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1.  HTLV-1 bZIP Factor Induces Inflammation through Labile Foxp3 Expression 
PLoS Pathogens  2013;9(9):e1003630.
Human T-cell leukemia virus type 1 (HTLV-1) causes both a neoplastic disease and inflammatory diseases, including HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). The HTLV-1 basic leucine zipper factor (HBZ) gene is encoded in the minus strand of the proviral DNA and is constitutively expressed in infected cells and ATL cells. HBZ increases the number of regulatory T (Treg) cells by inducing the Foxp3 gene transcription. Recent studies have revealed that some CD4+Foxp3+ T cells are not terminally differentiated but have a plasticity to convert to other T-cell subsets. Induced Treg (iTreg) cells tend to lose Foxp3 expression, and may acquire an effector phenotype accompanied by the production of inflammatory cytokines, such as interferon-γ (IFN-γ). In this study, we analyzed a pathogenic mechanism of chronic inflammation related with HTLV-1 infection via focusing on HBZ and Foxp3. Infiltration of lymphocytes was observed in the skin, lung and intestine of HBZ-Tg mice. As mechanisms, adhesion and migration of HBZ-expressing CD4+ T cells were enhanced in these mice. Foxp3−CD4+ T cells produced higher amounts of IFN-γ compared to those from non-Tg mice. Expression of Helios was reduced in Treg cells from HBZ-Tg mice and HAM/TSP patients, indicating that iTreg cells are predominant. Consistent with this finding, the conserved non-coding sequence 2 region of the Foxp3 gene was hypermethylated in Treg cells of HBZ-Tg mice, which is a characteristic of iTreg cells. Furthermore, Treg cells in the spleen of HBZ-transgenic mice tended to lose Foxp3 expression and produced an excessive amount of IFN-γ, while Foxp3 expression was stable in natural Treg cells of the thymus. HBZ enhances the generation of iTreg cells, which likely convert to Foxp3−T cells producing IFN-γ. The HBZ-mediated proinflammatory phenotype of CD4+ T cells is implicated in the pathogenesis of HTLV-1-associated inflammation.
Author Summary
Viral infection frequently induces tissue inflammation in the host. HTLV-1 infection is associated with chronic inflammation in the CNS, skin, and lung, but the inflammatory mechanism is not fully understood yet. Since HTLV-1 directly infects CD4+ T cells, central player of the host immune regulation, HTLV-1 should modulate the host immune response not only via viral antigen stimulation but also via CD4+ T-cell-mediated immune deregulation. It has been reported that Foxp3+CD4+ T cells are increased in HTLV-1 infection. It remains a central question in HTLV-1 pathogenesis why HTLV-1 induces inflammation despite of increase of FoxP3+ cells, which generally possess immune suppressive function. We have elucidated here that most of the increased Foxp3+ cells in HBZ-Tg mice or HAM/TSP patients is not thymus-derived naturally occurring Treg cells but induced Treg cells. Since the iTreg cells are prone to lose FoxP3 expression and then become cytokine-producing cells, the increase of iTreg cells could serve as a source of proinflammatory CD4+ T cells. Thus HTLV-1 causes abnormal CD4+ T-cell differentiation by expressing HBZ, which should play a crucial role in chronic inflammation related with HTLV-1. This study has provided new insights into the mechanism of chronic inflammation accompanied with viral infection.
PMCID: PMC3777874  PMID: 24068936
2.  Plasmodium falciparum–Mediated Induction of Human CD25hiFoxp3hi CD4 T Cells Is Independent of Direct TCR Stimulation and Requires IL-2, IL-10 and TGFβ 
PLoS Pathogens  2009;5(8):e1000543.
