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Cooperative Activation of TCRs by Enterotoxin Superantigens

Unité de Biologie des Interactions Cellulaires, Centre National de la Recherche Scientifique, Unité de Recherche Associée 1960, Institut Pasteur, Paris, France

	Abstract

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Staphylococcus enterotoxin superantigens are potent T cell activators. To gain new insights into the mechanism of T cell activation induced by these superantigens, we investigated the recruitment of signaling molecules in this process. Here, we show that enterotoxin superantigen activation can be transmitted to TCR-CD3 complexes that did not interact with their ligand. Indeed, by studying cells expressing two distinct TCRs, we found that enterotoxin superantigens induced tyrosine phosphorylation of TCR subunits, the recruitment and tyrosine phosphorylation of the protein tyrosine kinase ZAP-70, and an increase in protein tyrosine kinase activity of both directly stimulated and unstimulated TCR-CD3 complexes. As the involvement of unstimulated TCR-CD3 complexes in signal transduction would increase the number of signaling molecules and, therefore, the efficiency of T cell activation, these data provide a novel explanation for the ability of enterotoxin superantigens to potently activate T lymphocytes.

	Introduction

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Some Staphylococcus aureus exotoxins induce vigorous responses in T cells expressing particular Vß elements. These toxins, termed Staphylococcus enterotoxin (SE³) superantigens, activate T cells by binding simultaneously to TCR Vß domains and to MHC class II molecules on APCs (1). Although some of the enterotoxin superantigens have been extensively characterized structurally and functionally, the mechanism by which these toxins transduce activating signals to T cells is not fully understood.

One of the earliest biochemical events detected after TCR stimulation is the activation of the src family protein tyrosine kinases Lck and Fyn and, as a consequence, the phosphorylation of numerous substrates, including several TCR-CD3 subunits. This occurs on specific regions of their cytoplasmic tails, termed immunoreceptor tyrosine-based activation motifs (ITAMs). Tyrosine phosphorylation of ITAMs of the CD3 and TCR subunits promotes the recruitment of the protein tyrosine kinase ZAP-70, which binds to phosphorylated ITAMs via its src homology 2 (SH2) domains. Once bound to ITAMs, ZAP-70 becomes phosphorylated and activated. Genetic and biochemical evidence has revealed that ITAM phosphorylation and ZAP-70 activation are crucial events in TCR signaling. Therefore, the strength of TCR signal transduction depends on the amount of phosphorylated ITAMs and ZAP-70 molecules recruited (2, 3).

To gain new insights into the mechanism of signal transduction by Staphylococcus superantigens, we investigated the recruitment of signaling molecules in this process. We have previously shown that "cross-talk" between TCR complexes could occur upon enterotoxin superantigen activation in the absence of ligand cross-linking. To demonstrate this, we used cells expressing two different TCR sets: one belonging to the Vß3 and the other one to the Vß8 family. They can be distinguished by mAbs and can be specifically stimulated by enterotoxin superantigens. We showed that activation by enterotoxin superantigens specific for one set of receptors down-modulated not only stimulated TCRs, but also a significant number of unstimulated TCRs (4). We now address the question of whether enterotoxin superantigens could recruit, in addition to directly stimulated TCRs, unstimulated TCR-CD3 complexes for signaling. We found that several key events in TCR signal transduction, such as tyrosine phosphorylation of TCR subunits, recruitment and tyrosine phosphorylation of ZAP-70, and increased protein tyrosine kinase activity, occurred not only on stimulated TCR-CD3 complexes, but also on a large number of unstimulated TCR-CD3 complexes. The recruitment of TCR-CD3 complexes that did not directly interact with any ligand may improve the efficiency of TCR signaling during enterotoxin superantigen activation.

