Foxp3+ T regulatory (Treg) cells control all aspects of the immune response and play important roles in tumor immunity. Increased numbers of Treg cells are found in many tumors and it has been shown that the number of tumor infiltrating Treg cells correlate with adverse clinic outcomes. Therefore, modulating Treg cell recruitment, function, induction and migration can be of importance to control tumor growth and metastasis. In this review, we will focus on the development of Treg cells, Treg surface molecules, plasticity of Treg cells and mechanisms of Treg suppression to explore new therapeutic opportunities in the immunotherapy of cancer.
Treg CELLS DEVELOPMENT
CD4+CD25+ regulatory T cells, which are essential for the prevention of autoimmunity, were first described as a subpopulation of CD4+ T cells with high cell surface expression of the interleukin-2 receptor (IL-2R) α-chain (CD25) by Sakaguchi et al in 1995.1 Transcription factor Foxp3+ is one of the specific markers of CD4+CD25+Treg cells and indispensable for the development and function of CD4+CD25+Treg cells.2 Numerous studies have demonstrated that natural Treg (nTreg) cells are generated in the thymus through major histocompatibility complex (MHC) class II dependent T cell receptor interactions resulting in high avidity selection,3 although additional selection mechanisms may exist.4 In recent years it has become evident that Foxp3+ Treg cells could also be induced in the peripheral immune compartment by conversion of naïve CD4+ T cells into Foxp3+ T cells by transforming growth factor-β (TGF-β), vitamin D3, IL-10/Interferon α (IFNα) and other factors, and are referred to as induced Treg cells (iTreg) (Figure 1).5 The peripheral population of Foxp3+ Treg cells comprises both nTreg and iTreg cells. It is very likely that nTreg and Foxp3+ iTreg cells differ in their T cell receptor repertoire because iTreg cells are derived from mature peripheral naive CD4+ cells.6 Foxp3- iTreg cells, such as Tr1 and Th3 cells, will not be discussed in this review.
All T cells come from progenitor cells in the bone marrow which become committed to their lineage in the thymus. All T cells begin as CD4−CD8− T cell receptor− cells, the double-negative stage, where an individual cell will rearrange its T cell receptor genes to form a unique, functional molecule which they in turn test against cells in the thymic cortex for a minimal level of interaction with self-MHC. If they receive the necessary signals they proliferate and express both CD4 and CD8, becoming double-positive cells. The selection of Tregs occurs on radio-resistant haemopoietically-derived MHC class II expressing cells in the medulla or Hassal's corpuscles in the thymus. It seems that at the double-positive stage they are selected by their interaction with the cells within the thymus to begin the transcription of Foxp3+ and become Treg cells, although they may not begin to express Foxp3 until the single-positive stage, at which point they are functional Tregs.
