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International Immunology, Vol. 12, No. 3, 365-374, March 2000
© 2000 Japanese Society for Immunology

Comparison of Fas- versus perforin-mediated pathways of cytotoxicity in TCR- and Thy-1-activated murine T cells

Hidefumi Kojima1, Masahiro Toda2 and Michail V. Sitkovsky1

1 Biochemistry and Immunopharmacology Section, Laboratory of Immunology, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Building 10/llN3ll, 10 Center Drive, MSC 1892, Bethesda, MD 20892-1892, USA
2 Department of Physiology, Keio University School of Medicine, Tokyo 160-8582, Japan

Correspondence to: M. Sitkovsky


   Abstract
Top
 Abstract
Introduction
 Methods
 Results and discussion
 References
 
T cell-mediated cytotoxicity can be triggered by cross-linking of TCR or Thy-1 surface proteins. While the TCR-triggered signaling initiates both perforin- and Fas ligand (FasL)–Fas-mediated mechanisms of cytotoxicity, it was not clear which mechanism was utilized by Thy-1-triggered signals and which pathway of cytotoxicity was triggered at low levels of antigen expression. It is shown that glycophosphatidylinositol-linked surface glycoprotein Thy-1 preferentially activates FasL–Fas- but not perforin-mediated cytotoxicity. This is explained by the lesser intensity of Thy-1-mediated signaling in T cells. The data suggest that Thy-1-triggered Fas-mediated cytotoxicity is completely dependent on cross-talk between Thy-1 and TCR signals since mutations in TCR–CD3 complex molecules or inhibition of tyrosine kinases or calcineurin abolished or strongly inhibited Thy-1-triggered FasL–Fas-mediated cytotoxicity. Lower concentrations of antigenic peptide or levels of cross-linking with anti-TCR–CD3 mAb are required to trigger Fas-mediated than perforin-mediated cytotoxicity by different cytotoxic T lymphocyte (CTL) lines and clones, and it is shown that cross-linking of Thy-1 is much less efficient in triggering accumulation of second messengers (intracellular Ca2+) than cross-linking of TCR on CTL. Taken together, these data reflect the possibility of differential activation of FasL and/or perforin pathways of cytotoxicity depending on the nature of activating stimuli and surface receptor.

Keywords: cytotoxic T lymphocyte, Fas ligand, perforin, retargeting, TCR, Thy-1


   Introduction
Top
 Abstract
 Introduction
Methods
 Results and discussion
 References
 
Cytotoxic T lymphocytes (CTL) are considered to be important in immune responses against infections and in antitumor immunity (1,2). The recognition of antigen-expressing target cells by CTL is due to TCR cross-linking by antigenic peptide–MHC complexes on the target cells. This, in turn, results in TCR cross-linking and triggering of transmembrane signaling pathways in CTL that involve protein tyrosine or serine/threonine phosphorylation and activation of phospholipase C and calcineurin (3–7).

One important outcome of triggering TCR-dependent biochemical pathways in T cells is the activation of cytotoxic machinery that, in turn, initiates the lethal hit delivery to target cells. Two different mechanisms are able to account for T cell-mediated cytotoxicity (8–11). The lethal hit could be delivered by perforin molecules and by granzymes located in cytolytic granules (reviewed in 12,13). As a result of TCR cross-linking-triggered exocytosis, these molecules are released into the area between CTL and targets, and form pores in target cell membranes that lead to their death.

The existence of a second mechanism of cytotoxicity was predicted by experiments in which antigen-specific cytotoxicity was observed even after complete inhibition of the exocytic perforin-mediated pathway (14,15). This second mechanism was suggested to be mediated by Fas/Apo-1 molecules on the cell surface (16). Development of perforin gene-deficient mice provided conclusive confirmation of these data (9–11,17).

Molecules of Fas/Apo-1 were discovered in studies of mAb that were able to destroy tumor cells (18,19) and the mutations in Fas were shown to underlie the mechanism of lymphoproliferative disease (lpr) in mice (20).

Pathologies similar to lpr were observed in mice with gld mutations and it was subsequently shown that the mutation in Fas ligand (FasL) leads to a very similar phenotype. The TCR-triggered up-regulation of FasL (20) on the surface of CTL enables the second mechanism of cytotoxicity (16). FasL is not expressed constitutively and its expression is triggered by TCR cross-linking-triggered biochemical events (21,22).

It had been shown earlier that cytotoxicity was triggered not only by TCR but also by, for example, the glycophosphatidylinositol (GPI) -linked cell surface molecule Ly-6C (23,24), but it was not clear whether GPI-linked molecules and Thy-1 shared these properties.

The GPI-linked surface protein Thy-1 is a member of the Ig superfamily. Of importance, Thy-1 is able to transduce signals leading to increases in Ca2+ and to cellular responses (25,26). Thy-1-mediated T cell activation required co-expression of TCR–CD3 complex, as demonstrated in experiments with transformed T cells (27–29). Interest in the effects of Thy-1 rose following recent observations of the association of Thy-1 antigen with different G proteins (30), revealing the possible involvement of a G protein-mediated pathway. In mice, effects of Thy-1 cross-linking include thymocyte death through a Bcl-2-resistant mechanism that was observed after cross-linking of Thy-1 with signaling anti-Thy-1 mAb (25). Thy-1 could be involved not only in signaling but also in cell–cell interactions, since it was observed that anti-actin mAb cross-react with Thy-1 and block T cell aggregation (31). Thy-1 is expressed on a variety of cells in different species, including T cells and neurons in mice (32), but the exact functional role of Thy-1 in T cells remains to be determined. The intriguing possibility that Thy-1-mediated signaling may utilize a TCR-independent, G protein-mediated biochemical pathway was recently suggested by findings of associations of GPI-linked surface proteins on lymphocyte surface with different signal-transducing G proteins (30).