	Materials and Methods

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Reagents and cell lines

SE and anti-Vß mAbs were as previously described (4, 5). The anti- rabbit antiserum, N39 (6), was a gift of Dr. B. Alarcón (Centro de Biología Molecular, Madrid); anti-CD3 (2Ad2A2, IgM) mAb (7) was a gift from Dr. E. L. Reinherz (Dana-Farber Cancer Institute, Boston, MA); and anti-ZAP-70 rabbit antiserum (4.06) (8) was a gift from Dr. O. Acuto (Institut Pasteur, Paris). The anti-phosphotyrosine mAb (4G10, IgG2b) was from Upstate Biotechnlogy (Lake Placid, NY). Peroxidase- or alkaline phosphatase-linked anti-mouse or anti-rabbit Ig Abs, as well as enhanced chemiluminescence (ECL) and enhanced chemifluorescence (ECF) Western blotting detection reagents were from Amersham (Les Ulis, France). APC cell lines and Jurkat transfectants expressing one or two different TCRs have been previously described (4).

Immunoprecipitation and Western blotting

Cells, 10⁷ per immunoprecipitate, were incubated at 37°C in growth medium, together with MHC class II⁺ cells of the B cell line, Raji, as APCs (APC to T cell ratio, 1:1) or with APCs plus 10 µg/ml SEB or SEE, or with anti-CD3 mAb (2Ad2A2) at 1:200 dilution of ascites, for the times described in the figure legends. Cells were then centrifuged for 10 s at 15,000 x g, lysed in 1% Brij-97 (Sigma, St. Louis, MO) lysis buffer, and immunoprecipitated with anti-Vß3 (JOVI-3) or anti-Vß8 (17-34-14) mAbs. Cell lysis, immunoprecipitation, anti-phosphotyrosine Western blotting and ECL detection were conducted as previously described (9), except that we used poly(vinylidene difluoride) membranes (Amersham or Millipore, Bedford, MA). To determine the position of the TCR or ZAP-70 molecules on gels, anti- or anti-ZAP-70 immunoprecipitates were run in parallel with the anti-Vß immunoprecipitates. In addition, membranes were stripped according to the manufacturer’s instructions (Amersham) and a second immunodetection was conducted using specific Abs. Quantitative analysis of Western blots was conducted using ECF as detection method. In this case, the secondary Ab used for immunodetection was coupled to alkaline phosphatase. After washing, the membranes were incubated with Vistra (Amersham) reagent for 5–20 min and scanned on a Storm PhophorImager/FluorImager (Molecular Dynamics, Sunnyvale, CA). Phosphorylated bands were quantitated and the values obtained for each time point were expressed as multiples of the values measured in control lanes. The density values were in the linear range of the detection method.

Immune complex protein tyrosine kinase assay

The catalytic activity of protein tyrosine kinases associated with anti-Vß immunoprecipitates was assessed in vitro using the SignaTect Protein tyrosine kinase assay system (Promega, Madison, WI). The immunoprecipitations were conducted as described above. The protein Sepharose A beads were then washed with a 25 mM HEPES, pH 7.0, 10 mM MnCl₂, 10 mM MgCl₂ buffer and the kinase assay was performed according to the manufacturer’s instructions. Briefly, 20 µl of reaction buffer (8 mM imidazole hydrochloride, pH 7.3, 8 mM ß-glycerophosphate, 0.2 mM EGTA, 20 mM MgCl₂, 1 mM MnCl₂, 0.1 mM sodium vanadate, 20 mM ATP, 0.02 µCi/µl [-³²P]ATP, and 0.125 nM biotinylated peptide 1) were added to the bead pellets. Samples were incubated for 20 min at room temperature, and reactions were stopped with 12.5 µl of 7.5 M guanidine-HCl buffer. Samples were then centrifuged, and 12.5 µl of each reaction spotted onto the SAM² biotin-capture membrane. The membrane was then washed with 2 M NaCl and 2 M NaCl, 1% phosphoric acid, exposed overnight on a Kodak phosphor screen (Eastman Kodak, Rochester, NY) and quantitated on a Storm PhosphorImager.

