Lck-independent Triggering of T-cell Antigen Receptor Signal Transduction by Staphylococcal Enterotoxins*

(Received for publication, March 7, 1997, and in revised form, April 7, 1997)

Sho Yamasaki Dagger §, Makoto Tachibana , Nobukata Shinohara and Makio Iwashima Dagger §par

From the Dagger  Division of Cell and Information, Precursor Research for Embryonic Science and Technology, Japan Science and Technology Corporation, Tokyo 194, § Pharmaceutical Laboratory II, Yokohama Research Center, Mitsubishi Chemical Corporation, Yokohama 227, and the  Mitsubishi Kasei Institute of Life Sciences, Tokyo 194, Japan

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES


ABSTRACT

Superantigens (SAgs) activate T-cells in a manner specific to the Vbeta region of the T-cell antigen receptor. Stimulations by SAgs provoke drastic T-cell activation that leads to programmed cell death or the anergic state of responding cells. To characterize the signal transduction pathway initiated by SAgs, mutant lines derived from the human leukemic T-cell line Jurkat were tested for their reactivities against prototypic SAgs, staphylococcal enterotoxins. The J.CaM1.6 cell line, which lacks Lck expression and lost reactivity against T-cell antigen receptor-mediated stimulation, was activated by staphylococcal enterotoxins in a manner indistinguishable from the Jurkat cell line. In contrast, the J.45.01 cell line, which lacks expression of functional CD45, showed severely impaired reactivity. The role of Lck appears to be replaced by another Src family protein-tyrosine kinase, Fyn. In J.CaM1.6 cells, Fyn was rapidly phosphorylated and activated after staphylococcal enterotoxin treatment. The kinase-inactive mutant of Fyn significantly suppressed the reactivity against staphylococcal enterotoxin E in J.CaM1.6 cells, and the expression of the active form of Fyn reconstituted reactivity against staphylococcal enterotoxin E in J.45.01 cells. These results demonstrate that SAgs activate T-cells in an Lck-independent pathway and that Fyn plays a critical role in the process.


INTRODUCTION

Staphylococcal enterotoxins are prototypic superantigens (SAgs)1 that stimulate a large population of T-cells in a manner specific to the Vbeta region of the T-cell antigen receptor (TCR) (1, 2). SAgs have been implicated as a causative factor in a number of human diseases, such as toxic shock syndrome, rheumatoid arthritis, and diabetes mellitus (2, 3). Activation by staphylococcal enterotoxins induces rapid proliferation followed by the anergic state and programmed cell death of mature T-cells and thymocytes (1, 2). Although T-cell activation by SAgs is distinguishably unique, the precise signal transduction mechanism has not been well characterized.

Previous studies demonstrated that Lck, a member of the Src family protein-tyrosine kinases, plays an essential role in the TCR signal transduction pathway (4, 5). The T-cell line that lacks functional Lck is defective in TCR activation (6). Thymocyte differentiation is severely disturbed in mice lacking Lck expression and in mice expressing inactive forms of Lck (7-9). Biochemical analysis has shown that Lck plays at least two roles in the TCR signaling pathway, namely phosphorylation of the immunoreceptor tyrosine-based activation motifs (ITAMs) to recruit ZAP-70 and activation of membrane-recruited ZAP-70 (4, 5, 10-15). Another Src family protein-tyrosine kinase, Fyn, has also been implicated in playing a role in TCR signal transduction (4, 5, 16, 17). However, the phenotype of T-cells from Fyn knockout mice is much less dramatic than that of Lck knockout mice (18, 19).

CD45, a receptor-type protein-tyrosine phosphatase, also plays a critical role in TCR signal transduction (20-22). Loss of CD45 expression results in abrogation of the proximal TCR signaling process in both mature and immature T-cells (20-22). CD45 has been shown in vitro to up-regulate Src family protein-tyrosine kinases by dephosphorylating C terminus-negative regulatory tyrosine (23, 24). Impairment of the TCR signaling pathway in CD45-deficient cell lines appears to be due to the loss of function of certain populations of Src family protein-tyrosine kinases (25).

