Peroxisome proliferator-activated receptor α and γ agonists together with TGF-β convert human CD4+CD25- T cells into functional Foxp3+ regulatory T cells

Human peripheral CD4(+)CD25(-) T cells can be induced to express Foxp3 when activated in vitro by TCR stimulation with TGF-β and IL-2. However, these TGF-β-induced Foxp3(+) regulatory T cells (iTregs) lack a regulatory phenotype. From libraries of nuclear receptor ligands and bioactive lipids, we screened three peroxisome proliferator-activated receptor (PPAR)α (bezafibrate, GW7647, and 5,8,11,14-eicosatetraynoic acid) and two PPARγ agonists (ciglitazone and 15-deoxy-Δ-(12,14)-PG J(2)) as molecules that increased Foxp3 expression in human iTregs significantly compared with that in DMSO-treated iTregs (control). These PPARα and PPARγ agonist-treated iTregs maintained a high level of Foxp3 expression and had suppressive properties. There were no significant differences in the suppressive properties of iTregs treated with the three PPARα and two PPARγ agonists, and all of the treated iTregs increased demethylation levels of the Foxp3 promoter and intronic conserved noncoding sequence 3 regions. Furthermore, PPARα and PPARγ agonists, together with TGF-β, more strongly inhibited the expression of all three DNA methyltransferases (DNMTs) (DNMT1, DNMT3a, and DNMT3b) in activated CD4(+) T cells. These results demonstrate that PPARα and PPARγ agonists together with TGF-β elicit Foxp3 DNA demethylation through potent downregulation of DNMTs and induce potent and stable Foxp3 expression, resulting in the generation of functional iTregs. Moreover, trichostatin A and retinoic acid enhanced the generation of iTregs synergistically with PPARα and PPARγ agonists.     

TGF-β induces surface LAP expression on murine CD4 T cells independent of Foxp3 induction

Background

It has been reported that human FOXP3⁺ CD4 Tregs express GARP-anchored surface latency-associated peptide (LAP) after activation, based on the use of an anti-human LAP mAb. Murine CD4 Foxp3⁺ Tregs have also been reported to express surface LAP, but these studies have been hampered by the lack of suitable anti-mouse LAP mAbs.

Methodology/Principal Findings

We generated anti-mouse LAP mAbs by immunizing TGF-β^−/− animals with a mouse Tgfb1-transduced P3U1 cell line. Using these antibodies, we demonstrated that murine Foxp3⁺ CD4 Tregs express LAP on their surface. In addition, retroviral transduction of Foxp3 into mouse CD4⁺CD25⁻ T cells induced surface LAP expression. We then examined surface LAP expression after treating CD4⁺CD25⁻ T cells with TGF-β and found that TGF-β induced surface LAP not only on T cells that became Foxp3⁺ but also on T cells that remained Foxp3⁻ after TGF-β treatment. GARP expression correlated with the surface LAP expression, suggesting that surface LAP is GARP-anchored also in murine T cells.

Conclusions/Significance

Unlike human CD4 T cells, surface LAP expression on mouse CD4 T cells is controlled by Foxp3 and TGF-β. Our newly described anti-mouse LAP mAbs will provide a useful tool for the investigation and functional analysis of T cells that express LAP on their surface.     

Human CD8+CD25 +CD127 low regulatory T cells: microRNA signature and impact on TGF-β and IL-10 expression

