RNA-seq of activated mouse CD8 T cells treated with IL-4 and IFN-alpha
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE345167 | GEO | 2026/08/31
REPOSITORIES: GEO
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Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation Magnetically sorted untouched CD8+CD45R0-T cells from three different donors (named A, B and C) unstimulated or stimulated with IFN alpha 2b or with anti-CD3/CD28 beads alone or along with IFN alpha 2b or IFN alpha 5 for 48 hours.
2010-07-23 | E-GEOD-17301 | biostudies-arrayexpress
Project description:IFN alpha mediated gene expression pattern. The effect of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. This analysis examined the effects of IFN alpha on human CD8 T cells responding to antigen (signal 1) and costimulatory signals (signal 2) provided by beads coated with anti-CD3 and anti-CD28 mAbs. Magnetically sorted untouched CD8+CD45R0- T cells from three different donors were unstimulated or stimulated with IFNa2b or with anti-CD3/CD28 Beads alone or along with IFNa2b or IFNa5 for 48 hours. Individual mRNA samples were analyzed using HG-U133A 2.0 array gene chips. Keywords: Gene expression analysis after different stimulation
2010-07-23 | GSE17301 | GEO
Project description:CD8+ cytotoxic T lymphocytes (CTLs) play a major role in defense against intracellular pathogens, and their functions are specified by antigen recognition and innate cytokines. While effector CTLs eliminate the infection, a small population of memory cells are retained that yields more rapid and robust response upon re-infection. Antigen presenting cells secrete an array of innate cytokines including IL-12 and IFN-α after recognition of pathogens. Both IL-12 and IFN-α have been shown to act as the third signal regulating the development of CTLs. We have shown that these two cytokines have a non-redundant effect in generation of human effector CTL. IL-12 alone is sufficient for effector CTL genesis marked by IFN-γ and TNF-α production, as well as increased cytolytic activity. Even in the presence of IFN-α, IL-12 programs CTLs that express the chemokine receptor CXCR3 and effector cytokines. Using microarray analysis we have investigated how IL-12 and IFN-α differentially regulate the genetic programming pathways that give rise to effector CTLs among multiple human donors. We have also analyzed the gene expression patterns of cells sorted from healthy human peripheral blood that display surface markers of effector memory CTL (designated as ex vivo) samples. 5 healthy human donor samples were used for the in vitro cultures. For each donor the CFSE labeled cells (CD8+CD45RA+) were cultured in the presence of neutralized, IL-12, IFN-a, and IL-12+IFN-a conditions and plate-bound anti-CD3+anti-CD28 for 3.5 days. Total RNA from CFSEhi (Undiv) and CFSElo (Div) sorted cells were used for Illumina Bead Array. 4 healthy human donor samples were used for the ex vivo samples. Total RNA was collected from FACS sorted CD8+CCR7hiCXCR3lo and CD8+CCR7loCXCR3hi cells without any stimulation.
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Project description:Naive CD4+ CD62L+ CD25- T cells were differentiated under TH1 and TH2 conditions for 7 days, restimulated with anti-CD3 and anti-CD28 for 24h and sorted for IFN-gamma (TH1) and IL-4 (TH2) production using cytokine secretion assays.
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Project description:cAMP inhibits TCR signaling, T cell proliferation, cytokine production and T cell function. We used microarrays to detail the global programme of gene expression in TCR-activated WT or IFN-gR1-deficient CD4+ T cells by db-cAMP. CD4+ T cells were purified from spleens of WT or IFN-gR1-deficient mice by autoMACS, and activated with anti-CD3 plus anti-CD28 for 15 h. db-cAMP or vehicle control (med) was added into cultures for the last 3 h.
