T cell degranulation and payload delivery are modulated in space and time

Release of destructive payloads through degranulation is a hallmark of T cell cytotoxicity. Upon stimulation, tens to hundreds of granules are rapidly delivered to the interface with a target cell but only a small number of degranulation events occur. Two forms of degranulation have been observed and differ in the physical form of the payload released. The deposition of insoluble particles encapsulated in a proteinaceous shell may be more potent than release of purely soluble factors. The selection mechanisms and triggers for degranulation are still poorly understood, partly due to the lack of tools to dynamically map and distinguish each mode. Here we use high spatiotemporal resolution in vitro assays, coupled to a novel label-free strategy to characterize and track released particles, to show that degranulation is regulated not only by chemical but also physical aspects of the activation inputs. Furthermore, the released, insoluble particles are transported within the intermembrane junction with a preferred directionality. Immobilized stimulatory ligands favored the soluble degranulation mode suggesting that the particulate release is pertinent to mobile, physiological immune interfaces. Engineered chimeric antigen receptor (CAR) T cells have a defect in centralizing released particles, which may explain dampened toxicity and off-target tissue damage. Our results demonstrate how degranulation events are likely regulated by a combination of physical, chemical, and signaling mechanisms. We anticipate these findings will generate new strategies to bias degranulation outcomes and optimize the destructive capacity of single cytotoxic T cells. In the case of engineered T cells, efforts to restore the normal transport of insoluble particles could contribute to stronger therapeutics for cellular immunotherapy.