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Originally published online as doi:10.1189/jlb.0206095 on June 29, 2006

Published online before print June 29, 2006
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(Journal of Leukocyte Biology. 2006;80:458-470.)
© 2006 by Society for Leukocyte Biology

Development and function of naturally occurring CD4+CD25+ regulatory T cells

Akiko Toda and Ciriaco A. Piccirillo1

Department of Microbiology and Immunology, McGill University, Montreal, Canada

1 Correspondence: Department of Microbiology and Immunology, McGill University, Montreal, Canada, H3A 2B4. E-mail: ciro.piccirillo{at}mcgill.ca


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ABSTRACT
 
The immune system has evolved numerous mechanisms of peripheral T cell immunoregulation, including a network of regulatory T (Treg) cells, to modulate and down-regulate immune responses at various times and locations and in various inflammatory circumstances. Amongst these, naturally occurring CD4+CD25+ Treg cells (nTreg) represent a major lymphocyte population engaged in the dominant control of self-reactive T responses and maintaining tolerance in several models of autoimmunity. CD4+CD25+ Treg cells differentiate in the normal thymus as a functionally distinct subpopulation of T cells bearing a broad T cell receptor repertoire, endowing these cells with the capacity to recognize a wide range of self and nonself antigen specificities. The generation of CD4+CD25+ Treg cells in the immune system is genetically controlled, influenced by antigen recognition, and various signals, in particular, cytokines such as interleukin-2 and transforming growth factor-ß1, control their activation, expansion, and suppressive effector activity. Functional abrogation of these cells in vivo or genetic defects that affect their development or function unequivocally promote the development of autoimmune and other inflammatory diseases in animals and humans. Recent progress has shed light on our understanding of the cellular and molecular basis of CD4+CD25+ Treg cell-mediated immune regulation. This article discusses the relative contribution of CD4+CD25+ nTreg cells in the induction of immunologic self-tolerance and provides a comprehensive overview of recent finding regarding the functional properties and effector mechanism of these cells, as revealed from various in vitro and in vivo models.

Key Words: IL-2 • Foxp3 • TGF-ß1 • diabetes • colitis • infection


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INTRODUCTION
 
Multilevel immunoregulation of adaptive immune responses
Immunological tolerance requires a controlled balance between maintaining peripheral tolerance to autoantigens and preserving the potential to initiate protective immune responses against infectious agents [1 , 2 ]. To achieve a fine balance between these two drastically different immunological outcomes, a network of CD4+ regulatory T cells (Treg) exists to maintain this balance in various contexts and inflammatory settings. Thus, Treg cells must simultaneously suppress autoreactive T cells that escape thymic negative selection, maintain normal intestinal immunity toward enteric bacteria, and dampen the antipathogen effector mechanisms from inducing immune pathology [3 ].

CD4+ Treg cells exist in two general categories: induced (iTreg) and naturally occurring (nTreg; Table 1 ). iTreg CD4+ cells acquire their suppressive activity as a consequence of in vivo or ex vivo activation of CD4+ T cells, without intrinsic regulatory potential, under unique stimulatory conditions. A diverse number of iTreg cells capable of controlling peripheral T cell responses have been described, and their definition has largely been based on their potential to produce certain signature cytokines after antigen priming. Some of these iTreg cells include counter-regulatory IFN-{gamma}-producing Th1 cells and IL-4-producing Th2 cells, IL-10-producing Tr1 cells, and TGF-ß-secreting Th3 cells [4 5 6 7 ]. In contrast, CD4+CD25+ nTreg cells develop during the normal process of T cell maturation in the thymus, survive in the periphery poised for normal surveillance of self-antigens, and prevent potential autoimmune responses.


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  		Table 1. CD4+ Treg Cell Subsets

CD4+Foxp3+CD25+ nTreg cells
In 1995, a groundbreaking observation made by the laboratory of Shimon Sakaguchi [7 ] demonstrated that a unique subset of CD4+ T cells expressing CD25 [IL-2 receptor {alpha} chain (IL-2R{alpha})] in normal rodents displayed potent immunoregulatory functions in vitro and in vivo. Indeed, CD4+CD25+ Treg cells, termed nTreg, play a critical, master-switch role in dampening peripheral self-reactivity and contribute to the establishment of tolerance in several models of autoimmunity (Table 1) [8 , 9 ]. CD4+CD25+ Treg cells consist of an anergic lymphocyte population representing 1–10% of total CD4+ T cells in thymus, peripheral blood, and lymphoid tissues. CD4+CD25+ nTreg have a polyclonal T cell receptor (TCR) repertoire, based on diverse gene expression of various TCR-{alpha}/ß elements, and could conceivably recognize a wide spectrum of self and nonself antigens [10 11 12 13 ]. Functional abrogation of nTreg cells through depletion or genetic manipulation from the periphery results in the induction of multiorgan-specific autoimmunity and also increases immunity to tumors, grafts, allergens, and pathogens [14 ].

Most studies indicate that cell-surface CD25 and more particularly, the intranuclear forkhead transcription factor Foxp3 appear to be the most specific markers for nTreg in the normal T cell repertoire. Recent studies using multiparametric flow cytometry and gene microarray analysis have attempted to further define the phenotype of CD4+CD25+ nTreg cells (Fig. 1 ). The surface phenotype of nTreg cells partly resembles that of partially activated T cells, suggesting that nTreg cells may be stimulated continuously by self-antigens in vivo. nTreg cells not only constitutively express CD25 and Foxp3 but also preferentially express other surface markers including CD103, CD62 ligand (CD62L), glucocorticoid-induced tumor necrosis factor receptor (TNFR; GITR), cytotoxic T lymphocyte antigen-4 (CTLA-4), neuropilin-1, and lymphocyte activation gene-3 [15 16 17 18 ]. The functional significance of these various surface molecules in CD4+CD25+-mediated suppression remains elusive. Some suppressive activity has been documented in CD4+CD25+CD62Llow and CD4+CD25–CD45Rblow cell subsets in vitro and in vivo [19 , 20 ]. Furthermore, preferential expression of CC chemokine receptor 4 (CCR4), CCR5, CCR6, and CCR8, albeit not biomarkers of nTreg cells, may endow these cells with specific homing properties to secondary lymphoid tissues or sites of inflammation to control immune responses (refs. [21 , 22 ] and unpublished observations).


