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The Journal of Immunology, 1998, 160: 5253-5261.
Copyright © 1998 by The American Association of Immunologists

Two Types of Anti-TL (Thymus Leukemia) CTL Clones with Distinct Target Specificities: Differences in Cytotoxic Mechanisms and Accessory Molecule Requirements1

Kunio Tsujimura*, Toshitada Takahashi*, Shigeru Iwase*,{dagger}, Yasue Matsudaira*, Yoko Kaneko{ddagger}, Hideo Yagita§ and Yuichi Obata2,*

* Laboratory of Immunology, Aichi Cancer Center Research Institute, Nagoya; {dagger} Department of Chemical Hygiene and Nutrition, Faculty of Pharmaceutical Sciences, Nagoya City University, Nagoya; {ddagger} Department for Geriatric Research, National Institute for Longevity Sciences, Obu; and § Department of Immunology, Juntendo University School of Medicine, Tokyo, Japan


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
TCR{alpha}ß CTL clones recognizing mouse thymus leukemia (TL) Ags were established and categorized into two groups: those killing any TL+ target cells (type I) and those killing only TL+ Con A blasts (type II). Cold target inhibition assays showed that the antigenic determinant(s) recognized by type II clones are expressed not only on TL+ Con A blasts but also on other TL+ target cells. The relation of the target specificity to the killing machinery and the accessory molecules involved in cytotoxicity were therefore analyzed using four representative clones selected from each type. Of the target cells tested, Fas was only expressed on Con A blasts, indicating that Fas ligand (FasL)-dependent cytotoxicity is limited to such cells. All four type II and one of four type I clones expressed FasL on the surface, while both types contained perforin in the cytoplasm. Blocking studies using neutralizing anti-FasL mAbs and concanamycin A (CMA), a selective inhibitor of the perforin pathway, suggested that type I clones kill target cells by way of perforin, while type II clones kill TL+ Con A blasts through FasL together with perforin. For their cytotoxicity, type I CTLs require a signal through CD8, while type II require LFA-1/ICAM-1 interactions. Type II clones also need a costimulatory signal through an unknown molecule for perforin-dependent cytotoxicity. These results taken together suggest that the difference in the target specificity of anti-TL CTL clones is due to variation in the killing machineries and the dependence on accessory molecules.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Mouse thymus leukemia (TL)3 Ags belong to the family of nonclassical MHC class I Ags and have a unique manner of expression, i.e., in contrast to other classical or nonclassical MHC class I Ags, they are restricted to the intestines in all mouse strains and the thymus of TL+ strains (e.g., the A strain and BALB/c mice) (1, 2, 3, 4). Although TL- strains (e.g., C57BL/6 (B6) and C3H/He (C3H)) do not express TL in the thymus, a proportion of T lymphomas originating in these mice express TL as a tumor Ag. It has been known as a serologically defined Ag (1, 2), but recently we have succeeded in generating TCR{alpha}ß and {gamma}{delta} CTLs recognizing TL directly without Ag presentation by H-2 molecules and further showed that they are cytotoxic against syngeneic and allogeneic TL+ leukemia cells, indicating that TL serves as a tumor rejection Ag (5, 6).

In previous studies, we showed that TL-specific {gamma}{delta} CTLs are positively selected by Tlaa-3-TL expressed in the thymus of Tg.Tlaa-3 transgenic mice (C3H background) and characterized these {gamma}{delta} clones, since those with such a distinct CTL specificity are relatively rare (6, 7, 8). In the present study, to investigate the mechanisms of Ag recognition and cytotoxicity of anti-TL {alpha}ß CTL, which account for the major part of the anti-TL CTL population, TCR{alpha}ß clones were established from C3H and Tg.Tlaa-3-2 transgenic mice and characterized. They were categorized into two groups in terms of their target specificity, i.e., CTL clones that lyse any TL+ target cells (type I) and those that lyse only TL+ Con A blasts derived from Tg.Con.3-1 transgenic mice expressing T3b-TL ubiquitously (type II). Analysis of these CTL clones revealed that type I kills TL+ target cells preferentially by perforin, whereas type II kills Fas-expressing TL+ Con A blasts by Fas ligand (FasL) together with perforin. In addition, differences in the accessory molecule requirement for exertion of cytotoxicity were observed between type I and type II CTL clones.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Mice

The derivation of transgenic mouse strains has been described previously (7, 8). A transgenic strain, Tg.Con.3-1, with a chimeric gene in which the T3b gene (from B6) is driven by the H-2Kb promoter, expresses T3b-TL ubiquitously. Another strain, Tg.Tlaa-3-2, with a Tlaa-3 transgene (from A strain) with its own promoter, expresses Tlaa-3-TL predominantly on thymocytes and intestinal epithelial cells. These transgenic mice were generated on a C3H background, which does not express TL in the thymus, but expresses T3k-TL in the intestine. C3H mice were purchased from Japan SLC (Hamamatsu, Japan).

