Syntaxin 4 mediates endosome recycling for lytic granule exocytosis in cytotoxic T‐lymphocytes

Adaptive and innate immunity utilize the perforin‐killing pathway to eliminate virus‐infected or cancer cells. Cytotoxic T‐lymphocytes (CTLs) and natural killer cells mediate this process by releasing toxic proteins at the contact area with target cells known as immunological synapse (IS). Formation of a stable IS and exocytosis of toxic proteins requires persistent fusion of Rab11a recycling endosomes with the plasma membrane (PM) that may assure the delivery of key effector proteins. Despite the importance of the recycling endosomal compartment, the membrane fusion proteins that control this process at the IS remain elusive. Here, by performing knockdown experiments we found that syntaxin 4 (STX4) is necessary for cytotoxic activity and CD107a degranulation against target cells in a similar fashion to syntaxin 11, which is involved in lytic granule (LG) exocytosis and immunodeficiency when it is mutated. Using total internal reflection fluorescent microscopy we identified that STX4 mediates fusion of EGFP‐Rab11a vesicles at the IS. Immunoprecipitation experiments in lysates of activated CTLs indicate that endogenous STX4 may drive this fusion step by interacting with cognate proteins: Munc18‐3/SNAP23/VAMP7 and/or VAMP8. These results reveal the role of STX4 in mediating fusion of Rab11a endosomes upstream of lytic granules (LGs) exocytosis and further demonstrate the importance of this pathway in controlling CTL‐mediated cytotoxicity.      

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.     

Stepwise maturation of lytic granules during differentiation and activation of human CD8+ T lymphocytes

During differentiation, cytotoxic T lymphocytes (CTL) acquire their killing potential through the biogenesis and maturation of lytic granules that are secreted upon target cell recognition. How lytic granule load in lytic molecules evolves during CTL differentiation and which subsets of lytic granules are secreted following activation remains to be investigated. We set up a flow cytometry approach to analyze single lytic granules isolated from primary human CTL according to their size and molecular content. During CTL in vitro differentiation, a relatively homogeneous population of lytic granules appeared through the progressive loading of Granzyme B, Perforin and Granzyme A within LAMP1(+) lysosomes. PMA/ionomycin-induced lytic granule exocytosis was preceded by a rapid association of the docking molecule Rab27a to approximately half of the lytic granules. Activated CTL were found to limit exocytosis by sparing lytic granules including some associated to Rab27a. Our study provides a quantification of key steps of lytic granule biogenesis and highlights the potential of flow cytometry to study organelle composition and dynamics.     

Protein kinase C delta localizes to secretory lysosomes in CD8+ CTL and directly mediates TCR signals leading to granule exocytosis-mediated cytotoxicity

Lytic granule exocytosis is the major effector function used by CD8(+) CTL in response to intracellular pathogens and tumors. Despite recent progress in the field, two important aspects of this cytotoxic mechanism remain poorly understood. First, TCR-signaling pathway(s) that selectively induces granule exocytosis in CTL has not been defined to date. Second, it is unclear how Ag receptor-induced signals are converted into mobilization of lytic granules. We recently demonstrated that protein kinase C delta (PKC delta) selectively regulates TCR-induced lytic granule polarization in mouse CD8(+) CTL. To better understand how PKC delta facilitates granule movement, here we studied dynamics of intracellular localization of PKC delta in living CD8(+) CTL. Strikingly, we found that PKC delta localizes to the secretory lysosomes and polarizes toward immunological synapse during the process of target cell killing. Also, biochemical and structure-function studies demonstrated that upon TCR ligation, PKC delta becomes rapidly phosphorylated on the activation loop and regulates granule exocytosis in a kinase-dependent manner. Altogether, our current studies provide new insights concerning the regulation of TCR-induced lytic granule exocytosis by revealing novel intracellular localization of PKC delta, providing the first example of colocalization of a kinase with secretory lysosomes in CD8(+) CTL and demonstrating that PKC delta directly transduces TCR signals leading to polarized granule secretion.     

