The mechanism of cell-mediated cytotoxicity

CELLULAR

IMMUNOLOGY

81,9-21

(1983)

The Mechanism

of Cell-Mediated

Cytotoxicity

III. Protease-Specific Inhibitors Preferentially Block Later Events in Cytotoxic T Lymphocyte-Mediated Lysis Than Do Inhibitors of Methylation or Thiol-Reactive Agents’ DOUG

REDELMAN*

AND DOROTHY

HUDIG~

Department of Medicine, University of California, San Diego, La Jolla, Cal$ornia 92093 Received April 7, 1983; accepted June 17, I983 Highly active mouse cytotoxic T lymphocytes (CTL) generated in secondary mixed-lymphocyte responses were used to examine the manner in which adenosine derivatives, thiol-specific reagents, or protease-specihc probes affectedCTL-mediated lysis (CML). The adenosine deaminase inhibitor deoxycoformycin (dCF) enhanced inhibition by adenosine (AR) or by deoxyadenosine (AdR), but not by 7deamadenosine (tubercidin). L-Homocysteinethiolactone (L-Hey) acted synergistically with AR, but not with AdR or tubercidin, to block CML. Thus, AR derivatives may act both by affecting cellular methylation reactions, as demonstrated by the synergism between AR and L-Hey, and by inhibiting other events required for CML. Conditions were then established to determine whether these reagents preferentially atfected either the Ca*+-independent initial stage of cytolysis or the subsequent Ca2+dependent events. Methylation inhibitors blocked lysis most effectively if added before effector-target binding. Similarly, the nonpenetrating thiol-specific reagent quatemary ammonium monobromobimane (qBBr) was more inhibitory when added prior to the Ca*+dependent stage. Protease inhibitors such as a- 1-antichymotrypsin and protease substrates such as acetyltyrosine ethyl ester (ATEE) or tyrosine ethyl ester (TEE) also inhibited CML. But, in contrast to qBBr or methylation inhibitors, neither TEE nor ATEE was more effective when added prior to the initial effector-target interaction. Furthermore, TEE did not appreciably affect CTL binding to target cells at concentrations that nearly abrogated CML. Thus, the implicated protease step is unique in that it does not appear to participate in recognition or binding.

INTRODUCTION Target cell lysis by cytotoxic T lymphocytes (CTL) or by natural loller (NK) cells requires one or more steps involving molecules with cell surface thiol groups and one or more steps utilizing activable set-me-dependent proteases (l-4). We have also found that the source of the CTL can influence some of the apparent biochemical requirements for cytolysis. For example, effector populations with relatively low frequencies of CTL do not mediate cytolysis in the presence of azide or cyanide (5). In contrast, populations with higher CTL frequencies are refractory to inhibition by ’ This work was supported in part by USPHS Research Grants CA-24450 (D.R.) and CA-28 196 (D.H.) and grants from the UCSD Academic Senate. ’ To whom correspondence should be addressed: Division of Rheumatology, H-81 IG. r Member of the Theodore Gildred Cancer Facility. 9 0008-8749183 $3.00 Copyright 0 1983 by Academic Press. Inc. All rights of reproduction in any form reserved

