Interleukin 4 increases the production of induced regulatory T cells in C57BL/6 mice
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Naïve peripheral T cells can express the transcription factor Foxp3 and develop regulatory function. These “induced” regulatory T (iTreg) cells are essential for tolerance and complement the function of other regulatory populations. In this study we used the lymphopenia-associated model of colitis to compare the kinetics of iTreg cell production in both C57BL/6 and BALB/c mice, the prototypical Th1- and Th2-type strains. Following the adoptive transfer of naïve CD4+ T cells into Rag−/− recipients, we tracked the development of iTreg cells over a period of 40 days. Whereas iTreg cell production in C57BL/6 was minimal, by 10 days there was a relative five-fold increase in the number of iTreg cells in BALB/c mice that grew to 35 fold by 40 days. This dramatic increase in iTreg cells correlated with a similar increase in IL-17A producing cells in the mesenteric lymph nodes and gut of BALB/c mice. In contrast, the proportion of CD4+ T cells that produced IFN-γ was comparable between the two strains. Pretreatment of C57BL/6 Rag−/− recipient mice with IL-4 resulted in a six-fold increase in the number of iTreg cells compared to untreated mice. These results indicate that a Th2 milieu supports iTreg cell induction.Keywords:
Adoptive Cell Transfer
Mesenteric lymph nodes
Abstract Regulatory T cells (Treg) are negative regulators of the immune response. Whilst thymic Treg generation is well studied, it is not known whether and how Foxp3 transcription is induced and regulated in the periphery during immune responses. Here we use Foxp3 T imer o f c ell k inetics and activit y (Tocky) mice, which report real-time Foxp3 gene transcription by measuring the spontaneous maturation of Fluorescent Timer protein from Blue to Red fluorescence, to identify the flux of Foxp3 -to Foxp3 + T cells within the periphery and analyse the real-time activity of Foxp3 transcription. Using a murine model of skin allergy, we show that both the flux of new Foxp3 expressors and the rate of Foxp3 transcription are increased at inflamed sites. These persistent dynamics of Foxp3 transcription determine the effector Treg programme, and are dependent on a Foxp3 autoregulatory transcriptional circuit, as evidenced by analysis of T cells lacking functional Foxp3 protein. Such reactive and persistent Foxp3 transcriptional activity controls the expression of coinhibitory molecules including CTLA-4 and effector-Treg signature genes. Using RNA-seq, we identify two groups of surface proteins based on their relationship to the temporal dynamics of Foxp3 transcription, and we show proof-of-principle for the manipulation of Foxp3 dynamics by immunotherapy: new Foxp3 flux is promoted by anti-TNFRII antibody, and high frequency Foxp3 expressors are depleted by anti-OX40 antibody. Collectively, our study dissects time-dependent mechanisms behind Foxp3-driven T cell regulation, and establishes the Foxp3-Tocky system as a tool to investigate the mechanisms behind T cell immunotherapies.
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To evaluate the correlation between regulatory T cells and laryngeal squamous cells carcinoma(LSCC),peripheral blood samples were collected from 50 patients to determine the percentage of CD4+C25+Foxp3+Treg cells by flow cytometry and the cytokine levels by real-time PCR.It was observed that not only the percentage of CD4+C25+Foxp3+ Treg in peripheral CD4+T cells of LSCC patients but also the positive ratio of Foxp3 in CD4+CD25+CCR6+Tregs were much higher than those in healthy persons.By using real-time PCR,it was showed that the expression level of Foxp3 mRNA correlated well with clinical stages of LSCC and that the detectable levels of TGF-β and IL-10 mRNA were higher,and those of IL-2,IL-12 and IFN-γ mRNA were lower,in the patients when compared with those in healthy control.This suggests an involvement of the inducible regulatory T subset(Foxp3+iTreg) in the pathogenesis of LSCC.The increased percentage of the Foxp3+Tregs and the up-regulation of Foxp3 expression on CCR6+Treg in LSCC patients,therefore,may account for the down-regulation of anti-tumor immunity in the patients,which may be valuable for assessment of prognosis in LSCC treatment.
