OT-I T cell activation in Lrrc8af/f and Lrrc8af/f Cd4-Cre;OT-I mice
Ontology highlight
ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE243985 | GEO | 2023/09/26
REPOSITORIES: GEO
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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
Project description:Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. To identify transcriptional patterns and differentiation states induced by IL-6 in CD8 T cells, we performed RNAseq profiling of murine OT-I CD8+ T cells stimulated with SIINFEKL peptide in the presence of exogenous IL-6 (10 ng/ml) or anti-IL6R antibody (to block endogenous IL-6 signaling). Bulk OT-I splenocytes were stimulated with SIINFEKL peptide and CD8 T cells were FACS-sorted for RNA extraction after 2 and 7 days. We found that IL-6 potently repressed classical effector differentiation and promoted a gene expression pattern similar to that of memory precursor cells.
2022-11-04 | GSE199044 | GEO
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 55μM 2-ME with 1μM 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+Vα2+ (OT-1).
2009-02-08 | E-GEOD-13173 | biostudies-arrayexpress
Project description:OT-1 T-cells were co-cultured either alone in T-cell media supplemented with IL-2 (50IU/mL IL-2), with OVA loaded macrophages, or with both OVA loaded macrophages and CT2A-TRP2-β2mKO tumor cells. Cells were cultured at a 5:1 T-cell to tumor ratio, and 2:1 T-cell to macrophage ratio. After 24 h of co-culture, CD8 T-cells were FACS sorted, and RNA extracted (RNeasy Mini Kit, Qiagen). RNA was analyzed on an nCounter MAX Analysis System (Nanostring) with the PanCancer Immune profiling panel (Nanostring) according to manufacturer instructions. Expression dat OT-1 T-cells were isolated from OT-1 mice by culturing OT-1 splenocytes in T-cell media supplemented with 50IU/mL IL-2 and 1 μM OVA SIINFEKL peptide (Anaspec) for 48 h. Cells were purified for CD8 T-cells as described above and subsequently cultured in TCM with 50IU/mL IL-2, splitting every 24h for a total of 4 days.
2023-05-22 | GSE220960 | GEO
Project description:OT-1 RNA seq day36 MCMV-SIINFEKL vs RAE-1gMCMV-SIINFEKL
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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
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
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
Project description:Interleukin 6 (IL-6) is a pleiotropic cytokine with diverse roles in homeostasis, inflammation, and cancer. Here we performed transcriptional profiling of OT-I CD8+ T cells activated in the presence or absence of IL-6. In this study, splenocytes from wild type C57BL/6J mice were incubated with SIINFEKL peptide for two days to activate OT-I CD8+ T cells. SIINFEKL was then withdrawn, T cells were rested in medium with IL-2 for three days, and CD8+ T cells were restimulated with anti-CD3 + anti-CD28 antibodies and purified by FACS for RNA sequencing analysis on day 7. Throughout this culture period, some cells were treated with anti-IL6R antibody (clone MR16-1, 5 ug/ml), isotype control antibody (5 ug/ml), recombinant IL-6 (10 ng/ml), or recombinant hyper-IL-6 (IL-6 linked to IL6R; 20 ng/ml). Experimental conditions were run in triplicate, for a total of 15 samples. Conclusion: Neutralization of endogenous IL-6 signaling (with anti-IL6R antibody) enhances effector differentiation of CD8+ T cells, while addition of exogenous IL-6 or hyper-IL-6 suppresses effector differentiation.
2022-11-04 | GSE151157 | GEO
Project description: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.
2016-11-30 | GSE84781 | GEO