Cell-mediated Margaret University

K. Taylor

Progress is being made in determining factors

cloud the

cells, differences single cells

and J. John Cohen

of Colorado Medical School, Denver,

way the lethal hit is delivered Several

cytotoxicity

using

between

issue, including

Current

the

cells are activated,

the

events in the target

cell.

heterogeneity

of cytotoxic

fresh cells and cell lines, and the possibility

multiple

will be to define which

how cytotoxic

and the subsequent

Colorado, USA

cytotoxic cytotoxic

Opinion

mechanisms. mechanisms

in Immunology

Introduction This review focuses on recent advances in the understanding of cell-mediated cytotoxicity, a process of importance in resistance against viruses, certain other pathogens, grafts and tumors. As in past years, much research in this field centres on mechanisms of target cell injury. There is still no consensus about the way in which cytotoxic cells induce the death of their targets, but more workers are coming to the conclusion that there are multiple mechanisms and mediators. Some of the controversy undoubtedly comes from the natural tendency to simplify an experimental system so that only one mechanism can operate. The question of whether, in viva, a typical killer cell employs more than one means of attack at a time is still unresolved. We include in this review several studies which suggest that cytotoxic cells can become partially activated under certain circumstances, performing some but not all of their potential functions. A year ago Berke reviewed the field in these pages [ 11 and discussed two main competing models of cell-mediated cytotoxicity. These are generally called the internal disintegration and membrane attack models, which we sometimes think of as ‘suicide’ versus ‘murder’. The first model was based on observed differences between cell-mediated and complement-mediated cytotoxicity, especially in the target cell phenomena of zeiosis (violent membrane blebbing) and DNA fragmentation into nu cleosomes [24]. The second placed more emphasis on similarities to the effects of complement, with membrane pore formation followed by osmotic lysis [ 5,6]. In the ‘suicide’ model, the cytotoxic cell is assumed to trigger a self-destructive process in the target. Considerable interest now focuses on this process, because of its remarkable similarity to events seen in programed cell death, both morphological (zeiosis, nuclear collapse) and biochemical (internucleosomal DNA fragmentation) [ 7,8*,9*]. Thus, if all cells have the molecular mechanisms necessary to activate a self-destruction process when it

The

most

difficult

are significant

of

task

in vivo.

1992, 4:338-343

is physiologically appropriate for them to do so, it is reasonable to imagine that killer cells can switch this program on in any cell to which they are appropriately bound. This switch could involve transmembrane signaling with or without transfer of molecules, or the injection of signaling molecules. Another, attractive possibility is that the cells with the killer phenotype are able, when activated, to secrete toxic substances onto the target’s plasma membrane. The simplest of these molecules would be pore-forming, like the complement component C9. These models require a means by which the killer itself can escape destruction by its own secreted toxins. It has been suggested that killer cells are inherently resistant to these processes, but this remains a matter of controversy [ 10**,11,12].

Heterogeneity

of cells with the killer

phenotype There are two major problems in defining and studying the properties of cytotoxic cells. First, there are a number of naturally occurring cell types that are cytotoxic and have characteristics that frequently overlap; studies with primary cytotoxic cell cultures suffer from the heterogeneity and complex interactions of cells in the population. Second, the properties of cloned cytotoxic cell lines may easily drift from those of the parental cells, and this drift may differ in various laboratories according to the culture conditions used. A possible solution to some of these problems may come from recent studies in which cytotoxic clones were immortalized by infeating them with a retroviral vector containing the protein kinase C-y gene [ 13**]. These cells grew to a high density, and no longer required periodic antigen stimulation, although they remained interleukin (IL)-2-dependent. They were also fully active at antigen-specific cytolysis. It will be

Abbreviations ADCC-antibody-dependent

338

cell-mediated cytotoxicity; CTL-cytotoxic T lymphocyte; IL-interleukin; mA&monoclonal MHC-major histocompatibility complex; NK-natural killer; TCR-T-cell receptor. @ Current

Biology

Ltd ISSN 0952-7915

antibody;

Cell-mediated cytotoxicity Taylor and Cohen very useful if this approach can be used to immortalize lymphoid cells from primary cultures.