Treg CELL MARKERS
Foxp3, a forkhead winged helix family transcriptional regulator, is essential to establish a functional Treg cell lineage.2,7 Foxp3 is currently the most specific, reliable molecular marker, irrespective of CD25 expression, for thymic or peripheral Treg cells in rodents and humans.8 Foxp3 has been shown to operate as a homo-oligomer, and interacts with a variety of transcription factors; including, nuclear factor of activated T-cells (NFAT), acute myeloid leukemia-1 (AML-1), runt-related transcription factor 1 (Runx1), the histone acetyl transferase (HAT)/histone deacetyl transferase (HDAC) complex, and nuclear factor-kappa B (NF-κB). Following its activation, NFAT complexes with activator protein-1 (AP-1) and NF-κB and promotes the expression of several genes including IL-2, IL-4, cytotoxic T lymphocyte antigen-4 (CTLA-4) in normal, activated T cells. These in turn contribute to T cell activation and differentiation.9
CD25 continues to be the most useful cell surface marker for nTreg cells in the naïve immune system, although several other markers, like Foxp3+ and CD127,10 may be more specific. Mice deficient for IL-2, IL-2Ra (CD25), or IL-2Rb (CD122) have a drastically reduced pool of nTreg cells and paradoxically die prematurely from a severe lymphoproliferative and autoimmune syndromes (hemolytic anemia and inflammatory bowel disease), lymphocytic infiltration of multiple organs, and hyperreactivity to commensal microbes.11
CD39/ectonucleoside triphosphate diphosphohydrolase-1 is the dominant ecto-nucleotidase expressed by both endothelial cells and Treg cells which drives the sequential hydrolysis of adenosine 5′-triphosphate (ATP) and adenosine 5′-diphosphate (ADP) to adenosine 5′-monophosphate (AMP).12 The formation of extracellular adenosine from AMP is accomplished primarily through ecto-5′-nucleotidase (CD73), a glycosyl phosphatidylinositol-linked membrane protein found on the surface of a variety of cell types, including Treg cells.13 It was reported that CD39, together with CD73, efficiently distinguishes Treg cells from other resting or activated T cells in mice and humans.14
Treg cells also express other markers such as CTLA-4, folate receptor 4 (FR4), CD103, LAG-3, glucocorticoidinduced tumor necrosis factor receptor (GITR) and OX40 (CD134).15 CTLA-4 or CD152 is an essential receptor involved in the negative regulation of T cell activation.16 CTLA-4 interacts with two ligands (CD80 and CD86) found on antigen-presenting cells and delivers a cell-intrinsic negative signal, which inhibits T-cell activation.17 nTreg cells constitutively expressed high amounts of FR4. FR4highCD25+CD4+ T cells enriched from alloantigen-stimulated T cells suppressed graft rejection. Thus, specific manipulation of FR4highCD25+CD4+ Treg cells helps to control ongoing immune responses.18 A total of 20%-30% of the Treg cells in a normal animal express CD103. These CD103+ Tregs display an activated phenotype and have higher suppressive activity.19 LAG-3 on Treg cells can interact with MHC class II on antigen-presenting cells, such as dendritic cells (DC), and this binding results in an inhibitory signal that suppresses DC maturation and immunostimulatory capacity.20 The GITR molecule, which is expressed at high levels on Treg cells, mediates inhibitory signals to Treg cells and blocks their immunosuppressive function.21 OX40 (CD134), another member of the tumor necrosis factor receptor family, which is constitutively expressed on mouse Treg cells, is capable to mediate inhibitory signals and block Treg cell activity.22
PLASTICITY OF Treg CELLS
CD4+CD25+ regulatory T cells are a subpopulation of CD4+ T cells. Based on their functions, their pattern of cytokine secretion and their expression of specific transcription factors, Th cells, differentiated from naïve CD4 T cells, are classified into four major lineages, Th1, Th2, Th17 and Treg cells, although other Th lineages may exist.
Th cells, including Treg cells and Th17 cells, are heterogeneous and somewhat plastic. Many Tregs, just like naïve CD4 T cells, can differentiate into various types of Ths. It has been reported that Tregs can become IL-17-producing cells when they are cultured with IL-6 and this correlates with their upregulation of RORγt.23 Strober's group also reported that Tregs can be self-induced to become IL-17-producing cells in the absence of TGFβ when IL-6 is present.24 Transferring Tregs into a lymphopenic host also results in downregulation of Foxp3+ accompanied by the production of IL-17 and IFNγ.25 In studying the relationship between Th17 cells and iTregs cells, it was initially found that many differentiating cells co-express RORγt and Foxp3, and such cells were considered to be the progenitors of either Th17 or iTregs.26 Similarly, other studies showed the existence of IL-17-producing Foxp3+ cells, both in mice and humans.27 And also, Degauque et al28 has reported that agonist anti-Tim-1 (T cell, Ig, mucin) mAb prevents the induction of CD4+Foxp3+ Tregs from CD4+Foxp3− T cells and promotes the differentiation of Th17 cells.