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azide or cyanide (6), implying that oxidative phosphorylation is not a strict requirement for CTL function. In addition to differences related to CTL frequency, there may also be qualitative differences between effector cells generated in different ways. For example, cytolysis by effector populations obtained directly from immunized animals can be blocked by agents which elevate cyclic adenosine monophosphate (CAMP) (7) and partially blocked by inhibitors of protein synthesis such as cycloheximide (8). Conversely, CTL derived from mixed-lymphocyte responses (MLR) are refractory to both types of inhibitors, even when the apparent CTL frequency is lowered to approximate that in effector populations derived from immune animals (6, 9). These observations mean that a minimal mechanism of cytolysis may not require the biochemical events which have been inferred from studies using less active effector populations. Therefore, one must verify that a particular type of inhibitor blocks lysis by CTL independently of their level of activity before assuming that biochemical events blocked by the inhibitor are essential for CTL-mediated lysis (CML). CTG and NK-mediated lysis can be blocked by agents which interfere with cellular methylation reactions, implying that one or more methylation-dependent steps are required (10, 11). Since these earlier studies were done with effector populations having relatively low CTL frequencies, we retested the methylation requirement using highly active CTL populations derived from secondary MLR produced in vitro by cells from immunized responder animals. The present studies confirm the previous results from experiments done with freshly isolated CTL and show that one or more methylation events are required for CML regardless of the cytotoxic activity of the CTL population. Our current results also suggest that CML can be inhibited by interfering with one or more cellular processes unrelated to methylation that can be affected by adenosine (AR) or AR derivatives such as deoxyadenosine (AdR) or 7deazaadenosine (tubercidin). After establishing that these inhibitors affected highly active CTL, our major goal in the present studies was to define which stages of the cytolytic process were affected by various types of inhibitors. Several approaches to this problem have been reported, e.g., using lower temperatures to prevent the CML process from proceeding beyond the initial effector-target interaction ( 12), enumerating the frequency of effector cells which can bind target cells under conditions not permitting lysis (13), or using a medium without divalent cations and then pulsing for defined periods with Mg’+ and Ca2’ (14). We found that the temperature shift approach was not reliable because the effector populations we were using mediated significant cytolysis at room temperature. The cation pulse procedure worked well, although it was necessary to reduce the duration of the Ca2’ pulse to accommodate the higher activity of these CTL populations. Our data indicate that the methylation- and thiol-dependent steps occur concurrently with, and/or shortly after, the initial effector-target interactions. On the other hand, the protease-dependent events appear to occur later during the Ca2’dependent step(s). This latter observation was confirmed by demonstrating that effector-target binding, which is independent of Ca2’, was much less sensitive to the effects of protease substrates at concentrations which were able to inhibit CML effectively. MATERIALS AND METHODS Media and reagents. RPM1 1640 (UCSD Tissue Culture Core Facility) was used in MLR cultures to generate CTL and in CML assays. Medium for MLR cultures