Antibodies

The following mAbs were developed in our laboratories, provided by other investigators, or purchased: hamster mAbs against TCR{alpha}ß (H57-597) (9), TCR{gamma}{delta} (GL3; Cedarlane, Hornby, Canada), CD3 (145-2C11) (10), CD28 (37.51.1; Caltag, South San Francisco, CA), CD48 (HM48-1) (11), Fas (Jo2; PharMingen, San Diego, CA), FasL (MFL1) (12) and {alpha}5 integrin/CD49e (HM{alpha}5-1) (13), rat mAbs against TL (HD168 and HD177) (14), L3T4/CD4 (GK1.5) (15), Lyt-2/CD8{alpha} (53-6.7) (16), CD2 (RM2-1) (17), LFA-1/CD11a (KBA) (18), heat-stable Ag/CD24 (J11d) (19), CD45 (30F11.1; PharMingen), {alpha}4 integrin/CD49d (428; Seikagaku Kogyo, Tokyo, Japan), {alpha}V integrin/CD51 (RMV-7) (20), ICAM-1/CD54 (KAT-1) (21), CD62L (MEL-14; PharMingen), B7-1/CD80 (RM80) (22), B7-2/CD86 (PO.3) (22), Vß2 (B20.6; PharMingen), Vß8.1 and Vß8.2 (KJ-16) (23), Vß9 (MR10-2, PharMingen), Vß14 (14-2, PharMingen), perforin (P1-8) (24), and mouse mAbs against TL.2 (TT213) (5), FasL (K10) (12), Lyt-3.2/CD8ß (ID9P35) (6), Pgp-1/CD44 (NU5-50; Seikagaku Kogyo), and Vß8 (F23.1; PharMingen)

Flow cytometric analysis

Flow cytometric analysis was performed on a FACScan (Becton Dickinson, Mountain View, CA). For secondary reagents, phycoerythrin-conjugated streptavidin (Biomeda, Foster City, CA), FITC-labeled goat anti-hamster IgG (Caltag), goat anti-rat IgG (Chemicon, Temecula, CA), or rabbit anti-mouse Igs (Dakopatts, Glostrup, Denmark) were used. For staining perforin within the cytoplasm, CTL clones were fixed with cold acetone and 4% paraformaldehyde, treated with 0.5% HIO4, and then stained with rat anti-perforin mAb (P1-8) and FITC-conjugated goat anti-rat IgG as described previously (24). For staining FasL on the cell surface, CTL clones (1 x 105) were incubated with irradiated Tg.Con.3-1 spleen cells (1 x 106) and IL-2 (5 ng/ml) for 20 h, and then stained with anti-FasL mAb (K10) and FITC-conjugated rabbit anti-mouse Igs.

Skin grafts

Female Tg.Tlaa-3-2 and C3H mice (6–10 wk old) received full-thickness sections of the skin (1-cm disks) from the abdomens of Tg.Con.3-1 mice onto their backs (5). Plaster casts were removed on day 10.

Establishment and maintenance of CTL clones

CTL were generated by MLC as previously described with slight modifications (5, 6). Briefly, 4 to 6 wk after the rejection of grafted skin, MLC was performed in 24-well tissue culture plates. Spleen cells (2 x 106 cells/well) from the recipient mice were cultured with irradiated (20 Gy) Tg.Con.3-1 spleen cells (2 x 106 cells/well). Five days later, CTL bulk cultures obtained from primary MLC were restimulated with irradiated Tg.Con.3-1 spleen cells for 5 days (secondary MLC). CTL clones were established from secondary MLC by the limiting dilution method in the presence of 5 ng/ml human rIL-2 (Takeda Chemical Industries, Osaka, Japan). Established clones were maintained by weekly stimulation with irradiated Tg.Con.3-1 spleen cells. The CTL activity of the clones was tested by 51Cr release assay as previously described (5, 6). For cold target inhibition assays, the CTL activity of clones against Tg.Con.3-1 Con A blasts was tested in the presence of serially diluted cold inhibitor cells. For Ab blocking tests, the CTL activity of clones against Tg.Con.3-1 Con A blasts was tested in the presence of serially diluted mAbs.