T ceils with a y6 receptor The classical cytotoxic T lymphocyte (CTL) is antigenspecific, MHC class I-restricted, and bears CD3, CD8 and an afi T-cell antigen receptor (TCR). A minor population of T cells in blood are not WC-restricted and bear CD3 and a y6 receptor. Clones or bulk populations of these cells, maintained in high concentrations of IL-2, have cytoplasmic granules typical of cultured CTLs or natural killer (NK) cells [I4]. They also express perform and serine esterase mRNA, and stain with perform antiserum, all of which are markers for the cytotoxic phenotype. Freshly isolated human y6 cells lyse the NK target K562, and will lyse P815 cells if retargeted with an anti-CD3 antibody [15*-l. They also contain abundant granules and perforin. The y6 T-cell and NKcell systems, both minor components of the lymphoid cell mass, have been proposed as a primitive immune surveillance mechanism. In some studies yF T cells may be confused with NK cells unless precautions are taken to exclude the NK cells.

Natural

killer

cell-surface

markers

and

receptors NK cells are defined by the innate ability to kill YAC-1 (mouse) or K562 (human) targets. Although their receptors for target cells have not yet been defined, NK cells have a number of surface markers that can transduce an activating signal. Among these is CDl6, an Ig Fc receptor that allows NK cells to act as effecters in antibody-dependent cell-mediated cytotoxicity (ADCC). Another is CD2, which is also found on T cells. Crosslinking CD2 or CD16 on NK cells causes tyrosine phosphorylation of the 6 chain, which NK cells express in the absence of any other members of the TCRCD3 complex [ 16.1; this results in the complete activation profile, with calcium flux, granule exocytosis and enhanced cytotoxicity. Activation via CD16 is sufficient for ADCC, but the effects of CD2 in ADCC or NK processes have not yet been established. In an attempt to identify the NK cell antigen receptor, NK cell lines have been developed by growing CD3- ,CDI6+ cells in the presence of allogeneic tumor lines [ 171. These lines now lyse the allogeneic target, although they retain the NK phenotype, including the expression of CD56, another human NK cell marker, which is identified by the monoclonal antibodies (mAbs), NKHl and Leul9. Two other mAbs were raised to these cell lines; with Ieul9, they identify a family of CD56 isoforms, of which there are at least four and probably more [ 18-]. We believe that the isoforms arise from a combination of alternative message splicing and post-translational modifications. These are the first clonally variable molecules that have definitely been associated with NK cells, and are

therefore candidates for the NK ‘antigen’ receptors (terminology for the ligands engaged by NK cells is problematic). It seems very likely that ~~56 is itself one isoform of the neural adhesion molecule, N-CAM, which is implicated in homotypic and possibly heterotypic cellular interactions in the brain [ 191. Perhaps all NKlike interactions are of the cell adhesion molecule type. NKRPl is a 60 kD homodimer expressed on the surface of all rat NK cells. The crosslinking of antibodies to NKR Pl activates the NK cell, which subsequently kills the B-cell hybridoma producing these antibodies 1201. Although soluble antibody blocks this killing, suggesting that NKRPl is both necessary and sufficient, it does not block killing of the typical NK target, YACl. This suggests that NKRPl is not the NK cell receptor for ‘antigen’. Gene cloning shows that the mouse homologue of NKRPl is a member of a gene complex on chromosome 6 that includes another NK-specific marker, Ly-49 [ 21.1; the designation NKC (natural killer complex) is proposed for this region. It will be interesting to learn more about this complex and the genes it contains.

