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(Journal of Leukocyte Biology. 2001;70:18-29.)
© 2001 by Society for Leukocyte Biology

Unlocking the secrets of cytotoxic granule proteins

Mark J. Smyth*, Janice M. Kelly*, Vivien R. Sutton*, Joanne E. Davis*, Kylie A. Browne*, Thomas J. Sayers{dagger} and Joseph A. Trapani*

* Cancer Immunology Division, Trescowthick Laboratories, Peter MacCallum Cancer Institute, Melbourne, Australia; and
{dagger} Laboratory of Experimental Immunology, National Cancer Institute, FDR-DC, NIH, Frederick, Maryland

Correspondence: Mark J. Smyth, Cancer Immunology Division, Trescowthick Laboratories, Peter MacCallum Cancer Institute, Locked Bag 1, A’Beckett St, 8006, Melbourne, Australia. E-mail: m.smyth{at}pmci.unimelb.edu.au


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ABSTRACT
 
Cytotoxic lymphocytes largely comprise CD8+ cytotoxic T cells and natural killer cells and form the major defense of higher organisms against virus-infected and transformed cells. A key function of cytotoxic lymphocytes is to detect and eliminate potentially harmful cells by inducing them to undergo apoptosis. This is achieved through two principal pathways, both of which require direct but transient contact between the killer cell and its target. The first, involving ligation of TNF receptor-like molecules such as Fas/CD95 by their cognate ligands, results in mobilization of conventional, programmed cell-death pathways centered on activation of pro-apoptotic caspases. This review concentrates on the second pathway, in which the toxic contents of secretory vesicles of the cytotoxic lymphocyte are secreted toward the target cell, and some toxins penetrate into the target cell cytoplasm and nucleus. In addition to invoking a powerful stimulus to caspase activation, this "granule-exocytosis mechanism" provides a variety of additional strategies for overcoming inhibitors of the caspase cascade that may be elaborated by viruses. The key molecular players in this process are the pore-forming protein perforin and a family of granule-bound serine proteases or granzymes. The molecular functions of perforin and granzymes are under intense investigation in many laboratories including our own, and recent advances will be discussed. In addition, this review discusses the evidence pointing to the importance of perforin and granzyme function in pathophysiological situations as diverse as infection with intracellular pathogens, graft versus host disease, susceptibility to transplantable and spontaneous malignancies, lymphoid homeostasis, and the tendency to auto-immune diseases.

Key Words: apoptosis • lymphocyte • granzyme • perforin • granulysin


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THE GRANULE EXOCYTOSIS MODEL
 
Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells play a key role in immune responses toward virus-infected and malignant cells [1 ]. The granule exocytosis model describes one pathway by which these effector cells act upon target cells (reviewed in [2 3 4 5 6 7 ]). Recognition and binding between a cytotoxic lymphocyte and its target lead to vectorial exocytosis of specific cytoplasmic granules toward the target cell [5 6 7 ]. Delivery of this "lethal hit" is known to occur via proteins contained within the granules [8 9 10 ], many of which interact with or enter the target cell. This brings about cell death typified by cytosolic and nuclear-apoptotic changes. These cytotoxic granules are quite complex organelles that not only contain pro-apoptotic proteins restricted to CTL and NK cells [11 , 12 ] but also others ubiquitously expressed within lysosomes [11 ]. Thus, cytotoxic lymphocytes package secretory and lysosomal proteins within a specialized, cytotoxic organelle, which serves as a secretory lysosome (Table 1 )


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  		Table 1. Contents of Cytotoxic Granules of CTL and NK Cells


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DENSE CORE GRANULE COMPONENTS— A SUMMARY
 
The dense core of a cytotoxic granule contains the proteins responsible for target cell destruction [11 ]. These include perforin (pfp; otherwise termed pore-forming protein or cytolysin), calreticulin, and many serine proteases or granzymes (granule-associated enzymes; grz) [11 , 13 , 14 ], which are complexed with chondroitin sulfate-rich proteoglycans. Other than calreticulin, the expression of these proteins is largely restricted to activated CTL and NK cells [13 , 14 ].

Pfp
Pfp is a pore-forming protein with patchy homology to the C9 complement component [15 ] and is synthesized as a 70-kDa inactive precursor, which is cleaved at the C-terminus to yield a 60-kDa active form. This processing occurs in an acidic compartment (pH 5.5), and agents such as concanamycin A that disrupt granule acidification prevent pfp processing [16 , 17 ]. Cleavage of the pro-piece occurs at the boundary of a synaptotagmin-like C2 domain at the pfp carboxyl terminal; this domain is then capable of binding the plasma membrane and initiating pore formation in a calcium-dependent manner [16 ]. The clipped pro-piece is short (12–20 residues) but contains a bulky glycan attached at 528Asn. Its removal permits pfp monomers to undergo a conformational change in the presence of calcium ions, which allows binding of phosphorylcholine groups on membrane lipids and coalescence with other pfp molecules to form poly(pfp) pores [16 , 18 19 20 ]. Pfp insertion into the target cell membrane is a stimulus that amplifies the endocytic uptake of other granule constituents and their delivery into the target cell cytosol. Thus, now it seems that the formation of large transmembrane pores is not necessary for uptake of other granule constituents [21 ]. However, the specific molecular basis for this process is not well understood.

Granulysin
Granulysin is a member of the saposin family of lipid-binding proteins [22 23 24 ]. Although functionally related to other anti-bacterial peptides, defensins and magainins, granulysin is structurally distinct. It is active against a broad range of microbes, including Gram-positive and Gram-negative bacteria, fungi, and parasites. Two major protein products of 15 and 9 kDa are encoded by the granulysin gene. Recombinant 9-kDa granulysin disrupts artificial liposomes and cell membranes, damages mitochondria, and activates caspase-9 to induce apoptosis in nucleated cells [25 ]. Identification of this molecule indicates a broader and perhaps more significant role for killer cells in innate and acquired anti-microbial defenses. Thus far, granulysin has been shown to kill extracellular Mycobacterium tuberculosus directly by reducing its membrane integrity and to decrease the viability of intracellular M. tuberculosus by a pfp-dependent mechanism [26 ].