Target cells

The following cells were used as target cells for CTL assays: Con A blasts from Tg.Con.3-1 (expressing T3b) and C3H (TL-) mice, thymidine kinase-negative L cells (Ltk-, C3H-derived fibroblasts; TL-), H-2Kb/T3b Ltk- transfectants (T3b) (25), ERLD (B6-derived leukemia, T3b), and ASL1 (A strain-derived leukemia, Tlaa-1, -2, and -3). C3H-derived TL+ (T3k) leukemias, C3NB1 and C3NB2, and TL- leukemias, C3NB3 and C3NB4, were induced by N-butyl-N-nitrosourea (NBU) according to the method described by Nishizuka and Shisa (26) and were also used as target cells. All leukemias were maintained in vivo. Con A blasts were prepared from spleen cells as previously described (5, 6).

RNA extraction, RT-PCR, and nucleotide sequencing

Total RNA was extracted from CTL clones using TRIzol reagent (Life Technologies, Gaithersburg, MD). Ten micrograms of total RNA was reverse transcribed into first strand cDNA with 3 µg of random primers and 200 U of SuperScript II RT (Life Technologies). TCR ß-chains were amplified using Vß-specific primers and a single common Cß primer as described by Rao et al. (27). The RT-PCR products were separated by agarose gel electrophoresis, and Vß usage was determined. For nucleotide sequencing of the Vß-Dß-Jß junction, PCR products were cloned into a T-tailed M13 mp18 vector and sequenced by a cycle-sequencing method using an automated DNA sequencer (ABI 373A, Perkin-Elmer, Foster City, CA).

Inhibition of FasL-dependent and/or perforin-dependent cytotoxicity

For inhibition of FasL-dependent cytotoxicity, CTL activity was tested in the presence of a neutralizing anti-FasL mAb (MFL1). For inhibition of perforin-dependent cytotoxicity, CTL clones were pretreated with concanamycin A (CMA; Wako Pure Chemical Industries, Osaka, Japan) (28) for 2 h. CTL assays were also performed in the presence of both anti-FasL mAb and CMA.

N-{alpha}-benzyloxycarbonyl-L-lysine thiobenzoyl ester (BLT) esterase release assay

CTL clones (1 x 105) were incubated with various target cells (1 x 105) or immobilized mAbs for 4 h in 96-well round-bottom tissue culture plates, and the culture supernatants were collected. The BLT esterase activity of the supernatants was measured as described by Takayama et al. (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Anti-TL {alpha}ß CTL clones use a relatively limited Vß spectrum

Anti-TL CTL clones were established from seven C3H and nine Tg.Tlaa-3-2 transgenic mice expressing Tlaa-3-TL in the thymus (C3H background): T3b-TL positive skin derived from Tg.Con.3-1 transgenic mice was grafted onto both strains, and then TL-specific {alpha}ß CTL clones were established from spleen cells of mice that had rejected the skin grafts. Ab blocking tests showed that the CTL activity of all clones was inhibited by anti-TL Abs but not by anti-H-2k (data not shown), indicating that these CTL clones recognize TL directly without Ag presentation by H-2, in accordance with our previous results for bulk CTL lines (6, 7).

Vß usage of these {alpha}ß CTL clones was determined by RT-PCR, nucleotide sequencing, and flow cytometric analysis; the results are summarized in Table IGo. Relatively limited Vß (Vß2, -8.2, -8.3, -9, -14, and -15) were used, and especially, Vß8.3 and 14 were employed preferentially by CTL clones from both strains. All CTL clones derived from each individual mouse were found to use the same Vß except for one case (KC3 clones). Accordingly, heterogeneity in CDR3 regions of two KC2 clones and four KC4 clones were analyzed by nucleotide sequencing. All KC2 and KC4 clones used Vß8.3-Dß1.1-Jß1.3 and Vß8.2-Dß2.1-Jß2.7 with no N or P nucleotide heterogeneity, respectively (data not shown). These results suggest that the CTL clones established from each individual mouse, other than KC3 clones, may be derived from a single cell.