	Results

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Superantigen activation induced the phosphorylation of subunits of both stimulated and unstimulated TCR-CD3 complexes

Cells expressing two TCR sets (Vß3 and Vß8) were stimulated with either SEB or SEE in the presence of MHC class II⁺ APCs, lysed at various times, and each set of TCR-CD3 complexes was immunoprecipitated with anti-Vß3 or anti-Vß8 mAbs. SEB specifically stimulates Vß3 TCRs, whereas SEE stimulates Vß8 TCRs. The state of tyrosine phosphorylation of polypeptides associated with each TCR type was then analyzed by Western blotting (Fig. 1). Activation of cells with SEB resulted in increased tyrosine phosphorylation of TCR subunits associated with stimulated Vß3 receptors and also of those associated with unstimulated Vß8 receptors. Conversely, SEE, which activates Vß8 receptors, induced the tyrosine phosphorylation of TCR subunits associated with Vß8 TCRs and of those associated with Vß3 TCRs (Fig. 1B). Tyrosine phosphorylation of TCR subunits was more intense in directly stimulated TCR-CD3 complexes than in unstimulated ones (Fig. 1, A and B). As expected, stimulation of all surface TCR-CD3 complexes with an anti-CD3 mAb resulted in the equivalent phosphorylation of TCR polypeptides associated with Vß3 and with Vß8 TCR-CD3 complexes (Fig. 1C). Phosphorylation of TCR associated with stimulated and unstimulated TCRs followed the same kinetics, being observed at times as short as 30 s, reaching a maximum between 1–2 min, and decreasing later (data not shown). This suggests that the recruitment of stimulated and unstimulated TCR-CD3 complexes occurs simultaneously.

View larger version (26K): [in this window] [in a new window]   FIGURE 1. SEB or SEE induced the tyrosine phosphorylation of TCR subunits in stimulated and unstimulated TCR-CD3 complexes. Cells expressing both Vß3 and Vß8 TCRs were incubated with MHC class II+ APCs (APC:T cell ratio, 1:1), APCs plus enterotoxins, or anti-CD3 mAb for various times at 37°C. After lysing the cells, TCRs were immunoprecipitated with anti-Vß3 or anti-Vß8 mAbs. Immunoprecipitates were analyzed by 12% PAGE and Western blotting using anti-phosphotyrosine mAb and ECL detection. A, Cells were incubated with APCs alone for 5 min (lanes 1 and 4) or with APCs plus SEB for 2 min (lanes 2 and 5) or 5 min (lanes 3 and 6). B, Cells were incubated with APCs alone for 5 min (lanes 1 and 4) or with APCs plus SEE for 2 min (lanes 2 and 5) or 5 min (lanes 3 and 6). C, Cells were incubated with medium alone for 2 min (lanes 1 and 2) or with anti-CD3 mAb (2Ad2A2) for 2 min (lanes 3 and 4). Film exposures were chosen to be in the linear range of the film. D, Cells were activated with either SEB or SEE plus APCs and treated as above except that the anti-phosphotyrosine Western blots were revealed by ECF. The densities of tyrosine-phosphorylated bands corresponding to TCR were quantitated on a Storm FluorImager. Hatched bars represent directly stimulated TCRs, whereas black bars represent unstimulated TCRs. The plot shows the average values of four independent experiments.

Phosphorylation of TCR chains of unstimulated TCR-CD3 complexes was not the result of toxin cross-reactivity, because no increase in phosphorylation was ever observed when SEE was used to activate cells expressing only Vß3 TCRs (Fig. 2A, lane 3), nor when SEB was used to stimulate cells expressing only Vß8 TCRs (Fig. 2A, lane 5). Furthermore, no cross-reactivity of anti-Vß8 mAb for Vß3 TCR (Fig. 2B, lane 4) nor of anti-Vß3 mAb for Vß8 TCR (Fig. 2B, lane 7) could be revealed. Moreover, to rule out that phosphorylation of polypeptides associated with unstimulated TCR complexes could have happened after cell lysis, we conducted the following control experiment. Cells expressing only Vß8 TCRs were mixed with equal numbers of cells expressing only Vß3 TCRs and stimulated with APCs plus SEE. The cells were then centrifuged, lysed, and TCRs immunoprecipitated as in the case of cells expressing two distinct TCRs. Under these conditions, only TCR associated with Vß8-stimulated receptors showed increased tyrosine phosphorylation (Fig. 2C, lanes 2 and 3). This indicates that phosphorylation of subunits associated with unstimulated TCR-CD3 complexes did not take place in cell lysates after receptor solubilization. In addition, the experiment ruled out the possibility that a transfer of phosphorylated TCR subunits from stimulated to nonstimulated TCR-CD3 complexes could have occurred after receptor solubilization.

