Lack of DNA degradation in target cells lysed by granules derived from cytolytic T lymphocytes

It has been shown previously that fragmentation of target cell DNA is an early event in lysis mediated by cytolytic T lymphocytes (CTL). In this study, we have investigated whether CTL-derived granules that exhibit lytic activity also induce DNA fragmentation in murine target cells. Cytolytic granules isolated from three different alloreactive CTL clones were tested for the induction of DNA fragmentation in P815 and EL4 target cells, by using a Triton X-100-facilitated, radiolabeled DNA release assay. In contrast to the CTL clones from which they were derived, the cytolytic granules did not induce DNA fragmentation. Agarose gel electrophoretic analysis of DNA confirmed the lack of discrete DNA fragments in target cells lysed by CTL-derived granules. Possible explanations for the difference in the ability of CTL and CTL-derived granules to trigger DNA fragmentation are discussed.     

B cell stimulatory factor-2 is involved in the differentiation of cytotoxic T lymphocytes

The induction of cytotoxic T lymphocytes (CTL) from precursor T cells requires both antigen and lymphokine signals. Previous work from our laboratory has indicated that three lymphokines are required for the induction of CTL from murine thymocytes; interleukin 2, interferon-gamma (IFN-gamma), and a partially characterized factor referred to as cytotoxic differentiation factor (CDF). While attempting to clone CDF from the human T cell line C10-MJ2, we found that a gene encoding CDF-like activity is identical to the gene encoding the factor known variously as B cell stimulatory factor-2 (BSF-2), IFN-beta 2, and 26-kDa protein. We report here that BSF-2 can induce the differentiation of Ly-2+ CTL from murine thymocytes in the presence of interleukin 2 and that the level of cytotoxicity is augmented by the addition of murine IFN-gamma. Serine esterase, a marker for cytotoxic granules in CTL, was induced only in the presence of BSF-2, and the level of serine esterase activity correlated with the level of serine esterase activity correlated with the level of cytotoxicity. These data suggest that BSF-2 is a differentiation factor for CTL and that it functions in part by inducing proteins required for mediating target cell lysis.     

Cytotoxic lymphocyte-derived lytic granules do not induce DNA fragmentation in target cells

When target cells are exposed to CTL, they very quickly sustain nuclear damage, including DNA cleavage, and then they lyse. Nuclear damage of this type is not seen when cells are killed by antibody and C. The role of nuclear damage in the T cell-mediated killing process as well as the mechanism by which the killer cell induces this damage are unknown; however, accumulating evidence suggests that cytolysis may depend on induction of nuclear damage. The exocytosed contents of CTL granules are thought by many workers to mediate target cell lysis. We have now determined whether lytic granules also induce nuclear damage (DNA fragmentation) in cells which they lyse. They do not. In addition, no DNA fragmentation was detected in nuclei incubated with lytic granules or activated CTL. In summary, our results suggest that target cell DNA fragmentation induced by CTL is mediated neither by lytic granules nor by a CTL-derived endonuclease and support the view that the target cell is itself responsible for the internal damage it sustains.     

DNA Fragmentation Induced by Cytotoxic T Lymphocytes Can Result in Target Cell Death

Cytotoxic T lymphocyte (CTL)-mediated lysis is accompanied by fragmentation of target cell DNA into an oligonucleosome ladder, a hallmark of apoptosis. Is this a fortuitous coincidence, or could CTL be inducing lysis by activation of the suicide signal? In this report we demonstrate that CTL-mediated target cell death can be blocked with the drug aurintricarboxylic acid (ATA). The abrogation of death correlates with the inhibition of DNA fragmentation. While ATA prevented DNA fragmentation, it failed to significantly alter protein, RNA, or DNA synthesis in the cell lines over the dose range used. In addition, there was no inhibition of cell-cell interaction or granule exocytosis during CTL-mediated killing. ATA also significantly inhibited the cytolysis and DNA fragmentation mediated by isolated cytolytic granules, as well as the granular protein fragmentin. We developed an assay in which target cells could be separated from CTL after binding and programming for lysis. Once they had received the "kiss of death," target cells could be rescued from lysis (as indicated by inhibition of DNA fragmentation and increased target cell viability) by treatment with ATA. These results suggest that ATA blocks target cell death by inhibition of DNA fragmentation, and further, that chromatin degradation is a cause rather than a result of cell death in CTL-mediated lysis.     