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  		Table I. Vß usage of anti-TL CTL clones derived from C3H and Tg.Tlaa-3-2 mice

 
Anti-TL CTL clones show two distinct target specificities

We next investigated the target specificity of the CTL clones and found that they could be divided into two groups. As shown in Figure 1Go, one group of CTL clones (type I) lyses all target cells expressing TL Ag, and the other group (type II) lyses only TL+ Con A blasts (expressing T3b) derived from Tg.Con.3-1, but not TL+ leukemia cells or H-2Kb/T3b Ltk- transfectants (expressing T3b). As shown in Table IGo, three of seven clones from C3H and three of 11 clones from Tg.Tlaa-3-2 were classified as type I, and the other clones were classified as type II. The characteristics of eight CTL clones, consisting of four each from type I and type II, are summarized in Table IIGo. Since H-2Kb/T3b Ltk- transfectants express a greater amount of TL than TL+ Con A blasts, the difference in target specificity could not be explained by the difference in the amount of TL expression on the target cells. In addition, differences in Vß usage did not account for the target specificity. No apparent difference in the expression of a particular accessory molecule was found between type I and type II CTL clones (Table IIIGo) or between Con A blasts and other target cells (Table IVGo).



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  		FIGURE 1. Target specificity of representative {alpha}ß CTL clones. Type I and type II CTL clones were incubated with 51Cr-labeled target cells (2 x 104) for 3 h. Target cells were Con A blasts of Tg.Con.3-1 (•) and C3H ({circ}), H-2Kb/T3b Ltk- transfectants ({blacksquare}), Ltk- ({square}), C3NB1 TL+ leukemia cells ({blacktriangleup}), and C3NB3 TL- leukemia cells ({triangleup}). For the level of TL expression on these target cells, see Table IIGo.

 

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  		Table II. Target cell specificity of anti-TL CTL clones

 

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  		Table III. Ag profiles of type I and type II CTL clones

 

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  		Table IV. Ag profiles of TL+ target cells1

 
Type II CTL clones can also recognize TL expressed on target cells other than Con A blasts

To test whether CTL epitopes recognized by type II clones are expressed on TL+ target cells other than Con A blasts, cold target inhibition assays were performed. As shown in Figure 2Go, the CTL activity of both type I and type II clones against TL+ Con A blasts was inhibited by H-2Kb/T3b Ltk- transfectants and ASL1 leukemia cells (expressing Tlaa-1, -2, and -3) similarly to TL+ Con A blasts, indicating that type II CTL clones can recognize TL expressed on target cells other than Con A blasts, although they do not exert cytotoxicity against these TL+ cells.



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  		FIGURE 2. Cold target inhibition assay. Type I and type II CTL clones (1 x 105) were incubated with 51Cr-labeled Tg.Con.3-1 Con A blasts (2 x 104) for 3 h in the presence of various cold inhibitor cells: Con A blasts from Tg.Con.3-1 (•) and C3H ({circ}), H-2Kb/T3b Ltk- transfectants ({blacksquare}), Ltk- ({square}), and ASL1 ({blacktriangleup}); (see Table IIGo). The broken lines indicate the percentage of specific lysis obtained without these cold target inhibitor cells.

 
Type I CTL clones kill target cells by perforin, while type II kill target cells by both FasL and perforin

Two major effector molecules, perforin and FasL, have been shown to be responsible for T cell-mediated cytotoxic activity (30). We first analyzed the amount of perforin contained within the cytoplasm of type I and type II CTL clones. Flow cytometric analysis revealed that all CTL clones tested contained perforin; the amount did not vary significantly between the two types (Fig. 3GoA and Table IIIGo). Next, we analyzed the expression of FasL on CTL clones (Fig. 3GoA and Table IIIGo) and that of Fas on the target cells (Fig. 3GoB and Table IVGo). When CTL clones were stimulated with TL+ spleen cells, all type II CTL clones expressed FasL on their surfaces, whereas none of the type I clones did, except for TC4-1. FasL was induced even when these CTL clones were stimulated with TL+ transfectants or leukemia cells (data not shown). Among target cells, Fas is expressed only on Con A blasts on their surface but not on other target cells (Fig. 3GoB and Table IVGo). These results suggested that FasL-dependent cytotoxicity is effective only against Con A blasts, while perforin-dependent cytotoxicity is effective on Fas- target cells such as TL+ transfectants and leukemia cells.