Figure 1
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  		Figure 1. nTreg and iTreg CD4+ cells control T cell responses. nTreg and iTreg CD4+ populations potentially regulate the function of activated effector T cells in many different immunological contexts, and the mechanisms to achieve this regulation are diverse. Although CD4+ nTreg cells emerge from the thymus, CD4+ iTreg cells originate from the activation and differentiation of conventional CD4+ T cells in the periphery. CD4+ iTreg cells operate via the secretion of immunosuppressive cytokines including IL-10 and TGF-ß1, and nTreg cells might opt for cytokine-dependent (A), cell contact-dependent (B), or cytokine/cellular contact-dependent (A+B) modes of action to control similar T cell responses. The relative contribution of each subset in the overall regulation of immune responses is unclear, but both can conceivably synergize to achieve this outcome.

Lineage specificity of CD4+CD25+ Treg cells: natural or induced?
A common problem relates to the failure to discriminate CD4+CD25+ nTreg cells from iTreg cells, which emerge from nonregulatory CD4+ T cell precursors and induced to express markers such as CD25 during an immune response. Although CD4+CD25+ nTreg cells preferentially express the molecules described above in the resting immune system, these markers are not reliable indicators of nTregs, as most cell surface molecules become expressed at variable degrees on CD4+CD25– T cells upon TCR engagement [9 ]. The use of any of these markers as definitive markers of nTreg in a situation in which the immune system has been provoked by experimental conditioning, immunization, treatment, or disease may obscure data interpretation. Although nTreg cells differentiate intrathymically and are functional upon exit from the thymus, CD4+ iTreg cells typically require the activation of normal, peripheral CD4+ T cells under specific activation conditions (Fig. 1) . Indeed, Foxp3+ and Foxp3– iTreg cells can be generated in vitro or in vivo under various stimulation regimes and generally function in a cytokine-dependent manner in vitro and in vivo (Table 1 and Fig. 1 ) [23 24 25 26 27 28 29 ]. nTreg and iTreg cells likely cooperate with each other at suppressing inflammatory T cell responses [30 ].


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CD4+CD25+ nTreg CELLS POSSESS AN ANERGIC PHENOTYPE
 
The signals that induce the proliferation of nTreg cells differ from their nonregulatory CD4+CD25– counterparts. Although CD4+CD25+ Treg cells require TCR activation for their functional activation, they themselves are hypoproliferative or anergic to these signals [31 ]. CD4+CD25+ nTreg cells are refractory to TCR-induced proliferation, even in the presence of anti-CD28 agonistic antibodies, but retain their proliferative potential when activated by phorbol 12-myristate 13-acetate/ionomycin, suggesting a possible defect between proximal TCR signals and the protein kinase C-activating pathway [31 ]. This inability to proliferate upon TCR/CD28 engagement is a result of the failure of these cells to actively transcribe and secrete IL-2 [31 32 33 34 ]. It is interesting that a 300-base pair proximal region of the IL-2 promoter in Treg cells does not undergo nucleosomal alternations upon T cell activation [35 ]. As it was reported that dynamic DNA demethylation is necessary for IL-2 expression [36 ], the lack of chromatin remodeling within the IL-2 promoter may contribute to the lack of IL-2 production in nTreg cells. However, this apparent hyporesponsiveness to TCR-mediated signals is physiologically relevant, as studies using TCR transgenic T cells show that CD4+CD25+ nTreg cells could be activated to acquire suppressor function at peptide concentrations ten- to 100-fold lower that those needed to trigger proliferation in responding CD4+CD25– T cells [37 ].

The relationship between nTreg cell anergy and suppressive activity is unknown. Initial studies showed that abrogation of the anergic state by in vitro TCR stimulation in the presence of a high dose of IL-2 or CD28 ligation results in simultaneous loss of suppressive activity [38 ]. Moreover, these studies showed that this anergic state is a default state, as CD4+CD25+ nTreg cells spontaneously revert to the original anergic state and regain the suppressive activity once IL-2 or anti-CD28 costimulation is removed [39 ]. However, more recent studies have clearly indicated that nTreg cells can actively suppress IL-2 mRNA in responding T cells, even in the presence of exogenous IL-2 or anti-CD28 agonistic antibodies [32 ]. This reversible anergic state of CD4+CD25+ nTreg cells can also be seen in vivo, as CD4+CD25+ Treg cells are subject to lymphopenia- and antigen-induced proliferation [8 , 40 ]. It is interesting that a seemingly small fraction of CD4+CD25+ nTreg cells in normal, naive mice are proliferating continuously without exogenous antigenic stimulation, presumably by recognizing self-antigens in the periphery [8 ]. Thus, CD4+CD25+ nTreg cells can be adaptive in their proliferative capacity under certain instances of TCR engagement, such that they can potentially expand upon antigen-specific expansion to suppress immune responses.

A recent report showed that in vitro stimulation of CD4+CD25+ T cells with a high concentration of lipopolysaccharide (LPS) directly through Toll-like receptor 4 (TLR4) on nTreg cells induced their proliferation and augmented their in vitro suppressive activity, even in the absence of antigen-presenting cells (APC) [41 ]. It is interesting that the LPS/TLR4 pathway might stimulate immature dendritic cells (DC) to secrete IL-6 and another factor that blocked the suppressive function of nTreg cells [42 ]. Thus, antigen-specific TCR signals (self or nonself peptide antigens) as well as nonspecific TLRs for conserved molecular patterns may modulate CD4+CD25+ nTreg cell activation and function.