Regulatory T cells (Tregs) are central for maintaining immune balance and their dysfunction drives the expansion of critical immunologic disorders. During the past decade, microRNAs (miRNAs) have emerged as potent regulators of gene expression among which immune-related genes and their immunomodulatory properties have been associated with different immune-based diseases. The miRNA signature of human peripheral blood (PB) CD8⁺CD25 ⁺CD127 ^low Tregs has not been described yet. We thus identified, using TaqMan low-density array (TLDA) technique followed by individual quantitative real-time polymerase chain reaction (qRT-PCR) confirmation, 14 miRNAs, among which 12 were downregulated whereas two were upregulated in CD8 ⁺CD25 ⁺CD127 ^low Tregs in comparison to CD8 ⁺CD25 ⁻ T cells. In the next step, microRNA Data Integration Portal (mirDIP) was used to identify potential miRNA target sites in the 3′-untranslated region (3′-UTR) of key Treg cell-immunomodulatory genes with a special focus on interleukin 10 (IL-10) and transforming growth factor β (TGF-β). Having identified potential miR target sites in the 3′-UTR of IL-10 (miR-27b-3p and miR-340-5p) and TGF-β (miR-330-3p), we showed through transfection and transduction assays that the overexpression of two underexpressed miRNAs, miR-27b-3p and miR-340-5p, downregulated IL-10 expression upon targeting its 3′-UTR. Similarly, overexpression of miR-330-3p negatively regulated TGF-β expression. These results highlighted an important impact of the CD8 ⁺ Treg mirnome on the expression of genes with significant implication on immunosuppression. These observations could help in better understanding the mechanism(s) orchestrating Treg immunosuppressive function toward unraveling new targets for treating autoimmune pathologies and cancer.     

Increase in TGF-β Secreting CD4+CD25+ FOXP3+ T Regulatory Cells in Anergic Lepromatous Leprosy Patients

Background

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.

Conclusions/Significance

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.     

Segregated regulatory CD39+CD4+ T cell function: TGF-β-producing Foxp3- and IL-10-producing Foxp3+ cells are interdependent for protection against collagen-induced arthritis

Oral immunization with a Salmonella vaccine vector expressing enterotoxigenic Escherichia coli colonization factor Ag I (CFA/I) can protect against collagen-induced arthritis (CIA) by dampening IL-17 and IFN-γ via enhanced IL-4, IL-10, and TGF-β. To identify the responsible regulatory CD4(+) T cells making the host refractory to CIA, Salmonella-CFA/I induced CD39(+)CD4(+) T cells with enhanced apyrase activity relative to Salmonella vector-immunized mice. Adoptive transfer of vaccine-induced CD39(+)CD4(+) T cells into CIA mice conferred complete protection, whereas CD39(-)CD4(+) T cells did not. Subsequent analysis of vaccinated Foxp3-GFP mice revealed the CD39(+) T cells were composed of Foxp3-GFP(-) and Foxp3-GFP(+) subpopulations. Although each adoptively transferred Salmonella-CFA/I-induced Foxp3(-) and Foxp3(+)CD39(+)CD4(+) T cells could protect against CIA, each subset was not as efficacious as total CD39(+)CD4(+) T cells, suggesting their interdependence for optimal protection. Cytokine analysis revealed Foxp3(-) CD39(+)CD4(+) T cells produced TGF-β, and Foxp3(+)CD39(+)CD4(+) T cells produced IL-10, showing a segregation of function. Moreover, donor Foxp3-GFP(-) CD4(+) T cells converted to Foxp3-GFP(+) CD39(+)CD4(+) T cells in the recipients, showing plasticity of these regulatory T cells. TGF-β was found to be essential for protection because in vivo TGF-β neutralization reversed activation of CREB and reduced the development of CD39(+)CD4(+) T cells. Thus, CD39 apyrase-expressing CD4(+) T cells stimulated by Salmonella-CFA/I are composed of TGF-β-producing Foxp3(-) CD39(+)CD4(+) T cells and support the stimulation of IL-10-producing Foxp3(+) CD39(+)CD4(+) T cells.     