The physiologic ligand for Thy-1 is not yet known, but studies of Thy-1-mediated and other members of GPI-anchored Ig superfamily-mediated cytotoxicity may have important implications for the clinical use of CTL in immunoadoptive therapies (33,34) and for the potential use of retargeting antibody to destroy tumor targets by CTL (35–39).

In this study, we asked which pathway of cytotoxicity was triggered first at low levels of antigen expression and which mechanism of cytotoxicity was utilized by Thy-1-triggered signals. We report more stringent TCR signal intensity requirements for triggering perforin- versus Fas-mediated cytotoxicity and that Thy-1 preferentially activates FasL–Fas- but not perforin-mediated cytotoxicity.


   Methods
Top
 Abstract
 Introduction
 Methods
Results and discussion
 References
 
[Ca2+] measurement
For measurements of Ca2+ flux CTL were analyzed on a FACSVantage flow cytometer equipped with an argon laser tuned to 488 nm and a krypton laser tuned to 360 nm. Indo-1 fluorescence was analyzed at 390/20 and 530/20 nm for bound and free probe as was described (49,50). The percentage of cells that responded by an increase in intracellular Ca2+ after stimulation with antibody was determined using Multitime software for analysis of kinetic flow cytometry data (Phoenix Flow Systems, San Diego, CA).

BAK cells were pre-loaded with Indo-1 (final 3 µM) and Pleuronic ( 0.03%, Molecular Probes, Eugene, OR) in Ca2+ buffer (1% FCS, 10 mM HEPES and HBSS) for 45 min at 30°C. Then cells were washed with buffer. To stimulate BAK cells, combinations of biotinylated 2C11 anti-CD3 mAb (PharMingen, San Diego, CA) or biotinylated G7 (Southern Biotechnology Associates, Birmingham, AL) and Extra-Avidin (Sigma, St Louis, MO) were used. Optimal amount of antibodies were pre-determined by flow cytometry, then antibodies or avidin were titrated at Ca2+ measurement.

Cells
Cells were cultured in complete RPMI (cRPMI) (RPMI 1640 supplemented with 100 U/ml penicillin, 100 µg/ml streptomycin, 1 mM HEPES and 50 µM 2-mercaptoethanol). For CTL culture, cRPMI was further supplemented with 10% heat-inactivated FCS and 2.5% culture supernatant from rat spleen cells stimulated with concanavalin A for 48 h as T cell growth factors. For growth of other cells, including maintenance of hybridomas and functional assays, cRPMI was supplemented with 5% heat-inactivated FCS. OE4, anti-H-2d CTL, were cultured in modified DMEM with 10% FCS and 2.5% supernatant from phorbol myristate acetate-treated EL4 as described earlier (40). The anti-H-2k CTL lines BAK and B6K were derived from BALB/c and C57/BL6 mice respectively. The anti-H-2b CTL lines C3B and Gl-b were derived from normal C3H/He mice and functional FasL-deficient C3H-gld/gld mice respectively. A perforin-deficient CTL clone, P0K.3A8, was generated as previously described (9). The BA/CT-2 CTL line, which is specific for immunodominant peptide (SPSYVYHQF) derived from CT26 murine colon carcinoma in the context of the H-2Ld molecule, was established by immunization and in vitro stimulation (47).

CTL lines and clones were maintained by biweekly stimulation. TCR–CD3-deficient T hybridoma 2B4 variant, 21.2.2. cells, were kindly provided by Dr Jonathan Ashwell (29,39). A20.F0 is a Fas sensitivity variant of a B lymphoma A20.2J which was established by culturing A20.2J in the presence of anti-Fas antibody Jo2 (40).

Antibodies and reagents
Hybridomas producing, hamster anti-mouse CD3e mAb 2C11 (IgG), rat anti-mouse Thy-1 antigen antibody G7 (IgG2c) (kindly provided by Dr Nobukata Shinohara, Mitsubishi-kasei Institute of Life Sciences, Tokyo, Japan) and rat anti-mouse LFA-1 antibody FD441.8 (IgG2c) were cultured in 5% FCS/cRPMI, and culture supernatants were collected as the source of antibodies for retargeting assay. The used in retargeting assays 1:40 dilution of antibody was selected in preliminary experiments to determine maximal response of CTL to antibody treatment.

Anti-CD3 mAb 2C11 and anti-Thy-1 mAb G7 were also purchased from PharMingen. A tyrosine kinase inhibitor, herbimycin A (Calbiochem, San Diego, CA), was used as 1 mM stock solution in DMSO. Cyclosporin A (CsA; 1 mg/ml in HBSS) was provided by Dr Lixin Zhen (Laboratory of Immunology, NIAID, NIH). FITC-conjugated goat F(ab)'2 anti-rat IgG was purchased from Caltag (Burlingame, CA).