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Foxp3: MASTER-SWITCH FOR THE GENETIC PROGRAMMING OF CD4+ nTREG CELLS
 
Studies in recent years have clearly shown that Foxp3 is a functional marker of nTreg cells, playing a central role in their development and function [43 44 45 46 47 48 49 ]. CD4+CD25+ nTreg cells account for the vast majority of Foxp3 expression within murine T cells, and Foxp3–/– mice suffer from an autoimmune pathology, secondary to a loss of CD4+CD25+ nTreg cell function, a clinical outcome synonymous with what is believed to occur in Scurfy mice and immunodysregulation, polyendocrinopathy, enteropathy, X-linked patients [43 44 45 46 47 48 49 50 ]. Although primarily expressed in human CD4+CD25+ nTreg cells, conventional CD4+CD25– T cells can also up-regulate expression of this molecule. Foxp3-overexpressing mice have more Treg cells and can potently suppress the development of autoimmune disease [43 44 45 46 47 48 49 ]. Retroviral overexpression into nonregulatory CD4+CD25– or CD8+ T cells induces a functional phenotype similar to CD4+CD25+ nTreg cells possessing potent, suppressive functions in vitro and in vivo [43 44 45 46 47 48 49 ]. It is interesting that induction of Foxp3 has also been described in iTreg cells [51 ]. It remains to be seen what genes Foxp3 are modulating and what impact they have in Treg function; however, a recent study by Fontenot et al. clearly demonstrates that CD4+Foxp3+CD25– and CD4+Foxp3+CD25+ possess a similar gene expression signature compared with CD4+Foxp3–CD25+ T cells [49 ]. Collectively, these studies suggest that Foxp3 plays a determining role in the generation of CD4+CD25+ nTreg cells and illustrate how certain genetic pathways affect nTreg function such that autoreactive T cells are left uncontrolled and allowed to induce autoimmunity.


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MOLECULAR CONTROL OF CD4+CD25+ nTreg CELL DEVELOPMENT, ACTIVATION, AND FUNCTION
 
The complete array of signals needed for the induction and maintenance of CD4+CD25+ nTreg activity is not understood. In addition to TCR engagement, signals via costimulatory and cytokine receptors contribute to the development, activation, and function of nTreg cells and hence, the fine-tuning of suppression [31 ].

Functional and homeostatic functions for IL-2
IL-2 requirements for the development and function of nTreg cells
The cytokine signals required for nTreg cell development and function are not defined completely. Many studies show that IL-2 is an important molecular switch for the development, peripheral survival, and suppression function of nTreg cells. Mice deficient for IL-2, IL-2R{alpha}, or IL-2Rß have a drastically reduced pool of nTreg cells and die prematurely from a severe lymphoproliferative and autoimmune syndrome [52 , 53 ]. In IL-2Rß–/– mice with an IL-2Rß transgene, which is expressed predominantly in the thymus, CD4+CD25+ T cell development is restored, and lymphoadenopathy and autoimmunity are prevented [54 ], indicating that an intact IL-2/IL-2R pathway is also required for thymic generation of nTreg [54 ]. The adoptive transfer of wild-type (WT) nTreg cells can only prevent autoimmunity in mice lacking a functional IL-2R, in contrast to IL-2–/– mice, and with adoptive transfer of WT Treg cells into neonatal IL-2Rß–/– mice, these donor nTreg cells undergo rapid and extensive IL-2-dependent proliferation in lymph nodes and spleen [55 ]. These studies imply that the lack of nTreg cells contributes to the autoimmune phenotype of IL-2–/– and IL-2R–/– mice and that IL-2 is a critical growth and differentiation factor for Treg cells [56 , 57 ]. Similarly, CD4+ T cells from IL-2–/– mice can protect mice from spontaneous experimental autoimmune encephalomyelitis (EAE); however, CD4+ T cells from CD25–/– mice cannot protect from EAE [58 ]. Consistent with these observations, systemic IL-2 neutralization induces autoimmune gastritis in BALB/c mice, provokes spontaneous autoimmune neuropathy, and exacerbates diabetes in nonobese diabetic (NOD) mice [59 ]. In humans with cancer after chemotherapy-induced lymphopenia, peripheral expansion of Treg cells is augmented by recombinant human IL-2 therapy [60 ]. Lastly, another study by de la Rosa et al. [61 ] showed that blocking the IL-2R on Treg cells leads to a loss of their regulatory activity, suggesting a possible role for IL-2 for suppressor function. Collectively, this series of studies shows that IL-2 may be required in the production of nTreg and that early IL-2 production in sites of inflammation may drive CD4+CD25+ Treg cell-mediated suppression of T cell responses, and possible alterations in this pathway may block Treg cell development and provoke autoimmunity [57 ].

IL-2 triggers two signaling pathways that promote proliferation and survival in T cells by activating the signal transducer and activator of transcription 5 (STAT5) transcription factor, and by up-regulation of antiapoptotic molecules, Bcl-2 [62 63 64 ] and Bcl-2 deficiency does not affect CD4+ CD25+ Treg homeostasis, and transgenic expression of Bcl-2 does not rescue CD4+CD25+ Treg number in IL-2–/– or STAT5–/– mice, suggesting that abrogation of IL-2 signaling in Treg cells does not merely disrupt an essential survival pathway [65 ]. In contrast, STAT5A/5B–/– mice show autoimmune pathology similar to IL-2–/– and IL-2R–/– mice and a decreased number of CD4+ CD25+ Treg [66 ]. Furthermore, although STAT5–/– and IL-2–/– mice have few CD4+CD25+ nTreg cells, mice transgenic for the active form of STAT5 possess a greater frequency of these cells, thus confirming a requirement for IL-2 signaling in nTreg homeostasis [65 66 67 68 ].

