The role of protein kinase C in the cytotoxic T-cell lytic response

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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-

T-CELL-MEDIA TED CYTOTOXICITY thermore, we observed that over the course of hours, high concentrations of phorbol esters depleted some protein(s) associated with or required for lysis [5]. These data provided an alternative explanation for the apparent opposing effects of phorbol esters on proliferation and lysis. We hypothesized that phorbol esters in fact mimic a positive lytic signal from the antigen receptor. Essential to our hypothesis that phorbol esters mimic a positive signal in the lytic transduction pathway was direct, functional evidence in its support. We and others had failed to demonstrate such a positive effect on antigen-specific lytic function by phorbol esters. One explanation for this result could be that attempting to stimulate antigen-specific lysis with phorbol esters may be analogous to attempting to stimulate an enzymatic reaction under conditions where the enzyme is operating at Vmax' Therefore, we examined the effects of phorbol esters on lytic activity against weak and non-specific targets. Consistent with our stimulatory hypothesis, these experiments demonstrated that phorbol esters could, in fact, have a positive effect on the lytic response. Brief pretreatment experiments demonstrated that the effect was on the CTL rather than on the target [6]. Experiments in other systems had suggested that the phorbol ester receptor in cells was protein kinase C (PKC) which was responsible for most if not all of the biological effects of the phorbol esters [7]. Thus the next series of experiments were designed to determine whether the positive and/or negative effects of phorbol esters on lytic function were related to effects on this protein kinase. Comparison of a series of phorbol esters in concentration-effect experiments demonstrated a positive correlation between the stimulation of lysis and the capacity to cause translocation of PKC to the membrane fraction [8]. These data suggest that the ability of phorbol esters to stimulate lysis is related to their capacity to cause translocation of PKC to the membrane. Several laboratories have recently demonstrated that preincubation of cells

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with high concentrations of phorbol esters could deplete the cells of available PKC and make them unresponsive to subsequent stimulation by agonists requiring PKC for signal transduction [9]. A comparison of the various phorbol esters for their capacity to deplete CTL of PKC demonstrated a strong positive correlation between the ability of the ester to deplete the kinase and inhibit antigen-specific lysis. Thus, both the positive and negative effects of phorbol esters on lytic function appear to be related to their effects on PKC. The observations that PKC activation can stimulate lysis and that depletion of PKC inhibits subsequent antigen-driven lytic activity is strong evidence supporting the hypothesis that PKC plays an important role in the normal transduction of the lytic response. The next issue was to determine which CTL membrane protein was responsible for PKC activation. Nishizuka and his co-workers had shown that the natural activator of PKC was diacylglycerol, which is produced by phospholipase C activation. Nishizuka has proposed that many membrane receptor systems are coupled to phospholipase C activation, thus producing diacylglycerol for PKC activation [10]. Most cells have very active diacylglycerol kinases, so that it is difficult to directly measure diacylglycerol production by phospholipase C. However, the subsequent steps in the metabolic pathway (phosphatidic acid and phosphatidylinositol synthesis) are easily measured by prelabelling the cellular pool of ATP with 32P. Table I demonstrates that perturbation of the antigen receptor in a CTL clone results in increased labelling of phosphatidylinositol, which reflects diacylglycerol production. Thus, the antigen receptor has the metabolic potential to activate PKC through its production of diacylglycerol. This confirms and extends the observations of Imboden and Stobo [11], who demonstrated phospholipase C activation and subsequent increased intracellular Ca++ in the human tumour line Jurkat after stimulation by antibodies to the « receptor» or the T3

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Induction of phosphatidylinositol turnover in CTL by cross-linking various membrane proteins.

