Gene expression analysis in conventional and vitamin C treated iTreg cells
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
SUBMITTER: Kwon Ik Oh
PROVIDER: E-GEOD-67653 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Foxp3+ regulatory T cells (Treg), playing a crucial role in the maintenance of immune tolerance and prevention of autoimmune diseases, consist of thymus-derived naturally-occurring CD4+Foxp3+ Treg cells (nTreg) and another CD4+Foxp3+ Treg cells that can be induced ex vivo with TGF-β (iTreg). Although both Treg subsets share similar phenotypes and functional characteristics, they also have potential biologic differences on their biology. However, the role of iTreg in regulating B cells of lupus disease mice remains unclear so far. The lupus-prone New Zealand Mixed 2328 (NZM2328) mouse, a recombinant inbred strain that originated from the crosses among New Zealand Black and New Zealand White mice and their progenies also develops Lupus Glomerulonephritis. NZM2328 mice develop autoantibodies and glomerulonephritis with female predominance similarly to humans with systemic lupus erythematosus. The lupus disease onset is usually around 3-4 months age. In our experiments, iTreg induced from NZM2328 lupus-prone mice (10-12 weeks old) and nTreg sorted from the thymus of 10-12 weeks old NZM2328 lupus-prone mice were adoptively transferred into old NZM2328 mice (>4 months age) with established lupus for 32 days. Totally, there were three groups including NZM2328 mice as model (receive PBS), NZM2328 mice received iTreg, and NZM2328 received nTreg. The serum IgG and IgM autoantibody were detected by autoantibodies microarrays (UTSW Medical Center Autoantigen Array) at days 0, 14, 32 after cell transfer. Our results demonstrated that adoptive transfer of iTreg had a superior effect than nTreg subset on suppressing lupus B cell responses in vivo.
2017-01-26 | E-MTAB-4441 | biostudies-arrayexpress
Project description:The CD4+ regulatory T (Treg) cell lineage comprises thymus-derived (t)Treg cells and peripherally induced (p)Treg cells. As a model for Treg cells, studies employ TGF-β-induced (i)Treg cells generated from CD4+ conventional T (Tconv) cells in vitro. Here, we describe the relationship of iTreg cells to tTreg and Tconv cells. Proteomic analysis revealed that iTreg, tTreg and Tconv cell populations each have a unique protein expression pattern. iTreg cells had very limited overlap in protein expression with tTreg cells, regardless of cell activation status and instead shared signaling and metabolic proteins with Tconv cells. tTreg cells had a uniquely modest response to CD3/CD28-mediated stimulation. As a benchmark, we used a previously defined proteomic signature that sets ex vivo naïve and effector phenotype Treg cells apart from Tconv cells and includes unique Treg cell properties (Cuadrado et al., Immunity, 2018). This Treg cell core signature was largely absent in iTreg cells. We also used a proteomic signature that distinguishes ex vivo effector Treg cells from Tconv cells and naïve Treg cells. This effector Treg cell signature was partially present in iTreg cells. In conclusion, iTreg cells are distinct from tTreg cells and share limited features with ex vivo Treg cells at the proteomic level.
2022-11-28 | PXD038302 | Pride
Project description:Induced Treg (iTreg) cells are essential for tolerance and can be used therapeutically, yet their stability in vivo and mechanisms of suppression are unresolved. Here, we used a treatment model of colitis to examine the role of autologous IL-10 in iTreg cell function. Mice treated with IL-10+/+ iTreg cells in combination with IL-10–/– natural Treg (nTreg) cells survived and gained weight, even though iTreg cells were numerically disadvantaged and comprised just ~20% of all Treg cells in treated mice. Notably, ~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. The TCR repertoires of iTreg and ex-iTreg cells exhibited almost no overlap, which indicates that the two populations are clonally unrelated and maintained by different selective pressures. These data demonstrate a potent and critical role for iTreg cell produced IL-10 that can supplant the IL-10 produced by nTreg cells and compensate for the inherent instability of the iTreg population. BALB/c Rag1-/- mice were treated with 500,00 WT nTreg cells plus 500,000 WT in-vitro-derived iTreg cells. After 125 days cells were sorted by flow cytometry from spleens and mesenteric lymph nodes from 14 treated mice. EGFP+ Thy1.1+ iTreg cells, EGFP+ Thy1.1– nTreg cells, and EGFP–Thy1.1+ ex-iTreg cells were pooled and used to generate total RNA for each iTreg, nTreg, and ex-iTreg array set, which was labeled and hybridized to Affymetrix 430 2.0 GeneChips in accordance to the manufacturer’s protocol. Two sets of arrays were performed, and the results were averaged. Both iTreg and nTreg array sets were compared to a) naïve CD4+EGFP– Tconv cells from Foxp3EGFP. The subset of probe sets whose expression increased or decreased by twofold or more relative to Tconv cells as a common standard was identified and used for further analysis.
