Related Articles

Thymic stromal lymphopoietin (TSLP) is an essential cytokine for the initiation and development of allergic inflammation. Here, we have investigated the role of TSLP in the breakdown of immune tolerance and generation of inducible regulatory T cells (iTregs). Our results demonstrated that TSLP diverted airway tolerance against ovalbumin to Th2 sensitization and inhibited the generation of OVA-specific iTregs. TSLP exerted a direct inhibitory effect on both human and mouse iTreg development in vitro. Low doses of TSLP were capable of inhibiting iTreg induction without significantly promoting Th2 development, indicating that these two functions of TSLP are separable. Moreover, the TSLP-mediated inhibition of iTreg generation was only partially dependent on IL-4 and Stat6, and was effective when TSLP was present for the first 24 h of T cell activation. These results define a novel role for TSLP in regulating the balance of airway tolerance and allergic inflammation.

In addition to thymus-derived or natural T regulatory (nTreg) cells, a second subset of induced T regulatory (iTreg) cells arises de novo from conventional CD4+ T cells in the periphery. The function of iTreg cells in tolerance was examined in a CD45RBhighCD4+ T cell transfer model of colitis. In situ-generated iTreg cells were similar to nTreg cells in their capacity to suppress T cell proliferation in vitro and their absence in vivo accelerated bowel disease. Treatment with nTreg cells resolved the colitis, but only when iTreg cells were also present. Although iTreg cells required Foxp3 for suppressive activity and phenotypic stability, their gene expression profile was distinct from the established nTreg “genetic signature,” indicative of developmental and possibly mechanistic differences. These results identified a functional role for iTreg cells in vivo and demonstrated that both iTreg and nTreg cells can act in concert to maintain tolerance.

Regulatory T cells (Treg) express the forkhead box p3 (Foxp3) transcription factor and suppress pathological immune responses against self and foreign antigens, including commensal microorganisms. Foxp3 has been proposed as a master key regulator for Treg, required for their differentiation, maintenance, and suppressive functions. Two types of Treg have been defined. Natural Treg (nTreg) are usually considered to be a separate sublineage arising during thymus differentiation. Induced Treg (iTreg) originate upon T cell receptor (TCR) stimulation in the presence of tumor growth factor β. Although under homeostatic conditions most Treg in the periphery are nTreg, special immune challenges in the intestine promote more frequently the generation of iTreg. Furthermore, recent observations have challenged the notion that Treg are a stable sublineage, and they suggest that, particularly under lymphopenic and/or inflammatory conditions, Treg may lose Foxp3 and/or acquire diverse effector functions, especially in the intestine, which may contribute to uncontrolled inflammation.

Foxp3+ T regulatory cells (Tregs) consisting of natural and induced Treg subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The actions designed to correct defects in numbers or functions of Tregs may be therapeutic in the treatment of autoimmune diseases. While recent studies demonstrated that natural Tregs are instable and dysfunctional in the inflammatory condition, induced Tregs (iTregs) may have a different feature. Here we review the progress of iTregs, particularly focus on their stability and function in the established autoimmune diseases. The advantage of iTregs as therapeutics used under inflammatory conditions is highlighted. Proper generation and manipulation of iTregs used for cellular therapy may provide a promise for the treatment of many autoimmune and inflammatory diseases.

Background

We previously showed that co-immunization with a protein antigen and a DNA vaccine coding for the same antigen induces CD40low IL-10high tolerogenic DCs, which in turn stimulates the expansion of antigen-specific CD4+CD25-Foxp3+ regulatory T cells (CD25- iTreg). However, it was unclear how to choose the antigen sequence to maximize tolerogenic antigen presentation and, consequently, CD25- iTreg induction.

Results

In the present study, we demonstrated the requirement of highly antigenic epitopes for CD25- iTreg induction. Firstly, we showed that the induction of CD25- iTreg by tolerogenic DC can be blocked by anti-MHC-II antibody. Next, both the number and the suppressive activity of CD25- iTreg correlated positively with the overt antigenicity of an epitope to activate T cells. Finally, in a mouse model of dermatitis, highly antigenic epitopes derived from a flea allergen not only induced more CD25- iTreg, but also more effectively prevented allergenic reaction to the allergen than did weakly antigenic epitopes.