MECHANISM OF Treg-MEDIATED SUPPRESS
The cellular targets of Treg cell suppression can be CD4+, CD8+ T cells, DC, B cells, macrophages, osteoblasts, mast cells, natural killer (NK) cells and NKT cells. Current evidence suggests that CD4+CD25+Foxp3+ Treg cells contribute to immune suppression through five main mechanisms (Figure 2).
Treg cells may secrete suppressor cytokines, such as IL-10, TGF-β and IL-35,29 that can directly inhibit the function of responder T cells and myeloid cells. The secretion of suppressive cytokines is diverse in different diseases and the functional mechanisms are complex. Strauss et al30 reported that suppression in the tumor microenvironment is mediated by a unique subset of Treg cells, which produce IL-10 and TGF-β and do not require cell-to-cell contact between Treg cells and responder cells for inhibition. It was recently shown that IL-35 is constitutively expressed by mouse CD4+CD25+Foxp3+ Treg cells.31In vitro and in vivo studies suggested that the expression of IL-35 by mouse Treg cells contributed to their suppressive function.32 In another recent study, a single chain mouse IL-35-Fc fusion protein was demonstrated to enhance the proliferation of mouse Treg cells, while inhibiting the development of Th17 cells.33
One other potential mechanism for Treg-mediated suppression of responder T cells would be cytolysis of target cells. Human CD4+CD25+Foxp3+ Treg cells can be activated by a combination of antibodies to CD3 and CD46 to express granzyme A and kill activated CD4+ and CD8+ T cells and other cell types in a perforin-dependent, Fas-FasL-independent manner.34 Activation of mouse Treg cells also results in upregulation of granzyme B expression and one report has claimed that Treg cells kill responder cells by a perforin-independent, granzyme B-dependent mechanism35 and that granzyme B-deficient Treg cells had reduced suppressive activities in vitro. Cao et al36 demonstrated that 5%-30% of Treg cells in a tumor microenvironment express granzyme B and these Treg cells are lytic for NK cells and cytotoxic T lymphocytes in a granzyme B- and perforin-dependent manner.
Most studies have demonstrated that Treg cells mediate suppression by inhibiting the induction of IL-2 mRNA (and mRNA for other effector cytokines) in the responder Foxp3− T cells.37 Treg cells express all three components of the high-affinity IL-2R (CD25, CD122, and CD132) and IL-2 is essential not only for Treg cell homeostasis in vivo,38 but also for their efficient suppressor function in vitro.39 One study has raised the possibility that Treg cells may compete with Foxp3− T cells for IL-2, consume it, and inhibit the proliferation of Foxp3− T cells, resulting in a form of apoptosis dependent on the proapoptotic factor Bim.40
Extracellualr nucleotide metabolism
Extracellular ATP functions as an indicator of tissue destruction and exerts inflammatory effects on DCs. Catalytic inactivation of extracellular ATP by CD39 represents an anti-inflammatory mechanism that may be used by Treg cells to prevent the deleterious effects of ATP on antigen-presenting cell function.41 CD39 is the cell surface prototypic member of the ecto-nucleoside triphosphate diphosphohydrolase (E-NTPDase) family. Biological actions of CD39 are a consequence (at least in part) of the regulated phosphohydrolytic activity on extracellular nucleotides. This ecto-enzymatic cascade, in tandem with CD73, also generates adenosine and has major effects on both P2 and adenosine receptor signaling. Potential negative signals include upregulation of intracellular cyclic adenosine monophosphate, which leads to suppression of T cell proliferation and IL-2 secretion, or the generation of pericellular adenosine catalyzed by