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[30] SIMON, M.M., FRUTH, U., SIMON, H.-G. & KRAMER, M.D., A specific serine proteinase is inducible in Lyt-2+ ,L3T4- and Lyt-2- ,L3T4+ T cells in vitro but is mainly associated with Lyt-2+ ,L3T4- effector cells in vivo. Burop. J. Immunol., 1986 (in press). [31] KRAMER, M.D., BINNINGER, L., SCHIRRMACHER, V., MOLL, H., PRESTER, M., NERZ, G. & SIMON, M.M., Characterization and isolation of a trypsin-like serine protease from a long-term culture cytolytic T-cell line and its expression by functionally distinct T cells. J. Immunol., 1986, 136, 4644-4651. [32] SIMON, M.M., WELTZIEN, H.U., BOHRING, H.J. & EICHMANN, K., Aged murine killer Tcell clones acquire specific cytotoxicity for PS15 mastocytoma cells. Nature (Lond.), 1984, 308, 367-370. [33] ACHA-ORBEA, H., GROSCURTH, P., LANG, R., STITZ, L. & HENGARTNER, H., Characterization of cloned cytotoxic lymphocytes with NK-like activity. J. Immunol., 1983,130, 2952-2959. [34] PODACK, E.R. & KONIGSBERG, P.J., Cytolytic T-cell granules. Isolation, structural, biochemical, and functional characterization. J. expo Med., 1984, 160,695-710.

The authors would like to thank Gaby Nerz and Marlot Prester for their excellent technical assistance in all experiments, Rose Brugger and Gaby Eichhorn for secretarial assistance and Dr J. Langhorne for reading the manuscript.

THE ROLE OF LYMPHOTOXIN IN T-CELL-MEDIATED CYTOTOXICITY by N.H. Ruddle and D.S. Schmid

Department of Epidemiology and Public Health, Yale University, New Haven, CT (USA) The central thesis of this Forum contribution is that lymphotoxin (LT), a product of activated T cells, plays a role in T-cell-mediated cytotoxicity. The participation of soluble mediators in general, and LT in particular, has been a matter of some controversy. The work summarized in this manuscript indicates that this issue should no longer be under contention; the evidence supports the concept that several factors, including especially LT, are produced by cytolytic T cells (CTL) to carry out their cytotoxic activity. Lymphotoxin was first described [27] as a cytotoxic factor produced by rat lymphocytes after exposure to an antigen to which they had been

previously sensitized. Production of the factor, an in vitro correlate of delayed hypersensitivity (5, 26], was antigenspecific, though its effect, killing of syngeneic rat embryo fibroblasts, was neither antigen-specific nor species restricted. Almost simultaneously, a cytotoxic factor in the culture media of mitogen-activated lymphocytes was named lymphotoxin [12]. Though several size classes of LT have been described (15] Aggarwal and his colleagues isolated homogeneous LT of 20,000 to 25,000 MW from a human Blymphoblastoid line, RPMI -1788 [2]. Antibodies prepared against this factor neutralize the cytotoxic activity of nonadherent peripheral blood leukocytes

T-CELL-MEDIA TED CYTOTOXICITY [34], suggesting that the majority of Tcell-derived soluble cytotoxic activity is carried out by a single class of LT. The gene for human LT was cloned by Gray and his colleagues [13, 19], and we have recently cloned the gene for murine LT. Murine and human LT are remarkably similar with regard to gene organization [17], amino acid sequence (74 070 homology) [17] and chromosomallocation [16]. LT maps within or close to the respective major histocompatibility complexes of the two species. Murine LT is also slightly (35.5 070) homologous [17] to murine tumour necrosis factor (TNF) [21], a macrophage product. LT and TNF are functionally indistinguishable and compete for the same receptor [1]. It has been debated [25] whether the molecules should be called TNF-IX(TNF) and TNF-~ (LT). That nomenclature implies far too limited a biological role for these cytokines and will not be used here. Though it was originally suggested that LT could be a mediator of CTL killing, several arguments were advanced which appeared to invalidate such a role [3]. Recently, published data from several laboratories provide a more complete picture. The bulk of evidence to be summarized here indicates that lymphokines in general, and LT in particular. are involved in CTt. killing. LT is cytostatic and/or cytolytic for several cell lines. Though the L929 cell is the most frequently employed target. several other cell lines which are as sensitive, or even more sensitive to LT killing include rat embryo fibroblasts [271. mouse embryo fibroblasts [24], several lymphomas [33, 23J and thc mastocytoma, P815 [23]. a target frequently used in CTL killing assays. Rapidly proliferating normal Band T cells are also killed by LT [7, 23]. Thus. the gamut of cells [hat serve as targets for CTL are also killed by LT. CTL- and LT-producing cells are phenotypically identical and possess the same antigen and major histocompatibility (MHC) activation requirements. Though earlier data had indicated that CTL specific for transplantation antigens expressed the

