1. GENERAL INTRODUCTION

1.1. Cytotoxic lymphocytes and apoptosis

The immune system of high-order organisms is a highly specialized compartment that eliminates transformed cells and cells infected with viruses or bacteria through a controlled process of cell-mediated cytotoxicity. The immune cells responsible for mediating cell death are collectively called cytotoxic lymphocytes (CLs) and are made up of natural killer (NK) cells and cytotoxic T lymphocytes (CTL). CLs are distinguished primarily by their respective mechanism of antigen recognition. NK cells form part of the innate immune response, a generalized first line of defense. NK cells are generally CD3–CD56+ lymphocytes that recognize and respond to abnormal cells through an imbalance of facilitatory and inhibitory receptors (Bottino et al., 2004; Moretta et al., 2004). CTLs form part of the adaptive immune response, a more specific response that is generated subsequent to and as a consequence of the innate response. These cells use their clonotypic T-cell receptors (TcRs) to recognize a peptide antigen presented on the major histocompatability complex (MHC) proteins on the surface of the target cell. CTLs can be identified on the basis of expression of CD3 and CD8 (CD3+CD8+ ) on their cell surface. In addition, some CD4+ T cells (typically T-helper cells) can have limited cytotoxic capacity.

Although NK cells and CTLs recognize their targets through different receptors, both can kill their targets by one of two specific and directed processes: ligation of death receptors or granule exocytosis (Cohen et al., 1985; Lowin et al., 1994). NK cells primarily use the granule exocytosis pathway, whereas CD8+ CTLs typically use the granule pathway to kill virus or transformed cells, although not exclusively. CD4+ CTLs can also use either

pathway, depending on their subtype (Table 10-1). The purpose of this chapter is to provide an outline of the granule exocytosis pathway to cell death, as receptormediated apoptosis is outlined in detail elsewhere.

2. CYTOTOXIC GRANULES AND GRANULE EXOCYTOSIS

The idea of a granule exocytosis pathway was first formulated after the observations of effector/target conjugates seen under the electron microscope. It was seen that on conjugate formation, cytoplasmic granules in CLs became reoriented to the area of cell:cell contact (Bykovskaja et al., 1978), and pores were then observed to form on the target cell membrane (Dourmashkin et al., 1980). These observations brought about the hypothesis that pre-stored cytotoxic proteins could be released by the CLs in a vectorial fashion toward the target cell surface after antigen recognition (Dennert and Podack, 1983). Since then, it has become clear that CLs contain unique lysosome-like compartments that directly secrete their contents toward a target cell (Burkhardt et al., 1990; Yannelli et al., 1986). These compartments have aptly been named secretory granules and, when purified, have been shown to exhibit both membranolysis and apoptosis in a dose-dependent manner, with no particular target cell specificity.

Similar to secretory granules (or secretory lysosomes) from other hematopoietic cells, granules in CLs contain a uniform electron dense core surrounded by a thin cortex of membrane lamellae (Burkhardt et al., 1989). In common with other lysosomes, they have a low pH and harbor typical degradative lysosomal proteins; however, secretory granules have a dual function and also house specialized proteins involved in programmed cell death, which can be secreted in a regulated fashion (Bossi and Griffiths, 2005; Peters et al., 1991; Smyth et al.,

Table 10-1. Cytotoxic mechanism used by di erent lymphocyte subsets

Cytotoxic mechanism

Source: This table is adapted from a similar table in (Trapani, 1998).

2001). The proapoptotic proteins, including perforin and granzymes, have been shown, by means of colloid gold staining, to localize to the electron dense core, possibly by association with a proteoglycan, chondroitin sulfate (Burkhardt et al., 1989; Stevens et al., 1987). The low pH provides a favorable environment for the lysosomal hydrolases and protects the CLs from the action of the proteins involved in apoptosis that require a neutral pH for optimum activity (Persechini et al., 1989; Voskoboinik et al., 2005).