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  		FIGURE 3. FACS analysis of perforin, FasL, and Fas expression. A, Perforin and FasL expression of type I and type II CTL clones. Top, CTL clones were stained with anti-perforin mAb (P1-8) after appropriate fixation (see Materials and Methods). Bottom, CTL clones (1 x 105) were incubated with irradiated Tg.Con.3-1 spleen cells (1 x 106) and IL-2 (5 ng/ml) for 20 h, and then stained with anti-FasL mAb (K10). B, Fas expression on TL+ target cells. TL+ target cells were stained with anti-Fas mAb (Jo2).

 
To determine the killing machinery in type I and type II clones, CTL assays were performed in the presence of CMA, a blocker of perforin-dependent cytotoxicity (28). As shown in Figure 4GoA, CMA blocked the cytotoxicity of type I CTL clones completely, except for FasL+ TC4-1, while it only partially blocked those of type II clones and TC4-1. In addition, CTL activity of all four type I clones against target cells other than Con A blasts was completely inhibited by CMA (data not shown). FasL-dependent cytotoxicity of type I and type II CTL clones was tested next using a neutralizing mAb against FasL (MFL1). As shown in Figure 4GoB, MFL1 inhibited the cytotoxicity of type II CTL clones partially, but did not inhibit that of type I clones. Similar results were obtained with another anti-FasL mAb (K10) or brefeldin A, a blocker of the FasL-dependent pathway (28) (data not shown).



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  		FIGURE 4. Blocking analysis for determination of killing machineries used in type I and type II CTL clones. A, Effects of CMA, an inhibitor of perforin-dependent cytotoxicity, on CTL activity of type I and type II clones. CTL clones (1 x 105) were preincubated with various concentrations of CMA for 2 h and then incubated with 51Cr-labeled Tg.Con.3-1 Con A blasts (2 x 104) for 3 h in the presence of CMA. B, Effects of anti-FasL mAb on CTL activity of type I and type II clones. CTL clones (1 x 105) were incubated with 51Cr-labeled Tg.Con.3-1 Con A blasts (2 x 104) for 3 h in the presence of various concentrations of neutralizing anti-FasL mAb (MFL1). C, Effects of anti-FasL mAb and CMA on CTL activity of type I and type II clones. CTL clones (1 x 105) were incubated with 51Cr-labeled Tg.Con.3-1 Con A blasts (2 x 104) for 3 h in the presence of 10 µg/ml of anti-FasL mAb (MFL1) after preincubation with or without 100 nM CMA for 2 h. CMA was present in the culture throughout the CTL assay.

 
To study the roles of perforin- and FasL-dependent cytotoxicities further, a blocking test in the presence of both anti-FasL mAb and CMA was performed (Fig. 4GoC). CTL activity of type II clones and TC4-1 was completely blocked by anti-FasL mAb (MFL1) plus CMA. The CTL activities of other type II clones (KC1-4 and KC9-2) were also inhibited completely by anti-FasL or brefeldin A plus CMA (data not shown). These results suggested that type I CTL clones kill target cells by perforin, and CTL activities of type II clones and of TC4-1 against Con A blasts are mediated by FasL and perforin.

Different accessory molecules are involved in the activation of type I and type II CTL clones

Since type I and type II CTL clones use different killing machineries, the requirement for accessory molecules of both types was studied by mAb blocking of the cytotoxic activity against TL+ Con A blasts. The results for two clones of each type are shown in Figure 5Go. The CTL activity of type I, but not that of type II, clones was inhibited by anti-Lyt-2 (CD8{alpha}) mAb, although all CTL clones express CD8 on their cell surfaces. The CTL activity of type I clones was also inhibited by anti-Lyt-2.1 (31) and anti-Lyt-3.2 (CD8ß) mAbs (data not shown). On the other hand, the CTL activity of type II, but not that of type I, clones was inhibited by anti-LFA-1 and ICAM-1 mAbs, indicating that an LFA-1/ICAM-1 interaction is necessary for type II clones to kill TL+ Con A blasts. The CTL activity of both types was not inhibited by anti-CD2, -CD28, -CD48, -CD80, or -CD86 mAb or by combinations of these mAbs (data not shown). Thus, the accessory molecules required to exert CTL activity are different for type I and type II clones.