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Lyt-2 differentiation antigen, and that helper T cells were of [he Lyt-l phenotype, it is now clear that the Lyt phenotype is more clearly a reflection of the nature of MHC restriction than of function [35]. That is, Lyt-l + (or more accurately L3T4 +) cells generally recognize antigen within the context of class II MHC molecules, and Lyt-2+ cells recognize antigen within the context of class I molecules. Several workers [36, 37] have now demonstrated that Lyt-l + class II-restricted cells can behave as cytolytic T cells and effect 51Cr release from presenting B cells or macrophages. Tite and Janeway [37] demonstrated that L3T4 + IL-lmaintained ovalbumin (OVA)-specific class-II-restricted T-cell clones killed OVA-coupled AlO, a B lymphoma. Several of these clones also functioned as helper cells and all produced LT [38]. In fact, the ability of such « killer helper» clones to produce LT positively correlated with the ability of individual clones to effect killing. Thus, LT induction is just as exquisitely antigen-specific and MHC-restricted as is activation of CTL. We have also prepared several T-cell dones which are conventional TNPspecific CTL. They are specific for antigen within the context of class I MHC molecules, are Lyt-2 +, and their cytotoxicity against antigen-coupled targets can be evaluated by 51 Cr release. Comprehensive studies of the TNP-CTL cells demonstrated that, in every instance, cell lines and clones which are ::apable of killing in an antigen-specific, MHC class-I-restricted fashion, also secrete LT under stimula[ion by antigen or mitogen [6, 7, 30]. In fact, the requirements for LT production are absolutely identical to those of CTL killing. Not only are these cells ,estricted to class I antigens for both LT induction and CTL killing, they are also restricted to a particular class I antigen, K k. These results have been recently confirmed by Northern blot analysis. RNA isolated from activated CTL clones hybridizes with a murine LT eDNA probe (Ruddle and McGrath, in preparation). We have shown that LT can induce rapid 51 Cr release from targets with

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kinetics identical to CTL killing [30]. Previously observed differences in the kinetics of LT and CTL killing could be explained if CTL kill by injecting a toxin. It would simply take a longer time for the cells to take up LT in the absence of a CTL delivery system. We have demonstrated that LT preparations from cytotoxic T-cell lines can cause short-term release (4 h) of 51Cr from target cells if introduced into the cytoplasm [30). A technique for rapidly internalizing macromolecules into the cytoplasm of fibroblasts was used to introduce LT into L929 cells. Under the conditions of pinocytosis stimulation (hypertonic incubation in polythylene glycol) and hypotonic shock which lyses newly formed pinocytic vesicles but not the cells (20), LT released 51Cr from the cells. If growth medium alone was rapidly introduced into target cell cytoplasm, no appreciable 51Cr release over background levels was detected in

any of these experiments. Thus, LT can kill over a period of time comparable to that required by CTL if it is first forcibly internalized. This somewhat artifical system might have generated subtle abnormalities in target cell physiology unaccounted for by our controls. Nevertheless, the results are consistent with the concept that LT kills just as rapidly and dramatically as CTL if it can be rapidly introduced into the cytoplasm of target cells. We have demonstrated that bystander killing does occur both in the case of class II [38] and class I-restricted CTL [31]. Figure I summarizes data generated in the course of an experiment in which bystander killing was demonstrated. The TNP-specific H2Kk-restricted CTL killed TNP-L929 and also destroyed bystander P8I5 cells, as demonstrated by 51Cr release. The killing of P8I5 is somewhat delayed in time behind the 4-h peak 51Cr release