To understand the unique characteristics of SAg-induced T-cell activation, we analyzed the signal transduction pathway triggered by staphylococcal enterotoxins using the leukemic T-cell line Jurkat and its mutant derivatives. The results demonstrate that Lck is not required for T-cell activation by SAgs. In the absence of Lck, Fyn was clearly activated by SAg stimulation. In addition, loss of CD45 severely impaired SAg-induced activation. This impairment was partly reconstituted by the expression of the active form of Fyn. Thus, in place of Lck, SAgs appear to utilize Fyn for initial events in the signal transduction pathway.


EXPERIMENTAL PROCEDURES

Cell Lines and Culture Conditions

Jurkat and J.CaM1.6 (26), J.RT3.T3.5 (27), and J.45.01 (28) (gifts from Dr. Arthur Weiss, University of California, San Francisco) cells were maintained in RPMI 1640 medium supplemented with 5% fetal calf serum, 100 µg/ml penicillin, and 100 µg/ml streptomycin.

Expression and Reporter Plasmids

The NF-AT (nuclear factor of activated T-cells)/luciferase reporter construct (29) was a gift from Dr. Gerald Crabtree (Stanford University, Stanford, CA). The expression construct for the kinase-inactive form of Fyn, Fyn.K299M (mutated at Lys-299 to Met) (17) was a gift from Dr. Noemi Fusaki (Science University of Tokyo, Chiba, Japan). For the active form of Fyn, wild-type Fyn (thymus form) cDNA (a gift from Dr. Roger Perlmutter, University of Washington, Seattle) was mutated at Tyr-525 to Phe using polymerase chain reaction-based mutagenesis as described (12). cDNA from the active form of Fyn was cloned into the pREP3 expression vector (Invitrogen, Carlsbad, CA).

Cell Transfections, Stimulations, and Luciferase Assay

The Jurkat cell line and its mutant derivatives were transfected with the NF-AT/luciferase reporter construct as described (13). After transfection, each cell line was stimulated for 10 h with Raji cells pretreated with staphylococcal enterotoxin E or D (Toxin Technology, Sarasota, FL). Cells were harvested, and the activity of NF-AT from each transfectant was determined as described (13). The maximum response of each transfectant was determined by stimulation with 1.0 µM ionomycin (Calbiochem) and 10 ng/ml phorbol 12-myristate 13-acetate (Calbiochem). For experiments with Fyn.K394M and Fyn.Y525F, J.CaM1.6 and J.45.01 cells were transfected with the expression constructs (40 µg) 10 h prior to stimulation with staphylococcal enterotoxin E (SEE)-treated Raji cells. Normalization of the transfection efficiency was performed using the cytomegalovirus promotor-based LacZ expression vector (pCR3/LacZ, Invitrogen) and the beta -galactosidase-based luminescence system (Tropix Inc., Bedford, MA). Each experiment was performed more than once with either duplicate or triplicate samples, and the representative results are given.

Fluorescent Microscopic Analysis of Intracellular Free Calcium

Cells (5 × 105) were loaded with 5 µM Fura-2/AM (Dojindo, Kumamoto, Japan) in Hanks' balanced saline solution for 30 min at 37 °C and plated on a glass-bottom microwell dish (Mattek, Ashland, MA) coated with Cell-Tack (Collaborative Research, Bedford, MA). To cells attached to the plate were added 1 × 105 Raji cells incubated with 10 ng/ml SEE (30 min at 37 °C in Hanks' balanced saline solution). The ratio of emitted fluorescence signals at 340 and 360 nm excitation (F340/F360) was monitored and analyzed as described (30) using a SIT camera (C2400-08, Hamamatsu Photonics, Hamamatsu, Japan) and the Argus 50/CA system (Hamamatsu Photonics).