Subcellular localization of perforin and serine esterase in lymphokine-activated killer cells and cytotoxic T cells by immunogold labeling

CTL, NK cells, and lymphokine-activated killer (LAK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by forming large pores on the plasma membrane of the target cell. Other proteins besides perforin are found in the cytoplasmic granules of effector lymphocytes, and these include a family of serine esterases. Ultrastructural immunogold labeling studies with antibodies against perforin and a serine esterase (MTSP-1, also known as granzyme A and SE-1) show that all the granules of LAK cells and a CTL cell line contain perforin and serine esterase. For both LAK cells and CTL, perforin has been located mostly in the fine granular matrix of the granules, whereas gold particles corresponding to serine esterase have been found in both the matrix and the cap regions of the granules. Results from double immunogold labeling indicate that perforin and serine esterase colocalize to the same granules.     

Differences in target cell DNA fragmentation induced by mouse cytotoxic T lymphocytes and natural killer cells

Fragmentation of YAC-1 target cell DNA during cytolysis mediated by mouse natural killer (NK) cells and cytotoxic T lymphocytes (CTL) was compared. Cleavage of nuclear chromatin was always an extensive and early event in CTL-mediated cytolysis, whereas with NK cell-mediated killing the degree of DNA fragmentation showed an unexpected relationship to the effector:target (E:T) ratio. At low NK:YAC-1 ratios, DNA fragmentation and 51Cr release were equivalent and increased proportionately until a ratio of about 50:1 was reached; at higher ratios, 51Cr release increased as expected but DNA fragmentation decreased dramatically. Comparison of time course data at E:T ratios producing similar rates of 51Cr release showed that the target cell DNA fragmentation observed in NK killing was not nearly as rapid nor as extensive as that observed with CTL effectors. These results suggest that NK cells induce target cell injury via two different mechanisms. One mechanism would involve lysis mediated by cell-to-cell contact, while the other may induce DNA fragmentation via a soluble mediator. In support of this notion, cell-free culture supernatants containing NK cytotoxic factor (NKCF) induced DNA fragmentation in YAC-1 cells. The DNA fragments induced by NK cells and NKCF-containing supernatants consisted of oligonucleosomes indistinguishable from those induced by CTL. The results presented here show distinct differences in target cell DNA fragmentation induced by CTL and NK cells, and suggest that these two effectors use different mechanisms to achieve the same end. CTL seem to induce DNA fragmentation in their targets by direct signaling, whereas NK cells may do so by means of a soluble factor.     

Immunogold labeling of perforin and serine esterases in granulated metrial gland cells

Cytotoxic T lymphocytes (CTL) and natural killer (NK) cells are cytolytic lymphocytes known to produce a pore-forming protein, named perforin or cytolysin, that lyses target cells by creating large pores on the target plasma membrane. Besides perforin, the granules of CTL and NK cells contain a family of serine esterases. Perforin has also been localized in granulated metrial gland (GMG) cells of the murine embryo implantation site by light microscopic immunostaining. Ultrastructural immunogold labeling with antibodies against perforin and a serine esterase (MTSP 1 or granzyme A) shows that GMG cells contain both perforin and serine esterases in the fine granular matrix of their granules. Perforin has been located in all of the granules, whereas gold particles corresponding to serine esterases have been found in most of the granules. Results from the double immunogold technique indicate that perforin and serine esterases colocalize to most of the same granules in GMG cells. This study supports the view that GMG cells are related to cytolytic lymphocytes.     

The cytotoxic lymphocyte protease, granzyme B, targets the cytoskeleton and perturbs microtubule polymerization dynamics

Granzyme B, a serine protease derived from cytotoxic T lymphocyte (CTL) and Natural Killer (NK) cell granules, plays an important role in coordinating apoptosis of CTL and NK target cells. Here, we report that granzyme B targets the cytoskeleton by cleaving and removing the acidic C-terminal tail of alpha-tubulin. Consistent with this, Granzyme B markedly enhanced rates of microtubule polymerization in vitro, most likely by removal of an autoinhibitory domain within the tubulin C terminus. Moreover, delivery of Granzyme B into HeLa target cells promoted dramatic reorganization of the microtubule network in a caspase-independent manner. These data reveal that granzyme B directly attacks a major component of the cell cytoskeleton, which may contribute to the incapacitation of target cells during CTL/NK-mediated killing.     

