Foxp3 Expression In Treg Cells Research Articles

Analysis of FOXP3 DNA Methylation Patterns to Identify Functional FOXP3+ T-Cell Subpopulations.

Human regulatory CD4+CD25+FOXP3+ T cells (Tregs) are involved in the suppression of immune responses and play important roles in the maintenance of self-tolerance and immune homeostasis. Abnormal Treg function may result in disease states of varying severity. As FOXP3-expressing Treg cells are phenotypically and functionally heterogeneous, the success of Treg therapies depends on the ability to reliably distinguish subpopulations of T cells bearing a Treg-like phenotype. Methylation of cytosines within CpG dinucleotides is an important epigenetic mechanism involved in regulation (and suppression) of gene expression. On the other hand, demethylation of regulatory DNA sequences, such as promoters and enhancers, is essential for initiation of gene transcription. This protocol shows that bisulfite sequencing (BS) distinguishes methylated and unmethylated cytosines within DNA and reveals the methylation status of individual CpGs in cells within each population, identifying functionally different FOXP3+ subpopulations.

Bufei Decoction Improves Lung-Qi Deficiency Syndrome of Chronic Obstructive Pulmonary Disease in Rats by Regulating the Balance of Th17/Treg Cells.

Bufei decoction (BFD) has been applied to treat chronic obstructive pulmonary disease (COPD) for centuries as a recognized traditional Chinese herbal formula. However, mechanisms of BFD on COPD are unclear. This study conducts an inquiry into the underlying mechanisms of the therapeutic effect of BFD on COPD. A COPD rat model with qi deficiency in lungs was established through induction using cigarette and sawdust smoking combined with intratracheal instillation of lipopolysaccharide following BFD treatment for 28 days. Changes in Th17/Treg cells of COPD rats with the syndrome of lung qi deficiency after BFD administration were verified using pulmonary function, ELISA, flow cytometry, histopathology, and Western blotting assays. The findings showed that BFD protected COPD rats from decreased lung function and lung injury. BFD administration reduced proinflammatory cytokines IL-6 and IL-17 secretion, promoted inhibitory cytokines IL-10 and TGF-β secretion, decreased Th17/Treg cell ratio, markedly downregulated the Th17 cell transcription factor ROR-γt expression, and upregulated transcription factor Foxp3 expression in Treg cells. We speculate that lung tonic soup improved pulmonary qi deficiency in rats with COPD by regulating the balance of Th17/Treg cells.

BATF controls T cell differentiation during chronic antigen stimulation

Abstract CD4+ T cells preserve self-tolerance in the periphery by exerting active suppression as regulatory T cells (Treg) or entering an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. To identify transcription factors (TFs) underlying T cell anergy induction and its potential plasticity, we sorted polyclonal CD4+ T cell subsets from mice for downstream sequencing-based interrogation. Anergic CD44high Foxp3− CD73highFR4high Nrp1+ T cells were compared to effector-memory, Treg, and naïve T cell populations. Integrated ATAC-seq and RNA-seq analysis revealed a distinct program in the anergic T cell subpopulation. The anergy signature was driven by bZIP/AP-1 subfamily (Batf, Maf) and NFAT family (Nfatc1) transcription factors. Anergy-enriched TFs were then investigated in antigen-specific T cell tolerization systems where Batf knockdown led to altered expansion and minimal Treg cell differentiation. BATF was furthermore required for anergy-derived Foxp3+ Treg cell generation. Our data indicate that BATF orchestrates the transition to chronically-stimulated T cell differentiation states. Supported by grants from the NIH (T32 AI007313, P01 AI035296) and RRF (Future Physician Scientist Award)

Semaphorin-3A: a promising therapeutic tool in allergic rhinitis.

