine, which is endowed with strong tolerogenic effects. Also cAMP, similarly to adenosine, interferes with T_conv activation and survival. Granzyme and perforin induce the apoptosis of target cells by cytolysis. (3) T_reg secrete several immune-modulatory cytokines, which could directly modulate T_conv functions (TGF-β, IL-10, and IL-35), or indirectly promote the establishment of pro-tumorigenic microenvironment (VEGF).

Both human and mouse T_reg constitutively express on their surface cytotoxic T lymphocyte-associated antigen 4 (CTLA-4), a co-inhibitory member of the CD28/B7 family, endowed with strong immune-regulatory properties (17). The relevance of CTLA-4 in regulating T_reg function was demonstrated in several settings, including autoimmune diseases and different tumor types (18). A comparative gene expression profile between T_reg and T_conv revealed that T_reg specifically up-modulate lymphocyte activation gene 3 (LAG-3) (19), a homolog of CD4, that binds to MHC class II on antigen-presenting cells (APCs). LAG-3 is upregulated in tumor-infiltrating T_reg and experiments with anti-LAG-3 mAb demonstrated that functional LAG-3 is required for maximal T_reg suppressive function (20, 21). T_reg-DC interaction is also mediated by the transmembrane protein neuropilin-1 (Nrp-1), expressed on T_reg membrane, which ensures the stability of T_reg-DC interaction and allows T_reg to efficaciously suppress DC (22). A study conducted on patients with early-stage cervical tumor showed that T_reg infiltrating the tumor-draining lymph node of patients with metastasis have a higher expression of several immune-modulatory molecules, including Nrp-1 (23). The receptor activator of nuclear factor-kB ligand (RANKL), member of the tumor necrosis factor family, was found to be highly expressed on T_reg isolated from tumor-bearing hosts, and substantial evidence indicates that RANKL expressed by T_reg is involved in the onset of metastasis in both mammary (24) and prostate tumors (25).

Regulatory T cell suppression may be mediated by enzymatic activities, such as CD39/CD73 (26, 27), granzyme B, and perforin (28). CD39 and CD73 are two ecto-enzymes that dephosphorylate extracellular ATP and generate pericellular adenosine, which in turn exerts a strong pro-tumorigenic role modulating the function of numerous tumor-infiltrating immune cells. CD73-deficient mice develop a stronger anti-tumor immune response compared to CD73-sufficient mice (29). T_reg are also able to control the proliferation and function of different immune cells via the Perforin pathway (30). In mouse models of melanoma, lymphoma, and acute myeloid leukemia it has been demonstrated that tumor-infiltrating T_reg, but not naïve T_reg, secrete high amounts of both perforin and granzyme B, which in turn induce NK and CD8⁺ T cell death (28).

Immunosuppressive cytokines, like TGF-β and IL-10, are critical players in T_reg biology, being involved in both their differentiation and suppressive potential, especially in tumors. T_reg-derived TGF-β was found relevant in suppression of anti-tumor T cell response in both mouse (31) and human (32, 33) tumors. IL-10 is a well-known immunosuppressive mediator, and several pieces of evidence highlight the relevance of T_reg-derived IL-10 in controlling inflammation at the mucosal interfaces such as gut and lung (34, 35). Despite these data, little information is available about the roles of T_reg-derived IL-10 in tumor microenvironments. We have recently demonstrated that tumor-associated T_reg secrete high amounts of IL-10, which in turn impairs DC migration to the draining lymph nodes and the mounting of a specific anti-tumor immune response. This phenotype could be reverted by stimulating the receptor OX40 on the surface of intratumoral T_reg. Indeed, OX40-triggered T_reg showed reduced secretion of IL-10 as a consequence of the down-modulation of the interferon regulatory factor 1 (IRF1), a transcription factor active in the IL-10 promoter (36). Another cytokine recently described as critical for the full T_reg suppressive function is IL-35, formed by Epstein–Barr-virus-induced gene 3 (Ebi3) and IL-12α (p35) (37). In two different mouse transplantable tumor models (melanoma and colon carcinoma), it was observed that T_reg secrete abundant IL-35, thus promoting the differentiation of induced IL-35-secreting T_reg (37). It is well known that tumor growth is associated with a consistent process of new angiogenesis in response to hypoxia. A circuit involving tumor hypoxia, T_reg recruitment, and angiogenesis has been recently discovered (38). In the hypoxic tumor microenvironment, the chemokine axis CCL28–CCR10 plays a determinant role in the recruitment of T_reg, which secrete huge amounts of vascular endothelial growth factor (VEGF), further stimulating the new angiogenesis process and the establishment of a tolerogenic microenvironment (38).

Double Effects of T_reg on Prognosis

Since their discovery, T_reg were considered one of the main obstacles for tumor clearance, according to their tolerogenic properties and their accumulation along tumor progression. In this view, several anti-tumor immune-therapies focus on T_reg depletion or inhibition, in order to “contrasuppress” T_reg inhibitory functions and to block the conversion of non-regulatory cells (non-T_reg) into regulatory cells (15). Reduced T_conv/T_reg ratio was observed in patients with pancreatic tumor (39, 40), breast cancer (39), ovarian cancer (41), Hodgkin lymphoma (42), and melanoma (43). Increased T_reg frequency is generally associated to advanced tumor stage and poor prognosis, as recently demonstrated in a study on ovarian cancer (44). In the ascites of patients with advanced tumor, the percentage of T_reg was increased compared to the ascites of patients with early-stage tumor. Same results were obtained with the mouse WF-3 transplantable ovarian tumor model, showing augmented percentages of T_reg in both spleen and tumor-associated cells of mice with advanced tumors, compared to naïve or mice with early lesions. In addition, the treatment of tumor-bearing mice with the T_reg-depleting mAb PC61 (αCD25) reduced tumor growth and prolonged mice survival (44). Similarly, it has been demonstrated that T_reg number inversely correlated with the therapy outcome in melanoma patients treated with non-myeloablative chemotherapy, in combination or not with total body irradiation, followed by adoptive T cell transfer (45). Responder patients had a lower frequency of T_reg in peripheral blood compared to non-responder patients (45). A study conducted on patients affected by invasive ductal carcinoma showed a positive correlation among T_reg, Th17, and tumor aggressiveness. These data imply that T_reg and Th17 cells may concomitantly expand during tumor progression, with T_reg mainly suppressing protective effector T cell proliferation while sparing Th17 proangiogenic activities, fostering cooperatively tumor progression, and the metastatic process (46).

Nevertheless, recent data, in particular tumor systems, point out that T_reg may exert a protective role for the host (13, 47, 48). The connection between tumor and inflammation is a well-assessed process (49), but now it is clearly emerging that the type of tumor-associated inflammation imprints the behavior of T_reg, becoming detrimental or beneficial for the host. Type-1 inflammation, characterized by high concentration of IFN-γ and IL-12 and fully active cytotoxic cells, represents efficient anti-tumor immunity (49). In this setting, the inhibitory properties of T_reg may promote tumor escape and aggressiveness (47). On the contrary, immune responses dominated by cytokines like TNF-α, IL-1β, IL-6, IL-23, and IL-17 act as pro-tumoral mediators (47). In this environment T_reg may suppress a pro-tumoral inflammatory status, thus playing a protective role for the host (47).

These unexpected anti-tumoral T_reg properties were observed in patients with colorectal cancer (CRC) (50–52). In these patients, with different tumor stages, a better prognosis and an increased overall survival were associated with higher infiltration of FOXP3⁺ T cells compared to patients with a poor tumor outcome. These data suggested the hypothesis that FOXP3⁺ T cells could be considered as an independent prognostic factor for CRC. Following a strong activation, both conventional CD4⁺ (53) and CD8⁺ (54) lymphocytes up-regulate Foxp3 expression in colonic tissue. These observations indicated that the CRC prognosis positively correlated with non-regulatory FOXP3⁺ cells rather than to T_reg. However in vitro suppression assays demonstrated that FOXP3⁺ cells, isolated from CRC tissues, were endowed with suppressive functions, confirming their nature as regulatory cells (55). In a recent study conducted on 65 patients with different stages of CRC, FOXP3 expression was systematically evaluated in both tumor-infiltrating lymphocytes and neoplastic cells, and was correlated to tumor progression and clinical-pathological features (56). From this study the notion emerged that high FOXP3 expression in tumor cells correlated with poor tumor outcome, compared to tumors poorly expressing FOXP3; on the contrary, no correlation was observed between CRC prognosis and FOXP3 expression by T cells (56).

A protective role of T_reg was also found in head and neck squamous cell carcinoma (HNSCC) (57). Univariate and multivariate analysis demonstrated that the locoregional control of the tumor was positively associated with CD4⁺FOXP3⁺ regulatory cell infiltration (57). However, also for this type of neoplasia, there are some discordant data regarding the role of T_reg in tumor progression. Indeed, another study showed that T_reg frequency and suppressive function were higher in the peripheral blood of tumor-bearing patients than in healthy volunteers (58).

The discrepancies observed in these studies may be due to the number of patients included, different strategies of analysis and non-homogeneity of tumor samples (stage, metastasis, etiology). Certainly, to properly define the role of T_reg in tumor outcome, the new studies should take into account the tumor stage and the related inflammatory features, depending on the anatomical localization. In general, those tumors arising from chronic inflammation, almost at their initial stage, can benefit from the suppressive properties of T_reg. In fact, during the inflammatory process, T_reg highly accumulate in the site of inflammation such to prevent exacerbated immune responses and tissue damage, which are the prelude to neoplastic transformation. On the contrary, in the presence of an established tumor, T_reg may reduce anti-tumor immunity thus favoring tumor escape. A more specific definition of T_reg contribution in tumor development and progression is desirable for the design of new and more effective immunotherapies, allowing the discrimination among tumors that will benefit or not from T_reg depletion/inhibition.

Evidence for pT_reg or tT_reg Accumulation in Tumors

Distinguishing Features of pT_reg and tT_reg

Many efforts have been recently addressed to the identification of phenotypic, molecular, and functional features distinguishing tT_reg and pT_reg, besides their site of origin (11). Some markers have been proposed to distinguish pT_reg and tT_reg, even though with some limitations: the initial enthusiasm for the suggestion of Helios as able to identify tT_reg (59) has been soon moldered by the observation of Helios expression in pT_reg (60); the recent finding of the Nrp-1 as a marker of tT_reg (61, 62) has application limited to murine cells, being not expressed on human T_reg (63). Several attempts have been conducted to identify genetic (64–66) and/or epigenetic signatures distinguishing pT_reg and tT_reg. The T_reg-specific demethylated region (TSDR) is involved in the stable commitment of the T_reg lineage, and controversy still remains on whether iT_reg or pT_reg can efficiently demethylate this region and become fully committed T_reg (66–69). Despite this growing amount of information, distinguishing the relative contribution of pT_reg and tT_reg to immune suppression in physiological and pathological conditions remains hard. However, some pieces of evidence have accumulated in the last years that speak in favor of tT_reg or pT_reg prevalence or concomitance in tumors.

Evidence for tT_reg Accumulation in Cancer

One of the first attempts to distinguish between pT_reg conversion and tT_reg expansion in cancer was pursued by Bui and colleagues who adoptively transferred CD4⁺CD25⁺ cells, mixed at 1:10 ratio with CD25-depleted Thy1.1-congenic splenocytes, into immunodeficient mice bearing a progressive sarcoma (70). The analysis performed 10 days after tumor injection showed that the vast majority (around 80%) of tumor-infiltrating CD4⁺CD25⁺ cells derived from expansion/recruitment of the transferred T_reg, rather than from conversion of non-T_reg. This and other reports, appeared in the “pre-Foxp3” era, were biased by the idea that CD25 was the most stringent T_reg marker and that CD25-depleted cells represented a suitable precursor population to efficiently detect de novo generation of pT_reg. However, subsequent studies have demonstrated that the CD25⁺ subset of Foxp3⁻ T_conv is enriched in pT_reg precursors (69, 71), thus the extent of pT_reg differentiation from CD25-depleted cells represents probably an underestimation of the actual contribution of pT_reg induction in the tumor context. Other authors have shown that tT_reg may dominate pT_reg not only quantitatively but also qualitatively, in terms of suppressive function: indeed, IDO⁺ plasmacytoid dendritic cells, derived from mouse tumor-draining lymph nodes, were capable to induce Foxp3⁺ pT_reg at very high levels but were unable to activate the suppressive function of these cells to an extent comparable to tT_reg (72). Many studies have clearly shown T_reg proliferation (in terms of de novo DNA synthesis and/or cell division) in tumor-bearing mice or cancer patients, thus indirectly supporting the idea that expansion of pre-existing tT_reg might prevail over pT_reg differentiation in building the tumor-associated T_reg pool. For instance, T_reg have been shown to incorporate high levels of BrdU in tumor-draining lymph nodes and at cancer sites in several experimental models (73, 74). A study conducted in patients with multiple myeloma showed that the TREC content in naive cells was significantly lower in T_reg (identified as CD4⁺CD25^high cells) than CD4⁺CD25⁻ or CD25^low cells, suggesting that the T_reg pool mainly derived from peripheral expansion rather than recent thymic emigration (75). However, the observation of high T_reg proliferation at tumor sites cannot be considered as an unequivocal proof of tT_reg prevalence over pT_reg, since both subsets could be endowed with the same proliferative potential in vivo. Indeed, several pieces of evidence indicate that conversion and proliferation may represent uncoupled and independent events (see pTreg Development and tTreg Expansion as Independent Processes).