Pathogenesis
Regulatory T cell
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Purpose of review Foxp3 is the transcription factor that induces the regulatory T cell phenotype. This review will examine issues around Foxp3 induction and function as well as clinical data on tolerance and rejection. Recent findings Recent findings have included identification of the signals that drive naive T lymphocytes to express Foxp3 in the thymus and the signals peripherally that induce non-Foxp3 expressing T cells to express FOXP3. Further, the identification of the downstream targets of Foxp3 both by analysis of Foxp3 expressing cells and by analysis of gene promoters that bind Foxp3 has provided new insights into its function. Whereas Foxp3 T regulatory cells (Tregs) are associated with tolerance in a variety of animal transplant models, the human data show expansion of Foxp3 Tregs associated with rejection, though Tregs are also found in transplants in patients with mixed chimerism-induced tolerance. Further, there is a significant difference in the effect of the different immunosuppressive medications on Treg function and expansion that may be important in developing strategies to enhance Tregs in human trials. Conclusion Foxp3 CD4 T cells are frequently associated with rejection; however, this does not preclude their protective role and importance in tolerance induction.
Regulatory T cell
Identification
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To observe changes of pulmonary function, regulatory T cells (Treg), forkhead transcription factor protein 3 (Foxp3) in adjuvant-induced arthritis (AA) rats, and explore the roles of Treg and Foxp3 expression in immunological mechanisms of AA.Thirty rats were randomly divided into normal control (NC) group and model control (MC) group, with 15 in each. The rats of MC group were induced by intracutaneous injection of 0.1 mL complete Freund's adjuvant in the right posterior paw. After 30 d, we observed the changes of the toe swelling degree, arthritis index. Pulmonary function was detected by animal spirometry, Treg in the peripheral blood by flow cytometry, and Foxp3 mRNA and protein by RT-PCR, immunohistochemistry, Western blotting, respectively. RESULTS Compared with the NC group, the toe swelling degree and arthritis index significantly increased, and the parameters of pulmonary function, CD4(+);CD25(+); Treg, CD4(+);CD25(+);FoxP3(+); Treg, Foxp3 mRNA and protein levels decreased in the MC group (P<0.01 or P<0.05).AA rats suffer from reduced pulmonary function and immune dysfunction, which may be related to the decreased expressions of Treg and Foxp3.
Regulatory T cell
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Induction of Forkhead-box p3 (Foxp3) expression in developing T cells upon peptide-MHC encountering has been proposed to define a lineage of committed Treg cells. However, sustained expression of Foxp3 is required for Treg function and what maintains Foxp3 expression in peripheral Treg remains obscure. To address this issue, we monitored natural Treg phenotype and function upon adoptive transfer into lymphocyte-deficient mice. We first show that about 50% of Foxp3-GFP(+) Treg isolated from Foxp3(gfp) KI animals loose Foxp3 expression in severe lymphopenic conditions. We next evidence that the cytokine IL-2, either produced by co-transferred conventional T cells or administrated i.v. prevents Foxp3 downregulation. Moreover, we document that Treg that lost Foxp3 expression upon adoptive transfer produce IL-2 are not suppressive and promote tissue infiltration and damage upon secondary transfer into alymphoid mice. Our findings that Treg convert into pathogenic Th cells in absence of IL-2 provide new clues to the success of Treg-based immune therapies.
Adoptive Cell Transfer
Treg cell
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Objective:To investigate the Foxp3 expression in murine model of type 1 diabetes mellitus and the effects of Foxp3 in the pathogenic mechanism of type 1 diabetes mellitus.Methods:Type 1 diabetes mellitus of mouse was induced by STZ.The Foxp3 expression in the spleen cells was detected at the mRNA level by RT-PCR and at protein level by Western blot.The percentage of CD4+CD25+ Treg cells in the spleens were detected by Flow cytometry.Results:The expressing levels of Foxp3 mRNA and scurfin in the model group was higher than those of control group within the first week after induction,but the expressing level of Foxp3 mRNA and Scurfin began to decrease on day 7 and were lower than those of control group on day 30.The percentage of CD4+CD25+ Treg cells in model group was similar with that of control group within the first week after induction,but after day 7,the percentage of CD4+CD25+ Treg cells in model group began to get lower than contol group.Conclusion:The expressing level of Foxp3 is decreased,then the proportion of CD4+CD25+ Treg cells is decreased accordingly,which may contribute to the pathogenic mechanism in type 1 diabetes mellitus.
