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New twists of T cell fate: control of T cell activation and tolerance by TGF-β and NFAT
New twists of T cell fate: control of T cell activation and tolerance by TGF-b and NFAT Mark S Sundrud and Anjana Rao Protective and pathogenic immune responses were initially thought to be determined by the differentiation of naı¨ve T cells into Th1 and Th2 effector subsets and the immunosuppressive activity of thymic-derived regulatory T cells. It is now clear that naı¨ve T cells can also differentiate into ‘induced’ regulatory T cells or inflammatory T cells that secrete IL-17. These divergent T-cell subsets have opposing functions in imparting inflammation or tolerance, yet both developmental programs are controlled by the pluripotent cytokine transforming growth factor b and the transcription factor NFAT. Recent findings have begun to shed light on the mechanisms by which TGF-b and NFAT integrate multiple signaling inputs to determine the direction of naı¨ve T-cell differentiation. Addresses Department of Pathology, Harvard Medical School, and The CBR Institute for Biomedical Research, Inc, Boston, MA 02115, USA Corresponding author: Rao, Anjana ([email protected])
Current Opinion in Immunology 2007, 19:287–293 This review comes from a themed issue on Lymphocyte activation Edited by Ulrich von Andrian and Federica Sallusto Available online 12th April 2007 0952-7915/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2007.04.014
Introduction The immune system must balance the necessity of immune activation with the hazards of autoimmunity. Naı¨ve T cells recognize antigens through the T-cell antigen receptor (TCR), but distinguish self from foreign antigens and direct their subsequent differentiation on the basis of which cytokine and co-stimulatory receptors are simultaneously engaged. Pathogen recognition through Toll-like receptors (TLRs) on dendritic cells (DCs) results in inflammatory cytokine production and T-cell co-stimulation, promoting differentiation of effector T cells (Teffs). By contrast, self-antigens are normally presented in the absence of co-stimulation, which promotes tolerance through the induction of T-cell anergy and the gradual accumulation of regulatory T cells (Tregs). Tregs are central to the establishment of peripheral immune tolerance. Although Tregs encompass several diverse subsets that have unique properties [1], the best characterized Treg subset expresses the forkhead tranwww.sciencedirect.com
scription factor Foxp3 and imposes peripheral tolerance by suppressing Teff responses [2]. Foxp3-expressing Tregs were first described as arising through antigeninduced differentiation in the thymus (‘natural’ Tregs, nTregs), but it is now clear that they can also develop through the differentiation of naı¨ve T cells in peripheral lymphoid organs (‘adaptive’ or ‘induced’ Tregs, here abbreviated iTregs) [3,4]. Loss or mutation of FOXP3 (Foxp3) in both humans and mice is associated with multi-organ autoimmune disease, secondary to loss or decreased numbers of Tregs [2,5]. Recently, there has been a new appreciation of the pleotropic roles of the cytokine transforming growth factor b (TGF-b) in regulating T cell activation and tolerance (reviewed in [6]). TGF-b1-deficient mice develop a lethal wasting disease accompanied by multiorgan autoimmunity. Moreover, TGF-b regulates Foxp3 expression and iTreg differentiation in cell culture, potentially explaining the phenotypic similarities between TGF-b1-deficient and Foxp3-deficient mice. TGF-b also controls differentiation of the novel Th17 subset of effector T cells, characterized by production of the inflammatory cytokine interleukin (IL)-17 (reviewed in [7,8]). Given its dual role in driving iTreg and Th17 differentiation, TGF-b is a key player in inflammation and autoimmune disease. In this article, we review recent data on how differentiation of iTreg and Th17 is modulated by signals transduced through cell-surface receptors. We focus on the interplay between antigen, IL-2, TGF-b and co-stimulatory receptors, and the known or potential roles of downstream transcription factors in each of these pathways.
TGF-b: a jack of many trades TGF-b1, TGF-b2 and TGF-b3 constitute a family of biologically active polypeptides that modulate a diverse array of physiologic processes (reviewed in [9]). All three proteins bind multimers of type I and type II TGF-b receptors, transmembrane proteins that possess intrinsic serine/threonine kinase domains (reviewed in [10]). Phosphorylation of type I TGF-b receptors leads to recruitment and phosphorylation of receptor-associated Smad (R-Smad) proteins. Activated R-Smads form complexes with a single common Smad protein (Co-Smad4) and translocate to the nucleus where they directly activate or repress target gene transcription through cooperative interactions with other DNA-binding proteins [10,11]. Vertebrates also have two inhibitory Smads (I-Smad6 Current Opinion in Immunology 2007, 19:287–293
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and I-Smad7), which are transcriptionally upregulated by R-Smads and function in a negative-feedback loop to inhibit TGF-b receptor signaling, partly by competing with R-Smads for docking to TGF-b receptors and partly by recruiting E3 ubiquitin ligases to degrade components of the TGF-b receptor [10]. In addition, TGF-b receptors are coupled to the activation of several non-Smad signaling pathways, including ERK, SAPK/JNK and p70-S6 kinase [12]. The importance of these pathways for T-cell function is presently unclear. The role of TGF-b in Treg and Teff function has been studied in diverse genetic models (reviewed in [6]). Mice that lack the TGF-bRII receptor, or that express a ‘dominant-negative’ (signaling-incompetent) form of the TGFbRII receptor (TGF-bRII DN), only in CD4 and CD8 T cells, develop a severe autoimmune disease reminiscent of that observed in TGF-b1 / mice [13,14,15]. Thus T cells are key effectors of autoimmunity in these mice. The uncontrolled expansion of TGF-b-unresponsive T cells in peripheral lymphoid organs, and the resulting autoimmunity, is partly as a result of activation of Teffs through selfreactive TCR and IL2/IL15 receptors. Surprisingly, the mice are not impaired in development and function of nTregs, although they show a decreased number of Foxp3+ T cells in the periphery [14,16]. Loss of peripheral Tregs might be as a result of decreased exit of nTregs from the thymus, decreased survival of nTregs in the periphery, decreased differentiation of iTregs, failure to maintain Foxp3 expression in the absence of TGF-b signals, or a combination of these factors [13,14,16, 17]. TGF-b signaling is also required to render Teffs sensitive to suppression by Tregs: purified Teffs from TGF-bRII DN mice cause colitis in immunocompromised recipients even if adoptively transferred with wild-type nTregs [16]. Collectively, these data highlight TGF-b as a major regulator of T cell function (Figure 1), but, as is the case for many other pleotropic signaling molecules, the functional consequence of TGF-b signaling in T cells is context dependent, depending on the local extracellular milieu.