Conclusions

Our data thus indicate that efficient induction of CD25- iTreg requires highly antigenic peptide epitopes. This finding suggests that highly antigenic epitopes should be used for efficient induction of CD25- iTreg for clinical applications such as flea allergic dermatitis.

Interplay between Foxp3+ regulatory T cells (Treg) and dendritic cells (DCs) maintains immunologic tolerance, but the effects of each cell on the other are not well understood. We report that polyclonal CD4+Foxp3+ Treg cells induced ex vivo with transforming growth factor beta (TGFβ) (iTreg) suppress a lupus-like chronic graft-versus-host disease by preventing the expansion of immunogenic DCs and inducing protective DCs that generate additional recipient CD4+Foxp3+ cells. The protective effects of the transferred iTreg cells required both interleukin (IL)-10 and TGFβ, but the tolerogenic effects of the iTreg on DCs, and the immunosuppressive effects of these DCs were exclusively TGFβ-dependent. The iTreg were unable to tolerize Tgfbr2-deficient DCs. These results support the essential role of DCs in ‘infectious tolerance’ and emphasize the central role of TGFβ in protective iTreg/DC interactions in vivo.

Background

Induced regulatory T (iTreg) lymphocytes show promise for application in the treatment of allergic, autoimmune and inflammatory disorders. iTreg cells demonstrate advantages over natural Treg (nTreg) cells in terms of increased number of starting population and greater potential to proliferate. Different activation methods to generate iTreg cells result in iTreg cells that are heterogeneous in phenotype and mechanisms of suppression. Therefore it is of interest to explore new techniques to generate iTreg cells and to determine their physiological relevance.

Methods

Using phorbol myristate acetate (PMA)/ionomycin and anti-CD3 activation of CD4+CD25- cells we generated in vitro functional CD4+CD25+ iTreg (TregPMA) cells. Functionality of the generated TregPMA cells was tested in vivo in a mouse model of inflammatory bowel disease (IBD) - CD45RB transfer colitis model.

Results

TregPMA cells expressed regulatory markers and proved to ameliorate the disease phenotype in murine CD45RB transfer colitis model. The body weight loss and disease activity scores for TregPMA treated mice were reduced when compared to diseased control group. Histological assessment of colon sections confirmed amelioration of the disease phenotype. Additionally, cytokine analysis showed decreased levels of proinflammatory colonic and plasma IL-6, colonic IL-1 β and higher levels of colonic IL-17 when compared to diseased control group.

Conclusions

This study identifies a new method to generate in vitro iTreg cells (TregPMA cells) which physiological efficacy has been demonstrated in vivo.

Background

It has been documented all-trans retinoic acid (atRA) promotes the development of TGF-β-induced CD4+Foxp3+ regulatory T cells (iTreg) that play a vital role in the prevention of autoimmune responses, however, molecular mechanisms involved remain elusive. Our objective, therefore, was to determine how atRA promotes the differentiation of iTregs.

Methodology/Principal Findings

Addition of atRA to naïve CD4+CD25− cells stimulated with anti-CD3/CD28 antibodies in the presence of TGF-β not only increased Foxp3+ iTreg differentiation, but maintained Foxp3 expression through apoptosis inhibition. atRA/TGF-β-treated CD4+ cells developed complete anergy and displayed increased suppressive activity. Infusion of atRA/TGF-β-treated CD4+ cells resulted in the greater effects on suppressing symptoms and protecting the survival of chronic GVHD mice with typical lupus-like syndromes than did CD4+ cells treated with TGF-β alone. atRA did not significantly affect the phosphorylation levels of Smad2/3 and still promoted iTreg differentiation in CD4+ cells isolated from Smad3 KO and Smad2 conditional KO mice. Conversely, atRA markedly increased ERK1/2 activation, and blockade of ERK1/2 signaling completely abolished the enhanced effects of atRA on Foxp3 expression. Moreover, atRA significantly increased histone methylation and acetylation within the promoter and conserved non-coding DNA sequence (CNS) elements at the Foxp3 gene locus and the recruitment of phosphor-RNA polymerase II, while DNA methylation in the CNS3 was not significantly altered.

Conclusions/Significance

We have identified the cellular and molecular mechanism(s) by which atRA promotes the development and maintenance of iTregs. These results will help to enhance the quantity and quality of development of iTregs and may provide novel insights into clinical cell therapy for patients with autoimmune diseases and those needing organ transplantation.