CD4+CD25+Foxp3+ regulatory T cells (Tregs) regulate disease-associated immunity and excessive inflammatory responses, and numbers of CD4+CD25+Foxp3+ Tregs are increased during malaria infection. The mechanisms governing their generation, however, remain to be elucidated. In this study we investigated the role of commonly accepted factors for Foxp3 induction, TCR stimulation and cytokines such as IL-2, TGFβ and IL-10, in the generation of human CD4+CD25+Foxp3+ T cells by the malaria parasite Plasmodium falciparum. Using a co-culture system of malaria-infected red blood cells (iRBCs) and peripheral blood mononuclear cells from healthy individuals, we found that two populations of Foxp3hi and Foxp3int CD4+CD25hi T cells with a typical Treg phenotype (CTLA-4+, CD127low, CD39+, ICOS+, TNFRII+) were induced. Pro-inflammatory cytokine production was confined to the Foxp3int subset (IFNγ, IL-4 and IL-17) and inversely correlated with high relative levels of Foxp3hi cells, consistent with Foxp3hi CD4 T cell–mediated inhibition of parasite-induced effector cytokine T cell responses. Both Foxp3hi and Foxp3int cells were derived primarily from proliferating CD4+CD25− T cells with a further significant contribution from CD25+Foxp3+ natural Treg cells to the generation of the Foxp3hi subset. Generation of Foxp3hi, but not Foxp3int, cells specifically required TGFβ1 and IL-10. Add-back experiments showed that monocytes expressing increased levels of co-stimulatory molecules were sufficient for iRBC-mediated induction of Foxp3 in CD4 T cells. Foxp3 induction was driven by IL-2 from CD4 T cells stimulated in an MHC class II–dependent manner. However, transwell separation experiments showed that direct contact of monocytes with the cells that acquire Foxp3 expression was not required. This novel TCR-independent and therefore antigen-non specific mechanism for by-stander CD4+CD25hiFoxp3+ cell induction is likely to reflect a process also occurring in vivo as a consequence of immune activation during malaria infection, and potentially a range of other infectious diseases.
Author Summary
Infection with the malaria parasite Plasmodium falciparum affects 300–600 million people each year. Regulatory T cells (Tregs) expressing the transcription factor Foxp3, which drives genes involved in immunosuppression, are specialized immune cells that can inhibit both protective and harmful inflammatory responses during malaria. While Treg numbers are increased during malaria infection, little is known about how they are induced by the parasite. We addressed this question using an in vitro culture system to model the interaction of the malaria parasite with human immune cells. We found that the parasite induced soluble immune mediators, including the T cell growth-factor IL-2 and the regulatory proteins IL-10 and TGFβ, which drive the induction and expansion of Tregs. These Tregs expressed high levels of Foxp3 and suppressed the production of inflammation and protective immunity-driving mediators by concurrently induced effector T cells. Importantly, we demonstrate that induction of Tregs by the malaria parasite did not necessarily require direct contact with antigen-presenting cells. Our findings suggest that the parasite induces Tregs in an antigen non-specific manner, which may explain why malarial immunosuppression is not confined to malaria-specific immune responses, and provide new insights into the mechanisms governing Treg induction during malaria infection, and potentially other infectious diseases.
PMCID: PMC2718810  PMID: 19680449
3.  Regulatory T Cells in Many Flavors Control Asthma 
Mucosal immunology  2010;3(3):216-229.
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.
PMCID: PMC3039023  PMID: 20164832
Tregs; asthma; tolerance; TGF-β; IL-10
4.  Alterations of peripheral CD4+CD25+Foxp3+ T regulatory cells in mice with STZ-induced diabetes 
Complications arising from abnormal immune responses are the major causes of mortality and morbidity in diabetic patients. CD4+CD25+ T regulatory cells (Tregs) play pivotal roles in controlling immune homeostasis, immunity and tolerance. The effect of hyperglycemia on CD4+CD25+ Tregs has not yet been addressed. Here we used streptozotocin (STZ)-induced diabetic mice to study the effects of long-term hyperglycemia on CD4+CD25+ Tregs in vivo. Four months after the onset of diabetes, the frequency of CD4+CD25+Foxp3+ T regulatory cells was significantly elevated in the spleen, peripheral blood lymphocytes (PBLs), peripheral lymph nodes (pLNs) and mesenteric LNs (mLNs). CD4+CD25+ Tregs obtained from mice with diabetes displayed defective immunosuppressive functions and an activated/memory phenotype. Insulin administration rescued these changes in the CD4+CD25+ Tregs of diabetic mice. The percentage of thymic CD4+CD25+ naturally occurring Tregs (nTregs) and peripheral CD4+Helios+Foxp3+ nTregs were markedly enhanced in diabetic mice, indicating that thymic output contributed to the increased frequency of peripheral CD4+CD25+ Tregs in diabetic mice. In an in vitro assay in which Tregs were induced from CD4+CD25− T cells by transforming growth factor (TGF)-β, high glucose enhanced the efficiency of CD4+CD25+Foxp3+ inducible Tregs (iTregs) induction. In addition, CD4+CD25− T cells from diabetic mice were more susceptible to CD4+CD25+Foxp3+ iTreg differentiation than those cells from control mice. These data, together with the enhanced frequency of CD4+Helios−Foxp3+ iTregs in the periphery of mice with diabetes, indicate that enhanced CD4+CD25+Foxp3+ iTreg induction also contributes to a peripheral increase in CD4+CD25+ Tregs in diabetic mice. Our data show that hyperglycemia may alter the frequency of CD4+CD25+Foxp3+ Tregs in mice, which may result in late-state immune dysfunction in patients with diabetes.
