Cerottini, J.-C

Tschopp, J.

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Annals of Oncology 8 (Suppl. 2): S75-S78, 1997.© 1997 Kluwer Academic Publishers. Printed in the Netherlands.Original articleNew knowledge about T-cell cytotoxicityJ.-C. Cerottini1 & J. Tschopp2^Ludwig Institute for Cancer Research, Lausanne Branch, University of Lausanne; Institute of Biochemistry, University of Lausanne, Epalinges,SwitzerlandKey words: apoptosis, CD8+ T cells, cytotoxicity, Fas, perforinIntroductionEver since the initial reports that antigen-specific cyto-lytic T lymphocytes (CTLs) are generated during allograftrejection or viral infection [1], many attempts have beenmade to elucidate the precise mechanism(s) by whichCTLs destroy target cells. Earlier in vitro studies demon-strated that target-cell lysis requires intimate contactbetween the CTL and its target and is highly specific, i.e.,it does not affect antigenically irrelevant bystander targetcells. The assessment of target-cell lysis in vitro was facili-tated by the development of assays based on the extra-cellular release of radioactive markers from the cytoplasm(e.g., 51Cr) or the nucleus (e.g., 125I-UdR) of labeled targetcells. Moreover, it was clearly established that CTL-medi-ated lysis, in contrast to lysis induced by antibody andcomplement, is not simply caused by a loss of the integ-rity of the target cell plasma membrane (resulting incolloid-osmotic lysis), but involves an active process sim-ilar, if not identical, to apoptosis that includes nuclearevents such as chromatin condensation and extensiveDNA fragmentation.Recent work indicates that two major mechanisms areresponsible, at least in vitro, for CTL-mediated lysis [2-5]:(1) the exocytosis of cytolytic granules at the site of mem-brane contact between the CTL and its target, resultingin the local delivery of soluble lytic proteins such asperforin and granzymes, and (2) the expression on theCTL surface membrane of a molecule, the Fas ligand(FasL), which induces a death signal upon engagementwith its receptor, Fas, on the target cell membrane.The existence of two independent lytic pathways hasbeen clearly established in vitro by using CTL generatedin mice lacking perforin or with a nonfunctional Fasligand as well as target cells derived from Fas-deficientmice. Moreover, such mice have been used to assess therelative role of the two pathways in T-cell-mediated effec-tor mechanisms in vivo. Here, we summarize the recentadvances made in the understanding of the molecularmechanisms operating in CTL-mediated lysis in vitro. Therelevance of these findings to T-cell immunity is brieflydiscussed.The perforin/granzyme-dependent pathwayCD8+ CTLs possess specialized lytic granules which,although they share distinctive features with regulatedsecretory granules (such as those found in neuroendo-crine cells, for example), are unusual in their nature, i.e.,they are 'secretory lysosomes'. Indeed, while in most se-cretory cells lysosomal and secretory granule componentsare packaged into separate organelles, the CTL lyticgranules are organelles combining both secretory andlysosomal compartments. In addition to classical lyso-somal proteins, the granules contain several other pro-teins, including perforin and a set of serine proteasestermed granzymes. Following recognition of the targetcell antigen (a complex made of an antigenic peptidebound to a major histocompatibility complex class Imolecule) by the CTL antigen receptor (TCR), the gran-ules within the CTL reorient toward the CTL target-cellcontact site and then fuse with the plasma membraneresulting in the polarized delivery of their content.It should be emphasized that CD8+ (naive) T cells donot possess lytic granules before they are activated in aprimary response to antigen. Once they are activated intoCTLs, the presence of lytic granules with stored proteinsprecludes the need for protein synthesis in perforin-dependent CTL-mediated lysis. It appears, however, thatCTLs not only secrete lytic granules when they interactwith target cells, but may also be triggered to synthesizeadditional lytic proteins that are stored in newly formedgranules or secreted directly from the cell via a constitu-tive secretory pathway [4]. While this phenomenon mayexplain the ability of a single CTL to kill in vitro severaltarget cells sequentially, its significance remains to bedefined in vivo.Perforin, one of the soluble lytic proteins stored inCTL granules, is a glycoprotein with sequence homologyto the complement component C9. It is generally agreedthat, following granule exocytosis, perforin monomerscan bind and insert into the target-cell plasma membraneand subsequently aggregate to form homopolymeric porestructures [3-6]. While pores visible by electron micro-scopy contain 10-20 aggregated monomers, there is evi-76dence that 3-4 monomers may already constitute a func-tional channel. While such pores may perturb the perme-ability