Eur. J. Immunol. 1990.20: 179-184
Peggy StrackO, Carla Martin+, Saburu Saito+, Rosemarie H. Dekruyff* and Shyr-Te Juoo Arthritis Center, K510, Boston University School of Medicineo, and Department of Pathology, Harvard Medical School+, Boston, and Department of Pediatrics, Children’s Hospitala, Palo Alto
Cytolytic pathways of CD4 and CD8 clones
Metabolic inhibitors distinguish cytolytic activity of CD4 and CDS clones* The effect of various metabolic inhibitors on the expression of cytolytic activity of CD4 ( T H ~and ) CD8 (CTL) clones was studied. The cytolytic activity of CD4 clones, but not CD8 clones, was sensitive to the FWA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. Conversely, cholera toxin (CT) inhibited cytolytic activity of CD8, but not CD4 clones. Both mitomycin C, a DNA synthesis inhibitor, and cyclosporin A (CsA) failed to inhibit the cytolytic activity of either CD4 or CD8 clones. Although pretreatment with CsA or CTdid not inhibit the cytolytic activity of CD4 clones, lymphokine (interleukin 2, IL 2, interferon-y, IFN-y, and tumor necrosis factor, TNF) production was strongly inhibited. Similarly, pretreatment of a CD8 clone with actinomycin D or CsA inhibited lymphokine production without affecting cytolytic activity.The production of mRNA for TNF and IFN-y by concanavalin A-activated CD4 clones was also inhibited by CsA and CT. Moreover, perforin-specific mRNA was not detected in activated CD4 clones. Collectively, these observations demonstrated that de novo synthesis of RNA and protein is required for expression of cytolytic activity of CD4 clones, yet production of TNF, INF-y, IL 2 and perforin is not involved. In contrast, the cytolytic machinery of CD8 clones is present prior to activation and is quickly expressed following activation even when de novo synthesis of RNA, protein and lymphokines is blocked.
1 Introduction Both CD4 and CD8 Tcell subsets can express cytolytic activity upon activation [l-91. When activated by antigen and Ia-bearing APC, CD4 cells can be induced t o express cytolytic activity toward the APC. Similarly, the cytolytic activity of CD8 cells (CTL) can be induced by antigen and class I molecules on appropriate target cells. Nonspecific Tcell activators such as Con A or a anti-CD3 mAb can also induce the expression of cytolytic activity [ l , lo]. After antigen binding and cross-linking of TcR, membrane fluidity increases [ l l ] and signals are transduced through the cell membrane which increase intracellular calcium and activate PKC [12]. A calcium chelator, EGTA, inhibits cytolytic activity of CD4 clones by inhibiting T cell activation [8]. However, under defined conditions, CD8 cells express cytolytic activity in the presence of excess (Mg)z EGTA [13-151. The operationally identifiable stages of CTL killing, i. e. ,calcium-independent, magnesium-dependent binding, activation and lethal hit, have not been
[I 77671
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This work is supported by LO1 ACS-IN97, AI-24571 and CA-39790. Investigator of the American Heart Association.
Correspondence: Shyr-Te Ju, 71 E. Concord St., Arthritis Center, K510, Boston University School of Medicine, Boston, MA 02118, USA Abbreviations: AcD: Actinomycin D CX Cholera toxin Chx: Cycloheximide CsA: Cyclosporin A GLT: Random copolymer of L-Glu, L-LYS,L-Tyr MitC: Mitomycin C M T E 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
defined for CD4 clones [16]. Also, little is known about the intracellular pathway(s) of activation leading to expression of cytolytic activity.Variousmetabolic inhibitors have been used to study the intracellular elements important for the expression of cytolytic activity in heterogeneous NK and CD8 cells [17-201. These studies support the “stimulationsecretion” model for cytotoxicity and suggest that intracelMar CAMP, microtubules, phospholipase A2, calmodulin and lysosomal function are important for the expression of cytolytic activity [MI. The “stimulation-secretion’’ model agrees well with the observations that both CD8 and NK cells secrete perforin from their granules as a cytolytic mediator [21]. The cytolytic mechanisms of CD4 clones have not been well defined. It has been proposed that TNF and IFN-y are the soluble mediators for target lysis. However, many targets that are killed by activated CD4 clones are resistant to high concentrations of TNF and IFN-y. Although it remains possible that these targets are killed by very high local concentrations of these mediators, an alternative hypothesis is that a TNF (and IFN-y)-independent cytolytic mechanism is also induced to kill these targets upon activation [22]. In order to determine whether CD4 and CD8 clones use distinct cytolytic pathways and to understand better the relationship between lymphokine production (including the cytotoxickytostatic mediators of TNF/IFN-y) and the expression of T cell cytolytic activity, we compare the effect of metabolic inhibitors on cytolytic activity and lymphokine production by CD4 and CD8 clones. We demonstrate that (a) distinct cytolytic pathways are used by CD4 and CD8 clones, (b) separate activation pathways for lymphokine production and cytolytic activity exist in CD4 and CD8 clones, and (c) CD4 clones do not use perforin-dependent cytolytic mechanism of CD8 clones. 0014-2980/90/0101-0179$02.50/0
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2 Materials and methods 2.1 Animals
Female BALB/CByJ and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were maintained on acidified tap water and Purina Mouse Chow ad libitum.
Eur. J. Immunol. 1990.20: 179-184 MA), shaken every 5 min for 30-35 rnin and then washed 3 times before use. Antigen-pulsed LK cells (@/well in 1ml) were prepared by culturing overnight with the appropriate antigen (GLT for E10, KLH for C7 and E6) at 1 mg/ml. 51Cr-labeledEL4 cells, after washing once in PBS, pH 7.2, and resuspended in 1ml of PBS, pH 7.8,were mixed with 10 y1 NP-0-Su (2.5 mg/ml) for 4 min after which 10 ml PBS, pH 7.2, was added and immediately centrifuged. %r-labeled NP-EL4 cells were then washed 3 times in culture medium before use. All target cells were used at 104/well.
