T cell polarization and NFAT activation are stiffness dependent and differentially regulated by the channels PIEZO1...

T cell activation requires cell polarization and changes in gene expression. Target cell stiffness contributes to the activation of immune cells, and tumor cell softening is linked to cancer progression. We investigated how substrate stiffness influences T cell activation using functionalized, T cell-activating substrates of varying stiffness and softened target cells. Reorientation of the microtubule-organizing center (MTOC) toward the immunological synapse and nuclear translocation of the transcription factor NFAT1 were impaired on softer hydrogels or upon contact with softer target cells. The increase in intracellular Ca²⁺ induced by target engagement also depended on stiffness and was reduced on soft substrates. Stiffness-dependent Ca²⁺ signaling was crucial for both rapid (MTOC reorientation) and long-term (NFAT translocation) responses. Whereas MTOC reorientation depended on the mechanosensitive Ca²⁺-permeable channel PIEZO1, NFAT1 translocation depended on the Ca²⁺ channel ORAI1. Our results demonstrate that target stiffness directly influences MTOC reorientation and NFAT1 translocation in T cells, and these two processes are governed by different plasma membrane Ca²⁺ channels, indicating that these stiffness-regulated rapid and long-term responses can be decoupled. Our findings imply that tumor cell stiffness regulates T cell functionality and suggest that pathways regulated by PIEZO1 and ORAI1 might differentially control rapid and long-term responses to stiffness in other cell types.