Antigen receptor-triggered secretion of a trypsin-type esterase from cytotoxic T lymphocytes

Exocytosis of cytolysin-containing granules from cytotoxic T lymphocytes (CTL) was studied with the use of granule enzyme (BLT esterase) as a convenient biochemical marker. Using cloned CTL, we demonstrate here that BLT esterase secretion into the supernatant is specifically triggered by antigen-bearing target cells and that this secretion is inhibited by soluble monoclonal antibodies against the antigen-specific T cell receptor (TcR). Immobilized anti-receptor antibodies induced efficient enzyme secretion in the absence of target cells, thus implying a direct involvement of TcR complex in triggering exocytosis of granules. These results support the role of the granule exocytosis in CTL functions and provide a quantitative and direct assay of a rapid CTL functional response to antigenic stimulation.     

Cytotoxic T lymphocytes induce different types of DNA damage in target cells of different origins

Nuclear changes may be important in the mechanism of CTL-mediated lysis. Rapid cleavage of target cell DNA into oligonucleosomes has been demonstrated as a very early event in CTL-mediated killing of murine hematopoietic targets. However, the results presented herein and by other investigators have shown that this extensive dsDNA fragmentation does not occur in all CTL targets. In terms of actual DNA damage, there is a wide range in the extent and type of DNA cleavage in various targets. Differences exist at both the species and the cell lineage level. The extent of DNA damage generally corresponds to the efficiency of lysis; thus, murine hematopoietic cells, which undergo dsDNA fragmentation, are killed more rapidly and at lower E/T cell ratios than are murine nonhematopoietic cells, which sustain single-stranded nicks. Experiments using cloned CTL demonstrate that the same effector cell kills both hematopoietic and nonhematopoietic targets, producing different types of DNA damage. These observations indicate that the fate of the target cell DNA is determined by the nature of the target cell and not by the CTL. We propose that DNA damage results from an enzyme pathway inherent to the target, which is activated by, not transferred from, the CTL.     

Biology of cloned cytotoxic T lymphocytes specific for lymphocytic choriomeningitis virus. VI. Migration and activity in vivo in acute and persistent infection

Cloned cytotoxic T lymphocytes (CTL) specific for lymphocytic choriomeningitis virus (LCMV) were adoptively transferred to syngeneic mice acutely or persistently (carrier mice) infected with LCMV. Although infectious virus was cleared from the spleens during acute LCMV infection begun 24 hr earlier and the spleens remained clear of virus for the 4 days of testing, there was no concomitant reduction of viral titers in lymph nodes. In contrast, adoptive transfer of cloned CTL into animals with persistent rather than acute LCMV infection resulted in deaths of syngeneic but not allogeneic recipients. LCMV-immune spleen cells taken 30 to 50 days after a primary immunization and activated by in vitro stimulation before transfer also caused death of syngeneic carrier mice. However, LCMV-immune spleen cell per se provoked no clinical manifestations when transferred but cleared infectious virus and viral nucleic acid sequences from syngeneic carrier mice. The migration of 51Cr-labeled, LCMV-specific, H-2-restricted cloned CTL was assessed in vivo. The circulation of these CTL clearly differed from that of spleen cells freshly isolated from uninfected mice and from non-LCMV-specific CTL clone. Further, the circulatory pattern of LCMV-specific, H-2-restricted, cloned CTL in carrier mice was markedly different than in uninfected animals; only 7% of the injected cells remained in the lungs of uninfected mice 8 hr after injection, whereas 30% had accumulated in the liver. However, 55% of the cells injected into carrier mice still remained in their lungs 8 to 16 hr later. Hence, LCMV-specific, H-2-restricted, cloned CTL have unique trafficking patterns in the presence of LCMV antigens and immune activities in vivo.     