PMCID: PMC4002930  PMID: 21983870
CD4+CD25+Foxp3+ regulatory T cells;  diabetes; hyperglycemia; immune disorder; mice
5.  A dynamic dual role of IL-2 signaling in the two-step differentiation process of adaptive regulatory T cells1 
The molecular mechanism of the extrathymic generation of adaptive CD4+Foxp3+ regulatory T (iTreg) cells remains incompletely defined. We show that exposure of splenic CD4+CD25+Foxp3− cells to IL-2, but not other γc cytokines, resulted in Stat5 phosphorylation and induced Foxp3 expression in ~10% of the cells. Thus, IL-2/Stat5 signaling may be critical for Foxp3 induction in peripheral CD4+CD25+Foxp3− iTreg cell precursors. Herein, to further define the role of IL-2 in the formation of iTreg cell precursors as well as their subsequent Foxp3 expression, we designed a two-step iTreg cell differentiation model. During the initial “conditioning” step, CD4+CD25−Foxp3− naïve T cells were activated by TCR stimulation. Inhibition of IL-2 signaling via Jak3-Stat5 was required during this step to generate CD4+CD25+Foxp3− cells containing iTreg cell precursors. During the subsequent Foxp3-induction step driven by cytokines, IL-2 was the most potent cytokine to induce Foxp3 expression in these iTreg cell precursors. This two-step method generated a large number of iTreg cells with relatively stable expression of Foxp3, which were able to prevent CD4+CD45RBhigh cell-mediated colitis in Rag1−/− mice. Taken together, while initial inhibition of IL-2 signaling upon T cell priming generates iTreg cell precursors, subsequent activation of IL-2 signaling in these precursors induces the expression of Foxp3. These findings advance the understanding of iTreg cell differentiation, and may facilitate the therapeutic use of iTreg cells in immune disorders.
PMCID: PMC3608742  PMID: 23427250
6.  Role of conserved non-coding DNA elements in the Foxp3 gene in regulatory T-cell fate 
Nature  2010;463(7282):808-812.
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.
PMCID: PMC2884187  PMID: 20072126
7.  IL-10 produced by iTreg cells controls colitis and pathogenic ex-iTreg cells during immunotherapy1 
“Natural” regulatory T (nTreg) cells that express the transcription factor Foxp3 and produce IL-10 are required for systemic immunological tolerance. “Induced” Treg (iTreg) cells are non-redundant and essential for tolerance at mucosal surfaces, yet their mechanisms of suppression and stability are unknown. We investigated the role of iTreg cell-produced IL-10 and iTreg cell fate in a treatment model of inflammatory bowel disease. Colitis was induced in Rag1−/− mice by the adoptive transfer of naïve CD4+ T cells carrying a non-functional Foxp3 allele. At the onset of weight loss, mice were treated with both iTreg and nTreg cells where one marked subset was selectively IL-10-deficient. Body weight assessment, histological scoring, cytokine analysis, and flow cytometry were used to monitor disease activity. Transcriptional profiling and TCR repertoire analysis were used to track cell fate. When nTreg cells were present but IL-10 deficient, iTreg cell-produced IL-10 was necessary and sufficient for the treatment of disease, and vice versa. Invariably, ~85% of the transferred iTreg cells lost Foxp3 expression (ex-iTreg) but retained a portion of the iTreg transcriptome, which failed to limit their pathogenic potential upon retransfer. TCR repertoire analysis revealed no clonal relationships between iTreg and ex-iTreg cells, either within mice or between mice treated with the same cells. These data identify a dynamic IL-10-dependent functional reciprocity between Treg subsets that maintains mucosal tolerance. The niche supporting stable iTreg cells is limited and readily saturated, which promotes a large population of ex-iTreg cells with pathogenic potential during immunotherapy.
PMCID: PMC3537488  PMID: 23125413
8.  Natural and Induced T Regulatory Cells in Cancer 
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.
PMCID: PMC3708155  PMID: 23874336
Tregs; Foxp3; natural; induced; cancer; tumor; Interleukin-10; transforming growth factor β
9.  High TCR Stimuli Prevent iTreg Differentiation in an NF-κB-Dependent Manner1 
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.
PMCID: PMC3544303  PMID: 21411734
10.  Regulatory T-Cell (Treg) Hybridoma as a Novel Tool to Study Foxp3 Regulation and Treg Fate1 
Journal of autoimmunity  2011;37(2):113-121.
