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

ABSTRACT: 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.

INSTRUMENT(S): Orbitrap Fusion

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Primary Cell, T Cell

SUBMITTER: Anne Gonzalez de Peredo

LAB HEAD: Odile Schiltz

PROVIDER: PXD030080 | Pride | 2022-07-05

REPOSITORIES: Pride

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

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