View larger version (42K): [in this window] [in a new window]   FIGURE 2. Controls to rule out cross-reactivity of enterotoxins or mAbs. Cells expressing either Vß3 or Vß8 TCRs were activated and TCRs were immunoprecipitated and analyzed as in Fig. 1. A, Cells expressing only Vß3 TCR (lanes 1–3) or only Vß8 TCR (lanes 4–6) were activated with APCs alone (lanes 1 and 4), APCs plus SEB for 2 min (lanes 2 and 5), or APCs plus SEE for 2 min (lanes 3 and 6). B, Cells expressing only Vß3 TCR (lanes 1–4) or only Vß8 TCR (lanes 5–8) were activated in medium alone (lanes 1, 2, 5, and 6) or with anti-CD3 mAb (2AdA2) for 2 min (lanes 3, 4, 7, and 8). C, Equal numbers of cells expressing only Vß3 TCR or only Vß8 TCR were mixed with APCs alone (lane 1) or with APCs plus SEE for 2 min (lanes 2 and 3). The cells were lysed and immunoprecipitated as in Fig. 1.

Tyrosine-phosphorylated ZAP-70 is associated with stimulated and unstimulated TCR-CD3 complexes after superantigen activation

Tyrosine phosphorylation of TCR subunits does not always correlate with productive downstream signal transduction. Thus, partial TCR agonists induce TCR phosphorylation without ZAP-70 activation. In these cases, a different pattern of TCR phosphorylation was observed (10, 11). Our experiments showed a pattern of phosphorylated TCR in unstimulated TCR-CD3 complexes indistinguishable from that observed in stimulated ones (Fig. 1, A and B), suggesting that both types of TCR-CD3 complexes were equally involved in signal transduction. To formally prove this, we analyzed further events in TCR signaling, such as the recruitment of the protein tyrosine kinase ZAP-70 to TCR-CD3 complexes and its tyrosine phosphorylation. Cells expressing both Vß3 and Vß8 TCRs were stimulated with APCs plus either SEB or SEE, lysed at various times, and anti-Vß3 and anti-Vß8 immunoprecipitates were analyzed by anti-phosphotyrosine Western blotting. After activation of cells with bacterial superantigens, tyrosine-phosphorylated ZAP-70 was coimmunoprecipitated with stimulated as well as with unstimulated TCR-CD3 complexes (Fig. 3). The kinetics of tyrosine phosphorylation of ZAP-70 associated with stimulated and unstimulated TCR-CD3 were similar, reaching a maximum between 1 and 2 min and decreasing later. The phosphorylated ZAP-70 band associated with directly stimulated TCR-CD3 complexes was always more intense than that associated with unstimulated TCR-CD3 complexes (Fig. 3).

