P.A. HENKART AND M.V. SITKOVSKY CYTOTOXIC LYMPHOCYTES

Two ways to kill target cellsOf the two pathways implicated in target-cell damage by cytotoxic

lymphocytes, secretory granule exocytosis seems to dominate in anti-viralimmunity whereas the Fas pathway may regulate immune responses.

Cytotoxic lymphocytes are believed to be crucial for theefficient immunological control of many infections, andthey have the potential to be used as exquisitely selectivetherapeutic agents against tumors - for example, in theform of 'cytotoxic T lymphocyte vaccines' directedspecifically against tumor cells. There are two develop-mentally distinct lineages of cytotoxic lymphocytes: thecytotoxic T lymphocytes (CTLs) and the natural killer(NK) cells. These two types of cell recognize target cellsby using entirely different molecular interactions, and themolecules used by CTLs have recently been pinpointed.It is now clear that T-cell receptors borne on the surfaceof each CTL recognize antigen-derived peptides, incomplexes with class I molecules of the major histocom-patibility complex (MHC), which are carried on the sur-faces of target cells. In contrast, the receptors used byNK cells to recognize their targets remain undefined,although recent experiments suggest that, again, thetarget-cells' MHC molecules may be involved [1].

Lymphocyte-mediated cytotoxicity is a multi-stepprocess in which the effector cells (CTLs and NK cells)act by forming cell-cell contacts with potential targetcells and screening them for the expression of triggeringligands (Fig. 1). Thus, a CTL may contact numerouspotential target cells before finding one that carries aforeign peptide-MHC complex, which triggers deliveryof a 'lethal hit' by the CTL. Until recently, the onlymolecularly defined cytotoxicity pathway was thatinvolving secretory granule exocytosis of preformedeffector molecules - the cytolysin/perforin proteins andgranzyme proteases (as reviewed elsewhere [1,2]).Cytolysin/perforin forms pores in the target cell's mem-brane, allowing passage of large proteins, such asgranzymes, as well as ions into the target cell.

The recent development of mice in which the gene forcytolysin/perforin is disrupted has provided direct evi-dence for the involvement of secretory granule exo-cytosis in the immunological clearance of virus in vivo,as well as in cytotoxic activity in vitro [3]. The otherwisenormal activated CD8+ T cells of these 'gene knockout'mice were severely crippled in their ability to lyse spe-cific virus-infected or appropriate MHC-bearing targetcells in vitro. The activity of NK cells also seemedseverely reduced or eliminated in these knockout mice.Particularly striking were experiments involving virusinfection of these mice, which showed that inactiva-tion of the cytolysin/perforin gene abrogated thenormal ability of mice to clear a viral infection. As the

cytolysin/perforin gene is expressed only in cytotoxiclymphocytes, these experiments provide further evidencethat these cells play a crucial role in clearing at least someviruses in vivo.

Several recent studies have strongly implicated thegranzymes as additional important components in thedamage done to target cells by the granule exocytosispathway, as illustrated in Figure 2. Although they are notDNAses, granzymes can trigger the DNA fragmentationcharacteristic of apoptotic cell death, and they may act byderegulating normal cell-cycle control processes. One ofthe granzymes was recently shown to activate the cell-cycle regulator, the p34cdc2 protein kinase, inappropriatelyinitiating mitotic events such as the dissolution of thenuclear envelope to produce a 'mitotic catastrophe' [4].Moreover, transfection of a non-cytotoxic mast-cell linewith cytolysin/perforin and granzymes endows themwith potent cytolytic and nucleolytic activity againsttumor-cell targets [5]. Finally, CTLs from granzyme Bknockout mice were found to cause abnormally slow lysis

Fig. 1. The cycle of lysis by cytotoxic T lymphocytes (CTLs).Adhesion of a CTL to an antigen-bearing target cell allows T-cellreceptor triggering of the CTL, followed by CTL polarization andthen delivery of the 'lethal hit'. Dissociation of the CTL from thetarget cell is prompted by a yet-to-be-defined 'off' signal, allow-ing another cycle to begin. The same type of cycle is used by NKcells, although NK cells use different triggering receptors.