TGF-b and the iTreg/Th17 paradigm A striking feature of TGF-b is its ability to promote differentiation of two opposing T-cell lineages, iTregs and Th17 cells (Figures 1 and 2). Administration of TGFb to naı¨ve CD4+CD25 T cells in cell culture induces expression of Foxp3 mRNA and protein, but only if the cells are concomitantly stimulated through the TCR [3,4,18]. CD25 and cytotoxic T lymphocyte associated protein-4 (CTLA-4) are induced in parallel, and the resulting cells have regulatory function and can suppress immune responses in mice [4,19]. However, if the cultures contain the pro-inflammatory cytokine IL-6, Foxp3 induction is diminished or abrogated [20,21]. Instead, TGF-b and IL-6 cooperatively instruct expression of the orphan nuclear receptor RORgt, which in turn drives Current Opinion in Immunology 2007, 19:287–293
naı¨ve T-cell differentiation into the Th17 effector T-cell subset that produces IL-17, but not IL-4 or IFNg [22]. Both TGF-b and IL-6 are absolutely required for the development of Th17 cells in vivo, as evidenced by the paucity of Th17 cells in the periphery of mice that lack IL-6 [22] or that express TGF-bRII DN in T cells [23]. These mice are also significantly less susceptible to experimental autoimmune encephalitis, a disease thought to involve accumulation of Th17 cells in the central nervous system [23]. At the cellular level, IL-6 is primarily expressed by DCs, macrophages and B cells that are activated by TLR ligands, such as lipopolysaccharide or unmethylated (bacterial) CpG DNA [24,25]. By contrast, a prominent source of TGF-b is Treg cells themselves [6]. Thus, naı¨ve T cells exposed to antigen in the periphery are probably maintained in a tolerant state on account of suppressive contacts with Tregs that produce TGF-b. However, once ‘danger signals’ alert DCs to the presence of foreign pathogens in the form of TLR stimulation, IL-6 production by antigen-presenting DCs switches the tolerogenic program of TGF-b signaling to one that activates RORgt and fosters the development of inflammatory Th17 cells. Does IL-6 drive Th17 differentiation by acting directly on T cells, by enhancing co-stimulation, or both? Mice that lack IL-6 are defective in RORgt expression and Th17 cell development [22], but these responses have not yet been tested in naı¨ve T cells that lack components of the IL-6 receptor. However, IL-6 and STAT3 oppose Foxp3 expression and iTreg differentiation in cultured T cells in a cell-intrinsic manner, through a mechanism that does not appear to involve STAT3 binding to the Foxp3 promoter [20,26].
IL-2 and IL-23: cytokine modulation of the Treg/Th17 balance The role of IL-2 in Treg development is controversial. Tregs bear high levels of the IL2Ra subunit, CD25, and mice deficient in IL-2 or its receptor subunits develop autoimmune disease [27]. The bulk of the evidence indicates that, as with TGF-b, IL-2 is not essential for nTreg differentiation but is needed to maintain normal peripheral numbers of nTregs, perhaps by promoting thymic exit or peripheral survival [28,29]. IL-2 and IL-15, which both signal through a common receptor b chain, CD122, appear redundant in this respect. IL-2 and STAT5 also appear crucial for Foxp3 expression and iTreg differentiation, although this conclusion is qualified by incomplete deletion of STAT5 in naı¨ve T cells [26,30]. In contrast to STAT3, STAT5 binding to the Foxp3 locus can readily be demonstrated [26,30]. The influence of other cytokines on Th17 differentiation is more clear. Th17 differentiation is strongly inhibited by IL-12 or IFNg [21,22,24], establishing www.sciencedirect.com
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Figure 1
Pleotropic effects of TGF-b on T-cell activation, differentiation and tolerance. (a) TGF-b inhibits TCR-induced proliferation of naı¨ve T cells (TN) in cell culture and in mice, but inhibition can be overcome by co-stimulation through CD28 or by establishment of the IL-2/IL-2R autocrine growth pathway. (b) TGF-b interferes with the acquisition of Th1 and Th2 effector functions, even under optimal activation conditions where it no longer impedes T cell proliferation. (c) TGF-b is not required for nTreg development or function, but is needed to maintain Treg numbers in the periphery. (d) TGF-b induces Foxp3 and promotes the differentiation of induced Tregs (iTregs) in the absence of IL-6, whereas concomitant TGF-b and IL-6 signaling induces expression of the orphan nuclear receptor transcription factor RORgt, which drives the development of Th17 cells. (e) Intrinsic TGF-b signaling is essential for effector T cells to be suppressed by Tregs. Abbreviations: DP, double-positive (CD4+CD8+) thymocyte; SP, single-positive (CD4+CD8 or CD8+CD4 ) thymocyte. .