Treg cell
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Abstract NTregs exhibit higher homeostatic proliferation (HP) than Tconv which may provide a competitive advantage during Treg adoptive immunotherapy. Increased Treg HP could be 2° to a self-reactive TCR repertoire and/or Foxp3-mediated increase in survival/fitness. To address this, CFSE-labeled CD4 populations from Foxp3-reporter mice, were compared 10d after transfer into wt mice. We generated Foxp3+ iTregs by activating Tconv and adding TGFβ, or transducing with Foxp3-retrovirus (RV). iTregs did not increase HP over Tconv controls (no TGFβ; RV-vector). Thus, Foxp3 itself is not sufficient to augment HP. To determine the role of Foxp3, we examined GFP+ nTreg from Filig mice (F-nTreg; Foxp3 ~10% wt levels due to unstable mRNA). F-nTreg exhibited reduced HP (10%) vs. wt nTregs (50%). Foxp3-RV transduction increased HP of F-nTregs, while RV-control had no effect (40 vs. 12%). Thus, Foxp3 is necessary for high HP by nTreg. We also examined role of Foxp3 during thymic ontogeny, irradiated Thy1.1 hosts received Filig (Thy1.2) bone marrow that was transduced with Foxp3-RV vs. RV-control. Surprisingly, in this setting, Tconv cells (no endogenous Foxp3) constitutively expressing Foxp3, exhibited increased HP vs. Foxp3- RV-controls (32 vs. 12%). Thus, Foxp3 increases HP in nTreg and when expressed during thymic ontogeny but not in mature Tconv. Our findings provide new insight into the role of Foxp3 expression in Treg homeostasis and highlight additional distinctions between iTreg and nTreg.
Adoptive Cell Transfer
Homeostasis
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Objective To explore the phenotypic conversion of regulatory T cells (Tregs) in the lungs of mice with bronchopulmonary dysplasia (BPD)-affected mice. Methods A total of 20 newborn C57BL/6 mice were divided into air group and hyperoxia group, with 10 mice in each group. The BPD model was established by exposing the newborn mice to hyperoxia. Lung tissues from five mice in each group were collected on postnatal days 7 and 14, respectively. Histopathological changes of the lung tissues was detected by HE staining. The expression level of surfactant protein C (SP-C) in the lung tissues was examined by Western blot analysis. Flow cytometry was performed to assess the proportion of FOXP3
Hyperoxia
Bronchopulmonary Dysplasia
Regulatory T cell
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Viable bacteria were not cultured from the mesenteric lymph nodes, spleens, or livers of specific-pathogen-free (SPF) mice. Viable enteric bacteria, primarily indigenous Escherichia coli and lactobacilli, were present in the mesenteric lymph nodes of gnotobiotic mice inoculated intragastrically with the whole cecal microflora from SPF mice but not in the nodes of control SPF mice similarly inoculated. These indigenous E. coli also were cultured from the mesenteric lymph nodes of 96% of gnotobiotic mice monoassociated with E. coli but from none of the mesenteric lymph nodes of SPF mice inoculated with the E. coli. Furthermore, viable E. coli were detected in the mesenteric lymph nodes of these monoassociated gnotobiotes for as long as 112 days after inoculation. Indigenous Lactobacillus acidophilus also translocated to the mesenteric lymph nodes of gnotobiotic mice monoassociated with L. acidophilus. Apparently, there are mechanisms active in SPF mice inhibiting translocation of indigenous bacteria from the gastrointestinal tract to the mesenteric lymph nodes, spleens, and livers, whereas these mechanisms are either absent or reduced in gnotobiotic mice. Indigenous E. coli maintained higher population levels in the gastrointestinal tracts of the gnotobiotes compared with their population levels in SPF mice, suggesting that high bacterial population levels might promote translocation of certain bacteria from the gastrointestinal lumen to the mesenteric lymph nodes. Gnotobiotic and SPF mice, therefore, provide experimental models for determining the nature of the mechanisms operating to confine indigenous bacteria to the gastrointestinal tract in normal, healthy animals.
Mesenteric lymph nodes
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Abstract CD4 + FOXP3 + regulatory T (Treg) cells are essential for maintaining immunological self-tolerance. Treg cell development and function depend on the transcription factor FOXP3, which is present in several distinct isoforms due to alternative splicing. Despite the importance of FOXP3 in the proper maintenance of Treg cells, the regulation and functional consequences of FOXP3 isoform expression remains poorly understood. Here, we show that in human Treg cells IL-1β promotes excision of FOXP3 exon 7. FOXP3 is not only expressed by Treg cells but is also transiently expressed when naïve T cells differentiate into Th17 cells. Forced splicing of FOXP3 into FOXP3Δ2Δ7 strongly favored Th17 differentiation in vitro . We also found that patients with Crohn’s disease express increased levels of FOXP3 transcripts lacking exon 7, which correlate with disease severity and IL-17 production. Our results demonstrate that alternative splicing of FOXP3 modulates T cell differentiation. These results highlight the importance of characterizing FOXP3 expression on an isoform basis and suggest that immune responses may be manipulated by modulating the expression of FOXP3 isoforms, which has broad implications for the treatment of autoimmune diseases.
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