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was supplemented with 1% of a mixture containing 10 parts 100X penicillin-streptomycin (No. 17-603, Microbiological Associates, Bethesda, Md.) and 1 part 200 mM L-glutamine (PS/G), 5 X lop5 M 2-mercaptoethanol (2-Me), and 5% fetal bovine serum (FBS) (Lots 29101079 and 4055865, obtained from Flow Laboratories, Rockville, Md.; Lot 904243, Irvine Scientific, Irvine, Calif.; or Lot V56405, Reheis Chemical Co., Phoenix, Ariz.) Medium for CML assays contained PS/G, but no 2-Me, 1% heated FBS (56°C for 30 min), and 15 mM N-2-hydroxyethylpiperazine-l\r-2-ethanesulfonic acid (Hepes, Sigma Chemical Co. St. Louis, MO.). Concanavalin A (Con A) was prepared from Jack bean meal by affinity chromatography on Sephadex G-50 ( 16, 17). The nonpenetrating thiol-alkylating agent quaternary ammonium monobromobimane (qBBr) was purchased from Calbiochem (La Jolla, Calif.). Deoxycoformycin (dCF) was provided by the Developmental Therapeutics Program of the Chemotherapy Division of the National Cancer Institute. The human plasma protein (Y-1-antichymotrypsin was prepared ( 18) and generously provided by Dr. J. Travis, University of Georgia. The undiluted stock solution contained sufficient inhibitor to block 2.6 mg chymotrypsin/ml and had no detectable trypsin inhibitory activity (1). The thiol-alkylating agents N-iodoacetyl-N’-(8-sulfo-l-naphthyl)ethylenediamine (l8-IAEDANS) and the l-5 isomer were obtained from Pierce Chemical, Sigma, and Calbiochem. The D isomer of acetyltyrosine ethyl ester (D-ATEE) was obtained from Vega Biochemicals (Tucson, Ariz.). Unless otherwise stated, other chemicals and reagents were obtained from Sigma. Sterile balanced salt solution (SBSS, described in Ref. (19) and obtained from the UCSD Tissue Culture Core Facility) was used to solubilize inhibitors. Animals and tumor cells. CBA/J (H-2k), DBA/2 (H-2d), C57Bl/6 (B6, H-2b), and (BALB/c X DBA/2)F, (CD2F,) mice were obtained from the contractors supplying the Mammalian Genetics Animal Production Section, Division of Cancer Treatment, National Cancer Institute. EL4 thymoma and P8 15 mastocytoma cells were maintained as ascites tumors in B6 and DBA/2 or CD2FI mice, respectively. The CBA/J mice, used as the source of MLR responder cells, were immunized with - 1 X 10’ tumor cells one or more times with the final immunization at least 2 weeks before sacrifice. Culture conditions. CTL were generated in MLR cultures containing CBA/J responder spleen cells from immunized mice and mitomycin C-treated spleen stimulator cells, 5- 10 X lo6 each (1, 6). Cultures were incubated in 60-mm dishes containing 5 ml medium in an atmosphere of 10% COZ, 7% 02, and 83% N2 with continuous rocking for 4-8 days. In some cases, MLR-activated cells were recultured with Con A-conditioned medium containing T-cell growth factor (TCGF) activity (6). CML and binding assays. Cytotoxicity assays were performed as previously described (1, 6) except for the cation pulse procedure in which a special medium was used. The cation pulse medium consisted ofglucose (2 mgjml) and the inorganic components of RPM1 1640, but without the calcium and magnesium salts. This solution was supplemented with Hepes, 1% heated FBS, PS/G, and additionally with 0.25 mM EGTA and 0.50 mM MgS04. 7Hz0. Both the effecters and the targets were washed with this medium prior to beginning the assay. Effector-target interactions were initiated by room temperature centrifugation of the CTL and targets for 2-4 min at -400 rpm. The assay tubes were then warmed to 37°C and sufficient calcium was added to render the final concentration 0.50 m&Y Ca2+. The calcium pulse was terminated after 5, 10, or 15 min by adding EDTA to a final concentration of 1.O mM. Following the EDTA addition, the tubes were incubated for 60-120 min at 37°C to

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allow “Cr release to become complete. Inhibitors were solubilized in the same medium and added before or after centrifuging or just before the EDTA addition. In some experiments, the inhibitors were added after centrifugation and the CTL-target mixtures were incubated 5-20 min with the inhibitors prior to the Ca*’ pulse. All CML assays were terminated by adding cold medium (containing 1.O mi’t4 EDTA in the Ca2+ pulse experiments) and centrifuging at 4°C. Aliquots of supernatants were removed and counted, and the percentage specific release was calculated as done previously (1, 6). All experiments illustrated are representative of at least three similar protocols that produced comparable results with the exception of the tests of a-lantichymotrypsin which were only done twice because of limited amounts of the inhibitor. The percentage of CTL with binding activity was determined by mixing equal numbers (- 1 X 106) of CTL from MLR cultures, from which dead and damaged cells had been removed (20), and P815 cells that had been incubated with -5 pg/ ml fluorescein diacetate (FDA) for -30 min. The effectprs and FDA-labeled targets were mixed at room temperature, centrihtged for 4-5 min at 400 rpm, and resuspended by pipetting up and down 20 times with a 50 ~1 micropipet. An aliquot of the cells was immediately examined microscopically using fluorescence to identify the target cells unambiguously. The data are expressed as the percentage of the total lymphocytes counted (200-500) which were conjugated with fluorescent target cells. Comparable experiments were performed on three separate occasions with similar results and representative data are presented. RESULTS Adenosine Derivatives Inhibit

CML by at Least Two Mechanisms.