Stimulation of naïve mouse CD4+Foxp3− T cells in the presence of TGF-β results in the induction of Foxp3 expression and T suppressor function. However, Foxp3 expression in these induced T regulatory cells (iTreg) is unstable raising the possibility that iTreg would not be useful for treatment of autoimmune diseases. To analyze the factors that control the stability of Foxp3 expression in iTreg, we generated OVA-specific iTreg from OT-II Foxp3-GFP knock in mice. Following transfer to normal C57BL/6 mice, OT-II GFP+ cells maintained high levels of Foxp3 expression for 8 days. However, they rapidly lost Foxp3 expression upon stimulation with OVA in IFA in vivo. This unstable phenotype was associated with a strong methylation of the Treg-specific demethylated region (TSDR) within the Foxp3 locus. Administration of IL-2/anti-IL-2 complexes expanded the numbers of transferred Foxp3+ iTreg in the absence of antigen challenge. Notably, when the iTreg were stimulated with antigen, treatment with IL-2/anti-IL-2 complexes stabilized Foxp3 expression and resulted in enhanced demethylation of the TSDR. Conversely, neutralization of IL-2 or disruption of its signaling by deletion of Stat5 diminished the level of Foxp3 expression resulting in decreased suppressor function of the iTreg in vivo. Our data suggest that stimulation with TGF-β in vitro is not sufficient for imprinting T cells with stable expression of Foxp3. Administration of IL-2 in vivo results in stabilization of Foxp3 expression and may prove to be a valuable adjunct for the use of iTreg for the treatment of autoimmune diseases.

CD4+CD25+ regulatory T cells (Treg) are important mediators of immune tolerance. A subset of Treg can be generated in the periphery by TGF-beta dependent conversion of conventional CD4+CD25− T cells into induced Treg (iTreg). In chronic viral infection or malignancy, such induced iTreg, which limit the depletion of aberrant or infected cells, may be of pathogenic relevance. To identify potential targets for therapeutic intervention, we investigated the TGF-beta signaling in Treg. In contrast to conventional CD4+ T cells, Treg exhibited marked activation of the p38 MAP kinase pathway. Inhibition of p38 MAP kinase activity prevented the TGF-beta-dependent conversion of CD4+CD25− T cells into Foxp3+ iTreg in vitro. Of note, the suppressive capacity of nTreg was not affected by inhibiting p38 MAP kinase. Our findings indicate that signaling via p38 MAP kinase seems to be important for the peripheral generation of iTreg; p38 MAP kinase could thus be a therapeutic target to enhance immunity to chronic viral infection or cancer.

SUMMARY

Although both natural and induced regulatory T (nTreg and iTreg) cells can enforce tolerance, the mechanisms underlying their synergistic actions have not been established. We examined the functions of nTreg and iTreg cells by adoptive transfer immunotherapy of newborn Foxp3-deficient mice. As monotherapy, only nTreg cells prevented disease lethality, but did not suppress chronic inflammation and autoimmunity. Provision of Foxp3-sufficient conventional T cells with nTreg cells reconstituted the iTreg pool and established tolerance. In turn, acute depletion of iTreg cells in rescued mice resulted in weight loss and inflammation. Whereas the transcriptional signatures of nTreg and in vivo derived iTreg cells were closely matched, there was minimal overlap in their T cell receptor (TCR) repertoires. Thus, iTreg cells are an essential non-redundant regulatory subset that supplements nTreg cells, in part by expanding TCR diversity within regulatory responses.

In addition to naturally occurring regulatory T (nTreg) cells derived from the thymus, functionally competent Treg cells can be induced in vitro from peripheral blood lymphocytes in response to TCR stimulation with cytokine costimulation. Using these artificial stimulation conditions, both naïve as well as memory CD4+ T cells can be converted into induced Treg (iTreg) cells, but the cellular origin of such iTreg cells in vivo or in response to more physiologic stimulation with pathogen-derived antigens is less clear. Here, we demonstrate that a freeze/thaw lysate of Plasmodium falciparum schizont extract (PfSE) can induce functionally competent Treg cells from peripheral lymphocytes in a time- and dose-dependent manner without the addition of exogenous costimulatory factors. The PfSE-mediated induction of Treg cells required the presence of nTreg cells in the starting culture. Further experiments mixing either memory or naïve T cells with antigen presenting cells and CFSE-labeled Treg cells identified CD4+CD45RO+CD25− memory T cells rather than Treg cells as the primary source of PfSE-induced Treg cells. Taken together, these data suggest that in the presence of nTreg cells, PfSE induces memory T cells to convert into iTreg cells that subsequently expand alongside PfSE-induced effector T cells.