2.2 Reagents Culture medium was DMEM (Gibco, Grand Island, NY) supplemented with 1% penicillinlstreptomycin, 1% L-glutamine, 1% MEM vitamins, 1% nonessential amino acids, 1% sodium pyruvate (Gibco) and 7.5% FBS (Hyclone Laboratories, Inc., Logan, UT). A hybridoma (145-2C11) specific to C D ~ (CD3 E mAb) [23] was kindly provided by Dr. J. Bluestone at N.I.H. (Bethesda, MD). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTI'), Con A , mitomycin C (MitC), actinomycin D (AcD) and cycloheximide (Chx) were obtained from Sigma (St. Louis, MO). Cyclosporin A (CsA) was purchased from the University Hospital pharmacy. Cholera toxin (CT) was obtained from List Biol. Lab. Inc. (Campell, CA). 2.3 Derivation and maintenance of clones
The derivation of CD4 clone E l 0 (GLT specific, I-Ed restricted) has been described [24]. KLH-specific clones C7 (I-Ad restricted) and E6 (I-Ed specific) and Mlsa-reactive clones F3 and F8 (both are I-Ed restricted) were derived similarly from BALB/c mice. Clone 2G5 (Mlsa reactive) was derived from a MRL/MP-lpr mouse. These clones express CD4+, CD8- phenotype and secrete lymphokines of T Htype, ~ i . e . , IL 2 and IFN-./.They were maintained in 24-well plates (Costar 2424, Cambridge, MA) by periodic stimulation (every 10-15 days) of 0.25 x lo6clone cells with the appropriate antigen (20-30 pg/ml) and 3 x lo6 irradiated (3000 rds) spleen cells (SC) of appropriate mouse strains, and expanded with IL 2-containing Con A SN. CD8 clones NPSS-18, and NPSS-20 were derived by priming C57BL/6J mice with 1 mg (4-hydroxy-3-nitropheny1)acetyl (NF')-0-succinimide (NP-0-Su) solution in DMSO S.C. One week after priming, inguinal LN were removed and stimulated with NP-coupled SC. Three days later, LD microcultures at 1 cell/well were seeded with 6 x lo5 irradiated (2000 rds) C57BL/6J SC and 10% PMA-stimulated EL4 SN as a source of growth factors. Every 2 days 3 x 105 irradiated C57BL/6J SC and 10% EL4 SN were added to the culture. After 10-14 days growing cultures were expanded and then subcloned. The CD4-, CD8+ clones NPSS-18 and NPSS-20 are specific for NP in association with H-2Db and H-2Kb, respectively. Clones were maintained by culturing at 7-11-day intervals with NP-coupled irradiated SC.Viable cells of all clones were separated over a Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient and washed 3 times with culture medium before use. 2.4 Preparation of target cells Pelleted target cells were labeled with 100 pl W r by the addition of radioactive sodium chromate (NEN, Boston,
2.5 Effect of metabolic inhibitors on the cytolytic activity mediated by clones Clones were incubated with various metabolic inhibitors for 45 rnin in 96-well plates (total volume 100 pl) after which Wr-labeled target cells were added. When Wr-labeled P815 cells were used as targets, CD3-mAb (25%, v/v) was also added as a nonspecific activating agent to induce cytolytic activity. In some experiments, the clones (106/ml) were incubated 45 min with the various inhibitors,washed 3 times with medium, and then tested for cytolytic activity and lymphokine production. These treatments did not result in nonspecific toxicity of the effector clones, as judged by trypan blue dye exclusion or by MTT tests (added at the initiation of culture) and measured at 570 nm at the end of culture. After 4-6 h of culture, 100 yl of SN was carefully removed from each well and counted in a gamma-scintillation counter. Percent specific lysis was determined by the formula: % specific 51Cr release (or lysis) = (cpm of test sample - cpm of nonspecific release)/(cpm of total release - cpm of nonspecific release). The cpm of nonspecific release and total release were obtained by culturing target cells in medium and by lysing target cells with 0.5% NP40, respectively. 2.6 Generation and assays of lymphokines
Clones (106/ml) were incubated 45 min with the various inhibitors, washed 3 times and then cultured with Con A (2.5 pglml). SN were collected after 20 h. Assays for TNF, IFN-y and IL 2 were carried out as previously described [22]. Briefly, the assay for TNF was based on its ability to kill L-929 cells. The assay for IFN-y was based on its ability to induce Ia expression on P388D1 M@ tumor cell line. Ia expression was determined by cellular RIA. The assay for IL 2 was based on its ability to induce proliferation of CTLL cell, which was measured by [3H]dThd incorporation. In all cases, the specificity of the assay was confirmed by inhibition with specific monoclonal and polyclonal antibodies [22]. 2.7 Northern blot analysis
Analysis of mRNA for TNF, IFN-y, IL2R, actin and perforin was carried out with RNA extracts from 25 x 106 cloned cells that had been activated with Con A for 4 h in the presence or absence of metabolic inhibitors.The probes for TNF-a [25], TNF-P [26], IL 2R [27], actin [28] and perforin [21] were the kind gifts of Drs. A. Cerami, N. Ruddle, R. N. Germain, G . Freeman, and E. Poddack, respectively. The probe for IFN-y was prepared by and
Eur. J. Immunol. 1990. 20: 179-184
Cytolytic pathways of CD4 and CD8 clones
provided to us by Dr. F? Burd at the Department of Pathology, Harvard Medical School.
3 Results We compared the effects of several metabolic inhibitors on the cytolytic activity of CD4 and CD8 clones.The inhibitors used were: MitC for blocking DNA synthesis, AcD for blocking RNA synthesis, Chx for blocking protein synthesis, CT for increasing intracellular CAMP and CsA for selective inhibition of RNA and lymphokine synthesis [29, 30l.Three CD4 clones and two CD8 clones were tested with similar results. In the continuous presence of AcD or Chx, the cytolytic activity of CD4 clones were inhibited by > 80%. Both antigen-specific, Ia-restricted cytolytic activity on antigen-pulsed LK target cells and nonspecific cytolytic activity induced by CD3-mAb on P815 target cells were inhibited. In contrast, neither antigen-specific, H-2Kbrestricted lysis of NP-EL4 target nor CD3-mAb-induced nonspecific killing of P815 by CTL clones were inhibited. A reciprocal pattern of inhibition was found with CT.This reagent inhibited the cytolytic activity of CD8, but not CD4, clones. In agreement with previous studies [6, 311, CsA failed to inhibit the cytolytic activity of CD4 and CD8 clones. MitC did not inhibit the cytolytic activity of either CD4 or CD8 clones, although these treated cells failed to incorporate [3H]dThd into DNA in the presence of a high concentration of r I L 2 (10 BRMP U/ml). Under these conditions, none of the metabolic inhibitors caused significant cell damage to either effector or target cells (see Sect. 2.5). Moreover, the selective inhibition of cytolytic activity of CD4 or CD8 clones also ruled out nonspecific toxicity of these treatments. The generality of these observations was supported by the fact that similar results were obtained using three different CD4 clones, two separate CD8 clones and two different activation methods with each inhibitor (Table 1). The above effector clones were also pretreated with these metabolic inhibitors for 45 min and then washed three times before testing cytolytic activity.The results were essentially