The inclusion of adenosine, deoxyadenosine, or 7deazaadenosine inhibited cytolysis by murine CTL. The presence of deoxycoformycin at a concentration sufficient to inhibit murine lymphocyte adenosine deaminase (1 .O &4; D. Redelman, unpublished) enhanced the inhibitory effects of AR or AdR but not that of tubercidin. For example, CML assays containing threefold effector cell dilutions starting at an effectorkuget ratio (E:T) of - 13.1 were incubated with varying concentrations of AdR or AR alone, or with dCF (Fig. 1). When dCF was added, 100 to 1000 pit4 AdR or AR caused a reduction in cytolysis equivalent to a three- or ninefold reduction in the number of effkctor cells, respectively (Fig. 1). Tubercidin was inhibitory at similar concentrations, but dCF did not enhance its effectiveness (not shown), presumably because this derivative of AR is not degraded by adenosine deaminase (2 1). It should also be noted that these reagents when added alone or with dCF caused only a partial inhibition of cytolysis as is illustrated for AR and AdR in Fig. 1. One possible effect of AR derivatives would be to interfere with methylation reactions. These reactions, which use Sadenosylmethionine (SAM) as the methyl donor, are inhibited by accumulation of the demethylated product Sadenosylhomocysteine (SAH). Accumulation of SAH is normally prevented by the enzyme SAH hydrolase which hydrolyzes SAH to AR and homocysteine. The SAH hydrolase reaction is, however, readily reversible if the concentrations of the products are elevated. If SAH accumulation were contributing to the inhibition of CML by AR derivatives, then L-homocysteinethiolactone should act synergistically with the AR derivatives. To determine whether this synergy could be demonstrated, CTL were assayed with dCF

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-E.r=

lo

%

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12.8’1

AIR+

13

CML EVENTS

no AdRAlone

ao- A s-

BLOCK

a0 ARAlone

l.O!,M dCF

Ae

_

AR+ I.OIIM dCF

XE7=0”7’

E.7=0471

1.s)

I

0 "1.0

3.0

1

10

VW

1

30

8

100

I

300

W'4

I,,,, 10000

1.0

I

I

t

I

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3.0

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L lO(

l-1 GUM)

FIG. 1. Inhibition ofcytolysis by deoxyadenoine (AdR) or by adenosine (AR) is increased when adenosine deaminase is blocked by deoxycoformycin (dCF). GIL, at effector:target ratios (E:T) starting at 12.8: I, were incubated with target cells with or without 1.O PM dCF and with varying concentrations of AdR (A) or AR (B) for 2.5 hr. The three highest E:T ratios were tested with inhibitors but for simplicity only the effects of the inhibitors on the two highest ratios are depicted. CML by untreated effecters is depicted by the X’s in this and in Figs. 2 and 4 below. (Expt. 106.)

and AR derivatives alone, or with L-Hey. L-Hey alone, at concentrations as high as 300 to 1000 &f, caused very little diminution in cytolysis. However, the presence of L-Hey increased the effectiveness of AR such that 10 to 30 PM caused almost total inhibition of CML (Fig. 2). Neither AdR nor tubercidin was more inhibitory when L-Hey was added (not shown). These data suggested that AR derivatives could inhibit cytolysis by at least two mechanisms, one of which involved inhibition of methylation as indicated by the synergistic effects between AR and L-Hey. At least one other mechanism was probably involved since AdR and tubercidin could also block killing but did not synergize with L-Hey. Thiol-Reactive

Agents Inhibit

Cytolysis

Compounds which oxidize or alkylate thiol groups inhibit CML (1). Although the oxidizing agents must be present throughout the assay to exert an effect, alkylating agents block CML when the effecters are pretreated, demonstrating that the essential thiols are available on the CTL prior to interaction with the targets. The compound qBBr is of particular interest because it does not penetrate living cells, because it is a hapten to which antibody can be made, and because it becomes fluorescent only after it has reacted with a thiol. Small numbers of effecters (< - 1 X 106) are inhibited by - 1 mM qBBr. However, we found that pretreating larger numbers of CTL, such as 50- 100 X 106, required 1O-25 mM qBBr to cause inhibition of CML. CT’L treated with sufficient qBBr to be inhibited showed a faint, uniform fluorescence that was limited to the cell surface. We have additionally tested l-5 and I-%IAEDANS, which are also haptenic, fluorescent alkylating agents, and found that the l-5 isomer was much less effective than the l-8 form. The l-8 isomer inhibited CML both when included and when the CTL were pretreated as one would expect (not shown). However,