Grz
Grz are serine proteases belonging to the chymotrypsin superfamily [27 , 28 ]. The major features that define this family of serine proteases have been reviewed previously [29 , 30 ]. The crystal structure of grzB has been solved now, and the overall similarity between grz and chymotrypsin, verified [31 ]. Quite a number of grz and their genes have been characterized in mouse, rat, and human CTL and NK cells (Table 2 ) These can be divided into three subfamilies on the basis of their gene structure (indicating their evolutionary relationships), proteolytic specificity, and biological functions. GrzH is the product of a gene fusion unique to humans [32 ] and is the only known human grz with chymase activity [33 ]. As yet, human counterparts of mouse grzC to -G have not been isolated. All grz are produced as preproproteins, the "pro" segment usually comprising an acidic, inactivating dipeptide [13 , 14 , 34 , 35 ]. The leader sequence enables the nascent grz to be processed through the endoplasmic reticulum (ER) and Golgi apparatus during synthesis so that they may be ultimately targeted to the secretory pathway [13 , 14 ]. The glycosylation of grz is quite heterogeneous, but generally, their packaging into cytotoxic granules is dependent on the mannose 6-phosphate pathway [36 ]. Below, we will discuss how grz induce apoptotic death, however much experimental evidence indicates additional, potential functions for grz.


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  		Table 2. Properties of Human and Rodent Granzymes

Calreticulin
Calreticulin is a calcium-storage protein that carries a COOH-terminal KDEL sequence for retention in the lumen of the ER. However, in CTLs, calreticulin also colocalizes with pfp and is released with it on degranulation [37 ]. Pfp interacts with the P-domain of calreticulin, which has high, Ca2+-binding affinity and chaperone function, but the molecules dissociate upon exposure to higher, extracellular Ca2+ concentrations. Therefore, calreticulin may act as a Ca2+-regulated chaperone for pfp, potentially serving to protect the killer cell during biogenesis of granules and regulating pfp function after release [38 ].

Chondroitin sulfate proteoglycans
Chondroitin sulfate proteoglycans are protease-resistant, possess chondroitin sulfate A side-chains, and are specifically exocytosed upon contact with sensitive targets [39 , 40 ]. These negatively charged macromolecules may regulate the packaging and delivery of pfp and positively charged (basic) grz [41 ]. Recently, it has been proposed that polyvalent uptake of grz and other molecules by target cells may occur as a result of their attachment to serglycin-rich moieties [42 ].

Chemokines
Chemokines exocytosed by CTL are believed to be important in the noncytolytic inhibition of the human immunodeficiency virus (HIV)-1 replication. One study has shown that grzA, macrophage inflammatory protein (MIP)-1{alpha}, and RANTES (regulated on activation, normal T expressed and secreted) are localized in the cytolytic granules of HIV-1-specific CD8+ CTL [43 ]. These mediators are co-secreted after T-cell receptor (TCR) triggering, facilitating lysis of virion-producing cells and the inhibition of free virus. In addition, RANTES, MIP-1{alpha}, and MIP-1ß are secreted by CTL as a macromolecular complex containing sulfated proteoglycans, providing a potentially rapid response to pathogens, using preformed and prepackaged chemokines.


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GENERIC, LYSOSOMAL COMPONENTS
 
The multivesicular (peripheral) domains of cytolytic granules contain lysosomal hydrolases, including acid phosphatase, {alpha}-glucosidase, arylsulphatase, ß-glucoronidase, cathepsins B and D, cathepsin A-like protective protein (CAPP; which possesses serine carboxypeptidase and deamidase activities), and lysosomal membrane proteins, Lamp-1, Lamp-2, and CD63. In addition to their protein content, the lytic granules have other properties in common with lysosomes. This region has an acidic pH, comparable with that of endosomes and lysosomes. The multivesicular domains of the granules are rich in the 270-kD mannose-6-phosphate receptor, normally absent from mature lysosomes but present in earlier endocytic compartments. Thus, the granules represent an unusual, dual-function organelle, where a regulated secretory compartment, the dense core, is contained within a prelysosomal compartment, the multivesicular domain. How the biogenesis of the "secretory lysosome" differs from that of a conventional secretory granule is unclear, however a combination of lysosomal and other sorting signals appears to be required. Similar organelles are also found in other hemopoietic subsets of cells. Therefore, hemopoietic cells may possess specialized mechanisms that allow the correct sorting of secreted products to the lysosome, and these signals may differ from those in conventional secretory (e.g., neurosecretory) cells. Studies on Chediak-Higashi syndrome (CHS) patients support the idea that granules are specialized, secretory lysosomes, because their hemopoietic cells are unable to secrete their granule contents, and their conventional, secretory cells are able to do so [36 ]. CTL from CHS patients cannot secrete the giant granules in which their cytotoxic proteins are stored, thereby suggesting that the defect lies in protein sorting or membrane fusion.

Dipeptidyl peptidase I (DPPI; cathepsin C) is a lysosomal, cysteine protease and major posttranslational-processing enzyme responsible for generating activated myeloid and lymphoid granule serine proteases [44 ]. DPPI was first shown definitively to process and activate human grzB [45 ], and subsequently, the generation of DPPI-/- mice indicated that DPPI plays an essential role in the in vivo processing and activation of grzA and -B [46 ].