Flow cytometry analysis
Expression of Thy-1 on 2B4 cells was studied by flow cytometry. Flow cytometry data acquisition and analysis were done on FACScan using FACScan research software and CellQuest programs (Becton Dickinson, San José, CA). Cells (5x105) were analyzed in each assay by incubating with or without antibody (100 µl of hybridoma culture supernatant), then staining with FITC-conjugated goat F(ab)'2 anti-rat IgG. Detection of H2-Ld was accomplished using specific 30-5-7 mAb generously provided by Dr Nobu Shinohara (Japan). Detection of Fc receptors was accomplished by using 2.4G2 mAb (PharMingen).

Detection of FasL expression on T cells
A CD8 T cell line, BAK, was tested after stimulation with plate coated 2C11 or G7 antibody used at 10 µg/ml) for 1 h in the presence of 10 nM matrix metalloproteinase inhibitor (KB8301; PharMingen). After stimulation, cells were harvested and were fixed with paraformaldehyde. The staining procedure was performed by using anti-FasL mAb (MFL3; PharMingen) as first antibody and biotinylated anti-hamster IgG cocktail (PharMingen) as secondary antibodies followed by streptavidin–PE.

Cytotoxicity assay
CTL cytotoxicity was estimated in 51Cr-release assays of 51Cr-labeled target cells. Target cells were labeled by preincubation with 50 µCi Na251CrO4 for 1–2 h at 37°C followed by three washes. A retargeting assay was performed by incubating 2500 51Cr-labeled target cells with CTL in the presence of 2.5% (1:40 dilution) supernatant from mAb-producing hybridoma culture. For the peptide titration assay, target cells (1x106/ml) were incubated for 1 h at 37°C with antigenic peptide at indicated peptide concentrations before labeling. Assays were performed using V-bottomed 96-well plates for 4 h. Culture supernatants were collected using the Skatron harvesting system. Estimations of cytotoxicity were presented as percentage of specific 51Cr release and calculated as follows: (Exp – Spt)/ (Tot – Spt)x100, where Exp, Spt and Tot indicate experimental release, spontaneous release and total incorporation respectively. Spontaneous 51Cr release from target cells was usually <=15%.

The Northern blot analysis
2B4 cells (1.5x106) were incubated in a culture dish which was coated with anti-CD3 mAb or anti-Thy-1 mAb(10 µg/ml) for 6 h. Untreated or antibody-stimulated cells were used as the source of RNA. After incubation, cells were harvested and total RNA was extracted by TRIzol (Gibco/BRL, Rockville, MD). RNA (30 µg) was used for the Northern blot analysis after electrophoresis of RNA samples on a 1.1% agarose gel containing 5% formaldehyde, their transfer onto nitrocellulose membrane and hybridization with 32P-labeled DNA probes. The DNA probe for FasL and mouse ß-actin 3' untranslated region were kindly provided by Drs Lixin Zhen (Laboratory of Immunology, NIAID, NIH) and Takashi Saito (Chiba University, Chiba, Japan) respectively. DNA probes were labeled using RTG DNA labeling beads (Pharmacia, Piscataway, NJ).

After hybridization with FasL cDNA probe, the filter was reprobed with mouse ß-actin 3' untranslated region cDNA probe.

Granule exocytosis assay
CTL clone OE4 cells (1x105/well) were incubated in the 96-well plates pre-coated with either anti-CD3 (2C11, 10 µg/ml) or anti-Thy-1 (G7, 10 µg/ml) mAb antibodies for 4 h. After incubation supernatants were collected and BLT esterase activity was measured as previously described (14,15).


   Results and discussion
Top
 Abstract
 Introduction
 Methods
 Results and discussion
References
 
In preliminary experiments we observed that anti-Thy-1 mAb are less efficient than anti-TCR mAb in triggering CTL cytotoxicity. Explanations of these differences required better understanding of mechanisms of Thy-1- versus TCR-induced cytotoxicity. It was also required to determine whether lower levels of TCR cross-linking are required for triggering of Fas- versus perforin-mediated cytotoxicity.

Retargeting with anti-Thy-1 antibody G7 preferentially triggers the FasL–Fas- but not the perforin-based mechanism of cytotoxicity
To test whether Thy-1-mediated signal can induce cytotoxicity in CTL and the lethal hit delivery to T cells, the anti-Thy-1 antibody G7 was added into the retargeting assay (Fig. 1Go). It is shown that anti-Thy-1 mAb are similar to, although not as efficient as, anti-CD3 mAb in their ability to trigger lysis of antigen-non-expressing A20.2J target cells in a retargeting assay (Fig. 1AGo).



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  		Fig. 1. Retargeting with anti-Thy-1 antibody G7 preferentially triggers the FasL–Fas- but not the perforin-based mechanism of cytotoxicity. CTL were incubated with 51Cr-labeled target cells with or without antibodies (1:40 dilution). All assays lasted 4 h. (A) C57BL/6 anti-H-2k allospecific normal CTL line B6K were used as effectors and Fas-sensitive BALB/c origin A20.2J B lymphoma were used as targets. (B) C57BL/6 anti-H-2k allospecific normal CTL line B6K were used as effector and Fas-resistant variant A20.F0 were used as targets. (C) Perforin-deficient anti-H-2k CTL clone P0K.3A8 were used as effectors and Fas-sensitive A20.2J B lymphoma were used as targets. (D) C3H/He-gld/gld mouse-derived anti-H-2b CTL line Gl-b (Fas-ligand deficient) was used as effector and A20.2J were used as targets. •, Cells were retargeted with 2C11 (anti-CD3 mAb); {square}, cells were retargeted with G7 (anti-Thy-1 mAb); {blacktriangleup}, cells were retargeted with FD441.8 (anti-LFA-1 mAb).

