Pore formation at the lytic synapse triggers the canonical pyroptotic cell death pathway

Antigen-specific cytotoxic T lymphocytes ( CTLs ) kill targets through rapid release of perforin/granzymes into the lytic synapse. Lethal hit delivery activates programmed cell death in the target, which is classically considered apoptotic, caspase-mediated, and granzyme-dependent. Herein, we propose an unprecedented role for perforin in triggering target cell pyroptosis, complementing its well-documented role as a conduit for granzymes. Utilizing imaging and molecular approaches, we demonstrate that target perforation upon CTL attack elicits swift K ⁺ efflux, triggering the canonical inflammatory pyroptotic signaling cascade defined by activation of the NLRP3 inflammasome, caspase-1, and the pore-forming pyroptotic executioner, gasdermin D ( GSDMD ); this is followed by pyroptotic body formation, plasma membrane rupture, and release of intracellular contents. Disruption of perforin inhibited this pathway, while recombinant perforin activated caspase-1-dependent pyroptosis. These results reveal that perforin retains the pro-pyroptotic activity of ancestral pore-forming toxins, and highlight a previously unappreciated role for the canonical caspase-1/GSDMD pyroptotic pathway in adaptive immunity.