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WI WV FIG. 2. L-Homocysteinethiolactone (L-Hey) and adenosine (AR), which act synergistically to inhibit methylation, also act synergistically to inhibit CML. CTL, starting at an E:T of 7: 1, were preincubated with 1.OpM dCF, which had no effect on cytolysis, and assayedwith varying concentrations of AR included. In those assaysin which L-Hey was also included, the AR was 3- to lo-fold more inhibitory than in the absence of L-Hey. (Expt. 109.)

when the l-8 isomer was compared with qBBr, the latter compound was - lo-fold more effective. We also found that 1-8-IAEDANS was more toxic than qBBr. Therefore, qBBr was the most useful alkylating agent based on its ability to react selectively with surface thiol groups, its haptenic properties and its relatively low toxicity. Inhibition

of Cytolysis by Inhibitors

or Substrates of Chymotrypsin-like

Proteases

Inclusion of the macromolecular protease inhibitor human (Y-1-antichymotrypsin in CTL assays inhibited CML (Fig. 3). The presence of sufficient inhibitor to block 0.26 mg of chymotrypsin, i.e., the amount added in the dilution marked 1:4 in Fig. 3, caused a reduction in CML equivalent to a three- to ninefold reduction in the number of effector cells. It should also be noted that the inhibitor was more effective when the number of effector cells was reduced. For example, in the experiment depicted in Fig. 3, lysis by the highest number of CTL was only slightly affected by the protease inhibitor whereas lysis by the lower CTL concentrations was markedly reduced. This observation is consistent with the mechanism of action of the inhibitor since it attaches covalently and stoichiometrically to the active sites of chymotrypsinlike enzymes. Substrates of chymotrypsin-like enzymes were also able to block CML. Figure 4 illustrates experiments comparing the inhibitory activities of D- or L-acetyltyrosine ethyl ester and the hydrolysis product of L-ATEE, L-acetyl tyrosine (L-AT). Both the D and L forms of ATEE blocked CML but the L form was approximately twofold more active. The difference between the D and L forms and the inactivity of the

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Cultured Cells FIG. 3. The human plasma antiprotease (Y-I-antichymotrypsin blocks CML. CTL were titrated with control diluent or with dilutions of a-1-antichymotrypsin. Enzyme assays indicated that a 1:4 dilution contained sufficient inhibitory activity to inactivate 0.26 mg chymotrypsin. The preparation used had no trypsin inhibitory activity. (Expt. 92.)

-U I

0

E.T=03,.7 I

1

I

I

I

3

5

7

Inhibitor

I j

9

(mM)

FIG. 4. The L form of acetyltyrosine ethyl ester (L-ATEE) inhibits CML twice as effectively as DATEE. CTL, at E:T ratios starting at 8.5: 1, were assayed with varying concentrations of L-ATEE, D-ATEE, or the hydrolysis product of L-ATEE, L-acetyltyrosine (L-AT) included. These compounds required initial solubilization in ethanol so a control for these different ethanol concentrations is also shown. (Expt. 86.)