 
A20.2J targets could be killed by both perforin- and Fas-mediated pathways (40), and 2C11 anti-CD3 mAb are known to trigger both (16). We therefore considered the possibility that the lower intensity of anti-Thy-1 mAb-redirected lysis (Fig. 1AGo) is due to the inability of anti-Thy-1 mAb to trigger both pathways of cytotoxicity. This could explain the quantitative differences reported above. To test this possibility, we compared the effects of retargeting of CTL with anti-Thy-1 mAb on Fas-susceptible and Fas-resistant target cells.

While the activation of cells through the Thy-1-mediated pathway was sufficient to trigger lysis of Fas-susceptible A20.2J, no effect was seen in a parallel assay with Fas-resistant A20.F0 target cells (Fig. 1A and BGo). In a control assay, this Fas-resistant variant was killed by anti-CD3 mAb-redirected CTL (Fig. 1BGo). These results suggested that Thy-1 mAb preferentially trigger the Fas-mediated mechanism of cytotoxicity in CD8+ CTL. This was further confirmed in experiments using perforin gene-deficient CTL P0K.3A8 (Fig. 1CGo). These CTL are able to utilize only the Fas-mediated mechanism (40). Equally efficient levels of redirected cytotoxicity toward Fas-susceptible targets were accomplished by both anti-CD3 and anti-Thy-1 mAb.

Genetic evidence for the inability of Thy-1 to trigger perforin-mediated cytotoxicity was obtained using functional FasL-deficient anti-H-2b CTL lines. One such line, Gl-b, was established from C3H-gld/gld mice, which were demonstrated to have mutated and non-functional FasL (20). Using these CTL, we show that in the presence of 2C11 anti-CD3 mAb the Gl-b CTL were efficiently retargeted to lyse the non-antigen-bearing targets, thereby demonstrating that signaling through CD3 was sufficient to trigger the perforin-mediated pathway. In contrast, the anti-Thy-1 mAb G7 was not able to trigger sufficient signal to induce cytolytic activity in parallel assays (Fig. 1DGo).

It is most likely that the described Fas-mediated killing of targets in the retargeting assay with anti-Thy-1 mAb is due to the Thy-1-triggered inducible process of FasL up-regulation, and not to the facilitation of adhesion between CTL and targets by retargeting anti-Thy-1 mAb. Indeed, if CTL expressed the Fas-based mechanism constitutively, the CTL-target cell conjugate formation by anti-LFA-1 antibodies also would be expected to induce lysis of targets; however, no lysis of targets was observed in parallel controls in which retargeting anti-LFA-1 antibodies were present during the assay to facilitate conjugates between CTL and non-antigen-bearing targets (Fig. 1A–CGo). It was important to understand whether anti-Thy-1 and anti-TCR mAb employ the same biochemical mechanism for triggering cytotoxicity in CD8+ CTL.

The same phenomenon was observed in experiments with 2B4, a helper-type T hybridoma (Fig. 2Go). We show that these cells could lyse A20.2J in short-term assay (6 h) in the presence of anti-CD3 or anti-Thy-1 mAb. In the long-term assay (12–16 h), both CD3 stimulation and Thy-1 stimulation could induce the same degree of cytotoxicity against A20.2J cells (data not shown). However, Fas-resistant targets A20.F0 were not lysed by 2B4 in the presence of anti-Thy-1 mAb G7, even in long-term assay (16 h). The anti-CD3 mAb stimulation did induce low, but significant killing of these A20.F0. It is most likely that the cytotoxicity induced with anti-CD3 2C11 mAb in long-term assay is based on soluble factor, like tumor necrosis factor-{alpha}, since it was reported (48) that A20.2J cells are susceptible to soluble factor-mediated long-term lysis. These data suggest that the anti-Thy-1 antibody induced 2B4 cells cytotoxicity in the short-term assay is mediated by to Fas–FasL pathway.



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  		Fig. 2. Retargeting of FasL-expressing 2B4 cells with anti-Thy-1 or anti-CD3 mAb triggers Fas-based cytotoxicity. (A) 2B4 cells were incubated with 51Cr-labeled Fas-expressing A20.2J target cells for 6 h in the presence or absence of anti-CD3 mAb (2C11) or anti-Thy-1 mAb 9G7) as indicated. (B) 2B4 cells were incubated with 51Cr-labeled Fas-resistant A20.F0 target cells for 16 h in the presence or absence of anti-CD3 mAb (2C11) or anti-Thy-1 mAb (G7) as indicated. 2C11 mAb (•) and G7 ({square}) were added as 1:40 final dilution.

 
It was important to test whether stimulation with anti-Thy-1 mAb can induce FasL expression in T cells. The Northern blot experiment which is described in Fig. 3Go demonstrates that both anti-CD3 and anti-Thy-1 mAb can trigger FasL mRNA up-regulation in T cells. The effect of anti-Thy-1 mAb was, however, less profound than the effect of anti-CD3, 2C11 mAb stimulation. This result may explain observations which are described in Figs 1 and 2GoGo, where anti-CD3 2C11 mAb stimulation did induce higher cytotoxicity than stimulation with anti-Thy-1 mAb.



