Differentiation and function of Foxp3+ effector regulatory T cells

Review

Differentiation and function of Foxp3+ effector regulatory T cells Erika Cretney1,2, Axel Kallies1,2, and Stephen L. Nutt1,2 1 2

The Walter and Eliza Hall Institute of Medical Research, Parkville, Victoria, Australia Department of Medical Biology, The University of Melbourne, Parkville, Victoria, Australia

Regulatory T (Treg) cells are essential for immunological tolerance and homeostasis. Although forkhead box (Fox)p3 is continually required to reinforce the Treg cell program, Treg cells can also undergo stimulus-specific differentiation that is regulated by transcription factors typically associated with the differentiation of conventional CD4+ T cells. This results in effector Treg (eTreg) cells with unique migratory and functional properties matched to the stimulus that elicited the initial response. Despite this functional and transcriptional heterogeneity, expression of the transcription factor B lymphocyte-induced maturation protein (Blimp)-1, a key player in late B cell and conventional T cell differentiation, is common to all eTreg cells. Here, we discuss the factors that control the differentiation of eTreg cells and their importance in disease settings. The phenotype and function of eTreg cells Our knowledge of Treg cell heterogeneity, functional specialization, and transcriptional programming has dramatically increased since Sakaguchi’s seminal discovery in 1995 that a population of CD4+CD25+ T cells (now known as Treg cells) could prevent autoimmunity in mice [1]. More recently it has become evident that a series of markers can be used to identify functionally distinct subpopulations of Treg cells. Similar to conventional T cells, the majority of Treg cells in lymphoid organs express the homing receptors CD62L and chemokine CC receptor (CCR)7, allowing them to recirculate through lymphoid tissues. Despite their expression of moderate levels of CD44, the majority of these cells appear to be naı¨ve thymus-derived Treg cells [2,3]. In addition, a fraction of Treg cells in lymphoid organs and most of the Treg cells in non-lymphoid organs show downregulation of CD62L, high expression of CD44, inducible T cell costimulator (ICOS), glucocorticoid-induced tumour necrosis factor receptor (GITR) and varying expression of markers such as CD69, CD103, and CD38, consistent with an effector phenotype, while maintaining Foxp3 expression [4–6]. We have recently shown that a large fraction of these cells, which we term eTreg cells, also express the transcription factor Blimp-1 and the anti-inflammatory cytokine interleukin (IL)-10 [7]. In humans, naı¨ve Treg cells are FOXP3loCD45RA+ CD45RO–, whereas eTreg cells are FOXP3hiCD45RA– CD45RO+ and express both the Fas receptor (CD95) and Corresponding authors: Cretney, E. ([email protected]); Kallies, A. ([email protected]) Keywords: regulatory T cell; transcription; B lymphocyte-induced maturation protein-1; interleukin-10; differentiation; autoimmune disease.

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cytotoxic T lymphocyte antigen (CTLA)4 [8], and increase in proportion with age [9]. The human eTreg cell compartment also contains a population of ICOS+IL-10+ Treg cells [10] similar to the eTreg cells described above. Foxp3-expressing Treg cells exert their immunoregulatory functions through a variety of effector mechanisms, such as upregulation of CTLA-4, direct killing of antigenpresenting cells or T cells, consumption of IL-2, and the production of immunosuppressive cytokines such as IL-10, transforming growth factor (TGF)-b, IL-35 and galectin-1 [11–13]. Importantly, the phenotype of Treg cells and the mechanisms they use to exert their suppressive activity differ depending on the disease setting, inflammatory status of the local environment, and their anatomical localization. Diverse differentiation potential of Treg cells The impact of the cytokine milieu on CD4+ T helper (TH) cell differentiation is well documented [14]. Interferon (IFN)-g and IL-12 drive the differentiation of naı¨ve CD4+ T cells into TH1 cells that are important in host defense against intracellular pathogens. IL-4 promotes TH2 cell differentiation that is important for the eradication of extracellular parasites, and IL-6 and TGF-b drive differentiation of TH17 cells that play important roles in the defense against yeast, fungi, and extracellular bacteria. Finally, T follicular helper (TFH) cells provide ‘help’ to B cells and are essential for the germinal center response and efficient plasma cell differentiation. Recent findings indicate that the cytokine signals that polarize conventional T cells not only have an impact on TH cell differentiation but also on the functional specialization of Treg cells under these conditions [15–18]. This emerging paradigm of paired differentiation between TH and Treg cells may also extend to lymphocytes in nonlymphoid tissues because the recently described fat-resident Treg cells differ markedly in their gene expression profile compared to Treg cells found at other sites [19]. Thus, Treg cells display extensive phenotypic and functional diversity that closely parallels the differentiation state of conventional T cells that occupy the same regulatory environment or tissue niche. Transcriptional diversity of Treg cells that restrain TH cell driven inflammatory responses The finding that IFN regulatory factor (IRF)4 (a transcription factor required for the differentiation of TH2 and TH17 cells) is essential for the control of TH2-driven autoimmunity by Treg cells was the first evidence that Treg cells can