Semaphorin-3A (Sema-3A), a secreted member of the semaphorin family, is well known for playing regulatory functions at all stages of the immune response. Sema-3A transduces signals by binding to its cognate receptors, namely, class A plexins (Plxns A1 to A4) and neuropilin-1 (Nrp-1). The downstream diverse signaling pathways induced by connecting Sema-3A to its receptors were found to be involved in the pathogenesis of different immunological disorders, ranging from cancer to autoimmunity and allergies. Recent studies have demonstrated that Sema-3A expression is diminished in the murine models and patients with allergic rhinitis (AR; a chronic inflammatory disorder of the nasal mucosa), suggesting the involvement of Sema-3A in AR pathogenesis. Investigations also revealed that treatment of these mice with exogenous Sema-3A protein alleviates the clinical symptom scores of AR, thereby compensating for the reduced expression of Sema-3A in AR. Indeed, Sema-3A treatment could suppress allergic responses in AR via inhibiting Th2/Th17 responses and boosting Th1/Treg responses. Also, Sema-3A could diminish dendritic cell (DC) maturation and T cell proliferation. Since it is implicated in the pathogenesis of AR; thus, Sema-3A turns to be a promising tool of therapy to be studied and utilized in this disease. This review intends to highlight the recent evidence on the role of Sema-3A in AR pathogenesis and summarizes the recent findings regarding the expression status of Sema-3A, as well as its therapeutic potential for treating this disease. HIGHLIGHTS: Sema-3A plays regulatory functions at all stages of the immune response. Sema-3A receptors are the class A plexins (A1-A4) and neuropilin-1 (Nrp-1). Sema-3A expression is reduced in murine models and patients with allergic rhinitis. Connecting Sema-3A to Nrp-1 increases Foxp3 expression in Treg cells. Injecting Sema-3A protein exerts therapeutic effects in mouse models of allergic diseases. Sema-3A shows promise as a therapeutic tool for the treatment of allergic rhinitis.

The relationship between IL-16 and Foxp3 expression in Treg cells of healthy individuals

The relationship between IL-16 and Foxp3 expression in Treg cells of healthy individuals

Protein phosphatase 6 (Pp6) is crucial for regulatory T cell function and stability in autoimmunity

Regulatory T (Treg) cells constitute a dynamic population that is critical in autoimmunity. Treg cell therapies for autoimmune diseases are mainly focused on enhancing their suppressive activities. However, recent studies demonstrated that certain inflammatory conditions induce Treg cell instability with diminished FoxP3 expression and convert them into pathogenic effector cells. Therefore, the identification of novel targets crucial to both Treg cell function and plasticity is of vital importance to the development of therapeutic approaches in autoimmunity. In this study, we found that conditional Pp6 knockout (cKO) in Treg cells led to spontaneous autoinflammation, immune cell activation, and diminished levels of FoxP3 in CD4+ T cells in mice. Loss of Pp6 in Treg cells exacerbated two classical mouse models of Treg-related autoinflammation. Mechanistically, Pp6 deficiency increased CpG motif methylation of the FoxP3 locus by dephosphorylating Dnmt1 and enhancing Akt phosphorylation at Ser473/Thr308, leading to impaired FoxP3 expression in Treg cells. In summary, our study proposes Pp6 as a critical positive regulator of FoxP3 that acts by decreasing DNA methylation of the FoxP3 gene enhancer and inhibiting Akt signaling, thus maintaining Treg cell stability and preventing autoimmune diseases.

Epigenetic and transcriptional programs of CD4+ T cell anergy

Abstract T cell tolerance is critical to avoid autoimmunity and resolve inflammation. CD4+ T cells that recognize self-antigens in the periphery can exert active suppression as regulatory T cells (Treg) or enter an inactive state known as anergy. Recent evidence suggests that anergy occurs naturally within a subpopulation of polyclonal CD4+ T cells and can generate precursors to Foxp3-expressing Treg cells. We aimed to identify transcription factors underlying T cell anergy induction and its potential plasticity. Polyclonal anergic CD4+ T cells from untreated wildtype mice were sorted based on previously reported markers and compared to naïve, effector-memory, and Treg cell populations identified within the same samples. Paired epigenome and transcriptome analysis revealed a unique anergy signature driven by bZIP family (Batf, Nfil3), NFAT family (Nfatc1), and EGR family (Egr3, Egr4) transcription factors. Following tolerization by repeated peptide infusions, antigen-specific CD4+ T cells from Batf-deficient mice demonstrated exaggerated expansion and minimal Foxp3+ Treg cell differentiation. Our data suggest that a unique transcription factor network, including BATF, generates and maintains peripheral CD4+ T cell anergy.