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FUNCTIONS FOR CYTOTOXIC GRANULE PROTEINS
 
Target cell death
Pfp
The crucial role of pfp became evident when several laboratories created pfp-deficient, gene knock-out mice (pfp-/-) [8 , 9 , 47 ]. These mice have normal T- and NK-cell development [8 ], however their cytolytic lymphocytes are compromised, and the mice are highly susceptible to certain intracellular pathogens [8 , 9 , 48 ]. In vitro, pfp-deficient CTL and NK cells are defective in their killing of allo-reactive or xeno-reactive tumor cell lines and NK-sensitive target cells, respectively [49 , 50 ]. Despite its clear biological importance, little is known about the molecular function of pfp, and only recently have dogmas concerning its mechanism of action been questioned. The inability thus far to assign pfp functions to discrete parts of the molecule represents a major gap in our understanding of effector lymphocyte biology. Pfp shares functional, antigenic, and ultrastructural similarities with complement proteins C6–C9, as described some time ago, and functions such as membrane insertion and polymerization have been tentatively explained on the basis of these similarities [51 52 53 54 ]. As already discussed, definitive evidence has emerged that the C-terminal domain of pfp is the site of calcium ion-binding and initiates lipid insertion [16 ]. Based on the structure of synaptotagmin and related molecules, it is postulated that after cleavage at the carboxy terminal, multiple aspartate residues of the C2 domain become approximated in three dimensions to bind a calcium ion electrostatically. The refolded pfp molecule becomes highly reactive with lipids as a result of exposure of amphipathic domains elsewhere in the molecule and is able to attach to and be inserted in the plasma membrane.

A pfp receptor?
Although pfp can form transmembrane channels in synthetic, lipid membranes with no protein content, it has long been suspected that, for example, on the basis of markedly differing susceptibility of various cell types to pfp, factors other than lipid composition can regulate the activity of pfp. The recent studies of Berthou et al. [55 ] have provided some evidence of this proposition. These investigators have proposed that the lysolipid PAF (known as platelet activating factor because it promotes platelet aggregation) is co-released with pfp when NK cells degranulate and may potentiate pfp lysis by forming a molecular bridge between pfp and the PAF receptor (PAF-R) Thus, ternary complexes containing PAF, PAF-R, and pfp may achieve membrane disruption more efficiently by enhancing the binding of pfp to phosphorylcholine. Furthermore, as PAF-R expression is inducible with interferons (IFNs), local, inflammatory mediators may modulate the sensitivity of cells to pfp at a focus of infection. If verified, the studies by Berthou et al. [55 ] may also provide an opportunity to manipulate pfp function for experimental and even therapeutic purposes.

What does pfp actually do?
As with complement, purified pfp can cause cell lysis by forming discrete pores 12–20 nm in diameter but, by itself, cannot account for the morphological changes of apoptosis, such as chromatin condensation and DNA fragmentation [56 ]. The nuclear changes occur before cell membrane damage [57 , 58 ] and are reproducible in vitro with concentrations of pfp that cause minimal, membrane-permeability changes [21 ]. Thus, the proposition that large membrane pores are necessary for grz access to the target cell cytosol has come into question recently. Despite the structural similarities of the pores formed by purified pfp and complement, grzB did not trigger apoptosis of target cells when delivered by complement [21 ]. However, listeriolysin (LLO), a virulence factor of Listeria monocytogenes (LM) that causes lysis of endosomes, was able to deliver grzB potently in the absence of measurable plasma-membrane damage [21 ]. The pro-apoptotic activity of LLP was inhibited when the pH of endo-lysosomes was raised to neutral with ammonium chloride or bafilomycin [21 ]. Direct support for an endosomolytic function of pfp came from the observation that brefeldin A (BFA) inhibited pfp-induced release of grzB from endosomes, blocked its translocation to the nucleus, and inhibited cell death. Consistent with BFA having no effect on receptor-mediated uptake via endocytosis, BFA had no effect in kinetic or absolute terms on grzB uptake into the cell in the absence of pfp [21 ].

Because the concentration of pfp delivered to the target cell surface by a CTL has not been determined, a key question is whether endosomolysis is relevant physiologically at pfp concentrations causing appreciable cell-membrane damage. We have found that when target cells are incubated with concentrations of pfp, which caused 100% 51Cr release, freely diffusible, fluorescent proteins of 9–13 kDa remain excluded for over 1 h, whereas 32-kDa grzB and 65-kDa grzA continued to be delivered to the cytoplasm and nucleus within a few minutes [21 ]. This indicates the delivery of apoptotic mediators by pfp is indeed highly selective, and thus, grz entry into cells cannot be ascribed to passive diffusion simply through pfp pores, even if target cells are placed under severe, osmotic stress.

Grz
Previous reviews have described the gene structure, chromosomal loci, and protease specificity of grz family members [29 , 30 , 59 , 60 ]. The major, recent advance in terms of grz structure has been the description of a crystal structure for grzB in complex with a macromolecular inhibitor. The primary specificity for Asp residues occurs through a side-on interaction with a "buried" side chain of Arg226 of granzyme B. A further nine amino acids make contact with the substrate and dictate the extended substrate-specificity profile [31 ]. There has also been considerable progress in further elucidating the role of grz in apoptosis [60 ]. Early studies unequivocally illustrated that grzA and B could collaborate with pfp to kill target cells [57 , 61 , 62 ]. The most potent granzyme in this context is grzB, and grzB-deficient mice have impaired ability to induce rapid DNA fragmentation in the target cell [63 ]. Two other grz were purified, which induce target cell DNA fragmentation with much slower kinetics, and these were the tryptases grzA and tryptase-2 [57 ]. However, grzA-deficient mice have no generalized defect of target cell DNA fragmentation, indicating that unlike grzB, the absence of grzA can be compensated by other grz [64 ]. We and others [65 66 67 68 ] have shown that grzB, abundant in cytolytic granules, is the protease largely responsible for eliciting the nuclear changes of apoptosis. We and several other groups [67 68 69 ] have shown that grz can enter cells independently of pfp but remain sequestered in endosomes and so do not damage cells unless pfp is also present. Thus, the provision of a membrane pore by pfp is not necessary for grz to enter the target cell cytoplasm. Cell-surface binding of 125I-grzB is saturable and can be competed by unlabeled grz [69 ], suggesting uptake through a specific receptor. Recently, the 270-kDa, cation-independent, mannose 6-phosphate receptor was demonstrated to be a receptor through which grzB can enter the endosomal compartment [70 ]. Most surprisingly, it was claimed that expression of the same receptor on H2k-expressing fibroblasts was required for their rejection by allogeneic T cells, suggesting a primary role for grzB in the allogenic-effector response and predicting a further, possible mechanism for immune escape by tumors [70 ].