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  		Fig. 3. Both anti-Thy-1 and anti-CD3 mAb induce expression of FasL mRNA. The Northern blot analysis of expression of FasL mRNA in T cells was performed after incubation of 2B4 cells for 6 h alone (control) or with immobilized anti-CD3 or Thy-1 mAb. ß-Actin 3' untranslated region cDNA was used as an internal control.

 
Comparison of Thy-1- and TCR-mediated signaling in triggering granule exocytosis and FasL expression in CTL
To further test whether Thy-1 are less efficient than TCR in triggering Fas-mediated cytotoxicity we addressed this question on the level of molecular mediators of these two types of CTL-mediated cytotoxicity. It was done by incubating CTL with immobilized anti-CD3 or anti-Thy-1 antibody at surface densities which were found in preliminary experiments to elicit the maximal CTL response.

It is shown (Fig. 4AGo) that stimulation by anti-CD3 mAb, but not by anti-Thy-1 mAb induces stronger granule exocytosis in CTL. In fact, the small increase in BLT esterase secretion which is observed after by Thy-1 cross-linking (Fig. 4AGo) may provide the important baseline as to which levels of granule exocytosis are not sufficient for CTL-mediated cytotoxicity, since in parallel experiments similar levels of Thy-1 cross-linking on CTL were not sufficient to trigger target cell death.



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  		Fig. 4. Comparison of Thy-1 and TCR-mediated signaling in triggering granule exocytosis and FasL expression in CTL. (A) Stimulation by anti-CD3, but not by anti-Thy-1 mAb induces stronger granule exocytosis in CTL. CTL line BAK were incubated in the 96-well plates pre-coated with either anti-CD3 (2C11) or anti-Thy-1 (G7) mAb for 4 h and exocytozed BLT esterase activity was measured in supernatants as described in Methods. (B–D) Stimulation by anti-CD3 is more efficient than stimulation with anti-Thy-1 mAb in triggering the expression of FasL in CTL. (B) Control, no stimulation, (C) stimulation with anti-CD3 mAb and (D) stimulation with anti-Thy-1 mAb. CTL line BAK cells were incubated in the 48-well plates pre-coated with either anti-CD3 (2C11) or anti-Thy-1 (G7) mAb for 1 h and expression of FasL was measured by flow cytometry as described in Methods. The same concentration of stimulating antibody (10 µg/ml) was used in experiments described in panels (A)–(D).

 
In contrast, strong differences between Thy-1- and TCR-mediated signaling were observed after comparison of FasL expression on CTL line BAK which were incubated in the 48-well plates pre-coated with either anti-CD3 or anti-Thy-1 mAb. It is shown that while both Thy-1 and TCR cross-linking were able to induce FasL expression (see Figs 4C and DGo), the stimulation by anti-CD3 was more efficient than stimulation with anti-Thy-1 mAb in triggering the expression of FasL in CTL. This is in good agreement with our observation that Thy-1-mediated FasL-mediated cytotoxicity is in general weaker than TCR-triggered and FasL-mediated cytotoxicity, as is illustrated in experiments described in Fig. 5Go(A–C) using the same CTL line as in Fig. 4Go. Thus, the Thy-1-mediated signaling is sufficient to trigger cytotoxic levels of expression of FasL, but is not sufficient to trigger sufficient levels of granule exocytosis to result in measurable perforin-mediated cytotoxicity.



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  		Fig. 5. Inhibitors of tyrosine kinases and of PP2b phosphatase block both Thy-1- and TCR–CD3-triggered cytotoxicity in CTL. BALB/c anti-H-2k CTL line BAK or 2B4 T cells were incubated at the indicated E:T ratio for 4 or 6 h respectively with 51Cr-labeled A20.2J (A, B, D and E) or A20.F0 targets (C) in the presence of either 2C11 anti-CD3 mAb or G7 anti-Thy-1 mAb or alone. (A) Effect of tyrosine kinase inhibitor herbimycin on redirected killing. CTL line BAK and A20.2J target cells were co-incubated in the presence of antibodies without (closed symbols) or with (open symbols) herbimycin at a final concentration of 10 mM. (B) Effect of PP2b phosphatase inhibitor CsA on redirected killing induced with anti-CD3 or anti-Thy-1 antibodies. BAK CTL cells were preincubated with CsA (250 ng/ml) for 2 h and then mixed with an equal volume of A20.2J target cells, thereby diluting CsA to 125 ng/ml during assay. Symbols are the same as in (A). (C) Control, BAK CTL do not kill Fas-resistant targets in retargeting assay with anti-Thy-1 mAb but lyse them when retargeted with anti-CD3 mAb. BAK CTL were incubated with 51Cr-labeled A20.F0 cells with 2C11 or G7 mAb for 4 h at 37°C. (D) Effect of tyrosine kinase inhibitor herbimycin on redirected killing. 2B4 T cells and A20.2J target cells were co-incubated in the presence of antibodies without (closed symbols) or with (open symbols) herbimycin at a final concentration of 10 mM. (E) Effect of PP2b phosphatase inhibitor CsA on redirected killing induced with anti-CD3 or anti-Thy-1 antibodies. 2B4 T cells were preincubated with CsA (300 ng/ml) for 2 h and then mixed with an equal volume of A20.2J target cells, thereby diluting CsA to 150 ng/ml during 6 h assay.