2012-07-23 | E-GEOD-39592 | biostudies-arrayexpress
Project description:The aim of the study was to compare the two different costimuli anti-CD28 (costimulus 2a) and IL-2 (costimulus 2b) – in the presence of a suboptimal anti-CD3 signal (signal 1) – on the de novo activation of freshly purified human naive T cells. To this end, purified human naive T cells were co-incubated for seven days with a tumor vaccine which consisted of gamma-irradiated and Newcastle Disease Virus (NDV)-infected MCF-7 cells (a well-defined mammacarcinoma cell line). The gamma-irradiation dose was 200 Gray (Gy) to suppress the proliferation capacity of the vaccine cells (although the vaccine cells are not killed by such a procedure but still survive for several days); NDV-infection was performed at a dose of 100 hemagglutination units (HU) per 10^7 vaccine cells. The latter led – among others – to the upregulation of the viral surface molecules hemagglutinin neuraminidase (HN) and the fusion protein (F), both of which can serve for the attachment of fusion proteins delivering the activation signals to the naive T cells. The bispecific (bs) fusion proteins used in this study were of the single chain (scFv) format, binding with one arm to the viral HN molecule (for the attachment to the vaccine cells), and with the other arm either to the T cell receptor (TCR)-associated CD3 complex [as a surrogate of a major histocompatibility complex (MHC)-presented peptide recognized by the TCR] (bsHN-CD3), or to the costimulatory molecule CD28 (bsHN-CD28). Furthermore, a bispecific fusion protein containing the human interleukin (IL)-2 (bsHN-IL-2, a so-called immunocytokine) was used. The gene expression analysis of the differentially activated naive T cells revealed that in the presence of a suboptimal dose of anti-CD3 the two costimuli anti-CD28 (sample d7-S2_280705) and IL-2 (sample d7-S3_280705) led to a highly redundant pattern of gene upregulation in comparison to control samples in which naive T cells received only anti-CD3 (sample d7-S4_280705), anti-CD28 (sample d7-S5_280705) or IL-2 (sample d7-S6_280705) signals alone, or no signal at all (i.e. only vaccine cells; sample d7-S7_280705). All signals were compared to the reference sample which consisited of freshly isolated, non-activated naive T cells (sample d0-S1_280705). Global analysis of the gene expression showed that full naive T cell activation by anti-CD3/anti-CD28 and anti-CD3/IL-2 led to a significant upregulation of 76 and 71 genes, respectively (upregulation was considered to be significant if the relative gene expression was more than 5-fold than in the reference sample). However, the suboptimal anti-CD3 stimulus alone upregulated only 31 genes, the anti-CD28 stimulus alone only 6 genes, and the IL-2 stimulus alone only 22 genes. Naive T cells that reveived virus-modified vaccine cells alone upregulated 18 genes. Equally upregulated genes in anti-CD3/anti-CD28 and anti-CD3/IL-2 activated naive T cells comprised genes coding for both (i) signalling and (ii) effector molecules. Concerning signalling molecules, both costimuli induced the nuclear factor (NF)-kappa B and nuclear factor of activated T cells (NF-AT) pathway via the key enzymes protein kinase C (PKC)-theta and phospholipase C (PLC)-gamma 1, respectively. Of special note was that signalling molecules of the CD28-mediated stress-activated protein kinase (Sapk) pathway, namely Vav-1, Rac-1 and Jnk-2, were also upregulated by IL-2 costimulation. Upregulated genes coding for effector molecules could be classified into different functional groups: namely regulators of apoptosis, cell adhesion, receptors/ligands and cytokines/chemokines. Some examples of effector molecule-coding genes similarly upregulated in anti-CD3/anti-CD28 and anti-CD3/IL-2 stimulated naive T cells, respectively, were for instance the cell adhesion molecules LFA-1 and CD2, or members of the TNF and TNF receptor superfamilies such as CD27, CD70, CD40 ligand, Fas ligand, as well as TNF-alpha and TNF-beta (and their corresponding receptors). Among apoptosis-related genes, the anti-apoptotic gene bcl-XL as well as the pro-apoptotic gene caspase 9 were upregulated. The latter – together with the observed upregulation of cytotoxic T lymphocytes-associated protein (CTLA)-4 – might be interpreted as an indication of cellular events occurring seven days after stimulation which are aiming at termination of the response. Furthermore, the microarray analysis also revealed and upregulation of the receptor for interferon (IFN)-gamma inducible protein of 10 kDa (IP-10), namely CXCR3, of the IL-12 receptor beta-chain and of the IL-15 receptor alpha-chain; these are often seen in close association with T cell activation in general, with T cell proliferation and with generation of memory T cells. It is also of interest to mention the upregulation of beta-actin which is required for a re-organization of the cytoskeleton and the 40-fold upregulation of the metabolic enzyme glyceraldehye-3-phosphate dehydrogenase (GAPDH) which provides ATP necessary for the increased metabolism of the activated cells. A summary of the interesting gene subset is attached as a supplementary table (including the pairwise compared normalized data). In summary, these data suggest a synergism and cross-talk between the two costimulatory pathways mediated by anti-CD28/CD28 and IL-2/IL-2 receptor, respectively. Keywords: Human naive T cells, activation, costimulation, anti-CD28, IL-2, signal transduction, tumor vaccine, immunocytokine
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2008-12-01 | E-GEOD-12218 | biostudies-arrayexpress
Project description: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 µg/ml anti-CD3 and either anti-CD28 or anti-CD46. Purity was assessed by flow cytometry and was in all cases ⥠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.
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