unambiguously that Th17 cells represent a different cellular lineage than Th1 cells, which require both these cytokines. Th17 cells express high levels of the multimeric IL-23 receptor, and IL-23-deficient mice display a selective reduction in peripheral Th17, but not Th1, cell numbers [7]. IL-23 is not required for Th17 cell differentiation, but rather serves a vital function in Th17 cell maintenance or function [21,31]. www.sciencedirect.com
Co-stimulation as a determining factor in iTreg and Th17 differentiation Co-stimulation is a key factor in converting TGF-b signals from anti- to pro-inflammatory. Th17 cells develop in mice only under conditions that promote inflammation (e.g. immunization with agonist peptides in the presence of adjuvant), and in cell culture only in the context of full TCR/CD28 activation [21,22,24,25,31]. Among the Current Opinion in Immunology 2007, 19:287–293
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Figure 2
Signaling requirements for iTreg/Th17 cell development. Intracellular signaling in response to TCR and co-stimulatory receptors (i.e. CD28, CTLA-4) results in the activation of NFAT and AP-1 transcription factors and promotion of Th17 development. TGF-bR signals promote both iTreg and Th17 development, activating both Smad and non-Smad pathways. Smad complexes upregulate I-Smad7 expression, which is opposed by Foxp3 at the level of transcription. Whether TGF-bR-derived non-Smad pathways modulate naı¨ve T cell differentiation to iTreg or Th17 cells is not known. IL-6R-mediated STAT3 signaling is important for RORgt expression and repression of Foxp3 expression through an unknown mechanism. Additional activation of STAT5 through the IL-2R enhances Foxp3 expression by direct STAT5 binding to the Foxp3 promoter.
relevant co-stimulatory receptors are CD28 and ICOS, which are expressed on resting and activated T cells, respectively: ICOS-deficient murine T cells do not produce IL-17 if cultured with DCs that lack the two CD28 ligands B7.1 (CD80) and B7.2 (CD86) [32]. By contrast, iTreg differentiation is preferentially evoked by noninflammatory conditions [33,34]. A slow conversion of Foxp3 cells to Foxp3+ iTregs occurs in TCR-transgenic mice during the course of repeated administration of antigen, in low doses and in the absence of adjuvant stimulation or TLR activation [34,35]. Phenoconversion of monospecific Foxp3 T cells to Foxp3+ iTregs also occurs during homeostatic repopulation of lymphopenic hosts [33]. In both these circumstances, T cells are unliCurrent Opinion in Immunology 2007, 19:287–293
kely to receive full TCR/CD28 stimulation, suggesting strongly that co-stimulation is not required for Foxp3 expression during iTreg differentiation. This scenario, however, does not apply in the thymus, because nTreg differentiation requires cell-intrinsic signals delivered through CD28 [36]. Co-stimulatory receptors (i.e. integrins, CD28 and ICOS) potentiate TCR-induced signaling pathways leading to activation of the transcription factors NFkB and activator protein-1 (AP-1) (Fos-Jun), but do not generally activate nuclear factor of activated T cells (NFAT), a calciumregulated transcription factor that drives distinct biological programs depending on its partner transcription www.sciencedirect.com
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factors on DNA [37,38]. T cell activation requires NFAT–AP-1 cooperation, whereas T-cell anergy occurs when NFAT is activated in the absence of AP-1 or NFkB, and T-cell tolerance mediated by regulatory T cells relies on formation of a cooperative NFAT–FOXP3–DNA complex [39,40,41]. Several anergy-associated NFAT target genes are known to have roles in immune tolerance: for instance, deletion of Itch, Cblb or the gene encoding the Itch and Nedd4-interacting protein is associated with autoimmune/inflammatory diseases in mice and rats [42–44].
