Effect of TGFb on TCR-induced OT-I T cell gene expression
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE205654 | GEO | 2022/06/10
REPOSITORIES: GEO
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Project description:Effect of TGFb on TCR-induced OT-I T cell gene expression
| PRJNA846784 | ENA
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:In mice and humans, an immature CD4-CD8- double negative (DN) thymocyte subset expresses early, mature αβTCR. These early αβTCR+ DN (EADN) cells are susceptible to leukemogenesis in both species. Using the OT-1 TCR transgenic system to model EADN-stage αβ TCR expression, we found that EADN cells show coreceptor-independent reactivity to MHC, and their TCR:MHC dependent signals can drive T-ALL transformation. We generated gene expression data from OT-1 leukemic cells and assessed their developmental stage by comparing to wild-type thymocyte subsets.
2022-06-28 | GSE180556 | GEO
Project description:The biological functions of histone demethylases Jmjd3 and Utx remain poorly understood. We assessed such functions in developing T cells, using conditional (CD4-Cre-mediated) gene disruption, by inactivating Kdm6a and Kdm6b, respectively encoding Utx and Jmjd3, in immature CD4+CD8+ thymocytes. We compared microarray gene expression in mature (Va2hi CD24lo) mutant and wild-type CD4+CD8- thymocytes carrying the OT-II TCR transgene. We show that Jmjd3 and Utx redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress. Mature (Va2hi CD24lo) CD4 thymocytes were sorted from freshly prepared single-cell suspensions OT-II TCR transgenic thymocytes deficient for Utx and Jmjd3 (dKO, CD4-Cre conditional deletion of floxed Kdm6a and Kdm6b alleles), and from Cre-negative controls (wild-type). Total RNA was extracted from sorted thymocytes using the RNeasy Plus Mini Kit (Qiagen) and processed for microarray analyses (Affymetrix Mouse Exon 1.0 ST array) at the NCI microarray facility, following the manufacturer’s recommendation. Data is generated from 3 replicates from each experiment.
2015-09-17 | E-GEOD-70363 | biostudies-arrayexpress
Project description:The biological functions of histone demethylases Jmjd3 and Utx remain poorly understood. We assessed such functions in developing T cells, using conditional (CD4-Cre-mediated) gene disruption, by inactivating Kdm6a and Kdm6b, respectively encoding Utx and Jmjd3, in immature CD4+CD8+ thymocytes. We compared microarray gene expression in mature (Va2hi CD24lo) mutant and wild-type CD4+CD8- thymocytes carrying the OT-II TCR transgene. We show that Jmjd3 and Utx redundantly promote H3K27Me3 removal at, and expression of, a specific subset of genes involved in terminal thymocyte differentiation, especially S1pr1, encoding a sphingosine-phosphate receptor required for thymocyte egress.
2015-09-17 | GSE70363 | GEO
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:Much is known concerning the cellular and molecular basis for CD8+ T memory immune responses. Nevertheless, conditions that selectively support memory generation have remained elusive. Here we show that an immunization regimen that delivers TCR signals through a defined antigenic peptide, inflammatory signals through LPS, and growth and differentiation signals through the IL-2R initially favors antigen-specific CD8+ T cells to rapidly and substantially develop into tissue-residing T effector-memory cells by TCR transgenic OVA-specific OT-I CD8+ T cells. Amplified CD8+ T memory development depends upon a critical frequency of antigen-specific T cells and direct responsiveness to IL-2. A homologous prime-boost immunization protocol with transiently enhanced IL-2R signaling in normal mice led to persistent polyclonal antigen-specific CD8+ T cells that supported protective immunity to Listeria monocytogenes. These results identify a general approach for amplified T memory development that may be useful to optimize vaccines aimed at generating robust cell-mediated immunity. Gene expression analysis was performed for OT-I T cells on day 3 and day 5 after activation with ovalbumin and LPS in vivo with and without treatment with IL-2 using an agonists IL-2/anti-IL-2 complexes (IL2/Jes-6.1) OT-I T cells were purified and adoptively transferred into congenic syngenic mice. 24 hours later mice were immunization with ovalbumin and LPS. 24 hr later some mice received agonist IL2/anti-IL2. 3 and 5 days after immunization, the activated OT-I T cells were purifed by FACS and total RNA was isolated for genome wide expression analysis using Affymetrix Mouse Gene ST1.0 arrays
2012-10-03 | E-GEOD-39110 | biostudies-arrayexpress
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: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
Project description:We report the application of TCRβ sequencing to understand T cell repertoire of matched blood and tumor samples pre and post treatment in EG7.OVA tumor bearing mice with OT-1 cell transfer. TCRβ sequencing demonstrated that OT-1 (CASSRANYEQYF) was the most abundant T-cell clone in both blood and tumors of mRBC‑OVA-4-1BBL-IL-12 treated mice. To investigate the effects of mRBC‑OVA-4-1BBL-IL-12 on immune memory, the T cell repertoire was also analyzed before and after EG7.OVA and EL4 tumor rechallenge in cured and treatment-naive mice. TCRβ sequencing on T cells in peripheral blood showed that OT-1 clones increased in frequency after EG7.OVA challenge in previously cured mice. OT-1 frequency did not increase in treatment-naïve mice after tumor challenge, indicating that the tumor alone is insufficient to drive OT-1 cell expansion. We also evaluated the frequencies of unique TCRβ sequences in T cell clones that significantly expanded post EL4 challenge (EL4 responsive TCR). Increased frequency of EL4-responsive TCRs upon each tumor challenge (EG7.OVA and EL4) was associated with complete responders (mice that rejected EL4 challenge), suggesting that T-cell-mediated protection against parental tumor antigens was generated prior to EL4 challenge. Partial responders (delayed tumor growth compared with naïve mice) had increases in EL4-responsive TCR frequencies after EL4 challenge but not during the EG7.OVA rechallenge, whereas the non-responder (tumor growth similar to naïve mice) had minimal increases in TCR frequencies upon EL4 challenge. Overall, the ability to control EL4 tumors correlated with the expansion of EL4-responsive TCR clones.
2021-03-13 | GSE168826 | GEO