 
Tyrosine phosphorylation and CsA-inhibited PP2b phosphatase-mediated pathway are required for both Thy-1- and TCR–CD3-triggered cytotoxicity in CTL
Since tyrosine phosphorylation was shown to be a necessary event in TCR-triggered T cell activation (4), the tyrosine kinase inhibitor herbimycin A was added to the retargeting assay to test the involvement of these enzymes in Thy-1-mediated cytotoxicity. It was found (Fig. 5A and DGo) that this inhibitor completely blocked both anti-CD3 mAb (2C11)- and anti-Thy-1 (G7) mAb-induced cytotoxicity of CTL (Fig. 5AGo) and 2B4 cells (Fig. 5DGo). These results demonstrates that Thy-1-transduced signals employ tyrosine phosphorylation. This is in agreement with the recently published observation that protein tyrosine kinase Syk is involved in Thy-1 signaling in rat basophilic leukemia cells (41). In another series of experiments, an immunosuppressant CsA known to inhibit the TCR-triggered pathway by targeting calcineurin (42) was found to inhibit both anti-CD3 (2C11) mAb- and anti-Thy-1 (G7) mAb-induced cytotoxicity of CTL to the same degree (Fig. 2BGo). Also, addition of CsA abrogated both anti-CD3 mAb- and anti-Thy-1 mAb-triggered cytotoxicity of 2B4 (Fig. 5EGo). The results of these experiments support the previous reports (27) indicating that Thy-1 requires the TCR–CD3 complex to induce activation signals leading to IL-2 production by T cells. This point was further confirmed by experiments with TCR–CD3 complex-mutated T cells (Fig. 6Go). Since BAK CTL were used in experiments described in Fig. 5Go(A and B), it was important to determine the mechanism of retargeted lysis by this CTL. It is shown (Fig. 5CGo) that BAK CTL do not kill Fas-resistant targets in a retargeting assay with anti-Thy-1 mAb but do lyse them when retargeted with anti-CD3 mAb, thereby confirming that the Thy-1-mediated lysis of retargeted cells in Fig. 5Go(A and B) is completely Fas mediated.



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  		Fig. 6. Requirement in expression of TCR–CD3 complex for anti-Thy-1 mAb-triggered cytotoxicity. Controls for expression levels of Thy-1 antigen on effector cells, both 2B4 T cells (A) and 2B4 T cell mutant 21.2.2 (B). Expression of Thy-1 was determined by flow cytometry analysis. Cells were stained with FITC-conjugated goat anti-rat IgG F(ab)'2 directly (solid histograms) or after pretreatment with G7 (open histograms). TCR–CD3-expressing but not mutant T cells are able to kill Fas-sensitive targets in a retargeting assay. 51Cr-labeled A20.2J were co-cultured with 21.2.2. (C) at the indicated E:T ratio in the presence of 2C11 culture supernatant or G7 culture supernatant. Cytotoxicity was measured after 6 or 16 h of incubation, as indicated.

 
Expression of functional TCR–CD3 complex is required for anti-Thy-1 mAb-triggered FasL–Fas-mediated cytotoxicity in retargeting assays
It was shown previously with the 2B4 T cells used here that the intact TCR–CD3 complex signaling pathway was required to observe the anti-Thy-1 mAb-induced cellular responses (27–29,39), but it was still possible that the Thy-1-induced Fas-mediated cytotoxicity we describe in Figs 1 and 2GoGo was not dependent on TCR–CD3. To test whether the TCR–CD3 complex was also required for successful Thy-1 signaling and Fas-mediated killing, we used the parent 2B4 line and TCR–CD3 complex lacking 2B4 variant, 21.2.2. In control experiments, we established that parent 2B4 and variant 21.2.2. expressed very similar cell surface levels of Thy-1 antigen (Fig. 6A and BGo), while only parent 2B4 cells expressed TCR–CD3 cell surface molecules (28). It was found that TCR–CD3-deficient mutant 21.2.2 could not be retargeted by anti-Thy-1 mAb to lyse target cells in short-term (data not shown) and even long-term (Fig. 6CGo; 16 h) assays. Taken together, the results described in Figs 1 and 2GoGo established that Thy-1-triggered activation signal and FasL-mediated killing by CTL and T hybridoma cells required the intact TCR–CD3 complex.

Differential signaling requirements for the triggering of Fas- and perforin-based cytolysis by CTL
The above data also raised questions as to why the anti-TCR–CD3 mAb were able to trigger both the Fas- and perforin-mediated pathways of cytotoxicity, while anti-Thy-1 mAb were able to trigger only the FasL–Fas pathway. The most straightforward hypothesis was to assume that the Thy-1-mediated signaling was less intense then the TCR-triggered signaling and that the exocytosis of perforin-containing cytolytic granules required a higher intensity of transmembrane signaling than that needed for FasL up-regulation in CTL. If this were the case, then the anti-Thy-1 mAb-triggered signaling would be sufficient only for induction of Fas-mediated but not of perforin-mediated cytotoxicity. This model was tested and confirmed by studies of dose–response dependence of triggering the perforin- versus Fas–mediated pathway of CTL cytotoxicity with different concentrations of antigenic peptide. Quantitative differences in triggering Fas- versus perforin-based CTL cytotoxicity were demonstrated by using specific antigenic peptide and peptide-specific CTL. CTL line BA/CT-2, which specifically recognizes murine colon cancer line CT-26 peptide in the context of the H-2Ld molecule, was incubated with Fas-sensitive or -resistant targets. It is shown that the BA/CT-2 CTL line is efficiently retargeted by both anti-CD3 mAb and anti-Thy-1 mAb to kill Fas-sensitive targets (Fig. 7AGo), but only anti-CD3 mAb retarget these CTL to kill Fas-resistant targets (Fig. 7BGo). Since H-2Ld molecules are required to present antigenic peptide to BA/CT-2 CTL we tested in control experiment whether A20.2J and A20.F0 cells had similar levels of expression of H-2Ld (Fig. 7C and DGo). Similar levels of H-2Ld molecules are demonstrated in both cells, thereby confirming their suitability for quantitative evaluation of peptide-induced effector functions in CTL.