Syntaxin7 is required for lytic granule release from cytotoxic T lymphocytes

SNARE proteins are essential fusion mediators for many intracellular trafficking events. Here, we investigate the role of Syntaxin7 (Stx7) in the release of lytic granules from cytotoxic T lymphocytes (CTLs). We show that Stx7 is expressed in CTLs and is preferentially localized to the region of lytic granule release, the immunological synapse (IS). Interference of Stx7 function by expression of a dominant-negative Stx7 construct or by small interfering RNA leads to a dramatic reduction of CTL-mediated killing of target cells. Real-time visualization of individual lytic granules at the IS by evanescent wave microscopy reveals that lytic granules in Stx7-deprived CTLs not only fail to fuse with the plasma membrane but even fail to accumulate at the IS. Surprisingly, the accumulation defect is not caused by an overall reduction in lytic granule number, but by a defect in the trafficking of T cell receptors (TCRs) through endosomes. Subsequent high-resolution nanoscopy shows that Stx7 colocalizes with Rab7 on late endosomes. We conclude from these data that the accumulation of recycling TCRs at the IS is a SNARE-dependent process and that Stx7-mediated processing of recycling TCRs through endosomes is a prerequisite for the cytolytic function of CTLs.     

Behavior and Properties of Mature Lytic Granules at the Immunological Synapse of Human Cytotoxic T Lymphocytes

Killing of virally infected cells or tumor cells by cytotoxic T lymphocytes requires targeting of lytic granules to the junction between the CTL and its target. We used whole-cell patch clamp to measure the cell capacitance at fixed intracellular [Ca²⁺] to study fusion of lytic granules in human CTLs. Expression of a fluorescently labeled human granzyme B construct allowed identification of lytic granule fusion using total internal reflection fluorescence microscopy. In this way capacitance steps due to lytic granule fusion were identified. Our goal was to determine the size of fusing lytic granules and to describe their behavior at the plasma membrane. On average, 5.02 ± 3.09 (mean ± s.d.) lytic granules were released per CTL. The amplitude of lytic granule fusion events was ~ 3.3 fF consistent with a diameter of about 325 nm. Fusion latency was biphasic with time constants of 15.9 and 106 seconds. The dwell time of fusing lytic granules was exponentially distributed with a mean dwell time of 28.5 seconds. Fusion ended in spite of the continued presence of granules at the immune synapse. The mobility of fusing granules at the membrane was indistinguishable from that of lytic granules which failed to fuse. While dwelling at the plasma membrane lytic granules exhibit mobility consistent with docking interspersed with short periods of greater mobility. The failure of lytic granules to fuse when visible in TIRF at the membrane may indicate that a membrane-confined reaction is rate limiting.     

Signal strength controls the rate of polarization within CTLs during killing

Cytotoxic T lymphocytes (CTLs) are key effector cells in the immune response against viruses and cancers, killing targets with high precision. Target cell recognition by CTL triggers rapid polarization of intracellular organelles toward the synapse formed with the target cell, delivering cytolytic granules to the immune synapse. Single amino acid changes within peptides binding MHC class I (pMHCs) are sufficient to modulate the degree of killing, but exactly how this impacts the choreography of centrosome polarization and granule delivery to the target cell remains poorly characterized. Here we use 4D imaging and find that the pathways orchestrating killing within CTL are conserved irrespective of the signal strength. However, the rate of initiation along these pathways varies with signal strength. We find that increased strength of signal leads to an increased proportion of CTLs with prolonged dwell times, initial Ca²⁺ fluxes, centrosome docking, and granule polarization. Hence, TCR signal strength modulates the rate but not organization of effector CTL responses.     

