The cytolytic T lymphocyte and its mode of action

While the binding step of cytolytic T lymphocyte (CTL) target cell interaction resulting in conjugate formation is a well-characterized event, there seems to be more than one mechanism whereby lymphocytes kill the target. In recent years, infliction of complement (C)-like "holes" (I.D. 10-20 nm) on the target cell membrane, believed to be produced by the Ca2+-dependent lytic protein(s) perforin/cytolysin of secretory lytic granule origin has been proposed to be the mechanism of lymphocytotoxicity. More recent evidence, however, suggests that Ca2+-dependent exocytosis of lytic granules (where detectable) is not involved in lymphocyte-mediated cytolysis. Furthermore, neither formation of C-like "holes" in targets exposed to CTL, nor higher-than-background levels of lytic granules, perforin or BLT-esterases, have been detected in highly potent, peritoneal exudate CTL (PEL) derived directly from the animal or in cytocidal PEL-hybridomas. Hence exocytosis of perforin and formation of the above pores may apply to certain effector cells, particularly those grown in vitro in IL-2, but not to in vivo primed CTL such as PEL. On the other hand, work from this laboratory with Ca2+ probes has shown that lysis induced by CTL such as PEL-not involving lytic granules, perforin or formation of the above "holes"-is preceded by a marked prelytic elevation of cytosolic Ca2+ in the target. CTL-induced target cell membrane perturbation--a direct result of receptor-mediated effector-to-target interaction or through a membrane-bound or secreted effector component(s)--may be responsible for triggering the prelytic influx of Ca2+ from external sources, or its mobilization from internal stores in the target. We propose that CTL-induced, persistent elevation of cytosolic Ca2+, above a critical level, rather than formation of 10-20 nm pores, is responsible for the catastrophic prelytic events observed in the target, such as bleb formation, metabolic exhaustion and DNA degradation, ultimately leading to lysis.     

The lysis of cytotoxic T lymphocytes and their blasts by cytotoxic T lymphocytes.

After binding to their targets, cytotoxic T lymphocytes (CTL) deliver a lethal hit signal, ultimately leading to target cell lysis, and then can recycle to lyse additional targets, without themselves being destroyed. If non-specific secreted lytic mediators are involved in such lysis. CTL survival would not be expected unless the effectors are immune to CTL-mediated lysis. Therefore the lytic susceptibilities of alloimmune peritoneal exudate lymphocytes (PEL), containing up to 50% CTL, and of the cytolytic PEL blasts (PEB), obtained by culturing with interleukin-2 (IL-2), were examined. 51Cr-labelled BALB/c (H-2d) anti-EL4 (H-2b) (d alpha b) PEL were lysed 88%, 78%, and 48% by C3H/eb (H-2k) anti-P815 (H-2d) (k alpha d) PEL, C57BL/6 (H-2b) anti-P815 (b alpha d) PEL and b alpha d PEB, respectively. Similarly, b alpha d PEL were lysed 82% and 21% by d alpha b PEL and PEB, respectively. b alpha d PEB were lysed 82%, 28-39% and 39-51% by k alpha d PEL, b alpha d PEL and b alpha d PEB, respectively, b alpha d PEB were lysed 29-55% by d alpha b PEL. Furthermore, the CTL-containing populations were no less susceptible to lysis than normal lymphocytes. Since the majority (80-90%) of cells in these two types of CTL-containing populations can be directly and specifically lysed by appropriately immunized PEL CTL, we conclude that both the lytic granule and perforin lacking (PEL) and containing (PEB) CTL are not a priori immune to CTL-mediated lysis. These findings are in accord with theories proposing lysis to be induced by receptor-mediated contact between effector CTL and target cells, and challenge those suggesting the involvement of secreted lytic mediators.     

