Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Polysome profiling: Mouse OT-1 T cells WT vs Lck-/- (OFF)

ABSTRACT: In order to identify gene targets of translational regulation during T cell activation, a polysome analysis was performed in a T cell receptor (TCR) transgenic model (OT-1) possessing a homogenous alpha-beta CD8 T lymphocyte population. Cells with defective TCR signal tranduction (Lck-/-, OFF) were compared to WT cells to visualise targets regulated by TCR signalling. Total cytoplasmic ribonucleoprotein was extracted from ex vivo OT-1 transgenic T lymphocytes stimulated with SIINFEKL peptide antigen for 24h, extracts were fractionated using a sucrose density gradient, and separated into a sub-polysomal and a polysomal fraction. Total RNA from each fraction was extracted, equal volume of RNA from each fraction was individually labelled (Sub-poly: Cy3; Poly: Cy5), and hybridised onto a Agilent SurePrint G3 Mouse GE v2 8x60K Microarray.

ORGANISM(S): Mus musculus

PROVIDER: GSE84781 | GEO | 2016/11/30

SECONDARY ACCESSION(S): PRJNA331134

REPOSITORIES: GEO

ACCESS DATA

Json Xml

Dataset's files

Source:

			Action	DRS
		Other

Items per page:

1 - 1 of 1

Similar Datasets

Total RNA: 24h activated mouse OT-1 T lymphocytes, WT vs Lck-/- (OFF)

Project description:This experiment aims to study the transcriptome of activated T cells at 24h, using the same biological samples as the polysome profiling in GSE84781. A T cell receptor (TCR) transgenic mouse model (OT-1) posessing a homogenous alpha-beta CD8 T lymphocyte population was used, and cells with intact TCR signalling pathways (WT) were compared to those with defective signal transduction (Lck-/-, OFF). Ex vivo OT-1 T cells were isolated from the superficial cervicals, axillary, brachial, mesenteric, and inguinal lymph nodes and then activated for 24h with cognate peptide antigen SIINFEKL. Total RNA was extracted from the cells, labelled with Cy3, and hybridised onto an Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray.

2016-11-30 | GSE84856 | GEO

Analysis of the interactomes of ZAP70, CD247 and SLP76 upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR

Project description:T cells have the remarkable ability to sense and discriminate between a wide range of antigenic peptides bound to major histocompatibility complex molecules (pMHC). Here, we use interactomics to monitor in a time dependent manner the early molecular events taking place upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR. We used the mouse OT-I model in which T cells specifically recognize the ovalbumin OVA 257-264 peptide (SIINFEKL, also named N4) bound to the MHC-I molecule H-2Kb (pMHC). The OT-I TCR also recognizes altered peptide ligands amongst which SIITFEKL (T4) and SIIGFEKL (G4) with sub-optimal binding capacities (ligand affinity N4>T4>G4). For affinity purification (AP) of signalling complexes, we crossed OT-I TCR transgenic mice onto mice expressing an endogenous tagged (One-Strep-Tag: OST) version of the CD3z (also called CD247), ZAP70 or SLP76 molecules. CD8+ T cells were purified from pooled lymph nodes and spleens, shortly expanded, and were left unstimulated or stimulated with N4, T4 and G4 soluble pMHC tetramers for 30, 120 or 300 s. Complexes formed around either CD247, ZAP70 or SLP76 were then purified and analysed by mass spectrometry. Each AP-MS purification is associated with a corresponding control (purification from OT-I CD8+ T cells expressing only the endogenous version of each bait, cultured and stimulated in the same conditions). The number of replicate biological experiments was n=3 for all time-points, and each sample was analyzed twice by single-run nano LC-MS. In addition, the whole proteome of OT-I cell was analyzed by label-free shotgun proteomics in order to calculate cellular protein abundances for each interactor.

2022-07-05 | PXD029974 | Pride

Transcription profiling of mouse splenocytes from OT-1 TCR transgenic mice to assess the effect of IL-12 on CTL gene expression

Project description:The goal was to determine how IL-12 affects gene expression by murine CTL. Experiment Overall Design: Following RBC lysis, splenocytes from OT-1 TCR transgenic mice (5 million/ml) were cultured in six well-plates (5-6ml/well) in IMDM supplemented with 10% FCS and 55Î¼M 2-ME with 1Î¼M SIINFEKL. After 4 days, the cells were harvested by purification with Ficoll-Paque PLUS and placed in fresh medium containing 0 or 20ng/ml rmIL-12. Twenty-four hours later the cells were harvested and used in experiments. The live cells harvested were >98% CD8+VÎ±2+ (OT-1).

2009-02-08 | E-GEOD-13173 | biostudies-arrayexpress

Single-cell RNA sequencing of OT-I CD8+ T cells after stimulation with different affinity ligands

Project description:Activation of CD8+ T cells depends exquisitely on the affinity of the T cell receptor (TCR) for a peptide MHC (pMHC) ligand complex. Here, we activated OT-I transgenic CD8+ T cells with pure peptide and examined early activation responses by single-cell RNA-sequencing. T cells were activated with the high affinity OT-I cognate peptide (N4=SIINFEKL) for 1, 3 or 6 hours, or with reduced affinity peptides (T4=SIITFEKL and G4=SIIGFEKL) or the non-binding peptide (NP68=ASNENMDAM) for 6 hours. Cells were then sorted into 96-well plates by FACS and RNA was sequenced following an adapted Smart-Seq2 protocol.