Inhibition of cytotoxic T cell lysis by anti-CD8 monoclonal antibodies: studies with CD3-targeted cytolysis of nominal antigen-negative targets

The function of the CD8 molecule in lympholysis mediated by cytotoxic T cells was investigated by examining possible contributions of ligands on the target cell to the inhibition of lysis observed with CD8-specific mAb. In order to evaluate a variety of target cells, including those not expressing the nominal Ag (NA) for which the CTL was specific, lysis was effected by cross-linking the CTL and the target cells with anti-CD3 mAb. Such CD3 redirected cytotoxicity was demonstrated to be inhibited by anti-CD8 mAb when low anti-CD3 mAb concentrations were used. The possibility that inhibition by anti-CD8 mAb resulted for competition for the FcR between the anti-CD3 mAb and anti-CD8 mAb was eliminated by targeting TNP-modified cells with an antibody heteroconjugate prepared from Fab fragments of anti-CD3 and anti-DNP antibodies. Inhibition of the lysis of target cells not expressing NA including those deficient in class I expression, demonstrated that neither NA nor class I expression was required for anti-CD8 mAb inhibition. Whether the anti-CD8 mAb inhibition required CD8 Ag interaction with any ligand on the target cell was further investigated by measuring exocytosis of enzyme granule from CTL activated with CD3-coated poly-styrene beads. CD8-specific mAb inhibited such CTL activation in this target cell-free system. A CD8(+), MHC class II-specific CTL clone, was used to show differential inhibition by anti-CD8 mAb, depending on the target cell, therefore providing evidence that anti-CD8 mAb binding does not generate an absolute off signal. These data are consistent with the hypothesis that anti-CD8 mAb affect the lytic process independent of the recognition of a ligand on the target cell by CD8.     

Protein kinase Cdelta regulates antigen receptor-induced lytic granule polarization in mouse CD8+ CTL

Lytic granule exocytosis is the major pathway used by CD8+ CTL to kill virally infected and tumor cells. Despite the obvious importance of this pathway in adaptive T cell immunity, the molecular identity of enzymes involved in the regulation of this process is poorly characterized. One signal known to be critical for the regulation of granule exocytosis-mediated cytotoxicity in CD8+ T cells is Ag receptor-induced activation of protein kinase C (PKC). However, it is not known which step of the process is regulated by PKC. In addition, it has not been determined to date which of the PKC family members is required for the regulation of lytic granule exocytosis. By combination of pharmacological inhibitors and use of mice with targeted gene deletions, we show that PKCdelta is required for granule exocytosis-mediated lytic function in mouse CD8+ T cells. Our studies demonstrate that PKCdelta is required for lytic granule exocytosis, but is dispensable for activation, cytokine production, and expression of cytolytic molecules in response to TCR stimulation. Importantly, defective lytic function in PKCdelta-deficient cytotoxic lymphocytes is reversed by ectopic expression of PKCdelta. Finally, we show that PKCdelta is not involved in target cell-induced reorientation of the microtubule-organizing center, but is required for the subsequent exocytosis step, i.e., lytic granule polarization. Thus, our studies identify PKCdelta as a novel and selective regulator of Ag receptor-induced lytic granule polarization in mouse CD8+ T cells.     

Antigen sensitivity of CD22-specific chimeric TCR is modulated by target epitope distance from the cell membrane

We have targeted CD22 as a novel tumor-associated Ag for recognition by human CTL genetically modified to express chimeric TCR (cTCR) recognizing this surface molecule. CD22-specific cTCR targeting different epitopes of the CD22 molecule promoted efficient lysis of target cells expressing high levels of CD22 with a maximum lytic potential that appeared to decrease as the distance of the target epitope from the target cell membrane increased. Targeting membrane-distal CD22 epitopes with cTCR(+) CTL revealed defects in both degranulation and lytic granule targeting. CD22-specific cTCR(+) CTL exhibited lower levels of maximum lysis and lower Ag sensitivity than CTL targeting CD20, which has a shorter extracellular domain than CD22. This diminished sensitivity was not a result of reduced avidity of Ag engagement, but instead reflected weaker signaling per triggered cTCR molecule when targeting membrane-distal epitopes of CD22. Both of these parameters were restored by targeting a ligand expressing the same epitope, but constructed as a truncated CD22 molecule to approximate the length of a TCR:peptide-MHC complex. The reduced sensitivity of CD22-specific cTCR(+) CTL for Ag-induced triggering of effector functions has potential therapeutic applications, because such cells selectively lysed B cell lymphoma lines expressing high levels of CD22, but demonstrated minimal activity against autologous normal B cells, which express lower levels of CD22. Thus, our results demonstrate that cTCR signal strength, and consequently Ag sensitivity, can be modulated by differential choice of target epitopes with respect to distance from the cell membrane, allowing discrimination between targets with disparate Ag density.     

