Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells vectorially release perforin (PFN) and granzymes (Gzms) into their tumoral or virally infected target cells in order to induce apoptosis. Perforin, a pore forming protein, delivers granzyme serine proteases into the target cell cytosol where GzmA or GzmB by themselves can independently activate cell death although some viruses or tumors can be resistant to either pathway.

GzmB induces both caspase-dependent and caspase-independent cell death. The mitochondria play a key role in this pathway. Indeed the mitochondrial functions are altered as shown by the loss of mitochondrial transmembrane potential and the generation of reactive oxygen species (ROS). The mitochondrial outer membrane (MOM) is disrupted, resulting in the release of apoptogenic factors such as cytochrome c, HtrA2/Omi, endonuclease G, Smac/Diablo and apoptosis inducing factor (AIF) that are otherwise located in the mitochondrial intermembrane space. To date, the research on mitochondrial-dependent apoptosis has focused on mitochondrial outer membrane permeabilization (MOMP), leaving unclear whether the generation of ROS is incidental or essential to the execution of apoptosis.

We have shown that GzmA triggers a new rapid mitochondrial death pathway characterized by a robust ROS production and loss of the mitochondrial transmembrane potential independently of MOM disruption; consequently there is no release of apoptogenic factors from the mitochondria. GzmA-induced mitochondrial ROS and more specifically the superoxide anions (O2.-) is required for granule-mediated cytotoxicity since superoxide scavengers completely abrogate cell death induced by both GzmA loading with perforin or CTL attack by CTL expressing both GzmA and B. These results suggesting that the GzmB pathway is also ROS-dependent.

The focus of our Laboratory is to investigate the function of the ROS in CTL killing. We use a combination of cell biology, genetic, biochemistry, proteomics and electron spin resonance spectroscopy to characterize the ROS and their molecular target during cytotoxic lymphocytes mediated cell death.

The figure shows Hela cells treated with PFN and GzmA. To and T5 minutes of incubation are shown (left and right panel respectively). The mitochondria are in green and superoxide anions flash in red.