of the target cell membrane, it is clear that thiseffect is not sufficient to explain the various apoptoticchanges associated with CTL-mediated lysis. The searchfor additional soluble mediators stored in CTL granuleshas led to the identification of a family of serine protei-nases termed granzymes [3, 7]. Granzyme B is by far themost abundant granzyme in CTL granules and cleavesproteins to the carboxyl site of acidic amino acids (e.g.,aspartic acid). This substrate specificity is very unusualamong serine proteinases. Another granzyme, granzymeA, exhibits a trypsinlike activity and thus cleaves proteinsto the carboxyl side of basic residues (e.g., arginine orlysine). Following the demonstration that granzyme A orB can induce DNA fragmentation in target cells permea-bilized with a sublytic dose of perforin, it has been pro-posed that granzymes are involved in target-cell apopto-sis. As these soluble mediators have to enter the targetcell in order to activate the apoptotic program, perforinmay promote their entry, either directly through the poly-perforin pores, or indirectly by stimulation of endocytosis.The absolute requirement for perforin and granzymeB in rapid CTL-mediated lysis has been confirmed un-equivocally by studies using CTL generated in mice inwhich the perforin gene or the granzyme B gene has beendestroyed by homologous recombination [7, 8]. Perforin-or granzyme B-deficient mice develop normally and pro-duce activated CD8+ cells upon appropriate antigenicstimulation. However, activated perforin-deficient CD8+cells are unable to lyse in vitro antigenically relevanttarget cells (provided these target cells do not expressFas; see below). Activated granzyme B-deficient CD8+cells, although they induce 51Cr release from target cells,are unable to induce rapid target cell DNA fragmenta-tion. While these findings do not rule out that other CTLgranule proteins may play a supplementary role, theypoint to the essential role of both perforin and granzymeB in the triggering of target cell apoptosis resulting fromCTL granule exocytosis.DNA damage induced by the perforin/granzyme-dependent pathway does not depend on new protein syn-thesis in the target cell. Thus, all the molecules necessaryfor this pathway are already present, in contrast to someother pathways leading to apoptosis. Although DNAfragmentation is a biochemical hallmark of apoptosis, acentral role for DNA cleavage in the initiation of apop-tosis has not been convincingly demonstrated. Indeed,it appears that intracellular proteases, and not endonu-cleases, are the primary initiators of apoptosis [9-11].Accordingly, recent attention has focused on the identi-fication of intracellular proteases that may serve as sub-strate^) for granzyme B. While interleukin 1 (3 convertingenzyme (ICE) has been proposed as a putative substrate,proteases related to ICE may be more relevant than ICEitself. For example, a recently identified member of ICE-related proteases involved in programmed cell death hasbeen shown to be activated by granzyme B. As this pro-tease, termed apopain/Yama/CPP32, is also involved inFas-dependent apoptosis (see below), it may represent atarget cell component essential for CTL-mediated lysis.The Fas ligand/Fas-dependent pathwayIn addition to the regulated and polarized exocytosis oflytic granules, another mechanism of CTL-mediated lysisinvolves the interaction of membrane-bound Fas ligand(FasL) on the CTL with its cognate receptor Fas on thetarget-cell surface membrane, resulting in a signal leadingto apoptotic cell death [12-14].FasL is a type II membrane protein belonging to thetumor-necrosis-factor family. Interestingly, FasL is hardlydetectable on the surface of CTLs before they interactwith target cells. It is therefore likely that engagement ofthe CTL TCR with its ligand on the target cell triggersFasL expression at the CTL surface, in addition to exocy-tosis of granules. In contrast to the latter phenomenon,however, inhibition of RNA or protein synthesis in aCTL prevents its ability to mediate target-cell lysis viathe FasL/Fas-dependent pathway, thus suggesting theneed for continuous FasL synthesis (or the production ofa putative FasL-activating protein).Fas (CD95) is a type I membrane protein belonging tothe tumor-necrosis-factor-receptor family. Upon properengagement (including its cross-linking), Fas can trans-duce an apoptotic signal. A 70-amino-acid-long segmentof the Fas cytoplasmic domain is necessary and sufficientfor transduction of the apoptotic signal and, hence, iscalled a death domain. There is considerable variability inthe expression patterns of Fas in various tissues. Abun-dant expression is found in liver, heart, kidney, and ovary.Lymphocytes transformed with human T-cell leukemiavirus, human immunodeficiency virus, or Epstein-Barrvirus, as well as activated lymphocytes, express a rela-tively large amount of Fas on the cell surface.The biochemical mechanisms involved in the