Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

PBMC LNA-array microRNA profiling

ABSTRACT: T cells were collected from four healthy donors using heparin anti-coagulant, diluted with equal amounts of PBS EDTA, then layered over Lymphoprep and spun to collect PBMCs. CD4+ T cells were purifed using the Miltenyi positive selection magnetic labeling kit. Cells were resuspended in RPMI (Invitrogen) with 10% FCS amd 50 U/ml IL-2 (Immunotools), and 3.5x105 plated out per well in 48 well plates, coated overnight with 2 Âµg/ml anti-CD3 and either anti-CD28 or anti-CD46. Purity was assessed by flow cytometry and was in all cases â¥ 95%. RNA was purified from a set of unactivated cells before plating, to represent the resting, 0 hr time point samples. For early activation samples, CD28 or CD46 co-stimulated cells were harvested 2 hrs after plating. Then, 36 hours after plating, the remaining CD46-stimulated cells were harvested and labeled for IL-10 or IFN-g secretion using the Miltenyi cytokine detection kits, according to manufacturerâs instructions. Cells were then sorted into separate cytokine-secreting populations (IFN-g+/IL-10â, IFN-g+/IL-10+, and IFN-gâ/IL-10+) using a BD biosciences FACS machine.RNA from separate populations was labeled using the Hy3 microRNA (miRNA) Power labeling kit (Exiqon) and hybridised to an LNA (locked nucleic acid)-based miRCURY miRNA microarray slides (Exiqon, version 11) using a Maui hybridisation chamber (BioMicrosystems). Spot signals were acquired with an Agilent array scanner and GenePix Pro 4.1 was used for annotation (mirBase 14.0), signal processing and quantification using default background subtraction.

ORGANISM(S): Homo sapiens

SUBMITTER: Jonathan Lou Esguerra

PROVIDER: E-MTAB-2658 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

CD46 Activation Regulates miR-150-Mediated Control of GLUT1 Expression and Cytokine Secretion in Human CD4+ T Cells.

King Ben C BC Esguerra Jonathan L S JL Golec Ewelina E Eliasson Lena L Kemper Claudia C Blom Anna M AM

Journal of immunology (Baltimore, Md. : 1950) 20160108 4

CD46 is a cell surface complement inhibitor widely expressed in human tissues, in contrast to mice, where expression is limited to the testes. In humans, it has been identified as an important T cell costimulatory receptor, and patients deficient in CD46 or its endogenous ligands are unable to mount effective Th1 T cell responses. Stimulation of human CD4(+) T cells with CD3 and CD46 also leads to the differentiation of a "switched" Th1 population, which shuts down IFN-γ secretion and upregulate ...[more]

