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The Journal of Immunology, 1998, 161: 5179-5186.
Copyright © 1998 by The American Association of Immunologists

Selective Induction of CD8+ Cytotoxic T Lymphocyte Effector Function by Staphylococcus Enterotoxin B1

Claudette L. Fuller and Vivian Lam Braciale2

Department of Microbiology and Beirne B. Carter Center for Immunology Research, Health Sciences Center, University of Virginia, Charlottesville, VA 22908


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Upon encounter with its antigenic stimulus, CTL characteristically proliferate, produce cytokines, and lyse the Ag-presenting cell in an attempt to impede further infection. Superantigens are extremely efficient immunostimulatory proteins that promote high levels of proliferation and massive cytokine production in reactive T cells. We compared the activation of murine influenza-specific CD8+ CTL clones stimulated with either influenza peptide or the superantigen staphylococcus enterotoxin B (SEB). We found that influenza peptide/MHC and SEB appeared equally capable of eliciting proliferation and IFN-{gamma} production. However, while influenza peptide/MHC elicited both perforin- and Fas ligand (FasL)/Fas (CD95L/CD95)-mediated cytolytic mechanisms, SEB was unable to trigger perforin-mediated cytolysis or serine esterase release. Examination of intracellular Ca2+ mobilization events revealed that the ability to trigger intracellular Ca2+ flux was not comparable between influenza peptide and SEB. SEB stimulated only a small rise in levels of intracellular Ca2+, at times indistinguishable from background. These findings indicate that the short-term cytolytic potential of superantigen-activated CD8+ CTL clones appears to be restricted to FasL/Fas (CD95L/CD95) mediated cytolysis.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Superantigens are potent immunostimulatory proteins that lead to profound proliferative events and massive cytokine production, followed in vivo by a deletion of reactive T cells (1). Produced by bacteria or viruses, superantigens are the known etiologic agents behind several human illnesses, including food poisoning, scarlet fever, and toxic shock syndrome. They are also the suspected cause of a number of autoimmune diseases such as Kawasaki’s disease, lupus, multiple sclerosis, and arthritis (2, 3, 4). The ability of any T cell to respond to an Ag, i.e., peptide, on a given Ag-presenting cell is dependent on tertiary interactions between the peptide-MHC complex and the TCR. A consequence of this MHC restriction is that only a minute portion of the T cell repertoire will be able to respond to any given peptide Ag (5). On the other hand, the novel specificity of superantigen for the semipolymorphic Vß element of the TCR correlates with its ability to activate oligoclonal populations of the T cell repertoire (2, 5, 6, 7, 8, 9, 10).

The dissimilarities between peptide Ag and superantigen, epitomized by the physical differences in TCR engagement, led us to speculate whether influenza peptide and superantigen might also elicit differential activation events and/or effector functions. While the majority of work has focused on CD4+ T cells, both CD4+ and CD8+ T cells are known to be superantigen reactive (10). Superantigen-induced proliferation and secretion of inflammatory cytokines is characteristic of both CD4+ and CD8+ T cells in vivo and in vitro and is integral to superantigen pathology (5, 11). CD8+ T cells, or cytolytic T lymphocytes (CTL),3 are, as their name implies, primarily concerned with the lysis of infected target cells. Short-term cytolysis for CD8+ T cells is conducted via the perforin granule or the recently discovered FasL/Fas (CD95L/CD95) mechanisms (12, 13, 14). The perforin mechanism, believed to account for two-thirds of CTL cytolysis in vivo (15), involves preformed cytolytic granules that release perforin, which inserts into the target cell membrane, and granzymes (16, 17), which activate target cell caspases leading to target cell apoptosis. Perforin is considered to be the primary mechanism of short-term cytolysis directed against virus-infected or tumorigenic cells. FasL/Fas-mediated cytolysis depends upon the induction of FasL expression on T cells after activation and the presence of Fas, a member of the TNFR family, on the target cells (17, 18, 19, 20). Upon ligation with FasL, Fas sends a signal through its death domain that results in the activation of caspases, which leads to target cell apoptosis (21). FasL/Fas-mediated cytolysis is thought to be a mechanism for down-regulation of immune responses (15, 22), although recent evidence has raised the possibility of a greater role for FasL/Fas in viral immunity (23). Both mechanisms of cytolysis can be elicited in CD8+ T cells by peptide Ag/MHC. The recent delineation of short-term cytolysis into perforin granule exocytosis and FasL/Fas induction prompted us to examine whether superantigen could elicit both mechanisms of cytolysis.