IL-1β and TGF-β act antagonistically in induction and differentially in propagation of human proinflammatory precursor CD4+ T cells

Cytokines are critical messengers that control the differentiation of Th cells. To evaluate their impact on the fate of human naive CD4(+) T cells from cord and adult blood, early T cell differentiation was monitored after T cell activation in the presence of pro- and anti-inflammatory cytokines. Interestingly, the analysis of Th cell lineage-specific molecules revealed that IL-1β on its own mediates differentiation of Th cells that secrete a wide range of proinflammatory cytokines and stably express CD69, STAT1, IFN-γ, and IL-17. Notably, our data suggest that IL-1β induces Th17 cells independent of RORC upregulation. In contrast, TGF-β that triggers RORC prevents Th17 cell development. This suppressive function of TGF-β is characterized by inhibition of STAT1, STAT3, and CD69. However, after repeated anti-CD3 and anti-CD28 stimulation, we observe that TGF-β provokes an increase in Th17 cells that presumably relies on reactivation of a default pathway by preferential inhibition of IFN-γ. Hence, our data extend the view that the principal cytokines for determining Th cell fate are IL-12 for the Th1 lineage, IL-4 for the Th2 lineage, and TGF-β in conjunction with IL-6 for the Th17 lineage. We propose that IL-1β induces a general proinflammatory Th cell precursor that, in the presence of the lineage-specifying cytokines, further differentiates into one of the specific Th cell subpopulations.     

Are CD4+CD25-Foxp3+ cells in untreated new-onset lupus patients regulatory T cells?

Introduction  

Our previous study has reported that, in patients with untreated new-onset lupus (UNOL), there was an abnormal increase in the number of CD4⁺CD25^-Foxp3⁺ T cells that correlated with disease activity and significantly decreased after treatment. However, little is known about the nature of this cell entity. The aim of this study was to explore the nature of abnormally increased CD4⁺CD25^-Foxp3⁺ T cells in UNOL patients.     

Activated mouse CD4+Foxp3? T cells facilitate melanoma metastasis via Qa-1-dependent suppression of NK-cell cytotoxicity

The regulatory activities of mouse CD4⁺Foxp3⁺ T cells on various immune cells, including NK cells, have been well documented. Under some conditions, conventional CD4⁺Foxp3⁻ T cells in the periphery are able to acquire inhibitory function on other T cells, but their roles in controlling innate immune cells are poorly defined. As a potential cellular therapy for cancer, ex vivo activated CD4⁺Foxp3⁻ effector T cells are often infused back in vivo to suppress tumor growth and metastasis. Whether such activated T cells could affect NK-cell control of tumorigenesis is unclear. In the present study, we found that mitogen-activated CD4⁺Foxp3⁻ T cells exhibited potent suppressor function on NK-cell proliferation and cytotoxicity in vitro, and notably facilitated B16 melanoma metastasis in vivo. Suppression of NK cells by activated CD4⁺Foxp3⁻ T cells is cell-cell contact dependent and is mediated by Qa-1:NKG2A interaction, as administration of antibodies blocking either Qa-1 or NKG2A could completely reverse this suppression, and significantly inhibited otherwise facilitated melanoma metastasis. Moreover, activated CD4⁺Foxp3⁻ cells from Qa-1 knockout mice completely lost the suppressor activity on NK cells, and failed to facilitate melanoma metastasis when transferred in vivo. Taken together, our findings indicate that innate anti-tumor response is counter regulated by the activation of adaptive immunity, a phenomenon we term as “activation-induced inhibition”. Thus, the regulatory role of activated CD4⁺Foxp3⁻ T cells in NK-cell activity must be taken into consideration in the future design of cancer therapies.     

An islet-specific pulse of TGF-β abrogates CTL function and promotes β cell survival independent of Foxp3+ T cells

Effective therapies that prevent chronic inflammation from developing into type 1 diabetes remain elusive. In this study, we show that expression of TGF-β for just 1 wk in inflamed islets of NOD mice significantly delays diabetes development. Time course studies demonstrated that the brief TGF-β pulse protects only if administered when extensive β cell destruction has occurred. Surprisingly, TGF-β-mediated protection is not linked to enhanced Foxp3(+) regulatory T cell activity or to decreased intrapancreatic presentation of islet Ags. Instead, TGF-β disables the transition of primed autoreactive CD8(+) T cells to cytotoxic effectors and decreases generation, or maintenance, of CD8(+) memory T cells within the pancreas, significantly impairing their diabetogenic capacity.     