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.
PMCID: PMC3173573  PMID: 21621978
Foxp3; Hybridoma; Cytokines; Regulatory T-cell fate
11.  TGF-β-induced Myelin Peptide-Specific Regulatory T Cells Mediate Antigen-Specific Suppression of Induction of Experimental Autoimmune Encephalomyelitis1 
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.
PMCID: PMC2882517  PMID: 20483764
12.  IL-12 Converts Foxp3+ Regulatory T Cells to Foxp3+IFN-γ+ T Cells with Inhibitory Functions During Induction of Colitis 
Gastroenterology  2011;140(7):2031-2043.
Regulatory T (Treg) cells are plastic, but the in vivo mechanisms by which they are converted into Foxp3+interferon (IFN)-γ+ T cells, and whether these converted cells retain the ability to inhibit colitis, are not clear.
Foxp3+ Treg cells were generated by culture of naïve CD4+ T cells from Foxp3GFP CBir1 T-cell receptor (TCR) transgenic (CBir1-Tg) mice, which are specific for CBir1 flagellin (an immunodominant microbiota antigen), with transforming growth factor (TGF)-β. Foxp3GFP+ CBir1-Tg Treg cells were isolated by fluorescence-activated cell sorting and transferred into TCRβxδ−/− mice. Colitis was induced by transfer of naïve CBir1-Tg CD4+ T cells into immunodeficient mice.
Microbiota antigen-specific Foxp3+ Treg cells were converted, in the intestine, to IFN-γ+ T-helper (Th)1 cells, interleukin (IL)-17+ Th17 cells, and Foxp3+ T cells that coexpress IFN-γ and/or IL-17. Conversion of Treg cells into IFN-γ-producing Th1 cells and Foxp3+IFN-γ+ T cells required innate cell production of IL-12 in the intestine; blocking IL-12 with an antibody inhibited their conversion to Th1 and Foxp3+IFN-γ+ T cells in the intestines of mice that were recipients of Treg cells. Addition of IL-12, but not IL-23, promoted conversion of Treg cells into Th1 and Foxp3+IFN-γ+ T cells, in vitro. Foxp3+IFN-γ+ T cells had regulatory activity, because they suppressed proliferation of naïve T cells, in vitro, and inhibited induction of colitis by microbiota antigen-specific T cells. IFN-γ+ Th1 cells were not converted into Treg cells; Foxp3+IFN-γ+ T cells differentiated into IFN-γ+ but not Foxp3+ T cells.
IL-12 promotes conversion of Treg cells into IFN-γ-expressing cells; Foxp3+IFN-γ+ T cells retain their regulatory functions and develop during the transition of Foxp3+ Treg cells into IFN-γ+ Th1 cells.
PMCID: PMC3109200  PMID: 21419767
IBD; immune regulation; inflammation; Crohn’s disease
13.  Regulation of Foxp3+ Inducible Regulatory T Cell Stability by SOCS2 
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.
PMCID: PMC3607399  PMID: 23455506
14.  Induced T Regulatory Cells Suppress Osteoclastogenesis and Bone Erosion in Collagen-induced Arthritis Better than Natural T Tegulatory Cells 
Annals of the rheumatic diseases  2012;71(9):1567-1572.
Osteoclasts are responsible for bone destruction in rheumatoid arthritis (RA) and natural CD4+Foxp3+regulatory T cells (nTregs) can inhibit osteoclastogenesis. This study aims to determine whether TGF-β-induced CD4+Foxp3+regulatory T cells (iTregs) also suppress osteolastogenesis and bone erosion in collagen induced arthritis (CIA).
Osteoclasts were induced from bone-marrow CD11b+ cells with RANKL and macrophage colony-stimulating factor (M-CSF), and assessed with tartrate-resistant acid phosphatase (TRAP) staining. CD4+ iTregs were generated with TGF-β and added to cultures with different ratios with CD11b+ cells. Transwell and antibody blockade experiments were performed to define the mechanism of action. NF-kB activation was determined by western blot. 3×106 CD4+ iTregs, nTregs or control cells were adoptively transferred to DBA1/J mice on day 14 after immunization with CII/CFA. CIA onset and severity were monitored and bone erosion was examined by CT scan.
Both CD4+ Tregs almost completely suppressed osteoclastogenesis but only iTregs sustained the effect in the presence of IL-6 in vitro. CD4+ iTregs but not nTregs and control cells injected after immunization and before of onset of CIA significantly suppressed disease development. Of note, CT scan showed that the joints in CD4+ iTregs but not nTregs or control cells infused CIA had less bone erosion. Treatment with CD4+ iTregs but not other cells dramatically decreased the levels of NF-kB p65/p50 in osteoclasts in vitro and P65/50 and RANKL expression by synovial tissues in vivo.
