While it is known that Granzyme B (GrzB) plays an important role for the cytotoxicity of NK cells, much less is known about the role of the other four human granzymes A, H, K, and M. To investigate this, we created fluorescent localization reporters to compare the activity of GrzB to the other four granzymes inside individual target cells upon NK cell-mediated killing. Our data show that NK92 cells kill targets mainly via GrzB with partial support of GrzA. In GrzB^-/- NK92 cells GrzA acts as a backup and can induce apoptosis on its own. GrzA/B^-/- NK92 cells show only death-receptor-mediated killing, suggesting that GrzH, K, and M are not involved in NK92-mediated cytotoxicity. Primary human NK cells also mostly rely on GrzB-mediated cytotoxicity, however, the activity of other granzymes is also detected inside target cells upon killing. GrzA activity is increased in targets killed by activated NK cells and may be capable of inducing cell death without the assistance of other granzymes. GrzK is mostly used by CD56-bright NK cells, however, these cells still kill predominately via GrzB.

We modified our fluorescent localization reporters to investigate how GrzB enters target cells upon NK cell-mediated killing. We predominantly found GrzB activity directly at the plasma membrane (PM) with very little activity at endosomes or the ER, supporting a model where GrzB enters directly via perforin pores at the PM. The amount of endosomal GrzB activity was dependent on the target cell, suggesting that membrane repair processes could influence this pathway of GrzB uptake. Interestingly, we did not observe any GrzB activity at mitochondria of target cells, suggesting that there is very little diffusion of GrzB from the PM into the cell before cell death is induced.