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  		Fig. 7. Quantitative differences in triggering Fas- versus perforin-based CTL cytotoxicity. Specific antigenic peptide and peptide-specific CTL were used in these experiments. CTL line BA/CT-2, which specifically recognizes murine colon cancer line CT-26 peptide in the context of the H-2Ld molecule, was incubated with Fas-sensitive or -resistant targets. BA/CT-2 CTL line is efficiently retargeted by both anti-CD3 mAb (•) and anti-Thy-1 mAb ({square}) to kill Fas-sensitive targets (A), but only anti-CD3 mAb retarget these CTL to kill Fas-resistant targets (B). (C and D) Expression of H-2Ld molecules on A20.2J (C) and A20.F0 (D) was estimated by flow cytometry. (E) Cytotoxicity of specific antigenic peptide-triggered (34 nM to 100 mM) CTL BA/CT-2 were tested in a 4 h 51Cr-release assay with Fas-sensitive targets (•) and Fas-resistant targets ({triangleup}). Target cells were preincubated with antigenic peptide at the indicated concentrations for 1 h before labeling.

 
Control assays showed that peptide-specific CTL line BA/CT-2 is efficiently retargeted by both anti-CD3 mAb and anti-Thy-1 mAb to kill Fas-sensitive targets (Fig. 7AGo), but only anti-CD3 mAb retarget these CTL to kill Fas-resistant targets (Fig. 7BGo). Much higher concentrations of peptide are required to activate the perforin pathway of cytotoxicity than to trigger the Fas pathway (Fig. 7EGo). Similar results were obtained in experiments where lower concentrations of anti-CD3 mAb were required to induce the Fas rather than the perforin pathway of cytotoxicity (Fig. 8Go). These results support the view that the threshold of activation of Fas-mediated cytolysis is lower than that of perforin-mediated killing.



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  		Fig. 8. Lower levels of TCR cross-linking are required for triggering Fas- versus perforin-mediated cytotoxicity in CTL. CTL line C3B cells were co-incubated with Fc receptor-expressing, 51Cr-labeled Fas-sensitive A20.2J or its Fas-resistant variant A20.F0 for 4 h in the absence or presence of varied concentration of 2C11 culture supernatant. Serial dilutions 2.5% of stock 2C11 mAb culture supernatant were used in these assays. The ratio of CTL to target cells was 2. (A and B) Similar levels of expression of Fc receptors on both A20.2J (A) and A20.F0 (B) cells were determined by flow cytometry analysis. Cells were stained with FITC-conjugated goat anti-rat IgG Fab directly (solid histograms) or after pre-treatment with 2.4G2 (open histograms). (C) Dependence of Fas- versus perforin-mediated cytotoxicity on density of cross-linking anti-TCR–CD3 complex mAb. A CTL line C3B cells were co-incubated with 51Cr labeled Fas-sensitive A20.2J (•) or its Fas-resistant variant, A20.F0 ({triangleup}) for 4 h in the absence or presence of varied concentration of 2C11 culture supernatant at a CTL:target cell ratio of 2:1 and cytotoxicity was measured in 51Cr release assay.

 
It is important to note that all CTL used in these studies, which employ both perforin- and Fas-based pathways to lyse T cells, were much more efficient in lysing target cells if retargeted by 2C11 mAb than by G7 anti-Thy-1 mAb; however, and in agreement with the Fas-based mechanism of Thy-1-redirected cytotoxicity described here, there were no differences between the retargeting efficiencies of anti-CD3 mAb (2C11) and anti-Thy-1 mAb (G7) when perforin-deficient P0K.3A8 CTL were used (Fig. 1CGo). It is shown in Figs 2Go(A) and 5Go(D and E) that Thy-1 stimulation of 2B4 results in cytotoxicity which was lower in comparison to CD3-stimulated 2B4. This suggests that 6 h of Thy-1-mediated signaling is not sufficient to induce the maximal levels of Fas-mediated killing during the assay shown in Fig. 2Go(A). This interpretation is supported by observations of 2B4 expressing the same degree of cytotoxicity after stimulation with anti-CD3 mAb and anti-Thy-1 mAb during long-term assays (12–16 h, data not shown). On the other hand, the 4 h long Thy-1 stimulation appears to be sufficient to induce maximal levels of Fas-mediated cytotoxicity in CTL POK.3A8 and, therefore, the same degree of cytolytic activity was observed by stimulation with CD3 and with Thy-1 in Fig. 1Go(C).

Thus, we conclude that the ability of Thy-1-mediated signaling to preferentially trigger only Fas-mediated cytotoxicity may reflect the more stringent signal intensity requirements for triggering perforin-mediated cytotoxicity.

The described above data raised intriguing possibilities that in addition to Thy-1 other GPI-linked surface proteins may trigger T cells cytotoxicity. In future studies, we will examine whether GPI-linked proteins on the surface of cells in non-lymphoid tissues (43) may transduce signals leading to FasL up-regulation, thereby creating an immunopriviliged site with the potential to lyse approaching Fas-expressing lymphocytes.