Using influenza-specific CD8+ murine CTL clones, we compared the ability of influenza peptide Ag and the bacterial superantigen staphylococcus enterotoxin B (SEB) to induce proliferation and CTL effector functions, namely IFN-{gamma} secretion and cytolysis. We report here that SEB stimulation of the TCR is qualitatively distinct from influenza peptide Ag in that it selectively induces only a subset of CD8+ CTL effector functions.


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

CD8+ CTL clones were maintained as previously described (24). Briefly, all clones were stimulated weekly with A/JAP/57-irradiated infected syngeneic splenocytes from BALB/c mice in the presence of 20 U/ml recombinant human IL-2 (Chiron Pharmaceuticals, Emeryville, CA), 10% FCS, 2 mM Gln, and 50 mM ß-mercaptoethanol in Iscove’s complete media (Life Technologies, Gaithersburg, MD). CTL were used on days 4–7 after stimulation and were washed before use in all assays. LB15.13 Fas+ (H-2bxd) (25), L1210 Fas- (14) and Fas+ (13) (H-2d), and A20.2J Fas+ (Iad) and A20.FO Fas- (an anti-Fas-resistant variant of A20.2J, both kindly provided by Dr. M. Sitkovsky, National Institutes of Health, Bethesda, MD) were used as target and APCs and maintained in culture in RPMI 1640 (Life Technologies), supplemented with 10% FCS, 2 mM Gln, 1% NEAA, and 0.5% sodium pyruvate.

Proliferation

Proliferation assays were performed as described previously (26). Briefly, 106 irradiated splenocytes per well were plated with 2 x 104 CTLs per well in a 96-well plate. Irradiated splenocytes were infected with A/JAP/57 influenza virus or incubated with SEB. Plates were incubated for 66 h at 37°C in a CO2 incubator. At 66 h, [3H]TdR was added to the wells and allowed to incorporate for 5–6 h. The assay was then harvested using a Tomtec Mach II Harvester (Wallac, Gaithersburg, MD) and counted using a 1205 Betaplate scintillation counter (Wallac). All experiments were performed in quadruplicate, with error bars representing the SD of quadruplicate samples.

IFN-{gamma} production

Assays were performed in 96-well microtiter plates using 5 x 104 T cells and 104 LB15.13 target cells and were mock treated or sensitized with influenza peptides HA204–212 (CTL 11-1) at 0.01 µg/ml final, HA529–537 (CTL 14-7) or NP147–155 (CTL 14-13), both at 0.1 µg/ml final, or SEB at 10 µg/ml final. Cells were stimulated at 37°C for 5–6 h, at which time supernatants were harvested and IFN-{gamma} levels determined using a standard IFN-{gamma} ELISA (PharMingen, San Diego, CA) as described (27). Error bars represent the SD of quadruplicate samples.

Flow cytometry

Flow cytometric analyses were performed as described previously (28). CTLs were separated from splenocytes and dead cells by Isopaque-Ficoll gradient, and 5–7 x 105 cells/well were added to a microtiter plate in media containing 1–2.5% FCS. Cells were stimulated with 10 µg/ml SEB or influenza peptide, HA204–212 (CTL 11-1) at 0.01 µg/ml final, HA529–537 (CTL 14-7) or NP147–155 (CTL 14-13) both at 0.1 µg/ml final, (29) presented by LB15.13 APCs, and stained on ice at 1:100 with either anti-CD3{epsilon} (145.2C11), anti-CD25{alpha} (PC61), anti-Vß Abs (all directly conjugated, except for anti-CD25{alpha}, which was biotinylated; SA-PE was used as a secondary), or anti-FasL (MFL3) (29) (PharMingen). The anti-FasL Ab was used with a goat F(ab')2 anti-hamster FITC-conjugated secondary (Southern Biotechnology Associates, Birmingham, AL). Cells were analyzed on a FACScan (Becton Dickinson, Mountain View, CA).