View larger version (31K): [in this window] [in a new window]   FIGURE 3. Tyrosine-phosphorylated ZAP-70 is associated with stimulated and unstimulated TCR-CD3 complexes after activation with SEB or SEE. Cells expressing both Vß3 and Vß8 TCRs were activated with enterotoxins, lysed, and their TCRs were immunoprecipitated with anti-Vß3 or anti-Vß8 mAbs. Immunoprecipitates were analyzed by anti-phosphotyrosine Western blotting as in Fig. 1, except that samples were separated on 8% polyacrylamide gels. A, Cells were incubated with APCs alone for 5 min (lanes 1 and 2) or with APCs plus SEB for 1 min (lanes 3 and 4) or 5 min (lanes 5 and 6). B, Cells were incubated with APCs alone for 5 min (lanes 1 and 2) or with APCs plus SEE for 1 min (lanes 3 and 4) or 5 min (lanes 5 and 6). C, Cells were activated with either SEB or SEE plus APCs and treated as described in Fig. 1 except that the anti-phosphotyrosine Western blots were revealed by ECF. The densities of tyrosine-phosphorylated bands corresponding to ZAP-70 were quantitated on a Storm FluorImager. Hatched bars represent directly stimulated TCRs, whereas black bars represent unstimulated TCRs. The plot represents the average ± SD of five independent experiments.

These data demonstrate that, after activation with enterotoxin superantigens, the protein tyrosine kinase ZAP-70 can be recruited to stimulated as well as to unstimulated TCR-CD3 complexes and becomes phosphorylated on tyrosine residues. The enzymatic activity of ZAP-70 depends on its binding to tyrosine-phosphorylated TCR and its own subsequent tyrosine phosphorylation (12). Therefore, our data indicate that ZAP-70 associated with unstimulated TCR-CD3 complexes is in its active form and may participate in signal transduction.

Stimulated and unstimulated TCR-CD3 complexes display increased protein tyrosine kinase activity after enterotoxin superantigen stimulation

Signal transduction by TCRs occurs via changes in protein tyrosine kinase activity (2). Therefore, we measured the changes in protein tyrosine kinase activity associated with stimulated and unstimulated TCR-CD3 complexes after enterotoxin superantigen stimulation. As shown in Fig. 4, both stimulated and unstimulated TCR-CD3 complexes displayed comparable increased tyrosine kinase activity after enterotoxin superantigen stimulation, as measured in vitro using an exogenous substrate. This increase in protein tyrosine kinase activity associated with TCR immunoprecipitates is likely the result of the recruitment to TCR complexes as well as the increase in specific activity of several protein tyrosine kinases involved in TCR signaling, such as ZAP-70, Lck, and Fyn (2, 3, 13).

View larger version (18K): [in this window] [in a new window]   FIGURE 4. Stimulated and unstimulated TCR-CD3 complexes display increased protein tyrosine kinase activity after enterotoxin superantigen stimulation. Cells expressing both Vß3 and Vß8 TCRs were activated with APCs plus SEB, lysed, and their TCRs were immunoprecipitated with anti-Vß3 or anti-Vß8 mAbs. Protein tyrosine kinase activity was assessed in vitro using a synthetic peptide as an exogenous substrate, as described in Materials and Methods.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
We report here that enterotoxin superantigens induce the cooperative activation of TCRs. Thus, key events in TCR signaling, such as tyrosine phosphorylation of TCR subunits, recruitment and tyrosine phosphorylation of the protein tyrosine kinase ZAP-70, and increase in protein tyrosine kinase activity, occurred not only in directly stimulated TCR-CD3 complexes, but also in a considerable amount of unstimulated ones. Therefore, our data indicate that both stimulated and unstimulated TCR-CD3 complexes are involved in enterotoxin superantigen-induced T cell signaling. This would increase the number of signal transduction molecules involved and, therefore, the magnitude of the intracellular signal.

Quantitative analysis of anti-phosphotyrosine Western blots showed that the amount of tyrosine-phosphorylated TCR associated with unstimulated receptors was 50% of that associated with stimulated receptors (Fig. 1D), whereas phosphorylated ZAP-70 associated with unstimulated TCR-CD3 complexes represented 80% of that associated with directly stimulated receptors (Fig. 3C). This suggests that the tyrosine-phosphorylated TCR subunits detected in unstimulated TCR-CD3 complexes efficiently bind tyrosine phosphorylated forms of ZAP-70 that are putatively active. Consistent with this, protein tyrosine kinase activity associated with unstimulated receptors was close to that associated with directed stimulated TCR-CD3 complexes (Fig. 4, and data not shown). Therefore, our data indicate that the superantigen-induced signals can propagate to TCR-CD3 complexes that had not bound the ligand.