I. -

Membrane protein

(I)

Antigen receptor (F23.I) Thy-I.2 IL-2 receptor (7D4) Lyt-2 T200 LFA-I

Stimulation index 5.8 1.0 1.0 1.3 1.0 1.0

± ± ± ± ± ±

(2)

1.62 0.04 0.04 0.07 0;01 0.02

(I) Cells mixed with 10 fLg/ml of monoclonal antibody to this protein followed by 50 fLg/ml of an appropriate secondary antibody. (2) Cells prelabelled with 32p were incubated 10 min with antibodies followed by lipid extraction. The ratio of phosphatidylinositol in stimulated and control cells is reported as the mean ± SEM of three experiments.

antigen. Similar perturbation of a number of other cell surface proteins has no such effect on lipid metabolism. While this negative data on other proteins is not conclusive, the stimulation of phosphatidylinositol turnover by perturbation of the antigen receptor clearly demonstrates that it has the metabolic potential to activate PKC. We would propose that one of the earliest events in the lytic pathway is the antigen receptor-coupled activation of phospholipase C. In addition to increasing intracellular Ca++, this locally stimulated lipid metabolism causes the translocation of PKC to the CTL membrane in the region of CTL-target interaction. This PKC redistribution provides the mechanism for the apparent vectorial delivery of the «lethal hit» (fig. 1). However, it should be noted that our experiments demonstrating the capacity of phorbol esters to stimulate receptor-independent lysis suggest that once the kinase is activated, the lytic process is antigen-non-specific. Thus, while it is likely that the local stimulation of PKC gives a vectorial preference to the lytic response, a strong lytic signal could produce a more global

activation of the lytic machinery. Pagano and Longmuir have demonstrated that once formed, diacylglycerol can diffuse to other membrane areas [12]. Once the phospholipase is activated, PKC could be translocated to cellular sites distant from the original antigen stimulus. If an antigenically unrelated third party cell were maintained in close proximity to such a distant cellular site, it may be lysed. This is the likely biochemical basis of the «backdoor» lysis of CTL «A anti-B» killing a third party «C anti-A» CTL in the presence of «B» targets [13]. A number of important experiments remain to test our hypothesis. Direct evidence for the preferential association of PKC with the CTL membrane in the region of CTL-target contact is important. Further, it is important to identify the relevant substrates for PKC in this area and how their phosphorylation contributes to the activation of the lytic process. A better understanding of these relatively short-term events will provide a framework for an understanding of the mechanism of receptor-regulated long-term processes such as growth and tolerance.

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TARGET

PKC

eTl FIG. 1. -

Model jor the receptor-directed translocation of PKC by receptor-coupled phospholipase activity.

References. [1) WANG, l.L., MCCLAIN, D.A. & EDELMAN, G.M., Modulation of lymphocyte mitogenesis. Proc. nat. A cad. Sci. (Wash.), 1975,72, 1917-1921. [2) ANDREOTTI, P.E., Phorbol ester tumor promoter modulation of alloantigen-specific T lymphocyte responses. J. Immunol., 1982, 129, 91-95. [3) OROSZ, C.G., ROOPENIAN, D.C. & BACH, F.H., Phorbol ester mediates reversible reduction of cloned T lymphocyte cytolysis. J. Immunol., 1983, 130, 2499-2501. [4) RUSSELL, 1 .H., Phorbol esters inactivate the lytic apparatus of cytotoxic T lymphocytes. J. Immunol., 1984, 133, 907-912. [5) RUSSELL, l.H. & HOWE, R.C., Evidence for the molecular dissociation of binding and post-binding functions in cytotoxic lymphocytes. Advanc. expo BioI. Med., 1985, 184, 453-469. [6) RUSSELL, 1.H., Phorbol-ester stimulated lysis of weak and non-specific target cells by cytotoxic T lymphocytes. J. Immunol., 1986, 136,23-27. [7) KIKKAWA, D., TAKAI, Y., TANAKA, Y., MIYAKE, R. & NISHIZUKA, Y., Protein kinase C as a possible receptor protein of tumor-promoting phorbol esters. J. bioI. Chem., 1983, 258, 11442-11445. [8) RUSSELL, l.H., MCCULLEY, D.E. & TAYlOR, A.S., Antagonistic effects ofphorbol esters on lymphocyte activation: evidence that protein kinase C provides an early signal associated with lytic function. J. bioI. Chem., 1986, 261, 12643-12648. [9) BLACKSHEAR, P.l., WINTERS, L.A., GIRARD, P.R., Kuo, l.F. & QUAMO, S.N., Growth factor stimulated protein phosphorylation in 3T3-Ll cells. J. bioI. Chem., 1985, 260, 13304-13315. [10) KISHIMOTO, A., TAKAI, Y., MORI, T., KIKKAWA, D. & NISHIZUKA, Y., Activation of calcium and phospholipid-dependent protein kinase by diacylglycerol: its possible relation to phosphatidylinositol turnover. J. bioI. Chem., 1980, 255, 2273-2276.