CTL-mediated killing of intracellular Mycobacterium tuberculosis is independent of target cell nuclear apoptosis

Two subsets of human CTL have been defined based upon phenotype and function: CD4(-) CD8(-) double-negative (DN) CTL lyse susceptible targets via Fas-Fas ligand interaction and CD8(+) CTL via the granule exocytosis pathway. CD8(+) CTL, but not DN CTL, can mediate an antimicrobial activity against Mycobacterium tuberculosis-infected target cells that is dependent on cytotoxic granules that contain granulysin. We investigated the role of nuclear apoptosis for the antimicrobial effector function of CD1-restricted CTL using the caspase inhibitor N:-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone. We found that DN CTL-induced target cell lysis was completely dependent on caspase activation, whereas the cytolytic activity of CD8(+) CTL was caspase independent. However, both DN and CD8(+) CTL-induced nuclear apoptosis required caspase activation. More important, the antimicrobial effector function of CD8(+) CTL was not diminished by inhibition of caspase activity. These data indicate that target cell nuclear apoptosis is not a requirement for CTL-mediated killing of intracellular M. tuberculosis.     

Localization of Fas ligand in cytoplasmic granules of CD8+ cytotoxic T lymphocytes and natural killer cells: participation of Fas ligand in granule exocytosis model of cytotoxicity

Fas ligand (FasL) has been implicated in cytotoxic T lymphocyte (CTL)- and natural killer (NK) cell-mediated cytotoxicity. In the present study, we investigated the localization of FasL in murine CTL and NK cells. Immunocytochemical staining showed that FasL was stored in cytoplasmic granules of CD8+ CTL clones and in vivo activated CTL and NK cells, where perforin and granzyme A also resided. Immunoelectron microscopy revealed that FasL was localized on outer membrane of the cytoplasmic granules, while perforin was localized in internal vesicles. Western blot analysis showed that the membrane-type FasL of 40 kDa was stored in CD8+ CTL clones but not in CD4+ CTL clones. By utilizing a granule exocytosis inhibitor (TN16), we demonstrated that FasL translocated onto cell surface upon degranulation of anti-CD3-stimulated CD8+ CTL clones. Moreover, TN16 markedly inhibited the FasL-mediated cytotoxicity by CD8+ T cell clones and NK cells. These results suggested a substantial contribution of FasL to granule exocytosis-mediated target cell lysis by CD8+ CTL and NK cells.     

Lipids are a constitutive component of cytolytic granules

Cytolytic granules are specific organelles of activated cytotoxic lymphocytes mediating storage and regulated excretion of lytic molecules for killing of target cells. A variety of the other granule components may also participate in granule-mediated cytotoxicity. In this study, the subcellular localization of lipids in the granules of human decidual CD56+ natural killer-like cells was determined by staining with malachite green aldehyde and imidazole-buffered osmium tetroxide. Lipids were shown, for the first time, to be a constitutive component of cytolytic granules. Lipids formed an additional structural microdomain, located between the granule-limiting membrane and the granule core. Images of the granules on serial sections suggested that intragranular lipids wrap the core. We speculate that granule lipids participate in packing of lytic molecules inside the granules, in autocrine signaling ending granule secretion, and in the killing process.     