Evidence for pT_reg Induction in Cancer

Some studies support the idea that pT_reg conversion actually occurs in tumor-bearing hosts at higher efficiency than in physiological conditions, even if controversy still exists on whether pT_reg may prevail numerically over tT_reg at the tumor site. We have in the past demonstrated that thymectomized and CD25-depleted mice, subsequently transplanted with carcinoma cells, showed a significantly higher T_reg recovery in tumor-draining than in contralateral lymph nodes, suggesting that in tumor-bearing mice the T_reg pool might be replenished mostly by newly derived pT_reg than by proliferation of residual T_reg. To prove this possibility, CD25-depleted CD4-purified T cells were transferred into immunocompetent, Thy1.1-congenic, CT26 tumor-bearing mice. In this setting, we could show that the transferred cells acquired CD25 and Foxp3 at significantly higher levels in draining lymph nodes, compared to contralateral lymph nodes of tumor-bearing mice, or to the lymph nodes of tumor-free mice (76). This result clearly showed that tumor progression actively promoted the conversion of non-regulatory precursors into pT_reg. Some tumor-derived molecular signals were found to be involved in tumor-associated conversion. For instance, in different mouse models, tumor cells have been shown to induce in vitro T_reg conversion through TGF-β, and TGF-β neutralization abrogated T_reg accumulation at the tumor site (77). Human leukemic cells converted in vitro non-T_reg into T_reg through the tumor cell-restricted IDO activity, and IDO blockade prevented pT_reg induction in vivo in a leukemia mouse model (78). A confirmation of extensive pT_reg infiltration in murine tumors has been recently obtained thanks to the recent discovery of Nrp-1 as a tT_reg-restricted marker (61, 62). The analysis of Nrp-1 expression has indeed revealed that Nrp-1-negative bona fide pT_reg cells were significantly enriched at tumor site compared to spleen, ranging around 40–90% of total tumor-infiltrating T_reg depending on the tumor type (61). These Nrp-1-negative cells also presented a gene signature (Helios^low, SWAP-70^low, and Dapl1^high) compatible with the pT_reg identity (61). Unfortunately, human T_reg do not express Nrp-1 (63), thus this marker cannot be used to estimate the relative contribution of tT_reg or pT_reg in human cancers.

Developmental and Functional Relations between pT_reg and tT_reg in Cancer

pT_reg Development and tT_reg Expansion as Independent Processes

Many attempts have been made to understand whether tT_reg accumulation and pT_reg development are mutually exclusive or rather cooperative in establishing immune suppression. The evidence that tT_reg may “educate” T_conv to convert into T_reg through the secretion of cytokines, such as TGF-β and IL-10 (79), may support the latter possibility. This event would generate a cascade of suppressive function transmitted from T_reg to bystander cells, establishing a loop of immunosuppression, reminiscent of a phenomenon called as “infectious tolerance” (80). Zhou and coworkers have addressed this issue in the tumor setting, and have demonstrated that tumor-antigen-specific pT_reg could indeed arise from T_reg-depleted cells (adoptively transferred in mice carrying the cognate antigen-expressing tumor), but that the extent of pT_reg induction was not affected by the concomitant presence of tT_reg, either exogenous (adoptively co-transferred) or endogenous (pre-existing in the host) (81, 82). This result indicated that tT_reg and pT_reg accumulate in tumors in a reciprocally independent fashion and that “infectious tolerance” may play minor roles in shaping the tumor-associated T_reg pool.

A comprehensive scenario of T_reg accumulation in tumors should take into account, beside de novo conversion, the active proliferation of not only tT_reg but also pT_reg. Proliferation plays opposite roles in the differentiation of T_conv into pT_reg versus the expansion of already differentiated pT_reg. Regarding the former aspect, we have demonstrated that T_conv proliferation was not required for their conversion into pT_reg, since CD25⁺Foxp3⁺ cells could develop in tumor-bearing mice from CD25-depleted cells treated with an anti-proliferative agent (76). A seminal study by Kretschmer and colleagues showed that T_conv proliferation was not only dispensable but also detrimental to conversion: indeed, low levels of T cell proliferation, in conditions of suboptimal antigen presentation, lack of co-stimulation, and IL-2 paucity, favored TGF-β-mediated pT_reg induction, thus suggesting that an inverse relationship might exist between T_conv proliferation and conversion into pT_reg (83). However, once developed, pT_reg promptly proliferated in response to experimental antigens (83) and, more importantly, in response to tumor antigens (81, 82). Experiments performed in CNS1-mutated mice, which are genetically unable to generate pT_reg, have shown that pT_reg and tT_reg may occupy distinct “niches”: indeed, the efficiency of pT_reg differentiation from T_conv was higher when those T_conv were co-transferred, in lymphopenic recipients, with a CNS1-deficient (non-containing pT_reg) compared to a CNS1-sufficient (containing pT_reg) counterpart, suggesting that not only the tT_reg pool, but also the pT_reg niche, may be controlled by autonomous homeostatic mechanisms (84).

Division of Labor between tT_reg and pT_reg in Cancer

Both tT_reg and pT_reg have been generally recognized as immune suppressive cells in a variety of in vivo and in vitro experimental settings (12). However, whether the two subsets are endowed with peculiar activities remains unclear and is a matter of intense investigation.

Gene expression profiling revealed that the pT_reg and tT_reg signatures were mostly overlapping but also presented some differentially expressed genes, encoding for proteins involved in T_reg suppressive function, suggesting that pT_reg may preferentially exploit different molecules and related mechanisms to exert suppression (64–66). The Nrp-1 itself is not only a marker discriminating murine tT_reg from pT_reg, but also plays a role in T_reg suppression: since this molecule prolongs T_reg interactions with immature dendritic cells, tT_reg may benefit from this pathway in preferentially modulating dendritic cell and cognate T cell activation (22). Many data suggest that pT_reg may be specialized suppressors of immune responses at interfaces with external environments, such as airways, gut, and maternal-fetal interface (64, 84–87). Of note, a peculiar T_reg suppressive molecule, IL-10, plays crucial roles at environmental interfaces, therefore pT_reg may perform their specialized activity through IL-10 secretion (34, 88). IL-10 is critically involved in the establishment of tumor-associated immune suppression, and we have clearly demonstrated IL-10 production by around 40% of tumor-infiltrating T_reg in murine transplanted tumors (36). It would be interesting to understand whether the fraction of IL-10-producing T_reg is enriched in pT_reg, rather than tT_reg, in tumors. One study has directly addressed the issue of induced T_reg functional specialization in tumors, by generating in vitro tumor-specific iT_reg and co-culturing these cells, or tT_reg as control, with NK cells: these authors found that iT_reg and tT_reg equally suppressed IL-2-induced NK activation, but only iT_reg were endowed with the surprising ability not to suppress, but to enhance, NK cytotoxicity induced by tumor target cell contact (89). This observation may speak in favor of differential roles played by tT_reg and pT_reg in cancer, with the former more involved in preventing target cell-independent, and possibly self-directed, unwanted immune responses, and the latter concurrently enhancing tumor-specific immunity.

This division of labor may result in the progressive shaping of the immune response toward an effective anti-tumor immunity with minimal side effects. Such dichotomy is also reminiscent of the double role played by T_reg in different cancers, according to the hypothesis that high T_reg frequency is associated to poor or good prognosis in non-inflammatory or inflammatory cancer onset, respectively (13, 47). In the former case, i.e., non-inflammatory cancers in which protective type-1 responses are suppressed by high T_reg infiltration, T_reg may mainly recognize tumor-associated self-antigens, and mostly include tT_reg; conversely, in the case of inflammatory cancers, related to chronic low-dose type-17 cytokines, which are typical of mucosal tissues, high numbers of pT_reg may suppress pro-tumoral inflammation through IL-10, relevantly produced by pT_reg at those sites. We are tempting to speculate that tT_reg may dominate in suppressing anti-tumor type-1 responses, while pT_reg may prevail in shaping pro-tumor type-2 and type-17 inflammatory responses. Notably, the prototypical example of an inflammation-related tumor in which T_reg accumulation associates to good prognosis is CRC (50), developing in the gastrointestinal mucosa, in which immune tolerance is under the control of pT_reg (84).

Antigen Specificity of tT_reg Versus pT_reg in Cancer

Antigen recognition may play a crucial role in dictating whether tT_reg or pT_reg will prevail in the tumor context. Controversy still exists on the antigen specificity of these two populations. On the one side, tT_reg are generally believed to recognize self-antigens encountered during thymic selection (90). On the other side, pT_reg, deriving from T_conv, are thought to display the same TCR repertoire of T_conv and thus to mainly recognize foreign antigens. Indeed, only a small overlap exists between TCR repertoires of pT_reg and tT_reg (66), and pT_reg are believed to recognize non-self-antigens such as commensal microbiota, allergens, and fetal alloantigens (84, 87).

Tumor cells can express a variety of antigens that can be broadly classified into: (i) self-antigens physiologically expressed as in the tissue of origin, (ii) self-antigens aberrantly expressed, in terms of expression level, developmental stage, or histotype (called tumor-associated antigens or TAAs), and (iii) neo-antigens uniquely expressed by tumor cells, mostly derived from oncogenic mutations (named tumor-specific antigens or TSAs). Based on the above considerations, self-antigens and TAA should be recognized by tT_reg, while TSA would induce and activate pT_reg. However, a complex picture arises from studies analyzing the TCR specificity of tumor-associated T_reg.

T_reg can recognize TAA and TSA in tumors

In different tumor models, TCR-transgenic T_reg have been shown to promptly proliferate in response to the cognate antigen specifically expressed by tumor cells, suggesting that T_reg can undergo tumor-antigen-driven activation and expansion (74, 81, 82, 91). Antigen presentation in the tumor context may favor T_reg expansion: in a mouse model of spontaneous prostate cancer, an efficient T_reg induction/expansion occurred only when TCR-transgenic, antigen-specific CD4 T cells encountered the cognate antigen expressed in the context of prostate cancer cells, rather than non-transformed cells or viral vector-infected cells (91). In this model, TAA-specific T_reg were recognized as pT_reg induced in vivo in a TGF-β-independent fashion.

This evidence of TAA-responding T_reg has been confirmed in human tumors. CD4 clones derived from cancer patients resulted to be regulatory cells and to recognize peptides derived from TAAs, such as LAGE1 (92), ARTC1 (93), TRAG-3, NY-ESO-1 (94–96), Melan-A (97), survivin, TRP1, and gp100 (94) in melanoma patients, and WT-1 in leukemia patients (98). By using MHCII/peptide tetramer technology, other authors failed to detect T_reg specific for NY-ESO-1 in the peripheral blood of ovarian cancer patients (99). Bonertz et al. developed an in vitro system to screen the suppressive function of T_reg in response to single peptides and, with this approach, could detect T_reg specific for several TAA in the peripheral blood of colon carcinoma patients but not in healthy donors; notably, T_reg depletion in vitro unveiled TAA-specific T_conv responses (100).

The possibility that tumor-associated T_reg may recognize not only TAA but also TSA is demonstrated by the observation that T_reg specific for exogenous viral antigens, acting as TSA, may arise in virus-related cancers. T_reg clones specific for human papilloma virus (HPV), and suppressing the cognate antigen-directed T cell response, have been obtained from tumor-draining lymph nodes and tumor biopsies of cervical cancer patients (101). T_reg clones specific for antigens of the Epstein–Barr virus (EBV), associated to several hematological and solid malignancies, can be generated from the peripheral blood of healthy donors (102).

T_reg can recognize self-antigens in tumors

Several data in mouse models confirm that T_reg responding to self-antigens can play a role in suppressing the anti-tumor responses. Immunization with serologically defined auto-antigens was found to enhance tumor growth in different mouse models, and this event was dependent on the expansion of T_reg responding to those self-antigens (103). This study confirmed that self-antigens-specific T_reg could suppress anti-tumor immunity, but did not explore the T_reg response to self-antigens expressed by tumor cells themselves during tumor progression. This issue has been instead addressed in an experimental model in which a foreign antigen, artificially expressed in transgenic mice under tissue-specific promoter, was seen (peripherally and/or thymically) by the immune system as a self-antigen and elicited the generation of a pool of memory T_reg specific for that antigen (74). If those mice were injected with the cognate antigen-bearing tumor, the memory T_reg pool specific for that self-antigen was hugely expanded in tumors and tumor-draining lymph nodes, confirming that self-specific T_reg can respond to self-antigens expressed by tumor cells (74). A seminal paper has recently reported the immunoscope analysis of T_reg infiltrating spontaneous prostate tumors in a mouse transgenic model, and described the clonal enrichment of a single T_reg specificity that was directed not to a unique TSA but to a self-antigen expressed also by normal prostate cells (104). The development of T_reg specific for peripheral tissue-restricted self-antigens occurred in the thymus under the control of the Aire molecule, which allows the expression of peripheral antigens in thymic epithelial cells (104). These findings clearly demonstrate that T_reg can recognize self-antigens in cancer and suggest that maintaining self-antigen expression may help transformed cells to overcome the immune surveillance through self-specific T_reg expansion.