Cell death mediated by alloreactive cytotoxic T cells via the granule exocytosis or the Fas pathway is independent of p34cdc2 kinase: Fas dependent killing of cells arrested in the cell cycle

Inappropriate activation of p34cdc2 kinase has been shown to occur during apoptosis induced by cytotoxic T-cell derived perforin and fragmentin. We analysed the effect of two inhibitors of p34cdc2 kinase on alloreactive Tc-cell-mediated lysis and DNA fragmentation of P815 and L1210 target cells. Olomoucine, a specific inhibitor of cyclin dependent kinases, did not affect DNA fragmentation in the target cells. Lysis of olomoucine-treated target cells as assessed by 51Cr release over a typical 8-h period was also unaffected. We also examined the effects of thapsigargin on target cell death. This toxin causes increased intracellular calcium rises that then result in irreversible inhibition of cyclin dependent kinases, including p34cdc2 kinase. The same extent of specific cell lysis was induced by cytotoxic T cells from perforin(-/-), granzyme B(-/-), granzyme A(-/-), perforin(-/-) X granzymeB(-/-) X granzymeA(-/-) KO mice or normal mice in untreated target cells or target cells treated with either olomoucine or thapsigargin. Similarly DNA fragmentation measured by release of tritiated DNA was also unaffected. Thus inhibition of p34cdc2 kinase affects neither the Fas nor the perforin/granzyme pathways of alloreactive cytotoxic T-cell killing as measured by DNA fragmentation or chromium release. P815 cells treated with olomoucine were arrested in the cell cycle after 12-16 h exposure to the toxin. After cell cycle arrest, target cells now showed enhanced 51Cr release induced by effector cytotoxic T cells (CTL) derived from perforin(-/-) mice compared to untreated cells. This lysis was accompanied by an increase in cell surface Fas expression. Olomoucine induced cell cycle arrest and expression of Fas was reversible and when cells re-entered the cell cycle, surface expression of Fas was lost.     

Serpin-6 expression protects embryonic stem cells from lysis by antigen-specific CTL

The immune response to embryonic stem (ES) cells is still poorly understood. In this study, we addressed the adaptive cellular immune response to undifferentiated and differentiated ES cells infected with lymphocytic choriomeningitis virus (LCMV), a vertically transmitted pathogen in mice and humans. In contrast to the prevailing view, we found that undifferentiated and differentiated murine ES cells express MHC class I molecules, although at low levels. When cocultured with LCMV-infected ES cells, syngeneic but not allogeneic LCMV-specific CTL secrete IFN-gamma. Strikingly, LCMV-specific CTL do not efficiently kill LCMV-infected ES cells. ES cells showed high-level expression of the serine protease inhibitor 6, an endogenous inhibitor of the CTL-derived cytotoxic effector molecule granzyme B. Down-regulation of serpin-6 by RNA interference sensitized ES cells for CTL-induced cell death. The results of this study suggest that LCMV-infected murine ES cells present viral Ags and are recognized by LCMV-specific CTL in a MHC class I-restricted manner, yet resist CTL-mediated lysis through high-level expression of serine protease inhibitor 6.     

Virus specificity of cytotoxic T lymphocytes generated during acute lymphocytic choriomeningitis virus infection: role of the H-2 region in determining cross-reactivity for different lymphocytic choriomeningitis virus strains

We have compared the relatedness of five different strains of lymphocytic choriomeningitis virus (LCMV) as assessed by LCMV-specific cytotoxic T lymphocytes (CTL). Several different mouse strains were injected with each of the five LCMV strains, and the cross-reactivity of virus-specific CTL generated during the acute infection was tested by killing on a panel of target cells infected with the various LCMV strains. We found that the cross-reactivity pattern of LCMV-specific CTL generated in mice of H-2d haplotype (BALB/c WEHI and DBA/2) was strikingly different from that in mice of H-2b haplotype (C57BL/6 and C3H.Sw/Sn), suggesting that the fine specificity of LCMV-specific CTL is a function of the H-2 region. The characteristic cross-reactivity patterns were also observed in (C57BL/6 X DBA/2)F1 mice, demonstrating that the repertoire of the H-2b- and H-2d-restricted LCMV-specific CTL is not changed as a result of complementation by gene products of the other major histocompatibility haplotype. Studies with congenic BALB.B10 and (BALB.B10 X BALB/c)F1 mice firmly established that the characteristic cross-reactivity patterns of LCMV-specific CTL map to the H-2 region and are not influenced by background genes outside the major histocompatibility locus. These results suggest that LCMV determinants seen in the context of H-2d-restricting elements are different from those seen in the context of H-2b-restricting elements. Moreover, our studies show that CTL can be used as probes for dissecting differences among various LCMV strains, but the degree of relatedness between the different LCMV strains is not absolute when measured by CTL recognition. Since the H-2 region regulates the fine specificity of CTL generated during LCMV infection in its natural host, the degree of cross-protective immunity developed during a viral infection apparently depends on the major histocompatibility haplotype. The importance of these findings lies in understanding susceptibility or resistance of various host populations to viral infections and in designing vaccination programs to provide immunity.     