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T-CELL-MEDIA TED CYTOTOXICITY from comparable cultures of effector cells and prelabelled specific targets. More time, on the order of an additional 6 to 12 h, is needed to readily demonstrate high levels of non-specific killing in the course of an ongoing specific cytotoxic reaction. This is consistent with the concept that specific targets are killed rapidly because of high local concentrations of toxic factors. Once the extra or intracellular receptors of the specific target cells are occupied by LT, additional LT diffuses to adjacent cells. We have previously demonstrated that the ability of a T cell to cause bystander kill is positively correlated with its ability to produce LT under comparable circumstances [38]. Furthermore, the ability of a cell to serve as an effective target in bystander kill is proportional to its LT sensitivity [23, 38]. When CTL kill targets, they themselves are not usually killed in the 4-h course of the experiment. Thus, the concept arose that CTL killing is unidirectional and that CTL and LTproducing cells were immune to the killing process. We have now shown that this is not the case [4, 23, 31] and that LT-producing cells kill themselves. If minimal numbers of target cells are used to present antigen and large amounts of LT are produced, the producing cells are killed. It is not known whether the cytotoxic effect of cultures grown under conditions of high amounts of antigen and low amounts of feeders is due to destruction from within or to bystander killing by LT after its release. Autodestruction of CTL was probably previously not commonly seen because, in that case, the antigen was an integral part of the target. It is likely that most LT induced in such circumstances is delivered to the target and does not act back on the T cells in the course of the experiment. If minimal numbers of targets cells are presented to absorb the LT, the phenomenon is easily demonstrated. LT-producing cells contain granules antigenically similar to those described by Dennert and Podack in CTL [8]. Several proteins have been isolated from these granules [22]. Recently,

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Konigsberg and Podack have demonstrated that isolated cytolytic granules kill L929 cells and induce target cell DNA fragmentation [18], properties expressed by LT, suggesting that LT may be one of the 7 proteins associated with such structures. The granules have been found fairly consistently in activated CTL and NK clones [4, 8, 14, 22], and now we have found them invariably present in several mitogenactivated LT-secreting L3T4 + clones [22]. The postulated presence of LT in these granules, coupled with the formation of pore-like complexes by other components in the granules [8] has led to the suggestion that the granules provide killer cells with a mechanism for rapidly introducing LT into the target cell. CTL induce target cell DNA degradation [28], and we have now shown that LT-containing supernatant fluids, recombinant-derived LT and the functionally similar molecule, TNF, also induce DNA fragmentation of both lymphoma (A-20, BW5l47) [29] and fibroblast (L929) targets [32]. When DNA from lymphokine-treated targets is analysed electrophoretically, the characteristic laddered pattern is seen [29, 32]. This phenomenon is not observed in targets which have been lysed by antibody and complement. Since DNA fragmentation has been postulated to reflect the mechanism of CTL killing [28], these experiments suggest that LT, which also induces DNA degradation, contributes to CTL killing. In this manuscript, we have summarized the data which implicate soluble mediators in general and LT in particular in CTL killing. The evidence is still largely circumstantial. Several key experiments remain to be done. One is the demonstration that anti-LT antibody reproducibly inhibits CTL killing. Such experiments have been carried out repeatedly over the years with varying results. In several experiments, anti-LT inhibited CTL killing [39]; in others, it did not [11]. The use of recombinant derived LT and monoclonal antibodies may resolve some of the discrepancies. However, if

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LT is delivered to its target by CTL in association with granules, it might be unavailable for neutralization with an antibody. Nevertheless, the bystander killing seen slightly later (vide supra) would be susceptible to inhibition with anti-LT antibody. The mechanisms of CTL and LT killing must be completely delineated before complete understanding of their relationship is reached. DNA fragmentation is one early common event. How does this occur? It is unlikely the LT itself or any other factor delivered by the CTL is an endonuclease. The more reasonable explanation is that activation of a cellular endonuclease occurs. Does LT enter the target cell and induce a cascade of target cell events? How does the target cell participate in its own destruction?