Activation

of cytotoxic

cells

Although the subject of lymphocyte activation is dealt with elsewhere in this issue (Desiderio, pp 2X+-256), certain studies showing partial or split activation of cytotoxic cells are relevant to this review. A subpopulation of activated B cells (about the stage of CD38 expression) in the human tonsil has been identified [22-j which strongly stimulates NK cells to release interferon. These cells are resistant to lysis by NK cells, suggesting another role for NK cells in immunity; they might respond to antigen-stimulated lymphocytes thereby enhancing the inflammatory process. This phenomenon may be relevant to the observation that, at least with some cytotoxic cells retargeted by antibodies to a non-specific target, no lysis was seen after 8 h in vitro, nevertheless, tumor cell growth was effectively suppressed in vivo in a Winn-type assay [23]. When cytotoxic cells engage their targets, there is often a shape change and reorientation of granules towards the killer-target interface, followed by granule-content exocy tosis. As a number of authors have noted [1,8*,9*,24,25], this sequence is not always required for cytotoxic effector function, but when it occurs it seems to be relevant to the lytic process. The components of the sequence have now been functionally separated from one another. Thus, inhibitors of ADP-ribosyl transferase do not interfere with target binding or granule reorientation, but effectively inhibit calcium flux, granule exocytosis and target killing [26]. Ionomycin, which causes calcium flux from both internal stores and the extracellular space, caused granule movement but did not lead to secretion, unless phorbol ester was also present [27-l. This study also showed that both sources of calcium were important, as blocking either independently prevented granule movement and secretion; the cell seems to be able to distinguish between internally and externally-derived calcium.

339

340

lymphocyte

activation

and effector

functions

A subpopulation of CD8 cells secretes IL-2 when activated; these cells may be of importance in allograft rejection [28] and may also function as helpers for other CD8 cells [ 291. A clone of this phenotype, which requires antigen and accessory cells for proliferation, was preincubated for 2 days with antigenic peptides and fixed accessory cells [IO**]. After this, it would not proliferate in response to correctly presented antigen, nor secrete IL-2. However, it was not fully anergized, as it retained all its ability to kill targets in the presence of antigen. Further studies are required to determine what signaling pathways are affected in these cells,

Cytotoxic cell resistance

to self-killing

As mentioned above, the exocytosis or ‘murder’ model of cytotoxic effector function implies a mechanism for keeping the killer unharmed by its own secretions. This subject has been discussed recently [30*], As a number of laboratories have reported that cytotoxic cells are resistant to cell-mediated cytolysis, it is interesting that two T-cell lines are described, both of which are human, MHC class I-restricted and specific for viral peptides. One of these does not self-kill in the presence of the appropnate peptide, although it does become anergized [ 121. The other is completely sensitive to self-killing [ 111. The significant difference between these two lines is not clear. There is always the possibility of an in vitro artifact when using cell lines; any cell that is capable of self-killing would be expected to do so during culture, and either disappear or eventually select for resistant variants, unless careful experimental design avoids that outcome.

Apoptosis

in target cells

Targets of CTIs and NK cells are usually observed to un dergo morphologic changes reminiscent of apoptosis. In the first example of apoptosis to be studied in detail, thymocytes dying from exposure to glucocorticoids were shown to initiate transcription of genes whose products brought about their death [31-l. From this it has been assumed that apoptosis always requires protein synthesis [26], but this is clearly not so. There are several examples of apoptosis actually being induced by protein synthesis inhibitors [ 32-341. The rapidity of CTL-induced DNA fragmentation, which begins within minutes of killer cell-target cell interaction, suggests that gene activation does not affect the process. Although cell-mediated cyto~ toxicity, as measured in a standard 4 hour assay, is not generally affected by inhibitors of transcription or translation [4], there are exceptions, apparently; a variety of agents inhibited lysis of YAK-1 by lymphocyte-activated killer cells, and P815 by a CTL line [35]. It is not clear why some authors lind that target cell apoptosis requires new macromolecular synthesis and others do not. In a comparison between CD8 and CD4 killers, only the lat-