GrzB function in cell death: a crucial role for Bid-cleavage rather than direct caspase activation
Sublytic pfp can induce the redistribution of grzB or dimeric grzA (65 kDa) from endosomes into the cytosol and can amplify greatly cellular uptake of grz [67 ]. Apoptotic changes are apparent within only 2 min, and migration of grzB out of endosomes and its appearance in the nucleus are precise predictors of apoptotic death [65 ]. In addition to inducing programmed, cell-death pathways operating through caspases, grzB can directly cleave cytoplasmic substrates such as the actin-binding protein, filamin [71 ], and nuclear poly(ADPribose) polymerase (PARP) and nuclear matrix antigen at sites different than those preferred by caspases [72 ]. Grz entry into the nucleus occurs before apoptotic, nuclear-membrane disruption [58 ] and is dependent on an unknown cytosolic carrier protein but does not require expenditure of energy [67 ].

Through its unique ability to cleave after aspartate residues, grzB can cleave many pro-caspases in vitro [73 74 75 ]. However, in intact cells, there is a requirement for mitochondrial perturbation [76 , 77 ], without which direct caspase activation occurs only very slowly [78 ] (Fig. 1 ) We showed recently that a vital part of the apoptotic signal imparted by grzB to mitochondria is through direct cleavage of the pro-apoptotic, BH3-only, Bcl-2 family member Bid, which is specifically cleaved at a site 16 amino acids down-stream of that used by caspases [78 ]. Surprisingly, grzB can also induce death through a caspase-independent mechanism that involves damage to non-nuclear structures and is probably mediated by direct, grz-mediated disruption of mitochondria [66 , 79 , 80 ]. These caspase-independent pathways may safeguard against viruses that delay programmed cell death by expressing serpins such as the caspase-8 inhibitor of cowpoxvirus, cytokine response modifier A (crmA) [81 ].



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  		Figure 1. A simplified scheme for cell death mediated by grzB. Following the entry of grzB into the target cell by endocytosis and its liberation into the cytosol by pfp, the principal role of grzB (thick arrow) is to cleave the pro-apoptotic, Bcl-2-like molecule Bid, specifically at the grzB cleavage site at amino acid 75, which is strongly conserved between mouse and humans. Direct processing of caspases by grzB is inefficient in the absence of mitochondrial perturbation (dotted line). Truncated Bid disrupts mitochondria, resulting in the release of pro-apoptotic mediators, the precise nature of which is unknown at present (dashed arrow). These mediators may include Diablo/Smac and posssibly cytochrome c and induce efficient caspase processing and generic caspase-mediated cell death. Insertion of truncated Bid and release of apoptotic mediators from mitochondria can be negatively regulated by overexpression of Bcl-2. Cell death in response to grzB can also occur without efficient caspase activation through a non-nuclear mechanism, which is poorly understood but may involve cleavage of cytoskeletal elements (dashed arrow; for a deeper explanation, see text and ref. [76 ]).

GrzA function in cell death
GrzA is a specific tryptase, which becomes concentrated in the nucleus of targeted cells and synergistically enhances DNA fragmentation induced by grzB and pfp [58 ]. Little is known about grzA-induced cell death. Using recombinant, mutant, inactive grzA, two grzA-binding proteins, PHAP (putative HLA-associated protein) II and heat shock protein (hsp)27, were isolated [82 ], however neither of these has been demonstrated to be important yet in apoptosis mediated by grz. GrzA-induced membrane perturbation, nuclear condensation, and DNA damage are unimpaired by caspase blockade, however entry of grA to the nucleus is totally inhibited by Bcl-2 overexpression [83 ]. Recently, grzA was shown to induce single-strand DNA breaks rather than oligonucleosomal fragmentation [84 ]. GrzA enhances DNA accessibility to exogenous endonucleases and degrades histone H1 in vitro into ~16-kDa fragments. Histone digestion may provide a mechanism for unfolding compacted chromatin to facilitate endogenous DNase access to DNA [85 ]. CTL and NK cells of mice deficient in grzA and grzB (grzAB-/-) induced 51Cr-release in target cells at levels and with kinetics similar to those of normal mice [86 ]. This contrasts with their inability to induce apoptotic nuclear damage in target cells, suggesting that grzA and -B are critical for CTL/NK granule-mediated nucleolysis, with grzB being the main contributor, and target cell death is pfp-dependent and -independent of both proteases.