2012-12-06 | E-GEOD-35543 | biostudies-arrayexpress
Project description:To test whether human in vitro primed Th9 cells recapitulate the core pathogenic Th2 cell phenotype, we differentiated naïve T cells into Th1 (IL-12), Th2 (IL-4), Th9 (IL-4+TGF-β), and iTreg (TGF-β). After 7 days transcriptomic profiling by bulk RNA-seq was performed.
2022-10-31 | E-MTAB-12199 | biostudies-arrayexpress
Project description:A microarray study performed in iTreg of miR-31fl/fl/CD4Cre and control mice to identify genes that are regulated by the miR-31. CD4+ naM-CM-/ve T cells from miR-31fl/fl mice and miR-31fl/flCD4Cre mice were used to induce iTreg in vitro. Four independent experiments were performed. MiR-31fl/fl/CD4Cre mice (6 wk old) and age-matched control mice were sacrificed, and the spleen were removed and teased into cell-single suspensions and filtered through a 40 M-NM-<m cell strainer. NaM-CM-/ve CD4+CD25-Foxp3gfp-CD62Lhi T cells were sorted by FACSAria III (BD bioscience).The medium used for T cell cultures was RPMI-1640 (Gibco) supplemented with 10% heat-inactivated FBS (Gibco), 2 mM L-glutamine (Gibco), 100 U/ml penicillin, 100 M-NM-<g/ml streptomycin, and 5 mM M-NM-2-mercaptoethanol (Gibco). The CD4+ NaM-CM-/ve T cells were stimulated with plate-bound anti-CD3 (5 M-NM-<g/ml) plus soluble anti-CD28 (2 M-NM-<g/ml) under iTreg cell differentiation conditions (mTGF-M-NM-21, 5 ng/ml and rmIL-2, 40 ng/ml; R&D Systems). After 4 days, harvested cell ,the RNA was extracted in Trizol and then purified using the miRNeasy Micro Kit (Qiagen). This submission shows the data obtained from 3 individual miR-31fl/fl/CD4Cre mice mice measured against 3 control mice.
2014-10-02 | E-GEOD-61938 | biostudies-arrayexpress
Project description:PolyA+ mRNA-seq was performed on naive, Th1, Th2, Th17, splenic Treg, and in vitro-induced Treg (iTreg) cells.
2015-05-14 | E-MTAB-2582 | biostudies-arrayexpress
Project description:Differentiation of naïve CD4+ T cells into effector (Th1, Th2 and Th17) and induced regulatory (iTreg) T cells requires lineage-specifying transcription factors and epigenetic modifications that allow appropriate repression or activation of gene transcription. The epigenetic silencing of cytokine genes is associated with the repressive H3K27 trimethylation mark, mediated by Ezh2 or Ezh1 methyltransferase components of the polycomb repressive complex 2 (PRC2). EZH2 over-expression and activating mutations are implicated in tumorigenesis and correlate with poor prognosis in several tumor types 35. This spurred the development of EZH2 inhibitors which, by inducing tumor cell growth arrest and cell death, show therapeutic promise in cancer. A role for Ezh2 in suppressing Th1 and Th2 cytokine production and survival has recently been reported. It is not entirely clear whether Ezh2-PRC2 plays a role in H3K27me3 in cytokine loci in naïve CD4+ T cells and whether H3K27me3 has a non-redundant role in T helper cell lineage differentiation and survival. Here, we investigate the effects of T cell-specific Ezh2 deletion to determine the role that Ezh2-PRC2 plays in regulating the fate of differentiating naïve CD4+ T cells. Loss of Ezh2 altered the expression of 1328 genes in Th0 and 1979 genes in iTreg cells. Gene expression changes were positively correlated in both cell types, indicating that Ezh2 targets similar genes in these cells. As expected, Ifng was one of the genes most increased in expression by following loss of Ezh2. In addition, expression of Tbx21 homolog Eomes, a transcription factor that regulates IFNG production, was also significantly increased. We then performed H3K27me3 ChIP-seq on Ezh2fl/fl and Ezh2fl/fl.CD4Cre Th0 cells. Consistent with cellular phenotype and RNA-seq data, we observed a loss of the H3K27me3 at Eomes, Il4 and Il10 loci . Very low levels of H3K27me3 marks were present at Ifng and Tbx21 loci in differentiated Ezh2fl/fl Th0 cells, suggesting that upon differentiation, upregulation or activation of transcription factors accounts for IFNG overproduction. A significant loss of H3K27me3 was observed >2kb upstream of Gata3 locus , however this did not result in increased transcription . Of the 22381 genes tested for changes in H3K27me3, 1360 showed a statistically significant decrease in Ezh2fl/fl.CD4Cre Th0 cells, compared to wildtype. Furthermore, 404 of these genes also showed a concomitant gain in expression in Ezh2fl/fl.CD4Cre Th0 cells, suggesting that these loci are likely direct Ezh2-PRC2 targets. There are 3 biological replicates each of Ezh2fl/fl.CD4Cre and Ezh2fl/fl in both Th0 and iTreg cells for the RNA-seq experiment. There are 2 biological replicates each of Ezh2fl/fl.CD4Cre and Ezh2fl/fl in Th0 cells for the ChIP-seq experiment.