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the same with the exception that pretreatment with 2.5-100 yg/ml of Chx did not inhibit the cytolytic activity of CD4 clones (data not shown). This result pinpoints the effect of metabolic inhibitors on the effector clones and indicate that de novo synthesis of DNA, RNA and protein by target cells is not very critical for target lysis. To determine the relationship between lymphokine production and cytolytic activity, clones were pretreated with various inhibitors, washed and tested for cytolytic activity and the production of various lymphokines. As shown in Fig. 1, pretreatment with AcD, but not CsA or CT,strongly inhibited the cytolytic activity of CD4 clone E10.Yet both CsA and CT block production of IL 2 (> 97%), caused marked (> 90%) inhibition of IFN-y secretion and inhibited TNF production by approximately 80%.The degree of inhibition is comparable to that achieved with AcD treatment. Similarly, lymphokine production by the CD8 clone NPSS-20 was inhibited to a comparable degree by AcD or CsA treatment. These treatments did not inhibit cytolytic activity. The dissociation of lymphokine production from cytolytic activity indicates that TNF and IFN-y are not important for the killing of these target cells. ) AcD (2.5 yg/ml) on the The effect of CsA (10 y ~ and expression of RNA for TNF-a, IFN-y, IL 2R and actin by Con A-activated El0 clone was also determined (Fig. 2). Again, AcD, but not CsA, caused a significant inhibition of cytolytic activity on either GLT-pulsed LK cells or CD3mAb-induced lysis of P815 target (not shown). As shown in Fig. 2, after maintenance in IL 2-containing medium the E l 0 clone expresses residual levels of I F N y RNA. This level is increased after activation with Con A , but a 4-h incubation with either inhibitor strongly inhibited the Con A-induced expression of IFN-y RNA (Fig. 2b). Similar results were obtained with RNA that hybridized with the TNF-a probe (Fig. 2a). Both the 1.7-kb and the 1.6-kb bands that hybridized withTNF-a probe were enhanced by C o n A activation. Both AcD and CsA inhibited the expression of TNF-a mRNA t o control levels. This pattern of inhibition has been observed with 2.5 yM CsA on clones E10, F3 and 2G5 (not shown). The induction of TNF-p
Table 1. Effects of metabolic inhibitors on cytolytic activities of CD4 and CD8 clones % Specific 5'Cr releasea)
Inhibitor Concentrationb)
Antigen-specific killing)
CD3-mAb-induced killing
El0 c7 NPSS-20 NPSS-18 (GLT-LK) (KLH-LK) (NP-ELA) (NP-EU) Medium AcD Chx
CT CsA MitC
-
2.5 pglml 2.5 pglml 0.5pglml 1.5 pM
lO~g/ml
21f2 0k0 4f1 18 -t 2 19f1 22fO
41+2 9+1 8 f 2 35 k 3 32f1 40+3
30f2 25f1 28f2 3fl 26f2 27f2
33fl 32f2 28fO 5 f 2 30f2 32f3
c7
El0 (P815)
(P815)
F8 (P815)
NPSS-20 (P815)
NPSS-18 (P815)
34f2 3+0 6 f l 29f3 26f2 33f3
42f3 7 f 2 6 f 3 38k2 35f2 4Of3
50f3 7+2 4+2 ND 42+2 54+4
48+1 42+2 47+2 6 f 1 39f3 43+l
43f2 39+3 39f1 ND 33+l 41fO
a) 'hrgets were prepared as described in Sect. 2.4.6. Assays were determined 4-6 h after culture. b) Cloned cells (3 x 104 for CD4 clones E10, C7 and F8 or 2 x 104 CD8 clones NPSS-20 and NPSS-18) were cultured with metabolic inhibitors in 0.1 ml for 45 min, followed by the addition of cells and activation agents to a final volume of 0.2 ml.Values indicated represent the final concentrations of 0.2 ml culture. c) The Mlsa-reactiveclone F8 has not been tested for antigen-specific killing.
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CD4 Clone E l 0
Eur. J. Immunol. 1990. 20: 179-184
I
CD8 Clone NPSS-20
60
20
10
Cloned Cell x
Cloned Cells x 10'
lo'
T E
30 16
3 16 76 pl of Supernatants
:"oq
'O
g
3 0 4 IFN-y
=
20
f
0
3 16 76 PIof Supernatants
/
IFN--y
10
0.6 pl
3 16 76 of Supamatants
3
15
76
pl of Supernatants
0.6 3 16 76 PIof Supernatants
3
is
Figure 2. Northern blot analysis for the production of RNA for TNF-a (a), IFN-y (b), IL 2R (c) and actin (d) by clone E l 0 that had been activated with Con A for 4 h in the presence or absence of metabolic inhibitors. Ten pg of RNA extracts from control E l 0 clone (lane l ) , E l 0 clone activated with Con A in the presence of 10 p~ CsA (lane 2), Con A-activated E l 0 clone (lane 3) and E l 0 clone activated with Con A in the presence of 2.5 pg/ml AcD (lane 4) were used. Background expression of these specific RNA could be observed with control E l 0 clone cultured in medium alone (lane 1). Con A activation increases their expression (lane 3). Both CsA and AcD inhibit the Con A-induced expression of RNA for TNF-a and IFN-y, but not the RNA expression for IL 2R and actin.
7
P I of Supernatants
Figure 1. Effect of metabolic inhibitors on lymphokine production by a CD4 clone (E10, left panels) and a CD8 clone (NPSS-20, right panels). Clones (lo6) were cultured with medium, AcD (2.5 pg/ml), CsA (3 p ~ or) C T (1 pg/ml) for 45 min and then washed 3 times with medium. Aliquots of washed cells were tested for cytolytic activity against Wr-labeled P815 cells in the presence of CD3-mAb in a 4-h assay. Aliquots of washed cells were also activated with Con A for 20 h for lymphokine production. Lymphokines in culture SN were determined as described in Sect. 2.6.
(lymphotoxin) mRNA was also inhibited in a similar experiment under similar conditions (see Fig. 3b, below). Under identical conditions, mRNA for I L 2 R and actin were not significantly inhibited by either CsA or AcD treatment (Fig. 2 lower panels). The data indicate that TNF and IFN-y contribute minimally during the 4 h of cytolytic attack by activated CD4 clones. Moreover, the putative cytolytic machinery of CD4 clones is sensitive to AcD inhibition, but resistant to CsA. Although the above observations are consistent with the idea that CD8 clones, but not CD4 clones, use pre-formed cytolytic mediators, such as perforin for cytolytic activity, the possibility that CD4 clones synthesize perforin only during the short period of activation for cytolytic activity has not been ruled out.Therefore, the role of perforin in the cytolytic activity of CD4 and CD8 clones was investigated. Using a perforin-specific cDNA probe, it was shown that Con A-activated CD8 clone NPSS-20 expresses perforin-
Figure 3. Northern blot analysis for perforin mRNA in CD8 clone NPSS-20 and 5 CD4 clones that had been cultured with either medium (med) or Con A for 4 h in the presence or absence of inhibitors (a).The concentrations of CsA, Cr,and AcD used were 2.5 p ~ 1, pg/ml, and 2.5 pg/ml, respectively. As a control, the expression of TNF-P mRNA in CD4 clones was also determined (b). Ten pg of RNA extracts from CD4 clones and 4 pg of FWA extract from clone NPSS-20 were applied. The direction of electrophoresis was from right to 1eft.The sizes of perforin mRNA and TNF-P mRNA were 2.6 kb and 1.6 kb, respectively.
Eur. J. Immunol. 1990. 20: 179-184
specific mRNA (Fig. 3a). In contrast, CD4 clones E10, F3, 2G5, C7 and E6 did not express detectable perforin-specific mRNA upon activation with Con A. As controls, these activated CD4 clones were found to produce TNF-P mRNA, which was inhibited by the presence of 2.5 VM CsA or 1 pg/ml CT (Fig. 3b).The data indicate that in the short period of activation, perforin is not produced by CD4 clones and can not be responsible for CD4 clone-mediated cytolytic activity.
