Lck-independent Triggering of T-cell Antigen Receptor Signal Transduction by Staphylococcal Enterotoxins
1997; Elsevier BV; Volume: 272; Issue: 23 Linguagem: Inglês
10.1074/jbc.272.23.14787
ISSN1083-351X
AutoresSho Yamasaki, Makoto Tachibana, Nobukata Shinohara, Makio Iwashima,
Tópico(s)T-cell and B-cell Immunology
ResumoSuperantigens (SAgs) activate T-cells in a manner specific to the Vβ 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. Superantigens (SAgs) activate T-cells in a manner specific to the Vβ 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. Staphylococcal enterotoxins are prototypic superantigens (SAgs) 1The abbreviations used are: SAgssuperantigensTCRT-cell antigen receptorITAMimmunoreceptor tyrosine-based activation motifSEEstaphylococcal enterotoxin ESEDstaphylococcal enterotoxin DPHAphytohemagglutinin that stimulate a large population of T-cells in a manner specific to the Vβ region of the T-cell antigen receptor (TCR) (1Herman A. Kappler J.W. Marrack P. Pullen A.M. Annu. Rev. Immunol. 1991; 9: 745-772Google Scholar, 2Kotzin B.L. Leung D.L.M. Kappler J. Marrack P. Adv. Immunol. 1993; 54: 99-166Google Scholar). SAgs have been implicated as a causative factor in a number of human diseases, such as toxic shock syndrome, rheumatoid arthritis, and diabetes mellitus (2Kotzin B.L. Leung D.L.M. Kappler J. Marrack P. Adv. Immunol. 1993; 54: 99-166Google Scholar, 3Conrad B. Weidmann E. Trucco G. Rudert W.A. Behboo R. Ricordi C. Rodriquez-Rilo H. Finegold D. Trucco M. Nature. 1994; 371: 351-355Google Scholar). Activation by staphylococcal enterotoxins induces rapid proliferation followed by the anergic state and programmed cell death of mature T-cells and thymocytes (1Herman A. Kappler J.W. Marrack P. Pullen A.M. Annu. Rev. Immunol. 1991; 9: 745-772Google Scholar, 2Kotzin B.L. Leung D.L.M. Kappler J. Marrack P. Adv. Immunol. 1993; 54: 99-166Google Scholar). Although T-cell activation by SAgs is distinguishably unique, the precise signal transduction mechanism has not been well characterized. superantigens T-cell antigen receptor immunoreceptor tyrosine-based activation motif staphylococcal enterotoxin E staphylococcal enterotoxin D phytohemagglutinin Previous studies demonstrated that Lck, a member of the Src family protein-tyrosine kinases, plays an essential role in the TCR signal transduction pathway (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar). The T-cell line that lacks functional Lck is defective in TCR activation (6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Google Scholar). Thymocyte differentiation is severely disturbed in mice lacking Lck expression and in mice expressing inactive forms of Lck (7Molina T.J. Kishihara K. Siderovski D.P. van Ewijk W. Narendran A. Timms E. Wakeham A. Paige C.J. Hartmann K.-U. Veillette A. Davidson D. Mak T.W. Nature. 1992; 357: 161-164Google Scholar, 8Levin S.D. Anderson S.J. Forbush K.A. Perlmutter R.M. EMBO J. 1993; 12: 1671-1680Google Scholar, 9Hashimoto K. Sohn S.J. Levin S.D. Tada T. Perlmutter R.M. Nakayama T. J. Exp. Med. 1996; 18: 931-943Google Scholar). 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 (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar, 10Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Google Scholar, 11Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Google Scholar, 12Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Google Scholar, 13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar, 14Chan A.C. Dalton M. Johnson R. Kong G.-H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Google Scholar, 15Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Google Scholar). Another Src family protein-tyrosine kinase, Fyn, has also been implicated in playing a role in TCR signal transduction (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar,5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar, 16Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Google Scholar, 17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar). However, the phenotype of T-cells from Fyn knockout mice is much less dramatic than that of Lck knockout mice (18Appleby M.W. Gross J.A. Cooke M.P. Levin S.D. Qian X. Perlmutter R.M. Cell. 1992; 70: 751-763Google Scholar, 19Stein P.L. Lee H.-M. Rich S. Soriano P. Cell. 1992; 70: 741-750Google Scholar). CD45, a receptor-type protein-tyrosine phosphatase, also plays a critical role in TCR signal transduction (20Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Google Scholar, 21Alexander D. Shiroo M. Robinson A. Biffen M. Shivnan E. Immunol. Today. 1992; 13: 477-482Google Scholar, 22Penninger J.M. Wallace V.A. Kishihara K. Mak T.W. Immunol. Rev. 1993; 135: 183-214Google Scholar). Loss of CD45 expression results in abrogation of the proximal TCR signaling process in both mature and immature T-cells (20Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Google Scholar, 21Alexander D. Shiroo M. Robinson A. Biffen M. Shivnan E. Immunol. Today. 