ECH, an epoxycyclohexenone derivative that specifically inhibits Fas ligand-dependent apoptosis in CTL-mediated cytotoxicity

CTL eliminate cells infected with intracellular pathogens and tumor cells by two distinct mechanisms mediated by Fas ligand (FasL) and lytic granules that contain perforin and granzymes. In this study we show that an epoxycyclohexenone derivative,(2R,3R,4S)-2,3-epoxy-4-hydroxy-5-hydroxymethyl-6-(1E)-propenyl-cyclohex-5-en-1-one (ECH) specifically inhibits the FasL-dependent killing pathway in CTL-mediated cytotoxicity. Recently, we have reported that ECH blocks activation of procaspase-8 in the death-inducing signaling complex and thereby prevents apoptosis induced by anti-Fas Ab or soluble FasL. Consistent with this finding, ECH profoundly inhibited Fas-mediated DNA fragmentation and cytolysis of target cells induced by perforin-negative mouse CD4+ CTL and alloantigen-specific mouse CD8+ CTL pretreated with an inhibitor of vacuolar type H+-ATPase concanamycin A that selectively induces inactivation and proteolytic degradation of perforin in lytic granules. However, ECH barely influenced perforin/granzyme-dependent DNA fragmentation and cytolysis of target cells mediated by alloantigen-specific mouse CD8+ CTL. The components of lytic granules and the granule exocytosis pathway upon CD3 stimulation were also insensitive to ECH. In conclusion, our present results demonstrate that ECH is a specific nonpeptide inhibitor of FasL-dependent apoptosis in CTL-mediated cytotoxicity. Therefore, ECH can be used as a bioprobe to evaluate the contributions of two distinct killing pathways in various CTL-target settings.     

Resistance of primary CD8+ cytotoxic T lymphocytes to lysis by cytotoxic granules from cloned T cell lines

Recent evidence has shown that cloned, murine CTL cell lines are resistant to the cytotoxic components of the toxic granules they release upon specific interaction with their target cells. Inasmuch as the resistance might be due to selection in culture over many months by repeated exposure to these cytolytic components (which are released repeatedly as a result of the cultured CTL being periodically stimulated by target cells), we asked whether primary CTL are also resistant. The primary CTL were elicited in vivo by i.p. injection of allogeneic tumor cells or in vitro by 5- to 6-day MLC or by 48-h exposure to the lectin Con A. The responding cells were separated into purified CD8+ (i.e., CD4-, CD8+) and purified CD4+ (i.e., CD4+, CD8-) T cell populations that were analyzed for cytolytic activity and for resistance to lysis by toxic secretory granules derived from cloned CTL cell lines. The CD8+ T cells were highly cytolytic and relatively resistant; they retained their cytolytic activity and were lysed to a minimal extent (0 to 10%) by quantities of isolated granules that lysed 80 to 90% of the P815 tumor cell line (tested as a representative standard cell line). The CD4+ T cells, in contrast, had only minimal cytolytic activity and were far more susceptible to granule-mediated lysis. Although the resistance of primary CD8+ T cells is impressive, it is not as pronounced as the resistance of the cloned CTL cell lines, indicating that during long-term culture there is some selection for increased resistance to granule-mediated lysis. In contrast to T cells (especially CD8+ T cells), Ia+ macrophages, isolated from primary immune peritoneal exudates, were highly susceptible to granule-mediated lysis.     

The role of Ca2+ and Mg2+ in the cytotoxic T lymphocyte reaction and in the secretion of N alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester-serine esterase by human T cell clones