Cloned cytotoxic T lymphocyte target cells fail to induce early activation events in effector cytotoxic T lymphocytes

We have explored further the basis for resistance of cloned cytotoxic T lymphocytes (CTLs) to cell-mediated cytotoxicity. We find that most cloned CTLs recognized as specific target cells by other cloned CTLs used as effector cells fail to activate three early events that may be critical in triggering lysis in the effector CTLs: Ca2+ influx, microtubule organizing center (MTOC) reorientation, and serine esterase release. To the extent that any or all of these events are involved in activation or expression of the lytic pathway in effector CTLs, our results suggest that in addition to being inherently resistant to cytotoxic granule extracts, many CTLs are also unable to induce lytic function in other (effector) CTLs. We have found one CTL clone that can respond to recognizable cloned CTL target cells with at least MTOC reorientation and serine esterase release, although the target CTLs are still not lysed. In this case, the resistance of the target CTL to lysis may be due solely to its resistance to cytoplasmic granule contents.     

CTLs contain and use intracellular stores of FasL distinct from cytolytic granules

CTL lyse target cells through the release of cytolytic granule contents and cell surface expression of Fas ligand (FasL). Current models suggest that FasL is stored in cytolytic granules and that FasL cell surface expression would be subject to the same controls as degranulation. We demonstrate that murine CTLs undergo two waves of FasL cell surface expression after stimulation. The first wave is from a pre-existing pool of FasL, and the second wave requires new protein synthesis. Signaling for FasL expression appears to be finely tuned as a weak signal preferentially induced surface translocation of the stored FasL, whereas a strong signal preferentially triggered the expression of de novo synthesized FasL. The early FasL is differentially regulated from degranulation, as there were multiple circumstances whereby rapid FasL cell surface expression and FasL-dependent killing occurred in the absence of detectable degranulation. Furthermore, we found through confocal microscopy that stored FasL resides in vesicles distinct from cytolytic granules. Our data clearly show that CTL degranulation and FasL lytic mechanisms are fully independent with respect to stored component localization and regulation.     

The role of the T cell receptor, CD8, and LFA-1 in different stages of the cytolytic reaction mediated by alloreactive T lymphocyte clones

The cytotoxic reaction mediated by cytotoxic T lymphocytes (CTL) consists of three phases: first, the CTL binds to the target cell; next, the CTL is triggered to lyse the target cell; and in the third phase, the CTL detaches from the target cell which is lysed in the absence of the CTL. Recently, we obtained evidence that human alloreactive CTL clones initially adhere to target cells without the involvement of the interaction between the T cell receptor (Tcr) and its specific target antigen. In the present study, we investigated the effect of monoclonal antibodies specific for the Tcr on the cytotoxic reaction of three CD8+ HLA-A2-specific CTL clones, using a single cell assay in which the binding event can be distinguished from the post-binding (lytic) phase of the cytolytic reaction. It was found that monoclonal antibodies directed at a variable part of the Tcr do not affect the binding phase but strongly block the lytic phase of the cytotoxic reaction. An anti-constant region Tcr antibody and an anti-CD3 reagent had a similar effect on the two phases of the reaction as the anti-variable part Tcr antibodies. In contrast, antibodies specific for LFA-1 strongly blocked the adhesion phase but did not affect the lytic phase. Antibodies specific for CD-8 had intermediate effects. They could block both the adhesion as well as the lytic phase. The effect of anti-CD8 appeared to be dependent on the CTL clone tested. One clone was found to be inhibited in the adhesion phase, but not in the lytic phase, whereas anti-CD8 hardly blocked the adhesion phase of two other CTL clones, but affected the lytic step of those clones. Our data indicate that LFA-1 is a major adhesion molecule in the CTL reaction, whereas the Tcr/CD3 complex is implicated in a phase after the initial formation of conjugates. CD8 is associated with both steps in the cytolytic reaction. In addition to its minor role in the adhesion phase, our data suggest strongly that CD-8 is involved in the triggering phase of the cytolytic reaction.     

Poxvirus-encoded serpins do not prevent cytolytic T cell-mediated recovery from primary infections.