Antibodies, Immunoprecipitation, Western Blot Analysis, and in Vitro Kinase Assay

J.CaM1.6 cells (1 × 108) were combined with Raji cells (1 × 107) pretreated with 100 ng/ml SEE and incubated at 37 °C for 20 min. Cells were harvested, and cell lysates were immunoprecipitated as described (31) with 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-zeta (Transduction Laboratories, Lexington, KY), and anti-Fyn (Transduction Laboratories). Each sample was analyzed by Western blotting using 4G10 or anti-Fyn, horseradish peroxidase-conjugated anti-mouse IgG (Zymed, Laboratories, Inc., South San Francisco, CA), and the ECL system (Amersham International, Buckinghamshire, United Kingdom). For in vitro kinase assay, J.CaM1.6 cells (1 × 107) were mixed with Raji cells (5 × 106) pretreated with or without 100 ng/ml SEE. After 20 min of incubation at 37 °C, cell lysates were prepared, and Fyn was immunoprecipitated. In vitro kinase assay was performed as described previously (31).

Flow Cytometric Analysis

J.CaM1.6 and Jurkat cells (1 × 106) were stimulated with 2 µg/ml phytohemagglutinin (PHA)-P (Honen, Tokyo) or 100 ng/ml SEE and Raji cells (5 × 104) for 16 h. After wash with staining buffer (Hanks' balanced saline solution containing 5% calf serum and 0.02% sodium azide), cells were stained with fluorescein isothiocyanate-conjugated anti-human CD69 (Pharmingen, San Diego, CA) in staining buffer. Samples were analyzed by flow cytometry using FACScan (Becton-Dickinson, San Jose, CA).


RESULTS

Jurkat cells, which express Vbeta 8, were previously shown to respond strongly to staphylococcal enterotoxins A, D, and E presented by Raji cells, a leukemic B-cell line (32). To analyze the detailed mechanism of T-cell activation by staphylococcal enterotoxins, NF-AT-dependent transcriptional activity was measured after stimulation. As shown in Fig. 1A, treatment with SEE plus Raji cells strongly induced the NF-AT activity of Jurkat cells. Surprisingly, J.CaM1.6 cells, which are Lck-deficient (6), showed equivalent reactivity against SEE compared with Jurkat cells. This activation by SEE is TCR-dependent since the TCR-negative Jurkat mutant, J.RT3.T3.5 (27), did not show any response. SED also activated J.CaM1.6 and Jurkat cells equally well (Fig. 1B). There was no difference between Jurkat and J.CaM1.6 cells in reactivity against limited amounts of SEE (Fig. 1C). On the other hand, the anti-idiotypic antibody against Jurkat TCR, C305 (27), completely failed to activate J.CaM1.6 cells (Fig. 1D). It should be noted that the CD45-deficient mutant, J.45.01 (28), showed a greatly reduced response to SEE (Fig. 1A).


Fig. 1. Lck-independent activation of NF-AT by SEE and SED. A, NF-AT activation by SEE in mutant lines derived from Jurkat cells. Jurkat cells and mutant derivatives were stimulated with Raji cells pretreated with SEE (100 ng/ml). Each cell line, except J.RT3.T3.5, expresses an equivalent level of TCR (not shown). SEE-induced NF-AT activity is presented as the percent activity against maximum responses induced by stimulation with phorbol 12-myristate 13-acetate plus ionomycin. The J.CaM1.6 cell line is the Lck kinase-deficient derivative of the Jurkat cell line. J.RT3.T3.5 cells lost expression of TCR due to the mutation of the TCR beta  chain. J.45.01 cells are deficient in expressing CD45 protein-tyrosine phosphatase. B, comparison of stimulatory ability of SEE and SED in J.CaM1.6 and Jurkat cells. Jurkat (open bars) and J.CaM1.6 (closed bars) cells were stimulated with Raji cells pretreated with 100 ng/ml SEE or SED. NF-AT activity in cells stimulated for 10 h was determined and is presented as the -fold increase over unstimulated Jurkat cells. C, reactivity of Jurkat and J.CaM1.6 cells against limited amounts of SEE. The sensitivity of Jurkat and J.CaM1.6 cells to SEE was tested by serially diluting SEE. NF-AT activity under each condition was determined and is presented as the percent activity of the maximum response of Jurkat cells. D, anti-receptor antibody-induced NF-AT activity in Jurkat cells, but not in J.CaM1.6 cells. Jurkat and J.CaM1.6 cells were stimulated for 10 h with serially diluted ascites of anti-TCR monoclonal antibody C305. NF-AT activity was determined and is presented as described for C.
[View Larger Version of this Image (25K GIF file)]

