Characterization of the T Cell Receptor (TCR) pathway in primary T lymphocytes
|One of our research areas is to decipher, at the cellular and molecular levels, the functional mechanisms of T lymphocytes using proteomic approaches. In collaboration with teams bringing complementary expertise in immunopathology and T cell signaling (B. Malissen, CIML, Marseille; R. Lesourne, CPTP, Toulouse; A. saoudi, CPTP, Toulouse), we are involved in the detailed characterization of the T Cell Receptor (TCR) pathway, by analyzing the signalosome of several key components of this complex system using AP-MS strategies, or the signaling pathways engaged upon activation of T-cells using phosphoproteomics.
In collaboration with B. Malissen and R. Roncagalli (CIML, Marseille), we use KI mouse model expressing different bait proteins of the TCR pathway with a C-terminal One-Strep-tag to enrich endogenous signaling complexes at different time points following TCR activation. Time resolved proteomics allows to obtain dynamics information and to gain insight into the mechanisms of action of several proteins and the internal organization of the interactomes (1-4).
1-Reginald et al. Revisiting the Timing of Action of the PAG Adaptor Using Quantitative Proteomics Analysis of Primary T Cells. J Immunol 2015.
2-Voisinne et al. Co-recruitment analysis of the CBL and CBLB signalosomes in primary T cells identifies CD5 as a key regulator of TCR-induced ubiquitylation. Mol Syst Biol 2016.
3. Gaud et al. The costimulatory molecule CD226 signals through VAV1 to amplify TCR signals and promote IL-17 production by CD4+T cells. Sci. Signal. 2018.
4. Voisinne et al. Quantitative interactomics in primary T cells unveils TCR signal diversification extent and dynamics. Nat. Immunol, in press.
AP-MS workflow for analysis of the TCR signalosome. We analyze by mass spectrometry signalling complexes that form around canonical proteins used by the proximal TCR signal transduction pathway, and monitor their dynamic of assembly in the first minutes following engagement of the TCR. For that, we use genetically engineered mice models, which express at endogenous level a bait protein with a OneStreptag at their C-terminus, allowing the purification of physiological complexes, directly from primary cells, in a very efficient way.
We could identify a global interaction network involving more than 200 unique proteins and more than 300 high-confidence protein-protein interactions.