Immunity, Granzymes and Cell Killing

Abstract

Cytotoxic lymphocytes protect us from viral infection and cancer by directly killing tumour cells or cells harbouring a virus. One crucial mechanism they use to kill their targets is known as the ‘granule exocytosis’ pathway. This involves secretion of a potent mix of toxins, resulting in transfer of granule proteases (granzymes) from the killer cell into the target cell, where they cleave various intracellular substrates to activate diverse signalling pathways to cell death. Access to the target cell cytosol depends on a pore‐forming protein toxin, perforin. Because granule exocytosis is a crucial part of the body's natural defence against such dangerous cells, understanding how granzymes kill their targets may yield novel strategies and identify new molecular targets for anticancer or antiviral therapies. Indeed, recent advances in cancer immunotherapy such as checkpoint blockade and adoptive transfer of chimeric antigen receptor (CAR) T cells all ultimately rely on activating the granule exocytosis pathway to kill cancer cells.

Key Concepts

  • The critical role of cytotoxic T lymphocytes and natural killer cells is to eliminate abnormal cells, thereby defending higher organisms against intracellular infection.
  • An important additional function of cytotoxic lymphocytes also encompasses their role in surveillance against transformed cells.
  • Perforin and granzymes synergise to kill target cells. Perforin is critical to this process and its role is to enable granzyme proteases to access key substrates in the target cell.
  • Perforin functions by punching large pore‐like defects in the target cell membrane, permitting direct granzyme diffusion into its cytosol.
  • Granzyme B is the most potent proapoptotic granzyme, as it shares with caspases the capacity to cleave its substrates after key aspartate residues.
  • Cytotoxic lymphocytes have developed a plethora of different signalling pathways to bring about target cell death. This is critically important as viruses are adept at blocking cell death to maximise their opportunity for viral replication and spread to uninfected cells.
  • These granzyme‐dependent cell death pathways are mechanistically distinct and arranged in a hierarchy in terms of their potency and kinetics.
  • Apart from inflicting cell death, granzymes have additional functions such as directly interfering with viral replication and in cytokine processing, leading to amplification of the inflammatory response to pathogens.
  • Dysregulation of cytotoxic T‐cell function can sometimes lead to adverse effect such as autoimmune tissue damage.

Keywords: granzyme; cytotoxic T lymphocyte; cell death; perforin; NK cell

Figure 1. Cytotoxic lymphocytes kill their targets through granule exocytosis, and granzyme B initiates apoptosis via two main pathways. Granzyme B cleaves Bid to induce MOMP and subsequent release of cytochrome c from the mitochondria results in caspase activation and ‘classic’ apoptosis. However, granzyme B may also activate caspases directly (particularly in the mouse) and can also cleave many other substrates directly, potentially leading to caspase‐independent death. Granzyme B also shows species variation in substrate specificity, with human granzyme B preferentially cleaving Bid, and mouse granzyme B favouring direct caspases cleavage.
Figure 2. Mouse NK (natural killer) cells kill their targets via diverse pathways. Mouse NK cells were incubated with plastic‐adherent mouse embryonic fibroblasts (MEF) at a 2:1 ratio for 2 h. The NK cells were then washed off, and the target cells fixed, permeabilised and stained for cytochrome c. The cells were then visualised by confocal microscopy for morphological changes and cytochrome c localisation. MEF cells showed rounding and blebbing consistent with apoptosis and released cytochrome c from the mitochondria indicating that MOMP has occurred. Although the apoptotic phenotype was being blocked by the caspase inhibitor zVAD‐fmk, clonogenic survival was not rescued (data not shown) indicating that caspase‐independent death was taking place.
Figure 3. Granzymes cleave many substrates to initiate diverse pathways to cell death. Of the granzyme family, granzyme B is well characterised and initiates apoptosis of target cells (Figure). The other granzymes have also been shown to induce target cell death when applied directly to target cells in vitro; however, the morphology of cell death is not classically apoptotic and does not involve caspase activation. Although these other granzymes cleave a diverse range of substrates, granzymes A, C, K and M also lead to reactive oxygen species generation as a key feature. Many of the noncaspase substrates include critical cytoskeletal proteins and DNA (deoxyribonucleic acid) repair proteins, cleavage of which is implicated in cell death.
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Trapani, Joseph A(Jan 2017) Immunity, Granzymes and Cell Killing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0021982.pub2]