A few lines of evidence, however, do not entirely support a mandatory role for IL-2 in the genesis of Foxp3+ Treg cell function. Recently, Fontenot and Rudensky [69 ] examined whether IL-2 was required for the development of Foxp3+ nTreg cells. In this study, mice containing a Foxp3 knock-in allele [Foxp3green fluorescent protein (gfp) mice], encoding a GFP-Foxp3 fusion protein, were generated, thus enabling the identification of Foxp3+ Treg cells by GFP. From the analysis of Foxp3gfp mice genetically deficient in IL-2 or CD25, IL-2 signaling does not appear to be required for the induction of Foxp3 expression in developing thymocyte, and IL-2–/– and IL-2R{alpha}–/– Treg cells are unexpectedly able to suppress T cell proliferation in vitro. It is interesting that this study showed that IL-2 signaling was nonetheless necessary for maintaining the expression of genes involved in controlling cell growth and metabolism, possibly indicating that IL-2 signaling may be required for sustaining the homeostasis and competitive fitness of nTreg cells in vivo [70 ]. The reasons for the detection of Treg activity in IL-2- and IL-2R{alpha}-deficient mice are not thoroughly understood but could be explained by the inability to enrich for Treg cells in the midst of the large peripheral pool of the preactivated CD4+ T cell pool. The few CD25+ T cells that could be purified from these mice were likely effector T cells and thus, nonsuppressive in vitro. In Fontenot et al. [70 ], the apparent in vitro regulatory function in Foxp3+ Treg cells from IL-2- and IL-2R{alpha}-deficient mice could be explained by a preferential enrichment of Foxp3+ CD4+ T cells (based on GFP expression and not CD25) from the total CD4+ T cell population and thus, an exclusion of potentially preactivated effector T cells from the final preparation. These findings do not exclude the possibility that IL-2 may still mediate a pro-fitness property to peripheral Treg cells considering the autoimmune-prone phenotype of these mice. Similarly, analysis of an agonist-induced population of TCR-transgenic Foxp3+Treg cells in IL-2–/– or CD25–/– mice has shown that intrathymic nTreg development is not IL-2-dependent, and peripheral survival is IL-2-dependent [71 ], although in the stark opposition to the findings of Malek et al [54 ]. These observations are also consistent with the regulatory function seen in CD25+ Treg cells transferred into lymphopenic hosts and whose CD25 is down-regulated post-transfer, although sufficient IL-2 signaling may have been given prior to CD25 down-regulation in the lymphopenic environment [16 ]. Alternatively, other lymphopenia-associated signaling may have compensated or overcome the need for IL-2 in this system [16 ]. Lastly, the peripheral detection of Treg function in IL-2Rß-deficient mice after thymic reconstitution of IL-2Rß [54 ], despite the lack of IL-2Rß in peripheral Treg cells, is difficult to interpret but could be explained by increased IL-2 responsiveness in developing Treg cells in the thymus as a result of forced expression of the IL-2Rß chain, in turn, possibly circumventing the requirement for this critical signal in the periphery. Overall, this apparent discrepancy in the functional requirement of IL-2 in thymic development, Foxp3 expression, function acquisition, and peripheral fitness of Treg cells may be explained by a number of reasons, including the major histocompatibility complex (MHC) and non-MHC genetic background, costimulatory burden, and degree of lymphopenia in each of the mouse models used. Variation in these factors may ultimately influence IL-2 availability in vivo and the IL-2 dependency of Treg cells, possibly by influencing the antigenic repertoire, TCR avidity, and activation thresholds in developing T cells. It also remains unknown whether compensatory signals, including cytokines, may actually drive Treg development or function in the functional absence of IL-2, as shown with other {gamma}c signaling cytokines, such as IL-4, which can compensate for IL-2 in vitro and in vivo [70 , 72 ].

Costimulatory molecules in nTreg cell development and homeostasis
The relative contribution of CD28 signals in the induction of suppressor activity is unclear. Following antigen stimulation, CD28 provides naive T cells with a costimulatory signal for promoting IL-2 synthesis and cell expansion as well as preventing anergy induction and cell death [73 ]. CD4+CD25+ nTreg cells do not need CD28 for their activation, as CD4+CD25+ nTreg cells from CD28–/– or WT mice exhibit comparable, in vitro, suppressive activity [31 , 74 ]. CD4+CD25+ nTreg cells can be expanded with DC in the absence of exogenous cytokines, and expansion and induction of suppressive function required B7 costimulation [75 , 76 ]. Recently, Thornton et al. [72 ] showed that when CD4+CD25+ cells were preactivated with WT or B7.1–/–/B7.2–/– APC or in the presence of CTLA-4-immunoglobulin, CD4+CD25+ T cells retained potent suppressor activity under each stimulatory condition. CD4+CD25+ cells were fully capable of inhibiting the remaining response, despite the lack of CD28 or CTLA-4 costimulation, although costimulation through CD28-like molecules is conceivable. It remains to be examined at molecular levels how the balance between signals through CTLA-4 and CD28, both of which interact with B7.1 and B7.2 on APC, contributes to the tuning of the regulatory activity of nTreg cells. Collectively, B7-CD28 interactions do not seem to be required for the in vitro activation of suppressor function in peripheral Treg cells.

The role of B7/CD28 interactions in thymic development and peripheral homeostasis of the nTreg cells has been highlighted recently [77 , 78 ]. In addition to proper costimulation to developing immature, thymic precursors, CD28 engagement might be required to sustain a stable peripheral pool of nTreg cells by promoting their survival and self-renewing potential, possibly through the expression antiapoptotic molecules or through cytokines, which function as growth, survival, or suppressor activity maintenance factors. CD28 seems to play a key role in the generation of CD4+CD25+ nTreg cells in the thymus and presumably in their survival in the periphery, as CD28–/– mice develop a substantially reduced number of CD4+CD25+ nTreg cells in the thymus and periphery [77 , 78 ]. It is interesting that the abrogation of the B7/CD28 costimulatory pathway results in quantitative and qualitative defects in CD4+CD25+ nTreg cells in lymphoid tissues and a consequential induction of autoimmunity, as illustrated by CD28–/– and B7.1-B7.2–/–NOD mice, which develop an exacerbated form of diabetes compared with their NOD control littermates [79 ]. Similarly, CD4+CD25+CD45RBlow T cells are reduced in CD40–/– mice, and transfer of CD40–/– T cells into nude mice induces autoimmune disease. However, blocking of CD40/CD40 ligand (CD40L) interaction does not affect nTreg-suppressive activity in vivo [80 ]. IL-2 administration to CD40–/– mice normalizes nTreg number by promoting their survival and homeostatic proliferation, suggesting that the CD40/CD40L pathway is also needed for the genesis of nTreg cells [81 ]. Efficient generation of nTreg cells in the thymus requires CD28 costimulation, as expression of Foxp3 and the induction of the nTreg cell differentiation program are specifically induced in double-positive thymocytes, signaled in vitro by simultaneous coengagement of TCR and CD28 surface molecules [82 ]. However, CD28 signaling is essential for IL-2 production, so it has not been cleared whether reduction of nTreg in CD28-deficient mice may be IL-2-independent. Generation of nTreg cells and production of IL-2 require an identical Lck-binding motif in the CD28 cytosolic domain. Tai et al. [82 ] shows that the function of CD28 in nTreg cell generation and in IL-2 production was independent of each other. Thus, as B7/CD28 engagement appears to be required for thymic development and peripheral survival, the activation of nTreg cell function is CD28-independent.