Immunomodulatory Effects of Rhinovirus and Enterovirus Infections During the First Year of Life.

Early childhood infections have been implicated in the development of immune-mediated diseases, such as allergies, asthma, and type 1 diabetes. We set out to investigate the immunomodulatory effects of early viral infections experienced before the age of one year on the peripheral regulatory T cell population (Treg) and circulating cytokines in a birth-cohort study of Estonian and Finnish infants. We show here a temporal association of virus infection with the expression of FOXP3 in regulatory T cells. Infants with rhinovirus infection during the preceding 30 days had a higher FOXP3 expression in Treg cells and decreased levels of several cytokines related to Th1 and Th2 responses in comparison to the children without infections. In contrast, FOXP3 expression was significantly decreased in highly activated (CD4+CD127−/loCD25+FOXP3high) regulatory T cells (TregFOXP3high) in the infants who had enterovirus infection during the preceding 30 or 60 days. After enterovirus infections, the cytokine profile showed an upregulation of Th1- and Th17-related cytokines and a decreased activation of CCL22, which is a chemokine derived from dendritic cells and associated with Th2 deviation. Our results reveal that immunoregulatory mechanisms are up-regulated after rhinovirus infections, while enterovirus infections are associated with activation of proinflammatory pathways and decreased immune regulation.

The Role of Regulatory T and B Cells in the Etiopathogenesis of Idiopathic Granulomatous Mastitis

ABSTRACT Background and objectives The aim of this study was to evaluate the role of T- and B-regulatory cells (Tregs and Bregs) in the pathogenesis of idiopathic granulomatous mastitis (IGM). Methods This study includes 47 patients with pathologically proven IGM (Group P) and 26 healthy subjects (Group C). The patients in Group P were divided into two groups according to whether their lesions were active (Group PA, n: 21) or in remission (Group PR, n: 26). By using flow-cytometry, the frequencies of CD3+CD4+CD45RA−Foxp3high activated Tregs (aTregs), CD3+CD4+CD45RA−Foxp3low non-suppressive Tregs, CD3+CD4+CD45RA+Foxp3low resting Tregs (rTregs), CD3+CD4+CD25+Foxp3− T-effector cells (Teff), total Tregs and Bregs were analyzed in all subjects. Results The frequency of the Teff cells was statistically higher in Group P when compared with Group C (p =.004). The Foxp3 expression of Treg cells and the frequency of non-suppressive Tregs in Group P were statistically lower than Group C (p =.032 and p =.02, respectively). In addition, Group PR’s Foxp3 expressions were statistically lower than Group C (p =.027); Group PR’s aTregs ratio was statistically lower than Group PA (p =.021); and the non-suppressive Tregs ratio of Group PR was lower than both Group PA and Group C (p =.006 and p <.0001). No significant differences were seen Bregs and B cell subsets. Conclusion Significant changes in Foxp3 expression and Treg subsets were seen in patients with active IGM lesion and in remission. This study shows an intrinsic defect of Tregs in patients with IGM.

TNF receptor 2 signaling prevents DNA methylation at the Foxp3 promoter and prevents pathogenic conversion of regulatory T cells