ter were found to require new macromolecular synthesis by their targets for DNA fragmentation to occur [36]. The nature of DNA damage in target cells depends on the cell type, and not on the effector; some cells rapidly undergo internucleosomal DNA fragmentation as seen in apoptosis, while others develop frequent single-stranded nicks or rarer double-stranded breaks, which can be observed on alkaline and neutral sucrose-stabilized centrifugation [ 37**]. Whether this reflects fundamental differences in the mechanism remains to be determined. However, all target cells sustain some DNA damage during cytolysis. Although apoptosis is one outcome of target ceUCTL interaction, it does not seem to be obligatory for lysis. Sheep erythrocytes, lacking nuclei, cannot undergo at least the most obvious phenomena of apoptosis. However, when these cells were derivatized with antiCD3, they were effectively lysed by cloned CTIs or peritoneal exudate lymphocytes. Only the former released serine esterase, suggesting that these enzymes are not essential for the lytic process [38]. Lysis was prevented by agents that inhibit granule exocytosis, implicating lytic gran ules and perform in this form of cytotoxicity, although it is not clear whether peritoneal exudate lymphocytes contain perform. Although serine esterases and perform are known to be located in the same granules [ 391, they appear to be regulated independently [40]. Not all CTL clones can lyse sheep erythrocytes in a retargeted assay, and some CD4+ cytotoxic clones seem to be able to induce apoptosis but lack a direct membrane attack pathway [36,40]. It appears that a mixture of mechanisms is used by different killer cell types [ 411. This may be useful in viva. Any mechanism that required de nova protein synthesis by the target could be easily subverted by viruses that shut down host cell protein synthesis; in such cases, a membrane attack pathway would be essential in apoptosis pathway, not requiring protein synthesis, would allow rapid inactivation of viral nucleic acids within the target cell [42], while eventual lysis might be too late for safety.

Mediators

of cytotoxicity

Several molecules have been proposed as mediators of cellular cytotoxicity, including perform (or cytolysin, PFP), granule-associated serine esterases (‘granzymes’), other granule-associated proteins and ATP. Little is known about target-cell responses to these mediators, and’often the death of a target cell exposed to an isolated mediator is not very similar to its death when engaged by a killer cell. As discussed above, this uncertainty is a product of the (necessarily) reductionist approach to cytotoxicity. The mouse perform gene has been cloned [43] and is similar in structure to the human gene. Its 5’ regulatory region contains a number of interesting motifs, but does not yet reveal a killer-cell specific pattern. Freshly isolated

Cell-mediated

human peripheral blood y6 T cells contained perform protein [ 14,15**], while $3 thymocytes did not. Human granzyme B has been purified with full enzy matic activity, and characterized [44]. It is not inhibited by antithrombin III, which does inhibit the BLT (Ncr-benzyloxycarbonyl-L-lysine thiobenzyl) esterase commonly associated with lytic granules. Although this enzyme has not yet been shown to affect cytotoxicity, the availability of a purified preparation will facilitate these studies. Granzyme A binds to and cleaves nucleolin, a nuclear protein of unknown function [45]. It is difficult to see how this could be involved in DNA changes in target cells, because isolated granules do not induce DNA fragmentation in most workers’ hands. A curious new protein from cytotoxic cell granules has been cloned and sequenced [46*]. This protein, called TIAI, occurs in I5 and 40 kD forms. It does not cause lysis of intact cells, but will induce DNA fragmentation in digitonin-permeabilized thymocytes after several hours. This may mm out to be an important cytotoxic mediator, although the assay that demonstrates its DNA fragmenta tion capability has potential artifacts.

cytotoxicity

Taylor and Cohen

3.

RUSSEU JH: Internal Disintegration Model of Cytotoxic Lymphocyte-mediated Cytotoxicity. Biol Rev 1981, 56:153-197.

4.

EndonucleD~JKE RC, CHER~ENAKR, COHEN JJ: Endogenous ase-Induced DNA Fragmentation: An Early Event in CeU-mediated Cytolysis. Proc Nat1Acud Sci USA 1983, 8063616365.