Cellular sources of IL-2
As CD4+CD25+ nTreg cells suppress T cell responses by down-regulating IL-2 synthesis and paradoxically require IL-2 for their function and fitness, the dynamics of IL-2 production and suppression remain enigmatic in vivo. As IL-2 is likely to originate from activated T cells, a direct demonstration of the cellular source of IL-2 for CD4+CD25+ nTreg cell function in vivo has yet to be formally demonstrated. What cells produce IL-2 to stimulate nTreg has remained unclear. Neighboring cells such as activated T cells and DC are thought to be candidates. Matured DC (mDC) show a stimulation effect on nTreg cell proliferation [83 , 84 ]. However, whether this effect is dependent on IL-2 is controversial. No differences are shown in nTreg proliferation activity between WT and IL-2–/– mDC [33 , 83 , 84 ]. In contrast, CD40–/– DC are impaired in nTreg proliferation, but additional exogenous IL-2 is able to restore the normal number of nTreg cells in CD40–/– mice [80 ], suggesting that DC-derived IL-2 plays a critical role in nTreg cell activation [85 ]. Thymic stromal lymphopoietin (TSLP) expressed by human Hassall’s corpuscles activated DC maturation. In addition, these TSLP-conditioned DC induce the proliferation and differentiation of CD4+CD8–CD25– thymic T cells into CD4+CD25+Foxp3+ nTreg cells [86 ].

A model for IL-2-mediated T cell regulation: inflammation drives regulation
The attribution of IL-2 as an essential molecular determinant for nTreg suppressive activity has two important implications. It is known that TCR triggering alone does not induce full suppressive activity in nTreg cells. This likely represents a fail-safe system, as most nTreg probably recognize autoantigens with high affinity, would constantly be activated by antigen in vivo, and would likely result in permanent systemic suppression. Thus, productive nTreg activation of function may depend on another coactivating signal, such as IL-2, provided by another cell type, as nTreg cells do not produce it themselves. Under physiological conditions, IL-2 is largely produced by activated CD4+ T cells and to a smaller extent, CD8+ T cells [63 ]. Some studies have also reported DC as being a possible IL-2 source [80 ]. Thus, the activation status of effector T cells may modulate the induction of T cell suppression, such that the level of secreted IL-2 in inflammatory microenvironments may have a functional impact on nTreg cells. In normal, noninflammed mice, low-level IL-2 production, resulting from low-affinity TCR stimulation within the heterogeneous T cell repertoire, may determine nTreg cell peripheral homeostasis without necessarily inducing overt nTreg cell effector functions (see Go Fig. 3 ). Under such conditions, nTreg suppressive activity may not be required, as the lack of costimulation prevents efficient effector T cell activation. Conversely, inflammation may result in higher IL-2 levels within inflammatory settings, subsequently permitting efficient activation of responder T cells and simultaneously allowing for the induction of nTreg suppressor activity (see Fig. 3 ). This model would suggest that the temporal lag between effector T cell activation and induction of Foxp3+ nTreg cell-suppressive activity avoids hindering T cell priming but would permit control of activated effector T cells within inflamed tissues. It remains to be seen whether selective tissues alter the magnitude or kinetic of IL-2 production. However, the activated nTreg state is restricted only to the short time window of active immunity, such that nTreg cells are induced to suppress by IL-2 only transiently, and when the immune response is terminated, and external IL-2 levels decline, the activity of nTreg cells wanes, thus preventing systemic immunosuppression. It remains to be determined whether IL-2 drives various nTreg effector functions, including secretion of suppressive cytokines such as IL-10.


Figure 2
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  		Figure 2. IL-2 regulates peripheral homeostasis of CD4+ nTreg cells. IL-2 is essential in peripheral homeostasis and suppressor function of nTreg cells. Under normal, noninflammatory conditions, low-level IL-2 produced by T cells or DC maintains nTreg cell survival and peripheral homeostasis. Under inflammatory settings such as autoimmunity, infection, or tumorigenesis, high-level IL-2 produced by activated T cells or DC may promote the induction of nTreg cell function and subsequently inhibit clonal expansion and effector T cell function.


Figure 3
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  		Figure 3. Pathways to CD4+CD25+ Treg cell-mediated suppression. (A) To mediate its contact-dependent suppression, CD4+CD25+ nTreg cells may act by down-modulating APC function or by competing for APC-derived costimulatory signals such as adhesion and costimulation (A) or by a direct action on responding T cells (B). (B) CD4+CD25+ nTreg cells may mediate their suppressor function via an APC function-independent, T–T cell interaction, termed suppressor synapse (SS). This direct T–T cell interaction may require cognate contact with APC for initiation and stabilization of these T–T conjugates.


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MOLECULAR BASIS OF CD4+CD25+Treg CELL-MEDIATED SUPPRESSION
 
The molecular basis of the mechanism by which CD4+CD25+ nTreg cells suppress T cell-mediated immune responses is unknown [87 ]. Some studies support roles of cytokines in the regulation, and others support the contribution of cell-to-cell cognate interactions with effector T cells on APC (Figs. 2 and 3 ).

Inflammatory context-dependent roles for suppressive cytokines
Several immunosuppressive cytokines have been implicated in nTreg effector functions. Most murine and human in vitro studies conclude that nTreg-mediated suppression adopts a cytokine-independent mechanism of suppression not involving the action of suppressive cytokines such as IL-4, IL-10, and TGF-ß1, as in vitro neutralization of these cytokines alone or in combination failed to abrogate suppression [31 , 88 ]. In addition, CD4+CD25+ nTreg cells isolated from IL-4–/–, TGF-ß1–/–, or IL-10–/– mice are as effective as WT CD4+CD25+ T cell mice in their ability to suppress T cell proliferation in vitro [31 , 89 ]. Supernatants derived from activated CD4+CD25+ T cells do not possess suppressive properties, and suppression was not observed when CD4+CD25+ cells were separated from their target cells via Transwell© chambers, supporting the view that CD4+CD25+-mediated suppression is not mediated by secreted cytokines [31 ]. The possibility remains that contact-dependent suppression might be dominant in vitro, circumventing any requirements for long-range suppressive cytokines. The contribution of nTreg-derived cytokines in the mechanism via which CD4+CD25+ nTreg cells regulate responses in vivo is unknown and remains controversial. Their involvement might be affected by many context-dependent, physiological factors including the nature and magnitude of inflammation.