Manipulation of CD4+ iTregs may have therapeutic effects on rheumatoid arthritis and other bone erosion related diseases.
PMCID: PMC4038329  PMID: 22764040
15.  FOXP3 Promoter Demethylation Reveals the Committed Treg Population in Humans 
PLoS ONE  2008;3(2):e1612.
Naturally occurring thymus derived regulatory T cells (Tregs) are central in the maintenance of self-tolerance. The transcription factor FOXP3 is crucial for the suppressive activity of Tregs and is considered the most specific marker for this population. However, human non regulatory T cells upregulate FOXP3 transiently upon activation which calls for other means to identify the Treg population. Since epigenetic mechanisms are involved in the establishment of stable gene expression patterns during cell differentiation, we hypothesized that the methylation profile of the FOXP3 promoter would allow the distinction of truly committed Tregs.
Methodology/Principal Findings
Human CD4+CD25hi Tregs displayed a demethylated FOXP3 promoter (1.4%±0.95% SEM methylated) in contrast to CD4+CD25lo T cells which were partially methylated (27.9%±7.1%). Furthermore, stimulated CD4+CD25lo T cells transiently expressed FOXP3 but remained partially methylated, suggesting promoter methylation as a mechanism for regulation of stable FOXP3 expression and Treg commitment. In addition, transient FOXP3 expressing cells exhibited suppressive abilities that correlate to the methylation status of the FOXP3 promoter. As an alternative to bisulphite sequencing, we present a restriction enzyme based screening method for the identification of committed Tregs and apply this method to evaluate the effect of various culturing conditions. We show that a partial demethylation occurs in long-term cultures after activation, whereas the addition of TGF-β and/or IL-10 does not induce any additional change in methylation level.
The unique FOXP3 promoter methylation profile in Tregs suggests that a demethylated pattern is a prerequisite for stable FOXP3 expression and suppressive phenotype. Presently, FOXP3 is used to identify Tregs in several human diseases and there are future implications for adoptive Treg transfer in immunotherapy. In these settings there is a need to distinguish true Tregs from transiently FOXP3+ activated T cells. The screening method we present allows this distinction and enables the identification of cells suitable for in vitro expansions and clinical use.
PMCID: PMC2238816  PMID: 18286169
16.  IL-2 Controls the Stability of Foxp3 expression in TGF-β-induced Foxp3+ T cells in vivo 
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.
PMCID: PMC3098943  PMID: 21525380
17.  Tumor antigen specific iTreg accumulate in the tumor microenvironment and suppress therapeutic vaccination 
Oncoimmunology  2012;1(5):642-648.
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.
PMCID: PMC3429568  PMID: 22934256
FoxP3; iTreg; nTreg; regulatory T cell; tumor microenvironment
18.  Blockade of LFA-1 augments in vitro differentiation of antigen-induced Foxp3+ Treg cells 
Adoptive transfer of antigen-specific, in vitro-induced Foxp3+ Treg (iTreg) cells protects against autoimmune disease. To generate antigen-specific iTreg cells at high purity, however, remains a challenge. Whereas polyclonal T cell stimulation with anti-CD3 and anti-CD28 antibody yields Foxp3+ iTreg cells at a purity of 90–95%, antigen-induced iTreg cells typically do not exceed a purity of 65–75%, even in a TCR-transgenic model. In a similar vein to thymic Treg cell selection, iTreg cell differentiation is influenced not only by antigen recognition and the availability of TGF-β but also by co-factors including costimulation and adhesion molecules. In this study, we demonstrate that blockade of the T cell integrin Leukocyte Function-associated Antigen-1 (LFA-1) during antigen-mediated iTreg cell differentiation augments Foxp3 induction, leading to approximately 90% purity of Foxp3+ iTreg cells. This increased efficacy not only boosts the yield of Foxp3+ iTreg cells, it also reduces contamination with activated effector T cells, thus improving the safety of adoptive transfer immunotherapy.
•iTreg cells can be generated in an antigen-specific manner, even if specific Tconv cells are present at low frequency.•Blockade of anti-LFA-1 during iTreg cell differentiation augments Foxp3 induction.•The blockade of LFA-1 alters the iTreg cell phenotype but does not impair stability or function.
PMCID: PMC4265731  PMID: 25108241
Foxp3; LFA-1; Treg cell; Immunotherapy; Autoimmunity
19.  Epigenetic Control of the foxp3 Locus in Regulatory T Cells  
PLoS Biology  2007;5(2):e38.