Interplay among signaling pathways and transcription factors in iTreg and Th17 differentiation How do the same stimuli (antigen, IL-2 and TGF-b) signaling through the same cell surface receptors orchestrate two qualitatively different cellular responses depending on the context? We propose that co-stimulation is a critical factor. When naı¨ve T cells are exposed to low-dose antigen without co-stimulation, together with TGF-b and IL-2/IL-15 in the microenvironment, they induce STAT5, Smad and non-Smad signaling pathways as well as NFAT without AP-1 or NFkB. Together, this combination of transcription factors induces Foxp3, which in turn suppresses I-Smad7 expression, thus potentiating TGF-b signaling [18]. At the same time, NFAT in the absence of AP-1 upregulates Cbl-b [45], which downregulates TCR signals while promoting Foxp3 expression in TGF-b-stimulated cells [45,46]. Together, these pathways have the potential to set up a positive feedback loop that heightens responsiveness to TGF-b, thus maintaining Foxp3+ iTregs in the periphery (Figure 2). In the opposite scenario, antigen presentation by mature TLRactivated DCs would provide full co-stimulation to naı¨ve T cells, leading to activation of NFAT, AP-1 and NFkB. Under these circumstances, NFAT–AP-1 complexes would be formed, and Cblb and Foxp3 gene expression would be reduced. Additional instructive signals provided by DCs, through IL-12, IL-6, Notch ligands and costimulatory proteins, would then specify differentiation of the naı¨ve T cells into Th1, Th2 or Th17 effector lineages. There is emerging evidence for the hypothesis that NFAT directly or indirectly induces Foxp3. FOXP3 is induced in activated naı¨ve human [47] and mouse (MS and V Heissmeyer, unpublished) T cells, even in the absence of overt TGF-b stimulation. This induction is prevented by the calcineurin inhibitor cyclosporine A suggesting involvement of NFAT [47]. Mitogen activated protein kinase inhibitors — which would be expected to block AP-1 induction — have little effect, suggesting that Foxp3 induction might not require AP-1 or NFAT–AP-1 cooperation and in fact might preferentially occur in its absence. CTLA-4, a cell-surface inhibitor that opposes co-stimulation through CD28, is required www.sciencedirect.com
for naı¨ve T cells to express Foxp3 in response to TGF-b [48]. Ctla4 is a target of the cooperative NFAT–FOXP3 complex, and deletion of Ctla4 is associated with flagrant autoimmunity similar to that observed in Foxp3-deficient or TGF-b1-deficient mice [49]. These data further support the hypothesis that TGF-b responsiveness correlates with diminished co-stimulation and diminished NFAT– AP-1 cooperation in the nucleus of activated cells (Figure 2). On the basis of our recent finding that NFAT and Foxp3 can functionally cooperate on DNA [41], we suggest that activation-induced Foxp3 expression leads to autoamplification of Foxp3 gene expression, through mechanisms that involve cooperative interactions among Smad, Foxp3 and NFAT at regulatory regions of the Foxp3 gene. For example, an initial NFAT-dependent burst of Foxp3 transcription in naı¨ve T cells could be stabilized by TGF-b-activated R-Smad–Co-Smad4 complexes, either directly or through recruitment of chromatin-modifying enzymes, and could subsequently be sustained either by Foxp3 or by cooperative complexes of NFAT and Foxp3 (Figure 2). It is also noteworthy that the developmental cues that promote Foxp3 expression and regulatory function in iTregs do not appear to be conserved in nTregs. Several recent studies have highlighted significant differences between nTregs and iTregs: nTregs preferentially display TCR repertoires with high affinity for self-ligands, whereas iTregs, which are derived from naı¨ve precursors, show no such preference [50]. Moreover, the two cell types have reciprocal co-stimulatory requirements for their development. Whereas CD28 / mice have reduced numbers of thymic Tregs [36], CTLA-4 is required for iTreg development [48]. Even the epigenetic regulation of Foxp3 expression differs in nTregs and iTregs. In contrast to the extensive DNA methylation observed throughout the Foxp3 locus in iTregs, nTregs show widespread Foxp3 demethylation at conserved CpG islands [51]. Collectively, these differences suggest that nTregs and iTregs might have non-overlapping roles in immune regulation. Whereas nTregs are responsible for T-cell tolerance under resting conditions, it is probable that iTregs function during antigen-specific responses, perhaps during immune contraction. The identification of genes that are selectively expressed in nTregs or iTregs will be useful to validate this hypothesis.
Conclusions The lineage specification programs underlying T-cell differentiation involve the transcriptional integration of multiple extracellular signals. The divergent iTreg and Th17 lineages have a common requirement for stimulation through antigen and TGF-b receptors, but the direction of differentiation is determined by the presence or absence of co-stimulatory inputs and the cytokines Current Opinion in Immunology 2007, 19:287–293
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IL-2, IL-6 and IL-23. The transcription factors that read out co-stimulation are AP-1 and NFkB, and we propose that NFAT and NFAT–AP-1 complexes have opposing functions in orchestrating iTreg versus Th17 differentiation. Furthermore, the combination of activation-induced transcription factors that regulate thymic differentiation of nTregs has yet to be resolved. Although the iTreg/Th17 cell paradigm has provided exciting new insight into how we view T cell activation and tolerance, several major questions remain to be addressed. These include understanding the combinatorial nature of transcription factors that drive nTreg, iTreg and Th17 cell differentiation, and further elucidating the relative contributions of nTregs and iTregs to immune tolerance in vivo.
Acknowledgements This work was supported by National Institutes of Health grants CA42471 and AI48213, and Juvenile Diabetes Research Foundation (JDRF) grant 04-2004-368 to AR. MSS is a fellow of the Irvington Institute for Immunological Research.
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50. Hsieh CS, Zheng Y, Liang Y, Fontenot JD, Rudensky AY: An intersection between the self-reactive regulatory and nonregulatory T cell receptor repertoires. Nat Immunol 2006, 7:401-410.