The balance has shifted in favour of a role for soluble factors in CTL killing. The demonstration of LT involvement opens up the question of the role of other postulated mediators of CTL killing. These include TNF and other cytotoxins, leukoregulin [10] and interferon. Northern blot analysis with a TNF cDNA indicates that only a restricted range of CTL clones make TNF mRNA and that this mRNA production is not kinetically related to the conditions of activation. The role of other proteins produced by CTL, such as the serine esterases, is addressed by others in the Forum and is not considered here. In fact, the interactions of these various proteins made by CTL may be of the utmost significance and will be an interesting future focus of research.

References. [1] AGGARWAL, B.B., EESSALU, T.E. & HASS, P.E., Characterization of receptors for human tumor necrosis factor and regulation by y-interferon. Nature (Lond.), 1985,318,665. [2] AGGARWAL, B.B., HENZEL, W.J., MOFFAT, B., KOHR, W.J. & HARKINS, R.N., Primary structure of human lymphotoxin derived from 1788 lymphoblastoid cell line. J. bioI. Chem., 1985, 260, 2334. [3] BALLAS, Z.K. & HENNEY, C.S., The relationship between lymphokines and cell-mediated cytotoxicity, in «Biology of the Lymphokines» (Cohen, S., Pich, E. & Oppenheim, J.) (p. 165). Academic Press, London, New York, 1979. [4] BLUMENTHAL, R., MILLARD, P.J., HENKART, M.P., REYNOLDS, C.W. & HENKART, P.A., Liposomes as targets for granule cytolysin from cytotoxic large granular lymphocyte tumors. Proc. nat. A cad. Sci. (Wash.), 1984, 81, 5551. [5] BARRY, W.B. & RUDDLE, N.H., The delayed-type hypersensitivity response to (4-hydroxy-3-nitrophenyl) acetyl (NP) coupled proteins is carrier specific: in vivo and in vitro demonstrations. J. Immunol., 1983, 131, 70. [6] CONTA, B.S., POWELL, M.B. & RUDDLE, N.H., Production of lymphotoxin, lFN-y, and lFN-(X,p by murine T cell lines and clones. J. Immunol., 1983, 130, 2231. [7] CONTA, B.S., POWELL, M.B. & RUDDLE, N.H., Activation of Lyt-l+ and Lyt-2+ T cell cloned lines: stimulation of proliferation, lymphokine production, and selfdestruction. J. Immunol., 1985, 134, 2185. [8] DENNERT, G. & PODACK, E.R., Cytolysis by H-2 specific T killer cells: assembly of tubular complexes on target membranes. J. expo Med., 1983, 157, 1483. [9] EARDLEY, D.D., SHEN, F.W., GERSHON, R.K. & RUDDLE, N.H., Lymphotoxin production by subsets of cells. J. Immunol., 1980, 124, 1199. [10] EVANS, C.H., Leukoregulin, lymphotoxin and interferon transmembrane signals and regulation of cell growth. J. cell. Biochem., 1986 (Supp!. 10 B), 57. [11] GATELY, M.K., MAYER, M.M. & HENNEY, C.S., Effects of anti-lymphotoxin on cellmediated cytotoxicity. Cell. Immunol., 1976, 27, 82. [12] GRANGER, G.A. & WILLIAMS, T.W., Lymphocyte cytotoxicity in vitro: activation and release of a cytotoxic factor. Nature (Lond.), 1968, 218, 1253. [13] GRAY, P.W., AGGARWAL, B.B., BENTON, C.V., BRINGMAN, T.S., HENZEL, W.S., JARRETT, J.A., LEUNG, D.W., MOFFAT, B., NG, P., SVERDSKY, L.P., PALLADINO, M.A. & NEDWIN, G.E., Cloning and expression of the eDNA for human lymphotoxin: a lymphokine with tumor necrosis activity. Nature (Lond.), 1984, 312, 721.