IL-10
Although addition of neutralizing anti-IL-10 monoclonal antibodies (mAb) failed to abrogate the ability of CD4+CD25+ Treg cells to mediate suppression, its role in CD4+CD25+ T cell-mediated suppression appears to be context-dependent in vivo. An essential role for IL-10 in CD4+CD25+ T cell-mediated suppression has been shown primarily in a murine inflammatory bowel disease (IBD) model, where disease is induced by the transfer of CD4+CD25–CD45RBhigh T cells to severe combined immunodeficiency (SCID) mice and prevented by cotransfer of CD4+CD25+ or CD4+CD45RBlowT cells. In this system, administration of neutralizing anti-IL-10R mAb to recipient mice abrogated the suppression, resulting in the development of IBD [90 ]. In addition, CD4+CD25+ or CD4+ CD45RBlow T cells from IL-10–/– mice failed to prevent IBD in this model when disease was induced with antigen-experienced effector T cells [91 ]. In addition, IL-10–/– T cells effectively prevented autoimmune gastritis produced by depletion of CD4+CD25+ nTreg cells in BALB/c mice but are insufficient to suppress bacterial-driven IBD in the same recipient animal [92 ]. It is interesting that IL-10–/– BALB/c mice spontaneously developed IBD but not gastritis. Contribution of IL-10 to CD4+CD25+ nTreg cell-mediated suppression was also shown in murine models of transplantation tolerance, graft-versus-host disease, and a rat model of Type 1 diabetes (reviewed in refs. [9 , 30 ]). CD4+CD25+ nTreg cells control the persistence, and concomitant immunity to reinfections of the intracellular pathogen, Leishmania major, was also partially dependent on IL-10 [93 ]. The control of pathogen persistence in sites of infection occurred independently of nTreg-derived IL-10 in the initial phase of disease establishment and appeared to be IL-10-dependent in chronic phases of disease, confirming the notion that nTreg can adapt to their inflammatory microenvironments for efficient and timely disease control [93 ]. This apparent requirement for IL-10 in chronic stages of infection might reflect possible roles of IL-10 in nTreg activity or in the induction of IL-10 from other cellular sources.

TGF-ß1
The potential effector role of TGF-ß1 in CD4+CD25+ nTreg cell-mediated suppression in vitro is controversial. TGF-ß1 is a logical candidate, as it plays a critical role in the down-regulation of immune responses, as illustrated by the development of a severe autoimmune-like syndrome in TGF-ß1–/– mice, characterized by the spontaneous and progressive multiorgan infiltration of mononuclear cells and pathogenic autoantibodies [94 ]. This is further corroborated by the observation that genetic disruption of TGF-ß1 signaling in T cells by overexpression of a dominant-negative TGF-ß Type II receptor (DNRIITg), conditional deletion of the TGF-ß Type II receptor in hematopoietic progenitors [95 ], or inactivation of the gene encoding the receptor-activated Smad3 [96 ] alters the sensitivity of T cells to the inhibitory effects of TGF-ß and leads to aberrant T cell responses.

One study reported that activated CD4+CD25+ T cells express an inactive form of TGF-ß1 complexed to its latency-associated peptide (LAP), which is retained as a membrane-bound complex by an undefined surface receptor, a complex hypothesized to account for the enhanced, suppressive capacity of activated CD4+CD25+ T cells in vitro [97 , 98 ]. Nonetheless, most murine and human in vitro studies conclude that neither secreted nor membrane-bound forms of active or latent TGF-ß1 are responsible for contact-dependent suppression mediated by resting or activated CD4+CD25+ T cells. DNRIITg and Smad3–/– T cells, which are resistant to the growth-inhibitory effects of exogenous TGF-ß1, remain susceptible to suppression by CD4+CD25+ T cells [89 , 99 100 101 102 ]. It is more important that CD4+CD25+ T cells isolated from neonatal TGF-ß1–/– mice are anergic to TCR signals and display comparable suppressive activity to WT nTreg cells in vitro and in contrast to their CD4+CD25– counterparts, are positive for CTLA-4, GITR, CD103, and Foxp3, a phenotype consistent with WT CD4+CD25+ nTreg cells [89 , 103 ].

A consensus view on the role of TGF-ß1 in CD4+CD25+ nTreg cell function in vivo is currently lacking. Some reports have suggested that secretion of TGF-ß1 by CD4+CD25+ nTreg cells is required to protect SCID mice from IBD induced by CD4+CD45RBhigh effector T cells, as treatment of recipients of CD4+CD45RBhigh and CD4+CD25+ nTreg cells with neutralizing anti-TGF-ß antibody reversed suppression [19 ]. Similarly, one recent study reported a requirement for TGF-ß1 in CD4+CD25+ nTreg cell-mediated control of CD8+ T cell antitumor activity [104 ]. Administration of anti-TGF-ß antibody neutralized suppressive activity of CD4+CD45RClow T cells in rat T1D and thyroiditis produced by adult thymectomy and irradiations [105 ]. However, the cellular source of the bioactive TGF-ß1 was not determined in these experiments and remains largely unknown. The regulatory role for TGF-ß1 in CD4+CD25+ T cell-mediated suppression is complicated by the fact that a variety of cell types can produce TGF-ß1 and might include nTreg cells themselves or activated effector T cells or is possibly induced by nTreg cells in nonlymphoid target tissues, which are inflammed or in the process of healing [89 , 106 ].