Repertoire analysis as an estimation of pT_reg/tT_reg balance

The direct comparison between the repertoires of tumor-associated T_reg and T_conv may help understanding the processes underlying T_reg enrichment in cancer. Some authors have reported that the analysis of TCR diversity (performed with the immunoscope technology) showed that T_reg infiltrating murine transplanted tumors displayed a biased TCR repertoire toward “public” CDR3 sequences (i.e., shared by different mice), suggesting T_reg intra-tumor clonal expansion driven by the recognition of dominant antigens (105). Also tumor-infiltrating activated T_conv showed a biased TCR repertoire, but it was distinct from the T_reg spectrum, and the minimal overlap between the two subsets was mainly confined to “private” specificities (105). By using a similar approach, others have reported distinct and not overlapping TCR repertoires of T_reg and T_conv infiltrating prostate tumors in a genetically engineered mouse model of spontaneous prostate carcinogenesis (104). Lack of overlap between T_conv and T_reg repertoires was also found in tumors and tumor-draining lymph nodes in a mouse model of chemical carcinogenesis (106). Overall, the lack of overlap between T_reg and T_conv has been interpreted in many cases as the result of negligible pT_reg conversion at the tumor site; however, pT_reg and tT_reg may share more specificities than expected, since tT_reg-associated antigens may preferentially drive T_conv fate decision toward the conversion into pT_reg rather than toward the conventional activation; moreover, already established pT_reg may then undergo intra-tumor clonal expansion together with tT_reg in response to the same antigens. Therefore, the overall overlap between T_conv and T_reg specificities may not accurately estimate the extent of pT_reg induction in tumors. Indeed, in one study performed in advanced melanoma patients, TAA-specific TCRs, expressed by tumor T_reg clones, could be detected in both T_reg and T_conv populations, demonstrating that TAA-specific T_reg may be comprised of pT_reg derived from the conversion of T_conv (95).

Indirect data support the notion that TAA-specific T_reg may contain pT_reg. TAA-specific T_reg clones, obtained from patients with advanced melanoma, suppressed in vitro the cognate antigen-specific T cell response, but produced high levels of Th1 and/or Th2 cytokines (95), and showed low FOXP3 expression and TSDR demethylation, indicating that these cells may represent an incompletely uncommitted T_reg population, which more likely belongs to the pT_reg than to the tT_reg pool (95).

A recent study has directly evaluated the consequences of pT_reg and tT_reg antigen specificities in tumor-bearing hosts. Indeed, Schreiber et al. have shown that, if purified polyclonal tT_reg and T_conv, differentially labeled with green or red fluorescence, were co-transferred in CD4-null mice, the tT_reg progeny exceeded the newly T_conv-derived pT_reg population in tumor-draining lymph nodes as well as in the spleen; conversely, when transgenic, tumor-antigen-specific, tT_reg and T_conv were injected, tT_reg and pT_reg reached comparable frequencies in tumor-draining lymph nodes (107). These results suggest that tT_reg and pT_reg are mostly specific for self- or tumor-antigens respectively, and that the balance between pT_reg and tT_reg may be fine-tuned by the relative prevalence of TSAs versus self-antigens expressed by tumor cells.

Heterogeneity and Plasticity of tT_reg and pT_reg

T_reg Heterogeneity in Cancer: Relations with the pT_reg/tT_reg Dichotomy

During the latest years, it has become increasingly clear that T_reg, meant as Foxp3-positive cells, are not a homogeneous lineage, but rather represent a mixture of subpopulations. Indeed, beside the tT_reg/pT_reg distinction based on their developmental origin, diverse T_reg subsets can be identified endowed with peculiar features in terms of suppression, proliferation, and stability, even though not properly classifiable as developmentally distinct lineages (Figure 2). Tumor microenvironmental signals may differentially affect these subsets, thus shaping T_reg heterogeneity to the advantage of tumor progression.

FIGURE 2

Figure 2. Functional dynamics of tT_reg and pT_reg in cancer. This picture summarizes development, heterogeneity, plasticity, antigen specificity, and function of pT_reg and tT_reg in cancer. Activated T_reg, which are epigenetically committed and mostly self- and TAA-specific, can transiently lose Foxp3 without methylating TSDR thus becoming latent T_reg; in some conditions, they can acquire T-bet expression thus becoming specialized suppressors, detrimental to the anti-tumor type-1 response. Activated T_conv, mostly foreign (TSA) antigen-specific, can promiscuously express Foxp3 without demethylating TSDR. However, a fraction (CD25⁺, or CD39⁺) of activated T_conv can convert into pT_reg, progressively moving from an uncommitted to a committed stage. Through IL-10, committed pT_reg can suppress pro-tumoral inflammatory and type-17 responses, thus exerting beneficial roles for the host in some cancer types. In some contexts, uncommitted pT_reg (and possibly activated T_conv) can move back to exT_reg stage, acquiring the ability to produce inflammatory cytokines. Therefore, in some tumors such as colon cancer, Th17-like T_reg may foster type-17 inflammation thus supporting tumor growth; in other tumor contexts, Th1-like T_reg can favor type-1 responses that rather block tumor growth. Green, cells specific for self-antigens and TAA; light blue, cells specific for foreign antigens including TSA. Yellow dash, demethylated TSDR; blue dash, methylated TSDR. Red “F” in yellow circles, stable Foxp3; yellow “F” in empty circles, unstable Foxp3. Dashed arrows, unclear events. Orange rounded arrows, proliferation in the tT_reg or the pT_reg homeostatic niche. Light green frames, conditions in which T_reg are beneficial to the host; light orange frames, conditions in which T_reg are detrimental to the host.

T_reg stability and epigenetic commitment in cancer

Foxp3 inherent stability, rather than Foxp3 expression in a given moment and tissue, is intimately linked to an actual commitment to the T_reg lineage and therefore to the maintenance of immune suppression. Pioneer studies have demonstrated that Foxp3 stability is strictly related to an epigenetic imprinting of CpG demethylation in the TSDR region of the Foxp3 locus (67, 86, 108). TSDR demethylation was then recognized as the mechanism featuring the distinction between committed (demethylated) and uncommitted (methylated) T_reg, irrespective of Foxp3 expression (9). Controversy exists on whether pT_reg show complete or partial TSDR demethylation and can then be considered as committed T_reg. Many studies show that iT_reg have a partially or completely methylated TSDR (9, 67–69), while pT_reg have been described as TSDR-demethylated (68), TSDR-methylated (66), or as a mixed population of stable and unstable cells, characterized by CD25 high or low expression respectively (69).

Few data exist on the extent of TSDR demethylation in tumor-associated T_reg. The frequency of TSDR-demethylated cells is higher in peripheral and intratumoral leukocytes of lung, colon, prostate, or breast cancer patients, in relation to a higher T_reg frequency as determined by flow cytometry or immunohistochemistry (109). Of note, the extent of TSDR demethylation in CRC patients was only slightly higher than in healthy volunteers, in contrast to the significantly increased T_reg frequency in these samples shown by previous studies (110, 111). This discrepancy may be explained with the peculiar nature of this inflammation-related and mucosal tissue-located cancer, in which the inflammatory response may specifically involve pT_reg, possibly containing more uncommitted (TSDR-methylated) cells than tT_reg.

The evaluation of TSDR demethylation has been used as a reliable analytical tool for the estimation of committed T_reg in some tumor conditions and therapies. An increased frequency of epigenetically committed (TSDR-demethylated) T_reg has been determined in tumor-infiltrating cells of ovarian, colorectal, and bronchial cancers compared to non-tumoral tissue counterparts (112). TSDR demethylation was decreased in the peripheral blood of metastatic renal carcinoma patients receiving tumor vaccination (113), and increased in patients treated with dendritic cell vaccination and cytokine therapy (114).

T_reg functional dynamics in cancer

The idea of Foxp3 as the master transcription factor of T_reg lineage has been challenged by the observation that some T_reg features are Foxp3-independent, and that Foxp3 plays T_reg-unrelated functions (115). This is especially true for human FOXP3-positive cells, since human activated T_conv can transiently express this transcription factor that acts as an intrinsic T cell regulator (116). The concomitant analysis of CD45RA and FOXP3 in human T_reg in both physiological and pathological contexts allowed delineating a classification into three subsets: CD45RA⁺FOXP3^low resting T_reg (rT_reg), CD45RA⁻FOXP3^low non-T_reg, and CD45RA⁻FOXP3^high (CD45RO⁺) activated T_reg (aT_reg), endowed with different potentials of proliferation, suppression, stability, and plasticity (117). Whether each subset mainly contains tT_reg or pT_reg is unclear. While rT_reg were recognized as CD31⁺ recent thymic emigrants, thus belonging to the tT_reg pool, aT_reg can be considered as a mixed population of activated tT_reg (derived from rT_reg) and pT_reg (derived from non-T_reg or T_conv). The CD45RA⁻FOXP3^low non-T_reg subset may represent a mixture of activated T_conv (promiscuously and unstably expressing FOXP3), latent T_reg (transiently downregulating FOXP3), and recently converted pT_reg (117).

The three human T_reg subsets can be differentially expanded in distinct pathologies. In conditions characterized by exacerbated immune responses, such as autoimmune diseases, rT_reg and non-T_reg are expanded; conversely, in diseases associated to immune unresponsiveness, such as sarcoidosis, the aT_reg subset is instead enriched in the peripheral blood (117). The tumor context, which conceivably belongs to the latter category, should be characterized by aT_reg expansion. In line with this hypothesis, CD45RO⁺FOXP3^high aT_reg were found significantly expanded in the peripheral blood, and much more at the tumor site, in patients with malignant melanoma (118). Also the non-T_reg and the rT_reg fractions were increased, but only in the peripheral blood, in cancer patients compared to healthy controls, and both subsets positively correlated with tumor progression (118). The non-T_reg pool produced some IFN-γ and its frequency returned to normal levels after tumor removal, thus probably representing aberrantly activated T_conv, or T_reg with attenuated FOXP3 activity (118). A much deeper knowledge on T_reg dynamics in cancer is needed to better understand the role played by each specialized component in suppressing anti-tumor type-1, or pro-tumor inflammatory, responses.

T_reg subsets specified by functional/developmental markers

Several surface or intracellular markers have been suggested to identify T_reg subsets endowed with peculiar abilities other than suppressive functions. A portion of human T_reg with an effector/memory-like phenotype (26, 119, 120) expresses CD39, which has been proposed as a target to enrich human suppressive T_reg (119). CD39 was found overrepresented in peripheral and tumor-infiltrating T_reg from HNSCC, and was further increased in patients with advanced-stage disease or after radiochemotherapy (120, 121). CD39 is also expressed by a T_conv subset, which produces lower levels of pro-inflammatory cytokines than the bulk T_conv population, and is more prone to convert, at least in vitro, into T_reg (120). Both CD39⁺ T_reg and CD39⁺ T_conv were enriched in peripheral blood, and further increased at tumor site, in HNSCC patients, and a positive correlation existed between frequencies of these two populations (120). Therefore, these data suggest that tumor-associated CD39⁺ T_conv may represent a reservoir of CD39⁺ T_reg precursors. As a consequence, it could be suggested that CD39⁺ T_reg may include both tT_reg and pT_reg, and that both T_reg subsets can exploit the CD39-mediated suppressive mechanisms of ATP degradation and adenosine generation.

Not only the functional arms of suppression, but also the activation requirements may differ in tT_reg and pT_reg: for instance, TNFR2 expression is needed to activate tT_reg but not iT_reg suppressive ability in experimental colitis (122). Of note, TNFR2-positive T_reg have been found enriched in murine tumors, in association with a higher suppressive ability, ex vivo, in the standard suppression assay (123). In a mouse model of metastatic melanoma, TNF-α caused enhanced tumor progression through the TNFR2-mediated T_reg expansion at the site of metastasis (124). These data suggest that TNFR2 expression may label tumor-infiltrating T_reg of thymic origin, and that TNF-α at the tumors site may preferentially expand and activate tT_reg. Supporting the idea that tT_reg may represent more stable cells, TNFR2 was found to be involved in the maintenance of Foxp3 stability in mouse models of inflammation (125). Also in human peripheral blood, CD25 and TNFR2 co-expression identifies cells highly expressing FOXP3, showing an effector/memory phenotype and strong suppression, ex vivo (126). The TNF-α/TNFR2 pathway may amplify T_reg activation also through the induction of a NF-kB-driven transcriptional program enriched for other members of TNF superfamily, such as 4-1BB, FAS, and OX40 (127).

The early idea that Helios could differentiate tT_reg from pT_reg (59, 128) prompted the use of this marker in delineating tT_reg accumulation in cancer. The vast majority of tumor-infiltrating T_reg were found to express Helios in a mouse model of glioblastoma (129), in glioblastoma multiforme patients (129), and renal cell carcinoma patients (130). However, the value of Helios as univocal marker of tT_reg has been questioned by other studies that showed Helios also expressed in pT_reg (60, 131), and in association to T_reg suppression (128, 131) and commitment. Indeed, Helios⁻FOXP3⁺ cells freshly isolated from healthy donors or autoimmune disease patients showed decreased TSDR demethylation compared to Helios⁺FOXP3⁺ (132, 133), and also displayed a higher plasticity in terms of cytokine production (133). In a murine transplanted tumor model, tumor-infiltrating T_reg were enriched in Helios⁺ cells, representing the subset with the highest proliferative potential (as shown by Ki67 staining) (131). In summary, the well-recognized enrichment of Helios⁺ T_reg in several human and mouse tumors may be attributed, rather than to preferential attraction and expansion of tT_reg, to the tumor-driven local activation and/or commitment of both tT_reg and pT_reg.