38. Li W, Whaley CD, Bonnevier JL, Mondino A, Martin ME, Aagaard-Tillery KM, Mueller DL: CD28 signaling augments Elk-1-dependent transcription at the c-fos gene during antigen stimulation. J Immunol 2001, 167:827-835. 39. Macian F, Garcia-Cozar F, Im SH, Horton HF, Byrne MC, Rao A: Transcriptional mechanisms underlying lymphocyte tolerance. Cell 2002, 109:719-731. 40. Macian F: NFAT proteins: key regulators of T-cell development and function. Nat Rev Immunol 2005, 5:472-484.
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51. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E et al.: Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007, 5:e38. This article shows the Foxp3 locus in natural versus induced Tregs is differentially demethylated. In contrast to the highly stable pattern of Foxp3 expression observed in nTregs, the authors show that TGF-binduced Foxp3 expression can be silenced upon subsequent activation in the absence of TGF-b.
Current Opinion in Immunology 2007, 19:287–293
Current Opinion in Immunology 2007, 19:287–293 This review comes from a themed issue on Lymphocyte activation Edited by Ulrich von Andrian and Federica Sallusto Available online 12th April 2007 0952-7915/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. DOI 10.1016/j.coi.2007.04.014
Introduction The immune system must balance the necessity of immune activation with the hazards of autoimmunity. Naı¨ve T cells recognize antigens through the T-cell antigen receptor (TCR), but distinguish self from foreign antigens and direct their subsequent differentiation on the basis of which cytokine and co-stimulatory receptors are simultaneously engaged. Pathogen recognition through Toll-like receptors (TLRs) on dendritic cells (DCs) results in inflammatory cytokine production and T-cell co-stimulation, promoting differentiation of effector T cells (Teffs). By contrast, self-antigens are normally presented in the absence of co-stimulation, which promotes tolerance through the induction of T-cell anergy and the gradual accumulation of regulatory T cells (Tregs). Tregs are central to the establishment of peripheral immune tolerance. Although Tregs encompass several diverse subsets that have unique properties [1], the best characterized Treg subset expresses the forkhead tranwww.sciencedirect.com
scription factor Foxp3 and imposes peripheral tolerance by suppressing Teff responses [2]. Foxp3-expressing Tregs were first described as arising through antigeninduced differentiation in the thymus (‘natural’ Tregs, nTregs), but it is now clear that they can also develop through the differentiation of naı¨ve T cells in peripheral lymphoid organs (‘adaptive’ or ‘induced’ Tregs, here abbreviated iTregs) [3,4]. Loss or mutation of FOXP3 (Foxp3) in both humans and mice is associated with multi-organ autoimmune disease, secondary to loss or decreased numbers of Tregs [2,5]. Recently, there has been a new appreciation of the pleotropic roles of the cytokine transforming growth factor b (TGF-b) in regulating T cell activation and tolerance (reviewed in [6]). TGF-b1-deficient mice develop a lethal wasting disease accompanied by multiorgan autoimmunity. Moreover, TGF-b regulates Foxp3 expression and iTreg differentiation in cell culture, potentially explaining the phenotypic similarities between TGF-b1-deficient and Foxp3-deficient mice. TGF-b also controls differentiation of the novel Th17 subset of effector T cells, characterized by production of the inflammatory cytokine interleukin (IL)-17 (reviewed in [7,8]). Given its dual role in driving iTreg and Th17 differentiation, TGF-b is a key player in inflammation and autoimmune disease. In this article, we review recent data on how differentiation of iTreg and Th17 is modulated by signals transduced through cell-surface receptors. We focus on the interplay between antigen, IL-2, TGF-b and co-stimulatory receptors, and the known or potential roles of downstream transcription factors in each of these pathways.
TGF-b: a jack of many trades TGF-b1, TGF-b2 and TGF-b3 constitute a family of biologically active polypeptides that modulate a diverse array of physiologic processes (reviewed in [9]). All three proteins bind multimers of type I and type II TGF-b receptors, transmembrane proteins that possess intrinsic serine/threonine kinase domains (reviewed in [10]). Phosphorylation of type I TGF-b receptors leads to recruitment and phosphorylation of receptor-associated Smad (R-Smad) proteins. Activated R-Smads form complexes with a single common Smad protein (Co-Smad4) and translocate to the nucleus where they directly activate or repress target gene transcription through cooperative interactions with other DNA-binding proteins [10,11]. Vertebrates also have two inhibitory Smads (I-Smad6 Current Opinion in Immunology 2007, 19:287–293
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and I-Smad7), which are transcriptionally upregulated by R-Smads and function in a negative-feedback loop to inhibit TGF-b receptor signaling, partly by competing with R-Smads for docking to TGF-b receptors and partly by recruiting E3 ubiquitin ligases to degrade components of the TGF-b receptor [10]. In addition, TGF-b receptors are coupled to the activation of several non-Smad signaling pathways, including ERK, SAPK/JNK and p70-S6 kinase [12]. The importance of these pathways for T-cell function is presently unclear. The role of TGF-b in Treg and Teff function has been studied in diverse genetic models (reviewed in [6]). Mice that lack the TGF-bRII receptor, or that express a ‘dominant-negative’ (signaling-incompetent) form of the TGFbRII receptor (TGF-bRII DN), only in CD4 and CD8 T cells, develop a severe autoimmune disease reminiscent of that observed in TGF-b1 / mice [13,14,15]. Thus T cells are key effectors of autoimmunity in these mice. The uncontrolled expansion of TGF-b-unresponsive T cells in peripheral lymphoid organs, and the resulting autoimmunity, is partly as a result of activation of Teffs through selfreactive TCR and IL2/IL15 receptors. Surprisingly, the mice are not impaired in development and function of nTregs, although they show a decreased number of Foxp3+ T cells in the periphery [14,16]. Loss of peripheral Tregs might be as a result of decreased exit of nTregs from the thymus, decreased survival of nTregs in the periphery, decreased differentiation of iTregs, failure to maintain Foxp3 expression in the absence of TGF-b signals, or a combination of these factors [13,14,16, 17]. TGF-b signaling is also required to render Teffs sensitive to suppression by Tregs: purified Teffs from TGF-bRII DN mice cause colitis in immunocompromised recipients even if adoptively transferred with wild-type nTregs [16]. Collectively, these data highlight TGF-b as a major regulator of T cell function (Figure 1), but, as is the case for many other pleotropic signaling molecules, the functional consequence of TGF-b signaling in T cells is context dependent, depending on the local extracellular milieu.