CD39 and CD73 expressed by Foxp3+ Treg cells.42 The cyclic adenosine monophosphate signal pathway was also found to be involved in apoptosis of various lymphoma cells.43 Dwyer et al14 reported CD39 serves as an integral component of the suppressive machinery of Treg cells, acting, at least in part, through the modulation of pericellular levels of adenosine. They have also shown that the coordinated regulation of CD39/CD73 expression and of the adenosine receptor A2A activates an immuno-inhibitory loop that differentially regulates Th1 and Th2 responses. These data indicate the potential of CD39 and modulated purinergic signaling in the co-ordination of immunoregulatory functions of DC and Treg cells.14
Targeting dendritic cells
CTLA-4 on the surface of Treg cells downregulates or prevents the upregulation of CD80 and CD86, the major costimulatory molecules on antigen-presenting cells. Similarly, LAG-3 on Treg cells can interact with MHC class II on antigen-presenting cells, and binding of LAG-3 to MHC class II molecules expressed by immature DCs results in an inhibitory signal that suppresses DC maturation and immunostimulatory capacity. Indoleamine-2,3-dioxygenase (IDO) has been implicated as a normal, endogenous mechanism of peripheral tolerance and immunosuppression in a number of settings.20 Grohmann and colleagues showed that Tregs can trigger high levels of functional IDO expression in mouse DCs in vitro.44 This occurred through binding of CTLA-4 on Tregs to CD80 and CD86 on DCs, which transduced a signal in the DCs that upregulated IDO protein expression and functional enzymatic activity.45 A similar induction of IDO following interaction of CTLA4-CD80 and/or CD86 has been shown in human monocyte-derived DCs.46 Therefore, IDO might function as one downstream mechanism by which CTLA4+ Tregs mediate immunosuppression.47
TREG CELLS IN CANCERS
Increased frequencies of Foxp3+ Treg cells have been documented in tumor tissues and peripheral blood of patients with several types of cancer consistent with a role in tumor escape from immunological control. And also, not only Treg cell numbers, but also their functions were different between tumor patients and healthy control. Kobayashi et al48 did immunohistochemical comparative studies to examine the infiltration of Foxp3+ Treg cells. Their results showed the prevalence of Treg cells was significantly higher in hepatocellular carcinoma than in the nontumorous liver and the patient group with a high prevalence of Treg cells infiltrating hepatocellular carcinoma showed a significantly lower survival rate. It was also reported there was CD4+CD25+Foxp3+ surround epithelial tumor aggregates.49 Griffiths et al50 investigated the presence of Treg cells systemically and in situ in previously untreated patients with renal cell carcinoma. They showed that there is a significant increased frequency of CD4+CD25high T cells in renal cell carcinoma patients compared to normal donors. The early follow up data also showed an association between higher peripheral blood regulatory T-cell count and adverse overall survival. These data confirm the increase of CD4+CD25+ Treg cells in malignant tumor patients and provide impetus to further investigate modulation of Treg cells activity in these patients as part of therapy.
There is experimental evidence to support a grim scenario in which T cells in tumor tissue or draining lymph nodes can be perverted into regulatory T cells.22 When stimulated with malignant pleural effusion or tumor cellderived supernatants, a fraction of natural CD4+CD25− T cells was converted into CD4+CD25+Foxp3+T cells which had the functional activity resembling natural CD4+CD25+ Treg cells with anergy and immunosuppression.