1992; 13: 477-482Google Scholar, 22Penninger J.M. Wallace V.A. Kishihara K. Mak T.W. Immunol. Rev. 1993; 135: 183-214Google Scholar). CD45 has been shownin vitro to up-regulate Src family protein-tyrosine kinases by dephosphorylating C terminus-negative regulatory tyrosine (23Mustelin T. Coggeshall K.M. Altman A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 6302-6306Google Scholar, 24Mustelin T. Pressa-Morikawa T. Autero M. Gassman M. Anderson L.C. Gahmberg C.G. Burn P. Eur. J. Immunol. 1992; 22: 1173-1178Google Scholar). 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 (25Biffen M. McMichael-Phillips D. Larson T. Venkitaraman A. Alexandar D. EMBO J. 1994; 13: 1920-1929Google Scholar). 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. Jurkat and J.CaM1.6 (26Goldsmith M.A. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6879-6883Google Scholar), J.RT3.T3.5 (27Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Google Scholar), and J.45.01 (28Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Google Scholar) (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. The NF-AT (nuclear factor of activatedT-cells)/luciferase reporter construct (29Northrop J.P. Ullman K.S. Crabtree G.R. J. Biol. Chem. 1993; 268: 2917-2923Google Scholar) 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) (17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar) 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 (12Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Google Scholar). cDNA from the active form of Fyn was cloned into the pREP3 expression vector (Invitrogen, Carlsbad, CA). The Jurkat cell line and its mutant derivatives were transfected with the NF-AT/luciferase reporter construct as described (13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar). 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 (13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar). 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 β-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. 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 (30Ogura A. Akita K. Kudo Y. Neurosci. Res. 1990; 9: 103-113Google Scholar) using a SIT camera (C2400-08, Hamamatsu Photonics, Hamamatsu, Japan) and the Argus 50/CA system (Hamamatsu Photonics). 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 (31Irving B. Weiss A. Cell. 1991; 64: 891-901Google Scholar) with 4G10 (Upstate Biotechnology, Inc., Lake Placid, NY), anti-ζ (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 (31Irving B. Weiss A. Cell. 1991; 64: 891-901Google Scholar). 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). Jurkat cells, which express Vβ8, were previously shown to respond strongly to staphylococcal enterotoxins A, D, and E presented by Raji cells, a leukemic B-cell line (32Herman A. Croteu G. Sekaly C.L. Kappler J. Marrack P. J. Exp. Med. 1990; 172: 709-717Google Scholar). 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. 1 A, treatment with SEE plus Raji cells strongly induced the NF-AT activity of Jurkat cells. Surprisingly, J.CaM1.6 cells, which are Lck-deficient (6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Google Scholar), 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 (27Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Google Scholar), did not show any response. SED also activated J.CaM1.6 and Jurkat cells equally well (Fig. 1 B). There was no difference between Jurkat and J.CaM1.6 cells in reactivity against limited amounts of SEE (Fig.1 C). On the other hand, the anti-idiotypic antibody against Jurkat TCR, C305 (27Weiss A. Stobo J.D. J. Exp. Med. 1984; 160: 1284-1299Google Scholar), completely failed to activate J.CaM1.6 cells (Fig. 1 D). It should be noted that the CD45-deficient mutant, J.45.01 (28Koretzky G.A. Picus J. Thomas M.L. Weiss A. Nature. 1990; 346: 66-68Google Scholar), showed a greatly reduced response to SEE (Fig.1 A). Superantigen stimulation, as well as other activation through TCR, results in several changes in the cell phenotype such as expression of surface antigens (33Hamel M.E. Eynoon E.E. Savelkoul H.F.J. van Oudenaren A. Kruisbeek A.M. Int. Immunol. 1995; 7: 1065-1077Google Scholar). PHA is a strong T-cell mitogen and activates T-cells in a TCR-dependent manner (26Goldsmith M.A. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6879-6883Google Scholar). 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. 2M. Tachibana and M. Iwashima, unpublished observation. To further characterize the mechanism of SEE-induced T-cell activation, we investigated early signaling events that followed TCR engagement (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar,5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar). 