CTLANDROS · Reactive Oxygen Species in CTL-mediated Cell Death: from Mechanism to Applications

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells release granzyme and perforin from cytotoxic granules into the immune synapse to induce apoptosis of target cells that are either virus-infected or cancerous. Granzyme A activates a caspase-independent apoptotic pathway and induces mitochondrial damage characterized by superoxide anion production and loss of the mitochondrial transmembrane potential, without disrupting the integrity of the mitochondrial outer membrane; while causing single-stranded DNA damage. GzmB induces both caspase-dependent and caspase-independent cell death. In the caspase-dependent pathway, mitochondrial functions are altered as evidenced 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. To date, research on mitochondrial-dependent apoptosis has focused on mitochondrial outer membrane permeabilization (MOMP) however whether the generation of ROS is incidental or essential to the execution of apoptosis remains unclear. Like human GzmA, human GzmB promotes cell death in a ROS-dependent manner. Preliminary data suggest that human GzmB can induce ROS in a MOMP-independent manner as Bax and Bak double knockout MEF cells treated with human GzmB and perforin still display a robust ROS production and dye in an ROS-dependent manner. Since GzmA and GzmB induce cell death in a ROS-dependent manner, we hypothesize that oxygen free radicals are central to the execution of programmed cell death induced by the cytotoxic granules. Therefore, the goal of this proposal is to dissect the key molecular events triggered by ROS that lead to Citotoxic Tcell-induced target cell death. A combination of biochemical, genetic and proteomic approaches in association with Electron Spin Resonance (ESR) spectroscopy methodology will be used to unravel the essential role ROS play in CTL-mediated killing.