Tumor cells produce a variety of soluble factors involved in tumor progression and in Treg-related immunosuppression. These factors include several cytokines such as the TGF-β and IL-10. It has been proposed that TGF-β can induce the differentiation of peripheral CD4+CD25− precursors into functional CD4+CD25+ Treg cells through the induction of Foxp3+ transcription factor.51 Local production of TGF-β is a key factor in transforming effector T cells locally into suppressive Treg cells. The experimental results supplied by Zheng et al52 indicate that TGF-β secreted by ovarian carcinoma cells owns vital function in the process of converting peripheral CD4+CD25− T cells into CD4+CD25+ regulatory T cells, which may provide one immunotherapeutic target for ovarian cancer. Another convincing data concerning the role of CD4+CD25+ regulatory T cells in human cancer comes from the work of Curiel et al,53 who showed that the presence of such Treg cells in advanced ovarian cancer correlated with reduced survival. In addition, TGF-β is one of the factors produced by Treg and by Tr1 cells and is involved in their ability to suppress T cell proliferation and function.54
IL-10 is an immune-suppressive and anti-inflammatory cytokine, which is produced by several tumor cell types55 and is also produced by other cells with immunosuppressive activity such as tumor-associated macrophage (TAM) and Treg cells or by Th2 cells. Thus tumor-infiltrating or peripheral blood lymphocytes from cancer patients have been reported to secrete high levels of IL-10,56 which may be involved in tumor related immunosuppression through the stimulation of Treg cell activity.57 Moreover, IL-10 is involved in the development of a subset of Treg cells termed Tr1 which develop in the periphery following antigen binding in a tolerogenic environment and is also produced by Tr1 cells, thus mediating their immune-suppressive activity.58 Blockade of IL-10 by anti-IL-10 antibodies allowed to overcome tumor-induced immunosuppression in a mouse myeloma model.59
Over the last years a number of reports have described Treg cells can migrate into tumors and suppress effective anti-tumor responses in the tumor microenvironment, thus contributing to the prosperity and growth of human tumors. Chemokines are a superfamily of chemotactic cytokines that control leukocyte migration via G protein-coupled receptors expressed on target cells.60 Treg migration was dependent on the chemokine receptors CCR2, CCR4, and CCR5 and P- and E-selectin ligands.61 The chemokine receptors CCR4 and CCR8 were found to be functionally expressed on human peripheral blood CD4+CD25+ Treg cells.62 In patients with ovarian carcinoma, the host response to the tumor was shown to be inhibited by Foxp3+CCR4+ Treg cells that were recruited into the tumor as a result of the tumor-derived CCR4 ligands, CCL17 and CCL22.53 CCR4 plays an important role in Treg cell trafficking in LNs and that this is critical for Treg cell suppressive function in vivo.63 Immunohistochemical analysis of FOXP3 in primary breast tumors showed that a high number of tumor-infiltrating regulatory T cells which could be selectively recruited through CCL22/CCR4.64
Treg CELLS IN CANCER IMMUNOTHERAPY
Treg cells are considered inhibitors of anti-tumor immunity and CD4+CD25+Foxp3+ regulatory T cells have been considered a candidate for cancer immunotherapy for over a decade. Attempts to block or eliminate Treg cells have been made by the use of chemotherapy and strategies aimed at blocking Treg cells induction and migration may be clinically applicable, as suggested by experimental evidences in tumor models.
Blockade or elimination of Treg cells
One new approach to cancer therapy is based on the adoptive transfer of tumor-specific cytotoxic T cells and anti-CD25 antibodies. A combination of Treg cell-depletion using anti-CD25 mAbs and cytotoxic T lymphocytes administration is a feasible approach for treatment of cancers which warrants further exploration in the clinical setting.65 Administration of FR4 monoclonal antibody specifically reduced Treg cells, provoking effective tumor immunity in tumor-bearing animals, whereas similar treatment of normal young mice elicited autoimmune disease.18 CTLA-4 functions as a negative regulator of endogenous and vaccine-induced antitumor immunity. Periodic infusions of anti-CTLA-4 antibodies after vaccination with irradiated, autologous tumor cells engineered to secrete GM-CSF (GVAX) generate clinically meaningful anti-tumor immunity without grade 3 or 4 toxicity in a majority of metastatic melanoma patients.66 Triggering of GITR in vivo by means of anti-GITR agonistic antibodies67 or by gene transfer of GITR-L by adenoviral vectors68 is capable of evoking anti-tumor immunity and of eradicating established tumors. In addition, co-administration of agonistic anti-GITR and antagonistic anti-CTLA-4 antibodies could act synergistically and trigger immune rejection of more advanced tumors.67 Agonistic anti-OX40 antibodies blocked the immunosuppressive activity of Treg cells in vitro and in vivo in a GVHD model22 and also triggered tumor immunity.69
Increased Treg activity facilitates tumor growth70 whereas depletion of Tregs allows effective antitumor immune responses that would otherwise be undetectable or ineffectual.71 Imai et al72 reported that IL-2-induced antitumor immunity is enhanced by Treg-cell depletion and is due to expansion of the tumor-infiltrating cytotoxic CD8+ T cell population in mouse colon adenocarcinoma model.