Fas-dependent cytotoxic pathway are still poorly understood.As mentioned earlier, there is increasing evidence thatactivation of ICE-related cytoplasmic proteases such asCPP32 and/or ICE-LAP3 [15] is an important step in themediation of apoptotic death. Recent studies suggest thatsuch an activation could be triggered by the associationof the Fas cytoplasmic domain with an intracellularadaptor molecule termed FADD, leading to the assemblyof other proteins that participate in a cascade of enzy-matic reactions resulting in the cleavage of several cyto-plasmic and nuclear substrates. It is not yet clear whethera single or several of the substrates of ICE-related pro-teases may play a critical role in Fas-dependent cytotox-icity.The relative role of the FasL/Fas-dependent pathwayin CTL-mediated lysis has been clarified by the use ofCTL or target cells derived from mice with loss-of-func-tion mutations in FasL or Fas, respectively [12]. In micecarrying the gld (for generalized lymphoproliferative dis-ease) mutation, there is a replacement of a phenylalanineby a leucine in the extracellular C-terminus of FasL. This77substitution abolishes the ability of FasL to bind to Fas.In mice with the Ipr (for lymphoproliferative) mutation,transcription of the Fas gene is impaired by the insertionof a transportable element in an intron of the gene.Another allelic mutation (lprcg) in the Ipr locus results inthe replacement of an isoleucine by an asparagine in theFas death domain, which affects the ability of Fas totransduce the apoptotic signal.With the availability of such mice, it is now feasible toperform comparative in vitro assays in which normalCTL, perforin-deficient CTL, and FasL-deficient CTLare tested for lysis of Fas-positive or -negative target cells[16]. Studies along this line indicate that there is littlecross-talk between the two mechanisms of CTL-mediatedlysis: perforin-deficient CTL can perfectly lyse Fas-pos-itive target cells by means of the FasL/Fas-dependentpathway, whereas FasL-deficient CTL and Fas-negativetarget cells can lyse or be lysed, respectively, by means ofthe perforin-dependent pathway. Moreover, while there isdirect evidence that the two pathways are operative inCD8+ CTLs, it appears that CD4+ CTLs utilize primar-ily, although not exclusively, the FasL/Fas-dependentpathway, in contrast to NK cells, which utilize mainlythe perforin/granzyme-dependent pathway, although theyexpress FasL after activation [17,18].Significance for T-cell immunityIt is generally assumed that T-cell-mediated cytotoxicityis one of the major effector mechanisms responsible forimmunity to viruses, intracellular bacteria, and tumorcells. With few exceptions, this assumption is mainlybased on circumstantial evidence, i.e., lack or loss ofspecific immunity after depletion of CD8+ cells in vivo,or indirect detection (i.e., after expansion in vitro) ofspecific CTLs in immune mice. Since most CTLs alsosecrete lymphokines, including interferon-y, upon target-cell recognition, it is evident that effector mechanismsother than cell-mediated cytotoxicity may be responsiblefor CD 8+ cell-dependent immunity.The availability of perforin-deficient mice now allowsthe direct assessment of the role played by perforin-dependent cytotoxicity in immune reactions in vivo [8].The results reported so far indicate that the perforin-dependent cytotoxic pathway plays a crucial role in theimmunological control of some viral infections, but notof others (in spite of similar CTL responses). Similarly,perforin-dependent cytotoxicity has been shown to play amajor role in immune protection against Listeria mono-cytogenes, a model system for immunity against intra-cellular bacteria. Impaired immune rejection of tumorcells in perforin-deficient mice has been reported, butfurther work is needed to ascertain the role of perforin-dependent cytotoxicity in various model systems of tumorimmunity. An important role of perforin-dependentcytotoxicity has also been established in model systemsof autoimmune tissue damage [19]. As far as rejection ofallogeneic grafts is concerned, the role of perforin-dependent cytoxicity may be greatly influenced by thetissue or organ involved as well as by the participation, orlack thereof, of the graft vascular bed in the rejectionprocess.In contrast to perforin-deficient mice, lymphocyte de-velopment is abnormal in mice with Ipr ox gld mutations.Indeed, these mice develop lymphadenopathy and sple-nomegaly characterized by the accumulation of T lym-phocytes which do not express the CD4 or CD8 surfacemarkers. It is likely that these lymphocytes correspond tomature T cells which have not been eliminated afterantigen-induced activation because of the lack of a Fas-based mechanism normally involved in the regulation ofT-cell homeostasis. Because of such abnormality, the useof Ipr or gld mice to determine the role of FasL/Fas-dependent cytotoxicity in T-cell immunity is relativelylimited. As