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Molecular profiling of cultured CD8+ T cells from mice and treated with either IL2, IL2/SIIKFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

ABSTRACT: Molecular profiling of cultured CD8+ T cells from mice and treated with either IL2, IL2/SIIKFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

PROVIDER: PRJNA429800 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR6468350.fastq.gz	Fastqsanger.gz
	SRR6468360.fastq.gz	Fastqsanger.gz
	SRR6468370.fastq.gz	Fastqsanger.gz
	SRR6468351.fastq.gz	Fastqsanger.gz
	SRR6468361.fastq.gz	Fastqsanger.gz

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Transcriptomic profiling of cultured CD8+ T cells from mice and treated with either IL2, IL2/SIINFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

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Molecular profiling of cultured CD8+ T cells from mice and treated with either IL2, IL2/SIIKFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

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Transcriptomic profiling of cultured CD8+ T cells from mice and treated with either IL2, IL2/SIINFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

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Project description:T cell antigen-receptor (TCR) and cytokine receptor engagement trigger large changes in Serine/Threonine kinase signalling networks to drive T cell activation and differentiation. The role of only few kinase signalling pathways have been studied in detail, and in this context, Pim kinases are an interesting, yet understudied, family of Serine/Threonine kinases, with reported roles in key processes including survival, proliferation, metabolism across a range of cell types. T lymphocytes predominantly express PIM1 and PIM2, which are rapidly induced by TCR, costimulation and cytokine signalling. Using single shot DDA mass spectrometry we examine the impact of 24 hours treatment with two different pan-Pim kinase inhibitors PIM447 and AZD1208 on in vitro IL2 differentiated effector cytotoxic T lymphocytes. The Jak1/3 inhibitor tofacitinib was included as a control as this also blocks production of Pim kinases downstream of IL2. We find that treatment with pan-Pim kinase inhibitors has a similar phenotype to Pim1/Pim2 double deficiency, showing a reduction in proteins that are key for effector cell function: glucose transporters SLC2A1 and SLC2A3 and key effector molecule Granzyme B and an increase in the translational repressor PDCD4.

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ChIP-Seq of three H3 Acetylation epitopes in cultured CD8+ T cells from mice and treated with either IL2, IL2/SIINFEKL(TCR), IL2/IL4, or IL2/IL4/SIINFEKL(TCR)

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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.

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