Compelling evidence suggests that the transcription factor Foxp3 acts as a master switch governing the development and function of CD4+ regulatory T cells (Tregs). However, whether transcriptional control of Foxp3 expression itself contributes to the development of a stable Treg lineage has thus far not been investigated. We here identified an evolutionarily conserved region within the foxp3 locus upstream of exon-1 possessing transcriptional activity. Bisulphite sequencing and chromatin immunoprecipitation revealed complete demethylation of CpG motifs as well as histone modifications within the conserved region in ex vivo isolated Foxp3+CD25+CD4+ Tregs, but not in naïve CD25−CD4+ T cells. Partial DNA demethylation is already found within developing Foxp3+ thymocytes; however, Tregs induced by TGF-β in vitro display only incomplete demethylation despite high Foxp3 expression. In contrast to natural Tregs, these TGF-β–induced Foxp3+ Tregs lose both Foxp3 expression and suppressive activity upon restimulation in the absence of TGF-β. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to allow the development of a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of Tregs and for the understanding of long-term cell lineage decisions.
Author Summary
Regulatory T cells play a pivotal role in the maintenance of self-tolerance within the immune system by preventing autoimmunity or excessive activation of the T cells that respond to pathogens (naïve and effector T cells). They differentiate within the thymus, but can also be de novo induced in the rest of the body. Mechanisms determining development of a stable regulatory T cell lineage are unknown. Our study provides evidence for a critical role of epigenetic modifications in the locus coding for the forkhead transcription factor Foxp3, which acts as a master switch controlling regulatory T cell development and function: An evolutionarily conserved region within the non-coding part of the gene contains CpG motifs, which are completely demethylated in regulatory T cells, but methylated in naïve and effector T cells, whereas we observed an inverse occurrence of acetylated histones, another epigenetic chromatin modification. Regulatory T cells induced in vitro—which, in contrast to natural regulatory T cells, do not display a stable regulatory T cell phenotype—display only incomplete DNA demethylation despite high Foxp3 expression. Our data suggest that expression of Foxp3 must be stabilized by epigenetic modification to result in a permanent suppressor cell lineage, a finding of significant importance for therapeutic applications involving induction or transfer of regulatory T cells and for the understanding of long-term cell lineage decisions.
The transcription factor Foxp3 is a master switch for the regulatory T cell lineage. The authors show that the Foxp3 locus is epigenetically modified in stable regulatory T cells.
PMCID: PMC1783672  PMID: 17298177
20.  Commitment to the Regulatory T Cell Lineage Requires CARMA1 in the Thymus but Not in the Periphery 
PLoS Biology  2009;7(3):e1000051.
Regulatory T (Treg) cells expressing forkhead box P3 (Foxp3) arise during thymic selection among thymocytes with modestly self-reactive T cell receptors. In vitro studies suggest Foxp3 can also be induced among peripheral CD4+ T cells in a cytokine dependent manner. Treg cells of thymic or peripheral origin may serve different functions in vivo, but both populations are phenotypically indistinguishable in wild-type mice. Here we show that mice with a Carma1 point mutation lack thymic CD4+Foxp3+ Treg cells and demonstrate a cell-intrinsic requirement for CARMA1 in thymic Foxp3 induction. However, peripheral Carma1-deficient Treg cells could be generated and expanded in vitro in response to the cytokines transforming growth factor beta (TGFβ) and interleukin-2 (IL-2). In vivo, a small peripheral Treg pool existed that was enriched at mucosal sites and could expand systemically after infection with mouse cytomegalovirus (MCMV). Our data provide genetic evidence for two distinct mechanisms controlling regulatory T cell lineage commitment. Furthermore, we show that peripheral Treg cells are a dynamic population that may expand to limit immunopathology or promote chronic infection.
Author Summary
In mammals, CD4+ T cells are essential for controlling infections, but have the potential to attack host tissues as well, resulting in autoimmune disease. A subset of CD4+ T cells, regulatory T cells (Treg)—identified by the expression of the forkhead transcription factor Foxp3—serve to prevent immunopathology by dampening immune responses. These cells are unique among CD4+ T cell subsets, as only the Treg lineage can develop in both the thymus and periphery. Using a genetic approach, we identified a mutation in the gene Carma1, a key component of T and B cell signaling, which in mice distinguishes Treg cells derived from the periphery from thymic-derived regulatory T cells. The mutation caused an absence of thymic Treg cells. However, a small population of Treg cells was observed in the spleen, lymph nodes, and colon of Carma1-mutant mice that expanded after viral infection, suggesting that peripheral development of Treg cells could still occur. Indeed, Carma1-mutant CD4+ T cells could be converted into the Treg lineage in vitro. Our results demonstrate an organ-specific requirement for the CARMA1 signaling pathway that developing thymocytes need in order to become Treg cells, but that naïve CD4+ T cells can bypass in the periphery. This dichotomy suggests that Treg cells of thymic or peripheral origin may have different specificities or functions in vivo.