Superantigen stimulation, as well as other activation through TCR, results in several changes in the cell phenotype such as expression of surface antigens (33). PHA is a strong T-cell mitogen and activates T-cells in a TCR-dependent manner (26). Treatment of Jurkat cells with PHA induced a high level of CD69 expression (Fig. 2, upper left panel). In contrast, PHA stimulation of J.CaM1.6 cells resulted in minimum, if any, induction of CD69 expression. Treatment with C305 also resulted in induction of CD69 in Jurkat cells, but not in J.CaM1.6 cells (data not shown). On the contrary, SEE induced CD69 to the same level in Jurkat and J.CaM1.6 cells (Fig. 2, right panels). CD25 was also induced in both Jurkat and J.CaM1.6 cells by SEE (data not shown). In addition, programmed cell death was observed when J.CaM1.6 cells were cultured with SEE and Raji cells.2


Fig. 2. Cell-surface expression of CD69 in Jurkat and J.CaM1.6 cells stimulated with SEE or PHA. J.CaM1.6 and Jurkat cells were stimulated with PHA-P (left panels) or SEE and Raji cells (right panels) for 16 h. Stimulated (thick lines) and unstimulated (thin lines) cells were analyzed with fluorescein isothiocyanate-conjugated anti-CD69.
[View Larger Version of this Image (20K GIF file)]

To further characterize the mechanism of SEE-induced T-cell activation, we investigated early signaling events that followed TCR engagement (4, 5). First, the increase in intracellular Ca2+ levels in Jurkat and J.CaM1.6 cells was determined. It was previously shown that anti-TCR antibody (C305) treatment results in no increase in Ca2+ in J.CaM1.6 cells (6). To measure the increase in Ca2+ induced by SEE presented by Raji cells, microscopic fluorometric analysis was applied. Jurkat and J.CaM1.6 cells were compared at the single cell level with regard to the change in intracellular free Ca2+. As shown in Fig. 3A, both cell lines responded to SEE-treated Raji cells in an identical manner. An increase in Ca2+ was observed 10-15 min after the addition of Raji cells. Equivalent increases in Ca2+ levels were observed in both Jurkat and J.CaM1.6 cells. In addition, F340/F360 ratios were maintained at the maximum level over a 10-min period in both Jurkat and J.CaM1.6 cells. This demonstrates that SEE induces an increase in intracellular free Ca2+ in a manner distinctive from antibody-mediated stimulation. A comparison between the pseudo-color F340/F360 ratio image and the bright-field image confirmed that cells that show an increase in Ca2+ colocalize with the cells forming de novo cell-cell complexes, presumably composed of SEE-presenting Raji cells (identified as cells not showing any fluorescence) and J.CaM1.6 cells (Fig. 3B).


Fig. 3. Microscopic fluorometric analysis of cytoplasmic Ca2+ increase induced by SEE. A, changes in the cytoplasmic free Ca2+ concentration induced by SEE in individual Jurkat and J.CaM1.6 cells. Jurkat and J.CaM1.6 cells were loaded with Fura-2/AM and stimulated with Raji cells pretreated with SEE. Changes in the F340/F360 ratio were monitored by microscopic fluorometry. Data from five representative cells for each cell line are presented. B, de novo formation of the cell-cell complex between J.CaM1.6 and Raji cells. The bright-field images (left panels) and the pseudo-color images of the F340/F360 ratio (right panels) are from the same view. The upper panels are images taken at the beginning of measurement (0 min), and lower panels are images taken at the end of measurement (30 min). Added Raji cells are indicated by the green arrows.
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Protein tyrosine phosphorylation is an essential step in TCR signal transduction. Lck plays a major role in phosphorylating two tyrosines in ITAMs, recruiting ZAP-70, and initiating downstream events (4, 5, 10-15). Recruitment of ZAP-70 to the zeta  chain by SED stimulation was previously described (34). Thus, it was likely that staphylococcal enterotoxins activate T-cells through a pathway similar to that of antigenic stimulation via ITAM tyrosine phosphorylation and that a kinase other than Lck plays a major role in the process. As shown in Fig. 4A (left panel), SEE stimulation induced tyrosine phosphorylation in various cellular proteins. Fyn, an Src family protein-tyrosine kinase that associates with the TCR complex, was previously indicated as playing a role in the TCR signal transduction pathway (4, 5, 16-19). An increase in tyrosine phosphorylation on Fyn and the zeta  chain was confirmed (Fig. 4A, upper and middle right panels). An equivalent amount of the zeta  chain (data not shown) and Fyn was detected in the immunoprecipitates from unstimulated and stimulated J.CaM1.6 cells (Fig. 4A, lower right panel). Moreover, the in vitro kinase assay of anti-Fyn immunoprecipitates showed strongly induced kinase activity in SEE-activated J.CaM1.6 cells (Fig. 4B).