TGF-b and the iTreg/Th17 paradigm A striking feature of TGF-b is its ability to promote differentiation of two opposing T-cell lineages, iTregs and Th17 cells (Figures 1 and 2). Administration of TGFb to naı¨ve CD4+CD25 T cells in cell culture induces expression of Foxp3 mRNA and protein, but only if the cells are concomitantly stimulated through the TCR [3,4,18]. CD25 and cytotoxic T lymphocyte associated protein-4 (CTLA-4) are induced in parallel, and the resulting cells have regulatory function and can suppress immune responses in mice [4,19]. However, if the cultures contain the pro-inflammatory cytokine IL-6, Foxp3 induction is diminished or abrogated [20,21]. Instead, TGF-b and IL-6 cooperatively instruct expression of the orphan nuclear receptor RORgt, which in turn drives Current Opinion in Immunology 2007, 19:287–293
naı¨ve T-cell differentiation into the Th17 effector T-cell subset that produces IL-17, but not IL-4 or IFNg [22]. Both TGF-b and IL-6 are absolutely required for the development of Th17 cells in vivo, as evidenced by the paucity of Th17 cells in the periphery of mice that lack IL-6 [22] or that express TGF-bRII DN in T cells [23]. These mice are also significantly less susceptible to experimental autoimmune encephalitis, a disease thought to involve accumulation of Th17 cells in the central nervous system [23]. At the cellular level, IL-6 is primarily expressed by DCs, macrophages and B cells that are activated by TLR ligands, such as lipopolysaccharide or unmethylated (bacterial) CpG DNA [24,25]. By contrast, a prominent source of TGF-b is Treg cells themselves [6]. Thus, naı¨ve T cells exposed to antigen in the periphery are probably maintained in a tolerant state on account of suppressive contacts with Tregs that produce TGF-b. However, once ‘danger signals’ alert DCs to the presence of foreign pathogens in the form of TLR stimulation, IL-6 production by antigen-presenting DCs switches the tolerogenic program of TGF-b signaling to one that activates RORgt and fosters the development of inflammatory Th17 cells. Does IL-6 drive Th17 differentiation by acting directly on T cells, by enhancing co-stimulation, or both? Mice that lack IL-6 are defective in RORgt expression and Th17 cell development [22], but these responses have not yet been tested in naı¨ve T cells that lack components of the IL-6 receptor. However, IL-6 and STAT3 oppose Foxp3 expression and iTreg differentiation in cultured T cells in a cell-intrinsic manner, through a mechanism that does not appear to involve STAT3 binding to the Foxp3 promoter [20,26].
IL-2 and IL-23: cytokine modulation of the Treg/Th17 balance The role of IL-2 in Treg development is controversial. Tregs bear high levels of the IL2Ra subunit, CD25, and mice deficient in IL-2 or its receptor subunits develop autoimmune disease [27]. The bulk of the evidence indicates that, as with TGF-b, IL-2 is not essential for nTreg differentiation but is needed to maintain normal peripheral numbers of nTregs, perhaps by promoting thymic exit or peripheral survival [28,29]. IL-2 and IL-15, which both signal through a common receptor b chain, CD122, appear redundant in this respect. IL-2 and STAT5 also appear crucial for Foxp3 expression and iTreg differentiation, although this conclusion is qualified by incomplete deletion of STAT5 in naı¨ve T cells [26,30]. In contrast to STAT3, STAT5 binding to the Foxp3 locus can readily be demonstrated [26,30]. The influence of other cytokines on Th17 differentiation is more clear. Th17 differentiation is strongly inhibited by IL-12 or IFNg [21,22,24], establishing www.sciencedirect.com
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Figure 1
Pleotropic effects of TGF-b on T-cell activation, differentiation and tolerance. (a) TGF-b inhibits TCR-induced proliferation of naı¨ve T cells (TN) in cell culture and in mice, but inhibition can be overcome by co-stimulation through CD28 or by establishment of the IL-2/IL-2R autocrine growth pathway. (b) TGF-b interferes with the acquisition of Th1 and Th2 effector functions, even under optimal activation conditions where it no longer impedes T cell proliferation. (c) TGF-b is not required for nTreg development or function, but is needed to maintain Treg numbers in the periphery. (d) TGF-b induces Foxp3 and promotes the differentiation of induced Tregs (iTregs) in the absence of IL-6, whereas concomitant TGF-b and IL-6 signaling induces expression of the orphan nuclear receptor transcription factor RORgt, which drives the development of Th17 cells. (e) Intrinsic TGF-b signaling is essential for effector T cells to be suppressed by Tregs. Abbreviations: DP, double-positive (CD4+CD8+) thymocyte; SP, single-positive (CD4+CD8 or CD8+CD4 ) thymocyte. .