Specialization and Plasticity of tT_reg/pT_reg in Cancer

It is now well established that T_reg (or better, their specific subsets) adapt their molecular programs to optimize their in vivo suppressive function in distinct inflammatory milieus, which may be alternatively dominated by Th1, Th2, Th17, or T_FH responses. Strikingly, these T_reg specialized programs are orchestrated by the same transcription factors that drive the polarization of the targeted T-helper subset: therefore, T-bet, IRF4, Stat3, and Bcl6 expression are respectively and selectively required for the T_reg specialized suppression of Th1 (134, 135), Th2 (136), Th17 (137), and T_FH (138, 139) responses. Indeed, by acquiring master T-helper genes, T_reg may gain the expression of chemokine receptors driving the recruitment of specialized T_reg into inflamed tissues. However, in some contexts, T_reg (or, again, some T_reg subsets) can express not only T-helper-related transcription factors and migratory molecules, but also cytokines such as IFN-γ or IL-17, thus turning from specialized suppressors into so-called Th1-like or Th17-like T_reg that may rather contribute to inflammation (140). Some data, mostly from mouse experimental models, suggest that such T_reg plasticity is not a lineage reprograming of committed T_reg, which appear instead quite stable; rather, Th1-like or Th17-like T_reg may derive from uncommitted cells expanded in inflammatory conditions (69, 141). However, other studies have shown that in both mouse and human pathologies T_reg can produce relevant amounts of type-1 and type-17 cytokines even though preserving Foxp3 expression (142–146).

Th17-like T_reg in cancer

Regulatory T cells may shift to a Th17-like phenotype in inflamed microenvironments dominated by type-17 cytokines, thus favoring, rather than suppressing, pro-tumoral mechanisms of chronic inflammation. According to this idea, human T_reg have been found to spontaneously secrete IL-17 in the intestine of patients carrying inflammatory bowel disease (145, 147) and colon carcinoma (147). In epithelial ovarian cancer, a malignancy associated to chronic inflammation, T_conv were found to secrete high levels of IL-17 (and other cytokines) when cultured ex vivo with IL-2; under similar conditions, tumor-infiltrating T_reg were prone to FOXP3 downregulation, attenuation of suppressive function, and prompt IL-17 production (148). In human lung adenocarcinoma, FOXP3 message amounts correlated with Th17-related transcripts enriched at the tumor site, where IL-17 antagonized the development of the anti-tumor, T-bet-dependent, Th1 response (149). Myeloid antigen-presenting cells and cytokines such as IL-2, TGF-β, IL-1, IL-23, and IL-6 may initiate T_reg polarization into Th17-like cells in these tumor contexts (147–149). In a mouse model of hereditary colon polyposis, as well as in human colon cancer, the Th17-like T_reg co-expressed the Th17-related transcription factor ROR-γt, and fostered tumor progression, also through the promotion of mast cell local expansion (150, 151). This study clearly demonstrated that microenvironmental signals could direct T_reg plasticity toward pro-inflammatory and pro-tumoral activities.

One group has demonstrated that Th17-like T_reg can also arise in experimental tumors as an outcome of vaccination strategies (152). In this study, vaccination with antigen plus TLR-9 ligand induced T_reg reprograming into polyfunctional T-helper-like cells, producing a wide array of cytokines including IL-2, TNF-α, and IL-17, and expressing cell-surface CD40L, thus providing efficient T cell help for tumor-antigen cross-presentation and development of anti-tumor response (152). The IDO immunosuppressive enzymatic activity was responsible for preventing this anti-tumor T_reg polarization, which was instead enhanced using an IDO blocker (152).

Little data exist on the precursors of Th17-like T_reg in cancer. In the peripheral blood of healthy subjects, the CD45RA⁻FOXP3^low non-T_reg subset was found enriched in Th17-related transcripts and in cells actively secreting IL-17, even at higher levels than naïve or memory T_conv, a data suggesting that this population contains Th17 or Th17-like precursors (117). It would be interesting to understand whether the Th17 potential resides, within the non-T_reg gate, in activated T_conv, in latent T_reg, and/or in recently induced pT_reg, possibly co-expressing FOXP3 and RORγt and thus pre-committed to the Th17 lineage.

Th1-like T_reg in cancer

Pioneer studies from Koch and colleagues demonstrated that, following exposure to IFN-γ in Th1-dominated microenvironments, a subset of T_reg can up-regulate the Th1-related transcription factor T-bet, which drives T_reg expansion, migration (CXCR3-mediated), and function specifically during type-1 inflammation (134). Further experiments have shed light on the developmental requirements and the alternative fates of murine T-bet⁺ T_reg: following IFN-γ stimulation, T_reg could gain T-bet expression but failed to fully polarize into IFN-γ-producing Th1-like T_reg, due to an impaired T_reg susceptibility to IL-12. Indeed, IL-12 receptor β2, which is inducible in T_conv in an IFN-γ- and T-bet-dependent fashion, is epigenetically inaccessible in T_reg (135). Only long-lasting IFN-γ preconditioning could unlock IL-12 responsiveness, thus allowing the complete T_reg polarization into Th1-like cells (135). Presumably, in contexts characterized by chronic IFN-γ and IL-12 abundance, such as autoimmune, inflammatory, and viral diseases, T_reg will be oriented to a full reprograming into Th1-like cells. Supporting this idea, IFN-γ-producing T_reg have been reported in mouse models of graft-versus-host disease (153), viral (154) or parasite (142) infection, in human multiple sclerosis (144), and diabetes (146, 155). In one of these systems, IFN-γ-producing T_reg were recognized to be specific for a foreign (viral) antigen (154). Whether such Th1-like T_reg can be yet considered as classical regulatory cells is still debated. One study has shown that in vitro polarized Th1-like T_reg were less suppressive than conventional T_reg in the standard in vitro suppression assay, and that suppression could be partially rescued with concomitant IL-10 and IFN-γ neutralization (144). Another study has proven that IFN-γ-producing human iT_reg were equally functional as natural T_reg in suppressing both proliferation and cytokine production of responder T cells (156). In a mouse model of graft-versus-host disease, IFN-γ produced by stable (TSDR-demethylated) T_reg was shown to be even required for T_reg protective effect (153), suggesting that IFN-γ-releasing T_reg can display in vivo unexpected functions depending on the context.

Conversely, it could be envisaged that, in the tumor context, the low levels of IFN-γ derived from T_conv, NK, and CD8 cells, and the paucity of IL-12 production by tumor-associated APCs, may concur to induce a pool of T_reg expressing T-bet but not secreting IFN-γ, thus specialized in suppressing anti-tumor type-1 immunity. In line with this possibility, TAA-specific T_conv, but not TAA-specific T_reg, produced IFN-γ in patients with epithelial ovarian cancer (99). In both healthy subjects (117) and malignant melanoma patients (118), IFN-γ-producing cells were enriched within the circulating CD45RA⁻FOXP3^low (CD45RO⁺) non-T_reg subset, mostly including activated T_conv and/or uncommitted T_reg. Conversely, in ovarian cancer, tumor-infiltrating T_reg were enriched in CXCR3⁺ cells, highly expressing T-bet but poorly producing IFN-γ, and strongly suppressing Th1 response ex vivo (157). Tumor-associated CXCR3⁺ T_reg were mostly Helios-positive, and T-bet⁺ T_reg could be generated in vitro by culturing CD45RA⁺CCR7⁺ rT_reg (mostly containing tT_reg) under Th1-polarizing conditions (157), suggesting their derivation from committed tT_reg. This finding was in accordance to Koch et al. who showed that T-bet⁺ T_reg derived from T-bet⁻ T_reg, rather than from activated T_conv (134). These data support the idea that tT_reg, rather than pT_reg, may contain the precursors for Th1-specialized suppressors, thus playing critical roles in suppressing protective responses in tumors whose high T_reg frequency correlates with poor prognosis (13).

Some therapeutic interventions can force tumor-associated T_reg toward a fully differentiated Th1-like phenotype. For instance, circulating T_reg from melanoma patients showed significantly higher IFN-γ secretion following a protocol of tumor peptide vaccination plus IL-2 and cyclophosphamide, in line with enhanced serum IL-12 (158). On the whole, these data suggest that, especially in the human system, the transition from T-bet⁺ T_reg, specialized Th1 suppressors, into T-bet⁺ IFN-γ⁺ T_reg, Th1-like plastic cells, may not only depend on the availability and the responsiveness to exogenous stimuli, but may differentially occur in distinct T_reg precursors: on the one side, tT_reg, enriched in committed and self-specific cells, may be forced to arrest to the specialization (T-bet⁺) endpoint; on the other side, pT_reg, containing less committed and foreign antigens-specific cells, may be more prone to the complete reprograming into pro-inflammatory (T-bet⁺ IFN-γ⁺) cells. Future studies will elucidate the mechanisms by which different growing tumors may favor the expansion of pro-tumoral specialized Th1 suppressors or the induction of Th1-like plastic T_reg.

Implications for Cancer Immunotherapy

The initial enthusiasm on the use of therapeutic cancer vaccines has been soon disappointed by the observation of a low response rate in many trials (159). After the discovery of T_reg as potent immune suppressive cells hampering the establishment of anti-tumor immunity, it soon became clear that anti-tumor vaccination might fail to elicit an effective immune response and to achieve successful tumor eradication, because of the immune suppressive barrier created by T_reg. In addition, since T_reg may recognize TAA and TSA at higher frequency than T_conv, tumor-antigen-based vaccines may expand/induce T_reg rather than effector cells, thus inhibiting rather than boosting the anti-tumor response. Indeed, Zhou et al. first demonstrated that TCR-transgenic CD4 T cells specific for a TAA, adoptively transferred into mice bearing TAA-expressing tumor cells, proliferated extensively after administration of a therapeutic tumor vaccine (in the form of a recombinant vaccinia virus encoding the antigen), but tumor-antigen-experienced cells were mostly regulatory cells, ex vivo suppressive, and anergic to subsequent stimulation (81).

In cancer patients receiving tumor-antigen vaccination, the expansion of antigen-specific T_reg has been documented. Circulating NY-ESO-1-specific T_reg spontaneously develop in late-stage melanoma patients and are expanded following immunization with NY-ESO-1 protein supplemented with adjuvants (96). Therapeutic vaccination with an HPV synthetic long peptide vaccine, administered to patients with HPV-positive cervical carcinoma, induced both CD8 and CD4 T cell immunity, but also enhanced the HPV-specific T_reg pool (160). The pool of vaccine-specific T_reg may derive not only from the expansion of pre-existing tumor-antigen-specific clones, but also from de novo generation of vaccine-specific pT_reg. This is suggested by results obtained vaccinating melanoma patients with an HLA-DP4-restricted MAGE-A3 peptide: in this setting, a subset of vaccine-specific T_reg becomes detectable only after vaccination (161). Vaccine-elicited T_reg showed some degree of heterogeneity: out of five CD25⁺ regulatory clones isolated from vaccinated patients, four expressed high FOXP3 mRNA levels, produced TGF-β, and showed demethylated TSDR; one clone expressed less FOXP3, had methylated TSDR and produced some Th2 cytokines (161). These data suggest that antigen-specific T_reg, induced in the periphery following antigen exposure and thus recognizable as pT_reg, can contain both committed and uncommitted cells.

The concomitant and detrimental T_reg expansion in anti-tumor vaccination can be avoided by using CD8 T cell-targeted approaches. A melanoma vaccination protocol based on an MHCI-restricted Melan-A peptide significantly decreased the frequency of Melan-A-specific T_reg, in association with an improved and more diverse Th1 response (97).

Some attempts have also been made to combine active immunotherapy with T_reg depletion or functional blockade. Several studies showed that depletion of CD25⁺ cells in vivo in cancer patients could enhance the tumor-specific T cell responses induced by cancer vaccines (15). However, CD25-directed strategies may fail to achieve sustained results, since activated effector cells may be concomitantly eliminated and pT_reg may replenish the T_reg pool after depletion (15). Interestingly, a recent study has demonstrated that different T_reg-depleting agents, either CD25-targeted (IL-2/diphtheria toxin fusion protein, or anti-CD25 antibody) or not (low-dose cyclophosphamide), failed to consistently eliminate more than 50% of committed T_reg, as identified by TSDR demethylation (162).

Therefore, alternative strategies are needed to counteract the “hard core” of immune suppression that is represented by epigenetically committed T_reg. We have proposed in the past that T_reg functional inactivation, rather than depletion, may represent a successful strategy to prevent massive pT_reg induction and concomitantly block T_reg suppression (15). This idea may be corroborated by the observation that markers associated to T_reg suppressive functions, and therapeutically targetable, may show enriched or restricted expression in epigenetically committed T_reg. For instance, GITR stimulation has been shown to attenuate T_reg suppression and favor the rejection of experimental tumors (163). A recent study has demonstrated that GITR engagement in vivo led to the downregulation of Foxp3 expression in intratumoral T_reg (164). Of note, GITR⁺ T_reg were found enriched in Helios⁺ cells, representing highly committed T_reg (131), thus GITR targeting may preferentially block the strongest suppressors among the T_reg pool. A similar possibility could be envisaged for therapeutic strategies aimed at TNF-α/TNFR2 blockade, since this axis may be mainly involved in the activation of more committed and stable cells (122–126). Committed T_reg may also be targeted by virtue of their high proliferative potential: indeed, high proliferation rates, in terms of Ki67 positivity, were detected in healthy subjects within the aT_reg subset, enriched in stable and committed T_reg (117), and also in murine tumor-infiltrating Helios⁺ T_reg (131). Therefore, treatments based on the depletion of proliferating cells, such as low-dose cyclophosphamide, may efficiently target committed T_reg.