Regulatory T (Treg) cells expressing the transcription factor Foxp3 play an important role in maintaining immune homeostasis. Chronic inflammation is associated with reduced Foxp3 expression, function, and loss of phenotypic stability. Previous studies have established the importance of TNF receptor 2 (TNFR2) in the generation and/or activation of Treg cells. In this study, we assess the importance of TNFR2 in healthy mice and under inflammatory conditions. Our findings reveal that, in health, TNFR2 is important not only for the generation of Treg cells, but also for regulating their functional activity. We also show that TNFR2 maintains Foxp3 expression in Treg cells by restricting DNA methylation at the Foxp3 promoter. In inflammation, loss of TNFR2 results in increased severity and chronicity of experimental arthritis, reduced total numbers of Treg cells, reduced accumulation of Treg cells in inflamed joints, and loss of inhibitory activity. In addition, we demonstrate that, under inflammatory conditions, loss of TNFR2 causes Treg cells to adopt a proinflammatory Th17-like phenotype. It was concluded that TNFR2 signaling is required to enable Treg cells to promote resolution of inflammation and prevent them from undergoing dedifferentiation. Consequently, TNFR2-specific agonists or TNF1-specific antagonists may be useful in the treatment of autoimmune disease.

The transcription factor Foxp1 preserves integrity of an active Foxp3 locus in extrathymic Treg cells

Regulatory T (Treg) cells, which are broadly classified as thymically derived (tTreg) or extrathymically induced (iTreg), suppress immune responses and display stringent dependence to the transcription factor Foxp3. However precise understanding of molecular events that promote and preserve Foxp3 expression in Treg cells is still evolving. Here we show that Foxp1, a forkhead transcription factor and a sibling family member of Foxp3, is essential for sustaining optimal expression of Foxp3 specifically in iTreg cells. Deletion of Foxp1 renders iTreg cells to gradually lose Foxp3, resulting in dramatically reduced Nrp1−Helios− iTreg compartment as well as augmented intestinal inflammation in aged mice. Our finding underscores a mechanistic module in which evolutionarily related transcription factors establish a molecular program to ensure efficient immune homeostasis. Furthermore, it provides a novel target that can be potentially modulated to exclusively reinforce iTreg stability keeping their thymic counterpart unperturbed.

Changes in CDKN2A/2B expression associate with T-cell phenotype modulation in atherosclerosis and type 2 diabetes mellitus

Previous studies indicate a role of CDKN2A/2B/2BAS genes in atherosclerosis and type 2 diabetes mellitus (T2DM). Progression of these diseases is accompanied by T-cell imbalance and chronic inflammation. Our main objective was to investigate a potential association between CDKN2A/2B/2BAS gene expression and T cell phenotype in T2DM and coronary artery disease (CAD) in humans, and to explore the therapeutic potential of these genes to restore immune cell homeostasis and disease progression. Reduced mRNA levels of CDKN2A (p16Ink4a), CDKN2B (p15Ink4b), and CDKN2BAS were observed in human T2DM and T2DM-CAD subjects compared with controls. Protein levels of p16Ink4a and p15Ink4b were also diminished in T2DM-CAD patients while CDK4 levels, the main target of p16Ink4a and p15Ink4b, were augmented in T2DM and T2DM-CAD subjects. Both patient groups displayed higher activated CD3+CD69+ T cells and proatherogenic CD14++CD16+ monocytes, while CD4+CD25+CD127 regulatory T (Treg cells) cells were decreased. Treatment of primary human lymphocytes with PD0332991, a p16Ink4a/p15Ink4b mimetic drug and a proven CDK4 inhibitor, increased Treg cells and the levels of activated transcription factor phosphoSTAT5. In vivo PD0332991 treatment of atherosclerotic apoE-/- mice and insulin resistant apoE-/-Irs2+/- mice augmented Foxp3-expressing Treg cells and decreased lesion size. Thus, atherosclerosis complications in T2DM associate with altered immune cell homeostasis, diminished CDKN2A/2B/2BAS expression, and increased CDK4 levels. The present study also suggests that the treatment with drugs that mimic CDKN2A/2B genes could potential be considered as a promising therapy to delay atherosclerosis.