Calcium mobilization studies

CTL clones were separated from splenocytes on an Isopaque-Ficoll gradient on the third day after in vitro stimulation. CTL were resuspended at 106 cells/ml and were incubated with 1 µM (final) Indo-1/AM and 1 µl Pluronic F-127 20% w/v per 106 cells (Molecular Probes, Eugene, OR) for 1 h at 37°C in a 5% CO2 incubator. Free dye was removed by washing CTL three times in sterile filtered buffered saline solution. After the first wash, L1210 or LB15.13 APCs were added at an E:T of 1:1. CTL were stimulated at 30 s with 0.01 µg/ml HA204–212 (11-1), 0.10 µg/ml NP147–155 (14-7), or HA529–537 (14-13), or 10 µg/ml SEB. Intracellular calcium was measured quantitatively on an SLM 8000 spectrofluorometer (SLM Aminco, Urbana, IL) as described previously (30). Excitation was at a wavelength of 340 nm, and fluorescence intensity was measured at 398 and 480 nm with the instrument in the "T" format. Data are representative of at least three individual experiments.

Cytotoxicity

The JAM test, as described by Matzinger (31), was employed to assay perforin- and FasL/Fas-mediated cytolysis. Target cells were incubated overnight with [3H]TdR at 37°C. Targets were washed two times, mock treated or sensitized with influenza peptides HA204–212 (CTL 11-1) at 0.01 µg/ml final, HA529–537 (CTL 14-7), or NP147–155 (CTL 14-13) both at 0.1 µg/ml final, (32), staphylococcal enterotoxin B (Sigma, St. Louis, MO) or highly purified SEB (Toxin Technology, Sarasota, FL) at 10 µg/ml final. Emetine at 5 µg/ml (Sigma) and cyclosporin A (Sigma) at 5 µg/ml were added to CTL for 30 min before plating at 37°C in a CO2 incubator. Anti-FasL mAb MFL3 (29) (PharMingen) was used at 10 µg/ml and was incubated with CTLs for 30 min at room temperature. Targets were plated at 104 cells/well. CTL clones were added at an E:T of 5:1, spun for 2 min at 400 rpm, and incubated at 37°C in a CO2 incubator. After 5 to 6 h, assays were harvested using a Tomtec Mach II Harvester (Wallac) and counted using a 1205 Betaplate scintillation counter (Wallac). All experiments were performed in quadruplicate. Percent specific lysis was calculated as follows: % DNA loss = 100 x [(label retained without CTL cpm) - (label retained with CTL cpm)]/(label retained without CTL cpm). Label retained without CTL was never less than 90% of target cpm at time = 0. Error bars in the figures represent the SD of quadruplicate samples. Data are representative of at least six individual experiments.

Serine esterase release assay

A total of 105 LB15.13 or A20.2J target cells was plated along with 5 x 105 CTL in 96-well plates along with either influenza peptide HA204–212 (CTL 11-1) at 0.01 µg/ml final, HA529–537 (CTL 14-7), or NP147–155 (CTL 14-13), both at 0.1 µg/ml final (29), or 10 µg/ml SEB. Control wells contained CTL alone in 0.1% (final) Triton X-100 detergent or media and target cells alone in detergent. Plates were spun for 2 min at 400 rpm before incubating at 37°C in a CO2 incubator. At 5 h, plates were spun at 4°C for 5 min at 1500 rpm, and 100 ml of supernatant was collected. Granzyme A activity was determined as described (28, 33). After 30 min, absorbance values at 405 nm were determined by reading on a Dynatech Laboratories ELISA reader (Chantilly, VA). All experiments were performed in quadruplicate. Percent serine esterase release was calculated as follows: % serine esterase release = 100 x [(experimental release) - (spontaneous release)]/[(total release) - (spontaneous release)]. Error bars in the figures represent the SD of quadruplicate samples.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Influenza peptide and SEB stimulation of proliferation, IFN-{gamma} production, and IL-2R{alpha} expression in reactive CD8+ CTL clones

To compare influenza virus peptide Ag and SEB stimulation of CD8+ (CTL) effector function, three CD8+ influenza-specific CTL, 11-1, 14-13, and 14-7, (24) were utilized. As mentioned earlier, superantigen-reactive T cells are defined by TCR Vß usage. Flow cytometric analysis of the reactive clones demonstrated that the Vß usage of both 11-1 and 14-13 was consonant with an SEB-compatible TCR, Vß8 (data not shown). The third clone, 14-7, was not positive by flow cytometric analysis for any of the known SEB-reactive Vß elements (Vß3, 7, 8.1–8.3, 11, and 17) (5) and thus served as a negative control for SEB activation as well as for any nonspecific contaminating mitogens that might be present in the SEB stock.