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Fig. 2. The two defined CTL cytotoxicitypathways. On the right is the granuleexocytosis pathway, consisting of amodified classical receptor-triggereddegranulation pathway using pre-exist-ing secretory granules in the CTL: theirhighly directed degranulation into thesynapse-like gap between the two cellsreleases water soluble cytolysin/perforinmolecules, which undergo a Ca2+-trig-gered conformational change renderingthem amphitropic (straight) so that theyinsert into lipid bilayers, aggregate andform pores capable of passing bothions and macromolecules, includinggranzymes. The pore-damaged mem-brane is repaired by endocytosis orshedding, but the granzymes enter thecytoplasm (their physiological target isstill unknown). On the left is the Faspathway, in which T-cell receptor sig-nalling again induces polarization, butalso up-regulates expression of the Fasligand, FasL; this cross-links Fas mol-ecules on the target cell, which stimulusmay or may not lead to cell death,depending on the target cell.

and damage to target-cell DNA compared to controls [6].Thus, the combined 'one-two punch' normally providedby cytolysin/perforin and the granzyme proteases may bethe strategy used by CTLs to overcome the target cells'ability to repair membrane damage [7].

It has been clear for some time that the granule exocyto-sis pathway does not account for all the cytotoxic activi-ties of cytotoxic lymphocytes. In the absence of Ca2+ inthe medium, no granule exocytosis or membranedamage by cytolysin/perforin can occur, but CTLs arenevertheless able to kill some target cells normally andthey retain significant activity against many other targets[8,9]. This Ca2 +-independent lysis has recently beenshown to be accounted for by the ability of CTLs tocross-link the Fas antigen that is carried on the surface oftarget cells [10]. Fas (also known as APO-1) is an unusualmembrane protein, in that its cross-linking by antibodiescan trigger cell death without the involvement of com-plement. This Fas-induced death occurs within hours,does not require the synthesis of RNA or protein, and isCa2+-independent.

The involvement of Fas in a second pathway of lympho-cyte-mediated cytotoxicity has received strong supportwith the identification on the surface of some CTLs of aligand that binds to Fas. This ligand, FasL, a member ofthe tumor necrosis factor (TNF) protein family, isexpressed on activated, but not on resting, T cells[11,12]. Transfection of monkey COS cells with FasLrenders them able to kill Fas-bearing target cells, andsoluble FasL also has potent cytotoxicity against suchtargets. It turns out that the effects on mice of mutationsaffecting the function of both Fas and FasL had alreadybeen described. The pr mutation of mice was found tomap in the gene encoding Fas, and FasL is encoded by a

different gene thatgld mutation maps.

is located in the region to which the

In both Ipr and gld mutant mouse strains, the Fas cyto-toxicity pathway is non-functional, and target cellsderived from Ipr mice can be killed by CTLs using onlythe Ca2 +-dependent granule exocytosis pathway. Whenthe killing ability of CTLs from cytolysin/perforinknockout mice was tested using target cells from Ipr(Fas) mutant mice, cytotoxic activity was not detectablein the CTLs. When both the cytotoxicity pathways areinactivated, as in this situation, no cytolytic activityremains in CTLs. Both Ipr and gld mutant mice arecharacterized by a striking lymphadenopathy andautoimmunity which become manifest as the mice age.When young, the mice have relatively normal immunesystems, and young gld mutant mice mount a typicalCTL response to MHC-bound antigens. But there is aclear defect in T-cell receptor-triggered programmedcell death in mature T cells from Ipr mutant mice [13],and it is possible that expression of both Fas and FasL onT cells (after activation) down-regulates T-cell expansionin response to antigen,

Thus, it seems likely that the Fas pathway is principallyinvolved in regulating immune responses rather than indestroying cells infected by intracellular mircoorganisms.Molecular approaches have allowed two distinct lympho-cyte cytotoxicity pathways to be defined, and have facili-tated assessment of their relevance both in vitro and invivo. The regulation of cell-cell contacts that mediateboth pathways, and the identification of downstreamevents that lead to cell death, remain to be clarified inboth cases, and it will be interesting to discover theextent to which the two death pathways share furtherelements in common.