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GRANZYME SERPINS
 
Serprins contol many physiological processes by the balance of serine-protease activities and their regulated blockade. Serpins, such as crmA, form irreversible complexes with their cognate proteases by acting as pseudo-substrates. The inhibitory loop of these serpins contains sequences recognized specifically by the protease. Primary sequence recognition is encoded by the P1 residue, but neighboring residues upstream (P2, P3, etc.) or downstream (denoted P1', P2', etc.) of P1 can also influence recognition and cleavage. The two ends of the inhibitory loop are flexible, hinge-like structures, which enable the loop to become mobile after cleavage by the cognate protease, commencing a conformational change that locks the two molecules into a complex often strong enough to withstand boiling in sodium dodecyl sulfate (SDS)-containing buffer. Evidence has emerged recently that cytotoxic lymphocytes synthesize their own serpins, which act within the cytosol, to safeguard against missorting or mispackaging of pro-apoptotic granzymes. The proteinase inhibitor (PI) residue of the CTL/NK serpin PI-9 is Glu, allowing it to inhibit grzB specifically [87 ]. The choice of Glu at the P1 residue seems puzzling at first because grzB prefers cleaving at Asp in most instances [88 ]. Bird and colleagues [89 ] showed that mutation of the P1 residue to Asp resulted in poor complex formation with grzB and, furthermore, that the mutated molecule acquired a crmA-like ability to inhibit caspases, which wild-type PI-9 does not possess. Therefore, PI-9, which is absent from cytotoxic granules but present in high concentrations in the cell cytosol, can block toxic grzB molecules, which leak out of granules, without inhibiting physiological death of the CTL occurring through the Fas pathway [89 ]. Many new intracellular serpins have been described recently [90 ] in CTL/NK cells, and it is likely that effector cells are armed with appropriate, protective serpins specific for all of their grz.

Previous observations that the highly conserved, poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways were taken to indicate their involvement in immune evasion by poxviruses. The striking similarities between crmA and PI-9 suggest that viral products such as crmA may have arisen from the capture and mutation of homeostatic elements such as PI-9 or a related serpin expressed in infected cells. The data in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection question the role of serpins in the evasion of CTL killing [91 92 93 ]. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression affects the Fas-mediated and, to a lesser extent, the granule exocytosis pathway primarily and that serpin-2 is the main contributor to suppression for both killing pathways [93 ].


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WHAT TYPES OF IMMUNE RESPONSE ARE DEPENDENT ON CYTOTOXIC GRANULE PROTEINS?
 
Control of virus infections
Analysis of pfp-deficient mice has identified pfp as the preeminent effector molecule in T-cell-mediated control of many virus infections. Lymphocytic choriomeningitis virus (LCMV)-specific CTL are responsible for virus eradication and the onset of pathology associated with the disease, depending on the timing, route of entry, and strain of the virus [8 ]. CD8+ T cells are activated by LCMV in pfp-/- mice but fail to clear the virus effectively. Rather, a large, proliferative expansion and persistence of antigen-reactive T and B cells and antigen-presenting cells (APC) occur in pfp-deficient mice challenged with LCMV. This presentation bears a striking resemblance to that of pediatric patients with the autosomal-recessive immunodeficiency, familial hemophagocytic lymphohistiocytosis (FHL), about half of whom have been shown to lack NK cell activity as a result of inherited, structural mutations in the pfp gene [94 ]. It is possible that human FHL is triggered by a virus, which is normally cleared in a pfp-dependent way. Previous studies have established that NK/CD8+ T cytotoxicity was not essential to resolve most cytopathic poxvirus infections and that secretion of IFN-{gamma} by CD4+ and CD8+ T cells was crucial in immunity against poxviruses. However, a lack of pfp renders the relatively resistant C57BL/6 mice highly susceptible to the natural mouse pathogen ectromelia, a cytopathic orthopoxvirus [91 ]. Pfp-deficient mice showed increased mortality, elevated virus titers, increased cytopathic damage in their liver and spleen, and increased circulating liver transaminase levels [91 ]. It is interesting that mice deficient in grzA and -B were virtually as susceptible as pfp-deficient mice to this virus, and mice deficient in either grz alone were susceptible only partially [92 ]. Death of grzAB-/- mice occurred despite the expression of functionally active pfp and the absence of an intrinsic defect in generating splenic, cytolytic T cells.

The increased sensitivity of grzAB-/- mice to ectromelia is the most significant phenotype that grz-deficient mice display. It has been suggested that grz are important effector molecules in this setting, but it remains unclear why grz are so important in host protection from ectromelia. In contrast to ectromelia, cowpox virus is more virulent in the presence of pfp than in its absence. An additional lack of grzA increases the virulence of cowpox virus. NK cells and CD8+ CTL also have a protective role against cytopathic murine cytomegalovirus (MCMV) infection. Spleen NK cells control MCMV infection in a pfp-dependent manner [95 ], however in the liver, production of IFN-{gamma} by NK cells was the predominant mechanism that regulated MCMV DNA synthesis. More recent data extend previous studies on the critical role of NK/CD8+ T cells in the early control of MCMV infection by showing that pfp and grzA and -B contribute to viral elimination in the salivary glands, however none of these molecules alone was essential for final control of infection [96 ]. Control of ganglionic herpes simplex virus (HSV) infection depends on CD8+ cells but not on the death of infected neurons. It has been shown recently that grzA restricts the interneuronal spread of HSV and influences ganglionic virus load significantly. Thus, several other virus models are emerging where grz may play a more subtle role in controlling viral load, and these mechanisms may not involve cell death of virus-infected cells. Theiler’s virus, a murine picornavirus, infects the central nervous systems of C57BL/6 mice and is cleared in a pfp-dependent process, which requires CD8+ cytotoxic T cells [97 ]. Some controversy exists as to the role of pfp in HSV infection. One study indicates pfp is essential for host protection against ocular HSV challenge, but not herpetic stromal keratitis (HSK), an inflammatory disease of the cornea that often results in blindness [98 ]. Another study suggested that pfp-dependent cytotoxicity is an important effector mechanism in the production of HSK, but viral clearance from the eyes of pfp-/- mice was not impaired [99 ]. By contrast, these findings show that pfp is sometimes important in the pathogenesis of viral infection rather than viral clearance per se. Further, supporting an important role for pfp in the pathogenesis of infection is the exacerbation of Coxsackievirus B3-induced myocarditis by pfp [100 ] and pfp-mediated immunopathology in IFN-{gamma}-/- mice infected with LCMV [101 ].