Tumor specific antigens (TSA) provide an opportunity to mobilize therapeutic immune responses against cancer. To evade such responses, tumor development in immunocompetent hosts is accompanied by acquisition of both active and passive mechanisms of immune suppression, including recruitment of CD4+FoxP3+ regulatory T cells (Treg). Thymic derived Treg (nTreg) may recognize self-antigens in the tumor microenvironment, while peripherally induced Treg (iTreg) may preferentially recognize the same TSA which provide an opportunity for therapeutic immunity from peripheral T cells. In this study we provide a systematic analysis of nTreg and iTreg accumulation in the tumor microenvironment (TME) at the cellular level. iTreg accumulation to the TME was influenced by the abundance of a known TSA, and in the absence of a known TSA intratumoral Treg displayed a unique TCR repertoire from peripheral Treg. In vivo suppression assays demonstrate that cognate-antigen matched iTreg are more potent suppressors of CD4+ than are polyclonal iTreg or nTreg, but were unable to suppress CD8+ T cell proliferation. Suppression occurred only locally at the site of immunization, and correlated with decreased expression of CD80 and CD86 on CD11c positive cells. Although established tumors facilitated the induction of TSA-specific iTreg, these iTreg suppressed CD4+ T cell accumulation only locally to the TME. Tumor mediated suppression of CD8+ T cell immunity appeared independent of TSA-specific iTreg.

The low number of natural regulatory T cells (nTreg cells) in the circulation specific for a particular antigen and concerns about the bystander suppressive capacity of expanded nTregs presents a major clinical challenge for nTreg-based therapeutic treatment of autoimmune diseases. In the present study, we demonstrate that naïve CD4+CD25-Foxp3- T cells specific for the myelin proteolipid protein (PLP)139-151 peptide, can be converted into CD25+Foxp3+ induced Treg cells (iTreg cells) when stimulated in the presence of transforming growth factor-β (TGF-β), retinoic acid and interleukin-2. These PLP139-151-specific iTreg cells (139-iTreg cells) have a phenotype similar to natural Treg cells, but additionally express an intermediate level of CD62L and a high level of CD103. Upon transfer into SJL/J mice, 139-iTreg cells undergo antigen-driven proliferation and are effective at suppressing induction of experimental autoimmune encephalomyelitis induced by the cognate PLP139-151 peptide, but not PLP178-191 or a mixture of the two peptides. Furthermore, 139-iTregs inhibit delayed-type-hypersensitivity (DTH) responses to PLP139-151, but not PLP178-191, MOG35-55 or OVA323-339 in mice primed with a mixture of PLP139-151 and the other respective peptides. Additionally, 139-iTreg cells suppress the proliferation and activation of PLP139-151-, but not MOG35-55-specific CD4+ T cells in SJL/B6 F1 mice primed with a combination of PLP139-151 and MOG35-55. These findings suggest that antigen-specific-iTreg cells are amplified in vivo when exposed to cognate antigen under inflammatory conditions, and these activated iTreg cells suppress CD4+ responder T cells in an antigen-specific manner.

Adoptive transfer of thymus-derived natural regulatory T-cells (nTregs) effectively suppresses disease in murine models of autoimmunity and graft-versus-host disease (GVHD). TGFβ induces Foxp3 expression and suppressive function in stimulated murine CD4+25- T cells, and these induced Treg (iTregs), like nTreg, suppress auto- and allo-reactivity in vivo. However, while TGFβ induces Foxp3 expression in stimulated human T-cells, the expanded cells lack suppressor cell function. Here we show that Rapamycin (Rapa) enhances TGFβ-dependent Foxp3 expression and induces a potent suppressor function in naïve (CD4+25-45RA+) T cells. Rapa/TGFβ iTregs are anergic, express CD25 at levels higher than expanded nTregs, and few cells secrete IL-2, IFNγ or IL-17 even after PMA and Ionomycin stimulation in vitro. Unlike other published methods of inducing Treg function, Rapa/TGFβ induces suppressive function even in the presence of memory CD4+ T-cells. A single apheresis unit of blood yields an average ~240×109 (range ~70–560×109) iTregs from CD4+25- T-cells in ≤ 2 weeks of culture. Most importantly, Rapa/TGFβ iTregs suppress disease in a xenogeneic model of GVHD. This study opens the door for iTreg cellular therapy for human diseases.