1471-4906/$ – see front matter ß 2012 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.it.2012.11.002 Trends in Immunology, February 2013, Vol. 34, No. 2

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Figure 1. Differentiation of effector CD4 T cells and regulatory T (Treg) cells. Upon T cell receptor (TCR) stimulation, naı¨ve CD4 T cells can differentiate into functionally distinct effector cell subsets [T helper (TH)1, TH2, and TH17] depending on the specific inflammatory conditions present. This differentiation is driven by key transcriptional regulators including T-bet for TH1, GATA binding protein 3 (GATA3) and interferon regulatory factor (IRF)4 for TH2 and RAR-related orphan receptor gamma (RORgt) for TH17 differentiation. T follicular helper (TFH) cells also differentiate from naı¨ve T cells; a process driven by the transcriptional regulator B cell lymphoma 6 (Bcl6). B lymphocyte-induced maturation protein (Blimp)-1 is expressed in many effector T cell subsets including TH1 and TH2. Under inflammatory conditions naı¨ve Treg cells can differentiate into distinct effector Treg (eTreg) subsets (including TH1-Treg, TH2-Treg, and TH17-Treg cells), by co-opting expression of transcription factors including T-bet, IRF4 and signal transducer and activator of transcription (STAT)3 that were previously considered exclusive for conventional T cells. Similarly, naı¨ve Treg cells can also differentiate into Follicular regulatory T (TFR) cells and fat-resident Treg cells in processes driven by Bcl6 and peroxisome proliferator-activated receptor (PPAR)-g, respectively. A requirement for IRF4 expression has been documented for TH1-Treg and TH2-Treg cells, but by analogy to TH cell subsets, IRF4 may also be needed for TH17Treg cells and TFR cells. A feature common to all eTreg cells is the expression of interleukin (IL)-10 and Blimp-1.

co-opt expression of transcription factors previously considered to be exclusive for conventional T cells [15] (Figure 1). This concept was extended by the subsequent observation that T-bet, a transcription factor essential for the differentiation of TH1 cells, can be upregulated by Treg cells [17], challenging the paradigm that master regulatory factors such as T-bet and Foxp3 are expressed in a mutually exclusive fashion. T-bet controls Treg cell migration [via chemokine CXC receptor (CXCR)3], homeostasis, and function during type 1 inflammation, thereby promoting suppression of TH1 responses in vivo. Recently, GATA-binding protein 3 (GATA3), a transcription factor essential for TH2 cell differentiation, was found to play important roles in peripheral Treg cell differentiation [20,21]. In the steady state, GATA3 is expressed relatively selectively in Treg cells of the gastrointestinal tract and dermis [20]. GATA3-deficient Treg cells develop normally but are defective in their ability to restrain both spontaneous and lymphopenia-induced inflammatory responses, resulting in increased TH2 and TH17 cell differentiation and tissue damage [20–22]. GATA3 appears to function in a large molecular weight

complex with Foxp3 to control a subset of Foxp3 targets as well as to maintain high Foxp3 gene expression in Treg cells [22]. Treg-specific deletion of signal transducer and activator of transcription (STAT)3, a transcription factor that is required for TH17 effector cell differentiation, results in selective dysregulation of TH17 responses, with mice succumbing to fatal colitis [16]. Expression of CCR6, which mediates the migration of TH17 and Treg cells to inflammatory sites, is decreased in STAT3-deficient Treg cells, potentially impairing their ability to migrate to the gut to suppress inflammation. Alternatively, development of colitis in mice harboring STAT3-deficient Treg cells could be attributed to suboptimal Treg IL-10 production [23]. Foxp3 itself is required to restrain STAT3-mediated expression of TH17 genes through its interaction with the phosphorylated form of STAT3 and recruitment to the Il6 and Tgfb1 promoters. Based on chemokine receptor expression, distinct populations of human TH1-, TH2-, TH22-, and TH17-like eTreg cells have recently been identified [24]. These populations differ in their cytokine production and ability to respond to 75