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  		FIGURE 5. Ab blocking for characterization of type I and type II CTL clones. Type I and type II CTL clones (1 x 105) were incubated with 51Cr-labeled Tg.Con.3-1 Con A blasts (2 x 104) for 3 h in the presence of mAbs against Lyt-2 (53-6.7, {circ}), LFA-1 (KBA, {blacksquare}), or ICAM-1 (KAT-1, {blacktriangleup}). Purified mAbs were serially diluted and added to the culture. The broken lines indicate the percentage of specific lysis obtained without Abs.

 
Next, we attempted to determine whether CD8 and LFA-1 deliver the costimulatory signal for perforin/granzyme secretion to type I and type II CTL clones. CTL clones were cultured in the wells precoated with anti-Lyt-2 (CD8{alpha}) or LFA-1 mAb together with or without anti-CD3 mAb, and the amount of perforin/granzyme secreted was estimated by measurement of BLT esterase activity. The BLT esterase is stored in the cytoplasmic granules together with perforin/granzymes, and its secretion is considered to reflect the exocytosis of perforin/granzyme-containing granules (29). The results for two clones of each type are shown in Figure 6Go. Type I CTL clones released a larger amount of BLT esterase than type II when stimulated by an optimal dose of immobilized anti-CD3 mAb (10 µg/ml). When type I CTL clones were stimulated with a suboptimal dose (0.01 µg/ml), anti-Lyt-2 (CD8{alpha}), but not LFA-1, mAb showed synergistic effects. In the case of type II CTL clones, no significant synergistic stimulation was observed under the same conditions. Abs to other accessory molecules, such as CD28, CD2, and CD48, even in the presence of a suboptimal concentration of anti-CD3 mAb, did not show any stimulatory effects on the perforin/granzyme secretion by type I or type II CTL clones (data not shown). These results indicate that CD8 delivers a costimulatory signal to type I, but not to type II, CTL clones and that LFA-1 does not contribute to perforin-dependent cytotoxicity of type II clones as a costimulatory signal mediator.



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  		FIGURE 6. Release of BLT esterase by type I and type II CTL clones stimulated with various mAbs. CTL clones (1 x 105) were incubated for 4 h in 96-well tissue culture plates coated with anti-Lyt-2 (53-6.7) or LFA-1 (KBA) mAb alone (10 µg/ml) or in combination with the indicated doses of anti-CD3 mAb (145-2C11), and then the BLT esterase activity of the supernatants was measured.

 
Type II CTL clones require a certain signal mediated by Con A blasts to exert perforin-dependent cytotoxicity

Because type II CTL clones showed the perforin-dependent cytotoxicity against TL+ Con A blasts but not other TL+ target cells, Con A blasts must mediate an additional signal for perforin/granzyme secretion to type II clones. Therefore, we next attempted to compare the amounts of BLT esterase secretion of type I and type II CTL clones when they were stimulated by various TL+ target cells. The results for two clones of each type are shown in Figure 7Go. TL+ target cells stimulated type I CTL clones to secrete a large amount of BLT esterase, whereas TL- target cells did not. TL+ Con A blasts also stimulated type II CTL clones to secrete a detectable amount of BLT esterase, although lower than that by type I. However, when type II CTL clones were stimulated by TL+ target cells other than Con A blasts, they showed little or no BLT esterase secretion, confirming the finding that type II CTL clones do not kill these target cells. These results suggested that type II CTL clones require more stringent conditions for the perforin-dependent cytotoxicity than type I and, furthermore, need a certain costimulatory molecule that is present on Con A blasts but absent from transfectants and leukemia cells.



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  		FIGURE 7. Release of BLT esterase by type I and type II CTL clones stimulated with TL+ target cells. CTL clones (1 x 105) were incubated with TL+ or TL- target cells (1 x 105) for 4 h in 96-well tissue culture plates, and then the BLT esterase activity of the supernatants was measured.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we showed the presence of two types of anti-TL {alpha}ß CTL clones with distinct target specificity. Type I CTL clones kill any TL+ target cells by a perforin-dependent pathway, while type II kill only TL+ Fas+ Con A blasts by FasL- and perforin-dependent pathways. The results also indicated that the target specificity is determined by the killing machinery of the clones and by the accessory molecules involved in the interactions between CTL and TL+ target cells, as illustrated in Figure 8Go.