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CONTROL OF BACTERIAL INFECTION
 
Granule protein-mediated control of bacterial infections has not been widely studied, however many intracellular bacteria are controlled in part in a pfp-dependent way. Kagi et al. [8 ] first demonstrated that the absence of pfp-mediated cytotoxicity resulted in the delayed clearance of LM from the spleen but not the liver after primary infection. Clearly, pfp-dependent and -independent mechanisms of CD8+ T-cell-mediated clearance of Listeria were evident, however protection against a secondary infection was impaired drastically in pfp-/- mice. Subsequently, immunity for several Listeria antigens was shown to be mediated by pfp-expressing CD8+ T cells. Analysis of epitope-specific CD8+ T-cell expansion by major histocompatibility complex (MHC) class I tetramer staining and enzyme-linked immunospot (ELISPOT) revealed no deficiency in the primary or secondary response to LM infection in pfp-/- mice [102 ]. These data show that reduced resistance to LM observed with pfp-/- mice is a consequence of a deficiency in effector function and not a result of suboptimal, CD8+ T-cell priming. Recently, the same group has demonstrated that pfp-independent immunity in the spleen requires CD8+ T cell-derived tumor necrosis factor (TNF) [103 ]. The second microorganism of great interest has been M. tuberculosus. CTL have been shown to be protective against M. tuberculosus infections in the mouse, and these effectors are cytolytic toward M. tuberculosus-infected cells and release IFN-{gamma} in response to mycobacterial antigen. CTL have also been shown to kill intracellular pathogens by a granule-dependent mechanism involving perforin and granulysin, as described above [26 ].

Pfp plays a role in host protection against several other microorganisms. For example, Encephalitozoon cuniculi is a protozoan parasite shown recently to cause opportunistic infection in immunocompromised individuals. Protective immunity in the normal host is CD8+ T-cell-dependent, and pfp-/- mice are particularly sensitive to parasite challenge [104 ]. CD8+ T cells have also been shown to be required for acute resistance to infection with the protozoan parasite, Trypanosoma cruzi, the causative agent of Chagas’ disease. Although pfp-dependent cytolytic mechanisms can clearly affect acute resistance to T. cruzi infection, this contribution may be strain- and challenge-dose-dependent [105 ]. Pfp also plays a limited role in host resistance to Toxoplasma gondii, particularly during the chronic stage of infection [106 ]. The clearance of several other microorganisms tested including Chlamydia pneumoniae and Chlamydia trachomatis was not pfp-dependent.


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CONTROL OF TUMOR GROWTH
 
Tumor cells of different tissue origins have now been characterized for their sensitivity to pfp-mediated cytotoxicity, and in general, cytolytic lymphocytes kill the majority of these tumors in a pfp-dependent manner [107 , 108 ]. Some tumor cells, such as acute myeloid leukemias, may be able to protect themselves from pfp-mediated cell death through impaired binding of pfp [109 ]. Pfp-/- mice were challenged with syngeneic lymphoid tumors that were MHC class I-deficient and were shown to be controlled by NK cells in a pfp-dependent manner [110 ]. Subsequently, we supported this study by demonstrating that pfp accounts for all the effector function of the NK cells mediating rejection of MHC class I-deficient lymphoid tumors in the peritoneal cavity [111 ]. van den Broek et al. [112 ] also challenged naive pfp-/- mice with syngeneic tumor cell lines of various tissue origin. Most of the tumors were rejected ten- to 100-fold more efficiently by wild-type mice, and the difference between wild-type and pfp-/- mice was more marked following priming. Others have demonstrated the relative importance of pfp in graft versus leukemia effects following transplantation [113 114 115 ]. Adoptive transfer or biological immunotherapies that stimulate NK/NKT and CTL responses have also been demonstrated to mediate their anti-tumor activities via pfp [116 117 118 ]. These studies in experimental settings underline the importance of tumor rejection via the pfp pathway.

Despite the abundance of information that supports a key role for pfp in host immunity against experimental tumors, the role of CTL and NK cells, and in particular cytotoxicity, in tumor immune surveillance has remained a controversial question. Pfp-/- mice have provided an ideal model in which to revisit this issue. Approximating models of spontaneous tumor formation, van den Broek et al. [112 ] demonstrated that pfp-/- mice were more susceptible to sarcoma induction than wild-type mice after receiving the chemical carcinogen, methylcholanthrene (MCA) or oncogenic Moloney sarcoma virus. Subsequently, we have explored MCA-induction of sarcoma in pfp-/- and other gene-targeted mice. Clearly, there is a role for NKT and NK cells in host protection, and pfp is not the only protective effector mechanism used by the immune system [119 ]. We also examined whether tumorigenesis would be accelerated in tumor-prone, p53-deficient mice that also lacked pfp expression. From this study, we were able to show that pfp-deficient mice have a high incidence of malignancy in several distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis [120 ]. These highly malignant neoplasms were strongly rejected by pfp-expressing mice, indicating that the problem in pfp-deficient mice was poor tumor destruction rather than inefficient tumor cell recognition or antigen processing. The susceptibility to lymphoma was accelerated by a simultaneous lack of expression of the p53 gene. This was the first study to demonstrate that lymphocyte-mediated cytotoxicity plays an important role in promoting host resistance to spontaneous tumor formation. Although the pfp-/- mice in our previous aging studies have not been exposed to LCMV nor do they develop histiocytic infiltrates early in life, it is possible that other microorganisms might provide antigenic stimulation also leading to lymphoproliferation, extended cell survival, and an increased pool of cells vulnerable to oncogenesis in pfp-/- mice. Alternatively, some recent experimental evidence suggests that pfp may normally control B cell hyperplasia [121 ], suggesting that pfp also plays an immunoregulatory role (see below). In any event, the increased lymphoma incidence associated with pfp deficiency might have its genesis in an increased number of premalignant cells and the absence of the cytotoxic mechanism that normally eliminates them.


