Dynamic modulation of cortical actin at the immunological synapse controls lytic granule secretion in cytotoxic T lymphocytes

Abstract

Abstract CD8+ cytotoxic T lymphocytes (CTLs) are critical for eliminating virally infected cells. Upon target recognition, CTLs reorganize their actin cytoskeleton to facilitate the polarized secretion of specialized granules containing lytic proteins that kill cells. Because a single CTL can kill multiple targets, granule secretion must be tightly regulated, ensuring only appropriate cells are killed, and preserving granules for serial killing. However, how CTLs regulate secretion remains unclear. Here, we used live confocal and TIRF microscopy to evaluate how cortical actin affects granule secretion at the membrane. While previous work has shown that clearance of actin at the synapse precedes granule secretion, we find that after granule fusion, cortical actin recovers and no further secretion is observed. Depolymerization of recovered actin led to resumed granule fusion, suggesting that recovered actin acts as a barrier to prevent further lytic granule secretion. We further show that CTLs from mice unable to secrete granules due to a Rab27a mutation that prevents granule fusion, failed to recover cortical actin at the synapse. This suggests that granule fusion itself triggers actin recovery in CTLs. Thus, cortical actin both regulates, and is regulated by, secretion. Finally, using fluorescent reporters, we correlated the clearance and recovery of actin with PIP2. PIP2 binds numerous actin regulatory proteins, suggesting phosphatidylinositol distribution in the membrane as a potential mechanism through which CTLs regulate the density of cortical actin during killing. Our work provides insight into actin-related mechanisms regulating CTL secretion that may serial killing capacity during immune responses.