Our recent structural studies have definitively shown that perforin monomers polymerize to form a range of pores, the majority having internal diameters of 120 to 170 Å, which easily allows for trans-lumenal diffusion of granzyme molecules, typically ∼50 Å in diameter. How perforin enables the delivery of granzymes into the cytosolic compartment of the target cell is a fundamentally important immunological process but has remained largely unresolved, despite significant research efforts. 9 Overall, perforin is essential for CL function in the immune surveillance of viral infections and cancer. 7 Humans deficient in functional perforin develop familial hemophagocytic lymphohistiocytosis (FHL), 8 and recent work suggests some individuals with incomplete loss of perforin function are predisposed to hematologic cancers. As a result, perforin-deficient mice are unable to clear many viral infections, and this is manifested as major immune dysregulation these mice also fail to reject transplanted tumors and develop spontaneous B-cell lymphomas as they age. Consistent with this, perforin-deficient CLs are unable to kill target cells through the granule pathway. 4 Although granzyme B applied to target cells alone can be internalized through endocytosis, it remains inert and unable to reach its cytosolic substrates 5, 6 perforin’s key function is thus to enable granzyme delivery into the cytosol to initiate apoptosis. Upon activation by a target cell, the CL triggers the exocytosis of secretory granules containing perforin and the proapoptotic serine proteases, including granzyme B, into the synaptic cleft, resulting in target cell death. 1-3 It is stored in the secretory granules of cytotoxic lymphocytes (CLs), which seek out and bind virus-infected or transformed target cells and form a transient immunologic synapse. Perforin is a pore-forming member of the membrane-attack-complex-perforin (MACPF) family of proteins. This study defines the final sequence of events controlling cytotoxic lymphocyte immune defense, in which perforin pores assemble on the target cell plasma membrane, ensuring efficient delivery of lethal granzymes. Rapid initiation of apoptosis was evident from caspase-dependent target cell rounding within 2 minutes of perforin permeabilization. Remarkably, even such a short time frame was sufficient for the delivery of lethal amounts of granzymes into the target cell. Repair of these pores was initiated within 20 seconds and was completed within 80 seconds, thus limiting granzyme diffusion. Within the synaptic cleft, target cell permeabilization by perforin resulted in the rapid diffusion of extracellular milieu–derived granzymes. We show that, on recognition of targets, calcium influx into the lymphocyte led to perforin exocytosis and target cell permeabilization in as little as 30 seconds. Using biochemical approaches combined with time-lapse microscopy of human primary cytotoxic lymphocytes engaging their respective targets, we defined the time course of perforin pore formation in the context of the physiological immune synapse. However, the mechanism of granzyme entry into cells remains unresolved. Cytotoxic lymphocytes serve a key role in immune homeostasis by eliminating virus-infected and transformed target cells through the perforin-dependent delivery of proapoptotic granzymes.

Scaffold protein perforin