Close Contacts Unlocked: Nanopore-Stabilized Microvilli Bypass T Cell Receptor-Ligand-Dependent T Cell Activation.

The biophysical microenvironment critically shapes T cell activation, yet how nanoscale geometry regulates signaling remains poorly understood. Here, we demonstrate that microvilli insertion into nanopores robustly activates primary human T cells in the absence of TCR ligands, in a pore-size-dependent manner. Nanopores of ∼240 nm in diameter elicit strong ERK phosphorylation, Ca2+ influx, and NFAT nuclear translocation, reaching levels comparable to biochemical stimulation using antibodies against the TCR complex and CD28. Although TCR knockdown attenuates responses, residual CD69 expression upon nanopore engagement indicates that nanoscale confinement lowers the activation threshold. Perturbation of membrane mechanics with GsMTx4 or methyl-β-cyclodextrin, as well as disruption of extracellular Ca2+-dependent interactions by EDTA, markedly impaired signaling, implicating extracellular calcium and membrane organization as key regulators of signaling. Together, these findings support a model in which ∼240 nm-sized nanopores promote stable close-contact patches that seed TCR signaling. Finally, we show that nanoporous stimulation combined with CD28 costimulation activates patient-derived T cells comparably to conventional methods, highlighting a strategy with translational potential for immunotherapy.