Review antigens associated with TH1 (human cytomegalovirus) and TH17 (Candida albicans) responses. Analogous to murine Treg cells that co-opt expression of TH cell transcription factors, TH1- and TH17-like human Treg cells expressed T-bet and RAR-related orphan receptor gamma (RORgt) respectively, and GATA-3 is more highly expressed in TH2-like Treg cells. Together, these findings indicate that during inflammation, by using the same TH cell transcription factors, the immune system generates polarized eTreg cells that are molecularly geared for optimal suppression under the specific inflammatory conditions in which they are generated. Follicular regulatory T (TFR) cells TFH cells are a population of CD4+CXCR5highprogrammed death 1 (PD-1)high cells that require the transcription factor B cell lymphoma 6 (Bcl6) for their differentiation [25,26]. As a result of their key role in promoting antibody responses, TFH cell function needs to be tightly regulated to prevent autoimmunity [27]. Recently, a population of eTreg cells termed TFR cells were identified [18,28]. These cells suppress T and B cells in the germinal center, and similar to TFH cells, express high levels of the follicular homing receptor CXCR5, as well as PD-1 and CXC chemokine ligand 13 (Cxcl13). Analogous to the eTreg cells described above that utilize components of TH cell transcriptional machinery to restrain polarized inflammatory responses, TFR cells require Bcl6 for their development (Figure 1). Furthermore, they share a common requirement for developmental cues with TFH cells, such as signaling lymphocyte activation molecule (SLAM)-associated protein (SAP), CD28, and B cells. Consistent with the features of eTreg cells described by our group, TFR cells express Blimp-1 and IL-10 as well as GITR, CTLA4, and ICOS, and derive from Foxp3+ thymic Treg cells [18,28]. By regulating TFH cell number, TFR cells can tune the germinal center response, thereby preventing unwanted production of autoantibodies and autoimmune destruction. Both IRF4 and STAT3 are also required for TFH cell differentiation, thus, it remains to be tested whether some of the ascribed roles of these factors in controlling TH2 and TH17 cell mediated autoimmune pathology result from impaired TFR cell function. Fat-resident Treg cells A population of adipose-tissue-resident Foxp3+ Treg cells has recently been described by Feuerer et al. [19]. Gene expression profiling has revealed that these fat Treg cells, while maintaining a core Treg cell signature, differ markedly from Treg cells found in other sites of the body. Fat Treg cells express high amounts of Il10 and Blimp1 suggesting that they are part of the growing eTreg cell family [29]. Strikingly, the fat Treg cells express the major adipocyte transcription factor peroxisome proliferator-activated receptor (PPAR)-g and several genes involved in lipid metabolism, migration, and extravasation [30]. Visceral fat Treg cell numbers are reduced in obese compared to lean mice, and decreased frequency of fat Treg cells correlates with increased insulin resistance [19,31,32]. Interestingly, fat-resident Treg cells exhibit a unique T cell receptor repertoire, suggesting that they do 76