Since proliferation of reactive T cells is a hallmark of SEB activation in vivo, we examined the concentration of SEB necessary to induce proliferative levels roughly equivalent to those induced by influenza-infected splenocytes. Fig. 1Go shows a comparison of the proliferation stimulated by either influenza virus-infected irradiated splenocytes or various concentrations of SEB presented on irradiated syngeneic splenocytes. CTL clones 11-1 and 14-13 proliferated strongly in response to both influenza virus peptide Ag and SEB (Fig. 1Go, A and B), with 10 µg/ml SEB giving a level of proliferation consistently comparable to influenza peptide Ag. There did appear to be a high dose inhibition of proliferation by SEB as seen with CTL 11-1 (Fig. 1GoA) and with CTL 14-13 (data not shown) at doses greater than 100 µg/ml.



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  		FIGURE 1. Induction of proliferation by A/JAP/57 influenza virus and SEB. Approximately 2 x 104 CD8+ CTL 11-1 (A), 14-13 (B), and 14-7 (C) were incubated with 1 x 106 irradiated syngeneic splenocytes that were either uninfected or infected with A/JAP/57 influenza virus. SEB stimulation consisted of irradiated syngeneic splenocytes and CTL, with 0.1, 1.0, 10.0, or 100.0 µg/ml SEB added to the well at plating. All conditions were in the presence of 20 U/ml (final) IL-2 for 72 h.

 
Activation of inflammatory cytokines is the mechanism by which superantigens affect a state of shock in both humans and mice (11, 34, 35, 36). Among the cytokines responsible for SEB-associated pathology is IFN-{gamma} (11). For this reason, we compared induction of IFN-{gamma} production by viral peptide and SEB (Fig. 2Go). At 10 µg/ml, SEB showed the same ability to stimulate IFN-{gamma} production as influenza peptide Ag.



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  		FIGURE 2. Comparison of IFN-{gamma} production by influenza peptide or SEB-induced clones. LB15.13 cells were either untreated (media), treated with peptide HA204–212 (11-1, solid bars) at 0.01 µg/ml final, HA529–537 (14-7, open bars), or NP147–155 (14-13, hatched bars) both at 0.1 µg/ml final, or 10 µg/ml SEB and incubated with 5 x 104 at an E:T of 5:1. After 6 h, culture supernatants were harvested and examined in an IFN-{gamma} ELISA.

 
Many of the activation events that occur in T cells are reflected by cell surface molecule regulation. The ability to cause up-regulation of the high affinity {alpha} subunit of the IL-2R is very well correlated with activation since it serves to allow progression of T cells into cell cycle, representative of a late stage of activation (37). We therefore examined cell surface expression of the IL-2R{alpha} as an indicator of influenza peptide and SEB activation of the clones (Fig. 3Go, A and B). After 48 h of stimulation of clones either with influenza peptide or SEB presented on LB15.13 target cells, Abs against IL-2R{alpha} were used to examine activation of clones by either stimulus. The reactive clones were induced to up-regulate IL-2R{alpha} in response to both viral peptide Ag and SEB in an equivalent manner. Again, this supports previous data (Figs. 1Go and 2Go) that suggest that influenza peptide and SEB are equal in their ability to cause activation of reactive T cells.



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  		FIGURE 3. Comparison of influenza peptide and SEB induced up-regulation of IL-2R{alpha} (CD25). Clones were incubated at 5:1 with LB15.13 targets cells and mock sensitized (dotted lines), treated with peptide (thin lines) 0.01 µg/ml HA204–212 (11-1), 0.10 µg/ml HA529–537 (14-13), 0.10 µg/ml NP147–155 (14-7), or 10 µg/ml SEB (thick lines). CTL were stained for IL-2R{alpha} after 48 h.

 
CTL clone 14-7 also responded to viral Ag by proliferating, producing IFN-{gamma} and up-regulating IL-2R{alpha} (Figs. 1GoC, 2, and 3C). However, 14-7 did not have any proliferative or IFN-{gamma} response to SEB. This supports flow cytometry data that found Vß element usage by 14-7 did not match any of those known to be SEB-reactive and therefore was not stimulated by SEB.