An innovative way to improve immunotherapy would be to reprogram tumor-associated T_reg into fully armed effector cells, which would then become “exT_reg.” Different from other vaccine-based approaches, T_reg reprograming is expected to trigger anti-tumor response very rapidly, since T_reg are already located at the tumor site and already tumor-antigen-experienced, thus not requiring a de novo T cell priming. Therefore, exT_reg may function in an “innate-like” manner, promptly providing co-stimulatory and pro-inflammatory signals when adequately modulated, before a novel adaptive anti-tumor response develops (140). An example of this approach has been proposed by Sharma et al. who demonstrated that reprograming of mature pre-existing tumor-associated T_reg into CD40L-expressing helper effector cells was needed to achieve tumor regression in a model of immunotherapy combining antigen vaccination, TLR-9 stimulation, and IDO blockade (152).

The above-discussed data overall indicate that tT_reg and pT_reg may not be equally susceptible to functional reprograming, but this dichotomy may turn into a benefit for the efficacy and safety of the evoked response. Indeed, on the one hand, tT_reg, predominantly self-specific, highly committed, and hard to be reprogrammed into T-helper-like cells, would be preserved, thus ensuring immune tolerance to self-antigens and maintaining systemic immune homeostasis. On the other hand, pT_reg, mainly representing tumor-specific and uncommitted cells, may be more easily converted into exT_reg, thus mounting an immediate helper and/or effector response in a mostly tumor-antigen-specific fashion.

Reprograming into exT_reg may be achieved by immunotherapies aimed at subverting the immune suppression mechanisms established by innate cells in tumor microenvironments. For instance, in the above-reported model of tumor vaccination, CD40L upregulation by T_reg following TLR-9 stimulation was strictly dependent on host-derived MyD88 and IL-6 signals (152). In melanoma patients, tumor peptide antigen vaccination combined with low-dose cyclophosphamide and low-dose IL-2 evoked Th1-like T_reg accumulation, in line with a less tolerogenic microenvironment and with enhanced IL-12 availability (158). Of note, in this system, depletion of proliferating (conceivably committed and thymus-derived) T_reg by means of cyclophosphamide allowed the functional reshuffling of innate cells that in turn unveiled the emergence of Th1-like exT_reg.

However, it is arguable that microenvironmental rearrangements would better accomplish full T_reg reprograming with the concomitant direct modulation of T_reg activities, aimed especially at enhancing T_reg susceptibility to external signals. For instance, expression of IL-12 receptor, which is epigenetically regulated in T_reg (135), could be artificially boosted by pharmacological approaches. Also, targeting with monoclonal antibodies some receptors expressed on T_reg surface and correlated with T_reg stability (such as TNFR2 and GITR) could result in enhancing T_reg propensity to reprograming. In line with this idea, treatment of murine melanomas with a GITR agonistic antibody resulted in the accumulation of exT_reg at the tumor site (164). Suppressor of cytokine signaling (SOCS) 1 and 2, which maintain Foxp3 stability and prevent T_reg polarization into effector cells (165, 166), may be pharmacologically inhibited to unlock T_reg responsiveness to pro-inflammatory microenvironmental cytokines.

Conclusion

Even though discrimination between pT_reg and tT_reg by simple surface phenotyping is not yet possible many pieces of evidence indicate that both subsets contribute to the T_reg pool conditioning the tumor microenvironment. Nevertheless, the development/expansion of pT_reg and tT_reg are independent processes, possibly resulting from disparate antigens and signals, and their activities seem characterized by very peculiar features in terms of specificity, stability, and specialization. On the one side, tT_reg may expand at tumor site in response to self-antigens expressed by tumor cells, mostly include committed (TSDR-demethylated) Helios- and TNFR2-expressing cells, and contain the precursors of specialized T-bet⁺ Th1-suppressing cells, thus representing not only the guardians of systemic immune homeostasis but also the “hard core” of tumor immune escape. On the other side, pT_reg may mostly develop following local encounter with TAA or TSA antigens, possibly represent a mixed population of committed (TSDR-demethylated) and uncommitted (TSDR-methylated) cells, and are more prone to be reprogramed into Th1-like or Th17-like effector cells. We envisage that future successful immunotherapies may not only target committed T_reg but also favor “recycling” uncommitted T_reg into prompt anti-tumor effectors.

Conflict of Interest Statement

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

Acknowledgments

This work was supported by grants from Associazione Italiana Ricerca sul Cancro (MFAG 8726 to Silvia Piconese), from Ministero dell’Istruzione, dell’Università e della Ricerca (FIRB-Futuro in ricerca RBFR12I3UB_002 to Silvia Piconese), and Italian Ministry of Health. Alessia Burocchi is recipient of a FIRC fellowship.

References

1. Dunn GP, Old LJ, Schreiber RD. The three Es of cancer immunoediting. Annu Rev Immunol (2004) 22:329–60. doi:10.1146/annurev.immunol.22.012703.104803

CrossRef Full Text

2. Sakaguchi S, Sakaguchi N, Asano M, Itoh M, Toda M. Immunologic self-tolerance maintained by activated T cells expressing IL-2 receptor alpha-chains (CD25). Breakdown of a single mechanism of self-tolerance causes various autoimmune diseases. J Immunol (1995) 155:1151–64.

3. 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) 163:5211–8.

Pubmed Abstract | Pubmed Full Text

4. Fontenot JD, Gavin MA, Rudensky AY. Foxp3 programs the development and function of CD4+CD25+ regulatory T cells. Nat Immunol (2003) 4:330–6. doi:10.1038/ni904

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

5. Hori S, Nomura T, Sakaguchi S. Control of regulatory T cell development by the transcription factor Foxp3. Science (2003) 299:1057–61. doi:10.1126/science.1079490

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

6. Bennett CL, Christie J, Ramsdell F, Brunkow ME, Ferguson PJ, Whitesell L, et al. The immune dysregulation, polyendocrinopathy, enteropathy, X-linked syndrome (IPEX) is caused by mutations of FOXP3. Nat Genet (2001) 27:20–1. doi:10.1038/83713

CrossRef Full Text

7. Wildin RS, Ramsdell F, Peake J, Faravelli F, Casanova JL, Buist N, et al. X-linked neonatal diabetes mellitus, enteropathy and endocrinopathy syndrome is the human equivalent of mouse scurfy. Nat Genet (2001) 27:18–20. doi:10.1038/83707

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

8. Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B, Clark LB, Yasayko SA, et al. Disruption of a new forkhead/winged-helix protein, scurfin, results in the fatal lymphoproliferative disorder of the scurfy mouse. Nat Genet (2001) 27:68–73. doi:10.1038/83784

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

9. Ohkura N, Hamaguchi M, Morikawa H, Sugimura K, Tanaka A, Ito Y, et al. T cell receptor stimulation-induced epigenetic changes and Foxp3 expression are independent and complementary events required for Treg cell development. Immunity (2012) 37:785–99. doi:10.1016/j.immuni.2012.09.010

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

10. Abbas AK, Benoist C, Bluestone JA, Campbell DJ, Ghosh S, Hori S, et al. Regulatory T cells: recommendations to simplify the nomenclature. Nat Immunol (2013) 14:307–8. doi:10.1038/ni.2554

CrossRef Full Text

11. Bilate AM, Lafaille JJ. Induced CD4+Foxp3+ regulatory T cells in immune tolerance. Annu Rev Immunol (2012) 30:733–58. doi:10.1146/annurev-immunol-020711-075043

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

12. Curotto de Lafaille MA, Lafaille JJ. Natural and adaptive foxp3+ regulatory T cells: more of the same or a division of labor? Immunity (2009) 30:626–35. doi:10.1016/j.immuni

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

13. deLeeuw RJ, Kost SE, Kakal JA, Nelson BH. The prognostic value of FoxP3+ tumor-infiltrating lymphocytes in cancer: a critical review of the literature. Clin Cancer Res (2012) 18:3022–9. doi:10.1158/1078-0432.CCR-11-3216

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

14. Zou W. Regulatory T cells, tumour immunity and immunotherapy. Nat Rev Immunol (2006) 6:295–307. doi:10.1038/nri1806

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

15. Colombo MP, Piconese S. Regulatory-T-cell inhibition versus depletion: the right choice in cancer immunotherapy. Nat Rev Cancer (2007) 7:880–7. doi:10.1038/nrc2250

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

16. Feuerer M, Hill JA, Mathis D, Benoist C. Foxp3+ regulatory T cells: differentiation, specification, subphenotypes. Nat Immunol (2009) 10:689–95. doi:10.1038/ni.1760

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

17. Hodi FS. Cytotoxic T-lymphocyte-associated antigen-4. Clin Cancer Res (2007) 13:5238–42. doi:10.1158/1078-0432.CCR-07-0813

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

18. Wing K, Onishi Y, Prieto-Martin P, Yamaguchi T, Miyara M, Fehervari Z, et al. CTLA-4 control over Foxp3+ regulatory T cell function. Science (2008) 322:271–5. doi:10.1126/science.1160062

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

19. Huang CT, Workman CJ, Flies D, Pan X, Marson AL, Zhou G, et al. Role of LAG-3 in regulatory T cells. Immunity (2004) 21:503–13. doi:10.1016/j.immuni.2004.08.010

CrossRef Full Text

20. Camisaschi C, Casati C, Rini F, Perego M, De Filippo A, Triebel F, et al. LAG-3 expression defines a subset of CD4(+)CD25(high)Foxp3(+) regulatory T cells that are expanded at tumor sites. J Immunol (2010) 184:6545–51. doi:10.4049/jimmunol.0903879

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

21. Park HJ, Kusnadi A, Lee EJ, Kim WW, Cho BC, Lee IJ, et al. Tumor-infiltrating regulatory T cells delineated by upregulation of PD-1 and inhibitory receptors. Cell Immunol (2012) 278:76–83. doi:10.1016/j.cellimm.2012.07.001

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

22. 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) 28:402–13. doi:10.1016/j.immuni.2008.01.012

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

23. Battaglia A, Buzzonetti A, Baranello C, Ferrandina G, Martinelli E, Fanfani F, et al. Metastatic tumour cells favour the generation of a tolerogenic milieu in tumour draining lymph node in patients with early cervical cancer. Cancer Immunol Immunother (2009) 58:1363–73. doi:10.1007/s00262-008-0646-7

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

24. Tan W, Zhang W, Strasner A, Grivennikov S, Cheng JQ, Hoffman RM, et al. Tumour-infiltrating regulatory T cells stimulate mammary cancer metastasis through RANKL-RANK signalling. Nature (2011) 470:548–53. doi:10.1038/nature09707

CrossRef Full Text

25. Zhao E, Wang L, Dai J, Kryczek I, Wei S, Vatan L, et al. Regulatory T cells in the bone marrow microenvironment in patients with prostate cancer. Oncoimmunology (2012) 1:152–61. doi:10.4161/onci.1.2.18480

CrossRef Full Text

26. Borsellino G, Kleinewietfeld M, Di Mitri D, Sternjak A, Diamantini A, Giometto R, et al. Expression of ectonucleotidase CD39 by Foxp3+ Treg cells: hydrolysis of extracellular ATP and immune suppression. Blood (2007) 110:1225–32. doi:10.1182/blood-2006-12-064527

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

27. Deaglio S, Dwyer KM, Gao W, Friedman D, Usheva A, Erat A, et al. Adenosine generation catalyzed by CD39 and CD73 expressed on regulatory T cells mediates immune suppression. J Exp Med (2007) 204:1257–65. doi:10.1084/jem.20062512

CrossRef Full Text

28. Cao X, Cai SF, Fehniger TA, Song J, Collins LI, Piwnica-Worms DR, et al. Granzyme B and perforin are important for regulatory T cell-mediated suppression of tumor clearance. Immunity (2007) 27:635–46. doi:10.1016/j.immuni.2007.08.014

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

29. Stagg J, Divisekera U, Duret H, Sparwasser T, Teng MW, Darcy PK, et al. CD73-deficient mice have increased antitumor immunity and are resistant to experimental metastasis. Cancer Res (2011) 71:2892–900. doi:10.1158/0008-5472.CAN-10-4246

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

30. Grossman WJ, Verbsky JW, Barchet W, Colonna M, Atkinson JP, Ley TJ. Human T regulatory cells can use the perforin pathway to cause autologous target cell death. Immunity (2004) 21:589–601. doi:10.1016/j.immuni.2004.09.002

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

31. Chen ML, Pittet MJ, Gorelik L, Flavell RA, Weissleder R, von Boehmer H, et al. Regulatory T cells suppress tumor-specific CD8 T cell cytotoxicity through TGF-beta signals in vivo. Proc Natl Acad Sci U S A (2005) 102:419–24. doi:10.1073/pnas.0408197102

CrossRef Full Text

32. Hilchey SP, De A, Rimsza LM, Bankert RB, Bernstein SH. Follicular lymphoma intratumoral CD4+CD25+GITR+ regulatory T cells potently suppress CD3/CD28-costimulated autologous and allogeneic CD8+CD25− and CD4+CD25− T cells. J Immunol (2007) 178:4051–61.