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not derive from conventional T cells within adipose tissue. Decreased percentages of omental versus subcutaneous fat Treg cells have also been reported in obese humans, and an increased body mass index correlates with a decreased percentage of omental fat Treg cells. Together, these data suggest a role for fat-resident Treg cells in the suppression of inflammation in adipose tissue and show that the capacity of Treg cells to undergo peripheral diversification in response to the local environmental milieu also extends to nonhematopoietic tissues. Importance of IL-10 production by eTreg cells IL-10 is an immunosuppressive cytokine produced by a wide variety of innate and adaptive cells including Treg cells [33]. Although IL-10 production by Treg cells is not required to suppress systemic autoimmunity, it is essential to limit inflammation at environmental interfaces such as in the lung, skin, and colon [34]. Foxp3+IL-10+ cells are enriched in gut-associated lymphoid tissue of the small intestine and colon, and display a cell surface phenotype characteristic of eTreg cells [35]. This predominance of Foxp3+IL-10+ Treg cells in the gut may explain why mice with Treg cell specific ablation of IL-10 develop spontaneous intestinal inflammation [34]. Importantly, IL-10 acts as an effector molecule for eTreg cells that are polarized to suppress TH1, TH2, and TH17 inflammatory responses as well as TFR and fat-resident Treg cells, suggesting that all these eTreg subsets may use a common differentiation program (Figure 1). Blimp-1 expression by eTreg cells Blimp-1 is a transcriptional repressor and master regulator of terminal B cell differentiation [36,37], which also has important functions in T cells including Treg cells [7,38–41]. Our recent research has shown that Blimp-1 expression defines eTreg cells as a distinct population of mature Treg cells that produce IL-10 and display an effector phenotype [7] (Figure 2). Thus, we propose that Blimp-1 provides a common molecular signature for eTreg cells regardless of their localization or polarization. In lymphoid organs Blimp-1 is expressed in a minority of total Treg cells (10–20%), whereas in mucosal sites such as the gastrointestinal tract and lung fluid, most Treg cells express Blimp-1. Blimp-1-expressing Treg cells produce IL-10, show high expression of CD103, CD44, ICOS, GITR, CD38, and CD69, and low expression of CD62L consistent with the eTreg cell phenotype [7]. Blimp-1 is essential for Il10 expression by eTreg cells and regulates genes required for their function (ICOS), migration (CCR6), and survival [B cell lymphoma 2 (Bcl2)]. Blimp-1 limits Bcl2 and CCR6 expression by Treg cells, which may account for the accumulation of eTreg cells at mucosal sites in the absence of Blimp-1. Whether eTreg cells can be further divided in distinct differentiation stages is at present unclear, although it is noteworthy that some markers, including CD103 and killer cell lectin-like receptor subfamily G1 (KLRG1), are only present on a proportion of eTreg cells, and may delineate the most terminally differentiated state of a Treg cell [7,42].

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Figure 2. Requirement of interferon regulatory factor (IRF)4 and B lymphocyte-induced maturation protein (Blimp)-1 for effector regulatory T (eTreg) cell differentiation. Wild type (WT) naı¨ve Treg cells lack Blimp-1 expression and do not produce interleukin (IL)-10. Upon activation by T cell receptor (TCR) stimulation, IL-2, and inflammatory signals, IRF4 is required for the acquisition of an eTreg cell phenotype, characterized by IL-10 and Blimp-1 expression. Functionally mature Blimp-1+ eTreg cells have limited proliferative potential and lifespan and ultimately undergo apoptosis. Blimp-1-deficient Treg cells can differentiate into eTreg cells, but cannot express IL-10 and have elevated chemokine CC receptor (CCR)6 and B cell lymphoma 2 (Bcl2) expression, facilitating their survival and accumulation in inflammatory sites and nonlymphoid tissues. IRF4 is dispensable for naı¨ve Treg cell homeostasis, but is required for the induction of IL-10 and Blimp-1 expression as well as the differentiation of all eTreg cells.

Regulation of eTreg cell differentiation by IRF4 and Blimp-1 The observation that Blimp-1 expression defines eTreg cells raised the question of how the activation of the Blimp-1 gene is regulated in Treg cells. IRF4 represents an excellent candidate, because it activates Blimp-1 expression in late B cell differentiation [43]. Strikingly, IRF4-deficient Treg cells completely lack not only Blimp-1 expression, but also the entire eTreg cell compartment in all organs examined [7]. Although this finding demonstrates that IRF4 functions directly upstream of Blimp-1 in the differentiation of eTreg cells, it also suggests a much broader function of IRF4 in eTreg cells, beyond the role in TH2-polarized environments previously reported by Zheng et al. [15] (Figure 2). In that study, mice with a Treg cell specific deletion of IRF4 were shown to develop autoimmune disease, with increased production of IL-4 and IL-5, and elevated levels of serum IgG1 and IgE [15]. Although TH2 responses were undoubtedly dysregulated in these mice, the data were also consistent with unrestrained TFH, TH1, and TH17 responses, as seen by increased germinal center activity and elevated IFNg and IL-17 production. This is perhaps not surprising because IRF4 plays major roles in CD4+ T cells, not only in TH2 polarization but also in TH9, TH17, and TFH cell differentiation [44] (Figure 1). In keeping with this conclusion, IRF4deficient Treg cells failed to upregulate T-bet and generate eTreg cells under TH1 inflammatory conditions [7]. Gene expression analysis has revealed that IRF4 and Blimp-1 are required for Il10 expression and share common targets in eTreg cells, including Ccr6. Insights into how these factors program eTreg cell differentiation comes from the analysis of the chromatin structure of the Il10 gene in Treg cells, in which both IRF4 and Blimp-1 are required for active histone modifications, whereas IRF4 is specifically required for the

