Thymus-derived, naturally-occurring CD4+ FoxP3+ regulatory T cells (nTreg) have suppressive activity that is important for the establishment and maintenance of immune homeostasis in the healthy state. Abundant reports have shown that they can suppress pathogenic processes in autoimmune diseases and inhibit transplant rejection and graft-versus-host disease. Far less is known about induced regulatory T cells (iTreg) that are generated from naïve T cells in the periphery or in vitro, by directing naïve T cells to acquire suppressive function under the influence of transforming growth factor-β (TGF-β) and other factors. In this review, we describe mechanisms by which naïve T cells are thought to be converted into iTreg. We also discuss the suppressive potential of iTreg, particularly in comparison to their naturally-occurring counterparts, focusing on those reports in which direct comparisons have been made. Based on current knowledge, we consider the rationale for using iTreg versus nTreg in clinical trials.
Naturally-occurring regulatory T cells; induced regulatory T cells; immune suppression; tolerance; transplantation
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.
FOXP3; Regulatory T Cell; Th17
Regulatory T cells (Treg) that express the transcription factor Foxp3 are enriched within a broad range of murine and human solid tumors. The ontogeny of these Foxp3 Tregs - selective accumulation or proliferation of natural thymus-derived Treg (nTreg) or induced Treg (iTreg) converted in the periphery from naïve T cells - is not known. We used several strains of mice in which Foxp3 and EGFP are coordinately expressed to address this issue. We confirmed that Foxp3-positive CD4 T cells are enriched among tumor-infiltrating lymphocytes (TIL) and splenocytes (SPL) in B16 murine melanoma-bearing C57BL/6 Foxp3EGFP mice. OT-II Foxp3EGFP mice are essentially devoid of nTreg, having transgenic CD4 T cells that recognize a class II-restricted epitope derived from ovalbumin; Foxp3 expression could not be detected in TIL or SPL in these mice when implanted with ovalbumin-transfected B16 tumor (B16-OVA). Likewise, TIL isolated from B16 tumors implanted in Pmel-1 Foxp3EGFP mice, whose CD8 T cells recognize a class I-restricted gp100 epitope, were not induced to express Foxp3. All of these T cell populations - wild-type CD4, pmel CD8 and OTII CD4 - could be induced in vitro to express Foxp3 by engagement of their T cell receptor (TCR) and exposure to transforming growth factor β (TGFβ). B16 melanoma produces TGFβ and both pmel CD8 and OTII CD4 express TCR that should be engaged within B16 and B16-OVA respectively. Thus, CD8 and CD4 transgenic T cells in these animal models failed to undergo peripheral induction of Foxp3 in a tumor microenvironment.
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.
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.
Circulating Foxp3+ regulatory T cells (Treg) may arise in the thymus (natural Treg, nTreg) or through the adaptive upregulation of Foxp3 after T cell activation (induced Treg, iTreg). In this brief review, we explore evidence for the formation and function of iTreg during pathologic conditions. Determining the ontogeny and function of Treg populations has relied on the use of manipulated systems in which either iTreg or nTreg are absent, or lineage tracing of T cell clones through repertoire analyses. iTreg appear particularly important at mucosal interfaces. iTreg can also ameliorate tissue-specific autoimmunity and are a prominent source of tumor-infiltrating Treg in some models. However, under many conditions, including in CNS autoimmunity, diabetes, and some tumor systems, iTreg formation appears limited. The immunological contribution of iTreg is thus highly context dependent. Deciphering immune parameters responsible for iTreg formation and their role in modulating pathologic immune responses will be important.
regulatory T lymphocyte; nTreg; iTreg; EAE; diabetes; tumor; colitis; TCR repertoire
CD4+ CD25+ Foxp3+ regulatory T (Treg) cells are essential to the balance between pro- and anti-inflammatory responses. There are two major subsets of Treg cells, “natural” Treg (nTreg) cells that develop in the thymus, and “induced” Treg (iTreg) cells that arise in the periphery from CD4+ Foxp3− conventional T cells and can be generated in vitro. Previous work has established that both subsets are required for immunological tolerance. Additionally, in vitro-derived iTreg cells can reestablish tolerance in situations where Treg cells are decreased or defective. This review will focus on iTreg cells, drawing comparisons to nTreg cells when possible. We discuss the molecular mechanisms of iTreg cell induction, both in vivo and in vitro, review the Foxp3-dependent and -independent transcriptional landscape of iTreg cells, and examine the proposed suppressive mechanisms utilized by each Treg cell subset. We also compare the T cell receptor repertoire of the Treg cell subsets, discuss inflammatory conditions where iTreg cells are generated or have been used for treatment, and address the issue of iTreg cell stability.
Treg cells; Treg stability; immunotherapy; Treg function; gene expression profiling; TCR repertoire
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.