Monocyte activation and inflammation can exacerbate Treg/Th17 imbalance in infants with neonatal necrotizing enterocolitis

Necrotizing enterocolitis (NEC) is a potentially fatal disease that develops in the intestinal tract of newborn infants. An overactive immune system in the intestinal tract of NEC patients not only propagates inflammation and mediates tissue damage, but may also radiate the disease systemically and impose injury to distant organs. We previously identified impairment in the Treg compartment in NEC patients. Here, we hypothesized that monocytes, which could promote iTreg differentiation both in vitro and in vivo, were dysregulated in NEC infants. In age-, sex-, and weight-matched NEC infants and healthy control infants, the monocytes from PBMCs were investigated. Monocytes from NEC infants presented significantly higher TLR4 expression. With or without LPS stimulation, monocytes from NEC infants presented elevated TNF-α and IL-6 expression, together with reduced expression of TGF-β. When incubated with autologous CD4+ T cells, monocytes from NEC infants preferentially promoted the differentiation of RORγt-expressing Th17 cells, but not Foxp3-expressing Treg cells. However, using exogenous TGF-β and IL-10, the development of Foxp3 expression could be significantly elevated. Together, these results demonstrate that monocytes in NEC patients displayed a proinflammatory profile that might contribute to the production of proinflammatory cytokines and the suppression of Treg cells.

The effect of foxp3-overexpressing Treg cells on non-small cell lung cancer cells.

The aim of the present study was to investigate the novel mechanisms of forkhead box protein P3 (foxp3) in T regulatory (Treg) cells in lung cancer behavior. Treg cells were isolated from the peripheral blood of healthy volunteers and then co-cultured with 95D cells. A plasmid overexpressing foxp3 was constructed and transfected into Treg cells and an MTS assay was performed to assess cell viability. Flow cytometry was performed to evaluate cell apoptosis and reverse transcription-quantitative polymerase chain reaction was used to measure mRNA expression. A Transwell assay was used to assess cell invasion. Treg cells were successfully isolated from peripheral blood with purity of 94.26%. Foxp3 expression in Treg cells was significantly increased following co-culture with 95D cells, while matrix metalloproteinase-9 expression was upregulated in 95D cells co-cultured with Treg cells. The apoptosis, invasion and migration abilities of 95D cells were suppressed by co-culture with Treg cells, whereas the adhesive ability was enhanced. Foxp3 overexpression in Treg cells enhanced the viability and invasiveness of 95D cells, whereas cell adhesion and migration were decreased. The results of the present study demonstrate that the viability and invasiveness of 95D cells are enhanced by foxp3 overexpression in Treg cells, indicating that increased levels of foxp3 in the tumor microenvironment may promote tumor cell growth.

Loss of regulatory characteristics in CD4+ CD25+/hi T cells induced by impaired transforming growth factor beta secretion in pneumoconiosis.

Pneumoconiosis is caused by the accumulation of airborne dust in the lung, which stimulates a progressive inflammatory response that ultimately results in lung fibrosis and respiratory failure. It is possible that regulatory cells in the immune system could function to suppress inflammation and possibly slow or reverse disease progression. However, results in this study suggest that in pneumoconiosis patients, the regulatory T cells (Tregs) and B cells are functionally impaired. First, we found that pneumoconiosis patients presented an upregulation of CD4+ CD25+ T cells compared to controls, whereas the CD4+ CD25+ and CD4+ CD25hi T cells were enriched with Th1- and Th17-like cells but not Foxp3-expressing Treg cells and evidenced by significantly higher T-bet, interferon (IFN)-γ, and interleukin (IL)-17 expression but lower Foxp3 and transforming growth factor (TGF)-β expression. Regarding the CD4+ CD25hi T-cell subset, the frequency of this cell type in pneumoconiosis patients was significantly reduced compared to controls, together with a reduction in Foxp3 and TGF-β and an enrichment in T-bet, RORγt, IFN-γ, and IL-17. This skewing toward Th1 and Th17 types of inflammation could be driven by monocytes and B cells, since after depleting CD14+ monocytes and CD19+ B cells, the levels of IFN-γ and IL-17 were significantly decreased. Whole peripheral blood mononuclear cells and isolated monocytes and B cells in pneumoconiosis patients also presented reduced capacity of TGF-β secretion. Furthermore, monocytes and B cells from pneumoconiosis patients presented reduced capacity in inducing Foxp3 upregulation, a function that could be rescued by exogenous TGF-β. Together, these data indicated a potential pathway for the progression of pneumoconiosis through a loss of Foxp3+ Treg cells associated with impaired TGF-β secretion.