Serial killing by cytotoxic T lymphocytes: T cell receptor triggers degranulation,re-filling of the lytic granules and secretion of lytic proteins via a non-granule pathway

CD8⁺ cytotoxic T lymphocyte (CTL) clones begin to synthesize the lytic proteins granzyme A, granzyme B and perforin after stimulation with allogeneic target cells. The lytic proteins are stored in the secretory granules which are released after cross-linking of the T cell receptor (TcR) upon target cell recognition. During lytic granule biogenesis granzyme A protein synthesis can be detected between 2 and 10 days after allogeneic stimulation of the CTL. Although granzyme A is stored in the lytic granules over this period, the majority of granzyme A synthesized is secreted directly from the CTL. TcR triggering of degranulation also results in new synthesis of the lytic proteins, which can be inhibited by cycloheximide (CHX). Some of the newly synthesized lytic proteins can be stored in the cell and refill the granules. But up to one third of granzymes A and B can be secreted directly from the CTL via the constitutive secretory pathway as shown by granzyme A enzymatic activity and immunoblots of secreted granzyme B, where one third of the protein fails to acquire the granule targeting signal. Perforin is also secreted via the constitutive pathway, both from the natural killer cell line, YT, and from CTL clones after TcR cross-linking. Constitutive secretion of the lytic proteins can be blocked by both CHX and brefeldin A (BFA). While BFA does not affect the directional killing of recognized targets, it abrogates bystander killing, indicating that bystander killing arises from newly synthesized lytic proteins delivered via a non-granule route. These results demonstrate that the perforin/granzyme-mediated lytic pathway can be maintained while CTL kill multiple targets. We show that CTL not only re-fill their granules during killing, but also secrete lytic proteins via a non-granule-mediated pathway.     

Prevention of antigen-induced microtubule organizing center reorientation in cytotoxic T cells by modulation of protein kinase C activity

Lysis of target cells (TC) by cytotoxic T lymphocytes (CTL) is achieved by directional exocytosis of cytolytic molecules-perforin and granzymes. They are stored within lytic granules which can be readily released following antigenic stimulation. Secretion of lytic molecules appears to be controlled by protein kinase C (PKC) activity, since specific modulators of PKC activity abolish the lysis of TC. We have examined the effect of PKC modulation on some of the earliest events in the perforin/granzyme-mediated cytotoxicity. De novo synthesis of perforin mRNA, required for the refilling of granules and sustained cytotoxicity, seems to be unaltered in the presence of PKC modulators. Immunofluorescent studies of CTL-TC conjugates revealed that PKC modulation impairs reorientation of the microtubule organizing center toward the contact point with the TC, which accounts for the specific direction of lytic granules exocytosis. Thus, it appears that PKC regulates exocytosis of lytic granules by governing microtubule reorganization, one of the initial steps in perforin/granzyme-mediated cytotoxicity.      

Three-color flow cytometric assay for the study of the mechanisms of cell-mediated cytotoxicity

Cytotoxic lymphocytes kill tumor or virus-infected target cells utilizing two mechanisms (1) release of lytic granules (containing perforin and granzymes) and (2) Fas ligand (FasL)/Fas or TNF initiated apoptosis. We have examined mechanisms of target cell lysis using a new Flow Cytometric Cytotoxicity Assay (FC Assay). Target cells were labeled with PKH 67 dye. Cell death was estimated by 7-amino-actinomycin (7-AAD) inclusion and annexin V-PE binding. A strong direct correlation has been found between the percentage of dead target cells in the FC Assay and the results of 51Cr release assay when human LAK and CTL were used as a model system. We have shown that both NK and CTL kill tumor cells mostly by granule-mediated mechanisms, as lysis was blocked by a perforin inhibitor Concanamycin A (Folimycin) but was significantly less sensitive to zVAD-FMK caspase inhibition. The FC assay allows accurate measurement of cell-mediated cytotoxicity as individual target cell death is detected directly.     

Immune privilege and FasL: two ways to inactivate effector cytotoxic T lymphocytes by FasL-expressing cells