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HOMEOSTASIS OF THE LYMPHOID COMPARTMENT
 
To prevent uncontrolled expansion, the massive proliferation of T cells during an acute immune response has to be followed by their controlled deletion. Mounting evidence now supports an immunoregulatory role for the granule exocytosis pathway of cell death. The earliest indication of this role was the exacerbated lymphoid expansion of mice doubly deficient for pfp and the apoptosis-inducing Fas ligand [122 ]. These mice spontaneously develop infiltrates of highly activated CD8+ T cells in their kidneys, liver, pancreas, and uterus and die between 5 and 12 weeks of age [123 ]. Related studies, which examined the effects of combining pfp deficiency and Fas mutation, suggested that pfp-mediated cytotoxicity plays a specific role in regulating systemic autoimmunity [124 ]. These conclusions have been recently supported by studies in a model of graft versus host disease [121 ]. In the setting of B- and T-cell activation, pfp plays an important immunoregulatory role in the prevention of humoral autoimmunity through the elimination of autoreactive B cells and Ag-specific T cells. Moreover, an ineffective, initial CTL response can evolve into a persistent, antibody-mediated response and, with it, the potential for sustained, humoral autoimmunity.

A role for pfp in CD8+ T-cell homeostasis has also been investigated in the context of staphylococcal enterotoxin activation [123 , 125 ]. Previous in vitro studies have shown that these CTL effectively lyse MHC class II-expressing cells presenting the proper superantigen. Injection of staphylococcal enterotoxin B (SEB) into pfp-/- mice results in dramatically increased, selective expansion and prolonged persistence of CD8+, but not CD4+, SEB-reactive T cells [123 ]. Repeated injections of staphylococcal enterotoxin A to pfp-/- mice resulted in significantly less B-cell depletion compared with control mice [125 ]. This suggests that superantigen-activated CD8+ T cells lyse MHC class II+ APC in a pfp-dependent manner in vivo. In a model of LCMV infection, secondary immunization of TCR transgenic (for LCMV) pfp-/- mice with the LCMV-specific peptide led to an increased proliferation of transgenic CD8+ T cells, which was not explained by failure to deplete professional APC [126 ]. These results are supported by an additional study [127 ] and point to a novel mechanism of T-cell homeostasis in which the acquisition of pfp-dependent, cytotoxic activity regulates the expansion and persistence of CD8+ effector T cells in vivo.

T-cell memory depends on factors that regulate expansion and death of CD8+ effector T cells after antigenic stimulation. This differentiation of effector T cells into memory T cells is critical for an effective and controlled immune reaction [127 ]. There is now considerable interest in determining what effector molecules might control this differentiation step. Mice deficient in pfp and IFN-{gamma} exhibited increased expansion, altered immunodominance, and decreased death of antigen-specific CD8+ T cells after infection with an attenuated strain of LM, which was cleared from these mice [128 ]. Expansion of CD8+ T cells was controlled by pfp, whereas IFN-{gamma} was responsible for immunodominance and the death phase. Another study with Epstein-Barr virus (EBVP) infection supports this hypothesis, because that primary response is closely regulated, and the majority of cells are programmed to die via a cytokine-rescuable pathway, leaving only small populations of memory T cells surviving [129 ]. Thus, it is likely that pfp and IFN-{gamma} regulate distinct elements of CD8+ T-cell homeostasis, independent of their role as effector molecules.


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OTHER POTENTIAL grz FUNCTIONS?
 
Because of their many protease specificities, grz have been suggested to participate in lymphocyte functions such as antigen processing, extravasation, and migration of mature T cells. GrzA has been demonstrated to cleave several extracellular matrix proteins in vitro [130 ], and Sayers et al. [131 ] found that grzB inhibited the growth of adherent tumor cell lines by preventing their adhesion to extracellular matrix proteins. Perhaps a most striking finding is that the majority of autoantigens targeted across the spectrum of human systemic autoimmune diseases are cleaved efficiently by grzB in vitro and during cytotoxic lymphocyte, granule-induced death, generating unique fragments not observed during other forms of apoptosis [132 ]. The grzB cleavage sites in autoantigens contain amino acids in the P2 and P3 positions that are preferred by grzB but are not tolerated by caspase-8. In contrast, nonautoantigens are not cleaved by grzB or are cleaved to generate identical fragments arising in other forms of apoptosis. Systemic autoimmune diseases are a genetically complex, heterogeneous group of disorders in which the immune system targets a diverse but highly specific group of intracellular autoantigens. These targeted molecules are not unified by common structure, function, or distribution but become clustered and concentrated in surface blebs when cells undergo apoptosis. Collectively, these results focus attention on the role of the cytotoxic lymphocyte, granule-induced death pathway in the initiation and propagation of systemic autoimmunity. Future work in this area promises to be very revealing.

GrzA has also been demonstrated to induce cellular responses mediated by {alpha}-thrombin [133 ], however whether grzA plays any physiological role in these processes remains to be determined. Biological functions for all other grz, including grzM and grzH, have not been defined. The presence of active grz in serum and synovial fluid of rheumatoid joints has been described [134 ], but their pathophysiologic role remains unclear as yet. It still remains likley that the role of grz in functions other than cytolysis will be confirmed best in vivo in homozygous grz-deficient mice. The derivation of DPPI-/- mice and other grz-/- mice will be useful tools in this regard.