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[11] IMBODEN, J.B. & STOBO, J.D., Transmembrane signalling by the T-cell-antigen receptor: perturbation of the T3-antigen receptor complex generates inositol phosphates and releases calcium ions from intracellular stores. J. expo Med., 1985, 161, 446-456. [l2] PAGANO, R.E. & LONGMUIR, K.J., Phosphorylation, transbilayer movement, and facilitated intracellular transport of diacylglycerol are involved in the uptake of a fluorescent analog of phosphatidic acid by cultured fibroblasts. J. bioi. Chern., 1985, 260, 1909-1916. [13] LANZAVECCHIA, A., Is the T-cell receptor involved in T-cell killing. Nature (Lond.), 1986, 319, 778-780.

This work was supported by grant n° CA-28533. J .H.R. is the recipient of Research Career Development Award n° CA-00926. K.M.C. is supported by training grant n° HL-07275.

IS THERE EVIDENCE FOR TRANSFER OF MATERIALS FROM THE KILLER CELL TO THE TARGET REQUISITE FOR TARGET CELL LYSIS? by J .C. Hiserodt and W.H. Chambers Pittsburgh Cancer Institute, 230 Lothrop Street, Pittsburgh, PA 15213 (USA)

In 1976, Kramer and Granger reported evidence for the transfer of materials from a killer lymphocyte to a target, which was a requisite for target cell lysis (1]. Using an in vitro cytotoxicity assay employing human mononuclear leukocytes and PHAcoated mouse L-929 cells, these authors showed that trypsinization of the L-929 cells after a brief encounter with the human lymphocytes or after exposure to lymphotoxin (LT)-containing supernatants could protect the L-929 cells from subsequent lysis. This was confirmed by Hiserodt and Granger, who showed that antibodies against highly purified alpha-LT could also block L-929 lysis during the killer cell independent phase (KCIL) of this cytotoxic reaction [2]. In these studies, cytolysis was clearly mediated by defined cytotoxic factors (alpha-LT) and both release of LT from the killer lymphocyte and its binding on the L-929 plasma membrane appeared to be requisite events in lysis of L-929 cells. A rigorous examination of the role of factor(s) produced by more conventional cytotoxic populations (class-1-

restricted CTL and later NK cells) followed. Kalina and Berke examined whether the release of 51Cr could be detected from radiolabelled alloimmune CTL (PEL) [3]. Their results demonstrated no significant levels of 51Cr released from labelled effector cells (PEL) even when very high levels of target cell killing were achieved. The failure to detect release of 51Cr was used as an argument against a general secretion model for release of cytolytic factors. However, it could be argued that in those experiments, the relative contribution of total 51 Cr by the material involved in cytotoxicity may have been so small as to have been missed. Alternatively, the material would have been bound to the target cell surface and not detectable in the supernatant. There is evidence, however, in both CTL and NK cell killing systems for transfer of material(s) from the killer cell to the target. Using heterologous antibodies against purified alloimmune murine CTL (PEL), Hiserodt and Bonavida demonstrated potent blocking activity against CTL or NK cells without