CD4+CD25-Foxp3+ T cells: a marker for lupus nephritis?

Introduction

Systemic lupus erythematosus (SLE) is a heterogenous autoimmune disease, which can affect different organs. Increased proportions of CD4⁺CD25^-Foxp3⁺ T cells have been described in SLE patients. The exact role of this cell population in SLE patients still remains unclear. We therefore analyzed this T cell subset in a large cohort of SLE patients with different organ manifestations.

Methods

Phenotypic analyses, proportions and absolute cell numbers of CD4⁺CD25^-Foxp3⁺ T cells were determined by flow cytometry (FACS) in healthy controls (HC) (n = 36) and SLE patients (n = 61) with different organ manifestations. CD4⁺CD25^-Foxp3⁺ T cells were correlated with clinical data, the immunosuppressive therapy and different disease activity indices. In patients with active glomerulonephritis, CD4⁺CD25^-Foxp3⁺ T cells were analyzed in urine sediment samples. Time course analyses of CD4⁺CD25^-Foxp3⁺ T cells were performed in patients with active disease activity before and after treatment with cyclophosphamide and prednisone.

Results

CD4⁺CD25^-Foxp3⁺ T cells were significantly increased in active SLE patients and the majority expressed Helios. Detailed analysis of this patient cohort revealed increased proportions of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients with renal involvement. CD4⁺CD25^-Foxp3⁺ T cells were also detected in urine sediment samples of patients with active glomerulonephritis and correlated with the extent of proteinuria.

Conclusion

CD4⁺CD25^-Foxp3⁺ T cells resemble regulatory rather than activated T cells. Comparative analysis of CD4⁺CD25^-Foxp3⁺ T cells in SLE patients revealed a significant association of this newly described cell population with active nephritis. Therefore CD4⁺CD25^-Foxp3⁺ T cells might serve as an important tool to recognize and monitor SLE patients with renal involvement.     

HSP70 Enhances Immunosuppressive Function of CD4+CD25+FoxP3+ T Regulatory Cells and Cytotoxicity in CD4+CD25? T Cells

Human CD4⁺CD25⁺FoxP3⁺ T regulatory cells (Tregs) control effector T cells and play a central role in peripheral tolerance and immune homeostasis. Heat shock protein 70 (HSP70) is a major immunomodulatory molecule, but its effect on the functions of Tregs is not well understood. To investigate target-dependent and –independent Treg functions, we studied cytokine expression, regulation of proliferation and cytotoxicity after exposure of Tregs to HSP70. HSP70-treated Tregs significantly inhibited proliferation of CD4⁺CD25⁻ target cells and downregulated the secretion of the proinflammatory cytokines IFN-γ and TNF-α. By contrast, HSP70 increased the secretion of Treg suppressor cytokines IL-10 and TGF-β. Treatment with HSP70 enhanced the cytotoxic properties of Tregs only to a minor extent (4-fold), but led to stronger responses in CD4⁺CD25⁻ cells (42-fold). HSP70-induced modulation of T-cell responses was further enhanced by combined treatment with HSP70 plus IL-2. Treatment of Tregs with HSP70 led to phosphorylation of PI3K/AKT and the MAPKs JNK and p38, but not that of ERK1/2. Exposure of Tregs to specific inhibitors of PI3K/AKT and the MAPKs JNK and p38 reduced the immunosuppressive function of HSP70-treated Tregs as indicated by the modified secretion of specific target cell (IFN-γ, TNF-α) and suppressor cytokines (IL-10, TGF-β). Taken together, the data show that HSP70 enhances the suppressive capacity of Tregs to neutralize target immune cells. Thus HSP70-enhanced suppression of Tregs may prevent exaggerated immune responses and may play a major role in maintaining immune homeostasis.     