One study has concluded that CD4+CD25+LAP+ T cells expressing a membrane-bound form of TGF-ß1 were responsible for the control of IBD induced by CD4+CD45RBhigh effector T cells [98 ]. In contrast, Oida et al. [107 ] reported that TGF-ß1-dependent suppression was observed in the LAP+CD4+CD25– T cell subset. In addition, some studies have suggested a correlation between nTreg effector function and the apparent preferential expression of a latent, membrane-bound form of TGF-ß1 on CD4+CD25+ nTreg cells isolated from inflamed pancreatic lymphoid tissues [108 ]. However, these studies did not conclusively show functional evidence for a direct effect of nTreg cell-derived TGF-ß1 on responder T cells. Furthermore, additional studies have suggested that cell surface TGF-ß1, from autocrine or paracrine sources, may mediate its effects by actively signaling in CD4+CD25+ nTreg cells themselves, possibly by maintaining their survival, differentiation, expansion, or suppressive effector mechanism [103 , 104 ]. Furthermore, a recent study has shown that TGF-ß1 signaling in nTreg cells may promote Foxp3 expression and subsequent nTreg function [109 ]. We have recently demonstrated that CD4+CD25+ nTreg cells from TGF-ß1+/+ or neonatal TGF-ß1–/– mice can suppress the incidence and severity of IBD as well as colonic IFN-{gamma} mRNA expression induced by WT CD4+CD25– effector T cells [110 ]. Furthermore, TGF-ß-resistant Smad3–/– CD4+CD25+ nTreg cells are equivalent to WT nTreg cells in their capacity to suppress disease induced by WT and Smad3–/– CD4+CD25– effector T cells. Although CD4+CD25+ T cells from Smad3–/– mice appeared to function as efficiently as WT CD4+CD25+ T cells in our colitis model, it remains possible that TGF-ß could play a stimulatory role in the induction of CD4+CD25+ suppressor activity, perhaps in a Smad3-independent manner [96 ]. Thus, CD4+CD25+ nTreg cells are able to suppress intestinal inflammation by a mechanism not requiring nTreg cell-derived TGF-ß1 or effector T cell/Treg cell Smad3-dependent responsiveness to TGF-ß.

These findings strongly suggest that TGF-ß plays a critical role in the suppression of disease but that the TGF-ß is derived from host non-T cells or nonlymphoid cells or even from the effector T cells themselves. Furthermore, these results support the view that tissue/context-dependent factors may influence the mechanism of immune suppression by CD4+CD25+ nTreg cells in vivo. In autoimmune gastritis, CD4+CD25+ nTreg cells do not need suppressor cytokines and possibly suppress disease by resorting to a contact-dependent mechanism, a finding similar to what is observed in vitro. However, in the milieu of a bacteria-driven inflammation in the intestine, the contact-dependent pathway may not be sufficient to mediate disease protection and must be supplemented by suppressor cytokine production by the nTreg cells (e.g., IL-10) and by production of TGF-ß by non-nTreg cells. It is also possible that the CD4+CD25+ nTreg cells may facilitate the induction of TGF-ß production by host cells, which subsequently, promote the induction and differentiation of Foxp3+ nTreg cells from CD4+CD25– T cell precursors. We have recently demonstrated that TGF-ß1 can selectively promote the differentiation of IL-10-secreting, Foxp3+ CD4+ Treg cells from CD4+CD45RBlowCD25– T cell precursors (Michal Pyzik and C. A. Piccirillo, manuscript submitted). Thus, TGF-ß1 may enhance immunosuppression by sustaining Foxp3 expression/activity in nTreg cells and at the same time, promote the induction cytokine-secreting iTreg cells.


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REQUIREMENT OF CELL-TO-CELL INTERACTIONS FOR SUPPRESSION
 
Modulation of antigen-presenting function by CD4+CD25+ nTreg cells
The in vitro CD4+CD25+ nTreg cell-mediated suppression appears to depend on cell-to-cell cognate interactions in vitro. To mediate its contact-dependent suppression, nTreg cells may act by down-modulating APC function or by competing for APC-derived, costimulatory signals (Fig. 3) . nTreg cells can suppress CD4+ or CD8+ T cells in the presence of LPS-stimulated, chemically fixed blasts (metabolically inactive APC), suggesting that APC do not act as "suppression bridges," as modulation of costimulatory functions could not occur in this system [31 , 39 ]. Consistently, MHC Class I/II, B7.1/2, CD40, and intercellular adhesion molecule-1 expression on normal APC remains unaffected in suppressive cultures, although one study reported that CD4+CD25+ T cells down-regulated the expression levels of B7.1 and B7.2 on DC [111 ]. This in vitro, suppressive cell contact was not a result of killing of the responder population via a Fas/Fas ligand- or TNF/TNFR-dependent pathway, although a more recent study illustrates the importance of a granzyme B-dependent, perforin-independent mechanism in nTreg cell suppression [31 , 112 ]. In summary, although TCR activation of nTreg cells likely requires APC, the effector phase of CD4+CD25+ nTreg cell-mediated suppression in vitro is APC-independent.

Requirement for direct T–T cell interactions in suppression
We have addressed the functional relevance of nTreg-APC interactions in CD4+CD25+-mediated inhibition. To this end, we have made use of soluble MHC I H-2Kb/ovalbumin (OVA)257–264 tetramers to stimulate OT-I TCR transgenic, OVA-specific CD8+ T cells in a two-cell suppressor assay system to ask directly whether activated CD4+CD25+ T cells mediate their suppressor function via a direct T–T cell interaction or by modulating APC function (Fig. 3) [113 ]. With such a system, activated CD4+CD25+ nTreg cells, unlike CD4+CD25– T cells, can potently suppress the proliferation as well as IFN-{gamma} production by CD8+ T cells, thus conclusively demonstrating that nTreg cell-mediated suppression occurs via an APC-independent, T–T cell interaction, termed SS [113 ]. Similar results were also obtained in a study using anti-CD3/CD28-coated latex beads as surrogate APC [114 ]. The initial activation of nTreg is likely APC-dependent in vivo, and our proposed SS model of nTreg effector function does not exclude the possibility that nTreg cells might also inhibit APC function or use the APC surface as a stabilizing platform on which CD4+ or CD8+ effector and nTreg populations physically interact. T–T cellular conjugates can be readily observed and quantified by live cell imaging and fluorescein-activated cell sorter analysis (unpublished observation). It is interesting that these T–T cell interactions only occur when target T cells get activated via their TCR, suggesting that activated nTreg recognize a specific molecule(s) on the membranes of activated target T cells. Furthermore, as preactivated nTreg cells do not require TCR re-engagement for suppressor activity, one can infer that the suppressor mechanism operative in this SS remains stable and does not require de novo gene expression. It is also conceivable that the cognate receptor(s)-ligand(s) involved in this SS may be induced once initial contact between nTreg and responder T cells is engaged. Furthermore, this contact-dependent process may also require an active secretory component involving the action of short-range mediators, as shown by Gondek et al. [112 ]. The molecular nature of the SS and its significance are currently unknown. So far, no direct evidence for a contact-mediated suppression mechanism is available.

