Cytotoxic lymphocytes play a key role in immune homeostasis by eliminating virus infected and transformed target cells through the perforin-dependent delivery of pro-apoptotic serine proteases, granzymes. How perforin and granzymes synergise to bring about target cell death remained a mystery.
Using biochemical approaches combined with extensive quantitative time-lapse microscopy of primary human natural killer cells engaging their respective targets, we defined the precise moment of perforin pore formation in the context of the physiological immune synapse. We showed that upon recognition of targets, calcium influx into the lymphocyte led to a rapid effector molecule exocytosis into the synaptic cleft. There, target cell membrane was disrupted by perforin pores, resulting in the rapid diffusion of secreted granzymes. We also observed, in real-time, a rapid repair of these pores: the process was initiated within 20 sec and completed by 80 sec, thus offering granzymes only a short window of opportunity for diffusion into the target. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into every target cell hit by perforin. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization.
A subsequent comparative analysis of mouse and human NK and CD8+ T cells revealed an extraordinary consistency in the delivery of the “lethal hit”, irrespective of the genetic background of the donor. Finally, we demonstrated directly in real time that perforin pore delivery was unidirectional, occurring exclusively on the target cell membrane, but sparing the killer cell. Despite this, the CTL membrane was not intrinsically perforin-resistant, as intact CTLs presented as targets to effector CTLs, were capable of being killed by perforin-dependent mechanisms.
This study defines the secretory granule death pathway as a remarkably efficient cytotoxic mechanism, where rapid and unidirectional delivery of the giant perforin pore opens a gateway for granzyme diffusion into cognate target cells thereby providing effective immune defense against pathogens and cancer.