Inability of SEB to trigger perforin-mediated cytolysis or activate serine esterase release

Because cytolytic activity is arguably the most important of all CD8+ CTL effector functions, and since proliferation and IFN-{gamma} production appeared to be similar between influenza peptide and SEB, we examined whether cytolysis induced by influenza peptide and SEB would be equivalent as well. To examine the ability of SEB to elicit perforin-mediated cytolysis, we assayed for cytolytic effector function with the JAM test (31). The JAM test allows for quantitative analysis of cytolysis by the loss of intact [3H]TdR-labeled target cell DNA, indicative of the DNA fragmentation seen in both perforin- and FasL/Fas-mediated cytolysis (31, 38, 39, 40, 41, 42, 43). As shown in Fig. 4Go, influenza peptide Ag was able to stimulate all three clones to lyse class I+ L1210 Fas- (Fig. 4GoA) and class I and II+ A20.FO (Fig. 4GoB) target cells. SEB, however, was unable to stimulate any of the clones to lyse either class I+, or class I and II+ target cells within the 5–6 h of the assay (Fig. 4Go, A and B). 51Chromium release assays with Fas- targets, either class I+ or class I and II+, confirmed the results of Fig. 4Go in that SEB was unable to cause cytolysis of Fas- target cells (data not shown). Dose response assays (data not shown) indicate that the ability to trigger cytolysis was not a function of the concentration of SEB, since there was no cytolysis above background for doses of 0.01 to 1000 µg/ml SEB for either target cell.



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  		FIGURE 4. Lack of SEB-induced cytolysis of Fas- target cells. CTL clones 11-1 (solid bars), 14-13 (hatched bars), and 14-7 (open bars) were incubated at an E:T of 5:1 with 104 [3H]TdR-labeled L1210 Fas- (A) or A20.FO Fas- (B) target cells in a 5-h JAM assay. CTLs were mock stimulated, incubated with 0.01 µg/ml final HA204–212 (11-1), 0.1 µg/ml final HA529–537 (14-13), 0.1 µg/ml final NP147–155 (14-13), or 10 µg/ml final SEB. Percentage DNA loss was calculated as described in Materials and Methods.

 
To confirm the inability of SEB to trigger perforin cytolysis, SEB-stimulated release of the serine esterases associated with the granzymes of perforin-mediated cytolysis was examined (Fig. 5Go). In agreement with cytotoxicity data (Fig. 4Go), influenza peptide was able to trigger substantial serine esterase release in all clones, while SEB again failed to cause release above background levels. These results indicate that, although the pathways for induction of proliferation and IFN-{gamma} production were equally activated via influenza peptide and SEB, the ability to cause perforin-mediated cytolysis and serine esterase release was disparate.



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  		FIGURE 5. Release of serine esterase triggered by influenza peptide or SEB. LB15.13 target cells were mock stimulated (media), or treated with peptide 0.01 µg/ml HA204–212 (11-1), 0.1 µg/ml HA529–537 (14-13), 0.1 µg/ml NP147–155 (14-13), or 10 µg/ml SEB. Targets were incubated at an E:T of 5:1 with 5 x 105 of CTL 11-1 (solid bars), 14-13 (hatched bars), or 14-7 (open bars). After 5 h, supernatants were harvested, and serine esterase activity was assayed. Percent serine esterase release was calculated as described in Materials and Methods.

 
Ability of SEB to elicit an intracellular Ca2+ mobilization event in reactive CD8+ CTL clones