Currently, not much is known about the functional consequence of CD4+CD25+ nTreg cells on responding CD4+ or CD8+ T cells, other than the suppression of IL-2 mRNA transcription and consequential induction of cell cycle arrest [31 , 32 ]. One study reported the apparent induction of anergy in responding T cells, although this study needs confirmation [114 ]. Another study has reported that CD4+CD25+ nTreg cells facilitate the generation of other types of Treg cells, as coculture of nTreg cells with responder Th cells results in the differentiation of iTreg cells, which can suppress in vitro Th1 or Th2 cell responses via the production of TGF-ß1 and/or IL-10 [115 ]. CD4+CD25– T cells stimulated with cognate antigen in the presence of preactivated Treg cells become refractory to the mitogenic effect of IL-2. However the CD4+CD25–T cells cocultured with nTreg cells show STAT5 activation through an IL-2R signal [116 ]. In antigen-primed CD8+ CTLs, nTreg cells down-regulate Akt phosphorylation but have no effect on STAT5 activation [117 ]. Lastly, some reports demonstrate that suppression of effector T cells cocultured with CD4+CD25+ T cells is not abrogated in the presence of exogeneous IL-2, and it is supposed that IL-2 unresponsiveness may be induced in these T cells [113 , 116 ].

Adaptability versus diversification model of CD4+CD25+ Treg function
When taken together, these results indicate that more than one mechanism of CD4+CD25+ nTreg cell-mediated suppression is operative in vitro and in vivo. As discussed above, CD4+CD25+ nTreg cell function may resort to contact-dependent and/or cytokine-dependent mechanisms, depending on the nature and magnitude of the inflammatory response and the target tissue to which the immune response is directed. This context-dependent mode of regulation leads to an adaptability-diversification model of nTreg cell function (Fig. 4 ): nTreg cells may represent a functionally homogenous subset, which consists of precommitted cells capable of adapting to the inflammatory milieu such that one nTreg cell may exert suppression by more than one mechanism depending on the site of regulation and the intensity of the immune response. Alternatively, CD4+CD25+ nTreg cells may represent a functionally diversified group consisting of different nTreg subsets, such that some inhibit by a cell contact-dependent mechanism, and others are endowed with distinct cytokine production capabilities or possibly, a combination of both mechanisms. Currently, experimental evidence permitting discrimination between these two different models is lacking, but genomic and proteomic analysis of nTreg cells at the clonal level, in conjunction with functional correlates, would allow us to truly delineate possible molecular pathways underlying these functional differences.


Figure 4
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  		Figure 4. Adaptability versus diversification model for CD4+CD25+ Treg cells. The adaptability-diversification model of nTreg cell function may explain the context-dependent character of CD4+CD25+ nTreg cell activity depending on the nature, magnitude, and location of the inflammatory response. In the adaptability model, nTreg cells may represent a functionally homogenous subset, which consists of precommitted cells capable of adapting to the inflammatory milieu, such that one Treg cell may exert suppression by a variety of mechanisms, depending on the site of regulation and the intensity of the immune response. Alternatively, the diversification model suggests that CD4+CD25+ nTreg cells may represent a functionally diversified group consisting of different nTreg cell subsets, such that some inhibit by a cell contact-dependent mechanism, some endowed with distinct cytokine production capabilities, and others use a combination of mechanisms.


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SUMMARY AND CONCLUSION
 
There are multiple immune-tolerance pathways that regulate adaptive immune responses. Amongst these, CD4+CD25+ nTreg cells represent a master-switch of peripheral T cell tolerance, as abrogation of nTreg development or function can provoke autoimmunity and also increase immunity to tumor, allergens, grafts, and various pathogens. Thus, nTreg cells play a determining role in the balance between tolerance and immunity, and alterations in their development or function provoked by environmental or genetic triggers may represent a determining variable in disease resistance or susceptible (Fig. 5 ).


Figure 5
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  		Figure 5. CD4+Foxp3+ Treg maintain balance between tolerance and immunity. CD4+CD25+ nTreg cells represent a central master-switch of peripheral T cell tolerance and play a determining role in the balance between tolerance and immunity to self and foreign antigens (Ag). Alterations in the development or function of nTreg provoked by physical, chemical, environmental, or genetic triggers can provoke autoimmunity and also increase immunity to tumor, allergens, grafts, and various pathogens. In addition to antigen-specific signals, nTreg function may also be modulated by various nonspecific signals and thus, represent another determining variable in disease resistance or susceptibility.

The tolerogenic functions of nTreg cells in several mouse models of disease are well accepted. The ultimate goal of the treatment of autoimmune disease is to re-establish tolerance to the self-antigens targeted in autoimmune disease by potentiating nTreg cells to expand or bolster their suppressive activity and in turn, suppress the functions of autoimmune T cells in new-onset and established disease. Furthermore, given the potent immunosuppressive properties of CD4+CD25+ nTreg cells, their presence may also be detrimental during an immune response to tumors, pathogens, and in vaccination.

Finally, the mechanism underlying nTreg cell-mediated suppression still remains highly contentious, with noticeable differences between various in vitro and in vivo studies in mice and humans. The unreliable nature of CD25 as a stable and exclusive marker of nTreg cells in inflammatory contexts often complicates the interpretation of many studies. The discovery of more specific surface biomarkers for nTreg cells is imperative, as this will undeniably facilitate our ability to monitor nTreg cellular frequency and function in the context of a given disease and will serve to determine the clinical effectiveness of novel therapeutic strategies destined to modulate nTreg function in vivo.


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ACKNOWLEDGEMENTS
 
We acknowledge the Canadian Institutes for Health Research and Canadian Foundation for Innovation for financial support. C. A. P. is a recipient of a Canada Research Chair in "Regulatory Lymphocytes of the Immune System."

Received February 13, 2006; revised April 4, 2006; accepted April 26, 2006.


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