IL-1β promotes TGF-β1 and IL-2 dependent Foxp3 expression in regulatory T cells

Earlier, we have shown that GM-CSF-exposed CD8α- DCs that express low levels of pro-inflammatory cytokines IL-12 and IL-1β can induce Foxp3+ Tregs leading to suppression of autoimmunity. Here, we examined the differential effects of IL-12 and IL-1β on Foxp3 expression in T cells when activated in the presence and absence of DCs. Exogenous IL-12 abolished, but IL-1β enhanced, the ability of GM-CSF-exposed tolerogenic DCs to promote Foxp3 expression. Pre-exposure of DCs to IL-1β and IL-12 had only a modest effect on Foxp3- expressing T cells; however, T cells activated in the absence of DCs but in the presence of IL-1β or IL-12 showed highly significant increase and decrease in Foxp3+ T cell frequencies respectively suggesting direct effects of these cytokines on T cells and a role for IL-1β in promoting Foxp3 expression. Importantly, purified CD4+CD25+ cells showed a significantly higher ability to maintain Foxp3 expression when activated in the presence of IL-1β. Further analyses showed that the ability of IL-1β to maintain Foxp3 expression in CD25+ T cells was dependent on TGF-β1 and IL-2 expression in Foxp3+Tregs and CD25- effectors T cells respectively. Exposure of CD4+CD25+ T cells to IL-1β enhanced their ability to suppress effector T cell response in vitro and ongoing experimental autoimmune thyroidits in vivo. These results show that IL-1β can help enhance/maintain Tregs, which may play an important role in maintaining peripheral tolerance during inflammation to prevent and/or suppress autoimmunity.     

CD4+CD25+调节性T细胞

调节性T细胞(regulatory T cells,Treg)是机体维持自身耐受的重要组成部分。CD4^ CD25^ Treg细胞来源于胸腺,其主要功能是抑制自身反应性T细胞,并且其作用是通过直接的Treg-T效应细胞之间的相互接触方式来实现的。CD4^ CD25^ Treg细胞可分泌多种抑制性细胞因子,但与其抑制功能关系并不明确,目前有证据表明GITR和Foxp3与CD4^ CD25^ Treg细胞的抑制功能有关,并且Foxp3已作为CD4^ CD25^ Treg细胞的特异性标志。通过IL-10、TGF-β等抑制性细胞因子、imDC以及转基因技术可以产生具有免疫抑制功能的调节性T细胞。调节性T细胞在免疫相关性疾病、肿瘤免疫和抗感染免疫等方面具有重要意义。     

CD4+CD25+FOXP3+ T cells,Foxp3 gene and protein expression contribute to antiasthmatic effects of San’ao decoction in mice model of asthma

ObjectiveSan’ao decoction (SAD) is a commonly used traditional combinatorial formula composed of Herba Ephedrae, Radix Glycyrrhizae and Amygdalus Communis Vas. Early studies showed that in the OVA sensitization asthmatic mice model its compatibility could lower airway reactivity and airway inflammatory cell infiltration. Based on the above results, this study mainly discussed San’ao decoction's immunomodulatory effects on Tregs.MethodsUPLC–PDA–TOF-MS was applied to analyze chemicals of SAD, and under the optimized chromatographic and MS condition, the major components in SAD were well separated and detected within 22 min. An asthma model was established in BALB/c mice that were sensitized and challenged with ovalbumin. After 2 weeks’ treatment, peripheral blood and bronchoalveolar lavage fluid (BALF) were assessed for inflammatory cell counts; histological change of lung tissue were detected; flow cytometry detection of splenic CD4⁺CD25⁺Foxp3⁺ Treg cells of the mice were counted; Foxp3 expression in lung tissues were examined as well.Results22 Peaks signal chemical components in SAD were identified by UPLC–QTO-MS method. In terms of the percentage of eosinophile in peripheral blood and bronchoalveolar lavage fluid (BALF), SAD groups were significantly lower (p < 0.01) than model group. Compared with model group, lung histological changes of SAD groups were reduced; the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells in CD4⁺ cells of asthmatic mice also decreased; SAD significantly increased the proportion of CD4⁺CD25⁺Foxp3⁺ Treg cells and promoted Foxp3 expression in a mouse model of asthma.ConclusionThese results suggest that the antiasthmatic effects of SAD are at least partially associated with CD4⁺CD25⁺Foxp3⁺ Treg cells.     