4 Discussion In this study we demonstrated that metabolic inhibitors can selectively inhibit the expression of cytolytic activity of CD4 or CD8 clones.We found that AcD and Chx inhibit the cytolytic activity of CD4, but not CD8, clones. Conversely, CT inhibits the cytolytic activity of CD8, but not CD4, clones. This reciprocal inhibition pattern indicates that CD4 and CD8 clones utilize distinctly different cytolytic pathways for the expression of cytolytic activity. While resistance to MitC implies that DNA synthesis and cell division are not required for the expression of cytolytic activity by either CD4 or CD8 clones, the selective sensitivity of the cytolytic activity of CD4 clones to AcD and Chx indicates that CD4 clones, but not CD8 clones, require de novo synthesis of RNA and protein to express cytolytic activity. In contrast, the cytolytic principle is probably present in CD8 clones before activation. The fact that AcD and Chx do no inhibit the cytolytic activity of CD8'clones indicates that after activation, two separate pathways are followed; one leads to induction of RNA and protein synthesis, the other leads to activation of cytolytic activity; possibly by secretion of pre-formed cytotoxin-containing granules or other cytolytic mediators. One potential candidate to consider is perforin, which is found in granules of large granular lymphocytes and murine CD8 clones. Some, but not all, murine CD8 clones contain perforin [HI, although a recent study demonstrates that all murine CD8 cells express perforin-specific mRNA [21].The possibility that active synthesis of perforin by CD4 clones during activation was eliminated by the observation that no perforin mRNA could be detected in five individual clones that have been activated by Con A. These data argue convincingly that CD4 clones do not use perforin-mediated cytotoxicity. Perforin is secreted along with other enzymes, such as serine proteases, upon activation that induces granule exocytosis [32]. It has been shown that CsA and Mg2 EGTA, which block granule exocytosis, as is indicated by release of serine esterase activity, do not inhibit CTL cytolytic activity [13-15,331. Thus, although our inhibition data agrees with the concept of a pre-formed cytolytic machinery such as those identified in granules, it remains possible that additional [34] and unidentified pre-formed cytolytic components may be present in CD8 clones. CsA causes selective inhibition of certain mRNA species for lymphokines without affecting mRNA synthesis in general [29]. At the protein level, CsA inhibits I L 2 production, but not the expression of IL 2R [35, 361. We have found that mRNA production of TNF and IFN-y, but not actin and IL2R, to be inhibited (Figs. 2 and 3).
Cytolytic pathways of CD4 and CD8 clones
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Collectively, these observations suggest that the mRNA encoding the necessary components (including the putative cytolytic mediators) for the expression of cytolytic activity of CD4 clones is sensitive to AcD, but resistant to CsA. CT also inhibited lymphokine production (including IL 2), but not the cytolytic activity of CD4 clones. This agrees with a previous study that showed CT inhibits the proliferation of murine Tcell clones stimulated through TcR/CD3 complex but only weakly inhibits the IL 2-induced proliferation [37]. In our study, CT selectively inhibits the cytolytic activity of CD8, but not CD4 clones. After CT binding, the A subunit of CTcatalyzes ADP ribosylation of GTP-binding protein, activates membrane-bound adenylate cyclase and increases intracellular CAMP [38] to activate CAMP-dependent protein kinases [39]. How this signal transduction pathway inhibits lymphokine production of CD4 clones and selectively inhibits the cytolytic activity of CD8, but not CD4 clones, remains to be established. In a previous study [22], we have shown that a high-titered anti-TNF anti-serum completely inhibits the ability of CD4 clones to kill TNF-sensitive L-929 cells, but not TNFresistant targets such as those used in this study. These TNF-resistant targets were chosen so that cytolytic activity of CD4 and CD8 clones can be studied without TNF interference. The use of metabolic inhibitors to dissociate cytotoxic lymphokines (TNF and IFN-y) from expression of cytolytic activity not only rules out that these targets are killed by high local concentrations of TNF and IFN-y, but also indicates that both CD4 and CD8 clones can express a TNF-mediated (for TNF-sensitive targets) and TNF-independent cytolytic activity. In addition, these data also demonstrate that following activation of CD4 and CD8 clones, separate pathways for lymphokine production (including TNF and IFN-y) and expression of TNFindependent cytolytic activity are generated. Although we have shown that activated CD4 clones do not use TNF, EN-y and perforin as cytolytic mediators, the important challenge is to identify the putative cytolytic factor(s) responsible for the TNF-independent cytolytic activity of CD4 clones. Wethank Drs. A . Cerami, N . Ruddle, E. Poddack, R . N . Germain, l? Burd, G . Freeman and J. Bluestone for providing the probes and hybridomas used in this study. We also thank Dr. M . E. Dorf for his critical review of this manuscript.
Received June 28, 1989; in revised form September 28, 1989.
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€! Strack, C. Martin, S. Saito et al.
9 Ozaki, S.,York-Jolly, J., Kawamura, H. and Berzofsky, J. A., Cell. Immunol. 1987. 105: 301. 10 Leo, O., Sachs, D. H., Samuelson, L. E., Foo, M., Quinones, R., Gress, R. and Bluestone, J., J. Immunol. 1986. 137: 3874. 11 Utsunomiya, N., Nakanishi, M., Arata,Y.,Tasuda,T., Saito, S., Koyama, K. and Tadakuma, T., J. Immunol. 1988. 141: 1471. 12 Imboden, J. B. and Stobo, J. D., J. Exp. Med. 1985. 161: 446. 13 Ostergarrd, M. I., Kane, K. l?, Mescher, M. F. and Clark, W. R., Nature 1987. 330: 71. 14 Trenn, G., Takayama, H. and Sitkovsky, M. V , Nature 1987. 330: 72. 15 Young, J. D.-E., Clark, W. R., Liu, C.-C. and Cohn, Z. A., J. Exp. Med. 1987. 166: 1894. 16 Martz, E., Contemp. Top. Immunobiol. 1977. 7: 301. 17 Chang,T. and Eisen, H. N., J. Immunol. 1980. 124: 1028. 18 Quan, l?-C., Ishizada,T. and Bloom, B. R., J. Immunol. 1982. 128: 1786. 19 Wright, S. C. and Bonavida, B., J. Immunol. 1983. 130: 2960. 20 Redelman, D. and Hudig, D., J. Imrnunol. 1980. 124: 870. 21 Lowrey, D. M., Aebischer, T., Olsen, K., Lichtenheld, M., Rupp, F., Hengartner, H. and Podack, E. R., Proc. Natl. Acad. Sci. USA 1989. 86: 247. 22 Ju, S.-T., Strack, I?, Stromquist, D. and Dekruyff, R . H., Cell. Immunol. 1989. 117: 399. 23 Leo, O., Foo, M., Sachs, D. H., Samuelson, L. E. and Bluestone, J. A., Proc. Natl. Acad. Sci. USA 1987. 84: 1374.