Pubmed Abstract | Pubmed Full Text

33. Strauss L, Bergmann C, Szczepanski M, Gooding W, Johnson JT, Whiteside TL. A unique subset of CD4+CD25highFoxp3+ T cells secreting interleukin-10 and transforming growth factor-beta1 mediates suppression in the tumor microenvironment. Clin Cancer Res (2007) 13:4345–54. doi:10.1158/1078-0432.CCR-07-0472

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

34. Rubtsov YP, Rasmussen JP, Chi EY, Fontenot J, Castelli L, Ye X, et al. Regulatory T cell-derived interleukin-10 limits inflammation at environmental interfaces. Immunity (2008) 28:546–58. doi:10.1016/j.immuni.2008.02.017

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

35. Chaudhry A, Samstein RM, Treuting P, Liang Y, Pils MC, Heinrich JM, et al. Interleukin-10 signaling in regulatory T cells is required for suppression of Th17 cell-mediated inflammation. Immunity (2011) 34:566–78. doi:10.1016/j.immuni.2011.03.018

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

36. Burocchi A, Pittoni P, Gorzanelli A, Colombo MP, Piconese S. Intratumor OX40 stimulation inhibits IRF1 expression and IL-10 production by Treg cells while enhancing CD40L expression by effector memory T cells. Eur J Immunol (2011) 41:3615–26. doi:10.1002/eji.201141700

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

37. Collison LW, Workman CJ, Kuo TT, Boyd K, Wang Y, Vignali KM, et al. The inhibitory cytokine IL-35 contributes to regulatory T-cell function. Nature (2007) 450:566–9. doi:10.1038/nature06306

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

38. Facciabene A, Peng X, Hagemann IS, Balint K, Barchetti A, Wang LP, et al. Tumour hypoxia promotes tolerance and angiogenesis via CCL28 and T(reg) cells. Nature (2011) 475:226–30. doi:10.1038/nature10169

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

39. Liyanage UK, Moore TT, Joo HG, Tanaka Y, Herrmann V, Doherty G, et al. Prevalence of regulatory T cells is increased in peripheral blood and tumor microenvironment of patients with pancreas or breast adenocarcinoma. J Immunol (2002) 169:2756–61.

Pubmed Abstract | Pubmed Full Text

40. Liyanage UK, Goedegebuure PS, Moore TT, Viehl CT, Moo-Young TA, Larson JW, et al. Increased prevalence of regulatory T cells (Treg) is induced by pancreas adenocarcinoma. J Immunother (2006) 29:416–24. doi:10.1097/01.cji.0000205644.43735.4e

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

41. Woo EY, Chu CS, Goletz TJ, Schlienger K, Yeh H, Coukos G, et al. Regulatory CD4(+)CD25(+) T cells in tumors from patients with early-stage non-small cell lung cancer and late-stage ovarian cancer. Cancer Res (2001) 61:4766–72.

Pubmed Abstract | Pubmed Full Text

42. Marshall NA, Christie LE, Munro LR, Culligan DJ, Johnston PW, Barker RN, et al. Immunosuppressive regulatory T cells are abundant in the reactive lymphocytes of Hodgkin lymphoma. Blood (2004) 103:1755–62. doi:10.1182/blood-2003-07-2594

CrossRef Full Text

43. Correll A, Tuettenberg A, Becker C, Jonuleit H. Increased regulatory T-cell frequencies in patients with advanced melanoma correlate with a generally impaired T-cell responsiveness and are restored after dendritic cell-based vaccination. Exp Dermatol (2010) 19:e213–21. doi:10.1111/j.1600-0625.2009.01055.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

44. Chen YL, Chang MC, Chen CA, Lin HW, Cheng WF, Chien CL. Depletion of regulatory T lymphocytes reverses the imbalance between pro- and anti-tumor immunities via enhancing antigen-specific T cell immune responses. PLoS ONE (2012) 7:e47190. doi:10.1371/journal.pone.0047190

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

45. Yao X, Ahmadzadeh M, Lu YC, Liewehr DJ, Dudley ME, Liu F, et al. Levels of peripheral CD4(+)FoxP3(+) regulatory T cells are negatively associated with clinical response to adoptive immunotherapy of human cancer. Blood (2012) 119:5688–96. doi:10.1182/blood-2011-10-386482

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

46. Benevides L, Cardoso CR, Tiezzi DG, Marana HR, Andrade JM, Silva JS. Enrichment of regulatory T cells in invasive breast tumor correlates with the upregulation of IL-17A expression and invasiveness of the tumor. Eur J Immunol (2013) 43(6): 1518–28. doi:10.1002/eji.201242951

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

47. Gallimore AM, Simon AK. Positive and negative influences of regulatory T cells on tumour immunity. Oncogene (2008) 27:5886–93. doi:10.1038/onc.2008.269

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

48. Whiteside TL. What are regulatory T cells (Treg) regulating in cancer and why? Semin Cancer Biol (2012) 22:327–34. doi:10.1016/j.semcancer.2012.03.004

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

49. Chow MT, Moller A, Smyth MJ. Inflammation and immune surveillance in cancer. Semin Cancer Biol (2012) 22:23–32. doi:10.1016/j.semcancer.2011.12.004

CrossRef Full Text

50. Salama P, Phillips M, Grieu F, Morris M, Zeps N, Joseph D, et al. Tumor-infiltrating FOXP3+ T regulatory cells show strong prognostic significance in colorectal cancer. J Clin Oncol (2009) 27:186–92. doi:10.1200/JCO.2008.18.7229

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

51. Frey DM, Droeser RA, Viehl CT, Zlobec I, Lugli A, Zingg U, et al. High frequency of tumor-infiltrating FOXP3(+) regulatory T cells predicts improved survival in mismatch repair-proficient colorectal cancer patients. Int J Cancer (2010) 126:2635–43.

52. 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) 60:909–18. doi:10.1007/s00262-011-1046-y

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

53. Sinicrope FA, Rego RL, Ansell SM, Knutson KL, Foster NR, Sargent DJ. Intraepithelial effector (CD3+)/regulatory (FoxP3+) T-cell ratio predicts a clinical outcome of human colon carcinoma. Gastroenterology (2009) 137:1270–9. doi:10.1053/j.gastro.2009.06.053

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

54. Chaput N, Louafi S, Bardier A, Charlotte F, Vaillant JC, Menegaux F, et al. Identification of CD8+CD25+Foxp3+ suppressive T cells in colorectal cancer tissue. Gut (2009) 58:520–9. doi:10.1136/gut.2008.158824

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

55. Kryczek I, Liu R, Wang G, Wu K, Shu X, Szeliga W, et al. FOXP3 defines regulatory T cells in human tumor and autoimmune disease. Cancer Res (2009) 69:3995–4000. doi:10.1158/0008-5472.CAN-08-3804

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

56. Kim M, Grimmig T, Grimm M, Lazariotou M, Meier E, Rosenwald A, et al. Expression of Foxp3 in colorectal cancer but not in Treg cells correlates with disease progression in patients with colorectal cancer. PLoS ONE (2013) 8:e53630. doi:10.1371/journal.pone.0053630

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

57. Badoual C, Hans S, Rodriguez J, Peyrard S, Klein C, Agueznay Nel H, et al. Prognostic value of tumor-infiltrating CD4+ T-cell subpopulations in head and neck cancers. Clin Cancer Res (2006) 12:465–72. doi:10.1158/1078-0432.CCR-05-1886

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

58. Strauss L, Bergmann C, Gooding W, Johnson JT, Whiteside TL. The frequency and suppressor function of CD4+CD25highFoxp3+ T cells in the circulation of patients with squamous cell carcinoma of the head and neck. Clin Cancer Res (2007) 13:6301–11. doi:10.1158/1078-0432.CCR-07-1403

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

59. Thornton AM, Korty PE, Tran DQ, Wohlfert EA, Murray PE, Belkaid Y, et al. Expression of Helios, an Ikaros transcription factor family member, differentiates thymic-derived from peripherally induced Foxp3+ T regulatory cells. J Immunol (2010) 184:3433–41. doi:10.4049/jimmunol.0904028

CrossRef Full Text

60. Gottschalk RA, Corse E, Allison JP. Expression of Helios in peripherally induced Foxp3+ regulatory T cells. J Immunol (2012) 188:976–80. doi:10.4049/jimmunol.1102964

CrossRef Full Text

61. Weiss JM, Bilate AM, Gobert M, Ding Y, Curotto de Lafaille MA, Parkhurst CN, et al. Neuropilin 1 is expressed on thymus-derived natural regulatory T cells, but not mucosa-generated induced Foxp3+ T reg cells. J Exp Med (2012) 209:1723–42, S1. doi:10.1084/jem.20120914

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

62. Yadav M, Louvet C, Davini D, Gardner JM, Martinez-Llordella M, Bailey-Bucktrout S, et al. Neuropilin-1 distinguishes natural and inducible regulatory T cells among regulatory T cell subsets in vivo. J Exp Med (2012) 209:1713–22, S1–19. doi:10.1084/jem.20120822

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

63. Milpied P, Renand A, Bruneau J, Mendes-Da-Cruz DA, Jacquelin S, Asnafi V, et al. Neuropilin-1 is not a marker of human Foxp3+ Treg. Eur J Immunol (2009) 39:1466–71. doi:10.1002/eji.200839040

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

64. Haribhai D, Lin W, Edwards B, Ziegelbauer J, Salzman NH, Carlson MR, et al. A central role for induced regulatory T cells in tolerance induction in experimental colitis. J Immunol (2009) 182:3461–8. doi:10.4049/jimmunol.0802535

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

65. Feuerer M, Hill JA, Kretschmer K, von Boehmer H, Mathis D, Benoist C. Genomic definition of multiple ex vivo regulatory T cell subphenotypes. Proc Natl Acad Sci U S A (2010) 107:5919–24. doi:10.1073/pnas.1002006107

CrossRef Full Text

66. Haribhai D, Williams JB, Jia S, Nickerson D, Schmitt EG, Edwards B, et al. A requisite role for induced regulatory T cells in tolerance based on expanding antigen receptor diversity. Immunity (2011) 35:109–22. doi:10.1016/j.immuni.2011.03.029

CrossRef Full Text

67. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, et al. Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol (2007) 5:e38. doi:10.1371/journal.pbio.0050038

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

68. Polansky JK, Kretschmer K, Freyer J, Floess S, Garbe A, Baron U, et al. DNA methylation controls Foxp3 gene expression. Eur J Immunol (2008) 38:1654–63. doi:10.1002/eji.200838105

CrossRef Full Text

69. Miyao T, Floess S, Setoguchi R, Luche H, Fehling HJ, Waldmann H, et al. Plasticity of Foxp3(+) T cells reflects promiscuous Foxp3 expression in conventional T cells but not reprogramming of regulatory T cells. Immunity (2012) 36:262–75. doi:10.1016/j.immuni.2011.12.012

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

70. Bui JD, Uppaluri R, Hsieh CS, Schreiber RD. Comparative analysis of regulatory and effector T cells in progressively growing versus rejecting tumors of similar origins. Cancer Res (2006) 66:7301–9. doi:10.1158/0008-5472.CAN-06-0556

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

71. Schallenberg S, Tsai PY, Riewaldt J, Kretschmer K. Identification of an immediate Foxp3(-) precursor to Foxp3(+) regulatory T cells in peripheral lymphoid organs of nonmanipulated mice. J Exp Med (2010) 207:1393–407. doi:10.1084/jem.20100045

CrossRef Full Text

72. Sharma MD, Baban B, Chandler P, Hou DY, Singh N, Yagita H, et al. Plasmacytoid dendritic cells from mouse tumor-draining lymph nodes directly activate mature Tregs via indoleamine 2,3-dioxygenase. J Clin Invest (2007) 117:2570–82. doi:10.1172/JCI31911

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

73. Ghiringhelli F, Puig PE, Roux S, Parcellier A, Schmitt E, Solary E, et al. Tumor cells convert immature myeloid dendritic cells into TGF-beta-secreting cells inducing CD4+CD25+ regulatory T cell proliferation. J Exp Med (2005) 202:919–29. doi:10.1084/jem.20050463

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

74. Darrasse-Jeze G, Bergot AS, Durgeau A, Billiard F, Salomon BL, Cohen JL, et al. Tumor emergence is sensed by self-specific CD44hi memory Tregs that create a dominant tolerogenic environment for tumors in mice. J Clin Invest (2009) 119:2648–62. doi:10.1172/JCI36628

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

75. Beyer M, Kochanek M, Giese T, Endl E, Weihrauch MR, Knolle PA, et al. In vivo peripheral expansion of naive CD4+CD25high FoxP3+ regulatory T cells in patients with multiple myeloma. Blood (2006) 107:3940–9. doi:10.1182/blood-2005-09-3671

CrossRef Full Text

76. Valzasina B, Piconese S, Guiducci C, Colombo MP. Tumor-induced expansion of regulatory T cells by conversion of CD4+CD25− lymphocytes is thymus and proliferation independent. Cancer Res (2006) 66:4488–95. doi:10.1158/0008-5472.CAN-05-4217

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

77. Liu VC, Wong LY, Jang T, Shah AH, Park I, Yang X, et al. Tumor evasion of the immune system by converting CD4+CD25− T cells into CD4+CD25+ T regulatory cells: role of tumor-derived TGF-beta. J Immunol (2007) 178:2883–92.

78. Curti A, Pandolfi S, Valzasina B, Aluigi M, Isidori A, Ferri E, et al. Modulation of tryptophan catabolism by human leukemic cells results in the conversion of CD25− into CD25+ T regulatory cells. Blood (2007) 109:2871–7.