unambiguously that Th17 cells represent a different cellular lineage than Th1 cells, which require both these cytokines. Th17 cells express high levels of the multimeric IL-23 receptor, and IL-23-deficient mice display a selective reduction in peripheral Th17, but not Th1, cell numbers [7]. IL-23 is not required for Th17 cell differentiation, but rather serves a vital function in Th17 cell maintenance or function [21,31]. www.sciencedirect.com
Co-stimulation as a determining factor in iTreg and Th17 differentiation Co-stimulation is a key factor in converting TGF-b signals from anti- to pro-inflammatory. Th17 cells develop in mice only under conditions that promote inflammation (e.g. immunization with agonist peptides in the presence of adjuvant), and in cell culture only in the context of full TCR/CD28 activation [21,22,24,25,31]. Among the Current Opinion in Immunology 2007, 19:287–293
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Figure 2
Signaling requirements for iTreg/Th17 cell development. Intracellular signaling in response to TCR and co-stimulatory receptors (i.e. CD28, CTLA-4) results in the activation of NFAT and AP-1 transcription factors and promotion of Th17 development. TGF-bR signals promote both iTreg and Th17 development, activating both Smad and non-Smad pathways. Smad complexes upregulate I-Smad7 expression, which is opposed by Foxp3 at the level of transcription. Whether TGF-bR-derived non-Smad pathways modulate naı¨ve T cell differentiation to iTreg or Th17 cells is not known. IL-6R-mediated STAT3 signaling is important for RORgt expression and repression of Foxp3 expression through an unknown mechanism. Additional activation of STAT5 through the IL-2R enhances Foxp3 expression by direct STAT5 binding to the Foxp3 promoter.
relevant co-stimulatory receptors are CD28 and ICOS, which are expressed on resting and activated T cells, respectively: ICOS-deficient murine T cells do not produce IL-17 if cultured with DCs that lack the two CD28 ligands B7.1 (CD80) and B7.2 (CD86) [32]. By contrast, iTreg differentiation is preferentially evoked by noninflammatory conditions [33,34]. A slow conversion of Foxp3 cells to Foxp3+ iTregs occurs in TCR-transgenic mice during the course of repeated administration of antigen, in low doses and in the absence of adjuvant stimulation or TLR activation [34,35]. Phenoconversion of monospecific Foxp3 T cells to Foxp3+ iTregs also occurs during homeostatic repopulation of lymphopenic hosts [33]. In both these circumstances, T cells are unliCurrent Opinion in Immunology 2007, 19:287–293
kely to receive full TCR/CD28 stimulation, suggesting strongly that co-stimulation is not required for Foxp3 expression during iTreg differentiation. This scenario, however, does not apply in the thymus, because nTreg differentiation requires cell-intrinsic signals delivered through CD28 [36]. Co-stimulatory receptors (i.e. integrins, CD28 and ICOS) potentiate TCR-induced signaling pathways leading to activation of the transcription factors NFkB and activator protein-1 (AP-1) (Fos-Jun), but do not generally activate nuclear factor of activated T cells (NFAT), a calciumregulated transcription factor that drives distinct biological programs depending on its partner transcription www.sciencedirect.com
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factors on DNA [37,38]. T cell activation requires NFAT–AP-1 cooperation, whereas T-cell anergy occurs when NFAT is activated in the absence of AP-1 or NFkB, and T-cell tolerance mediated by regulatory T cells relies on formation of a cooperative NFAT–FOXP3–DNA complex [39,40,41]. Several anergy-associated NFAT target genes are known to have roles in immune tolerance: for instance, deletion of Itch, Cblb or the gene encoding the Itch and Nedd4-interacting protein is associated with autoimmune/inflammatory diseases in mice and rats [42–44].