The intracellular Ca2+ mobilization event initiated by TCR stimulus occurs within seconds of T cell-target cell engagement (44, 45). It is one of the earliest T cell signaling events and is an important second messenger for many of the pathways leading to CTL effector function, including proliferation (46), induction of cytokine genes (47), and perforin-mediated cytolysis (30, 48). Our lab has recently reported on a CD8+ T cell variant that was unable to mediate perforin-mediated cytolysis as a result of the failure to mobilize intracellular Ca2+ (28). Since SEB was unable to trigger perforin cytolysis, we decided to examine whether the SEB signal transduced through the TCR would result in a diminished Ca2+ profile, as compared with influenza peptide, which was able to trigger perforin cytolysis. When influenza peptide, at the same concentrations as in previous figures, was used to stimulate clones (Fig. 6Go, checked line), all three responded with an increase in intracellular Ca2+ well above background (grey line), which constituted a significant portion of the maximal Ca2+ response represented by addition of 100 µM ionomycin at 370 s. SEB (solid line), however, was not able to induce a similar Ca2+ profile in any of the clones. Although 11-1 (Fig. 6GoA) did have a Ca2+ response marginally above background, the response of 14-13 (Fig. 6GoB) was indistinguishable from background and was roughly equivalent to the response from 14-7 (Fig. 6GoC), the nonreactive clone, to SEB. These data indicate that, although SEB is able to trigger proliferation and IFN-{gamma} production in reactive clones, it is impaired in its ability to elicit a Ca2+ mobilization event, which may be indicative of differences in the signal transduced following influenza peptide or SEB stimulation.



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  		FIGURE 6. Intracellular Ca2+ mobilization triggered by influenza peptide or SEB. The change in intracellular Ca2+ was measured after mock (grey lines), peptide HA204–212 (11-1) at 0.01 µg/ml final, HA529–537 (14-7) or NP147–155 (14-13) both at 0.1 µg/ml final (checked lines), or 10 µg/ml SEB (black lines) presented on either L1210 Fas- or LB15.13 Fas+. Indo-1 AM loaded CTL clone 11-1 (A), 14-13 (B), and 14-7 (C) were incubated with LB15.13 APCs at an E:T of 1:1. At 30 s, peptide or SEB was added. Ionomycin (100 µM final) was added at 370 s to determine maximal Ca2+ release. Data are representative of at least three individual experiments, with clones 11-1 and 14-13 using either APC.

 
SEB-induced cytolysis of Fas+ target cells and up-regulation of FasL expression

Because the signaling events for perforin- and FasL-mediated cytolysis are very different in their requirement for Ca2+ (28, 49) and the loss of or failure to signal one pathway does not appear to impair the other (28), we explored the possibility that SEB could induce the FasL/Fas-mediated cytolysis of Fas+ target cells (Fig. 7Go). Dose response curves using SEB revealed that SEB was able to induce cytolysis of several different Fas+ target cells well above background for concentrations as low as 0.01 µg/ml SEB (data not shown). SEB-stimulated cytolysis was as much as 70% higher on Fas+ than Fas- target cells, implying that FasL/Fas was the chief mediator of the SEB-cytolysis observed. Flow cytometric data (Fig. 8Go) corroborated FasL as the mechanism of SEB-induced cytolysis since both the SEB reactive clones were induced to express FasL upon stimulation.



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  		FIGURE 7. SEB-induced cytolysis of Fas+ target cells. [3H]TdR-labeled LB15.13 Fas+ target cells were mock sensitized or treated with peptide 0.01 µg/ml final HA204–212 (11-1), 0.1 µg/ml final HA529–537 (14-13), 0.1 µg/ml final NP147–155 (14-13), or 10 µg/ml final SEB. Cytolysis was quantitated with the JAM test. Clones 11-1 (solid bars), 14-13 (hatched bars), and 14-7 (open bars) were incubated at an E:T of 5:1 for 5–6 h.

 


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  		FIGURE 8. Comparison of influenza peptide and SEB-induced up-regulation of FasL (CD95L). Clones were incubated at 5:1 with LB15.13 targets cells and mock sensitized (dotted lines), treated with peptide (thin lines) 0.01 µg/ml HA204–212 (11-1), 0.10 µg/ml HA529–537 (14-13), 0.10 µg/ml NP147–155 (14-7), or 10 µg/ml SEB (thick lines). CTL were stained for FasL after 5 h.