Lower levels of TCR cross-linking are required for triggering Fas- versus perforin-mediated cytotoxicity in CTL
This hypothesis was further tested using different experimental system in experiment described in Fig. 8Go using stimulating anti-TCR–CD3 mAb or anti-Thy-1 mAb which cross-linked their ligands on the CTL surface after being presented to CTL by Fc receptor expressing Fas-susceptible (A20.2J) and Fas non-susceptible(A20.F0) target cells in a retargeting assay. In control experiments (Fig. 8A and BGo) for expression levels of Fc receptors on both A20.2J (A) and A20.F0 (B) cells we determined by flow cytometry analysis that both Fas-sensitive and Fas-resistant cells express similar levels of Fc receptors, thereby justifying their use in these experiments.

It is shown (Fig. 8CGo) that higher concentrations of anti-TCR–CD3 mAb are required to trigger perforin-mediated cytotoxicity than for induction of Fas-mediated lysis. Indeed, 1/256 dilution of 2C11 mAb were required to induce 50% of lysis of Fas- and perforin-sensitive A20.2J targets, while 1/128 dilution of mAb was used to induce the equal degree of destruction of Fas-resistant A20.F0 cells. These results support the view that the threshold of activation of Fas-mediated cytolysis is lower than for perforin-mediated killing, and they confirm and expand the earlier findings (44,51) that triggering of Fas-mediated cytotoxicity has a lower threshold than activation of perforin-mediated cytotoxicity and with the concept of different responses of CTL to different levels of TCR occupancy (45,46).

Cross-linking of TCR molecules is more effective in causing increases of second messengers (intracellular [Ca2+]i) in CTL than cross-linking of Thy-1 molecules
To directly address the question as to whether weaker triggering of FasL-mediated cytotoxicity in CTL by Thy-1 cross-linking is due to weaker transmembrane signaling we tested anti-Thy-1 mAb- and anti-CD3 mAb-pretreated CTL for changes in concentration of intracellular Ca2+ which reflect the intensity of activating stimuli (49,50). CTL were analyzed by flow cytometry for increases in concentration of intracellular calcium after stimulation with antibody by first injecting buffer and then, 20 s later, by injecting biotinylated mAb followed by injection of avidin for cross-linking (Fig. 9Go).



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  		Fig. 9. Thy-1-mediated signaling is less efficient than TCR-mediated signaling in triggering increases in intracellular Ca2+ concentrations in CTL BAK cells. BAK cells were pre-loaded with Indo-1 (final concentration 3 µM) and Pleuronic (0.03%) in Ca2+ buffer (1% FCS, 10 mM HEPES, HBSS) for 45 min at 30°C. Then cells were washed 3 times with Ca2+ buffer. To stimulate BAK cells, combinations of biotinylated 2C11 (PharMingen) or biotinylated G7 (Southern Biotechnology Associates) followed by incubation with cross-linking reagent Extra-Avidin (Sigma) were used. Optimal amounts of antibodies which were required for maximal response were established in preliminary flow cytometry experiments where CTL were stained with these mAb. Several concentrations of antibodies or avidin were tested in [Ca2+]i measurement experiments to allow for a comparison between intensities of Thy-1- and TCR-induced signaling. (A) Time course of appearance of cells with increased concentrations of intracellular Ca2+ during incubation with anti-CD3 mAb. (B) Time course of appearance of cells with increased concentrations of intracellular Ca2+ during incubation with anti-Thy-1 mAb.

 
It is shown that incubation of CTL with cross-linking anti-TCR–CD3 complex mAb resulted in a dramatically larger response, with many more cells being activated and with higher levels of increases of [Ca2+]i in individual cells. The Ca2+ response of TCR-triggered CTL is both faster and more intensive than in parallel samples with anti-Thy-1 mAb. Since granule exocytosis is known to be dependent on stimuli-induced increases in intracellular Ca2+ (15) these data help to explain why Thy-1-induced signaling was not sufficient to trigger granule exocytosis (Fig. 4Go) and perforin-mediated cytotoxicity (Figs 1, 5 and 7GoGoGo).

Thus, we established that Thy-1 is able to trigger the Fas-mediated cytotoxicity of CTL by utilizing the TCR–CD3-triggered signal transduction pathway. The Thy-1-triggered signaling is weaker than TCR-induced signaling and it is reflected in a more limited repertoire of effector functions which are activated in Thy-1- versus TCR-stimulated CTL. Taken together, these data reflect the possibility of differential activation of FasL and/or perforin pathways of cytotoxicity depending on the nature of activating stimuli and surface receptor.


   Acknowledgments
 
The authors thank Drs John Ashwell, Nobukata Shinohara, Lixin Zheng and Takashi Saito for reagents, Brenda Marshall and Shirley Starnes for editorial assistance, and Dr Ronald L. Rabin, Laboratory of Clinical Investigation, NIAID, for help in measurements of intracellular Ca2+ in T cells.


   Abbreviations
 
CTL cytotoxic T lymphocytes
CsA cyclosporin A
FasL Fas ligand
GPI glycophosphatidylinositol

   Notes
 
Transmitting editor: T. Saito

Received 21 December 1998, accepted 24 November 1999.


   References
Top
 Abstract
 Introduction
 Methods
 Results and discussion
 References
 

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