removal of repressive histone 3 lysine 27 trimethylation marks [7]. Mice containing IL-10-deficient Treg cells [34] have a defect in controlling mucosal immune responses and develop spontaneous colitis but do not develop systemic autoimmune disease, such as in mice harboring IRF4deficient Treg cells. These differences in disease development indicate that eTreg cells have IL-10-independent functions critical for the protection against systemic autoimmune disease. This conclusion is also in line with earlier studies, which demonstrated that Blimp-1-deficient Treg cells were functional in a standard suppression assay in vitro and during T cell induced colitis in vivo [38]; two models that are independent from Treg cell derived IL-10 [45,46]. Thus, although Blimp-1 plays an important role in regulating IL-10 production of eTreg cells, IRF4 acts upstream of Blimp-1 and is critical for differentiation of eTreg cells (Figure 2). eTreg cells and Blimp-1 expression in humans As outlined above, human eTreg cells are FOXP3hiCD45RO+CD45RA– and can be distinguished from their naı¨ve counterparts that are FOXP3loCD45RA+CD45RO– [9]. Expression of CD127, the a chain of the IL-7 receptor [47], further allows FOXP3+ Treg cells to be distinguished from activated CD4+CD25+ T cells. Treg cells express low levels of CD127, whereas conventional T cells express high levels of CD127. This is paralleled by a distinct methylation status of the FOXP3 gene, which is demethylated in a conserved region of intron 1 in Treg cells but methylated in conventional T cells [48]. More detailed analysis of human FOXP3+ T cells have provided evidence for the existence of a subset of Treg cells with high expression of IL-10 and ICOS that may be the human counterpart of the mouse Blimp-1-expressing eTreg cell [10]. ICOS+FOXP3+ cells 77

Review have been identified in human peripheral blood, thymus, and secondary lymphoid tissues. These cells suppress dendritic cell function in an IL-10-dependent manner and T cells in a TGF-b-dependent manner. Blimp-1 expression has also recently been confirmed in a population of human peripheral blood Treg cells (Seddiki, unpublished). Similar to the eTreg cell phenotype described above, these cells were ICOShiFOXP3+ and expressed IL-10. Whether Blimp-1 expression is indeed a common signature of all human eTreg cells remains to be elucidated. Furthermore, it is now crucial to determine whether eTreg cells have distinct functions in different disease settings and how these cells can be targeted therapeutically. Treg cells and disease Treg cells play important roles in restraining autoimmune disease, preventing organ transplant rejection, and suppressing inflammation. The immunosuppressive activities of Treg cells can also play a detrimental role in the immune response against some cancers and during infection with pathogens such as Listeria monocytogenes and Mycobacterium tuberculosis. To date most studies have only examined the Treg cell population as a single entity, with little characterization of eTreg cells, which may represent the most biologically potent population. Only an in-depth understanding of the eTreg cell subsets involved and their functions in specific pathological situations may allow us to manipulate these cells effectively in patients to treat disease. Autoimmune diseases The central importance of Treg cells in controlling autoimmunity is highlighted by the lethal autoimmune disease in mice and humans that lack Treg cells, due to inherited mutations in Foxp3 [49]. More subtle functional defects have been uncovered in Treg cells isolated from patients with autoimmune diseases including rheumatoid arthritis (RA), psoriasis, type I diabetes (T1D), myasthenia gravis, and multiple sclerosis (MS) [50–54]. The model of parallel differentiation of conventional effector and eTreg cells in response to environmental queues such as inflammation predicts that the autoimmune responses outlined above would normally be suppressed by an appropriate eTreg cell population, although evidence to support this view is only now emerging. For example, some of the functional defects observed in Treg cells isolated from RA patients have been shown to be tumor necrosis factor (TNF) dependent, because anti-TNF monoclonal antibody (mAb) therapy (infliximab) induces a new population of eTreg cells that suppress via IL10 and TGF-b [50,55,56]. In mouse models, IL-12 has been shown to induce a population of IFN-g-expressing TH1-type eTreg cells during experimentally induced colitis [57]. Understanding how eTreg cell differentiation is induced and why in certain situations eTreg cells fail to prevent autoimmune disease should enable targeted treatment to restore effective Treg cell suppression. Transplantation Treg cells play an important role in transplantation. In tolerant transplant recipients, numbers of Treg cells are higher in the blood [58–60] and at the graft site [61], 78

