T cell polarization and NFAT translocation are stiffness-dependent and are differentially regulated by Piezo1 and Orai1

Effective T cell responses against tumor cells require diverse effector functions including polarization towards tumor cells to form immunological synapses and nuclear factor of activated T-cells (NFAT)-dependent gene transcription. While the role of tumor cell softening has been associated with malignancy, stemness, and metastasis, potentially contributing to immune evasion, its impact on cellular processes in T cells is not well understood. Here, we show that both T cell polarization and NFAT nuclear translocation are modulated by target stiffness in a Ca ²⁺ dependent manner. Using both anti-CD3 antibody-functionalized substrates with varying stiffness as surrogates for target cells or softened tumor cells, we found that both, reorientation of microtubule organizing center (MTOC) towards the tumor cells, a hallmark for T cell polarization, and NFAT translocation were impaired on softer hydrogels or following contact with softer cancer cells. The amplitudes of intracellular Ca ²⁺ signals were dependent on stiffness, and removal of extracellular Ca ²⁺ inhibited stiffness-dependent T cell responsiveness. While stiffness-dependent Ca ²⁺ signaling was crucial for both, T cell polarization and NFAT translocation, Ca ²⁺ influx through Piezo1, a mechanosensitive ion channel, mediated stiffness-dependent MTOC reorientation but not NFAT translocation. In contrast, Ca ²⁺ influx through store-operated Orai channels mediated NFAT translocation but not MTOC reorientation. Our results demonstrate that tumor cell stiffness directly influences T cell functionality through distinct Ca ²⁺ influx pathways, revealing cell softening as an essential mechanism employed by malignant cells to evade immune surveillance.