Endocytosis of cytotoxic granules in CD8 T lymphocytes is required for multiple killing of target cells (IRM14P.448)

Abstract

Abstract Cytotoxic (CD8+) T lymphocytes (CTLs) eliminate tumorigenic and virus-infected target cells by releasing the contents of cytotoxic granules (CGs) at the immunological synapse. Even though CTLs have only a limited number of pre-existing fusogenic CGs, they can efficiently kill multiple target cells upon activation. We therefore hypothesized that, following CG fusion, the remaining components are recycled and subsequently refilled with cytotoxic substances to become available for the next round of fusion, thus enabling efficient multiple killing. We visualized endogenous CGs and their recycling in real-time in CTLs from synaptobrevin2-mRFP knock-in mice by fluorescently-labeled anti-RFP antibodies. We found that endocytosis (recycling) of CGs was blocked by Dynasore, an inhibitor of dynamin. Additionally, clathrin was colocalized with endocytosing CGs at the plasma membrane following dynasore pretreatment. Thus, endocytosis of CGs is clathrin- and dynamin-dependent. Importantly, although CTLs pre-treated with Dynasore showed no immediate effect on CG exocytosis, they exhibited a 50% deficiency in killing target cells after 2 hours of incubation. Our data strongly suggest that recycling of CGs plays an important role in CTLs for executing multiple killing of target cells.