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hydrolysis product are consistent with a chymotrypsin-like enzyme being required. L-ATEE is an alternate substrate and as such can compete with the natural substrate. On the other hand, isolated chymotrypsin-like enzymes do not hydrolyze D-ATEE. Thus, D-ATEE is a competitive inhibitor for chymotrypsin-like proteases whose Ki is approximately twofold higher than the K,,, of L-ATEE for chymotrypsin (22). This relationship is similar to what we observed in experiments such as that shown in Fig. 4. (It should be noted that the Ki is equivalent to the Km when an alternate substrate is used as an inhibitor.) L-AT, which is not an effective protease inhibitor, had little effect on CML (Fig. 4). Glycyltyrosine and glycyltyrosinamide were also tested in this same experiment, but neither of these compounds had any detectable effect on CML at a concentration of 9 mM (not shown). ATEE required initial solubilization in ethanol which could itself cause some inhibition of CML as shown in Fig. 4. Therefore, both tyrosine ethyl ester (TEE) and tyrosine methyl ester (TME), which are readily soluble in aqueous solutions, were tested for inhibitory activity. Both compounds were effective CML inhibitors at 3-5 mM, causing a reduction in CML equivalent to a threefold decrease in the number of effector cells. (It should be noted that both of these agents require careful neutralization with base to avoid toxicity since the soluble hydrochloride salts produce acidic solutions.) Defining the Stages of Cytolysis Afected by Inhibitors CML can be blocked by interfering with the interaction between the effector and target cells or by interfering with one or more of the events following the initial interaction. We found that CTL generated in secondary MLR were able to mediate significant lysis at 20-22°C (not shown). These results meant that the stage ofcytolysis blocked by inhibitors could not be accurately defined by using temperature shift techniques (12). We then tested the cation pulse procedure devised by Golstein and Smith (14). We found that the CTL we were using killed effectively with Ca2’ pulses as short as 5- 15 min. Therefore, a pulse of 10 min was routinely used. There was no cytolysis if the Ca2+ was omitted. If an inhibitor were to interfere with events preceding the requirement for Ca 2+, then it would be expected to be more effective the earlier it was added. On the other hand, if an inhibitor were to act exclusively on events occurring during the Ca2+-dependent phase, then it should be equally effective when added at any point prior to the Ca2’ pulse. The only exception would be those agents which might require additional time to enter cells and/or to exert their effects. We controlled for this possibility by allowing additional incubation time with the inhibitor before adding the Ca2+ in those caSes in which inhibitors were less effective when added later. The Stages of Cytolysis Afleeted by Inhibitors or by Protease Substrates

of Methylation,

by Thiol Alkylation,

The Ca2’ pulse procedure was used to test the effects of adding inhibitors methylation (L-Hey, dCF, and AR) before or after centrifugation. Adding the inhibitors before centrifugation caused a total inhibition as shown in Fig. 5. Blocking methylation after the initial Ca2+-free interaction was less effective but still caused significant blockage of CML. Similarly, the nonpenetrating thiol-alkylating agent qBBr was also more effective when added before the Ca2+ pulse (Fig. 6). Although these agents caused virtually no inhibitory effect when added immediately before the Ca” pulse, their inhibitory activity was increased ,by allowing the CTL and targets to incubate

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105 Cultured Cells FIG. 5. Methylation inhibitors block CML most effectively if added before the initial interaction between effecters and targets. The methylation inhibitors (10 & dCF, 100 j&f AR, and 75 pM L-Hey) were added before or after binding of targets and effecters by the initial centrifugation. The CTL-target mixtures were then incubated with the inhibitors for 5-20 min before the addition of the Ca*+ pulse. (Expt. 93.)

with the inhibitors at room temperature or at 37°C before adding the Ca*+ as illustrated in Figs. 5 and 6. Nevertheless, these inhibitors were still not as effective as when they were added before the initial centrifugation. These data indicated that the events requiring methylation and those requiring a thiol group were initiated near the time of the effector-target interaction but were still partially susceptible to inhibitors added after binding.

X

= 40-

10

mm

a++

Pulse

r....:=

, 105 Cultured

t 80,

Cells

FIG. 6. Alkylation of cell surface thiols by qBBr inhibits cytolysis more effectively if added before the initial effector-target interaction. CTL-target mixtures were treated with 1.O mM qBBr as described above in Fig. 5. (Expt. 93.)