Blockade of Treg cell induction and migration
TGF-β can induce the differentiation of peripheral CD4+CD25− precursors into functional CD4+CD25+ Treg cells through the induction of Foxp3+ transcription factor, therefore, the blockade of TGF-β function may benefit anti-tumor immunity. Indeed, several studies have shown that suppression of TGF-β functions by the use of anti-TGF-β antibodies73 or soluble TGF-β RII74 may overcome TGF-β immunosuppressive effects and restore tumor immunity. SB-431542 is a small ATP-mimetic inhibitor of the kinase activity associated with members of the Activin Receptor-like Kinase family involved in TGF-β signaling.75 This drug can inhibit tumor growth and motility by blocking TGF activity.76 Another new approach to suppress TGF-β function in vivo is based on the use of adenoviral vectors, inducing the expression of Smad-7, an inhibitor of the TGF-β pathway, which inhibited metastatic tumor growth in nude mice.77
CD4+CD25+ regulatory T cells and Th17 cells are subpopulations of CD4+ T cells. Many Tregs, just like naïve CD4 T cells, can be differentiated into various types of helper T cells. T cells exposed to TGF-β upregulate Foxp3+ and become induced Treg cells; however, when cultured with TGF-β and IL-6, naïve T cells generate Th17 cells with highly morbific activities.78 The Tim gene family has recently been identified as being potentially involved in the regulation of effector T cell responses. In vitro, anti-Tim-1 mAb stimulation might amplify the proliferative response of activated Tregs while Foxp3+ transcription factor expression was downregulated in the presence of agonist anti-Tim-1 mAb.28 We can infer that the use of Th17 cells, IL-6 and Tim-1 may provide new immunotherapy methods for treatment of autoimmune disease and malignancies.
Tumors evade immune destruction by actively inducing immune tolerance through the recruitment of Treg cells. In both human pancreatic adenocarcinoma and a murine pancreatic tumor model, tumor cells produce increased levels of ligands for the CCR5 chemokine receptor and, reciprocally, CD4+Foxp3+ Tregs, compared with CD4+Foxp3− effector T cells, preferentially express CCR5. When CCR5/CCL5 signaling is disrupted, either by reducing CCL5 production by tumor cells or by systemic administration of a CCR5 inhibitor, Treg migration to tumors is reduced and tumors are smaller than in control mice.79
In conclusion, it is now clear that several mechanisms of immunosuppression are involved in Treg-mediated tumor immunity. Immunosuppressive cells, especially Treg cells, are found in the tumor microenvironment and in cancer patients. Distinct immunosuppressive molecules expressed by Treg cells or diverse molecules related to Treg cell induction or migration represent potential drug targets in cancer immunotherapy. The identification of molecular components of the Treg related immunosuppressive network is still ongoing and its complexity appears increasingly evident. Several drugs or biological approaches are now available to interfere in this network and to overcome immunosuppression, allowing the induction of effective immune responses to tumor antigens. However, whether approaches aimed at subverting immunosuppression, alone or in combination with immune-stimulatory cytokines, cancer vaccines or traditional chemotherapy, will be effective in cancer patients, is one of the crucial issues that are currently being addressed. The potential exploitation of some new molecules related to Treg cell function or migration, such as CD39, CD103 and CCL22/CCR4, in tumor immunotherapy still need to be explored.
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