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  		FIGURE 8. Possible difference in the cytotoxicity mechanism between type I and type II CTL clones. Type I CTL clones kill any TL+ target cells preferentially by the perforin-dependent cytotoxicity, whereas type II clones kill only TL+ Con A blasts by both FasL- and perforin-dependent cytotoxicities. Type II CTL clones cannot kill TL+ target cells other than Con A blasts by either pathway, because these targets express neither Fas nor an unknown costimulatory molecule to interact with a putative ligand on type II clones to release perforin/granzymes. The LFA-1/ICAM-1 interaction is essential for the cytotoxicity of type II CTL clones, but does not provide a costimulatory signal.

 
CD8 was essential for cytotoxicity of type I CTL clones, since it was blocked with anti-Lyt-2 mAb (CD8{alpha}) mAb, and anti-Lyt-2 mAb showed a synergistic effect on granule exocytosis when type I clones were stimulated with a suboptimal dose of anti-CD3 mAb. In contrast, the cytotoxicity of type II CTL clones, either FasL or perforin mediated, was independent of CD8 signaling, although they did express CD8 on their cell surfaces. Recent studies have suggested that TCR signaling first induces CD8-mediated adhesion to target cells, and then CD8 delivers a triggering signal for perforin/granzyme secretion (32). It has also been suggested that differences in the sensitivity of CTL to anti-CD8 mAb result from variation in the TCR affinity (33) and/or antigenic determinant density on the target cells (32, 34). To clarify the CD8 dependency of type I and type II CTL clones, their TCR characteristics need to be determined. In addition, a detailed analysis of CTL epitopes needs to be performed, as discussed below. Recently, it has also been demonstrated that integrins are involved in adhesion and/or perforin/granzyme secretion by CTL (20, 32, 35). When type I, but not type II, CTL clones were stimulated by a suboptimal dose of anti-CD3 mAb, {alpha}V integrin mAb showed a synergistic effect, similarly to Lyt-2 mAb (CD8{alpha}; our unpublished observations). However, anti-{alpha}V integrin mAb did not inhibit the CTL activity of type I clones, suggesting that an additional signal through {alpha}V integrin is not always necessary.

The present study also showed that an LFA-1/ICAM-1 interaction is essential for the cytotoxicity of type II CTL clones. The importance of this interaction has been observed in FasL-dependent cytotoxicity, such as Th1 cell-mediated B cell apoptosis (36) and Ag-specific CTL-mediated bystander killing (37). Our results showed that the LFA-1/ICAM-1 interactions are not essential for induction of FasL on the surface of type II CTL clones, since even ICAM-1- TL+ transfectants induced FasL, suggesting that these interactions may be necessary for enhancement of the Fas/FasL interactions for the formation of a death-inducing signaling complex. In this context, the report by Medema et al. is of interest, in that activation of caspases requires a certain period of stabilized engagement of Fas by FasL (38).

LFA-1/ICAM-1 also seems to be involved in perforin-dependent cytotoxicity by type II CTL clones, since the CTL activity against TL+ Con A blasts was almost completely inhibited by anti-LFA-1 or anti-ICAM-1 mAbs. In addition, these mAbs inhibited the remaining CTL activity in the presence of anti-FasL mAb or brefeldin A (our unpublished observations). However, it may not be sufficient for type II CTL clones to exert perforin-dependent cytotoxicity because 1) TL+ leukemia cells expressing ICAM-1 were not killed by type II clones; and 2) no or very little synergistic effect on perforin/granzyme secretion was observed when immobilized anti-LFA-1 mAb was combined with a suboptimal concentration of anti-CD3 mAb to stimulate type II clones. Ybarrondo et al. also reported that LFA-1 does not deliver a costimulatory signal for TCR-dependent perforin/granzyme secretion (39). Therefore, the role of LFA-1/ICAM-1 interactions in perforin/granzyme secretion in type II CTL clones is apparently different from that for CD8/TL in type I clones, and its major role is probably the enhancement of CTL binding to the target cells.

