Periodic Reporting for period 3 - BASILIC (Decoding at systems-level the crosstalk between the T cell antigen receptor, the CD28 costimulator and the PD-1 coinhibitor under physiological and pathological conditions.)
Okres sprawozdawczy: 2021-08-01 do 2023-07-31
Although the T cell antigen receptor (TCR) occupies a central place in the activation of T lymphocytes, it does not work in isolation and the key activation signals it triggers are tuned by several other receptors expressed at the T cell surface that deliver positive (costimulators) and negative (coinhibitors) signals that reflect the state of activation of antigen-presenting cells. We still lack a satisfying understanding of the way T cells integrate inputs from such multiple signaling pathways and use inter-pathway crosstalk to make informed decisions. To address the challenges described above, the Basilic ERC-ADG project combined mouse functional genomics and novel mass spectrometry and computational tools. At its conclusion, the project resulted in 11 publications that lead (1) to the quantitative description of the composition and dynamics of the protein signaling complexes (signalosomes) that assemble in mouse and human primary CD4+ T cells following physiologic TCR engagement, (2) to unravel the way T cell co-inhibitory receptors (PD-1, BTLA, and HVCR2) inhibit the activating signals delivered by the TCR and the CD28 co-stimulatory pathways, (3) to decipher the kinetic proofreading mechanisms through which the multi-step activation of the ZAP-70 protein tyrosine kinase underlies early T cell ligand discrimination, (4) to elucidate the molecular and cellular drivers of an autoimmune and type 2 inflammatory disorder that develops in mice with a loss-of-function mutation in the LAT signalosome, a key signaling hub of the TCR signaling pathway, and (5) to demonstrate using scRNAseq and functional genomics that the LAT signalosome pathology described under (4) constitutes a genuine preclinical model of a human inflammatory and autoimmune disorder called IgG4-related disease.
During the period covered by the Basilic ERC-ADG project, we have combined mouse functional genomics, advanced mass spectrometry and computational tools to describe (1) in a time-resolved and quantitative manner the dynamics of the protein signaling complexes (signalosomes) that assemble in mouse and human primary CD4+ T cells following physiologic T cell antigen receptor (TCR) engagement, (2) the way T cell co-inhibitory receptors (PD-1, BTLA, and HVCR2) inhibit the activating signals delivered by the TCR and the CD28 co-stimulatory pathways, (3) showed that kinetic proofreading through the multi-step activation of the ZAP70 kinase underlies early T cell ligand discrimination, and (4) elucidated using scRNAseq and functional genomics the etiology of the LatY136F immunological disorder and showed that in constitutes a novel model of a human inflammatory and autoimmune disorder called IgG4-related disease. During the Basilic ERC-ADG project, we further developed a unique reverse genetics approach that constitutes a novel decision support tool permitting to identify in 4 months and without mice breeding candidate genes the loss-of-function of which yield T cell phenotypes of interest at organismal levels. Moreover, the results of the Basilic ERC-ADG study illustrates the importance of distinguishing protein-protein interactions that occur at physiological levels and that do not disrupt the subtle stoichiometry of intracellular signalling complexes from those that are possible experimentally in conditions of overexpression or of disrupted cellular architecture. They also showed that by permitting pairwise comparison of co-inhibitory signalosomes in primary T cells, quantitative interactomics unveils whether they elicit redundant inhibitory signals, and helps deciding if a given pair of coinhibitory receptors has to be preferred over another during the design of combination of immunotherapeutic agents. Importantly we developed a novel integrated approach to prioritize and reduce cost and time to translate therapies targeting T cells into the clinic. Accordingly, the novel fast-track platform we developed during the Basilic ERC-ADG project uses Cas9/sgRNA RNP nucleofection and non-viral Homology-Directed Repair DNA templates renders primary human CD4+ and CD8+ T cells rapidly amenable to AP-MS. It allows to define at systems-level the composition, dynamics and stoichiometry of the human proximal TCR-signal transduction network and chart its similarities and differences between human CD4+ and CD8+ T cells and across the human and mouse species. In conclusion, the whole Basilic ERC-ADG project has proceeded on time and without rectification. In terms of result exploitation and dissemination, 11 publications have been published most of them in highly recognized journals. The results of the Basilic ERC-ADG have been also presented in close to 40 seminars given in Europe, Asia and the USA.
Many gaps remain to gain a full understanding of T cell activation. Accordingly, the BASILIC ERC-ADG project was intended to understand how T cells integrate a wealth of signals originating from key receptors expressed at their surface to make informed decisions. These receptors consist of the T cell antigen receptor (TCR), co-stimulatory molecules such as CD28 and co-inhibitory molecules such as PD-1 and CTLA-4. The results of the BASILIC ERC-ADG project are thus of direct clinical relevance since tumor infiltrating T cells are under multiple layers of co-inhibitor regulation and only a subset of patients responds to current checkpoint inhibitors. The BASILIC ERC-ADG project went beyond the state-of-the-art and provided the most comprehensive systems-level analysis yet on the composition, stoichiometry and dynamics of the proximal TCR signal-transduction network in primary human T cells. Our study revealed that the proximal TCR signal-transduction network has a high degree of qualitative and quantitative conservation across human and mouse T cells. The possibility to render primary human T cells rapidly amenable to quantitative interactomics also permitted to precisely define whether a drug acts upstream or downstream of a given signalosome of the human TCR signal transduction network. Therefore, the fast-track system-levels analysis of human T cell signaling pathways described in the BASILIC ERC-ADG project permit to optimize CAR T cell signaling and evaluate at systems-level the mechanisms of action and possible side effects of drugs targeting human T cell activation prior to translating them into the clinic. In line with its medical relevance, the BASILIC ERC ADG also elucidated the etiology of the LatY136F immunological disorder and showed that it constitutes a genuine preclinical model of a human inflammatory and autoimmune disorder called IgG4-related disease. In conclusion the BASILIC ERC ADG project went well beyond the state-of-the-art in that it constitutes a molecular foundation permitting to provide a rational for the function of co-inhibitory and co-stimulatory molecules that are the targets of therapeutic antibodies. Moreover, it permits to evaluate at systems-level the mechanisms of action and possible side effects of drugs targeting human T cell activation prior to translating them into the clinic.