Pubmed Abstract | Pubmed Full Text

79. Zheng SG, Wang JH, Gray JD, Soucier H, Horwitz DA. Natural and induced CD4+CD25+ cells educate CD4+CD25− cells to develop suppressive activity: the role of IL-2, TGF-beta, and IL-10. J Immunol (2004) 172:5213–21.

Pubmed Abstract | Pubmed Full Text

80. Qin S, Cobbold SP, Pope H, Elliott J, Kioussis D, Davies J, et al. “Infectious” transplantation tolerance. Science (1993) 259:974–7. doi:10.1126/science.8094901

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

81. Zhou G, Drake CG, Levitsky HI. Amplification of tumor-specific regulatory T cells following therapeutic cancer vaccines. Blood (2006) 107:628–36. doi:10.1182/blood-2005-07-2737

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

82. Zhou G, Levitsky HI. Natural regulatory T cells and de novo-induced regulatory T cells contribute independently to tumor-specific tolerance. J Immunol (2007) 178:2155–62.

Pubmed Abstract | Pubmed Full Text

83. Kretschmer K, Apostolou I, Hawiger D, Khazaie K, Nussenzweig MC, von Boehmer H. Inducing and expanding regulatory T cell populations by foreign antigen. Nat Immunol (2005) 6:1219–27. doi:10.1038/ni1265

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

84. Josefowicz SZ, Niec RE, Kim HY, Treuting P, Chinen T, Zheng Y, et al. Extrathymically generated regulatory T cells control mucosal TH2 inflammation. Nature (2012) 482:395–9. doi:10.1038/nature10772

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

85. Curotto de Lafaille MA, Kutchukhidze N, Shen S, Ding Y, Yee H, Lafaille JJ. Adaptive Foxp3+ regulatory T cell-dependent and -independent control of allergic inflammation. Immunity (2008) 29:114–26. doi:10.1016/j.immuni.2008.05.010

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

86. 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) 463:808–12. doi:10.1038/nature08750

CrossRef Full Text

87. Samstein RM, Josefowicz SZ, Arvey A, Treuting PM, Rudensky AY. Extrathymic generation of regulatory T cells in placental mammals mitigates maternal-fetal conflict. Cell (2012) 150:29–38. doi:10.1016/j.cell.2012.05.031

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

88. Schmitt EG, Haribhai D, Williams JB, Aggarwal P, Jia S, Charbonnier LM, et al. IL-10 produced by induced regulatory T cells (iTregs) controls colitis and pathogenic ex-iTregs during immunotherapy. J Immunol (2012) 189:5638–48. doi:10.4049/jimmunol.1200936

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

89. Bergmann C, Wild CA, Narwan M, Lotfi R, Lang S, Brandau S. Human tumor-induced and naturally occurring Treg cells differentially affect NK cells activated by either IL-2 or target cells. Eur J Immunol (2011) 41:3564–73. doi:10.1002/eji.201141532

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

90. Hsieh CS, Lee HM, Lio CW. Selection of regulatory T cells in the thymus. Nat Rev Immunol (2012) 12:157–67.

91. Getnet D, Maris CH, Hipkiss EL, Grosso JF, Harris TJ, Yen HR, et al. Tumor recognition and self-recognition induce distinct transcriptional profiles in antigen-specific CD4 T cells. J Immunol (2009) 182:4675–85. doi:10.4049/jimmunol.0803400

CrossRef Full Text

92. Wang HY, Lee DA, Peng G, Guo Z, Li Y, Kiniwa Y, et al. Tumor-specific human CD4+ regulatory T cells and their ligands: implications for immunotherapy. Immunity (2004) 20:107–18. doi:10.1016/S1074-7613(03)00359-5

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

93. Wang HY, Peng G, Guo Z, Shevach EM, Wang RF. Recognition of a new ARTC1 peptide ligand uniquely expressed in tumor cells by antigen-specific CD4+ regulatory T cells. J Immunol (2005) 174:2661–70.

Pubmed Abstract | Pubmed Full Text

94. Vence L, Palucka AK, Fay JW, Ito T, Liu YJ, Banchereau J, et al. Circulating tumor antigen-specific regulatory T cells in patients with metastatic melanoma. Proc Natl Acad Sci U S A (2007) 104:20884–9. doi:10.1073/pnas.0710557105

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

95. Fourcade J, Sun Z, Kudela P, Janjic B, Kirkwood JM, El-Hafnawy T, et al. Human tumor antigen-specific helper and regulatory T cells share common epitope specificity but exhibit distinct T cell repertoire. J Immunol (2010) 184:6709–18. doi:10.4049/jimmunol.0903612

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

96. Ebert LM, Macraild SE, Zanker D, Davis ID, Cebon J, Chen W. A cancer vaccine induces expansion of NY-ESO-1-specific regulatory T cells in patients with advanced melanoma. PLoS ONE (2012) 7:e48424. doi:10.1371/journal.pone.0048424

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

97. Jandus C, Bioley G, Dojcinovic D, Derre L, Baitsch L, Wieckowski S, et al. Tumor antigen-specific FOXP3+ CD4 T cells identified in human metastatic melanoma: peptide vaccination results in selective expansion of Th1-like counterparts. Cancer Res (2009) 69:8085–93. doi:10.1158/0008-5472.CAN-09-2226

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

98. Lehe C, Ghebeh H, Al-Sulaiman A, Al Qudaihi G, Al-Hussein K, Almohareb F, et al. The Wilms’ tumor antigen is a novel target for human CD4+ regulatory T cells: implications for immunotherapy. Cancer Res (2008) 68:6350–9. doi:10.1158/0008-5472.CAN-08-0050

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

99. Redjimi N, Duperrier-Amouriaux K, Raimbaud I, Luescher I, Dojcinovic D, Classe JM, et al. NY-ESO-1-specific circulating CD4+ T cells in ovarian cancer patients are prevalently T(H)1 type cells undetectable in the CD25+ FOXP3+ Treg compartment. PLoS ONE (2011) 6:e22845. doi:10.1371/journal.pone.0022845

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

100. Bonertz A, Weitz J, Pietsch DH, Rahbari NN, Schlude C, Ge Y, et al. Antigen-specific Tregs control T cell responses against a limited repertoire of tumor antigens in patients with colorectal carcinoma. J Clin Invest (2009) 119:3311–21.

101. van der Burg SH, Piersma SJ, de Jong A, van der Hulst JM, Kwappenberg KM, van den Hende M, et al. Association of cervical cancer with the presence of CD4+ regulatory T cells specific for human papillomavirus antigens. Proc Natl Acad Sci U S A (2007) 104:12087–92. doi:10.1073/pnas.0704672104

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

102. Voo KS, Peng G, Guo Z, Fu T, Li Y, Frappier L, et al. Functional characterization of EBV-encoded nuclear antigen 1-specific CD4+ helper and regulatory T cells elicited by in vitro peptide stimulation. Cancer Res (2005) 65:1577–86. doi:10.1158/0008-5472.CAN-04-2552

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

103. Nishikawa H, Kato T, Tanida K, Hiasa A, Tawara I, Ikeda H, et al. CD4+ CD25+ T cells responding to serologically defined autoantigens suppress antitumor immune responses. Proc Natl Acad Sci U S A (2003) 100:10902–6. doi:10.1073/pnas.1834479100

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

104. Malchow S, Leventhal DS, Nishi S, Fischer BI, Shen L, Paner GP, et al. Aire-dependent thymic development of tumor-associated regulatory T cells. Science (2013) 339:1219–24.

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

105. Sainz-Perez A, Lim A, Lemercier B, Leclerc C. The T-cell receptor repertoire of tumor-infiltrating regulatory T lymphocytes is skewed toward public sequences. Cancer Res (2012) 72:3557–69. doi:10.1158/0008-5472.CAN-12-0277

CrossRef Full Text

106. Hindley JP, Ferreira C, Jones E, Lauder SN, Ladell K, Wynn KK, et al. Analysis of the T-cell receptor repertoires of tumor-infiltrating conventional and regulatory T cells reveals no evidence for conversion in carcinogen-induced tumors. Cancer Res (2011) 71:736–46. doi:10.1158/0008-5472.CAN-10-1797

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

107. Schreiber TH, Wolf D, Bodero M, Podack E. Tumor antigen specific iTreg accumulate in the tumor microenvironment and suppress therapeutic vaccination. Oncoimmunology (2012) 1:642–8. doi:10.4161/onci.20298

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

108. Kim HP, Leonard WJ. CREB/ATF-dependent T cell receptor-induced FoxP3 gene expression: a role for DNA methylation. J Exp Med (2007) 204:1543–51.

Pubmed Abstract | Pubmed Full Text

109. Wieczorek G, Asemissen A, Model F, Turbachova I, Floess S, Liebenberg V, et al. Quantitative DNA methylation analysis of FOXP3 as a new method for counting regulatory T cells in peripheral blood and solid tissue. Cancer Res (2009) 69:599–608. doi:10.1158/0008-5472.CAN-08-2361

CrossRef Full Text

110. Clarke SL, Betts GJ, Plant A, Wright KL, El-Shanawany TM, Harrop R, et al. CD4+CD25+FOXP3+ regulatory T cells suppress anti-tumor immune responses in patients with colorectal cancer. PLoS ONE (2006) 1:e129. doi:10.1371/journal.pone.0000129

CrossRef Full Text

111. Ling KL, Pratap SE, Bates GJ, Singh B, Mortensen NJ, George BD, et al. Increased frequency of regulatory T cells in peripheral blood and tumour infiltrating lymphocytes in colorectal cancer patients. Cancer Immun (2007) 7:7.

112. Sehouli J, Loddenkemper C, Cornu T, Schwachula T, Hoffmuller U, Grutzkau A, et al. Epigenetic quantification of tumor-infiltrating T-lymphocytes. Epigenetics (2011) 6:236–46. doi:10.4161/epi.6.2.13755

CrossRef Full Text

113. Pohla H, Buchner A, Stadlbauer B, Frankenberger B, Stevanovic S, Walter S, et al. High immune response rates and decreased frequencies of regulatory T cells in metastatic renal cell carcinoma patients after tumor cell vaccination. Mol Med (2012) 18:1499–508. doi:10.2119/molmed.2012.00221

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

114. Schwarzer A, Wolf B, Fisher JL, Schwaab T, Olek S, Baron U, et al. Regulatory T-cells and associated pathways in metastatic renal cell carcinoma (mRCC) patients undergoing DC-vaccination and cytokine-therapy. PLoS ONE (2012) 7:e46600. doi:10.1371/journal.pone.0046600

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

115. Josefowicz SZ, Lu LF, Rudensky AY. Regulatory T cells: mechanisms of differentiation and function. Annu Rev Immunol (2012) 30:531–64. doi:10.1146/annurev.immunol.25.022106.141623

CrossRef Full Text

116. McMurchy AN, Gillies J, Gizzi MC, Riba M, Garcia-Manteiga JM, Cittaro D, et al. A novel function for FOXP3 in humans: intrinsic regulation of conventional T cells. Blood (2013) 121:1265–75. doi:10.1182/blood-2012-05-431023

CrossRef Full Text

117. Miyara M, Yoshioka Y, Kitoh A, Shima T, Wing K, Niwa A, et al. Functional delineation and differentiation dynamics of human CD4+ T cells expressing the FoxP3 transcription factor. Immunity (2009) 30:899–911. doi:10.1016/j.immuni.2009.03.019

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

118. Fujii H, Arakawa A, Kitoh A, Miyara M, Kato M, Kore-Eda S, et al. Perturbations of both nonregulatory and regulatory FOXP3+ T cells in patients with malignant melanoma. Br J Dermatol (2011) 164:1052–60. doi:10.1111/j.1365-2133.2010.10199.x

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

119. Schuler PJ, Harasymczuk M, Schilling B, Lang S, Whiteside TL. Separation of human CD4+CD39+ T cells by magnetic beads reveals two phenotypically and functionally different subsets. J Immunol Methods (2011) 369:59–68. doi:10.1016/j.jim.2011.04.004

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

120. Schuler PJ, Schilling B, Harasymczuk M, Hoffmann TK, Johnson J, Lang S, et al. Phenotypic and functional characteristics of CD4+ CD39+ FOXP3+ and CD4+ CD39+ FOXP3neg T-cell subsets in cancer patients. Eur J Immunol (2012) 42:1876–85. doi:10.1002/eji.201142347

CrossRef Full Text

121. Mandapathil M, Szczepanski MJ, Szajnik M, Ren J, Lenzner DE, Jackson EK, et al. Increased ectonucleotidase expression and activity in regulatory T cells of patients with head and neck cancer. Clin Cancer Res (2009) 15:6348–57. doi:10.1158/1078-0432.CCR-09-1143

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

122. Housley WJ, Adams CO, Nichols FC, Puddington L, Lingenheld EG, Zhu L, et al. Natural but not inducible regulatory T cells require TNF-alpha signaling for in vivo function. J Immunol (2011) 186:6779–87. doi:10.4049/jimmunol.1003868

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

123. Chen X, Subleski JJ, Kopf H, Howard OM, Mannel DN, Oppenheim JJ. Cutting edge: expression of TNFR2 defines a maximally suppressive subset of mouse CD4+CD25+FoxP3+ T regulatory cells: applicability to tumor-infiltrating T regulatory cells. J Immunol (2008) 180:6467–71.

