Entry mechanism of natural killer cell derived granzyme B into target cells
Loading...
Date
2025
Authors
Journal Title
Journal ISSN
Volume Title
Publisher
Alternative Title(s)
Abstract
During elimination of virally infected or malignant cells, NK cells release lytic granules. These contain perforin and granzyme B (GrzB), which synergistically trigger apoptosis of affected cells. However, despite intensive research, the mechanism by which GrzB traverses membrane barriers to gain access to the target cell cytosol is controversial. Two main entry models have been described: I) perforin pores in the plasma membrane (PM) act as passageways for GrzB to allow direct diffusion into the cytosol or II) target cells internalize GrzB via endocytosis followed by a release from endosomes. Yet, mutual exclusivity is not necessarily a given; both pathways could also support each other. To investigate how GrzB enters target cells during NK cell-mediated cytotoxicity, we developed fluorescent localization reporters that can detect GrzB activity in living cells. We can localize these reporters at the PM, the endoplasmic reticulum (ER), or other intracellular compartments of target cells. Therefore, it becomes possible to assess the GrzB entry site over time using live cell imaging. Using the NK cell line NK92 or different human primary NK cells as effectors and HeLa cells as targets, we could observe a predominant GrzB activity at the PM of target cells; but low GrzB activity was also detected at the ER. This could indicate either a mutual supportive role of both pathways or diffusion of GrzB from the PM to the ER. However, we did not observe GrzB activity at mitochondria arguing against general diffusion of GrzB. By reducing the amount of active perforin with concanamycin A (CMA), we were able to show that the preferential entry through PM pores is independent of the available amount of perforin. However, in the absence of perforin, no GrzB activity was detectable in target cells, confirming the essential role of perforin for GrzB to enter target cells. Taken together, our results demonstrate that during NK cell-mediated killing GrzB enters HeLa directly via the PM. However, we found differences between target cells. K562 were almost exclusively killed without GrzB, while we found rare events in MDA-MB #468 cells where GrzB was predominantly delivered via the endocytic pathway, which may be enhanced by altering membrane tension.
Description
Table of contents
Keywords
Natural killer Cell, NK cell, GrzB, Granzyme, Entry mechanism, Cytotoxicity
Subjects based on RSWK
Killerzelle