香菇多糖对急性弓形虫感染过程中CD4+CD25+Foxp3+调节性T细胞的调节作用研究

目的 探讨香菇多糖(Lentinan,Lent)对急性弓形虫感染BALB/c小鼠CD4+ CD25+ Foxp3+调节性T细胞(Tregs)数量和功能的调节作用.方法 对RH强毒株感染的BALB/c小鼠进行不同时间点的Lent预处理,动态观察用药后各组感染小鼠的生存率;在感染后第0、3、5、8和10天提取小鼠的脾细胞,FACS检测Tregs细胞数量的动态变化,ELISA法检测脾细胞培养上清中IL-10的分泌水平.结果 感染前6 d 1 mg/kg Lent用药组与药物未处理组相比显著提高了弓形虫感染小鼠的生存率;具有免疫抑制功能的Tregs数量于感染后8d达峰值,同时免疫应答中关键细胞因子IL-10的分泌水平也于感染后8d和10d显著增加.结论 对急性弓形虫感染的BALB/c小鼠采用Lent预处理之后能有效的调节Tregs的数量和功能,从而调控Th1/Th2之间的动态平衡达到治疗弓形虫的作用,为弓形虫病的临床治疗提供的新的理论依据.     

Negligible Effect of Sodium Chloride on the Development and Function of TGF-β-Induced CD4+ Foxp3+ Regulatory T Cells

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Murine CD4 T cells produce a new form of TGF-β as measured by a newly developed TGF-β bioassay

Background

It is generally assumed that T cells do not produce active TGF-β since active TGF-β as measured in supernatants by ELISA without acidification is usually not detectable. However, it is possible that active TGF-β from T cells may take a special form which is not detectable by ELISA.

Methodology/Principal Findings

We constructed a TGF-β bioassay which can detect both soluble and membrane-bound forms of TGF-β from T cells. For this bioassay, 293T cells were transduced with (caga)₁₂ Smad binding element-luciferase along with CD32 (Fc receptor) and CD86. The resulting cells act as artificial antigen presenting cells in the presence of anti-CD3 and produce luciferase in response to biologically active TGF-β. We co-cultured pre-activated murine CD4⁺CD25⁻ T cells or CD4⁺CD25⁺ T cells with the 293T-caga-Luc-CD32-CD86 reporter cells in the presence of anti-CD3 and IL-2. CD4⁺CD25⁻ T cells induced higher luciferase in the reporter cells than CD4⁺CD25⁺ T cells. This T cell-produced TGF-β is in a soluble form since T cell culture supernatants contained the TGF-β activity. The TGF-β activity was neutralized with an anti-mouse LAP mAb or an anti-latent TGF-β/pro-TGF-β mAb, but not with anti-active TGF-β Abs. An anti-mouse LAP mAb removed virtually all acid activatable latent TGF-β from the T cell culture supernatant, but not the ability to induce TGF-β signaling in the reporter cells. The induction of TGF-β signaling by T cell culture supernatants was cell type-specific.

Conclusions/Significance

A newly developed 293T-caga-Luc-CD32-CD86 reporter cell bioassay demonstrated that murine CD4 T cells produce an unconventional form of TGF-β which can induce TGF-β signaling. This new form of TGF-β contains LAP as a component. Our finding of a new form of T cell-produced TGF-β and the newly developed TGF-β bioassay system will provide a new avenue to investigate T cell function of the immune system.