Pubmed Abstract | Pubmed Full Text

124. Chopra M, Riedel SS, Biehl M, Krieger S, von Krosigk V, Bauerlein CA, et al. Tumor necrosis factor receptor 2-dependent homeostasis of regulatory T cells as a player in TNF-induced experimental metastasis. Carcinogenesis (2013) 34(6): 1296–303. doi:10.1093/carcin/bgt038

CrossRef Full Text

125. Chen X, Wu X, Zhou Q, Howard OM, Netea MG, Oppenheim JJ. TNFR2 is critical for the stabilization of the CD4+Foxp3+ regulatory T cell phenotype in the inflammatory environment. J Immunol (2013) 190:1076–84. doi:10.4049/jimmunol.1202659

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

126. Chen X, Subleski JJ, Hamano R, Howard OM, Wiltrout RH, Oppenheim JJ. Co-expression of TNFR2 and CD25 identifies more of the functional CD4+FOXP3+ regulatory T cells in human peripheral blood. Eur J Immunol (2010) 40:1099–106. doi:10.1002/eji.200940022

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

127. Nagar M, Jacob-Hirsch J, Vernitsky H, Berkun Y, Ben-Horin S, Amariglio N, et al. TNF activates a NF-kappaB-regulated cellular program in human CD45RA− regulatory T cells that modulates their suppressive function. J Immunol (2010) 184:3570–81. doi:10.4049/jimmunol.0902070

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

128. Getnet D, Grosso JF, Goldberg MV, Harris TJ, Yen HR, Bruno TC, et al. A role for the transcription factor Helios in human CD4(+)CD25(+) regulatory T cells. Mol Immunol (2010) 47:1595–600. doi:10.1016/j.molimm

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

129. Wainwright DA, Sengupta S, Han Y, Lesniak MS. Thymus-derived rather than tumor-induced regulatory T cells predominate in brain tumors. Neuro-oncology (2011) 13:1308–23. doi:10.1093/neuonc/nor134

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

130. Elkord E, Sharma S, Burt DJ, Hawkins RE. Expanded subpopulation of FoxP3+ T regulatory cells in renal cell carcinoma co-express Helios, indicating they could be derived from natural but not induced Tregs. Clin Immunol (2011) 140:218–22. doi:10.1016/j.clim.2011.04.014

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

131. Zabransky DJ, Nirschl CJ, Durham NM, Park BV, Ceccato CM, Bruno TC, et al. Phenotypic and functional properties of Helios+ regulatory T cells. PLoS ONE (2012) 7:e34547. doi:10.1371/journal.pone.0034547

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

132. Alexander T, Sattler A, Templin L, Kohler S, Gross C, Meisel A, et al. Foxp3+ Helios+ regulatory T cells are expanded in active systemic lupus erythematosus. Ann Rheum Dis (2013) 72:1549–58. doi:10.1136/annrheumdis-2012-202216

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

133. Kim YC, Bhairavabhotla R, Yoon J, Golding A, Thornton AM, Tran DQ, et al. Oligodeoxynucleotides stabilize Helios-expressing Foxp3+ human T regulatory cells during in vitro expansion. Blood (2012) 119:2810–8. doi:10.1182/blood-2011-09-377895

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

134. Koch MA, Tucker-Heard G, Perdue NR, Killebrew JR, Urdahl KB, Campbell DJ. The transcription factor T-bet controls regulatory T cell homeostasis and function during type 1 inflammation. Nat Immunol (2009) 10:595–602. doi:10.1038/ni.1731

CrossRef Full Text

135. Koch MA, Thomas KR, Perdue NR, Smigiel KS, Srivastava S, Campbell DJ. T-bet(+) Treg cells undergo abortive Th1 cell differentiation due to impaired expression of IL-12 receptor beta2. Immunity (2012) 37:501–10. doi:10.1016/j.immuni.2012.05.031

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

136. Zheng Y, Chaudhry A, Kas A, Deroos P, Kim JM, Chu TT, et al. Regulatory T-cell suppressor program co-opts transcription factor IRF4 to control T(H)2 responses. Nature (2009) 458:351–6. doi:10.1038/nature07674

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

137. Chaudhry A, Rudra D, Treuting P, Samstein RM, Liang Y, Kas A, et al. CD4+ regulatory T cells control TH17 responses in a Stat3-dependent manner. Science (2009) 326:986–91. doi:10.1126/science.1172702

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

138. Chung Y, Tanaka S, Chu F, Nurieva RI, Martinez GJ, Rawal S, et al. Follicular regulatory T cells expressing Foxp3 and Bcl-6 suppress germinal center reactions. Nat Med (2011) 17:983–8. doi:10.1038/nm.2426

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

139. Linterman MA, Pierson W, Lee SK, Kallies A, Kawamoto S, Rayner TF, et al. Foxp3+ follicular regulatory T cells control the germinal center response. Nat Med (2011) 17:975–82. doi:10.1038/nm.2425

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

140. Zhou X, Bailey-Bucktrout S, Jeker LT, Bluestone JA. Plasticity of CD4(+) FoxP3(+) T cells. Curr Opin Immunol (2009) 21:281–5. doi:10.1016/j.coi.2009.05.007

CrossRef Full Text

141. Rubtsov YP, Niec RE, Josefowicz S, Li L, Darce J, Mathis D, et al. Stability of the regulatory T cell lineage in vivo. Science (2010) 329:1667–71. doi:10.1126/science.1191996

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

142. Oldenhove G, Bouladoux N, Wohlfert EA, Hall JA, Chou D, Dos Santos L, et al. Decrease of Foxp3+ Treg cell number and acquisition of effector cell phenotype during lethal infection. Immunity (2009) 31:772–86. doi:10.1016/j.immuni.2009.10.001

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

143. Zhou X, Bailey-Bucktrout SL, Jeker LT, Penaranda C, Martinez-Llordella M, Ashby M, et al. Instability of the transcription factor Foxp3 leads to the generation of pathogenic memory T cells in vivo. Nat Immunol (2009) 10:1000–7. doi:10.1038/ni.1774

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

144. Dominguez-Villar M, Baecher-Allan CM, Hafler DA. Identification of T helper type 1-like, Foxp3+ regulatory T cells in human autoimmune disease. Nat Med (2011) 17:673–5. doi:10.1038/nm.2389

CrossRef Full Text

145. Hovhannisyan Z, Treatman J, Littman DR, Mayer L. Characterization of interleukin-17-producing regulatory T cells in inflamed intestinal mucosa from patients with inflammatory bowel diseases. Gastroenterology (2011) 140:957–65. doi:10.1053/j.gastro.2010.12.002

CrossRef Full Text

146. McClymont SA, Putnam AL, Lee MR, Esensten JH, Liu W, Hulme MA, et al. Plasticity of human regulatory T cells in healthy subjects and patients with type 1 diabetes. J Immunol (2011) 186:3918–26. doi:10.4049/jimmunol.1003099

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

147. Kryczek I, Wu K, Zhao E, Wei S, Vatan L, Szeliga W, et al. IL-17+ regulatory T cells in the microenvironments of chronic inflammation and cancer. J Immunol (2011) 186:4388–95. doi:10.4049/jimmunol.1003251

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

148. Leveque L, Deknuydt F, Bioley G, Old LJ, Matsuzaki J, Odunsi K, et al. Interleukin 2-mediated conversion of ovarian cancer-associated CD4+ regulatory T cells into proinflammatory interleukin 17-producing helper T cells. J Immunother (2009) 32:101–8. doi:10.1097/CJI.0b013e318195b59e

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

149. Reppert S, Boross I, Koslowski M, Tureci O, Koch S, Lehr HA, et al. A role for T-bet-mediated tumour immune surveillance in anti-IL-17A treatment of lung cancer. Nat Commun (2011) 2:600. doi:10.1038/ncomms1609

CrossRef Full Text

150. Blatner NR, Bonertz A, Beckhove P, Cheon EC, Krantz SB, Strouch M, et al. In colorectal cancer mast cells contribute to systemic regulatory T-cell dysfunction. Proc Natl Acad Sci U S A (2010) 107:6430–5. doi:10.1073/pnas.0913683107

CrossRef Full Text

151. Blatner NR, Mulcahy MF, Dennis KL, Scholtens D, Bentrem DJ, Phillips JD, et al. Expression of RORgammat marks a pathogenic regulatory T cell subset in human colon cancer. Sci Transl Med (2012) 4:164ra159. doi:10.1126/scitranslmed.3004566

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

152. Sharma MD, Hou DY, Baban B, Koni PA, He Y, Chandler PR, et al. Reprogrammed foxp3(+) regulatory T cells provide essential help to support cross-presentation and CD8(+) T cell priming in naive mice. Immunity (2010) 33:942–54. doi:10.1016/j.immuni.2010.11.022

CrossRef Full Text

153. Koenecke C, Lee CW, Thamm K, Fohse L, Schafferus M, Mittrucker HW, et al. IFN-gamma production by allogeneic Foxp3+ regulatory T cells is essential for preventing experimental graft-versus-host disease. J Immunol (2012) 189:2890–6. doi:10.4049/jimmunol.1200413

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

154. Zhao J, Fett C, Trandem K, Fleming E, Perlman S. IFN-gamma- and IL-10-expressing virus epitope-specific Foxp3(+) T reg cells in the central nervous system during encephalomyelitis. J Exp Med (2011) 208:1571–7. doi:10.1084/jem.20110236

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

155. Du W, Shen YW, Lee WH, Wang D, Paz S, Kandeel F, et al. Foxp3+ Treg expanded from patients with established diabetes reduce Helios expression while retaining normal function compared to healthy individuals. PLoS ONE (2013) 8:e56209. doi:10.1371/journal.pone.0056209

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

156. Chowdary Venigalla RK, Guttikonda PJ, Eckstein V, Ho AD, Sertel S, Lorenz HM, et al. Identification of a human Th1-like IFNgamma-secreting Treg subtype deriving from effector T cells. J Autoimmun (2012) 39:377–87. doi:10.1016/j.jaut.2012.06.004

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

157. Redjimi N, Raffin C, Raimbaud I, Pignon P, Matsuzaki J, Odunsi K, et al. CXCR3+ T regulatory cells selectively accumulate in human ovarian carcinomas to limit type I immunity. Cancer Res (2012) 72:4351–60. doi:10.1158/0008-5472.CAN-12-0579

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

158. Camisaschi C, Filipazzi P, Tazzari M, Casati C, Beretta V, Pilla L, et al. Effects of cyclophosphamide and IL-2 on regulatory CD4 T cell frequency and function in melanoma patients vaccinated with HLA-class I peptides: impact on the antigen-specific T cell response. Cancer Immunol Immunother (2013) 62(5): 897–908. doi:10.1007/s00262-013-1397-7

CrossRef Full Text

159. Rosenberg SA, Yang JC, Restifo NP. Cancer immunotherapy: moving beyond current vaccines. Nat Med (2004) 10:909–15. doi:10.1038/nm1100

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

160. Welters MJ, Kenter GG, Piersma SJ, Vloon AP, Lowik MJ, Berends-van Der Meer DM, et al. Induction of tumor-specific CD4+ and CD8+ T-cell immunity in cervical cancer patients by a human papillomavirus type 16 E6 and E7 long peptides vaccine. Clin Cancer Res (2008) 14:178–87. doi:10.1158/1078-0432.CCR-07-1880

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

161. Francois V, Ottaviani S, Renkvist N, Stockis J, Schuler G, Thielemans K, et al. The CD4(+) T-cell response of melanoma patients to a MAGE-A3 peptide vaccine involves potential regulatory T cells. Cancer Res (2009) 69:4335–45. doi:10.1158/0008-5472.CAN-08-3726

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

162. de Vries IJ, Castelli C, Huygens C, Jacobs JF, Stockis J, Schuler-Thurner B, et al. Frequency of circulating Tregs with demethylated FOXP3 intron 1 in melanoma patients receiving tumor vaccines and potentially Treg-depleting agents. Clin Cancer Res (2011) 17:841–8. doi:10.1158/1078-0432.CCR-10-2227

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

163. Ko K, Yamazaki S, Nakamura K, Nishioka T, Hirota K, Yamaguchi T, et al. Treatment of advanced tumors with agonistic anti-GITR mAb and its effects on tumor-infiltrating Foxp3+CD25+CD4+ regulatory T cells. J Exp Med (2005) 202:885–91. doi:10.1084/jem.20050940

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

164. Cohen AD, Schaer DA, Liu C, Li Y, Hirschhorn-Cymmerman D, Kim SC, et al. Agonist anti-GITR monoclonal antibody induces melanoma tumor immunity in mice by altering regulatory T cell stability and intra-tumor accumulation. PLoS ONE (2010) 5:e10436. doi:10.1371/journal.pone.0010436

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

165. Takahashi R, Nishimoto S, Muto G, Sekiya T, Tamiya T, Kimura A, et al. SOCS1 is essential for regulatory T cell functions by preventing loss of Foxp3 expression as well as IFN-{gamma} and IL-17A production. J Exp Med (2011) 208:2055–67. doi:10.1084/jem.20110428

Pubmed Abstract | Pubmed Full Text | CrossRef Full Text

166. Knosp CA, Schiering C, Spence S, Carroll HP, Nel HJ, Osbourn M, et al. Regulation of Foxp3+ inducible regulatory T cell stability by SOCS2. J Immunol (2013) 190:3235–45. doi:10.4049/jimmunol.1201396

CrossRef Full Text