Eur. J. Immunol. 1990.20: 179-184 24 Dekruyff, R. H., Ju, S.-T., Lanning, J. and Dorf, M. E., Eur. J. Immunol. 1987. 17: 1115. 25 Caput, D., Beutler, B., Hartog, K. ,Thayer, R., Brown-Shimer, S. and Cerami, A , , Proc. Natl. Acad. Sci. USA 1986. 83: 1670. 26 Sheehan, K. F., Ruddle, N. H . and Schreiber, R. D., J. Immunol. 1989. 142: 3884. 27 Miller, J., Malek,T. R., Leonard,W. J., Greene,W. C., Shevach, E. M. and Germain, R. N., J. Immunol. 1985. 134: 4212. 28 Enoch, T., Zinn, K. and Maniatis,T., Mol. Cell. Biol. 1986. 6: 801. 29 Elliott, J. F., Lin,Y., Mizel, S. B., Bleakly, R. C., Harnish, D. G. and Paetkau,V, Science 1984.226: 1439. 30 Bunjes, D., Hardt, C., Rollinghof, M. and Wagner, H., Eur. J. Immunol. 1981. 11: 657. 31 Shevach, E. M., Annu. Rev. Immunol. 1985. 3: 397. 32 Garcia-Sanz, J. A., Plactinck, G., Vellotti, F., Masson, D., Tschopp, J., MacDonald, H. R. and Nabholz, M., Eur. J. Immunol. 1987. 17: 933. 33 Lancki, D. W., Kaper, B. €! and Fitch, E W., J. Immunol. 1989. 142: 416. 34 Liu, C.-C., Steffan, M., King, K. and Young, J. D.-E., Cell 1987. 51: 393. 35 Palacios, R., J. Immunol. 1982. 128: 337. 36 Koizumi,T., Nakao,Y., Matsui,T., Katakami,Y., Katakami, N. and Fugita, T., Cell. Immunol. 1985. 103: 469. 37 Kim, D.-K., Nau, G.T., Lancki, D. W., Dawson, G. and Fitch, E W., J. Immunol. 1988. 141: 3429. 38 Van Heyningen, S., Biosci. Rep. 1982. 2: 135. 39 Smith, S. B.,White, H. D., Siegel, J. B. and Krebs, E. G., Proc. Natl. Acad. Sci. USA 78: 1591.
Peggy StrackO, Carla Martin+, Saburu Saito+, Rosemarie H. Dekruyff* and Shyr-Te Juoo Arthritis Center, K510, Boston University School of Medicineo, and Department of Pathology, Harvard Medical School+, Boston, and Department of Pediatrics, Children’s Hospitala, Palo Alto
Cytolytic pathways of CD4 and CD8 clones
Metabolic inhibitors distinguish cytolytic activity of CD4 and CDS clones* The effect of various metabolic inhibitors on the expression of cytolytic activity of CD4 ( T H ~and ) CD8 (CTL) clones was studied. The cytolytic activity of CD4 clones, but not CD8 clones, was sensitive to the FWA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide. Conversely, cholera toxin (CT) inhibited cytolytic activity of CD8, but not CD4 clones. Both mitomycin C, a DNA synthesis inhibitor, and cyclosporin A (CsA) failed to inhibit the cytolytic activity of either CD4 or CD8 clones. Although pretreatment with CsA or CTdid not inhibit the cytolytic activity of CD4 clones, lymphokine (interleukin 2, IL 2, interferon-y, IFN-y, and tumor necrosis factor, TNF) production was strongly inhibited. Similarly, pretreatment of a CD8 clone with actinomycin D or CsA inhibited lymphokine production without affecting cytolytic activity.The production of mRNA for TNF and IFN-y by concanavalin A-activated CD4 clones was also inhibited by CsA and CT. Moreover, perforin-specific mRNA was not detected in activated CD4 clones. Collectively, these observations demonstrated that de novo synthesis of RNA and protein is required for expression of cytolytic activity of CD4 clones, yet production of TNF, INF-y, IL 2 and perforin is not involved. In contrast, the cytolytic machinery of CD8 clones is present prior to activation and is quickly expressed following activation even when de novo synthesis of RNA, protein and lymphokines is blocked.
1 Introduction Both CD4 and CD8 Tcell subsets can express cytolytic activity upon activation [l-91. When activated by antigen and Ia-bearing APC, CD4 cells can be induced t o express cytolytic activity toward the APC. Similarly, the cytolytic activity of CD8 cells (CTL) can be induced by antigen and class I molecules on appropriate target cells. Nonspecific Tcell activators such as Con A or a anti-CD3 mAb can also induce the expression of cytolytic activity [ l , lo]. After antigen binding and cross-linking of TcR, membrane fluidity increases [ l l ] and signals are transduced through the cell membrane which increase intracellular calcium and activate PKC [12]. A calcium chelator, EGTA, inhibits cytolytic activity of CD4 clones by inhibiting T cell activation [8]. However, under defined conditions, CD8 cells express cytolytic activity in the presence of excess (Mg)z EGTA [13-151. The operationally identifiable stages of CTL killing, i. e. ,calcium-independent, magnesium-dependent binding, activation and lethal hit, have not been
[I 77671
*
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This work is supported by LO1 ACS-IN97, AI-24571 and CA-39790. Investigator of the American Heart Association.
Correspondence: Shyr-Te Ju, 71 E. Concord St., Arthritis Center, K510, Boston University School of Medicine, Boston, MA 02118, USA Abbreviations: AcD: Actinomycin D CX Cholera toxin Chx: Cycloheximide CsA: Cyclosporin A GLT: Random copolymer of L-Glu, L-LYS,L-Tyr MitC: Mitomycin C M T E 3(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide 0 VCH Verlagsgesellschaft mbH, D-6940 Weinheim, 1990
defined for CD4 clones [16]. Also, little is known about the intracellular pathway(s) of activation leading to expression of cytolytic activity.Variousmetabolic inhibitors have been used to study the intracellular elements important for the expression of cytolytic activity in heterogeneous NK and CD8 cells [17-201. These studies support the “stimulationsecretion” model for cytotoxicity and suggest that intracelMar CAMP, microtubules, phospholipase A2, calmodulin and lysosomal function are important for the expression of cytolytic activity [MI. The “stimulation-secretion’’ model agrees well with the observations that both CD8 and NK cells secrete perforin from their granules as a cytolytic mediator [21]. The cytolytic mechanisms of CD4 clones have not been well defined. It has been proposed that TNF and IFN-y are the soluble mediators for target lysis. However, many targets that are killed by activated CD4 clones are resistant to high concentrations of TNF and IFN-y. Although it remains possible that these targets are killed by very high local concentrations of these mediators, an alternative hypothesis is that a TNF (and IFN-y)-independent cytolytic mechanism is also induced to kill these targets upon activation [22]. In order to determine whether CD4 and CD8 clones use distinct cytolytic pathways and to understand better the relationship between lymphokine production (including the cytotoxickytostatic mediators of TNF/IFN-y) and the expression of T cell cytolytic activity, we compare the effect of metabolic inhibitors on cytolytic activity and lymphokine production by CD4 and CD8 clones. We demonstrate that (a) distinct cytolytic pathways are used by CD4 and CD8 clones, (b) separate activation pathways for lymphokine production and cytolytic activity exist in CD4 and CD8 clones, and (c) CD4 clones do not use perforin-dependent cytolytic mechanism of CD8 clones. 0014-2980/90/0101-0179$02.50/0
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2 Materials and methods 2.1 Animals
Female BALB/CByJ and C57BL/6 mice were purchased from Jackson Laboratory (Bar Harbor, ME). The mice were maintained on acidified tap water and Purina Mouse Chow ad libitum.