In addition to the LFA-1/ICAM-1 interaction, type II CTL clones apparently require a costimulatory signal to exert perforin-dependent cytotoxicity, since TL+ transfectants and leukemia cells did not stimulate type II clones to exert BLT esterase, in contrast to TL+ Con A blasts. For FasL induction on their surface, however, signals through TCR/CD3 in response to TL+ transfectants and leukemia cells appeared sufficient, similarly to TL+ Con A blasts, suggesting that perforin-dependent cytotoxicity may require stronger signals than FasL-dependent cytotoxicity. These observations appear consistent with previous findings by others that the FasL-dependent, but not the perforin-dependent, pathway, is selectively activated by partial agonists (40, 41) and that the FasL pathway is still intact in a variant CTL clone with a defect in the TCR-triggered Ca2+ flux (42). The degree of BLT esterase secretion by type II CTL clones on stimulation with Con A blasts or immobilized anti-CD3 mAb was lower than that of type I clones. Therefore, it is likely that they kill TL+ target cells predominantly by FasL, while they use perforin/granzymes in addition to FasL only when they are confronted with unique target cells with high Ag-presenting capability, such as Con A blasts.

It has been speculated that the perforin-dependent pathway is used by T cells to eliminate infected or transformed cells (43), whereas the role of FasL-dependent cytotoxicity is down-regulation of an immune response by eliminating activated T cells in the periphery (44). In future studies, it would be interesting to dissect the molecular components of signal transduction events leading to cytotoxicity and how FasL expression affects these mechanisms. Our preliminary results showed that bulk CTL against TL obtained from primary MLC kill target cells mainly by perforin-dependent cytotoxicity, suggesting that type II CTL clones are established in the process of limiting dilution in the presence of IL-2, as shown in Table IGo. Our preliminary results also showed that type II CTL clones undergo apoptosis more readily after soluble anti-CD3 mAb stimulation than type I clones. These observations, taken together, suggest that the growth and the death of type II CTL in vivo are more tightly regulated. We are currently investigating the conditions influencing the differentiation of anti-TL CTL precursors into type I or type II CTL.

We have shown by cold target inhibition assays that CTL epitopes of both type I and type II clones are expressed on all TL+ target cells tested. The Ab blocking test in the present study also demonstrated that both types of CTL clones recognize TL directly without Ag presentation by H-2 molecules. However, there still remains the possibility that CTL epitopes recognized by type I and type II clones are different. Some CTL clones may recognize TL plus peptides, while other may recognize the TL framework. To elucidate how these CTL clones recognize TL, they need to be tested against transfectants of Drosophila melanogaster cells expressing TL devoid of endogenous binding peptides (45). Since the CTL assay is by far the most sensitive method for detection of Ag peptide binding to MHC (46), its use will clarify the issue of whether TL binds peptides (47) or not (48) and will provide further insight into the physiologic significance of two types of anti-TL CTL.


   Acknowledgments
 
We thank Drs. E. Nakayama, T. S. Doi, and A. Morita for their valuable discussions and suggestions, and H. Hasegawa-Nishiwaki, H. Tamaki, and S. Ozeki for their expert technical assistance. We also thank Drs. J. A. Bluestone, R. T. Kubo, E. Nakayama, T. Nishimura, N. Shinohara, and S. Tonegawa for their kind gifts of mAbs. We thank Dr. M. A. Moore for his editorial assistance.


   Footnotes
 
1 This work was supported in part by a grant-in-aid for Encouragement of Young Scientists, a grant-in-aid for Scientific Research on Priority Areas, and a grant-in-aid for General Scientific Research from the Ministry of Education, Science, Sports, and Culture, Japan, by a grant-in-aid from the Ministry of Health and Welfare, Japan, and in part by a grant from the Imanaga Foundation and a Bristol-Myers Squibb Biomedical Research Grant. Back

2 Address correspondence and reprint requests to Dr. Yuichi Obata, Laboratory of Immunology, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464, Japan. E-mail address: Back

3 Abbreviations used in this paper: TL, thymus leukemia; FasL, Fas ligand; B6, C57BL/6; C3H, C3H/He; Ltk-, thymidine kinase-negative L cell; NBU, N-butyl-N-nitrosourea; CMA, concanamycin A; BLT, N-{alpha}-benzyloxycarbonyl-L-lysine thiobenzoyl ester. Back

Received for publication January 5, 1998. Accepted for publication January 29, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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