 
To directly demonstrate FasL/Fas-mediated cytolysis, we used the inhibitors emetine and CsA, as well as anti-FasL-blocking Abs. The induction of FasL expression following TCR stimulation is dependent on transcription and translation of the fasL gene (14, 50) and, thus, is sensitive to emetine, a protein synthesis inhibitor (49, 51). FasL is also sensitive to CsA, a calcineurin phosphatase inhibitor that has also been shown to inhibit FasL induction (28, 52, 53). Fig. 9Go represents a JAM test performed concurrently with the assays in Figs. 4Go and 7Go, except that these CTL were pre-treated with either emetine or CsA for 30 min before plating. Fig. 9GoA shows that the perforin cytolysis elicited by the influenza peptide is insensitive to inhibition with emetine or CsA. Fig. 9GoB represents the effects of the inhibitory drugs on SEB-induced cytolysis. We found that, as opposed to influenza peptide-stimulated cytolysis, SEB-stimulated cytolysis was substantially inhibited by emetine and CsA, with reduction of specific cytolysis ranging from 30 to 50%. This suggested that protein synthesis and calcineurin-dependent events were required for SEB-induced cytolysis, both of which are characteristic of FasL/Fas cytolysis. To further confirm the mechanism of cytolysis, we used anti-FasL Abs to specifically block the interaction of FasL/Fas (29). As with the inhibitory drugs, we saw a reduction of at least 40% for both 11-1 and 14-13. These data indicate that SEB does not trigger perforin-mediated cytolysis, but selectively induces FasL/Fas-mediated cytolysis.



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  		FIGURE 9. Inhibition of SEB-induced FasL/Fas-mediated cytolysis of target cells by emetine, CsA, and anti-FasL Abs. Clones 11-1 (solid bars), 14-13 (hatched bars), and 14-7 (open bars) were incubated at an E:T of 5:1 with [3H]TdR-labeled LB15.13 Fas+ target cells presenting (A) peptide at 0.01 µg/ml HA204–212 (11-1), 0.10 µg/ml HA529–537 (14-13), 0.10 µg/ml NP147–155 (14-7), or (B) 10 µg/ml SEB. CTL were pretreated for 15 min with 10 µg/ml anti-FasL Ab, or 30 min with 5 µg/ml emetine or 5 µg/ml CsA. Assays were harvested after 5 h.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The TCR is an intricate complex of multiple signal transduction subunits that are responsible for the activation of several signaling pathways involving dozens of molecules upon ligand engagement. Studies of altered peptide ligands have led to the understanding that activation is not a simple binary switch and that intrinsic to any ligand is the ability to selectively involve TCR signaling modules. A consequence of this is that the quality of the TCR ligation has implications for the induction of TCR-mediated effector functions. Our examination of CD8+ CTL effector functions induced by influenza peptide or SEB suggests that, although they are engaging the same TCR, the signal transduced by the receptor varies markedly as a result of the ligand engaged. Here we report that the signal transduced through the TCR by influenza peptide Ag and SEB are distinct in their ability to elicit CD8+ CTL effector function.

Despite the fact that peptide and superantigen are spatially and functionally distinct in the immunologic consequences of activation in vivo, our data point to several similarities in induction in vitro. Proliferative events induced by influenza-infected APC and SEB (Fig. 1Go) were notably similar on a per-cell basis. In vivo, a given viral peptide would be able to activate only a small subset of the total T cell population, less than 1% (5). Superantigens, on the other hand, are specific for the TCR Vß element, which has limited polymorphism, such that a single superantigen has the potential to activate as much as 20–30% of murine T cells (5, 54). The results seen with IFN-{gamma} production (Fig. 2Go) were similar to proliferation data in that SEB was not superior to influenza peptide on a per-cell basis. Our finding that, given equal numbers of reactive clones, SEB has no greater ability to induce proliferation or IFN-{gamma} production reinforces the idea that superantigen-related pathology in vivo is a consequence of the sheer number of T cells activated and not a "super" ability to activate a subset of cells.

On the other hand, the signals transduced by influenza peptide and SEB through the TCR, however, were not equal in ability to induce perforin-mediated cytolysis (Fig. 4Go). Dose response assays showed that this response was not dose dependent since SEB concentrations as high as 1000 µg/ml were unable to cause CTL lysis of Fas- targets (data not shown). Our lab has recently reported a correlation between calcium response and cytolysis, in which low levels of sustained calcium mobilization are able to induce FasL/Fas-mediated cytolysis, while a more "classical" Ca2+ mobilization is required for perforin cytolysis (49). Our findings with SEB, with its induction of a diminutive yet sustained Ca2+ response (Fig. 6Go), would support the notion that an absent or very diminished Ca2+ response correlates with the inability to elicit perforin-mediated cytolysis. The Ca2+ requirement for perforin-mediated cytolysis has been well established in that it is important in the movement of the cytolytic granules to the interface between the CTL and target cell (30). This implies that the signal for perforin cytolysis may involve activation of cytoskeletal elements to allow such movement. SEB, therefore, may be unable to induce reorientation of the cytoskeletal machinery necessary to cause perforin granule exocytosis.