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The effects of adding alternate protease substrates were different from the effects of the inhibitors discussed above. Figure 7 shows that ATEE added before centrifugation was no more effective than when added immediately before the Ca*’ pulse. Extending the incubation with ATEE before the Ca2+ addition did not increase its effectiveness, in contrast to the effects of inhibitors of methylation or thiol-alkylating agents described above. These data implied that the principal effect of ATEE was on events occurring after the initial CTL-target interactions. Similar results were obtained with TEE (not shown). None of these three types of inhibitors had any effect on target lysis if added at the time of the EDTA addition (not shown). Thus, none of these agents appeared to block the terminal, CTL-independent lytic events. The Efect of Protease Substrates on Efector-Target Cell Binding The results above (Fig. 7) suggested that the protease step probably followed the initial interaction between CTL and target cells. This assumption was tested by comparing the effects of TEE on effector-target binding and on cytolysis. The data in Fig. 8 show that the inclusion of TEE at 3-5 mM caused a reduction in cytolysis equivalent to a threefold reduction in the number of eflector cells. The inclusion of 7 mM TEE caused an inhibition of killing equivalent to a 1:27 dilution of the effector cells, i.e., -97% inhibition. Binding, however, was much less sensitive to the effects of TEE. The inclusion of 1, 3, or 5 mM TEE caused only a small decrease in the number of lymphocytes bound to target cells. TEE at 7 rnM, which caused a nearly total inhibition of cytolysis, reduced the fraction of effecters bound to targets by less than 50%. DISCUSSION Cytolysis by murine CTL was inhibited by agents which inhibit methylation, by compounds which alkylate cell surface thiol groups, and by inhibitors and alternate

Cultured

FIG. 7. L-ATEE blocks CML time of the CL?' pulse. L-ATEE was initiated by centrifugation time as that depicted in Fig. 6.

Cells

equally effectively when added before the initial centrifugation or at the at final concentrations of 3.0 or 5.0 mM was added before or after binding or at the time of the Ca*+ pulse. This experiment was done at the same (Expt. 93.)

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90 1

L-TEE

conjugates:

Effector Cells FIG. 8. L-TEE blocks cytolysis more effectively than it interferes with effector-target conjugate formation. CTL were titrated with 1.0, 3.0, 5.0, or 7.0 m&f L-TEE included in the assays.Aliquots of these effector cells were centrifuged with FDA-labeled target cells in the presence of the same concentrations of L-TEE. Conjugates were counted for 200-500 lymphocytes as described in the text and are expressed as the percentage of CTL with one of more target cells bound. (Expt. 150.)

substrates of chymotrypsin-like proteases. The first two classes of inhibitors were more effective when added before the initial CTL-target interaction, implying that they could affect the initial stages of the cytolytic process preceding the Ca*+-dependent events. In contrast, the protease-dependent steps did not appear to be involved in the initial interactions between effector and target cells since chymotrypsin substrates such as ATEE and TEE were equally effective when added before the initial interaction or at the same time as the Ca*+ in cation pulse procedures (Fig. 7). Furthermore, concentrations of these inhibitors which were sufficient to block more than 90% of the cytolytic activity had relatively little effect on the formation of effector-target conjugates (Fig. 8). It should be emphasized that the tight binding required to form detectable effector-target conjugates may not be required for completion of cytolysis ( 15). Therefore, it is not safe to assume that agents which inhibit or disrupt conjugates will actually block the interactions required for cytolysis (15). Conversely, virtually all other agents which block cytolysis also interfere with conjugate formation (5). The present findings make the protease inhibitors and alternate substrates appear to be unique since they can block cytolysis at concentrations that have little effect on CTLtarget conjugates. The effects of adenosine derivatives on cytolysis are probably mediated through at least two mechanisms,’ one of which is an inhibition of methylation. AR, in the presence of an inhibitor of adenosine deaminase such as dCF, acted synergistically with L-Hey to block cytolysis. AR and L-Hey are known to act synergistically to block SAH hydrolase (10, 23) and ultimately to inhibit methylation reactions which