Eur. J. Immunol. 1990.20: 179-184 MA), shaken every 5 min for 30-35 rnin and then washed 3 times before use. Antigen-pulsed LK cells (@/well in 1ml) were prepared by culturing overnight with the appropriate antigen (GLT for E10, KLH for C7 and E6) at 1 mg/ml. 51Cr-labeledEL4 cells, after washing once in PBS, pH 7.2, and resuspended in 1ml of PBS, pH 7.8,were mixed with 10 y1 NP-0-Su (2.5 mg/ml) for 4 min after which 10 ml PBS, pH 7.2, was added and immediately centrifuged. %r-labeled NP-EL4 cells were then washed 3 times in culture medium before use. All target cells were used at 104/well.
2.2 Reagents Culture medium was DMEM (Gibco, Grand Island, NY) supplemented with 1% penicillinlstreptomycin, 1% L-glutamine, 1% MEM vitamins, 1% nonessential amino acids, 1% sodium pyruvate (Gibco) and 7.5% FBS (Hyclone Laboratories, Inc., Logan, UT). A hybridoma (145-2C11) specific to C D ~ (CD3 E mAb) [23] was kindly provided by Dr. J. Bluestone at N.I.H. (Bethesda, MD). 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTI'), Con A , mitomycin C (MitC), actinomycin D (AcD) and cycloheximide (Chx) were obtained from Sigma (St. Louis, MO). Cyclosporin A (CsA) was purchased from the University Hospital pharmacy. Cholera toxin (CT) was obtained from List Biol. Lab. Inc. (Campell, CA). 2.3 Derivation and maintenance of clones
The derivation of CD4 clone E l 0 (GLT specific, I-Ed restricted) has been described [24]. KLH-specific clones C7 (I-Ad restricted) and E6 (I-Ed specific) and Mlsa-reactive clones F3 and F8 (both are I-Ed restricted) were derived similarly from BALB/c mice. Clone 2G5 (Mlsa reactive) was derived from a MRL/MP-lpr mouse. These clones express CD4+, CD8- phenotype and secrete lymphokines of T Htype, ~ i . e . , IL 2 and IFN-./.They were maintained in 24-well plates (Costar 2424, Cambridge, MA) by periodic stimulation (every 10-15 days) of 0.25 x lo6clone cells with the appropriate antigen (20-30 pg/ml) and 3 x lo6 irradiated (3000 rds) spleen cells (SC) of appropriate mouse strains, and expanded with IL 2-containing Con A SN. CD8 clones NPSS-18, and NPSS-20 were derived by priming C57BL/6J mice with 1 mg (4-hydroxy-3-nitropheny1)acetyl (NF')-0-succinimide (NP-0-Su) solution in DMSO S.C. One week after priming, inguinal LN were removed and stimulated with NP-coupled SC. Three days later, LD microcultures at 1 cell/well were seeded with 6 x lo5 irradiated (2000 rds) C57BL/6J SC and 10% PMA-stimulated EL4 SN as a source of growth factors. Every 2 days 3 x 105 irradiated C57BL/6J SC and 10% EL4 SN were added to the culture. After 10-14 days growing cultures were expanded and then subcloned. The CD4-, CD8+ clones NPSS-18 and NPSS-20 are specific for NP in association with H-2Db and H-2Kb, respectively. Clones were maintained by culturing at 7-11-day intervals with NP-coupled irradiated SC.Viable cells of all clones were separated over a Ficoll-Hypaque (Pharmacia, Piscataway, NJ) gradient and washed 3 times with culture medium before use. 2.4 Preparation of target cells Pelleted target cells were labeled with 100 pl W r by the addition of radioactive sodium chromate (NEN, Boston,
2.5 Effect of metabolic inhibitors on the cytolytic activity mediated by clones Clones were incubated with various metabolic inhibitors for 45 rnin in 96-well plates (total volume 100 pl) after which Wr-labeled target cells were added. When Wr-labeled P815 cells were used as targets, CD3-mAb (25%, v/v) was also added as a nonspecific activating agent to induce cytolytic activity. In some experiments, the clones (106/ml) were incubated 45 min with the various inhibitors,washed 3 times with medium, and then tested for cytolytic activity and lymphokine production. These treatments did not result in nonspecific toxicity of the effector clones, as judged by trypan blue dye exclusion or by MTT tests (added at the initiation of culture) and measured at 570 nm at the end of culture. After 4-6 h of culture, 100 yl of SN was carefully removed from each well and counted in a gamma-scintillation counter. Percent specific lysis was determined by the formula: % specific 51Cr release (or lysis) = (cpm of test sample - cpm of nonspecific release)/(cpm of total release - cpm of nonspecific release). The cpm of nonspecific release and total release were obtained by culturing target cells in medium and by lysing target cells with 0.5% NP40, respectively. 2.6 Generation and assays of lymphokines
Clones (106/ml) were incubated 45 min with the various inhibitors, washed 3 times and then cultured with Con A (2.5 pglml). SN were collected after 20 h. Assays for TNF, IFN-y and IL 2 were carried out as previously described [22]. Briefly, the assay for TNF was based on its ability to kill L-929 cells. The assay for IFN-y was based on its ability to induce Ia expression on P388D1 M@ tumor cell line. Ia expression was determined by cellular RIA. The assay for IL 2 was based on its ability to induce proliferation of CTLL cell, which was measured by [3H]dThd incorporation. In all cases, the specificity of the assay was confirmed by inhibition with specific monoclonal and polyclonal antibodies [22]. 2.7 Northern blot analysis
Analysis of mRNA for TNF, IFN-y, IL2R, actin and perforin was carried out with RNA extracts from 25 x 106 cloned cells that had been activated with Con A for 4 h in the presence or absence of metabolic inhibitors.The probes for TNF-a [25], TNF-P [26], IL 2R [27], actin [28] and perforin [21] were the kind gifts of Drs. A. Cerami, N. Ruddle, R. N. Germain, G . Freeman, and E. Poddack, respectively. The probe for IFN-y was prepared by and
Eur. J. Immunol. 1990. 20: 179-184
Cytolytic pathways of CD4 and CD8 clones
provided to us by Dr. F? Burd at the Department of Pathology, Harvard Medical School.
