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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.

Periodic Reporting for period 2 - 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.)

Reporting period: 2020-02-01 to 2021-07-31

Although the TCR occupies a central place in T cell physiology, it does not work in isolation and the signals it triggers are tuned by several other surface receptors that deliver positive (costimulators) and negative (coinhibitors) informations about the state of activation of antigen-presenting cells (APC). Therapeutic antibodies (immune-checkpoint inhibitors) blocking coinhibitors have become standard treatment for metastatic melanoma, leading to a revival in the study of T cell coinhibition and costimulation. We lack a satisfying comprehension of the way T cells integrate inputs from multiple signalling pathways and use inter-pathway crosstalk to make informed decisions. By combining advanced mass spectrometry and computational tools, we recently described in a time-resolved and quantitative manner the dynamics of the protein signaling complexes (signalosomes) that assemble in primary CD4+ T cells following physiologic T cell antigen receptor (TCR) engagement. This places us in a particularly favorable situation to investigate the crosstalk that exists between the signaling protagonists used by the TCR, the costimulators and the coinhibitors. Accordingly, the BASILIC project will tackle the challenging question of 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. The novelty of our proposal stems from (1) its use of primary T cells, (2) its capacity to probe the architecture and dynamics of signalosomes resulting from physiological T cell-APC encounters, (3) the possibility to benefit of recent breakthrough in proteomics, (4) the particular attention we pay to the stoichiometry of the corresponding signalosomes, an important parameter that has been largely ignored and even altered in most previous studies, and (5) its multidisciplinary nature that straddles molecular and organismal scales.
Deciphering how TCR signals are modulated by coinhibitory receptors is of fundamental and clinical interest. Using quantitative interactomics, we have already defined the composition and dynamics of the PD-1 and BTLA coinhibitory signalosomes in primary effector T cells and at the T cell-antigen-presenting cell interface. We also solved the existing controversy regarding the role of the SHP-1 and SHP-2 protein-tyrosine phosphatases in mediating PD-1 coinhibition. PD-1 predominantly recruits SHP-2, but when absent, it recruits SHP-1 and remains functional. In contrast, BTLA predominantly recruits SHP-1 and to a lesser extent SHP-2. By separately analyzing the PD-1-SHP-1 and PD-1-SHP-2 complexes, we show that both dampen the TCR and CD28 signaling pathways equally. Therefore, our study illustrates how comparison of coinhibitory receptor signaling via quantitative interactomics in primary T cells unveils their extent of redundancy and provides a rationale for designing combinations of blocking antibodies in cancer immunotherapy on the basis of undisputed modes of action. These results have been published in July 2019. Moreover, we are in the process of finalizing a key deliverable in which we show that quantitative interactomics in primary T cells unveils the extent and dynamics of TCR diversification.
Many gaps remain to gain a full understanding of T cell activation. Therefore, BASILIC is intended to provide a platform for generating new knowledge about key aspects of T cells with potentially major gains. For instance, the major goal of BASILIC is to understand how T cells integrate a wealth of signals originating from key receptors expressed at their surface to make informed decisions. The combination of our genetic, proteomics, computational and immunological approaches will create ideal experimental conditions for developing an integrated view of the complexity of adaptive immune responses at the molecular, cellular and organismal levels. Moreover, BASILIC is at the intersection of several lines of research and will impact on scientists investigating fundamental aspect of T cell biology. Owing to the present revival in the manipulation of T cells in clinical conditions, it will be of interest for clinicians. BASILIC will improve our understanding of the complexity of adaptive immunity with limited a priori assumptions using quantitative and comprehensive approaches, such as interaction proteomics, computational science, epigenomics and ‘genetic without crossing’ screens. Tumor infiltrating T cells are under multiple layers of negative regulation and only a subset of patients responds to current checkpoint inhibitors. Generating robust therapeutic immune responses also require the coincident delivery of costimulatory signals. As already illustrated by our first publication, the mechanistic data provided by BASILIC will improve the rational design of immunotherapies. Moreover, considering that we recently improved our capacity to probe lower abundant peptide species, we expect, in the course of BASILIC, to be able to characterize the signalosomes present even using the few pathogenic T cells that can be extracted from pathological tissue samples.
Quantitative Interactomics in Primary T Cells Provides a Rationale for Concomitant PD-1 and BTLA Coi