To effectively kill their targets by granule exocytosis, the death-inducing proteins of the cytotoxic granules (perforin and granzymes) must be delivered from the CL into the target cell. This is a multistage process involving

(1) synthesis and loading of the granule proteins into the secretory granules; (2) formation of an immunological synapse between the effector and target cell; (3) granule trafficking within the effector cell; (4) secretion of granule proteins into the immunological synapse; (5) their uptake into the target cell; and finally, (6) activation of death pathways in the target cell.

2.1. Synthesis and loading of the cytotoxic granule proteins into the secretory granules

Although both perforin and granzymes are constitutively expressed in NK cells, naive T lymphocytes do not express these cytotoxic proteins, nor are secretory granules found in their cytoplasm (Bou-Gharios et al., 1988; Olsen et al., 1990). On TcR engagement, an increase in intracellular calcium initiates signaling cascades that mediate the transcription of various lysosomal and proapoptotic proteins trafficked to the secretory granules, as well as lysosomal transmembrane proteins that help mediate cell signaling (Esser et al., 1998; Gray et al., 1987). Protein synthesis occurs within hours of TcR triggering and is accompanied by T-cell division and maturation and the appearance of secretory granules in the cytoplasm typically by 12 to 48 hours

(Bou-Gharios et al., 1988; Olsen et al., 1990; Podack and Kupfer, 1991).

Granzymes are processed like many proteases and are transported through the endoplasmic reticulum (ER) and Golgi as pre-pro proteins, where a signal peptide piece is removed. Granzymes are targeted to the lysosomes through the M6R pathway; however, an M6R-independent pathway also exists, as patients with I cell disease (a deficiency of enzyme-mediated mannose phosphorylation) still have active granzymes in their secretory granules (Griffiths and Isaaz, 1993; Masson et al., 1990). Within the secretory granules, an N-terminal acidic activation di-peptide is removed by DPP1/cathepsin C (Pham et al., 1996). The lymphocytes of cathepsin C-null mice have therefore been proposed to totally lack granzyme B (grB) activity and perforindependent cytotoxicity (Pham and Ley, 1999). Surprisingly however, cells targeted by allogeneic CD8+ CTL raised in cathepsin C-null mice can still die through perforin and grB-dependent apoptosis, albeit at a reduced rate (Sutton et al., 2007). Thus at least one other granule protease is capable of processing pro-grB.

Perforin is initially synthesized in the ER, and after cleavage of a 21-amino acid pro-piece, an intermediate form of perforin is glycosylated with complex glycan in the Golgi. In the secretory granules, an unknown cysteine protease is thought to cleave approximately 20 amino acids at the C-terminus (with the attached glycan), resulting in acquisition of lytic activity (Uellner et al., 1997). Although granzymes and perforin are stored in their active form in the secretory granules, their enzymatic function is restricted by the acidic pH of the granules, providing protection for the CL. Thus, once a CL becomes conjugated with a target cell, it is able to induce death almost immediately, because perforin and granzymes are active once they encounter the neutral extracellular pH.

2.2. The immunological synapse

Once CTL differentiation/activation has occurred, T cells recognize/interact with their targets by TcR engagement of antigen presented on MHC and form a tight seal between the effector and target cell. This junction is known as the immunological synapse (IS) (Stinchcombe and Griffiths, 2003). A mature synapse contains a specific outer ring of membrane-bound proteins mediating cell– cell adhesion and enclosing other proteins involved in signaling cascades required for protein synthesis and cell activation (Monks et al., 1998). This tight seal may prevent leakage of the cytotoxic granule proteins and facilitate their vectorial delivery to the target cell. The IS also

contains a unique secretory domain, which is the region within which secretory granules exocytose their cytolytic proteins (Stinchcombe et al., 2001). Although granules are maintained at an acidic pH, the environment in the IS has not been formally characterized (with respect to the pH and calcium concentration). It therefore remains unclear precisely how the environment of the IS supports the function of granule proteins secreted into this domain (Stinchcombe and Griffiths, 2007).