5.

DENNERTG, PODACK ER: Cytolysis by H-2 Specific T Wer CeUs. Assembly of Tubular Complexes on Target Mem.branes. / Exp Med 1983, 157:14831495.

6.

HENKART PA, M~ARD PJ, REYNOLDSCW, HENKART MP: Cytolytic Activity of Purified Cytoplasmic Granules from Cytotoxic Rat Large Granular Lymphocyte Tumors. / Exp Med 1984, 160~75-93.

7/.

COHEN

JJ, DUKE RC, CHERVENAK R, SEILINS KS, OISON LK: DNA Fragmentation in Targets of CTL: an Example of Programmed Cell Death in the Immune System. In Mechanisms of Cell-mediated Cytotoxicity II. Edited by Henkart P, Martz E. New York: Plenum Press; 1985:493-506.

DUKE RC: Apoptosis in Cell-mediated Immunity. In Apoptosis The Molecular Basis of Cell Death. Edited by Tomei ID, Cope FO. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press; 1991:209-226. This review discusses two examples of death of the ‘programed’ phenotype occurring in a cell-mediated immune response, in T cells dependent upon growth factor after its removal and in targets of CTL. 8. .

COHEN JJ, DUKE RC, FADOK VA SELLINS KS: Apoptosis and Programmed CeU Death in Immunity. Annu Rev Immunol 1992, 10: in press. This review discusses the currently understood morphology and me& anism ofapoptosis, and includes many examples of cell death in the immune’system that display apoptotic characteristics, 9. .

Conclusion

,

Although the phenomenon of cell-mediated cytotoxicityhas been known for about 30 years, there has been little success in unravelling its mechanisms, partly because there are too many different kinds of cells with the killer phenotype; it is not known whether they can all use the same mechanism, or even if those cells with similar phenotypes use the same mechanisms. There is clear evidence of different mechanisms; targets can be killed by membrane attack, by induced apoptosis and even by secreted toxins such as tumor necrosis factor (discussed elsewhere in this issue; Ruddle, pp 327-332). Finally, it is difficult to do biochemical analyses on what must be a two-cell system. If an inhibitor is effective, did it block killer cell activation, binding or a target cell event? Given all these problems, it is encouraging to be able to record some real progress, a tendency towards umlied approaches, and a lessening of the competing-model ethos.

OTTEN GR, GERMAIN RN: Split Anergy in a CDS+ T CeU: Receptor-dependent Cytolysis in the Absence of Interleukin-2 Production. Science 1991, 251:12281231. A Line of CD8+ T cells, othetwise known to be capable of both cy totoxicity and IL~2 secretion, lost the ability to secrete IL-2, but not to kill after 2 days of incubation with peptide and fixed antigen~presenting cells. This ‘split anergy’ demonstrates that these ceils may have different functions depending on activation conditions. 10. ..

11.

MOSS DJ, BUR&WS SR, BAXTERGD: tkVlN MF: T CeU-T CeU KiUing is In&rced by Specific Epitopes: Evidence for an Apoptotic Mechanism. J Exp Med 1991, 173681686.

12.

Recogmtkm of HLA ROBBINS PA, MCMICHAELAJ: Immune Molecules Downmodulates CD8 Expression of Cytotoxic T Lymphocytes. J Exp Med 1991, 173:221-230.

13. ..

FINN OJ, PERSONSDA, BENDT KM, PII?AMIL, RICC~ARDI P: Retroviral Transduction of Protein Kinase C-y Into Cytotoxic T Lymphocyte Clones Leads to Immortalization with Retention of Specific Function. J Immunol 1991, 146:10991103. A new technique is described, in which murine CTL clones are immortalized with a retrovirai vector containing the protein kinases C-y gene. These clones no longer require periodic antigen stimulation, yet remain functionally identical to the parental CTLs. 14.

References

and recommended

Papers of particular interest, published view, have been highlighted as: . of interest .. of outstanding interest 1.

2.