The theory that Fas ligand (FasL)-expressing tumours are immune-privileged and can directly counterattack Fas-expressing effector T lymphocytes has recently been questioned and several alternative mechanisms have been proposed. To address this controversial issue, we analysed the impact of FasL-expressing tumours on in vivo-primed cytotoxic T lymphocytes (CTLs) and the mechanisms involved. CTLs were obtained from the peritoneal cavity (PEL) after in vivo priming with syngeneic or allogeneic murine tumour cells. We have found that PEL populations undergo Fas-based apoptotic cell death when co-cultured with FasL-expressing tumour cells and that PEL destruction of cognate targets in a 51Cr-release assay was markedly inhibited by the pre-exposure to either cognate or non-cognate tumour cells expressing FasL. Furthermore, cytocidal function of PEL was markedly inhibited by preincubation with FasL-negative tumour cells, if and only if they were the cognate targets of the CTL; this CTL inhibition involved FasL-Fas interactions. The killing function of 'bystander' PELs, reactive to a third-party target cell, was inhibited by co-cultivation with PELs mixed with their cognate target. This activation-induced CTL fratricide was not influenced by the expression of FasL on the cognate target cells. These studies demonstrate the existence of two distinct pathways whereby FasL-expressing cells inhibit in vivo-primed FasL- and Fas-expressing CTLs: first, by FasL-based direct tumour counterattack, and second, by FasL-mediated activation-induced cell death of the CTLs, which is consistent with the concept that FasL expression in vivo could play a role in inducing immune privilege.     

Regulation of Fas(Apo-1/CD95)- and perforinmediated lytic pathways of primary cytotoxic T lymphocytes by the protooncogene bcl-2

Cytotoxic T cells (CTL) induce cell death of their target cells either by the surface interaction between Fas ligand and Fas or by the release of perforin and granzymes. Both lytic pathways induce apoptosis yet it is not known whether identical or distinct apoptotic pathways are activated. The protooncogene bcl-2 is known to protect various hematopoietic cells from apoptosis induced by diverse agents. Here we show that overexpression of the Bcl-2 protein in the murine mastocytoma line P815 or in concanavalin A-activated splenocytes suppresses apoptotic cell death induced by allospecific primary cytotoxic T lymphocytes (CTL) in which only the Fas lytic pathway was functional. Bcl-2 also reduced target cell killing induced by CTL whose lytic activity was dependent on the perforin/granzyme pathway only. These data provide evidence that, in the target cells studied here, both perforin/granzyme and Fas apoptotic pathways are modulated by Bcl-2 and suggest that these two pathways converge at a step prior to Bcl-2 inhibition.     

Cytoplasts from cytotoxic T lymphocytes are resistant to perforin-mediated lysis

Cytotoxic T lymphocytes (CTL) contain a potent cytolytic pore-forming protein (PFP, perforin or cytolysin) localized in their cytoplasmic granules. In the presence of calcium, perforin lyses a variety of target cells (TC) non-specifically. CTL, however, are generally resistant to the lytic effect of perforin. In this work, cytoplasts from CTL and susceptible TC were made by centrifuging cells on a Ficoll density gradient in the presence of cytochalasin B. Characterization by electron microscopy and a serine esterase assay established that both CTL and TC cytoplasts were completely devoid of nuclei and CTL cytoplasts contained essentially no granules. CTL cytoplasts are just as resistant to perforin-mediated lysis as the intact CTL, and both TC and their corresponding cytoplasts are very sensitive to lysis. Furthermore, CTL cytoplasts are less effective than TC cytoplasts in inhibiting hemolysis, a property shared by the respective intact cells. These results indicate that soluble granular components do not confer resistance on CTL, and suggest that the protective agent(s) acts by impeding perforin binding to the CTL membrane.     

Cytotoxic T lymphocyte perforin and Fas ligand working in concert even when Fas ligand lytic action is still not detectable

The reason(s) why individual cytotoxic T lymphocytes (CTL) possess a fast-acting, perforin/granzyme-mediated, as well as a much slower, Fas ligand (FasL) -driven killing mechanism is not clear, nor is the basis for wide variations in killing activity exhibited by individual CTL, ranging from minutes to hours. We show that perforin expression among individual, conjugated CTL varies widely, which can account for the heterogeneity in killing speeds exhibited by individual CTL. Despite a 2-hr lag in FasL-based killing, CTL lytic action is enhanced when the two mechanisms operate in concert. This is explained by finding that the two pathways in fact are jump-started simultaneously with the lag in FasL lytic action reflecting pre-lytic caspase-8 activation and BH3-interacting domain (BID) cleavage. The complementary action of the two lytic pathways, co-expressed at varying levels among individual CTL, facilitates the lytic action of late-stage poor perforin-expressing CTL, ensuring optimal cytocidal action throughout the CTL response.     