shown recently, however, short term studiesbased on the adoptive transfer of T cells from gld micemay bring direct information as to the participation ofFas-based immune mechanisms in vivo. In a model sys-tem of lethal graft-versus-host disease (GVHD), spleencells from perforin-deficient mice or from FasL-deficientmice were able to induce the disease, although mortalitywas delayed compared to that observed after transfer ofnormal T cells. In contrast, GVHD was abrogated whenspleen cells lacking both molecules were used as thesource of effector cells [20].ConclusionsIt is now clear that two independent pathways are in-volved in T-cell-mediated cytotoxicity. The first one isbased on the local delivery of lytic proteins, includingperforin and granzymes, normally stored in CTL gran-ules, whereas the second one depends on the expressionof both FasL on the CTL plasma membrane and Fas onthe target-cell membrane. Both pathways are triggered bythe engagement of the CTLTCR with its antigenic ligandand result in target cell apoptosis. While the biochemicalmechanisms involved in the apoptotic processes mediatedby either pathway are still poorly understood, activationof distinct target-cell cytoplasmic proteases belonging tothe ICE-like family may be a crucial step common toboth pathways. The relative role of the two pathwaysin T-cell-mediated immunological phenomena in vivois under intensive investigation thanks to the availabil-ity of mice deficient in one or the other molecule identi-fied in vitro as a key component of T-cell-mediatedcytotoxicity. Elucidation of the mechanisms leading totarget-cell apoptosis in vivo will further our understand-ing of the role of CTL in immunity and autoimmunity,and may provide new tools for manipulating their ac-tivity, positively or negatively, in a variety of immuneprocesses.78AcknowledgementsWe thank J. Dietschy for excellent secretarial assistance.Owing to the limited space allotted, review articles arecited wherever possible, resulting in the omission of mostprimary references.References1. Cerottini J-C, Brunner KT. Cell-mediated cytotoxicity, allograftrejection, and tumor immunity. Adv Immunol 1974; 18: 67-132.2. Berke G. The CTL's kiss of death. Cell 1995; 81: 9-12.3. Lowin B, Peitsch M, Tschopp J. Perforin and granzymes: Crucialeffector cell molecules in cytolytic T lymphocyte and natural killercell-mediated cytotoxicity. Curr Topics Microbiol Immunol 1995;198:1-24.4. Griffiths GM. The cell biology of CTL killing. Curr Opin Im-munol 1995; 7: 343-8.5. Moller G (ed). Molecular basis of T-cell cytotoxicity. Immunol Rev1995; 146: 5-266.6. Liu C-C, Walsh CM, Young JD-E. Perforin: Structure and func-tion. Immunol Today 1995; 16: 194-201.7. Smyth MJ, Trapani JA. Granzymes: Exogenous proteinases thatinduce target cell apoptosis. Immunol Today 1995; 16: 202-6.8. Kagi D, Ledermann B, Biirki K et al. Molecular mechanisms oflymphocyte-mediated cytotoxicity and their role in immunologicalprotection and pathogenesis in vivo. Annu Rev Immunol 1996; 14:207-32.9. Martin SJ, Green DR. Protease activation during apoptosis: Deathby a thousand cuts? Cell 1995; 82: 349-52.10. Kroemer G, Petit P, Zamzami N et al. The biochemistry ofprogrammed cell death. FASEB J 1995; 9:1277-87.11. Patel T, Gores GJ, Kaufmann SH. The role of proteases duringapoptosis. FASEB J 1996; 10: 587-97.12. Nagata S, Suda T. Fas and Fas ligand: Ipr and gld mutations.Immunol Today 1995; 16: 39-42.13. Nagata S, Golstein P. The Fas death factor. Science 1995; 267:1449-56.14. Takayama H, Kojima H, Shinohara N. Cytotoxic T lymphocytes:The newly identified Fas (CD95)-mediated killing mechanism anda novel aspect of their biological functions. Adv Immunol 1995; 60:289-321.15. Duan H, Chinnayan AM, Hudson PL et al. ICE-LAP3, a novelmammalian homology of the Caenorhabditis elegans cell deathprotein CED-3 is activated during Fas- and tumor necrosis factor-induced apoptosis. J Biol Chem 1996; 271:1621-5.16. Lowin B, Mattman C, Hahne M et al. Comparison of Fas (Apo-1/CD95)- and perforin-mediated cytotoxicity in primary T lympho-cytes. Int Immunol 1996; 8: 57-63.17. Williams NS, Engelhard VH. Identification of a population ofCD4+ CTL that utilizes a perforin- rather than a Fas ligand-dependent cytotoxic mechanism. J Immunol 1996; 156: 153-9.18. Tanaka M, Suda T, Haze K et al. Fas ligand in human serum.Nature Med 1996; 2: 317-22.19. Kagi D, Odermatt B, Ohashi PS et al. Development of insulitiswithout diabetes in transgenic mice lacking perforin-dependentcytotoxicity. J Exp Med 1996; 183: 2143-52.20. Braun MY, Lowin B, Acha-Orbea H et al. Cytotoxic T cells defi-cient in both functional Fas ligand and perforin show residualcytolytic activity yet lose their capacity to induce lethal acute graft-versus-host disease. J Exp Med 1996; 183: 657-61.Correspondence to:Prof. Jean-Charles CerottiniLudwig Institute for Cancer Research1066 EpalingesSwitzerland

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