Selective granzyme B replenishment dominates the molecular switch for sustained cytotoxicity in effector CD8+ T lymphocytes

Cytotoxic T lymphocytes (CTLs) are highly efficient multiple killer cells, yet how they sustain killing after the initial discharge of canonical lytic granules remains incompletely understood. Late-phase cytotoxicity is often attributed to death receptor signaling or cytokine-mediated mechanisms, but whether CTLs also remodel their intrinsic cytotoxic machinery during prolonged engagement remains unclear. Here, we show that human CTLs maintain cytotoxic resilience through selective remodeling of their lytic program, marked by preferential preservation of granzyme B (GzmB). Following acute degranulation and reduction of mature perforin, CTLs selectively replenish GzmB through de novo protein synthesis, whereas perforin and several other granule-associated components show limited restoration. Replenished GzmB becomes redistributed into vesicular compartments with reduced overlap with mature perforin and is delivered to successive targets during sustained cytotoxic responses. Under conditions of limited detectable mature perforin, CTLs continue to deliver enzymatically active GzmB, induce altered calcium responses in target cells, and support productive but kinetically heterogeneous killing. This GzmB-preserved effector state is maintained and further accentuated during engagement with resistant melanoma cells. Together, our findings identify selective GzmB renewal as a central feature of sustained CTL cytotoxicity and reveal an adaptive remodeling of the cytotoxic machinery that may support CTL resilience during prolonged immune responses.