A novel flow cytometric assay focusing on perforin release mechanisms of cytotoxic T lymphocytes

CD8(hi+) cytotoxic T lymphocytes (CTL) are major players in immune defense. In addition, they contribute to the maintenance of immune homeostasis. We now describe a hitherto unavailable, but simple assay to determine ex vivo lytic granule-based cytotoxic functions of human CD8(hi+) CTL subgroups in a clinical setting, under target cell free conditions. Ficoll-isolated peripheral blood lymphocytes from 17 healthy volunteers were stimulated either by phorbol 12-myristate 13-acetate (PMA) in combination with ionomycin or by antibody mediated crosslinking of the CD3 molecule on the T cell surface. Using perforin as a marker for lytic granules, the reduction of CTL granules over time intervals up to 120 min was quantified by FACScan flow cytometry. The kinetics of perforin reduction were compared to the kinetics of NA-CBZ-L-lysine-thiobenzyl ester hydrochloride (BLT)-esterase release and of CD63 upregulation. The reduction in the perforin(+) portion of CD8(hi+) CTLs was correlated inversely with BLT-esterase release and CD63 upregulation. At 30 and 120 min after PMA/ionomycin stimulation, 55 +/- 14% and 42 +/- 14%, respectively, of CD8(hi+) CTLs still stained perforin(+) (time point 0 min = 100%). Perforin-granule release induced by CD3-crosslinking occurred as fast within 30 min (55 +/- 17%), but over the 120 min time interval it was not as complete when compared to PMA/ionomycin-stimulated perforin-reduction. Thus, the combination of an established degranulation assay with the power of immuno flow cytometry allows one to investigate the cytotoxic capability of CTL-subtypes and the kinetics of perforin-granule release. In addition, the assay may prove useful in the elucidation of intracellular signaling cascades governing the perforin-granule release process.     

A new quantitative (two-photon extracellular polar-tracer imaging-based quantification (TEPIQ)) analysis for diameters of exocytic vesicles and its application to mouse pancreatic islets

Cytotoxic T lymphocytes kill targets via secretion of lytic agents including perforin and granzymes. Recently, new methods have been developed to monitor cytotoxic T lymphocyte degranulation. These include detecting the appearance of lysosome-associated membrane protein on the cell's surface, and monitoring decreases in cellular perforin content. We have combined these methods with microscopy and flow cytometry to provide the first analysis of how single cytotoxic T cells degranulate. We used TALL-104 human leukaemic cytotoxic T cells as a model system, and stimulated them with thapsigargin and PMA, soluble agents that mimic the two major signalling pathways activated by T cell receptor cross-linking. Our results indicate that essentially every TALL-104 cell responds to maximal stimulation by releasing about half of its lytic granule complement. This reflects complete release of the contents of half the cell's granules, rather than partial release of the contents of all of the granules. Sub-maximal stimulation reduces both the fraction of cells that respond and the magnitude of single cell responses. We find that individual cells respond to maximal stimulation with a variable latency, and provide evidence that, once it starts, degranulation is a slow process taking tens of minutes.     

The actin cytoskeleton and cytotoxic T lymphocytes: evidence for multiple roles that could affect granule exocytosis-dependent target cell killing

One important mechanism cytotoxic T lymphocytes (CTLs) use to kill virus-infected, transplanted or tumour targets is exocytosis of granules that contain cytotoxic agents such as perforin and granzymes. Granule exocytosis-dependent target cell killing is a complex process, involving initial T-cell receptor (TCR)-dependent signalling that includes Ca²⁺ influx and activation of protein kinase C, shape changes that serve to bind the CTL to the target and, finally, exocytosis of lytic granules at the site of contact with the target cell. Although there is reason to propose that multiple steps in the lytic process could involve the actin cytoskeleton of CTLs, few studies have examined this issue, and those that have do not allow the specific step(s) involved to be determined. We have used the potent membrane-permeant actin cytoskeleton-modifying drugs jasplakinolide and latrunculin A to investigate the actin dependence of defined processes that are expected to be important for granule exocytosis-dependent killing. Our results, obtained using TALL-104 human leukaemic CTLs as a model system, are consistent with the idea that a functional actin cytoskeleton is required for TCR/CD3-dependent signalling, for activation of store-dependent Ca²⁺ influx and for CTL shape changes. When cells were stimulated with solid-phase anti-CD3 antibodies, treatment with either jasplakinolide or latrunculin A abolished granule exocytosis. However, when cells were stimulated in a manner that bypasses TCR/CD3-dependent signalling, granule exocytosis was not significantly altered, suggesting that the actin cytoskeleton does not function as a barrier to exocytosis.     