Previous observations that the highly conserved poxvirus-encoded serpins inhibit cytotoxic activities of alloreactive CTL via granule and/or Fas-mediated pathways was taken to indicate their involvement in immune evasion by poxviruses. We now show that interference with 51Cr release from target cells by ectromelia and cowpoxvirus is limited to alloreactive but not MHC-restricted CTL. The data are in support of the paramount importance of CTL and its effector molecule perforin in the recovery from primary ectromelia virus infection and question the role of serpins in the evasion of poxviruses from killing by CTL. Further analysis of poxvirus interference with target cell lysis by alloreactive CTL revealed that suppression primarily affects the Fas-mediated, and to a lesser extent, the granule exocytosis pathway. Serpin-2 is the main contributor to suppression for both killing pathways. In addition, inhibition of lysis was shown to be both target cell type- and MHC allotype-dependent. We hypothesize that differences in TCR affinities and/or state of activation between alloreactive and MHC-restricted CTL as well as the quality (origin) of target cells are responsible for the observed phenomenon.     

The acquisition and maintenance of cytolytic activity by CD4+ murine T-lymphocyte clones.

The class II MHC antigen-specific CTL clones described in this report lose lytic activity when grown in exogenous rIL-2, but regain lytic activity when rIL-2 is removed from the culture medium. Using this cell model, we have investigated the metabolic activities (i.e., DNA, RNA, and protein synthesis) required for CTL to acquire or down-regulate lytic activity. DNA synthesis inhibitors (hydroxyurea and cytosine-arabinoside) and irradiation did not prevent CTL from gaining lytic activity. However, when protein or RNA synthesis was inhibited, these CTL could no longer acquire lytic activity. Furthermore, evidence showed that continuous RNA and protein syntheses were essential for CTL to exert their lytic function. Studies on cell surface antigen expression of CD3, CD4, Thy-1, and LFA-1 revealed no significant difference of antigen expression between a cloned CTL in its lytic and nonlytic states. Our data suggested that the synthesis of certain proteins and their encoded mRNA are essential for CTL to exert its lytic function and these proteins are not the cell surface antigens involved in CTL-target recognition or binding. Data also indicated that a granule enzyme, serine-esterase, was not involved in the expression of lytic activity in these CTL clones.     

Mechanisms of lysis by cytotoxic T lymphocyte clones. Lytic activity and gene expression in cloned antigen-specific CD4+ and CD8+ T lymphocytes.

Cloned murine Th having properties of either Th1 or Th2 cells as well as CD8+ CTL were tested for the capacity to lyse: 1) nucleated target cells bearing Ag or coated with anti-CD3 mAb, or 2) SRBC target cells coated with anti-CD3 mAb in a short term 51Cr-release assay. The lysis of SRBC occurs by a mechanism that does not involve nuclear degradation but presumably does involve membrane damage. Three patterns were observed: CTL and some Th2 cells lysed efficiently nucleated target cells and SRBC coated with anti-CD3 mAb. Th1 and some Th2 T cells lysed nucleated target cells but did not lyse efficiently the SRBC coated with anti-CD3 mAb. Finally, some Th2 cells failed to lyse efficiently either nucleated or SRBC targets. We also examined these clones for their expression of N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity, and for the expression of perforin or CTLA-1 (granzyme B) mRNA. Total N-alpha-benzyloxycarbonyl-L-lysin thiobenzyl esterase activity expressed by CTL and Th2 clones tended to be higher than that of Th1 cells. Perforin mRNA and CTLA-1 mRNA were readily detectable in CTL and some Th2 clones. Expression of perforin and CLTA-1 mRNA correlated well with the capacity of these clones to lyse SRBC coated with anti-CD3 mAb. Our results show that some but not all Th2 clones have lytic characteristics similar to those of CD8+ CTL. Two mechanisms appear to contribute to their lytic process, one mechanism of lysis involves membrane damage that correlates with the expression of perforin mRNA; a second mechanism involves the induction of DNA degradation in the target cells. In contrast, some CD4+ effector cells appear to lack the capacity to lyse efficiently via the mechanism involving membrane damage and may only have the lytic activity associated with the capacity to induce DNA degradation.     

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.