BERKE G: T-cell-mediated 1991, 3:32&325.

reading

within the annual period of re-

Cytotoxicity.

Curr

Cpin Immunol

SANDER~ONCJ: Morphological Aspects of Lymphocyte Mediated Cytotoxicity. In Mechanisms of Cell-mediated Cytotoxicity Edited by Clark WR, Golstein P. New York: Plenum Press; 1982%21.

KO~ZIJMIH, Lru CC, ZHENG LM, JOAGSW,BAYNENK, HOLOSHITZ J, YO~INCJD: Expression of Perforin and Serine Esterases by Human yF T Cells. .I Exp Med 1991, 173:493-502.

NAKATAM, SMYIX MJ, NOIUHISAY, KAWGAKIA, SHINKAI Y, OKUMUKA K, YAGITA H: Constitutive Expression of Poreforming Protein in Peripheral Blood y6 T Cells: Implication of Their Cytotoxic Role in viuo. J Exp Med 1990, 172:1877-1880. Fresh y6 T ceils isolated from peripheral blood were able to lyse NKsensitive target K562 but not the NKresistant, lymphocyte-activated killer-sensitive target P815. The peripheral blood y6 T cells also possessed granules containing perform. 15. ..

16. .

VMER E, MORIN PM, O’BRIEN C, SZHL~SSMANSF, ANDERSON P: CD2 is Functionally Linked to the Zeta-Natural Killer Receptor Complex. Eur J Immunol 1991, 21:1077-1080.

341

342

lymphocyte

activation and effector functions

NK cells are known to express the 6 subunit of the TCR complex in association with the Fc receptor CD16. In this study, CD2 activation via mAb led to the phosphorylation of 5 on tyrosine, implying that CD2 is involved in NK activation, leading to ADCC. 17.

N, BL%NCHIE, B.&s H, SUZUKI T, BENDERJ, PARDI R, BRENNERCA, L~RRICKJW, ENGELMANEG: Natural ICiIIer Lines and Clones with Apparent Antigen Specificity. J Eap Med 1990, 172:457462.

SUZUKIN, SUZUKIT, ENGELMANEG: Evidence for the Involvement of CD56 Molecules in Alloantigen-spectic Recognition by Human Natural Killer Cells. J E~CI Med 1991, 173:1451-1461. with different mAbs, the authors Using sequential immunoprecipitation identified several isofomx of CD56. The mAbs stained most peripheral blood NK cells, and staining patterns for aUospecific NK cell lines were distinct for each line. The mAbs blocked the tysis of the appropriate allogeneic target, but not of the non-specific target K562. It was there fore proposed that the CD56 isoforms have some function in a limited allorecognition scheme. 19.

IANIER LL, TESTI R, BINDLE J, PHILLIPS RJ: Identity of Leu19 (CD56) Leukocyte Ditferentiation Antigen and Neural Adhesion Molecule. J Exp Med 1989, 169:2233-2238.

20.

RXANRC, NIEMI EC, GOLDFEIN RD, HISERODTJC, SW WE: NKR-Pl, an Activating Molecule on Rat Natural Wer Cells, Stimulates Phosphoinositide Turnover and a Rise in Intracellular Calcium. J Immunol 1991, 147:3244-3250.

21. .

YOKOYAMAWM, RYAN JC, HUNTER JJ, SMITH HR, STARK M, SF,AMAN WE: cDNA Cloning of Mouse NKR-PI and Genetic Linkage with LY-49: Identilication of a Natural KiIIer Cell Gene Complex on Mouse Chromosome 6. J Immunoll991, 147~32293236. Monoclonal antibodies specific for the rat NK cell marker NKR-Pl have previously been shown to stimulate lytic activity by rat NK cells. In this study the mouse homologue of NKKPl was cloned and proved to be genetically linked to the locus of Ly-49, another NK marker. The authors termed this region of mouse chromosome 6 the NKC or natural killer complex. 22. .