2.3. Secretion of granule proteins

CLs do not exocytose their granules randomly; granules are directed specifically to the IS. Further, in an activated CTL, the IS can form within minutes of TcR stimulation (Grakoui et al., 1999; Stinchcombe et al., 2001), and only transient interaction with target cells may take place. The process of granule redistribution from the posterior to the leading edge of an effector cell (polarization) and their exocytosis is therefore a rapid, directed, and coordinated process (Kupfer and Dennert, 1984; Yannelli et al., 1986). It is unclear how many granules are released into the synapse, although it has been suggested that not all the granules are exocytosed, with some remaining in the effector cell to allow for the serial killing often seen with an individual CL (Stinchcombe et al., 2001).

Before secretory granule movement, the microtubule organizing center (MTOC) and Golgi compartment are redeployed to the point of contact between the two cells (Kupfer and Dennert, 1984; Kupfer et al., 1985). Secretory granules cluster around the MTOC by moving along microtubules by means of kinesinand dynein-based motors (Kamal and Goldstein, 2000). From the MTOC, secretory granules dock at the plasma membrane, fuse, and release their cytolytic proteins into the IS (Stinchcombe et al., 2004). More recently, granules have been shown to be delivered directly to the plasma membrane, a process that is believed to be dependent on centrosome placement at the plasma membrane, in particular at the central supramolecular activation cluster of the IS (Stinchcombe et al., 2006).

The various proteins involved in secretory granule migration, membrane docking, fusion, and subsequent secretion have been identified by examining the genetic defects underlying patients suffering from diseases of the secretory granules, such as Chediak-Higashi syndrome, Griscelli syndrome, Hermansky-Pudlack syndrome, and familial hemophagocytic lymphohistiocytosis (FHL) as is reviewed by Stinchcombe et al. (2004). Proteins such as Lyst, Rab27a, Munc13–4, and syntaxin 11 have all been identified to play a role in efficient

secretory granule transport to the IS (Menager et al., 2007; Stinchcombe and Griffiths, 2007).

2.4. Uptake of proapoptotic proteins into the target cell

Once released into the IS, cytolytic molecules must make their way into the target cell, and the mechanism by which this occurs remains one of the most controversial areas of granule-mediated killing. Originally, electron microscopy analysis of a killer/target conjugate showed close association of cytotoxic granules with the target membrane, suggesting that the granule contents of CL could be directly responsible for forming transmembrane channels (Dennert and Podack, 1983). Purified granules from CL showed a very high hemolytic and tumoricidal activity in the presence of calcium at 37◦C and at neutral pH, compared with whole intact cells from which granules were derived (Criado et al., 1985; Podack and Konigsberg, 1984), and the cytolytic activity in these purified granules was eventually attributed to the 66kDa protein, perforin (Masson and Tschopp, 1985). Generation of perforin-deficient mice confirmed the essential role for perforin in granule-mediated cell death (Kagi et al., 1994).

Purification of perforin revealed that it could polymerize and insert in lipid bilayers (Masson and Tschopp, 1985; Podack et al., 1985; Young et al., 1986), making

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pores with an internal diameter of approximately 160 A (16 nm). It was thus proposed that the perforin pore could trigger lysis by disrupting osmotic homeostasis or stimulate a calcium-regulated processes of internal disintegration by altering intracellular calcium flux (Duke et al., 1989; Kraut et al., 1990). However, various lines of evidence suggested that CL-induced death was distinct from perforin lysis. CL-induced death was shown to involve fragmentation of DNA into oligonucleosomalsized fragments, by a process that was explicitly dependent on granzymes, in particular grB (Shi et al., 1992). Importantly however, granzyme-dependent cell death is not evident unless perforin is present (Hayes et al., 1989; Shiver and Henkart, 1991). Therefore, perforin must function as a vehicle for the efficient delivery of granzymes into the apoptotic pathways of the target cell.

Originally, it was believed that the perforin pore acted simply as a conduit for granzyme diffusion into the cell; however, more recent studies have indicated a more complex process (Keefe et al., 2005; Trapani et al., 1998b). First, it was shown that grB could enter the target cell in an energy-dependent process without the need for perforin, but remained compartmentalized in endosomes and did not kill the target cell (Froelich