The organ-specific requirement for CARMA1-dependent signaling in the thymus suggests that regulatory T cells of thymic or peripheral origin may have different roles in vivo.
PMCID: PMC2650725  PMID: 19260764
21.  Generation and Function of Induced Regulatory T Cells 
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.
PMCID: PMC3685796  PMID: 23801990
Treg cells; Treg stability; immunotherapy; Treg function; gene expression profiling; TCR repertoire
22.  Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients 
Lepromatous leprosy caused by Mycobacterium leprae is associated with antigen specific T cell unresponsiveness/anergy whose underlying mechanisms are not fully defined. We investigated the role of CD25+FOXP3+ regulatory T cells in both skin lesions and M.leprae stimulated PBMC cultures of 28 each of freshly diagnosed patients with borderline tuberculoid (BT) and lepromatous leprosy (LL) as well as 7 healthy household contacts of leprosy patients and 4 normal skin samples.
Methodology/Principle Findings
Quantitative reverse transcribed PCR (qPCR), immuno-histochemistry/flowcytometry and ELISA were used respectively for gene expression, phenotype characterization and cytokine levels in PBMC culture supernatants. Both skin lesions as well as in vitro antigen stimulated PBMC showed increased percentage/mean fluorescence intensity of cells and higher gene expression for FOXP3+, TGF-β in lepromatous (p<0.01) as compared to tuberculoid leprosy patients. CD4+CD25+FOXP3+ T cells (Tregs) were increased in unstimulated basal cultures (p<0.0003) and showed further increase in in vitro antigen but not mitogen (phytohemaglutinin) stimulated PBMC (iTreg) in lepromatous as compared to tuberculoid leprosy patients (p<0.002). iTregs of lepromatous patients showed intracellular TGF-β which was further confirmed by increase in TGF-β in culture supernatants (p<0.003). Furthermore, TGF-β in iTreg cells was associated with phosphorylation of STAT5A. TGF-β was seen in CD25+ cells of the CD4+ but not that of CD8+ T cell lineage in leprosy patients. iTregs did not show intracellular IFN-γ or IL-17 in lepromatous leprosy patients.
Our results indicate that FOXP3+ iTregs with TGF-β may down regulate T cell responses leading to the antigen specific anergy associated with lepromatous leprosy.
Author Summary
Lepromatous leprosy is a generalized infectious disease caused by Mycobacterium leprae with the patients showing T cell mediated unresponsiveness to the pathogen and chronicity of lesions. The causation of unresponsiveness and anergy in this form of leprosy is not fully understood. The recent discovery of CD25+FOXP3+ cells with regulatory functions (Tregs) in mice and man have made it possible to study their role in the dampening of T cell responses in lepromatous leprosy. We investigated both skin and PBMC from leprosy patients for lineage specific molecular, and phenotypic markers of Tregs as well as cytokines in situ and in in vitro M.leprae stimulated PBMC cultures (iTreg). Our studies find an increase in lineage specific CD4+ iTregs in lepromatous leprosy as compared to the limited form of borderline tuberculoid leprosy. Such cells secrete TGF-β, an inhibitory cytokine and may play a role in negatively regulating the T cell immune responses in lepromatous disease.
PMCID: PMC3894184  PMID: 24454972
23.  1,25(OH)2 vitamin D3 promotes FOXP3 expression via binding to vitamin D response elements in its conserved non-coding sequence region 
Forkhead box P3 (FOXP3)-positive regulatory T cells (Treg) are a unique subset of T cells with immune regulatory properties. Treg cells can be induced from non-Treg CD4+ T cells (induced Treg, iTreg) by T cell receptor (TCR) triggering, IL-2 and TGF-β or retinoic acid. 1,25(OH)2 vitamin D3 (VD3) affects the functions of immune cells including T cells. 1,25(OH)2VD3 binds the nuclear vitamin D receptor (VDR) that binds target DNA sequences known as the vitamin D response element (VDRE). Although 1,25(OH)2VD3 can promote FOXP3 expression in CD4+ T cells with TCR triggering and IL-2, it is unknown whether this effect of 1,25(OH)2VD3 is mediated through direct binding of VDR to the FOXP3 gene without involving other molecules. Also, it is unclear whether FOXP3 expression in 1,25(OH)2VD3-induced Treg (VD-iTreg) cells is critical for the inhibitory function of these cells. Here we demonstrated the presence of VDREs in the intronic conserved non-coding sequence (CNS) region +1714 to +2554 of the human FOXP3 gene and the enhancement of the FOXP3 promoter activity by such VDREs in response to 1,25(OH)2VD3. In addition, VD-iTreg cells suppressed the proliferation of target CD4+ T cells and this activity was dependent on FOXP3 expression. These findings suggest that 1,25(OH)2VD3 can affect human immune responses by regulating FOXP3 expression in CD4+ T cells through direct VDR binding to the FOXP3 gene which is essential for inhibitory function of VD-iTreg cells.