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[14] HENKART, P.A., MILLARD, P.J., REYNOLDS, C.W. & HENKART, M.P., Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granulor lymphocyte tumors. J. expo Med., 1984, 160, 75. [15] HENKART, P.A., MILLARD, P.J., REYNOLDS, C.W. & HENKART, M.P., Cytolytic activity of purified cytoplasmic granules from cytotoxic rat large granulor lymphocyte tumors. J. expo Med., 1984, 160, 75. [16] HORNUNG, R.L., PRAVTCHEVA, D., RUDDLE, F.H., LI, C.B. & RUDDLE, N.H., The gene for lymphotoxin maps to murine chromosome 17. J. expo Zool., 1987 (in press). [17] LI, C.B., LIN, P.F., GRAY, P., McGRATH, K., HORNUNG, R. & RUDDLE, N.H., Cloning the gene for murine 1ymphotoxin. 6th International Congress of Immunology, Toronto, 1986. Abstract, p. 375. [18] KONIGSBERG, P. & PODACK, E., DNA damage of target cells by cytolytic T-cell granules. J. cell. Biochem., 1986 (Supp!. 10 B), 85. [19] NEDWIN, G., NAYLOR, S.L., SAKAGUCHI, A.Y., SMITH, D., NEDWIN, J.J., PENNICA, D., GOEDDEL, D.V. & GRAY, P.W., Human lymphotoxin and tumor necrosis factor genes: structure, homology and chromosome localization. Nucl. Acids Rev., 1985, 13, 6361. [20] OKADA, c.Y. & RECHSTEINER, M., Introduction of macromolecules into cultured mammalian cells by osmatic lysis of pinocytic vesicles. Cell, 1982, 29, 33. [21] PENNICA, D., HAYFLlCK, J.S., BRINGMAN, T.S., PALLADINO, M.A. & GOEDDEL, D.V., Cloning and expression in Escherichia coli of the eDNA for murine tumor necrosis factor. Proc. nat. A cad. Sci. (Wash.), 1985, 82, 6060. [22] PODACK, E.R. & KONIGSBERG, P.J., Cytolytic T cell granules: isolation, structural, biochemical and functional characterization. J. expo Med., 1984, 160, 695. [23] POWELL, M.B., CONTA, B.S., HOROWITZ, M. & RUDDLE, N.H., The differential inhibitory effect of lymphotoxin and immune interferon on normal and malignant lymphoid cells. Lymphokine Res., 1985, 4, 13. [24] RUDDLE, N.H., Cytotoxicity reactions mediated by antigen activated rat and mouse lymphocyte, in «Mechanisms of cell-mediated immunity» (R.T. McCluskey & S. Cohen, J.) (p. 401). John Wiley and Sons Ltd, Chichester, 1974. [25] RUDDLE, N.H., Letter to the Editor. J. Immunol., 1986, 136,2335. [26] RUDDLE, N.H. & WAKSMAN, B.H., Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. -II. Correlation of the in vitro response with skin reactivity. J. expo Med., 1968,128, 1255. [27] RUDDLE, N.H. & WAKSMAN, B.H., Cytotoxicity mediated by soluble antigen and lymphocytes in delayed hypersensitivity. - III. Analysis of mechanism. J. expo Med., 1968, 128, 1267. [28] RUSSELL, J.H., MASAKOWSKI, V.R. & DOBOS, C.B., Mechanisms of immune lysis. I. Physiological distinction between target cell death by cytotoxic T lymphocytes and antibody plus complement. J. Immunol., 1980, 124, 1100. [29] SCHMID, D.S., MCGRATH, K.M., HORNUNG, R., PAUL, N. & RUDDLE, N.H., Target cell DNA fragmentation is mediated by the cytokines, Iymphotoxin and tumor necrosis factor. Lymphokine Res., 1986 (submitted). [30] SCHMID, D.S., POWELL, M.B., MAHONEY, K.A. & RUDDLE, N.H., A comparison of lysis mediated by Lyt-2 + TNP-specific cytotoxic T lymphocyte (CTL) lines with that mediated by rapidly internalized Iymphotoxin-containing supernatant fluids: evidence for a role of soluble mediators in CTL-mediated killing. Cell. Immunol., 1985,93,68. [31] SCHMID, D.S. & RUDDLE, N.H., Production and function of lymphotoxin secreted by cytolytic T cells, in «Cytolytic lymphocyte clones and complement as effectors of the immune system» (E.R. Podack). CRC Press, West Palm Beach, 1986 (in press). [32] SCHMID, D.S., TITE, J.P. & RUDDLE, N.H., DNA fragmentation: manifestation of target cell destruction mediated by cytotoxic T cell lines, Iymphotoxin-secreting helper Tcell clones and cell-free Iymphotoxin-containing supernatant. Proc. nat. Acad. Sci. (Wash.), 1986, 83, 1881-1885. [33] SMITH, M.E., LAUD/CO, R. & PAPERMASTER, B.W., A rapid quantitative assay for lymphotoxin. J. Immunol. Methods, 1977, 14, 243. [34] STONE-WOLFF, D.S., YIP, Y.K., CHROHOCZEK KELKER, H., LE, J., HENRIKSEN-DESTAFANO, D., RUBIN, B.Y., RINDERNECHT, E., AGGARWAL, B.B. & VILCEK, J., Interrelationships of human interferon-gamma with lymphotoxin and monocyte cytotoxin. J. expo Med., 1984, 159, 828. [35] SWAIN, S.L., T cell subsets and the recognition of MHC class. Immunol. Rev., 1983, 74, 129. [36] SWAIN, S.L., DENNERT, G., WORMSLEY, S. & DUTTON, R.W., The Lyt phenotype of long term allowspecific T cell lines. Both helper and killer activities are mediated by Ly-1 cells. Europ. J. Immunol., 1981, 11, 175.