Human T cell clones contain enzymes that can cleave the substrate N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (BLT). All CTL clones tested in this study secreted BLT-serine esterase activity, whereas only one of three tested non-cytolytic T cell clones secreted this enzymatic activity upon Ag-specific activation. BLT-serine esterase secretion could also be induced by the Fc gamma+ target cell Daudi in the presence of mAb specific for the TCR/CD3 complex, CD2, or the T cell activation Ag Tp 103. In addition, anti-CD3 and a mitogenic combination of anti-CD2 mAb, induced secretion of BLT-serine esterase in the absence of target cells, whereas anti-Tp 103 failed to do so. The secreted BLT-serine esterase activity induced by the various ligands was inhibited by the serine esterase inhibitors PMSF and m-ABA, but not by N-alpha-p-tosyl-L-lysine chloromethyl ketone. Significant BLT-serine esterase activity was induced by target cells or soluble anti-CD3 in the absence of extracellular Ca2+ ions, provided that extracellular Mg2+ ions were present. The cytotoxic activities by the human CTL clones were completely blocked under these conditions. All ligands that induced BLT-serine esterase secretion in the absence of extracellular Ca2+, induced a transient rise of intracellular Ca2+. Soluble anti-CD3 mAb did not induce a transient rise in intracellular Ca2+ or secretion of BLT serine esterase in CTL preincubated for 2 h with 5 mM EGTA. These findings indicate that mobilization of intracellular Ca2+ in human CTL clones is required for induction of secretion of BLT-serine esterase.     

Generation of large granular T lymphocytes in vivo during viral infection

Cytolytic lymphocytes were isolated from the spleens of lymphocytic choriomeningitis virus (LCMV)-infected mice and were characterized in regards to function, cell size, antigen phenotype, and cell morphology. Only 2% of the Lyt-2+ cells from uninfected mice were large granular lymphocytes (LGL), whereas 21% of the Lyt-2+ cells isolated 7 days postinfection were LGL. The day 7 Lyt-2+ populations contained all of the LCMV-specific, class I histocompatibility antigen-restricted cytotoxic T lymphocyte (CTL) activity, but no natural killer (NK) cell activity. The NK cell activity was consistently recovered in Lyt-2- populations isolated from both control mice and mice on day 7 postinfection. The LGL isolated on day 7 postinfection were concluded to be predominantly T cells and not NK cells because 1) the proportions of LGL in fractionated cell populations 7 days postinfection correlated with levels of CTL-mediated lysis but not NK cell-mediated lysis, 2) they were recovered in the Lyt-2+ population, and 3) antibody to asialo GM1, known to eliminate NK cell-mediated lysis but not T cell-mediated lysis, dramatically reduced NK cell LGL numbers in vivo on day 3 postinfection but only marginally affected LGL numbers on day 7. Virus-induced inflammation elicited a 50-fold increase in LGL numbers in the peritoneum on day 7 postinfection. The peritoneal exudate LGL were also associated with CTL activity and were resistant to treatment with antibody to asialo GM1. These results indicate that in vivo-generated CTL have the morphology of LGL and that the appearance of cytoplasmic granules correlates with the ability of cells to mediate lysis. To focus on cells being stimulated during infections, activated blast cells were separated from small resting cells by centrifugal elutriation. Coincidental with the peak in overall spleen leukocyte cytotoxic activity, the peaks of blast NK cells and CTL were at days 3 and 7 postinfection respectively. More than 50% of the blast lymphocytes isolated on either day 3 or day 7 postinfection were LGL. The CTL activity in the blast populations on day 7 postinfection was mediated by Lyt-2+ cells, and 37 to 64% of these Lyt-2+ blast cells were LGL. Cytolytic NK cell and CTL LGL could not be distinguished by morphology or by cell densities, because they overlapped in low density Percoll gradient fractions. Since this technique has been used to enrich for LGL, these data indicate that heterogeneity in LGL populations may result from the presence of both CTL and NK cell LGL.     

Involvement of granule proteins in T-cell-mediated cytolysis

Cytolytic T lymphocytes (CTL) and large granular lymphocytes contain dense cytoplasmic granules which, when isolated, are lytic for a variety of target cells. Granule proteins are released from the effector cell upon target cell interaction, further suggesting that they play a role in the cytolytic mechanism. Major proteins in CTL granules are a family of serine esterases (granzymes) and a pore-forming protein called perforin (cytolysin). Despite structural similarities between functionally conserved regions of perforin and the ninth component of complement (C9), these two lytic molecules are clearly distinct in their mode of target cell recognition. Perforin, unlike C9, is not dependent on a protein receptor molecule but binds to the target cell membrane via phosphorylcholine in a Ca2(+)-dependent manner. Here, we discuss the stimulus-secretion model for T-cell-mediated cytotoxicity with respect to our current understanding of perforin and the granzyme proteases.