PMID: 26746193

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Project description:Several versions of SWI/SNF complexes, BAF and PBAF, have been described based on unique subunit composition. We find that T cell development in the thymus and lymphoid periphery is largely normal in the absence of the PBAF-specific component BAF180. However, BAF180-deficient Th2 cells express high levels of the immunoregulatory cytokine IL-10. BAF180 binds directly to regulatory elements in the Il-10 locus but is replaced by BAF250 BAF complexes in the absence of BAF180, resulting in increased histone acetylation and CBP recruitment to the IL-10 locus. These results demonstrate that BAF180 is a repressor of IL-10 transcription in Th2 cells and suggest that the differential recruitment of different SWI/SNF subtypes can have direct consequences on chromatin structure and gene transcription. mouse primary T helper lymphocytes, naïve and Th2 cells, resting and stimulated, comparison of wild-type and BAF180/Pbrm1 deficient cells RNA from T helper cells was compared in the presence (WT) and absence (KO) of the Pbrm1/BAF180 gene. Comparison was made in 2 T helper subsets (Naive and Th2), each under 2 condtions (resting and stimulated), in triplicate (A,B,C). Resting naïve CD4+ T cells (N) were purified to 95% purity from the spleens and lymph nodes of 4-6 week old Balb/c mice (WT, or BAF180 cKO) using CD4+CD62L+ T cell Purification Kit II per manufacturer’s instructions (Miltenyi). Stimulated Naive cells (S) were prepared from resting naive cells by stimulation for 24 hours on anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml. Resting Th2 cells (2U) were prepared from resting naive cells by plating onto anti-CD3 (1ug/ml), anti-CD28 (2 ug/ml) coated plates at 1-2 x 106/ml in the presence of 10ng/ml IL-4, 10ug/ml anti-IFN-gamma. IL-2 (100U/ml) was added 24 hours later. Cultures were expanded in IL-2 (100U/ml) three days after initial culture. Stimulated Th2 cells (2S) were prepared from resting Th2 cells, stimulated with PMA (50ng/ml) and Ionomycin (500ng/ml) for 1.5 hours. RNA was harvested from 3 replicates of the primary Th cells under each condition. RNA was isolated using RNAeasy Kit (Qiagen), Quality and quantity of the total RNA was checked with the Agilent 2100 bioanalyzer using RNA 6000 Nano chips. RNA was labeled using the standard Illumina protocol and Illumina TotalPrep RNA Amplification Kit (Ambion; Austin, TX, cat # IL1791) Biotin labeled cRNA was hybridized to Illumina’s Sentrix MouseRef-8,v2 Expression BeadChips.

Project description:In this work we present an analytical strategy to systematically identify early regulators by combining gene regulatory networks (GRN) with GWAS. We hypothesized that early regulators in T-cell associated diseases could be found by defining upstream transcription factors (TFs) in T-cell differentiation. Time series expression and DNA methylation profiling of T-cell differentiation identified several upstream TFs, of which TFs involved in Th1/2 differentiation were most enriched for disease associated SNPs identified by GWAS. NaÃ¯ve CD4+ T cells were isolated from buffy coat of four healthy donors using a naÃ¯ve CD4+ T cell isolation kit (Miltenyi, Bergisch-Gladbach, Germany). NaÃ¯ve CD4+ T cells were stimulated with plate-bound anti-CD3 (500 ng/mL), soluble anti-CD28 (500 ng/mL), in the presence of IL-12 (5 ng/mL), IL-2 (10 ng/mL) and antiâ??IL-4 (5 Âµg/mL) for Th1, IL-4 (10 ng/mL), IL-2 (10 ng/mL) and anti-IL-12 (5 Âµg/mL) and antiâ??IFN-g (5 Âµg/mL) for Th2. Cells were cultured for six days in Iscoveâ??s modified Dulbecco medium (IMDM) supplemented with 2 mM L-glutamine (PAA Laboratories, Linz, Austria), 10% heat-inactivated FCS (PAA Laboratories, Linz, Austria), 5 ÂµM Î²â??mercaptoethanol (Sigma-Aldrich, St. Louis, Missouri, USA) and 50 ug/mL gentamicin (Sigma-Aldrich, St. Louis, Missouri, USA). Cells were cultured for 6 days and then re-stimulated with plate-bound anti-CD3 and soluble anti-CD28 in the presence of corresponding polarizing cytokines and antibodies for another 2 days (Zhang et al. 2013). RNA was extracted using a miRneasy Mini Kit (Qiagen). The RNA concentrations were analysed with NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies). For the gene expression microarray analysis, The cRNA was prepared using a Low Input QuickAmp Labeling Kit. For Th1 and Th2 cells the gene expression microarray analysis was performed using SurePrint G3 Human Gene Expression 8x60K microarray kit, according to the manufactureâ??s instruction (Agilent Technologies).

Dataset Information

PBMC LNA-array microRNA profiling

Publications

CD46 Activation Regulates miR-150-Mediated Control of GLUT1 Expression and Cytokine Secretion in Human CD4+ T Cells.

Similar Datasets

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