Whereas TGF-β is essential for the development of peripherally induced Foxp3+ regulatory T cells (iTreg cells) and Th17 cells, the intracellular signaling mechanism by which TGF-β regulates development of both cell subsets is less understood. In this study, we report that neither Smad2 nor Smad3 gene deficiency abrogates TGF-β–dependent iTreg induction by a deacetylase inhibitor trichostatin A in vivo, although the loss of the Smad2 or Smad3 gene partially reduces iTreg induction in vitro. Similarly, SMAD2 and SMAD3 have a redundant role in development of Th17 in vitro and in experimental autoimmune encephalomyelitis. In addition, ERK and/or JNK pathways were shown to be involved in regulating iTreg cells, whereas the p38 pathway predominately modulated Th17 and experimental autoimmune encephalomyelitis induction. Therefore, selective targeting of these intracellular TGF-β signaling pathways during iTreg and Th17 cell development might lead to the development of therapies in treating autoimmune and other chronic inflammatory diseases.

Foxp3 reporter mice including DEREG (DEpletion of REGulatory T cells) mice have greatly helped in exploring the biology of Foxp3+ Tregs. DEREG mice express a DTR-eGFP fusion protein under the control of a bacterial artificial chromosome (BAC)-encoded Foxp3 promoter, allowing the viable isolation and inducible depletion of Foxp3+ Tregs. Adaptive Tregs differentiated in vitro to express Foxp3 (iTregs) are gaining high interest as potential therapeutics for inflammatory conditions such as autoimmunity, allergy and transplant rejection. However, selective isolation of Foxp3+ iTregs with a stable phenotype still remains to be a problem, especially in the human setting. While screening for culture conditions to generate stable CD4+Foxp3+ iTregs from DEREG mice, with maximum suppressive activity, we observed an unexpected dichotomy of eGFP and Foxp3 expression which is not seen in ex vivo isolated cells from DEREG mice. Further characterization of eGFP+Foxp3− cells revealed relatively lower CD25 expression and a lack of suppressive activity in vitro. Similarly, eGFP− cells isolated from the same cultures were not suppressive despite of a broad CD25 expression reflecting mere T cell activation. In contrast, eGFP+Foxp3+ iTregs exhibited potent suppressive activity comparable to that of natural eGFP+Foxp3+ Tregs, emphasizing the importance of isolating Foxp3 expressing iTregs. Interestingly, the use of plate-bound anti-CD3 and anti-CD28 or Flt3L-driven BMDC resulted in considerable resolution of the observed dichotomy. In summary, we defined culture conditions for efficient generation of eGFP+Foxp3+ iTregs by use of DEREG mice. Isolation of functional Foxp3+ iTregs using DEREG mice can also be achieved under sub-optimal conditions based on the magnitude of surface CD25 expression, in synergy with transgene encoded eGFP. Besides, the reported phenomenon may be of general interest for exploring Foxp3 gene regulation, given that Foxp3 and eGFP expression are driven from distinct Foxp3 loci and because this dichotomy preferentially occurs only under defined in vitro conditions.

Foxp3+T regulatory cell (Treg) subsets play a crucial role in the maintenance of immune homeostasis against self-antigen. The lack or dysfunction of these cells is responsible for the pathogenesis and development of many autoimmune diseases. Therefore, manipulation of these cells may provide a novel therapeutic approach to treat autoimmune diseases and prevent allograft rejection during organ transplantation. In the article, we will provide current opinions concerning the classification, developmental and functional characterizations of Treg subsets. A particular emphasis will be focused on transforming cell growth factor beta (TGF-β) and its role in the differentiation and development of induced regulatory T cells (iTregs) in the periphery. Moreover, the similarity and disparity of iTregs and naturally occurring, thymus-derived CD4+CD25+Foxp3+ regulatory T cells (nTregs) will also be discussed. While proinflammatory cytokine IL-6 can convert nTregs to IL-17-producing cells, peripheral Tregs induced by TGF-β are resistant to this cytokine. This difference may affect the role of each in the adaptive immune response.