Foxp3-independent mechanism by which TGF-β controls peripheral T cell tolerance

Peripheral T cell tolerance is promoted by the regulatory cytokine TGF-β and Foxp3-expressing Treg cells. However, whether TGF-β and Treg cells are part of the same regulatory module, or exist largely as distinct pathways to repress self-reactive T cells remains incompletely understood. Using a transgenic model of autoimmune diabetes, here we show that ablation of TGF-β receptor II (TβRII) in T cells, but not Foxp3 deficiency, resulted in early-onset diabetes with complete penetrance. The rampant autoimmune disease was associated with enhanced T cell priming and elevated T cell expression of the inflammatory cytokine GM-CSF, concomitant with pancreatic infiltration of inflammatory monocytes that triggered immunopathology. Ablation of the GM-CSF receptor alleviated the monocyte response and inhibited disease development. These findings reveal that TGF-β promotes T cell tolerance primarily via Foxp3-independent mechanisms and prevents autoimmunity in this model by repressing the cross talk between adaptive and innate immune systems.

Id3 Maintains Foxp3 Expression in Regulatory T Cells by Controlling a Transcriptional Network of E47, Spi-B, and SOCS3

The transcription factor Foxp3 dominantly controls regulatory T (Treg) cell function, and only its continuous expression guarantees the maintenance of full Treg cell-suppressive capacity. However, transcriptional regulators maintaining Foxp3 transcription are incompletely described. Here, we report that high E47 transcription factor activity in Treg cells resulted in unstable Foxp3 expression. Under homeostatic conditions, Treg cells expressed high levels of the E47 antagonist Id3, thus restricting E47 activity and maintaining Foxp3 expression. In contrast, stimulation of Id3-deficient or E47-overexpressing Treg cells resulted in the loss of Foxp3 expression in a subset of Treg cells invivo and invitro. Mechanistic analysis indicated that E47 activated expression of the transcription factor Spi-B and the suppressor of cytokine signaling 3 (SOCS3), which both downregulated Foxp3 expression. These findings demonstrate that the balance of Id3 and E47 controls the maintenance of Foxp3 expression in Treg cells and, thus, contributes to Treg cell plasticity.

Critical roles of adenosine A2A receptor in regulating the balance of Treg/Th17 cells in allergic asthma.

Deficiency of Treg cells and hyperactivity of Th17 cells together are involved in the immunological pathogenesis of asthma. The adenosine A2A receptor (A2AR) plays a critical role in the increased Foxp3 expression of Treg cells and the decreased Th17 generation. The study aimed to investigate A2AR expression in peripheral blood and its regulatory effect on balance of Treg/Th17 cells in asthma. Thirty-one patients with chronic persistent asthma were recruited and divided into 18 intermittent to mild asthma patients, 13 moderate to severe asthma patients. A2AR, Foxp3, and ROR-γt mRNA expression levels in peripheral blood mononuclear cells (PBMCs) were measured by quantitative polymerase chain reaction (qPCR). TGF-β, IL-17, and IgE in plasma were detected with enzyme-linked immunosorbent assay (ELISA). Forty-two BALB/c mice were randomly, equally assigned to control group, ovalbumin (OVA) group and OVA + CGS (CGS21680, A2AR agonist) group. The infiltration of lung inflammation cells were evaluated by HE, A2AR, Foxp3, and ROR-γt mRNA in lung tissues measured by qPCR, TGF-β, IL-17, and IgE in plasma measured with ELISA, and IL-17 and TGF-β protein in lung tissues analyzed with immunohistochemical. Our results showed that expression A2AR mRNA in PBMCs was associated with asthma severity. Foxp3 mRNA, TGF-β, and FEV1%pred positively correlated with A2AR mRNA in asthma. ROR-γt mRNA and IL-17 negatively correlated with A2AR mRNA in asthma. CGS could promote Foxp3 mRNA expression, TGF-β, and improve lung function while inhibit ROR-γt mRNA expression, IL-17, and the infiltration of lung inflammation cells. A2AR could regulate the balance of Treg/Th17 cells in asthma.