PMCID: PMC3358577  PMID: 22529297
24.  Targeting Tregs in Malignant Brain Cancer: Overcoming IDO 
One of the hallmark features of glioblastoma multiforme (GBM), the most common adult primary brain tumor with a very dismal prognosis, is the accumulation of CD4+CD25+Foxp3+ regulatory T cells (Tregs). Regulatory T cells (Tregs) segregate into two primary categories: thymus-derived natural Tregs (nTregs) that develop from the interaction between immature T cells and thymic epithelial stromal cells, and inducible Tregs (iTregs) that arise from the conversion of CD4+FoxP3− T cells into FoxP3 expressing cells. Normally, these Treg subsets complement one another’s actions by maintaining tolerance of self-antigens, thereby suppressing autoimmunity, while also enabling effective immune responses toward non-self-antigens, thus promoting infectious protection. However, Tregs have also been shown to be associated with the promotion of pathological outcomes, including cancer. In the setting of GBM, nTregs appear to be primary players that contribute to immunotherapeutic failure, ultimately leading to tumor progression. Several attempts have been made to therapeutically target these cells with variable levels of success. The blood brain barrier-crossing chemotherapeutics, temozolomide, and cyclophosphamide (CTX), vaccination against the Treg transcriptional regulator, FoxP3, as well as mAbs against Treg-associated cell surface molecules CD25, CTLA-4, and GITR are all different therapeutic approaches under investigation. Contributing to the poor success of past approaches is the expression of indoleamine 2,3-dioxygenase 1 (IDO), a tryptophan catabolizing enzyme overexpressed in GBM, and critically involved in regulating tumor-infiltrating Treg levels. Herein, we review the current literature on Tregs in brain cancer, providing a detailed phenotype, causative mechanisms involved in their pathogenesis, and strategies that have been used to target this population, therapeutically.
PMCID: PMC3654236  PMID: 23720663
malignant glioma; glioblastoma multiforme; regulatory T cells; Tregs; natural Tregs; tumor-induced Tregs; IDO (indoleamine 2,3-dioxygenase)
25.  Lung-resident tissue macrophages generate Foxp3+ regulatory T cells and promote airway tolerance 
Lung-resident antigen-presenting macrophages promote tolerance to inhaled antigens via the induction of regulatory T cells.
Airway tolerance is the usual outcome of inhalation of harmless antigens. Although T cell deletion and anergy are likely components of tolerogenic mechanisms in the lung, increasing evidence indicates that antigen-specific regulatory T cells (inducible Treg cells [iTreg cells]) that express Foxp3 are also critical. Several lung antigen-presenting cells have been suggested to contribute to tolerance, including alveolar macrophages (MØs), classical dendritic cells (DCs), and plasmacytoid DCs, but whether these possess the attributes required to directly promote the development of Foxp3+ iTreg cells is unclear. Here, we show that lung-resident tissue MØs coexpress TGF-β and retinal dehydrogenases (RALDH1 and RALDH 2) under steady-state conditions and that their sampling of harmless airborne antigen and presentation to antigen-specific CD4 T cells resulted in the generation of Foxp3+ Treg cells. Treg cell induction in this model depended on both TGF-β and retinoic acid. Transfer of the antigen-pulsed tissue MØs into the airways correspondingly prevented the development of asthmatic lung inflammation upon subsequent challenge with antigen. Moreover, exposure of lung tissue MØs to allergens suppressed their ability to generate iTreg cells coincident with blocking airway tolerance. Suppression of Treg cell generation required proteases and TLR-mediated signals. Therefore, lung-resident tissue MØs have regulatory functions, and strategies to target these cells might hold promise for prevention or treatment of allergic asthma.
PMCID: PMC3620360  PMID: 23547101

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