The phenomena described here are likely the result of superantigen-induced interactions between directly stimulated TCRs and unstimulated ones rather than the result of a stable association between Vß3 and Vß8 subunits. Indeed, immunoprecipitation experiments did not reveal any association between Vß3 and Vß8 subunits in our cells (data not shown), consistent with previously reported data (14). Moreover, we have previously shown that enterotoxin superantigens induce the cell surface comodulation of Vß3 and Vß8 TCRs in these cells and that the extent of comodulation varied with the stimulation conditions (4). These findings suggest that interaction between several TCR complexes could be an active phenomenon induced by superantigen stimulation.

Interactions between TCRs on the lymphocyte surface appear to be an important event in T cell Ag recognition and activation (15). Several mechanisms, such as cross-linking by MHC class II dimers presenting the appropriate peptide Ag or superantigen and/or lateral interactions between TCRs and CD4 coreceptors have been proposed to favor these interactions (15, 16). The data we present here provide new insights into this process by showing that functional interactions between TCRs can take place in the absence of ligand cross-linking. In addition, coreceptors are not absolutely required for these interactions, because our cells do not express CD4 or CD8. Therefore, other mechanisms probably account for TCR interactions in our experimental model. For instance, ligand binding-specific conformational changes within the TCR-CD3 complex could facilitate interactions between TCRs (15, 17). Moreover, interactions between TCR complexes could also be favored by anchoring the receptors to cytoskeletal structures and/or by reducing receptor mobility within membrane domains (18, 19, 20). In vivo, however, these processes may not be mutually exclusive and together could modulate TCR signal transduction.

Altogether, the data described here show that TCR signaling triggered by enterotoxin superantigens can be propagated to TCR-CD3 complexes that did not interact with their ligand. This effect would increase the sensitivity of TCR signaling and may explain how low-affinity TCR ligands, such as enterotoxin superantigens (21, 22), can be efficient T cell activators, even when present in small amounts (23). Interestingly, researchers have recently proposed a model to explain the high sensitivity of bacteria to chemical attractants (24, 25). From their theoretical work, these authors proposed that upon ligand binding, the change in activity of a receptor can spread to neighboring receptors, involving them in signaling. They also proposed that this "activity spread mechanism" could be extended to other cellular receptor responses. Bacteria, as T lymphocytes, can respond to low amounts of extracellular signals, even though just a minute fraction of receptors are expected to bind the ligand. The work we present here is consistent with the activity spread mechanism model and is, to our knowledge, the first in vivo evidence that this mechanism can be used by eukaryotic cells and in particular by T lymphocytes.

	Acknowledgments

 
We thank Drs. B. Alarcón and O. Acuto for helpful discussions and reagents, Dr. M. Thome for initial advice on phosphotyrosine experiments, Drs. M. J. Owen, E. L. Reinherz, and G. Spagnoli for the generous gift of cell lines, cDNAs, and mAbs, and Drs. G. Bismuth, D. Ojcius and P. Sarthou for suggestions and comments on the manuscript.

	Footnotes

 
¹ F.N. is a recipient of a teaching and research assistantship (ATER) from the University of Paris.

² Address correspondence and reprint requests to Dr. Andrés Alcover, Biologie des Interactions Cellulaires, Institut Pasteur, 28 rue du Dr. Roux, 75724 Paris Cedex 15, France; E-mail address:

³ Abbreviations used in this paper: ITAM, Immunoreceptor tyrosine-based activation motif; SE, Staphylococcus enterotoxin; ECF, enhanced chemifluorescence; ECL, enhanced chemiluminescence.

Received for publication May 19, 1998. Accepted for publication August 7, 1998.

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Top Abstract Introduction Materials and Methods Results Discussion References

 

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