Understanding the mechanism of altered cell signalling due to differential activation of ZAP-70 in T cells

SenGupta, Soumee (2026) Understanding the mechanism of altered cell signalling due to differential activation of ZAP-70 in T cells. PhD thesis, Indian Institute of Science Education and Research Kolkata.

Text (PhD thesis of Soumee SenGupta (20RS036)) 20RS036.pdf - Submitted Version Restricted to Repository staff only Download (14MB)

Abstract

The T cell antigen discrimination represents one of the most rigorously controlled cellular responses. A kinetic proofreading mechanism mediates T cell receptor signalling which is calibrated in response to the antigen-binding kinetics, thereby facilitating distinct self versus non-self antigen recognition. The recent breakthroughs suggest ionic flux of intracellular monovalent cations like Na⁺ and K⁺ are the key regulators of T cell effector functions and metabolism. Upon antigen interaction, T lymphocytes respond to the proximal T cell receptor signalling by influx of Na⁺ and Ca²⁺ along with subsequent efflux of cellular K⁺. Imbalance of such ionic flux in the tumor microenvironment renders the tumor- infiltrating T lymphocytes non- functional. Though the disparity of both the monovalent cations functions by altering cellular metabolism and T cell receptor signalling, elevated [K⁺] in the tumor microenvironment suppresses the T-cell effector functions while high intracellular [Na⁺] counterintuitively enhances the same. Here, we presented a molecular mechanism of how the two monovalent cations regulate the functional output of T lymphocytes by modifying the interactions of the T cell receptor and Zeta Chain Associated Protein Kinase 70 (ZAP70), key modulator of proximal T cell receptor signalling. We show how T cells are sensitive to high [K⁺] that disrupts the allosteric coupling of the two tSH2 domains of ZAP70 necessary for ligand binding. Our work delineates a proof-of-concept that fluctuating intracellular potassium levels correlates with T cell activity. Elevated potassium concentrations in T-cells prevents T-cell receptor signalling by impairing ZAP70 recruitment to T-cell receptor. Recreating these elevated potassium conditions in mice model showed impaired T-cell thymic developmental and regulatory functions and manifested symptoms similar to the SKG variant (ZAP70w¹⁶³c) murine model of autoimmune arthritis caused by improper T-cell receptor signalling in thymocytes. While antigen-discrimination lies central to T-cell response, our comprehensive in vitro and in vivo studies delved into the mechanisms of how ionic imbalance uncouples antigen binding and ZAP70-dependant downstream signalling. Identifying the critically affected kinetic proof-reading steps, we sought to engineer the ZAP70 kinase to generate a potassium-insensitive kinase. Altering the ligandbinding regulations of ZAP70 in the early T-cell receptor signalling cascade drives the signalling towards a more tunable response, offering plausible opportunities for controlled immunomodulation in the tumor microenvironment.

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