Decidual vascular endothelial cells promote maternal–fetal immune tolerance by inducing regulatory T cells through canonical Notch1 signaling

Adaptation of the maternal immune response to accommodate the semiallogeneic fetus is necessary for pregnancy success. However, the mechanisms by which the fetus avoids rejection despite expression of paternal alloantigens remain incompletely understood. Regulatory T cells (Treg cells) are pivotal for maintaining immune homeostasis, preventing autoimmune disease and fetus rejection. In this study, we found that maternal decidual vascular endothelial cells (DVECs) sustained Foxp3 expression in resting Treg cells in vitro. Moreover, under in vitro Treg cell induction condition with agonistic antibodies and transforming growth factor (TGF)-β, DVECs promoted Treg cell differentiation from non-Treg conventional T cells. Consistent with the promotion of Treg cell maintenance and differentiation, Treg cell-associated gene expression such as TGF-β, Epstein-Barr-induced gene-3, CD39 and glucocorticoid-induced tumor necrosis factor receptor was also increased in the presence of DVECs. Further study revealed that DVECs expressed Notch ligands such as Jagged-1, Delta-like protein 1 (DLL-1) and DLL-4, while Treg cells expressed Notch1 on their surface. The effects of DVECs on Treg cells was inhibited by siRNA-induced knockdown of expression of Jagged-1 and DLL-1 in DVECs. Downregulation of Notch1 in Treg cells using lentiviral shRNA transduction decreased Foxp3 expression in Treg cells. Adoptive transfer of Notch1-deficient Treg cells increased abortion rate in a murine semiallogeneic pregnancy model. Taken together, our study suggests that maternal DVECs are able to maintain decidual Treg cell identity and promote Treg cell differentiation through activation of Notch1 signal pathway in Treg cells and subsequently inhibit the immune response against semiallogeneic fetuses and preventing spontaneous abortion.

Loss- and Gain-of-Function Approaches Indicate a Dual Role Exerted by Regulatory T Cells in Pulmonary Paracoccidioidomycosis.

Paracoccidioidomycosis (PCM), is a pulmonary fungal disease whose severity depends on the adequate development of T cell immunity. Although regulatory T (Treg) cells were shown to control immunity against PCM, deleterious or protective effects were described in different experimental settings. To clarify the function of Treg cells in pulmonary PCM, loss-and gain-of-function approaches were performed with Foxp3GFP knock-in mice and immunodeficient Rag1-/- mice, respectively, which were intratracheally infected with 106 yeast cells. The activity of Foxp3-expressing Treg cells in pulmonary PCM was determined in Foxp3GFP transgenic mice. First, it was verified that natural Treg cells migrate to the lungs of infected mice, where they become activated. Depletion of Treg cells led to reduced fungal load, diminished pathogen dissemination and increased Th1/Th2/Th17 immunity. Further, adoptive transfer of diverse T cell subsets to Rag1-/- mice subsequently infected by the pulmonary route demonstrated that isolated CD4+Foxp3+ Treg cells were able to confer some degree of immunoprotection and that CD4+Foxp3- T cells alone reduced fungal growth and enhanced T cell immunity, but induced vigorous inflammatory reactions in the lungs. Nevertheless, transfer of Treg cells combined with CD4+Foxp3- T cells generated more efficient and balanced immune Th1/Th2/Th17 responses able to limit pathogen growth and excessive tissue inflammation, leading to regressive disease and increased survival rates. Altogether, these loss- and gain-of-function approaches allow us to clearly demonstrate the dual role of Treg cells in pulmonary PCM, their deleterious effects by impairing T cell immunity and pathogen eradication, and their protective role by suppressing exacerbated tissue inflammation.