2018-06-25 | E-MTAB-6051 | biostudies-arrayexpress

OT-I T cell activation in Lrrc8af/f and Lrrc8af/f Cd4-Cre;OT-I mice

Project description:Splenocytes were separated from Lrrc8af/f and Lrrc8af/f Cd4-Cre;OT-I mice. The splenocytes were stimulated with indicated peptides (unstime/ SIINFEKL-10pM(NL)/ SIINFEKL-1000pM(NH)/ SIIGFEKL-1000nM(G4)) for 6 hours. The OT-I CD8+ T cells were labeled with Va2-PE/CD8-A700 and sorted for the sequencing.

2023-09-26 | GSE243985 | GEO

Impact of secondary TCR engagement on the heterogeneity of pathogen-specific CD8+ T cell response during acute and chronic toxoplasmosis

Project description:Initial TCR-dependent engagement of CD8+ T cells results in T cell expansion, and the early events involved in priming influence the generation of diverse effector and memory populations. During infection, some of these activated T cells re-encounter cognate antigen, but whether this influences phenotypic heterogeneity is unclear. To address this issue, OT-I T cells, which are specific for the SIINFEKL peptide from OVA and express the Nur77-GFP reporter of recent TCR activation, were paired with transgenic T. gondii that express OVA to assess the impact of TCR activation on parasite-specific CD8+ T cell responses.

2021-11-11 | GSE188495 | GEO

Time-resolved phosphoproteomic analysis of OT-I CD8+ T cells stimulated with MHC-I tetramers loaded with N4 agonist peptide

Project description:T cell receptor (TCR) signaling is essential for the function of T cells. Here we monitor and quantify the dynamics of phosphorylation sites in OT-I CD8+ TCR transgenic T cells that have been stimulated with pMHC-I tetramers conjugated with the SIINFEKL (N4) peptide for 0, 15, 30, 120, 300 and 600 seconds. Cells were lysed in urea, proteins were digested into tryptic peptides, and phosphorylated peptides were enriched using titanium (TiO2) beads. A fraction of the eluate was analyzed by nanoLC-MS/MS, while the remaining sample was further enriched using phospho-Tyrosine (pTyr) immuno-precipitation. The eluate of the anti-pTyr immuno-precipitation was analyzed by nanoLC-MS/MS in a second serie of MS runs. We performed 3 independent experiments, each containing 6 time-points of stimulation. All samples were analyzed in triplicate MS runs. This dataset contains the raw LC-MS/MS files of the TiO2-enriched peptides (TiO2, 18 samples, 54 MS raw files), and the pTyr enriched peptides (Ptyr, 18 samples, 54 MS raw files).

2020-07-09 | PXD016583 | Pride

T cell receptor signal strength and antigen affinity differentially regulate memory CD8+ T cell development

Project description:IL-2 inducible tyrosine kinase (Itk) is a Tec family non-receptor tyrosine kinase that is known to regulate T cell receptor signal strength (TcR) and T cell development and differentiation. TcR signal strength and antigen affinity are parameters known to regulate the development of CD8+ T cell memory. However, the intersection between TcR signal strength and antigen affinity on CD8+ memory T cell development remains unclear. Therefore, we compared the transcriptomes of WT and Itk-/- OT-1/Rag-/- CD8+ T cells at day 0 and day 7 following infection with Listeria monocytogenes expressing the SIINFEKL (N4) or SIITFEKL (T4) OVA peptide variant (different affinities for TcR) by RNA sequencing.

2020-09-30 | GSE137406 | GEO

Time-resolved phosphoproteomic analysis of murine CD8+ T cells upon stimulation with ligands of different affinity to the TCR

Project description:T cells have the remarkable ability to sense and discriminate between a wide range of antigenic peptides bound to major histocompatibility complex molecules (pMHC). Here, we use phosphoproteomics to monitor in a time dependent manner the signalling cascade taking place upon stimulation of murine CD8+ T cells with ligands of different affinity to the TCR. We used the mouse OT-I model in which T cells specifically recognize the ovalbumin OVA257-264 peptide (SIINFEKL, also named N4) bound to the MHC-I molecule H-2Kb (pMHC). The OT-I TCR also recognizes altered peptide ligands amongst which SIITFEKL (T4) and SIIGFEKL (G4) with sub-optimal binding capacities (ligand affinity N4>T4>G4). CD8+ T cells were purified from pooled lymph nodes and spleens, shortly expanded, and were left unstimulated or stimulated with N4, T4 and G4 soluble pMHC tetramers for 30, 120, 300 or 600s. We conducted six independent multiplexed experiments, each containing either independent stimulations with the N4 and T4 peptides (N4-T4 experiments, 3 replicates) or with the N4 and G4 peptides (N4-G4 experiments, 3 replicates). Samples from each experiment were labeled by TMT10plex, pooled and subjected to phospho-enrichment using first TiO2 beads and then phospho-Tyrosine (pY) immuno-precipitation (IP). Samples were injected before TiO2 enrichment, after TiO2 and after TiO2 followed by pY IP for analysis of the proteome, serine/threonine phospho-proteome and tyrosine phospho-proteome, respectively.

2022-07-05 | PXD030080 | Pride

Total RNA: naïve ex vivo mouse OT-1 T lymphocytes, WT vs Lck-/- (OFF)

Project description:This experiment aims to study the transcriptome of naïve T cells at the steady state. A T cell receptor (TCR) transgenic mouse model (OT-1) posessing a homogenous alpha-beta CD8 T lymphocyte population was used, and cells with intact TCR signalling pathways (WT) were compared to those with defective signal transduction (Lck-/-, OFF). Naïve (CD8b+, CD44low) ex vivo T cells were isolated from the superficial cervicals, axillary, brachial, mesenteric, and inguinal lymph nodes. Total RNA was extracted from the cells, labelled with Cy3, and hybridised onto an Agilent-074809 SurePrint G3 Mouse GE v2 8x60K Microarray.

2016-11-30 | GSE84855 | GEO

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data