The CD25+Foxp3+ regulatory T-cells (Treg) that had lost CD25 and Foxp3 in vivo (ex-Treg) exist but are difficult to study. We generated antigen (Ag)-specific Treg hybridomas from iTreg clones (iTreg-hyb) using iTreg of DO11.10.Foxp3-GFP mice and presented evidence that they behave like ex-Treg. The iTreg-hyb displayed little CD25 and Foxp3-GFP but strong expression could be induced with OVA323–339 in the presence of Ag-presenting cells, rIL-2 and rTGF-β1. They displayed all of the iTreg-associated markers examined except CTLA-4, the latter was also absent in the ex-Treg. They lacked the Helios transcription factor, suggesting they were derived from iTreg. Similar to ex-Treg, the iTreg-hyb produced high level of IL-2 and Foxp3 under specific activation conditions. Two unusual properties were observed. First, the ability to induce Foxp3-GFP upon activation is progressively lost in culture over a period of 2 to 4 weeks. Second, Rag2−/− spleen cells alone selectively induced Foxp3-GFP expression albeit 30 times less efficient than Ag-specific activation. We identified cell-free supernatant, IL-6, IL-9, and IL-27 as Foxp3-inducing factors. Our study has significant implications to the stability, plasticity and fate of Treg. The usefulness and limitation of iTreg-hyb as a novel tool to study Foxp3 regulation and the fate of specific Treg subsets are discussed.

Tumors convert conventional CD4+ T cells into induced CD4+CD25+FoxP3+ T regulatory (iTreg) cells that serve as an effective means of immune evasion. Therefore, the blockade of conventional CD4+ T cell conversion into iTreg cells represents an attractive target for improving the efficacy of various immunotherapeutic approaches. Using a novel form of 4-1BBL molecule, SA-4-1BBL, we previously demonstrated that costimulation via 4-1BB receptor renders both CD4+and CD8+ T effector (Teff) cells refractory to inhibition by Treg cells and increased intratumoral Teff/Treg cell ratio that correlated with therapeutic efficacy in various preclinical tumor models. Building on these studies, we herein show for the first time, to our knowledge, that signaling through 4-1BB inhibits antigen- and TGF-β-driven conversion of naïve CD4+FoxP3− T cells into iTreg cells via stimulation of IFN-γ production by CD4+FoxP3− T cells. Importantly, treatment with SA-4-1BBL blocked the conversion of CD4+FoxP3− T cells into Treg cells by EG.7 tumors. Taken together with our previous studies, these results show that 4-1BB signaling negatively modulate Treg cells by two distinct mechanisms: i) inhibiting the conversion of CD4+FoxP3− T cells into iTreg cells and ii) endowing Teff cells refractory to inhibition by Treg cells. Given the dominant role of Treg cells in tumor immune evasion mechanisms, 4-1BB signaling represents an attractive target for favorably tipping the Teff:Treg balance toward Teff cells with important implications for cancer immunotherapy.

The ability of regulatory T cells (Treg) to traffic to sites of inflammation supports their role in controlling immune responses. This feature supports the idea that adoptive transfer of in vitro expanded human Treg could be used for treatment of immune/inflammatory diseases. However, the migratory behavior of Treg as well as their direct influence at the site of inflammation remains poorly understood. To explore the possibility that Treg may have direct anti-inflammatory influences on tissues, independent of their well-established suppressive effects on lymphocytes, we studied the adhesive interactions between mouse Treg and endothelial cells, as well as their influence on endothelial function during acute inflammation. We show that Foxp3+ adaptive/inducible Treg (iTreg) but not naturally occurring Treg (nTreg) efficiently interact with endothelial selectins and transmigrate through endothelial monolayers in vitro. In response to activation by endothelial antigen presentation or immobilized anti-CD3ε, Foxp3+ iTreg suppressed TNFα and IL-1β mediated endothelial selectin expression and adhesiveness to effector T cells. This suppression was contact independent, rapid acting, and mediated by TGFβ-induced activin receptor like kinase [ALK]5 signaling in endothelial cells. In addition, Foxp3+ iTreg adhered to inflamed endothelium in vivo and their secretion products blocked acute inflammation in a model of peritonitis. These data support the concept that Foxp3+ iTreg help to regulate inflammation independently of their influence on effector T cells by direct suppression of endothelial activation and leukocyte recruitment.