Fig. 4. Induction of tyrosine phosphorylation and Fyn kinase activity in J.CaM1.6 cells following stimulation with SEE. A, Western blot analysis of protein tyrosine phosphorylation in J.CaM1.6 cells treated with SEE and Raji cells. Immunoprecipitates (IP; antibodies used were anti-phosphotyrosine (anti-pY; left panel), anti-zeta (upper right panel), and anti-Fyn (middle right panel)) from unstimulated (-) and SEE-stimulated (+) samples were analyzed by Western blotting using anti-phosphotyrosine monoclonal antibody 4G10. Equivalent amounts of the zeta  chain (not shown) and Fyn (lower right panel) were detected for each immunoprecipitate. B, increase in the in vitro kinase activity of Fyn immunoprecipitated from SEE-stimulated J.CaM1.6 cells. Fyn was immunoprecipitated from unstimulated (-) or SEE-stimulated (+) J.CaM1.6 cells, and the in vitro kinase activity of autophosphorylation was determined.
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The functional role of Fyn was further tested by expressing the kinase-inactive form, Fyn.K299M (17), in J.CaM1.6 cells. As shown in Fig. 5A, the expression of Fyn.K299M in J.CaM1.6 cells significantly suppressed NF-AT activation induced by differing doses of SEE. This shows that Fyn.K299M functions in a dominant-negative manner in the signal transduction pathway activated by SEE. The role of Fyn was also tested using J.45.01 cells. Previously, it was shown that the loss of CD45 expression leads to greatly impaired reactivity of T-cells to anti-TCR antibody stimulation (20-22). Loss of CD45 expression in T-cells was associated with the increased level of tyrosine phosphorylation of Tyr-505 in Lck and Fyn (35-37). As shown in Fig. 1A, the reactivity of J.45.01 cells against SEE is also severely impaired (Fig. 1A). When the active form of Fyn (Tyr-528 to Phe mutation in Fyn) was expressed in J.45.01 cells, NF-AT activation by SEE was enhanced to a level equivalent to that in Jurkat cells (Fig. 4B; also see Fig. 1). In contrast, no enhancement was observed when wild-type Fyn was expressed, indicating that Tyr-528 of Fyn plays a critical function in relation to the function of CD45.


Fig. 5. NF-AT activation by SEE in cells expressing mutant forms of Fyn. A, SEE-induced NF-AT activation in J.CaM1.6 cells transfected with the expression construct for kinase-inactive Fyn. J.CaM1.6 cells was transiently transfected with the expression construct for the kinase-inactive form of Fyn and stimulated with various amounts of SEE. NF-AT activity was determined and is presented as the -fold induction over the unstimulated mock transfectant. B, NF-AT activation in J.45.01 cells expressing the active form of Fyn. J.45.01 cells were transfected with the expression construct for no insert (mock), wild-type Fyn, and the active form of Fyn, Fyn.Y528F, along with the NF-AT/luciferase reporter construct. Transfectants were stimulated with SEE (100 ng/ml) with Raji cells as describe for A, and NF-AT activity was determined.
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DISCUSSION