WYATT RM, DAWS~N JR: Characterization of a Subset of Human B Lymphocytes Interacting with Natural Wer Cells. J Immunol 1991, 147:3381-3388 A population of human tonsillar B cells, with a phenotype corresponding to a late activation stage, interacted with NK cells, results ing in interferon-y production. This suggests that NK cells may help to regulate the immune response, in addition to their cytotoxic effects. SEGAL DM, GARRIDO MA, QLW JH, ME~~ANANICAD, ANDREW S, PEREZ P, KURUCZ I, VALDAYOMJ, Trrus JA, WINKLER DF, WUNDERUCHJR: Effecters of Targeted Cellular Cytotoxicity. Mol Immunol 1990, 27:133%1342.

24.

GOSTEIN P, OJCIUS DM, YOUNG JD: CeU Death Mechanisms and the Immune System. Immunol Rev 1991, 121:29-65.

25.

BERKEG: Lymphocyte-triggered Internal Target Disintegration. Immunol Today 1991, 12:396399.

26.

NO~ICKI M, L/WDON C, SUGAWARA S, DENNERTG: Nicotinamide and 3-aminobenzamide Interfere with Receptor-mediated Transmembrane Signaling in Murine Cytotoxic T Cells: Independence of Golgi Reorientation from Calcium Mobilization and Inositol Phosphate Generation. Cell Immunol 1991, 132:115~126.

HAVERSTICKDM, ENGELHARDVH, GRAY Is: Three Intracellular Signals for Cytotoxic T Lymphocyte-mediated Killing. Independent Roles for Protein Kinase C, Ca*+ Influx and Ca*+ Release from Internal Stores. J Immunol 1991, 146:33063313. Using phorbol ester, ionomycin, and EGTA (ethylene-bis (oxyethylenenitrilo) tetraacetic acid), the authors showed that protein kinase C activation, the release of intracellular Ca* + and a Ca* + influx from the extracellular environment are all required for secretion of granules from. CTLs. Further, phorbol ester treatment alone produced shape changes in the CTLs, while ionomycin induced movement of granules within the CTLS.

27. .

ROSENBERGAS, MZOCHI T, SINGERA Evidence for Involvement of Dual-function T Cells in Rejection of MHC Class I Disparate Skin Grafts. J ./Zap Med 1988, 168:33-45.

29.

KUNG JT, CA~TILLOM, HEARD P, KERBACHER K, THOMASC% Subpopulations of CD8+ Cytotoxic T-cell Precursors Collaborate in the Absence of Conventional CD4+ Helper T-ceils. J Immunol 1991, 146~178%1790.

SUZUKI

18. ..

23.

28.

KRAHENBUHL 0, TSCHOPPJ: Perforin-induced Pore Formation. Immunol To&y 1991, 12:39’+402. i review of evidence for the perforidgranule exocytosis model of lymphocyte-mediated cytotoxicity. 30.

COHEN JJ: Programmed Cell Death in the Immune System. Adv Immunol 1991, 50~5585. this review of programed ceU death focuses on the current understanding of its mechanism, triggering and regulation, and provides examples in B and T lymphocytes.

31.

32.

MARTIN SJ, LENNONSV, BONHAMAM, COTIER TG: Induction of Apoptosis (Programmed CeU Death) in Human Leukemic HL-60 Cells by Inhibition of RNA or Protein Synthesis. J Immunol 1990, 145:185~1867.

33.

BANSALN, HOUE A, MELNYKOVYCH G: Apoptosis: Mode of CeU Death Induced in T CelI Leukemia Lines by Dexamethasone and Other Agents. FRSEB J 1991, 5:211-216.

34.

MCCALLCA, COHEN JJ: Programmed Cell Death in Terminally Differentiating Keratinocytes: Role of Endogenous Endonuclease. J Invest Dermatol 1991, 97:111-114.

35.

ZYCHIJNSKYA, ZHENG LM, LIU CC, YOUNG JD: Cytolytic Lymphocytes Induce Both Apoptosis and Necrosis in Target Cells. J Immunol 1991, 146:393$400.