That regulatory T cells (Tregs) have a crucial role in controlling allergic diseases such as asthma is now undisputed. The cytokines most commonly implicated in Treg-mediated suppression of allergic asthma are TGF-β and IL-10. In addition to naturally occurring Tregs, adaptive Tregs, induced in response to foreign antigens, have been demonstrated in recent studies. The concept of inducible/adaptive Tregs (iTregs) has considerable significance in preventing asthma if generated early enough in life. This is because cytokines such as IL-4 and IL-6 inhibit Foxp3 induction in naïve CD4+ T cells and therefore de novo generation of Tregs can be expected to be less efficient when it is concomitant with effector cell development in response to an allergen. However, if iTregs can be induced, the process of infectious tolerance would facilitate expansion of the iTreg pool as suggested in the recent literature. It is tempting to speculate that there is a window of opportunity in early life in the context of a relatively immature immune system that is permissive for the generation of iTregs specific to a spectrum of allergens that would regulate asthma lifelong. The focus of this review is the relevance of nTregs and iTregs in controlling asthma from early life into adulthood, the mechanisms underlying Treg function and the prospects for utilizing our current concepts to harness the full potential of Tregs to limit disease development and progression.

The concentration of antigen or mitogenic stimuli is known to play an important role in controlling the differentiation of naïve CD4+ T cells into different effector phenotypes. In particular, whereas TCR engagement at low antigen doses in the presence of TGF-β and IL-2 can promote differentiation of Foxp3-expressing induced regulatory T cells (iTregs), high levels of antigen have been shown in vitro and in vivo to prevent Foxp3 upregulation. This tight control of iTreg differentiation dictated by antigen dose likely determines the quality and duration of an immune response. However, the molecular mechanism by which this high dose-inhibition of Foxp3 induction occurs is not well understood. In this study, we demonstrate that when cells are in the presence of CD28 costimulation, TCR-dependent NF-κB signaling is essential for Foxp3 inhibition at high doses of TCR engagement in mouse T cells. Prevention of Foxp3 induction depends on the production of NF-κB-dependent cytokines by the T cells themselves. Moreover, T cells that fail to upregulate Foxp3 under iTreg-differentiating conditions and high TCR stimulation acquire the capacity to make TNF and IFN-γ, as well as IL-17 and IL-9, especially if IFN-γ signaling is antagonized. Thus, NF-κB helps T cells control their differentiation fate in a cell-intrinsic manner and prevents peripheral iTreg development under conditions of high antigen load that may require more vigorous effector T cell responses.

CD4 T cell lineages are marked by the signature transcription factor each lineage expresses. For example, regulatory T cells (Tregs) are characterized by expression of FOXP3, which is either induced during thymic development for natural Tregs (nTregs), or in the periphery in the presence of TGFβ and retinoic acid for induced Tregs (iTreg). Interestingly, recent work has shown that the signature transcription factor for Th17 cells, RORγt, is also induced by TGFβ, thus linking the differentiation of the Treg and Th17 lineages. In the absence of a second signal from a proinflammatory cytokine, FOXP3 can inhibit RORγt function and drive Treg differentiation. However, when the cell also receives a signal from a proinflammation cytokine (e.g., IL-6), FOXP3 function is inhibited and the Th17 differentiation pathway is induced. Therefore, it is the balance between FOXP3 and RORγt function that determines CD4 T cell fate and the type of immune response that will be generated.

Induced T regulatory (iTreg) cells can be generated by peripheral dendritic cells (DCs) that mediate T cell-unresponsiveness to re-challenge with antigen. The molecular factors required for the function of such iTreg cells remain unknown. We report a critical role for the transcription co-factor Homeodomain only protein (Hop, also know as Hopx) in iTregs cells to mediate T cell unresponsiveness in vivo. Hopx-sufficient iTreg cells down-regulate the expression of the AP-1 complex and suppress other T cells. In the absence of Hopx, iTreg cells express high levels of the AP-1 complex, proliferate and fail to mediate T cell-unresponsiveness to re-challenge with antigen. Thus, Hopx is required for the function of Treg cells induced by DCs and the promotion of DC-mediated T cell unresponsiveness in vivo.