3 Results We compared the effects of several metabolic inhibitors on the cytolytic activity of CD4 and CD8 clones.The inhibitors used were: MitC for blocking DNA synthesis, AcD for blocking RNA synthesis, Chx for blocking protein synthesis, CT for increasing intracellular CAMP and CsA for selective inhibition of RNA and lymphokine synthesis [29, 30l.Three CD4 clones and two CD8 clones were tested with similar results. In the continuous presence of AcD or Chx, the cytolytic activity of CD4 clones were inhibited by > 80%. Both antigen-specific, Ia-restricted cytolytic activity on antigen-pulsed LK target cells and nonspecific cytolytic activity induced by CD3-mAb on P815 target cells were inhibited. In contrast, neither antigen-specific, H-2Kbrestricted lysis of NP-EL4 target nor CD3-mAb-induced nonspecific killing of P815 by CTL clones were inhibited. A reciprocal pattern of inhibition was found with CT.This reagent inhibited the cytolytic activity of CD8, but not CD4, clones. In agreement with previous studies [6, 311, CsA failed to inhibit the cytolytic activity of CD4 and CD8 clones. MitC did not inhibit the cytolytic activity of either CD4 or CD8 clones, although these treated cells failed to incorporate [3H]dThd into DNA in the presence of a high concentration of r I L 2 (10 BRMP U/ml). Under these conditions, none of the metabolic inhibitors caused significant cell damage to either effector or target cells (see Sect. 2.5). Moreover, the selective inhibition of cytolytic activity of CD4 or CD8 clones also ruled out nonspecific toxicity of these treatments. The generality of these observations was supported by the fact that similar results were obtained using three different CD4 clones, two separate CD8 clones and two different activation methods with each inhibitor (Table 1). The above effector clones were also pretreated with these metabolic inhibitors for 45 min and then washed three times before testing cytolytic activity.The results were essentially
181
the same with the exception that pretreatment with 2.5-100 yg/ml of Chx did not inhibit the cytolytic activity of CD4 clones (data not shown). This result pinpoints the effect of metabolic inhibitors on the effector clones and indicate that de novo synthesis of DNA, RNA and protein by target cells is not very critical for target lysis. To determine the relationship between lymphokine production and cytolytic activity, clones were pretreated with various inhibitors, washed and tested for cytolytic activity and the production of various lymphokines. As shown in Fig. 1, pretreatment with AcD, but not CsA or CT,strongly inhibited the cytolytic activity of CD4 clone E10.Yet both CsA and CT block production of IL 2 (> 97%), caused marked (> 90%) inhibition of IFN-y secretion and inhibited TNF production by approximately 80%.The degree of inhibition is comparable to that achieved with AcD treatment. Similarly, lymphokine production by the CD8 clone NPSS-20 was inhibited to a comparable degree by AcD or CsA treatment. These treatments did not inhibit cytolytic activity. The dissociation of lymphokine production from cytolytic activity indicates that TNF and IFN-y are not important for the killing of these target cells. ) AcD (2.5 yg/ml) on the The effect of CsA (10 y ~ and expression of RNA for TNF-a, IFN-y, IL 2R and actin by Con A-activated El0 clone was also determined (Fig. 2). Again, AcD, but not CsA, caused a significant inhibition of cytolytic activity on either GLT-pulsed LK cells or CD3mAb-induced lysis of P815 target (not shown). As shown in Fig. 2, after maintenance in IL 2-containing medium the E l 0 clone expresses residual levels of I F N y RNA. This level is increased after activation with Con A , but a 4-h incubation with either inhibitor strongly inhibited the Con A-induced expression of IFN-y RNA (Fig. 2b). Similar results were obtained with RNA that hybridized with the TNF-a probe (Fig. 2a). Both the 1.7-kb and the 1.6-kb bands that hybridized withTNF-a probe were enhanced by C o n A activation. Both AcD and CsA inhibited the expression of TNF-a mRNA t o control levels. This pattern of inhibition has been observed with 2.5 yM CsA on clones E10, F3 and 2G5 (not shown). The induction of TNF-p
Table 1. Effects of metabolic inhibitors on cytolytic activities of CD4 and CD8 clones % Specific 5'Cr releasea)
Inhibitor Concentrationb)
Antigen-specific killing)
CD3-mAb-induced killing
El0 c7 NPSS-20 NPSS-18 (GLT-LK) (KLH-LK) (NP-ELA) (NP-EU) Medium AcD Chx
CT CsA MitC
-
2.5 pglml 2.5 pglml 0.5pglml 1.5 pM
lO~g/ml
21f2 0k0 4f1 18 -t 2 19f1 22fO
41+2 9+1 8 f 2 35 k 3 32f1 40+3
30f2 25f1 28f2 3fl 26f2 27f2
33fl 32f2 28fO 5 f 2 30f2 32f3
c7
El0 (P815)
(P815)
F8 (P815)
NPSS-20 (P815)
NPSS-18 (P815)
34f2 3+0 6 f l 29f3 26f2 33f3
42f3 7 f 2 6 f 3 38k2 35f2 4Of3
50f3 7+2 4+2 ND 42+2 54+4
48+1 42+2 47+2 6 f 1 39f3 43+l
43f2 39+3 39f1 ND 33+l 41fO
a) 'hrgets were prepared as described in Sect. 2.4.6. Assays were determined 4-6 h after culture. b) Cloned cells (3 x 104 for CD4 clones E10, C7 and F8 or 2 x 104 CD8 clones NPSS-20 and NPSS-18) were cultured with metabolic inhibitors in 0.1 ml for 45 min, followed by the addition of cells and activation agents to a final volume of 0.2 ml.Values indicated represent the final concentrations of 0.2 ml culture. c) The Mlsa-reactiveclone F8 has not been tested for antigen-specific killing.
P. Strack, C. Martin, S. Saito et al.
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CD4 Clone E l 0
Eur. J. Immunol. 1990. 20: 179-184
I
CD8 Clone NPSS-20
60
20
10
Cloned Cell x
Cloned Cells x 10'
lo'
T E
30 16
3 16 76 pl of Supernatants
:"oq
'O
g
3 0 4 IFN-y
=
20
f
0
3 16 76 PIof Supernatants
/
IFN--y
10
0.6 pl
3 16 76 of Supamatants
3
15
76
pl of Supernatants
0.6 3 16 76 PIof Supernatants
3
is
Figure 2. Northern blot analysis for the production of RNA for TNF-a (a), IFN-y (b), IL 2R (c) and actin (d) by clone E l 0 that had been activated with Con A for 4 h in the presence or absence of metabolic inhibitors. Ten pg of RNA extracts from control E l 0 clone (lane l ) , E l 0 clone activated with Con A in the presence of 10 p~ CsA (lane 2), Con A-activated E l 0 clone (lane 3) and E l 0 clone activated with Con A in the presence of 2.5 pg/ml AcD (lane 4) were used. Background expression of these specific RNA could be observed with control E l 0 clone cultured in medium alone (lane 1). Con A activation increases their expression (lane 3). Both CsA and AcD inhibit the Con A-induced expression of RNA for TNF-a and IFN-y, but not the RNA expression for IL 2R and actin.
7
P I of Supernatants
Figure 1. Effect of metabolic inhibitors on lymphokine production by a CD4 clone (E10, left panels) and a CD8 clone (NPSS-20, right panels). Clones (lo6) were cultured with medium, AcD (2.5 pg/ml), CsA (3 p ~ or) C T (1 pg/ml) for 45 min and then washed 3 times with medium. Aliquots of washed cells were tested for cytolytic activity against Wr-labeled P815 cells in the presence of CD3-mAb in a 4-h assay. Aliquots of washed cells were also activated with Con A for 20 h for lymphokine production. Lymphokines in culture SN were determined as described in Sect. 2.6.