CD4+Foxp3+ T regulatory (Treg) cells control many facets of immune responses ranging from autoimmune diseases, to inflammatory conditions, and cancer in an attempt to maintain immune homeostasis. Natural Treg (nTreg) cells develop in the thymus and constitute a critical arm of active mechanisms of peripheral tolerance particularly to self antigens. A growing body of knowledge now supports the existence of induced Treg (iTreg) cells which may derive from a population of conventional CD4+ T cells. The fork-head transcription factor (Foxp3) typically is expressed by natural CD4+ Treg cells, and thus serves as a marker to definitively identify these cells. On the contrary, there is less consensus on what constitutes iTreg cells as their precise definition has been somewhat elusive. This is in part due to their distinct phenotypes which are shaped by exposure to certain inflammatory or “assault” signals stemming from the underlying immune disorder. The “policing” activity of Treg cells tends to be uni-directional in several pathological conditions. On one end of the spectrum, Treg cell suppressive activity is beneficial by curtailing T cell response against self-antigens and allergens thus preventing autoimmune diseases and allergies. On the other end however, their inhibitory roles in limiting immune response against pseudo-self antigens as in tumors often culminates into negative outcomes. In this review, we focus on this latter aspect of Treg cell immunobiology by highlighting the involvement of nTreg cells in various animal models and human tumors. We further discuss iTreg cells, relationship with their natural counterpart, and potential co-operation between the two in modulating immune response against tumors. Lastly, we discuss studies focusing on these cells as targets for improving anti-tumor immunity.
Tregs; Foxp3; natural; induced; cancer; tumor; Interleukin-10; transforming growth factor β
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.
Immunoregulation; regulatory T cells; TGF-β; Foxp3; Th17 cells
Immune homeostasis is dependent on tight control over the size of a population of regulatory T (Treg) cells capable of suppressing over-exuberant immune responses. The Treg cell subset is comprised of cells that commit to the Treg lineage by upregulating the transcription factor Foxp3 either in the thymus (tTreg) or in the periphery (iTreg)1,2. Considering a central role for Foxp3 in Treg cell differentiation and function3,4, we proposed that conserved non-coding DNA sequence (CNS) elements at the Foxp3 locus encode information defining the size, composition and stability of the Treg cell population. Here we describe the function of three Foxp3 CNS elements (CNS1–3) in Treg cell fate determination in mice. The pioneer element CNS3, which acts to potently increase the frequency of Treg cells generated in the thymus and the periphery, binds c-Rel in in vitro assays. In contrast, CNS1, which contains a TGF-β–NFAT response element, is superfluous for tTreg cell differentiation, but has a prominent role in iTreg cell generation in gut-associated lymphoid tissues. CNS2, although dispensable for Foxp3 induction, is required for Foxp3 expression in the progeny of dividing Treg cells. Foxp3 binds to CNS2 in a Cbf-β–Runx1 and CpG DNA demethylation-dependent manner, suggesting that Foxp3 recruitment to this ‘cellular memory module’ facilitates the heritable maintenance of the active state of the Foxp3 locus and, therefore, Treg lineage stability. Together, our studies demonstrate that the composition, size and maintenance of the Treg cell population are controlled by Foxp3 CNS elements engaged in response to distinct cell-extrinsic or -intrinsic cues.
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) are needed in the control of immune responses and to maintain immune homeostasis. Of this subtype of regulatory lymphocytes, the most potent are Foxp3 expressing CD4+ T cells, which can be roughly divided into two main groups; natural Treg cells (nTreg), developing in the thymus, and induced or adaptive Treg cells (iTreg), developing in the periphery from naïve, conventional T cells. Both nTreg cells and iTreg cells have their own, non-redundant roles in the immune system, with nTreg cells mainly maintaining tolerance toward self-structures, and iTreg developing in response to externally delivered antigens or commensal microbes. In addition, Treg cells acquire tissue specific features and are adapted to function in the tissue they reside. This review will focus on some specific features of Treg cells in different compartments of the body.
regulatory T cell; tissue specificity; systems biology; tolerance; immunity; microbiota
Suppressor of cytokine signaling (SOCS) proteins are key regulators of CD4+ T cell differentiation, and in particular, we have recently shown that SOCS2 inhibits the development of Th2 cells and allergic immune responses. Interestingly, transcriptome analyses have identified SOCS2 as being preferentially expressed in both natural regulatory T cells (Tregs) and inducible Tregs (iTregs); however, the role of SOCS2 in Foxp3+ Treg function or development has not been fully elucidated. In this study, we show that despite having no effect on natural Treg development or function, SOCS2 is highly expressed in iTregs and required for the stable expression of Foxp3 in iTregs in vitro and in vivo. Indeed, SOCS2-deficient CD4+ T cells upregulated Foxp3 following in vitro TGF-β stimulation, but failed to maintain stable expression of Foxp3. Moreover, in vivo generation of iTregs following OVA feeding was impaired in the absence of SOCS2 and could be rescued in the presence of IL-4 neutralizing Ab. Following IL-4 stimulation, SOCS2-deficient Foxp3+ iTregs secreted elevated IFN-γ and IL-13 levels and displayed enhanced STAT6 phosphorylation. Therefore, we propose that SOCS2 regulates iTreg stability by downregulating IL-4 signaling. Moreover, SOCS2 is essential to maintain the anti-inflammatory phenotype of iTregs by preventing the secretion of proinflammatory cytokines. Collectively, these results suggest that SOCS2 may prevent IL-4–induced Foxp3+ iTreg instability. Foxp3+ iTregs are key regulators of immune responses at mucosal surfaces; therefore, this dual role of SOCS2 in both Th2 and Foxp3+ iTregs reinforces SOCS2 as a potential therapeutic target for Th2-biased diseases.