Interplay among signaling pathways and transcription factors in iTreg and Th17 differentiation How do the same stimuli (antigen, IL-2 and TGF-b) signaling through the same cell surface receptors orchestrate two qualitatively different cellular responses depending on the context? We propose that co-stimulation is a critical factor. When naı¨ve T cells are exposed to low-dose antigen without co-stimulation, together with TGF-b and IL-2/IL-15 in the microenvironment, they induce STAT5, Smad and non-Smad signaling pathways as well as NFAT without AP-1 or NFkB. Together, this combination of transcription factors induces Foxp3, which in turn suppresses I-Smad7 expression, thus potentiating TGF-b signaling [18]. At the same time, NFAT in the absence of AP-1 upregulates Cbl-b [45], which downregulates TCR signals while promoting Foxp3 expression in TGF-b-stimulated cells [45,46]. Together, these pathways have the potential to set up a positive feedback loop that heightens responsiveness to TGF-b, thus maintaining Foxp3+ iTregs in the periphery (Figure 2). In the opposite scenario, antigen presentation by mature TLRactivated DCs would provide full co-stimulation to naı¨ve T cells, leading to activation of NFAT, AP-1 and NFkB. Under these circumstances, NFAT–AP-1 complexes would be formed, and Cblb and Foxp3 gene expression would be reduced. Additional instructive signals provided by DCs, through IL-12, IL-6, Notch ligands and costimulatory proteins, would then specify differentiation of the naı¨ve T cells into Th1, Th2 or Th17 effector lineages. There is emerging evidence for the hypothesis that NFAT directly or indirectly induces Foxp3. FOXP3 is induced in activated naı¨ve human [47] and mouse (MS and V Heissmeyer, unpublished) T cells, even in the absence of overt TGF-b stimulation. This induction is prevented by the calcineurin inhibitor cyclosporine A suggesting involvement of NFAT [47]. Mitogen activated protein kinase inhibitors — which would be expected to block AP-1 induction — have little effect, suggesting that Foxp3 induction might not require AP-1 or NFAT–AP-1 cooperation and in fact might preferentially occur in its absence. CTLA-4, a cell-surface inhibitor that opposes co-stimulation through CD28, is required www.sciencedirect.com
for naı¨ve T cells to express Foxp3 in response to TGF-b [48]. Ctla4 is a target of the cooperative NFAT–FOXP3 complex, and deletion of Ctla4 is associated with flagrant autoimmunity similar to that observed in Foxp3-deficient or TGF-b1-deficient mice [49]. These data further support the hypothesis that TGF-b responsiveness correlates with diminished co-stimulation and diminished NFAT– AP-1 cooperation in the nucleus of activated cells (Figure 2). On the basis of our recent finding that NFAT and Foxp3 can functionally cooperate on DNA [41], we suggest that activation-induced Foxp3 expression leads to autoamplification of Foxp3 gene expression, through mechanisms that involve cooperative interactions among Smad, Foxp3 and NFAT at regulatory regions of the Foxp3 gene. For example, an initial NFAT-dependent burst of Foxp3 transcription in naı¨ve T cells could be stabilized by TGF-b-activated R-Smad–Co-Smad4 complexes, either directly or through recruitment of chromatin-modifying enzymes, and could subsequently be sustained either by Foxp3 or by cooperative complexes of NFAT and Foxp3 (Figure 2). It is also noteworthy that the developmental cues that promote Foxp3 expression and regulatory function in iTregs do not appear to be conserved in nTregs. Several recent studies have highlighted significant differences between nTregs and iTregs: nTregs preferentially display TCR repertoires with high affinity for self-ligands, whereas iTregs, which are derived from naı¨ve precursors, show no such preference [50]. Moreover, the two cell types have reciprocal co-stimulatory requirements for their development. Whereas CD28 / mice have reduced numbers of thymic Tregs [36], CTLA-4 is required for iTreg development [48]. Even the epigenetic regulation of Foxp3 expression differs in nTregs and iTregs. In contrast to the extensive DNA methylation observed throughout the Foxp3 locus in iTregs, nTregs show widespread Foxp3 demethylation at conserved CpG islands [51]. Collectively, these differences suggest that nTregs and iTregs might have non-overlapping roles in immune regulation. Whereas nTregs are responsible for T-cell tolerance under resting conditions, it is probable that iTregs function during antigen-specific responses, perhaps during immune contraction. The identification of genes that are selectively expressed in nTregs or iTregs will be useful to validate this hypothesis.
Conclusions The lineage specification programs underlying T-cell differentiation involve the transcriptional integration of multiple extracellular signals. The divergent iTreg and Th17 lineages have a common requirement for stimulation through antigen and TGF-b receptors, but the direction of differentiation is determined by the presence or absence of co-stimulatory inputs and the cytokines Current Opinion in Immunology 2007, 19:287–293
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IL-2, IL-6 and IL-23. The transcription factors that read out co-stimulation are AP-1 and NFkB, and we propose that NFAT and NFAT–AP-1 complexes have opposing functions in orchestrating iTreg versus Th17 differentiation. Furthermore, the combination of activation-induced transcription factors that regulate thymic differentiation of nTregs has yet to be resolved. Although the iTreg/Th17 cell paradigm has provided exciting new insight into how we view T cell activation and tolerance, several major questions remain to be addressed. These include understanding the combinatorial nature of transcription factors that drive nTreg, iTreg and Th17 cell differentiation, and further elucidating the relative contributions of nTregs and iTregs to immune tolerance in vivo.
Acknowledgements This work was supported by National Institutes of Health grants CA42471 and AI48213, and Juvenile Diabetes Research Foundation (JDRF) grant 04-2004-368 to AR. MSS is a fellow of the Irvington Institute for Immunological Research.
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51. Floess S, Freyer J, Siewert C, Baron U, Olek S, Polansky J, Schlawe K, Chang HD, Bopp T, Schmitt E et al.: Epigenetic control of the foxp3 locus in regulatory T cells. PLoS Biol 2007, 5:e38. This article shows the Foxp3 locus in natural versus induced Tregs is differentially demethylated. In contrast to the highly stable pattern of Foxp3 expression observed in nTregs, the authors show that TGF-binduced Foxp3 expression can be silenced upon subsequent activation in the absence of TGF-b.
Current Opinion in Immunology 2007, 19:287–293