Lytic granules, secretory lysosomes and disease

Lytic granules harbour many of the dangerous apoptosis-inducing molecules of the immune system, including perforin, granzymes and Fas ligand. Safe transport, storage and release of these lytic components is vital. As a secretory lysosome, the lytic granule is able to accomplish these roles, as well as conferring the lysosomal functions of cytotoxic T lymphocytes and natural killer cells. Secretory lysosomes are common to many other haemopoietic cells and also melanocytes. Many of the proteins used in lysosomal secretion are found in both melanocytes and hemopoietic cells, and are dysfunctional in genetic diseases with defects in these proteins. The genetically heterogeneous Hermansky-Pudlak syndrome represents an excellent model for revealing proteins involved in secretory lysosome functioning. However, studies of this disease reveal differences between the various different types of secretory lysosomes, including lytic granules.     

Cell death‐independent functions of granzymes: hit viruses where it hurts

Granule exocytosis by cytotoxic lymphocytes is the key mechanism of our immune response to eliminate virus‐infected cells. These lytic granules contain the pore‐forming protein perforin and a set of five serine proteases called granzymes (GrA, GrB, GrH, GrK, GrM) that display distinct substrate specificities. Granzymes have mostly been studied for their ability to induce cell death. However, viruses have evolved many inhibitors to effectively block apoptosis. Evidence is emerging that granzymes also use noncytotoxic strategies to inhibit viral replication and potential viral reactivation from latency. Granzymes directly cleave viral or host cell proteins that are required in the viral life cycle. Furthermore, granzymes induce a pro‐inflammatory cytokine response to create an antiviral environment. In this review, we summarize and discuss these novel strategies by which the immune system counteracts viral infections, and we will address the potential therapeutic applications that could emerge from this intriguing mechanism. Copyright © 2011 John Wiley & Sons, Ltd.     

Delivering the kiss of death: progress on understanding how perforin works

Killer lymphocytes release perforin and granzymes from cytotoxic granules into the immunological synapse to destroy target cells as a critical mechanism in the defense against viruses and cancer. Perforin, a Ca(2+)-dependent pore-forming protein that multimerizes in membranes, delivers granzymes into the target cell cytosol. The original model for perforin (acting by forming a cell membrane channel through which granzymes pass) does not fit the experimental data. Recently, an alternative model has been proposed that involves active target cell collaboration with perforin to deliver granzymes and direct the target cell to an apoptotic, rather than necrotic, death.     

The lymphocyte pore-forming protein perforin is associated with granules by a pH-dependent mechanism

A pore-forming protein (PFP, perforin or cytolysin) has been found in the cytoplasmic granules of cytotoxic T lymphocytes (CTL) and natural killer (NK) cells. Extraction of granules with high-salt buffers or by freezing-and-thawing results in the release of perforin, which occurs only when the buffer pH is above 7.0. While high-salt extraction and freezing-and-thawing of granules at low pH (below 7.0) do not result in perforin release, these treatments render granules susceptible to a subsequent incubation with low-salt buffers (pH 7-8) that then solubilizes perforin completely. Granules may thus have been made leaky by high-salt extraction or freezing-and-thawing that may occur regardless of the buffer pH, while dissociation of perforin from granules may be exquisitely pH-sensitive. Freezing-and-thawing intact CTL and NK cells in physiological buffers with pH in the range of 7-8 (but not below 7) also causes release of perforin activity to the cell supernatant, thus providing a simple procedure by which perforin activity can be quantitated in small cell samples. Our results suggest that during lymphocyte-mediated killing, the extracellularly released perforin may rapidly dissociate from granules as a result of pH change and, in the process, become cytolytically active.