Our results presented here demonstrate that SAgs activate T-cells in an Lck-independent manner and that Fyn may play a critical role in the signaling process. Two other reports support this. Migita et al. (38) demonstrated that staphylococcal enterotoxin B activates Fyn, but not Lck, in mouse splenic T-cells in vivo. In Fyn knockout mice, the proliferative response of splenocytes to staphylococcal enterotoxin B was substantially impaired (17). There are at least two roles that Lck plays in the TCR signal transduction pathway (4, 5, 10-15). One is tyrosine phosphorylation of ITAMs in the TCR complex to create binding sites for ZAP-70, and the other is activation of ZAP-70 by tyrosine phosphorylation. It was previously demonstrated in a heterologous system using COS cells that Fyn and Lck induce tyrosine phosphorylation of ITAMs and activation of ZAP-70 at an equivalent efficiency (10). Fyn was found in association with the zeta  chain (16), and the overexpression of Fyn induces hyperactivity of antigen-specific T-cells (17). Thus, it is likely that SAg stimulation leads to activation of Fyn in the absence of Lck and initiates the signaling process. Recent reports also indicate the functional redundancy of Fyn and Lck in T-cell development (39, 40).

It is still unclear, however, why SAgs, but not other TCR stimulators, activate T-cells independently of Lck. This is not due to the strength of SAg-induced activation since limited amounts of SEE activated J.CaM1.6 cells in an identical manner to Jurkat cells, whereas anti-TCR antibody and PHA completely failed to activate J.CaM1.6 cells. Thus, the differences between SAg stimulation and other stimulation derive from the qualitative differences in the manner in which TCR is triggered. One possibility is that the activation by SAgs involves a T-cell surface molecule other than TCR. Such a coreceptor could function in activating Fyn only in case of SAg stimulation. Another possibility is that SAgs induce a unique conformational change in the TCR complex so that Fyn can be activated. Recently, the crystal structure of a complex between the beta  chain of TCR and staphylococcal enterotoxin B was reported (41). The complex formed by SAg with TCR is distinctive in structure from the complex formed by TCR with the MHC+ peptides (42). In addition, antibody treatment of the CD3 epsilon chain induced Syk activation and a Ca2+ increase in J.CaM1.6 cells (26, 43). Thus, Fyn may be activated by the structural alteration within the TCR complex by SAg stimulation.

Finally, the biological responses that are unique to SAg activation may be contributed by the involvement of Fyn in its signal transduction process. A difference in the substrate specificity of Lck and Fyn is a factor that could contribute to the biological consequences. In addition, we have recently shown that the SH2 and SH3 domains of Lck play critical roles in T-cell activation initiated by membrane-localized ZAP-70 (13). It is likely that the SH2 and SH3 domains of Fyn also play roles in the signaling process initiated by SAg stimulation. These domains of Lck and Fyn may have different repertoires of targets in vivo (44). This implies that there can be differing molecules that are involved in the distal events of the signal transduction pathway when Fyn instead of Lck is involved. In such cases, the molecules associated specifically with the SH2 and SH3 domains of Fyn could contribute to the unique biological consequences provoked by SAg stimulation.


FOOTNOTES

*   The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
par    To whom correspondence should be addressed: Mitsubishi Kasei Inst. of Life Sciences, 11 Minamiooya, Machida, Tokyo 194, Japan. Tel./Fax: 81-427-25-8191; E-mail: makio{at}libra.ls.m-kagaku.co.jp.
1   The abbreviations used are: SAgs, superantigens; TCR, T-cell antigen receptor; ITAM, immunoreceptor tyrosine-based activation motif; SEE, staphylococcal enterotoxin E; SED, staphylococcal enterotoxin D; PHA, phytohemagglutinin.
2   M. Tachibana and M. Iwashima, unpublished observation.

ACKNOWLEDGEMENTS

We are very grateful to Gerald Crabtree, Noemi Fusaki, Roger Perlmutter, and Arthur Weiss for reagents; to Masako Takamatsu and Yi-Ying Huang for excellent technical assistance; to Hideyoshi Higashi for operation of the Argus system; to Fumie Sahira for secretarial assistance; and to Makoto Iwata and Julie Hambleton for critical reading of the manuscript. We also are grateful to Ko Okumura, Minoru Muramatsu, and Hiroto Hara for support.


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