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strongly implicating Treg cells in the maintenance of tolerance. In support of this idea, Treg cells adoptively transferred from tolerant into naı¨ve mice protect subsequent grafts in the recipient mice from undergoing rejection [62]. The immunosuppressive activity of Treg cells has been shown to be important in both the graft site and in the draining lymph nodes (dLNs) [63–65], suggesting that Treg cells need to undergo phenotypic changes consistent with effector differentiation that allow tissue migration and function. In line with this conclusion, a recent study using an islet allograft model has shown that adoptively transferred Treg cells migrate from the blood to the graft site and subsequently to the dLNs. During this process these cells acquire a phenotype consistent with eTreg cells and are capable of prolonging graft survival [66]. Treg cells that fail to migrate to the graft site due to chemokine receptor or P- and E-selectin deficiency fail to protect grafts from rejection, demonstrating that migration from the graft to the dLNs is essential for optimal eTreg cell function. Infectious diseases Although Treg cells can play a protective role in the host defense against some pathogen infections, in other infections they play a detrimental role [67]. A series of studies has revealed that Treg cells can protect from immune pathology during infection with herpes simplex virus 2, lymphocytic choriomeningitis virus, or West Nile virus [68,69]. Experiments in which IL-2/IL-2 mAb complex injection was used to expand Treg cells (including eTreg cells) in vivo have also revealed a protective role for Treg cells against Plasmodium berghei (murine malaria) and Toxoplasma gondii parasite infection [70,71]. By contrast, Treg cell expansion or adoptive transfer of Treg cells increases pathogen burden during infection with certain bacteria including L. monocytogenes, Salmonella enterica and M. tuberculosis, whereas Treg cell ablation improves clearance [72–75]. M. tuberculosis infection that induces a TH1 inflammatory response leads to the recruitment and expansion of T-bet-expressing eTreg cells to the site of infection alongside T-bet-expressing TH1 effector T cells [17], illustrating the concept of parallel differentiation of the appropriate TH and eTreg cell populations in vivo. Similarly, infection by the parasite Heligmosomoides polygyrus induces a TH2 inflammatory response and subsequently eTreg cells that express GATA-3 [20]. Interestingly, Treg cells recovered from mice infected by M. tuberculosis express high levels of ICOS at the site of infection [74], suggesting that differentiation of eTreg cells is critical during that infection. Cancer Treg cells are powerful inhibitors of antitumor immunity and their accumulation is generally associated with poor prognosis in human carcinomas [76,77]. Treg cells facilitate tumor growth by directly suppressing antitumor immunity, thus, enabling progression of the primary tumor. Treg cells can also induce apoptosis in dendritic cells located in tumor dLNs, potentially inhibiting the priming of CD8+ T cells [78], and stimulate metastatic progression of some tumors via receptor activator of nuclear factor

Review kappa-B (RANK) signaling [79]. In cancer patients, tumorinfiltrating Treg cells have been reported to produce IL-10 and to express high levels of ICOS [80–82], suggesting that they are eTreg cells. Although more research in this area is clearly required, it is plausible that strategies can be developed that specifically modulate eTreg cells within the tumor microenvironment, rather than target Treg cells systemically. Given the deleterious autoimmunity associated with global Treg cell depletion, this would be of tremendous clinical importance. Concluding remarks Treg cells play both beneficial and harmful roles in the body in healthy physiological and in disease settings, and it has become evident that eTreg cells are major executers of the multiple functions of the Treg cell lineage. In order to target and manipulate Treg cells effectively in a clinical setting it is critical to expand our knowledge about the diverse populations of eTreg cells generated under inflammatory conditions or in specific tissues. This may enable targeting of distinct populations of Treg cells, while leaving untouched populations important for normal immune homeostasis and for protection from autoimmunity. Elucidating how the diverse repertoire of eTreg cells is generated and defining their unique and common functions will be paramount to manipulate Treg cells safely and effectively in therapy. Acknowledgments This work is supported by a National Health and Medical Research Council (NHMRC) of Australia Biomedical (Peter Doherty) Fellowship to E.C., and Australian Research Council Future Fellowships to A.K and S.L.N. This work was made possible through Victorian State Government Operational Infrastructure Support and Australian Government NHMRC Independent Research Institute Infrastructure Support (IRIIS) scheme.

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