Measurement, inhibition, and killing mechanisms of cytotoxic granule serine proteases

Abstract

Natural killer (NK) cells and cytotoxic T lymphocytes (CTL) are critical for the protection of organisms against pathogens and cancer. The process by which these cells eliminate infected or transformed cells are through two basic mechanisms, receptor-mediated interactions, or delivery of contents from intracellular cytotoxic granules. Granules are comprised of perforin and a family of serine proteases, called granzymes. Upon entry into target cells, these proteins work together to initiate cellular death pathways. Previous and extensive biochemical studies had already established that granzyme B (GrB) was a powerful inducer of apoptosis, but sensitive assays to confirm its release from cytotoxic cells were lacking. We hypothesized that GrB release, measured by ELISPOT, directly assessed the lytic potential of antigen-specific cytotoxic cells. Indeed, data provided in this thesis established a strong correlation between GrB release and target cell lysis. Our results imply that GrB could be a promising tool to assess cell-mediated immunity during vaccine development. However, several other independent studies in grB-/- mice demonstrated that additional granzymes were capable of clearing viruses and tumorigenic cells. Granzyme H (GrH) is highly and constitutively expressed in human NK cells, and therefore, we hypothesized that it was also an effective cytotoxic molecule. Our experiments established that GrH-induced cell death by a mechanism distinct from those of GrB and Fas. We identified a GrH substrate, DFF45/ICAD, and showed that GrH induced mitochondrial damage through a Bid-independent mechanism. Furthermore, cell death was dependent on Bax and/or Bak, but independent of caspase activation. Hence, we have elucidated an alternative cytotoxic pathway that could be employed to eliminate target cells with immune evasion strategies targeted to GrB or Fas. Finally, control of serine proteases by endogenous inhibitors is important to numerous biological processes, including apoptosis. We hypothesized that as GrH displayed chymase activity, the serine protease inhibitor anti-chymotrypsin (ACT) would impair GrH function. Our data established that ACT effectively attenuated GrH cytotoxicity and prevented proteolysis of a GrH substrate. Collectively, this thesis describes a novel GrH inhibitor, provides a new tool to evaluate cell-mediated immunity, and provides evidence of an alternative mechanism of cytotoxicity.