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CONCLUDING REMARKS
 
The last decade of research into cytotoxic granule proteins allows us to take away several important messages and pose interesting questions for future investigation. Firstly, it is clear that the function of most granule proteins revolves around pfp. Mutations of pfp in mice and man verify the central role pfp plays in host-immune reaction and regulation. The functions of grz and granulysin, the best-defined effector molecules in granules, are pfp-dependent. Despite clearly defined, apoptotic pathways triggered by grzB, experiments in grz-deficient mice make us question whether grz play a generalized role in lymphocyte-mediated cell death. Combined with the healthy cytolytic activity of grzAB-/- CTL/NK cells, these studies illustrate the potential relevance of grz to the clearance of specific pathogens. Perhaps the ability of grz to target the nucleus and trigger DNA fragmentation is specific for viral infections in cytotoxic cells. In addition, we need to determine how CTL/NK cells can kill target cells in a pfp-dependent manner that is independent of grzA and grzB. Obviously, the key to regulating CTL/NK cell-granule function is to understand exactly how pfp facilitates granule-protein function. There is a great need to relate the structure of pfp to its molecular function to enable the rational design of agents that can control pfp action. Other proteins housed within the cytotoxic granules, which are undefined yet, will be important in the normal processing and control of pfp activity and may provide important clues to regulating pfp activity. This decade also promises many surprises as the secrets of cytotoxic granules are unraveled.


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ACKNOWLEDGEMENTS
 
M. J. S. and J. A. T. are Principal Research Fellows of the National Health and Medical Research Council (NHMRC) of Australia. J. M. K., V. R. S., and K. A. B. are supported by project grants from the NHMRC. J. E. D. is supported by a post-graduate scholarship from the Anti-Cancer Council of Victoria. T. J. S. is supported by the Intramural Research Support Program, SAIC, National Cancer Institute-Frederick, Frederick, MD. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the U.S. Government. This project has been funded in whole or in part with federal funds from the National Cancer Institute, under contract number N01-CO-56000.

Received February 5, 2001; revised February 12, 2001; accepted February 15, 2001.


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M. S. Bharhani, J. S. Grewal, M. J. Pilgrim, C. Enocksen, R. Peppler, L. London, and S. D. London
Reovirus Serotype 1/Strain Lang-Stimulated Activation of Antigen-Specific T Lymphocytes in Peyer's Patches and Distal Gut-Mucosal Sites: Activation Status and Cytotoxic Mechanisms
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J. A. Rutigliano and B. S. Graham
Prolonged Production of TNF-{alpha} Exacerbates Illness during Respiratory Syncytial Virus Infection
J. Immunol., September 1, 2004; 173(5): 3408 - 3417.
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D. Hongo, J. S. Bryson, A. M. Kaplan, and D. A. Cohen
Endogenous Nitric Oxide Protects against T Cell-Dependent Lethality during Graft-versus-Host Disease and Idiopathic Pneumonia Syndrome
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B. L. Shacklett, C. A. Cox, M. F. Quigley, C. Kreis, N. H. Stollman, M. A. Jacobson, J. Andersson, J. K. Sandberg, and D. F. Nixon
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J. Immunol., July 1, 2004; 173(1): 641 - 648.
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J. J. Zaunders, W. B. Dyer, B. Wang, M. L. Munier, M. Miranda-Saksena, R. Newton, J. Moore, C. R. Mackay, D. A. Cooper, N. K. Saksena, et al.
Identification of circulating antigen-specific CD4+ T lymphocytes with a CCR5+, cytotoxic phenotype in an HIV-1 long-term nonprogressor and in CMV infection
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S. Nikiforow, K. Bottomly, G. Miller, and C. Munz
Cytolytic CD4+-T-Cell Clones Reactive to EBNA1 Inhibit Epstein-Barr Virus-Induced B-Cell Proliferation
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F. Andrade, L. A. Casciola-Rosen, and A. Rosen
A Novel Domain in Adenovirus L4-100K Is Required for Stable Binding and Efficient Inhibition of Human Granzyme B: Possible Interaction with a Species-Specific Exosite
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I. Barao, D. Hudig, and J. L. Ascensao
IL-15-Mediated Induction of LFA-1 Is a Late Step Required for Cytotoxic Differentiation of Human NK Cells from CD34+Lin- Bone Marrow Cells
J. Immunol., July 15, 2003; 171(2): 683 - 690.
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L. Krenacs, M. J. Smyth, E. Bagdi, T. Krenacs, L. Kopper, T. Rudiger, A. Zettl, H. K. Muller-Hermelink, E. S. Jaffe, and M. Raffeld
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B. J. Johnson, E. O. Costelloe, D. R. Fitzpatrick, J. B. A. G. Haanen, T. N. M. Schumacher, L. E. Brown, and A. Kelso
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E. Wieckowski, G.-Q. Wang, B. R. Gastman, L. A. Goldstein, and H. Rabinowich
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N. Bidere, M. Briet, A. Durrbach, C. Dumont, J. Feldmann, B. Charpentier, G. de Saint-Basile, and A. Senik
Selective Inhibition of Dipeptidyl Peptidase I, Not Caspases, Prevents the Partial Processing of Procaspase-3 in CD3-activated Human CD8+ T Lymphocytes
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A. Kelso, E. O. Costelloe, B. J. Johnson, P. Groves, K. Buttigieg, and D. R. Fitzpatrick
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Z. Fan, P. J. Beresford, D. Zhang, and J. Lieberman
HMG2 Interacts with the Nucleosome Assembly Protein SET and Is a Target of the Cytotoxic T-Lymphocyte Protease Granzyme A
Mol. Cell. Biol., April 15, 2002; 22(8): 2810 - 2820.
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