2014-08-04 | E-GEOD-57500 | biostudies-arrayexpress
Project description:Recent data have shown that Hypermethylated in cancer 1 (HIC1) is an important contributor to iTreg cell development and function. Using affinity-purification and tandem mass spectrometry we systematically characterized the HIC-1 interactome in human iTreg cells. On the basis of these data, we have shown that HIC1 is a part of FOXP3-RUNX1-CBFβ protein complex that regulates Treg signature genes and is indispensable for the suppressive function of FOXP3+ regulatory T cells. SRM was used to validate HIC1 interactors
2023-10-16 | PXD039337 | Pride
Project description:Through their functional diversification, CD4+ T cells play key roles in both driving and constraining immune-mediated pathology. Transcription factors are critical in the generation and maintenance of cellular diversity and negative regulators antagonistic to alternate fates often act in conjunction with positive regulators to stabilize lineage specification1. Polymorphisms within the locus encoding a transcription factor BACH2 are associated with diverse immune-mediated diseases including asthma2, multiple sclerosis3, Crohn¹s disease4-5, coeliac disease6, vitiligo7 and type 1 diabetes8. A role for Bach2 in maintaining immune homeostasis, however, has not been established. Here, we define Bach2 as a broad regulator of immune activation that stabilizes immunoregulatory capacity while repressing the differentiation programmes of multiple effector lineages in CD4+ T cells. Bach2 was required for efficient formation of regulatory (Treg) cells and consequently for suppression of lethal inflammation in a manner that was Treg cell dependent. Assessment of the genome-wide function of Bach2, however, revealed that it represses genes associated with effector cell differentiation. Consequently, its absence during Treg polarization resulted in inappropriate diversion to effector lineages. In addition, Bach2 constrained full effector differentiation within Th1, Th2 and Th17 cell lineages. These findings identify Bach2 as a key regulator of CD4+ T-cell differentiation that prevents inflammatory disease by controlling the balance between tolerance and immunity. The role of Bach2t to regulate immune homeostasis was investigated by mapping DNA binding profiles of Bach2 in iTreg condition. The function of Bach2 was also evaluated by comparing transcriptome in WT and Bach2-deficient iTreg cells and further comparison was done with transcriptome in naive, Th1, Th2, and Th17 conditions.
2013-06-27 | E-GEOD-45975 | biostudies-arrayexpress
Project description:DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germline and may be mediated by Tet (ten-eleven-translocation) enzymes, which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Tet enzymes have been extensively studied in mouse embryonic stem (ES) cells, which are generally cultured in the absence of Vitamin C, a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of Vitamin C to ES cells promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by Vitamin C treatment. Importantly, Vitamin C, but not other antioxidants, enhances the activity of recombinant human Tet1 in a biochemical assay and the Vitamin C-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ES cells. Vitamin C has the strongest effects on regions that gain methylation in cultured ES cells compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) elements, which are resistant to demethylation in the early embryo, are resistant to Vitamin C-induced DNA demethylation. Collectively, this study establishes that Vitamin C is a direct regulator of Tet activity and DNA methylation fidelity in ES cells. Oct4-GiP mouse embryonic stem (ES) cells were cultured in the presence or absence of Vitamin C (L-ascorbic acid 2-phosphate, 200 M-NM-<g/ml) for 72 hours. RNA was harvested from biological triplicates for each condition and hybridized to Affymetrix microarrays. Cells were maintained in N2B27 medium supplemented with LIF (1000 U/ml), MEK inhibitor PD0325901 (1 M-NM-<M), and GSK3M-NM-2 inhibitor CHIR99021 (3 M-NM-<M).
2013-06-30 | E-GEOD-46319 | biostudies-arrayexpress