Acute graft versus host disease (GvHD) is a major cause of mortality in allogeneic bone marrow transplantation (BMT), for which administration of FoxP3+ Treg cells has been proposed as a therapy. However, the phenotypic stability of Treg cells is controversial and cytokines that signal through the transcription factor STAT3 can inhibit FoxP3 expression. We assessed whether the elimination of STAT3 in T cells could limit the severity of GvHD, and if so, what mechanisms were involved. We found STAT3 limited the numbers of FoxP3+ Treg cells following allogeneic BMT by two pathways: instability of nTregs and inhibition of iTreg cell polarization from naïve CD4+ T cells. Deletion of STAT3 within only the nTreg cell population was not sufficient to protect against lethal GvHD. In contrast, transfer of STAT3-deficient naïve CD4+ T cells increased FoxP3+ Treg cells post-BMT and prevented lethality, suggesting that the consequence of STAT3-signaling may be greater for inducible rather than natural Treg cells during GvHD.
Acute GvHD; STAT3; nTreg plasticity; FoxP3
Transforming growth factor-β (TGF-β) has been shown to play an essential role in the suppression of inflammation, yet recent studies have revealed the positive roles of TGF-β in inflammatory responses. For example, TGF-β induces Foxp3-positive regulatory T cells (iTregs) in the presence of interleukin-2 (IL-2), while in the presence of IL-6, it induces pathogenic IL-17 producing Th17 cells. TGF-β inhibits the proliferation of immune cells as well as cytokine production via Foxp3-dependent and -independent mechanisms. Little is known about molecular mechanisms involved in immune suppression via TGF-β; however, Smad2/3 have been shown to play essential roles in Foxp3 induction as well as in IL-2 and IFN-γ suppression, whereas Th17 differentiation is promoted via the Smad-independent pathway. Interaction between TGF-β and other cytokine signaling is important in establishing the balance of immunity and tolerance.
Immunity; tolerance; signal transduction; smad; T cell
Mice deficient for the adaptor Ndfip1 develop inflammation at sites of environmental antigen exposure. We show here that these animals contain fewer inducible regulatory (iTreg) cells. In vitro, Ndfip1-deficient T cells express normal levels of the transcription factor Foxp3 during the first 48 hours of iTreg cell differentiation, however this cannot be sustained. Abortive Foxp3 expression is because Ndfip1–/– cells produce interleukin 4 (IL-4). We demonstrate that Ndfip1 is transiently unregulated during iTreg cell differentiation in a transforming growth factor-β (TGF-β) dependent manner. Once expressed Ndfip1 promotes Itch-mediated degradation of the transcription factor JunB, thus preventing IL-4 production. Based on these data, we propose that TGF-β signaling induces Ndfip1 expression to silence IL-4 production, thus permitting iTreg cell differentiation.
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.
FoxP3; iTreg; nTreg; regulatory T cell; tumor microenvironment
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.
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.
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.
Foxp3; Hybridoma; Cytokines; Regulatory T-cell fate
Regulatory T cells (Tregs) are a subset of T cells that are responsible for maintaining peripheral immune tolerance and homeostasis. The hallmark of Tregs is the expression of the forkhead box P3 (FoxP3) transcription factor. Natural regulatory T cells (nTregs) are a distinct population of T cells that express CD4 and FoxP3. nTregs develop in the thymus and function in maintaining peripheral immune tolerance. Other CD4+, CD4-CD8-, and CD8+CD28- T cells can be induced to acquire regulatory function by antigenic stimulation, depending on the cytokine milieu. Inducible (or adaptive) Tregs frequently express high levels of the interleukin 2 receptor (CD25). Atypical Tregs express FoxP3 and CD4 but have no surface expression of CD25. Type 1 regulatory T cells (Tr1 cells) produce IL-10, while T helper 3 cells (Th3) produce TGF-β. The function of inducible Tregs is presumably to maintain immune homeostasis, especially in the context of chronic inflammation or infection. Induction of Tregs in coronaviral infections protects against the more severe forms of the disease attributable to the host response. However, arteriviruses have exploited these T cell subsets as a means to dampen the immune response allowing for viral persistence. Treg induction or activation in the pathogenesis of disease has been described in both porcine reproductive and respiratory syndrome virus, lactate dehydrogenase elevating virus, and mouse hepatitis virus. This review discusses the development and biology of regulatory T cells in the context of arteriviral and coronaviral infection.
regulatory T cell; arterivirus, porcine reproductive and respiratory syndrome virus; lactate dehydrogenase-elevating virus; coronavirus
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.
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.
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.
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.
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.