IN THE COMPANY OF KILLERS—INDUCTION OF APOPTOSIS BY CYTOTOXIC LYMPHOCYTES
The apoptosis of peripheral T lymphocytes is mirrored by the ability of cytotoxic lymphocytes (CL) to induce apoptosis. CD8+ cytotoxic T lymphocytes (CTLs) and natural killer (NK) cells are capable of inducing apoptosis by both the intrinsic and extrinsic pathways described above.45 Targets of CL are those that are transformed (such as cells with downregulated expression of MHC class I) and cells that present appropriate “foreign” antigen in the context of MHC I for deletion by CD8+ CTLs.
CD8+ CTLs remove unwanted or dangerous cells by inducing apoptosis by one of two known mechanisms: a calcium-dependent mechanism mediated by proteins contained within cytolytic granules of the CTL, namely, the concerted action of perforin and members of the granzyme family of serine proteinases; or a triggering of the apoptotic program in target cells by CTLs' death ligand, Fas-ligand, by engagement of the Fas death receptor expressed on the surface of the target cell. Each of these triggers engages the caspase protease cascade at some point to effect the morphological changes that we observe as apoptosis, but each engages the cascade at different points of the killing process. Based on in vitro studies, it has long been assumed that only the Ca2+-dependent mode of killing is dependent on interactions between CTL TCR and MHC class I-dependent presentation of corresponding antigen by the target cell. Fas-ligand-mediated apoptosis is viewed as a more “promiscuous” form of CTL-induced killing; this is based on the observation that many cell types that express Fas-ligand acquire the ability to kill Fas-bearing targets without the need for antigen presentation.41-42 However, in CTLs, Fas-ligand may be compartmentalized in cytolytic granules and, therefore, it may not be deployed until favorable TCR engagement occurs.
Upon engagement of the TCR, specialized CTL granules containing granzymes, perforin, and Fas-ligand migrate toward the site of contact between the two cells.46 Following the migration of granules to the site of contact, exocytosis of granule contents into the intercellular space between killer and target and uptake of granzymes, along with the activity of perforin, results in the generation of all biochemical features of apoptosis commonly studied in vitro. The kinetics of death is very rapid, within 0.25 to 1.7 h. The Ca2+-dependent aspect of CTL killing enlists the concerted actions of granzymes and perforin to engage the intrinsic apoptosis pathway characterized by mitochondrial disruption, release of cytochrome c and Smac/DIABLO, and activation of the caspase-9 containing apoptosome.
Granzymes make up a family of serine proteases expressed primarily, but not exclusively, by CTL after activation. Granzymes B and A are the principal family members believed to be responsible for causing target cell apoptosis. Granzyme B, the only known serine protease with caspaselike substrate specificity, is capable of engaging the caspase-dependent death pathway at several points, including cleavage and activation of Bid and caspase-3. However, recent reports suggest that direct activation of caspase-3 by granzyme B requires the additional regulatory step in which inhibition of caspase-3 by XIAP must be relieved by Smac/DIABLO, which must be released from mitochondria.47,48 Granzyme B preferentially cleaves and activates the proapoptotic Bcl-2 family member Bid,49-51 which acts with Bax or Bak to mediate the release of mitochondrial proteins. A recent report suggests that Bax and Bak are not necessary for granzyme B-induced apoptosis,52 which points to the possibility of multiple granzyme B-mediated effects.
Granzyme A is another constituent of cytotoxic granules of CTL. Granzyme A, when delivered with perforin, can induce target cell death, albeit with delayed kinetics and via different biochemical processes from granzyme B.
CTL lacking granzyme B induced a delayed target cell apoptosis (approximately 12 h) that is attributed to granzyme A.53 These data suggest that some compensation may occur at the level of target cell clearance, but they also indicate that the biochemical properties of the pathways engaged by each granzyme differ greatly. Whereas granzyme B engages the caspase pathway, albeit downstream of mitochondrial events, granzyme A acts in a manner independent of intracellular death proteases.54 Known substrates of granzyme A are associated with a large multimeric complex known as the SET complex (reviewed in Lieberman and Fan55). It was proposed that granzyme A-mediated cleavage of the SET complex proteins may actually result in a phenotype more closely resembling necrosis than apoptosis.56 In this model, generation of double-strand DNA breaks would result in activation of the DNA repair machinery, including poly-ADP ribose polymerase (PARP) rather than disabling it, as is often observed in apoptosis. Cleavage of PARP by caspase-3 is viewed as a hallmark of late-stage apoptosis.Given that necrotic cells and apoptotic cells have different effects on APC activation,11,12 it is possible that the mechanism by which a cell dies (i.e., granzyme B, granzyme A, or death ligands) will impact target cell clearance and have downstream consequences on immune cell activation and regulation. There is a strong possibility that the manner in which a target cell dies will also affect the way in which it becomes engulfed and stimulates cytokine production and activation of antigen presenting cells. Thus, the biochemical aspects of death triggered by components of CTL will contribute to an immunosuppressive or inflammatory milieu.