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[37] TITE, J.P. & JANEWAY, C.A., Cloned helper T cells can kill B-lymphoma cells in the presence of specific antigen: la-restriction and cognate vs. non-cognate interactions in cytolysis. J. Immunol., 1984, 14, 878. [38] TITE, J.P., POWELL, M.B. & RUDDLE, N.H., Protein-antigen specific la-restricted cytolytic T cells: analysis of frequency, target cell susceptibility, and mechanism of cytolysis. J. Immunol., 1985, 135, 25. [39] WALKER, S.M. & LUCAS, Z.J., Role of soluble cytotoxins in cell-mediated immunity. Transplant. Proc., 1973, 5, 137.

THE ROLE OF PROTEIN KINASE C IN THE CYTOTOXIC T-CELL LYTIC RESPONSE by J .H. Russell and K.M. Coggeshall

Department of Pharmacology, Washington University School of Medicine, 660 South Euclid Avenue, St. Louis, MO 63110 (USA) The interaction between the cytotoxic lymphocyte (CTL) and its antigenbearing target provides an interesting example of cellular interaction in which both partners may play an active role. As we learn more about the specific molecules involved, it becomes possible to develop testable models of the signal transduction pathways producing the biological responses (CTL growth and target cell lysis) which result from this interaction. In this report, we will review recent experiments from this laboratory directed at the hypothesis that protein kinase C (PKC) activation by the CTL antigen receptor plays an important role within the CTL in directing its lytic response. It has been recognized since the mid-70's that the tumour promoting phorbol esters provide a signal important in the proliferation of mixed lymphocyte cultures [1]. However, similar experiments on the effects of phorbol

esters on lytic activity indicated that these agents had no effect on lytic activity in short-term experiments and suppressed lytic activity in long-term experiments. These data were interpreted as suggesting that phorbol esters either failed selectively to stimulate the proliferation of CTL or activated suppressor pathways [2]. However, Orosz and his colleagues demonstrated that both the positive proliferative effects and the negative effects on lytic activity were direct influences of the ester on the CTL [3]. Because these experiments suggest that phorbol esters have opposing effects on growth and lysis, they could be interpreted to indicate that these responses are driven by distinct processes. Experiments from our laboratory demonstrated that CTL pretreated with phorbol esters retained functional target binding but were reduced in their capacity to lyse bound targets [4]. Fur-