Project description:Cryopreserved human PBMCs from six donors were stimulated with anti-CD3/CD28 beads in the presence or absence of 100ng/ml IL-23 for 22 hours. RNA samples were assessed for consistency with a Bioanalyzer (Agilent) and quantified by a Nanodrop® ND 1000 Spectrophotometer (Nanodrop Technologies, USA). Expression array data was quantile normalised, detection above background statistical testing performed, comparison between activated un-stimulated versus activated IL-23 stimulated conditions performed as a paired test using the Illumina Custom differential expression algorithm (all implemented in Illumina BeadStudio GeneExpression Module v3.2). Two sample groups were compared, each with 6 biological replicates. Group 1 was stimulated with CD3CD28 antibody coated beads as a control group for 22hours. Group 2 was stimulated as the control group and treated with IL-23 100ng/ml for the same time period. Keywords: Cytokine Treatment
Project description:Difference in gene expression profile between 30000 freshly isolated human PBMCs stimulated with the immobilized anti-CD3 mAb (0.25µg/ml) alone (lane 1), along with ALCAM-Fc (1µg/ml) (lane 2), IL-2 (2.5ng/ml) (lane 4) or both (lane 3) or compared to unstimulated cells (lane 5), incubated for 72 h.
Project description:Gene expression profiling of in vitro differentiated murine Th cell subsets. Flow cytometrically sorted naive Th cells (CD4+ CD44- Foxp3-) were polyclonally stimulated in vitro for 3 days using 4 µg/ml plate-bound antibody to CD3 (145-2C11) and 2 µg/ml soluble antibody to CD28 (PV-1). Th0 cells were cultured in the absence of exogenous cytokines. Th17 cells were differentiated with 50 ng/ml IL-6 plus 0.5 ng/ml TGF-β. Tr-1 cells were differentiated with 100 ng/ml IL-27 plus 0.5 ng/ml TGF-β.
Project description:Human naïve CD4 T cells were purified from healthy volunteers' blood and were differentiated into induced Tregs in vitro by culturing for 5 days in the presence of anti-CD3 and CD28 antibodies (2 µg/ml each), IL-2 (100 units/ml) and TGF-β1 (5 ng/ml) for 5 days, then in presence of anti-CD3 and CD28 antibodies for 2 days. Human iTregs were harvested on day 7 post differentiation, then treated with either vehicle or IL-21 (100 ng/ml) in serum-free RPMI media for 18 hours.
Project description:CD3+_DONOR 1: 72 Hour T cell activation (CD3.CD28 beads) + 50U/ml IL-2 For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
Project description:CD3+_DONOR 4: 72 Hour T cell activation (CD3.CD28 beads) + 50U/ml IL-2 For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODE_Data_Use_Policy_for_External_Users_03-07-14.pdf
Project description:Difference in gene expression profile between 30000 freshly isolated human PBMCs stimulated with the immobilized anti-CD3 mAb (0.25µg/ml) alone (lane 1), along with ALCAM-Fc (1µg/ml) (lane 2), IL-2 (2.5ng/ml) (lane 4) or both (lane 3) or compared to unstimulated cells (lane 5), incubated for 72 h. Organism used: Human Slides: Human 8x15k Arrays Starting material: Human peripheral blood mononuclear cells RNA Samples used: Pooled 1 â Unstimulated, Anti-CD3, Anti-CD3 + IL-2, Anti-CD3 + ALCAM, Anti-CD3 +ALCAM + IL-2. Labeling kit: Agilentâs Quick-Amp labeling Kit (p/n5190-0444) Labeling Method: T7 promoter based-linear amplification to generate labeled complementary RNA (One-Color Microarray-Based Gene Expression Analysis) Total RNA and cRNA Purification Kit: Hybridization Kit: Qiagenâs RNeasy minikit Cat#74104 RNA quality was checked using Bioanalyzer.
Project description:To broadly assess which pathways FRCs regulate in CD8+ T cells, we sorted CD8+ T cells for RNA-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). Here we demonstrate that FRC-derived signals, including IL-6, act in concert with TCR signaling and co-stimulation to promote the expression of MYC and HIF-1-dependent glycolytic genes and vital pro-survival genes (encoding BCL-2 family members, inhibitors of apoptosis [IAPs] members and Cflar) in activated CD8+ T cells.
Project description:To broadly assess which pathways FRCs regulate in CD8+ T cells, we sorted CD8+ T cells for ATAC-seq following activation in whole splenocyte mixtures via soluble anti-CD3/CD28 (0.25μg/ml) for 48 hours (Stim), activated with anti-CD3/CD28 in the presence of Nos2–/– FRCs (FRC) or activated with anti-CD3/CD28 plus 100 ng/ml recombinant IL-6 (IL-6). Here we demonstrate that FRC-derived signals, including IL-6, act in concert with TCR signaling and co-stimulation to remodel chromatin and render essential transcription factor binding motifs accessible in activated CD8+ T cells. Signals from FRCs led to increased binding regions for MYC, HIF-1α and HIF-1β, which are known to promote metabolic pathways following T cell activation. Additionally, FRC-derived signals promoted activity of transcription factors that regulate survival and memory programs in T lymphocytes such as BATF and BACH2.
Project description:As part of our study in understanding the role of SP140 in inflammatory pathways in macrophages, we inhibited SP140 mRNA using siRNA. Peripheral blood mononuclear cells (PBMCs) were obtained from whole blood of healthy donors (from Sanquin Institute Amsterdam or from GSK Stevenage Blood Donation Unit) by Ficoll density gradient (Invitrogen). CD14+ monocytes were positively selected from PBMCs using CD14 Microbeads according to the manufacturer’s instructions (Miltenyi Biotec). CD14+ cells were differentiated with 20 ng/mL of macrophage colony-stimulating factor (M-CSF) (R&D systems) for 3 days followed by 3 days of polarization into classically activated (inflammatory) M1 macrophages (100 ng/mL IFN-γ; R&D systems). M1 macrophages were transfected with siGENOME human smartpool SP140 siRNA or non-targeting scrambled siRNA for 48h with DharmaFECT™ transfection reagents according to manufacturer’s protocol (Dharmacon). The cells were left unstimulated or stimulated with 100 ng/mL LPS (E. coli 0111:B4; Sigma) for 4h (for qPCR) or 24h (for Elisa). The cells were lysed (ISOLATE II RNA Lysis Buffer RLY-Bioline) for RNA extraction.150 ng total RNA was labelled using the cRNA labelling kit for Illumina BeadArrays (Ambion) and hybridized with Ref8v3 BeadArrays (Illumina). Arrays were scanned on a BeadArray 500GX scanner and data were normalized using quantile normalization with background subtraction (GenomeStudio software; Illumina). This submission only contains processed data