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+ naïve T cells from miR-31fl/fl mice and miR-31fl/flCD4Cre mice were used to induce iTreg in vitro. Four independent experiments were performed.
Project description:To investigate the function of Embigin on LTA1 induced-CD4+TRM cells, we immunized CD4cre x Embfl/fl mice with OmpX and LTA1 at Day0 and Day21, and isolated CD4+ cells from Embfl/fl CD4cre-(WT) and Embfl/fl CD4cre+(KO) mice from the lung by magnetic beads. We then performed gene expression profiling analysis using data obtained from RNA-seq of CD4+cells from 5 WT and 5 KO mice.
Project description:To investigate the molecular mechanism how Uhrf1 regulate gene expression and cell cycle progression in Treg, we precipitated methylated DNA using recombinant MBD1 followed by genome-wide analysis of methylated gene loci by next generation sequencer Examination of methylated DNA in Treg and Tconv isolated from Cd4cre-Uhrf1+/+ and Cd4cre-Uhrf1flox/flox (Cd4cre-Uhrf1fl/fl)
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.
Project description:Cancer cells must evade immune responses at distant sites to establish metastases. The lung is a frequent site for metastasis. We hypothesized that lung-specific immunoregulatory mechanisms create an immunologically permissive environment for tumor colonization. We found that T cell-intrinsic expression of the oxygen-sensing prolyl-hydroxylase (PHD) proteins is required to maintain local tolerance against innocuous antigens in the lung, but powerfully licenses colonization by circulating tumor cells. PHD proteins limit pulmonary type helper (Th)-1 responses, promote CD4+-regulatory T (Treg) cell induction, and restrain CD8+ T cell effector function. Tumor colonization is accompanied by PHD protein-dependent induction of pulmonary Treg cells and suppression of IFN-g-dependent tumor clearance. T cell-intrinsic deletion or pharmacological inhibition of PHD proteins limits tumor colonization of the lung and improves the efficacy of adoptive cell transfer immunotherapy. Collectively, PHD proteins function in T cells to coordinate distinct immunoregulatory programs within the lung that are permissive to cancer metastasis. RNA expression was measured by RNA-Seq at day 4 following stimulation of naïve FACS-sorted CD4+ T cells with anti-CD3 and anti-CD28 antibodies in the presence of indicated doses of TGF-b. Gene expression was analysed separately in control Cd4Cre (WT) and Egln1fl/fl Egln2fl/fl Egln3fl/fl Cd4Cre (tKO) cells, or in cells treated with the pharmacological PHD inhibitor dimethyloxaloylglycine (DMOG) and control vehicle-treated cells.
Project description:To investigate the function of Bcl11b in activated C57Bl/6 mouse CD4+ T cells, we deleted Bcl11b in vivo using a Bcl11b-fl/fl;Cd4cre-ert2 tamoxifen-inducible strategy. We then cultured CD4 T cells isolated from these mice 3 days in resting or activating condtions. Finally, we performed gene expression profiling analysis using data obtained from RNA-seq of 3-5 samples per genotype per condition.
Project description:Tumor associated CD4+ and CD8+ T cells were sorted from B16f10 OVA expressing tumors in miR-155 flox, miR-155 flox CD4Cre+, and miR-155 flox CD4Cre+ mice treated with immune checkpoint blocking (ICB) antibodies by flow sorting on CD45+CD3+CD4+ cells and CD45+ CD3+CD8+ cells. RNA was collected from these cells to perform RNA sequencing of total RNA.
Project description:Disturbed expression of microRNAs (miRNAs) in regulatory T-cells (Tregs) leads to development of autoimmunity in experimental mouse models. However, the miRNA expression signature characterizing Tregs of autoimmune diseases, such as rheumatoid arthritis (RA) has not been determined yet. Moreover, the technical limitations prevented the analysis of such minute T-cell population as naive and memory Tregs. In this study we have used a microarray approach to comprehensively analyze miRNA expression signatures of naive Tregs (CD4+CD45RO-CD25++), memory Tregs (CD4+CD45RO+CD25+++), as well as conventional naive (CD4+CD45RO-CD25-) and memory (CD4+CD45RO+CD25-) T-cells (Tconvs) derived from peripheral blood of RA patients, and matched healthy controls. Differential expression of selected miRNAs was validated by TaqMan-based qRT-PCR. We found a positive correlation between increased expression of miR-451 in T-cells of RA patients and disease activity score (DAS28), ESR levels, and serum levels of IL-6. Moreover, we found characteristic, disease and treatment independent, global miRNA expression signatures defining naive Tregs, memory Tregs, naive Tconvs and memory Tconvs. The analysis allowed us to define miRNAs characteristic for a general naive phenotype (e.g. miR-92a), a general memory phenotype (e.g. miR-21, miR-155), and most importantly miRNAs specifically expressed in both naive and memory Tregs, defining as such the Treg phenotype (i.e. miR-146a, miR-3162, miR-1202, miR-1246a, and miR-4281). MicroRNA profiling was performed in four CD4+ T-cell subsets: naive Tconventional (CD3+CD8-CD45RO-CD25-), naive Tregulatory (CD3+CD8-CD45RO-CD25+), memory Tconventional (CD3+CD8-CD45RO+CD25-), and memory Tregulatory (CD3+CD8-CD45RO+CD25+) derived from 2 healthy controls, and 6 rheumatoid arthritis patients (total n=8).