(lymphotoxin) mRNA was also inhibited in a similar experiment under similar conditions (see Fig. 3b, below). Under identical conditions, mRNA for I L 2 R and actin were not significantly inhibited by either CsA or AcD treatment (Fig. 2 lower panels). The data indicate that TNF and IFN-y contribute minimally during the 4 h of cytolytic attack by activated CD4 clones. Moreover, the putative cytolytic machinery of CD4 clones is sensitive to AcD inhibition, but resistant to CsA. Although the above observations are consistent with the idea that CD8 clones, but not CD4 clones, use pre-formed cytolytic mediators, such as perforin for cytolytic activity, the possibility that CD4 clones synthesize perforin only during the short period of activation for cytolytic activity has not been ruled out.Therefore, the role of perforin in the cytolytic activity of CD4 and CD8 clones was investigated. Using a perforin-specific cDNA probe, it was shown that Con A-activated CD8 clone NPSS-20 expresses perforin-
Figure 3. Northern blot analysis for perforin mRNA in CD8 clone NPSS-20 and 5 CD4 clones that had been cultured with either medium (med) or Con A for 4 h in the presence or absence of inhibitors (a).The concentrations of CsA, Cr,and AcD used were 2.5 p ~ 1, pg/ml, and 2.5 pg/ml, respectively. As a control, the expression of TNF-P mRNA in CD4 clones was also determined (b). Ten pg of RNA extracts from CD4 clones and 4 pg of FWA extract from clone NPSS-20 were applied. The direction of electrophoresis was from right to 1eft.The sizes of perforin mRNA and TNF-P mRNA were 2.6 kb and 1.6 kb, respectively.
Eur. J. Immunol. 1990. 20: 179-184
specific mRNA (Fig. 3a). In contrast, CD4 clones E10, F3, 2G5, C7 and E6 did not express detectable perforin-specific mRNA upon activation with Con A. As controls, these activated CD4 clones were found to produce TNF-P mRNA, which was inhibited by the presence of 2.5 VM CsA or 1 pg/ml CT (Fig. 3b).The data indicate that in the short period of activation, perforin is not produced by CD4 clones and can not be responsible for CD4 clone-mediated cytolytic activity.
4 Discussion In this study we demonstrated that metabolic inhibitors can selectively inhibit the expression of cytolytic activity of CD4 or CD8 clones.We found that AcD and Chx inhibit the cytolytic activity of CD4, but not CD8, clones. Conversely, CT inhibits the cytolytic activity of CD8, but not CD4, clones. This reciprocal inhibition pattern indicates that CD4 and CD8 clones utilize distinctly different cytolytic pathways for the expression of cytolytic activity. While resistance to MitC implies that DNA synthesis and cell division are not required for the expression of cytolytic activity by either CD4 or CD8 clones, the selective sensitivity of the cytolytic activity of CD4 clones to AcD and Chx indicates that CD4 clones, but not CD8 clones, require de novo synthesis of RNA and protein to express cytolytic activity. In contrast, the cytolytic principle is probably present in CD8 clones before activation. The fact that AcD and Chx do no inhibit the cytolytic activity of CD8'clones indicates that after activation, two separate pathways are followed; one leads to induction of RNA and protein synthesis, the other leads to activation of cytolytic activity; possibly by secretion of pre-formed cytotoxin-containing granules or other cytolytic mediators. One potential candidate to consider is perforin, which is found in granules of large granular lymphocytes and murine CD8 clones. Some, but not all, murine CD8 clones contain perforin [HI, although a recent study demonstrates that all murine CD8 cells express perforin-specific mRNA [21].The possibility that active synthesis of perforin by CD4 clones during activation was eliminated by the observation that no perforin mRNA could be detected in five individual clones that have been activated by Con A. These data argue convincingly that CD4 clones do not use perforin-mediated cytotoxicity. Perforin is secreted along with other enzymes, such as serine proteases, upon activation that induces granule exocytosis [32]. It has been shown that CsA and Mg2 EGTA, which block granule exocytosis, as is indicated by release of serine esterase activity, do not inhibit CTL cytolytic activity [13-15,331. Thus, although our inhibition data agrees with the concept of a pre-formed cytolytic machinery such as those identified in granules, it remains possible that additional [34] and unidentified pre-formed cytolytic components may be present in CD8 clones. CsA causes selective inhibition of certain mRNA species for lymphokines without affecting mRNA synthesis in general [29]. At the protein level, CsA inhibits I L 2 production, but not the expression of IL 2R [35, 361. We have found that mRNA production of TNF and IFN-y, but not actin and IL2R, to be inhibited (Figs. 2 and 3).
Cytolytic pathways of CD4 and CD8 clones
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Collectively, these observations suggest that the mRNA encoding the necessary components (including the putative cytolytic mediators) for the expression of cytolytic activity of CD4 clones is sensitive to AcD, but resistant to CsA. CT also inhibited lymphokine production (including IL 2), but not the cytolytic activity of CD4 clones. This agrees with a previous study that showed CT inhibits the proliferation of murine Tcell clones stimulated through TcR/CD3 complex but only weakly inhibits the IL 2-induced proliferation [37]. In our study, CT selectively inhibits the cytolytic activity of CD8, but not CD4 clones. After CT binding, the A subunit of CTcatalyzes ADP ribosylation of GTP-binding protein, activates membrane-bound adenylate cyclase and increases intracellular CAMP [38] to activate CAMP-dependent protein kinases [39]. How this signal transduction pathway inhibits lymphokine production of CD4 clones and selectively inhibits the cytolytic activity of CD8, but not CD4 clones, remains to be established. In a previous study [22], we have shown that a high-titered anti-TNF anti-serum completely inhibits the ability of CD4 clones to kill TNF-sensitive L-929 cells, but not TNFresistant targets such as those used in this study. These TNF-resistant targets were chosen so that cytolytic activity of CD4 and CD8 clones can be studied without TNF interference. The use of metabolic inhibitors to dissociate cytotoxic lymphokines (TNF and IFN-y) from expression of cytolytic activity not only rules out that these targets are killed by high local concentrations of TNF and IFN-y, but also indicates that both CD4 and CD8 clones can express a TNF-mediated (for TNF-sensitive targets) and TNF-independent cytolytic activity. In addition, these data also demonstrate that following activation of CD4 and CD8 clones, separate pathways for lymphokine production (including TNF and IFN-y) and expression of TNFindependent cytolytic activity are generated. Although we have shown that activated CD4 clones do not use TNF, EN-y and perforin as cytolytic mediators, the important challenge is to identify the putative cytolytic factor(s) responsible for the TNF-independent cytolytic activity of CD4 clones. Wethank Drs. A . Cerami, N . Ruddle, E. Poddack, R . N . Germain, l? Burd, G . Freeman and J. Bluestone for providing the probes and hybridomas used in this study. We also thank Dr. M . E. Dorf for his critical review of this manuscript.
Received June 28, 1989; in revised form September 28, 1989.
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