DISPATCH 925

References1. Sitkovsky MV, Henkart PA: Cytotoxic Cells: Recognition,

Effector Function, Generation and Methods. Birkhauser; Boston:1993.

2. Podack ER, Hengartner H, Lichtenheld MG: A central role ofperforin in cytolysis? Annu Rev Immunol 1991, 9:129-157.

3. Kagi D, Ledermann B, Burki K, Seller P, Odermatt B, Olsen KJ,Podack ER, Zinkernagel RM, Hengartner H: Cytotoxicity mediatedby T cells and natural killer cells is greatly impaired in perforin-deficient mice. Nature 1994, 369:31-37.

4. Shi L, Nishioka WK, Th'ng J, Bradbury EM, Litchenfield DW,Grennberg AH: Premature p34cdc2 activation required for apopto-sis. Science 1994, 263:1143-1145.

5. Nakajima H, Henkart PA: Cytotoxic lymphocyte granzymestrigger a target cell internal disintegration pathway leadingto cytolysis and DNA breakdown. I Immunol 1994, 152:1057-1063.

6. Heusel JW, Wesselschmidt RL, Shresta S, Russell JH, Ley TJ:Cytotoxic lymphocytes require granzyme B for the rapid inductionof DNA fragmentation and apoptosis in allogeneic target cells. Cell1994, 76:977-987.

7. Morgan BP: Complement membrane attack on nucleated cells:resistance, recovery, and non-lethal effects. Biochem J 1989,264:1-1 4.

8. Trenn G, Takayama H, Sitkovsky MV: Exocytosis of cytolytic

granules may not be required for target cell lysis by cytotoxicT lymphocytes. Nature 1987, 330:72-74.

9. Ostergaard HL, Kane KP, Mescher MF, Clark WR: Cytotoxic T-lymphocyte mediated lysis without release of serine esterase.Nature 1987, 330:71-72.

10. Rouvier E, Luciani MF, Golstein P: Fas involvement in Ca2+-independent T-cell-mediated cytotoxicity. J Exp Med 1993,177:195-200.

11. Suda T, Takahashi T, Golstein P, Nagata S: Molecular cloning andexpression of the Fas ligand, a novel member of the tumor necrosisfactor family. Cell 1993, 78:1169-1178.

12. Suda T, Nagata S: Purification and characterization of the Fasligand that induces apoptosis. I Exp Med 1994, 179:873-879.

13. Russell JH, Rush B, Weaver C, Wang R: Mature T cells of auto-immune Ipr/Ipr mice have a defect in antigen-stimulated suicide.Proc Natl Acad Sci USA 1993, 90:4409-4413.

P.A. Henkart, Experimental Immunology Branch,National Cancer Institute, and M.V. Sitkovsky, Labora-tory of Immunology, National Institute of Allergy andInfectious Diseases, National Institutes of Health,Bethesda, Maryland 20814, USA.

THE JANUARY 1995 ISSUE (VOL. 7, NO. 1) OFCURRENT OPINION IN IMMUNOLOGY

will include the following reviews, edited by Harvey R. Colten and Jeff Ravetch, on Innate Immunity:

Complement: chemotaxis/chemotactic receptors by R. WetselComplement/Immunoglobulin interactions by M. Frank

Non-lymphoid cytokine receptors (shared subunits) by T. Kishimoto and T. TagaPentraxins by H. Gewurz

Innate immunity of insects by J. HoffmanPerforin knock-out mice by E. Podack

Macrophages by S. GordonThe role of NK cells in host-parasite interactions by G. Trinchieri and P. Scott

The same issue will also include the following reviews on Antigen Recognition,edited by Emil Unanue and Ken Rock:

Cell biology of class II MHC peptide presentation by E. UnanueChemistry of class I and II MHC presented peptides by H. Rammensee

Supply and transport of peptides presented by class I MHC molecules by J. HowardT-cell receptor recognition and antagonistic peptides by P. Allen and J. Sloan-Lancaster

NK cell recognition by W. YokoyamaMolecular chaperones in antigen presentation by D. Williams

New class I and II molecules and their role byJ. FormanB-cell tolerance and antigen presenting function by A. Basten