36.

Ju S-T: Distinct Pathways of CD4 and CDS CeUs Induce Rapid Target DNA Fragmentation. J Immunol 1991, 146:812+18.

SELLINSKS, COHEN JJ: Cytotoxic T Lymphocytes Induce Different Types of DNA Damage in Target CeUs of Different Origins. J Immunol 1991, 147:79%303. The nature of DNA damage in the targets of CT& varied widely between target types, from fragmentation into oligonucleosomes to single stranded nickincr: the oattem observed was determined bv the type ._ of target cell involved anh not by the CTL.

37. ..

38.

RATNER 4 CLARKWR: Lack of Target Cell Participation in Cytotoxic T Lymphocyte-mediated Lysis. J Immunol 1991, 147:55-59.

39.

PETERSPJ, BOR~T J, OOFXHOT V, FUKUDAM, KRAHENBUHL0, TSCHOPP J, SIOT JW, GEUZE HJ: Cytotoxic LymphocytesT Granules are Secretory Lysozymes, Containing Both Perforin and Granzymes. J E@ Med 1991, 173:109Fl109.

40.

IANCKI DW, HSIEH CS, FITCH I%: Mechanisms of Lysis by Cytotoxic T Lymphocyte Clones: Lytic Activity and Gene Expression in Cloned Antigen-Specific CD4+ and CD8+ T Lymphocytes. J Immunol 1991, 146:3242-3249.

41.

SMYTHMJ, NOREIISAY, ORTAUX) JR: Multiple Cytolytic Mechanisms Displayed by Activated Human Peripheral Blood T CeU Subsets. J Immunol 1992, 148:55-62.

42.

SELLINSKS, COHEN JJ: Polyoma Viral DNA is Damaged in Target Cells During Cytotoxic T Lymphocyte-mediated Killing. J Viral 1989, 63:572-578.

43.

YOUN BS, Ltu CC, KIM KK, YOUNG JD, KWON MH, KWON BS: Structure of the Mouse Pore-forming Protein (Perforin) Gene: Analysis of Transcription Initiation Site, 5’ Flanking Sequence, and Alternative Splicing of 5’ Untranslated Regions. J Eqb Med 1991, 173:813-822.

44.

POE M, BLAKEJT, BOULTON DA, GAMMONM, SIGNALNH, Wu JK, ZWEERINKHJ: Human Cytotoxic Lymphocyte Granzyme B: Its Putication from Granules and the Characterization of Substrate and Inhibitor Specificity. J Biol C&m 1991, 266:9%103.

Cell-mediated 45.

PASTERNACK MS, BIHER KJ, MCINERNEY TN:

Binding to Target Cell to and Cleaves Nucleolin 266:14703-14708.

Granzyrne

A

Proteins: Granzyme A Binds in vitro. J Biol Cbem 1331,

TLW Q, STREW M, SAITO H, FCHLOSSMANSF, ANDERSON P: A to the Granules Polyadenylate Biding Protein Localized of Cytotoxic Lymphocytes Induces DNA Fragmentation in Target Cells. Cell 1331, 67:62%639. Two isoforms (15 and 40 kD) of the granule protein m-1 were cloned and characterized. The 4OkD isoform is an RNA-binding protein with specificity for ply(A) and with sequence homology to a known family of RNA~binding proteins. The 15 kD protein, the predominant form in

46. .

cytotoxicity Taylor and Cohen

cytolytic granules, appears to be derived from the 40 kD carboxytemk nal auxiliary domain via proteoiyW processing. Neither the 15 or 40 !dI forms cause chromium release from K562 or Molt-4 targets, but both induced DNA fragmentation in digitonin-permeabilized thymocytes after 12 hours of incubation.

.MK Taylor and JJ Cohen, Department of Microbiology and Immtinology, B-175, University of Colorado Medical School, Denver, Colorado 80262, USA.

343