Interestingly, although Ca2+ has been implicated in the induction of IFN-{gamma} through activation of NF-AT (47, 55), SEB was able to cause IFN-{gamma} secretion in the absence of substantial Ca2+ mobilization. Similarly, the requirement for NF-AT in FasL induction has been reported (56), and two NF-AT-responsive elements have been found in the FasL promoter. Again, the fact that SEB was able to induce FasL suggests that the small yet sustained Ca2+ flux induced was enough to allow NF-AT import to the nucleus and cause transcription of both FasL and IFN-{gamma}. This is supported by other reports that have shown that superantigens, and SEB in particular, do cause NF-AT translocation (57, 58). These data point to the hypothesis that the quality of the Ca2+ response may be just as important to the signal transduced as the quantity, a finding that is also being examined for B cells (59, 60).

The ability of SEB to selectively induce FasL/Fas-mediated cytolysis is interesting in light of the fact that FasL/Fas is believed to be the primary mechanism of immunoregulation in vivo (15) and the mechanism by which superantigen reactive T cells are deleted in vivo (61, 62, 63). MRL-lpr/lpr mice have also been shown to be largely resistant to SEB-induced clonal deletion (34). The findings presented here would support the idea that one of the mechanisms of superantigen-related pathology may be lysis of Fas+ cells, whether they be APCs or other T cells. A large number of autoimmune diseases recently hypothesized to be the result of a superantigen activation of T and B cells (1, 2, 3, 64) could also be the result of SEB-induced bystander lysis, which has been shown to be characteristic of FasL/Fas-mediated cytolysis (65, 66). We would therefore propose that, in addition to cytokine-induced pathology, a potential mechanism of superantigen pathology may be FasL/Fas-mediated bystander cytolysis of tissue that constitutively expresses Fas, such as the gut, lungs, liver, and heart (67). The connection between SEB-induced cytolysis and the high Fas expression in these sites may help shed light on superantigen-related food poisoning and autoimmune diseases such as Kawasaki’s disease (2).

Taken together, our data support the idea of a TCR receptor capable of transducing a variety of signals based on the ligand that it engages. This is an intriguing idea for the modulation of the immune response in infection and situations like autoimmune disease or transplant rejection, since it would suggest that a ligand could be engineered to provide or suppress a given immune response. Recent crystal structure analysis of the TCR-pMHC complex (68), along with our data from SEB and peptide activation of T cells, 1) supports the notion that the site of ligand interaction with the TCR may be as important to the signal transduced as the nature of the ligand, 2) warrants further investigation, and 3) may provide a great deal of insight into the nature of T cell activation and the role of the ligand in modulating its response.


   Acknowledgments
 
We thank Rhoel Dinglasan for expert technical assistance, Dr. Doris M. Haverstick for assistance in the calcium fluorometry studies, Dr. M. Sitkovsky for the A20 target cells, and Dr. Victoria Camerini for the generous gift of Ab reagents. Also, thanks to Hua-Poo Su and Dr. Mark T. Esser for helpful discussion and technical assistance.


   Footnotes
 
1 This work was supported in part by the Beirne B. Carter Center for Immunology Research and grants from the U. S. Public Health Service (to Thomas J. Braciale). C.L.F. was supported by funds from the U. S. Public Health Service, National Institutes of Health Training Grant T32A107496, and University of Virginia Pratt and Hearst Fellowships. Back

2 Address correspondence and reprint requests to Dr. Vivian Lam Braciale at the current address: University of Texas Medical Branch, Department of Microbiology and Immunology, 301 University Blvd, Galveston, TX 77555-1070. E-mail address: Back

3 Abbreviations used in this paper: CTL, cytolytic T lymphocyte; FasL, Fas ligand; CsA, Cyclosporin A; Emet, emetine; SEB, staphylococcus enterotoxin B; HA, hemagglutinin; NP, nucleoprotein. Back

Received for publication April 30, 1998. Accepted for publication July 7, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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