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virtually ah use S-adenosylmethionine as the methyl donor (23). We cannot determine from our current results whether the essential methylated compounds are protein or phospholipid since SAM is the methyl donor for both types of molecules. Other investigators have suggested that phospholipid methylation is the essential process for mediator release (24) or for lysis by natural killer cells (11). There may be multiple events in the lytic process that require methylation since we have found methylation inhibitors, while most effective if added before the interaction between effector and target cells, can still cause partial CML inhibition if added after the initial CTLtarget interaction and prior to the addition of Ca*+ (Fig. 6). These results could mean that the early methylationdependent events are reversible or that additional methylation-dependent events occur after the initial interaction. Zimmerman et al. (10) have suggested that methylation of intracellular charged proteins may be a way of moving them through the CTL membrane and delivering them to the target cell surface where they could participate in cytolysis. This type of protein methylation could occur following the initial effector-target interactions and would be consistent with our results. In addition to the effects on methylation, AR derivatives appear to exert at least one other effect which can also inhibit cytolysis. Although both AdR (Fig. 1) and tubercidin (not shown) caused partial inhibition of CML when added alone, neither compound acted synergistically with L-Hey. These results implied that methylation was not involved with CML inhibition by tubercidin or AdR. One possible explanation is that AdR or tubercidin might cause an elevation in CAMP, which can block some types of CML (7). Although CAMP-mediated CML inhibition has been reported (7), Plaut (9) found that CTL generated in MLR cultures as we have done were refractory to inhibition by CAMP elevation. Thus, it is possible that AR derivatives could interfere with CML by an entirely different process that may be related neither to methylation inhibition nor to changes in cyclic nucleotide levels. The present results have also shown that the events in the lytic process which are blocked by nonpenetrating thiol-alkylating agents probably occur early since inhibition was most effective if qBBr was added prior to the initial interaction (Fig. 6). Cy-tolysis by human NK cells is also blocked by alkylating agents. In that system, we found that pretreatment with qBBr inhibited the binding of NK cells to K562 target cells (4) as one would predict from the current findings. The thiol-requiring events of CML appeared to be reversible and/or to occur at more than one stage of the lytic process since addition of qBBr after binding was able to block CML partially. The involvement of thiol-bearing molecules in the early events of cytolysis is consistent with our more recent characterization of the qBBr-reactive molecules. We have prepared antisera to qBBr and used the antibody to immunoprecipitate surface ‘ZSI-labeled molecules from CTL (25). There are only a limited number of bands detected on the SD!% PAGE autoradiograms. Two of these bands appear similar to the heavy and light chains recognized by antibody to the molecule termed lymphocyte function antigen (LFA) (26, 27). Antibody to LFA blocks CML with its principal effect being exerted during the early stages of cytolysis (26, 27). Experiments are currently underway to verify that qBBr does react with one or more cell surface thiol groups on LFA. The inhibition of CML with protease-reactive reagents indicates that a serinedependent protease having an aromatic amino acid cleavage specificity is essential for CML and may be part of the Ca*+dependent events. The plasma antiprotease a-1-antichymotrypsin reacts only with the serine-dependent class of proteases and

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only with those proteases having aromatic specificity ( 18). Its large molecular weight (-52,000) and acidic p1 (pH 4.5) suggest that it would not readily penetrate CTL and imply that the protease with which it reacts is likely to be on the cell surface or to be secreted during cytolysis. The inhibition of CML by low molecular weight substrates derived from aromatic amino acids is also consistent with one or more enzymes having chymotrypsin-like specificity being required for CML. Since these substrates did not inhibit when added after the Ca2+ pulse, we think it is unlikely that the CTL-independent phase of CML involves direct protease digestion of the target cells. We suggest that this protease is part of a cascade of biochemical events that follow the Ca’+-independent initial steps of target recognition. There are numerous precedents for protease participation, initiation, or amplification of biological reactions, e.g., the blood coagulation and complement systems. The protease could function by activating another zymogen, such as a prophospholipase, or by modifying prolytic molecules to become active as in the complement system. We favor the latter possibility based upon the similarities between CML and complement-mediated lysis. ACKNOWLEDGMENTS We thank Dr. N. J. Zvaifler and Dr. H. G. Bluestein for providing the laboratory space, Ms. E. Bussett for her dedicated technical assistance, and KayPro for manuscript preparation.

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