T lymphocytes are essential cells of the immune system, that specifically recognize a wide range of pathogens, but also malignant cancer cells in the body. In order to develop therapeutic approaches based on the modulation of the immune response, it is important to finely characterize their molecular mechanisms of action. Using high-throughput methods, a research team from IPBS-Toulouse (CNRS / Université Toulouse III-Paul Sabatier), in collaboration with immunologists from the CIML in Marseille, systematically identified, quantified, and characterized the thousands of phosphorylation sites that get “switched on” in primary T lymphocytes during the first 10 min of activation. These results are published in the journal Molecular Systems Biology on July 3rd 2020.
The T Cell receptor (TCR) is the entry point of a series of molecular events underlying T cell activation. Located at the lymphocyte cell surface, it recognizes a variety of major histocompatibility complex‐associated antigenic peptides, thereby encoding extracellular signals into an adaptive immune response. Upon binding to these antigens, it triggers a signaling cascade based on the rapid and successive phosphorylation of many intracellular proteins. This small chemical modification takes place on specific sites of the proteins, and promotes their conformational change, their association with other partners, and the activation of protein kinases that in turn also phosphorylate other protein targets. Upon TCR engagement, signaling is initiated by the phosphorylation of the CD3 protein chains, located at the cell membrane, by the LCK kinase. This provides docking sites for the ZAP70 kinase, which itself becomes phosphorylated and recruits key adaptors proteins such as LAT and SLP‐76, allowing signal diversification through the mobilization of additional proteins with diverse enzymatic activities This ultimately promotes the activation of T cells, which undergo morphological changes, synthetize new proteins, and secrete cytokines that activate other types of immune cells.
Recent advances in mass spectrometry and bioinformatics analysis now allow a better characterization of the signaling pathways based on protein phosphorylation. Using high-throughput phosphoproteomic methods, researchers from the “Proteomics and Mass Spectrometry of Biomolecules” group headed by O. Schiltz at IPBS, could monitor the phosphorylation kinetics of around 7,000 phosphosites during the first 10min following TCR engagement in primary T lymphocytes. This study provided a global, dynamic, and detailed picture of all the molecular events induced and propagated upon TCR activation. New players of this pathway could be identified such as the ITSN2 protein, that was further characterized and shown to regulate T‐cell effector functions by controlling TCR surface down‐regulation upon antigenic stimulation.
All this data was integrated in the LymphoAtlas database. To facilitate the exploration of this data set by the scientific community, a Web application that interactively displays phosphorylation kinetics of selected sites was created. By quantitatively capturing changes in phosphorylation dynamics, LymphoAtlas provides a rationale to the identification of therapeutic targets to advance immunotherapies based on T‐cell reprogramming.
Mapping of a subset of TCR‐regulated phosphosites on the canonical TCR signaling network. Phosphosites on proteins are color‐coded according to their kinetic of phosphorylation during the first 10 minutes of activation.
This work was funded by by the CNRS, the University Toulouse III-Paul Sabatier, the Ministry of Research, Higher Education and Innovation, the National Research Agency through the French National Infrastructures for Proteomics (ProFI) and for mouse Phenogenomics (PHENOMIN), and the European Research Council.
LymphoAtlas: a dynamic and integrated phosphoproteomic resource of TCR signaling in primary T cells reveals ITSN2 as a regulator of effector functions. Locard-Paulet M*, Voisinne G*, Froment C, Goncalves Menoita M, Ounoughene Y, Girard L, Gregoire C, Mori D, Martinez M, Luche H, Garin J, Malissen M, Burlet-Schiltz O, Malissen B, Gonzalez de Peredo A** and Roncagalli R**. Mol Syst Biol. 2020 Jul; 16(7):e9524. DOI: 10.15252/msb.20209524
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