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 (6Straus D.B. Weiss A. Cell. 1992; 70: 585-593Google Scholar). 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.3 A, 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-colorF340/F360 ratio image and the bright-field image confirmed that cells that show an increase in Ca2+ colocalize with the cells forming de novocell-cell complexes, presumably composed of SEE-presenting Raji cells (identified as cells not showing any fluorescence) and J.CaM1.6 cells (Fig. 3 B). 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 (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar,10Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Google Scholar, 11Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Google Scholar, 12Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Google Scholar, 13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar, 14Chan A.C. Dalton M. Johnson R. Kong G.-H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Google Scholar, 15Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Google Scholar). Recruitment of ZAP-70 to the ζ chain by SED stimulation was previously described (34Chan A.C. Irving B.A. Fraser J.D. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9166-9170Scopus (339) Google Scholar). 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. 4 A (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 (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar, 16Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Google Scholar, 17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar, 18Appleby M.W. Gross J.A. Cooke M.P. Levin S.D. Qian X. Perlmutter R.M. Cell. 1992; 70: 751-763Google Scholar, 19Stein P.L. Lee H.-M. Rich S. Soriano P. Cell. 1992; 70: 741-750Google Scholar). An increase in tyrosine phosphorylation on Fyn and the ζ chain was confirmed (Fig.4 A, upperand middle right panels). An equivalent amount of the ζ chain (data not shown) and Fyn was detected in the immunoprecipitates from unstimulated and stimulated J.CaM1.6 cells (Fig. 4 A, 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. 4 B). The functional role of Fyn was further tested by expressing the kinase-inactive form, Fyn.K299M (17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar), in J.CaM1.6 cells. As shown in Fig. 5 A, 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 (20Chan A.C. Desai D.M. Weiss A. Annu. Rev. Immunol. 1994; 12: 555-592Google Scholar, 21Alexander D. Shiroo M. Robinson A. Biffen M. Shivnan E. Immunol. Today. 1992; 13: 477-482Google Scholar, 22Penninger J.M. Wallace V.A. Kishihara K. Mak T.W. Immunol. Rev. 1993; 135: 183-214Google Scholar). Loss of CD45 expression in T-cells was associated with the increased level of tyrosine phosphorylation of Tyr-505 in Lck and Fyn (35Ostergaard H.L. Shackelford D.A. Hurley T.R. Johnson P. Hyman R. Sefton B.M. Trowbridge I.A. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 8959-8963Google Scholar, 36Sieh M. Bolen J.B. Weiss A. EMBO J. 1993; 12: 315-321Google Scholar, 37Burns C.M. Sakaguchi K. Appella E. Ashwell J.D. J. Biol. Chem. 1994; 269: 13594-13600Google Scholar). As shown in Fig. 1 A, the reactivity of J.45.01 cells against SEE is also severely impaired (Fig. 1 A). 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. 4 B; 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. 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. (38Migita K. Eguchi K. Kawabe Y. Nagataki S. Immunology. 1995; 85: 550-555Google Scholar) 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 (17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar). There are at least two roles that Lck plays in the TCR signal transduction pathway (4Weiss A. Littman D.R.. Cell. 1994; 76: 263-274Google Scholar, 5Wange R.L. Samelson L.E. Immunity. 1996; 5: 197-205Google Scholar, 10Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Google Scholar, 11Wange R.L. Malek S.N. Desiderio S. Samelson L.E. J. Biol. Chem. 1993; 268: 19797-19801Google Scholar, 12Iwashima M. Irving B.A. van Oers N.S.C. Chan A.C. Weiss A. Science. 1994; 263: 1136-1139Google Scholar, 13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar, 14Chan A.C. Dalton M. Johnson R. Kong G.-H. Wang T. Thoma R. Kurosaki T. EMBO J. 1995; 14: 2499-2508Google Scholar, 15Wange R.L. Guitian R. Isakov N. Watts J.D. Aebersold R. Samelson L.E. J. Biol. Chem. 1995; 270: 18730-18733Google Scholar). 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 (10Chan A.C. Iwashima M. Turck C.W. Weiss A. Cell. 1992; 71: 649-662Google Scholar). Fyn was found in association with the ζ chain (16Samelson L.E. Phillips A.F. Luong E.T. Klausner R.D. Proc. Natl. Acad. Sci. U. S. A. 1990; 87: 4358-4362Google Scholar), and the overexpression of Fyn induces hyperactivity of antigen-specific T-cells (17Fusaki N. Semba K. Katagiri T. Suzuki G. Matsuda S. Yamamoto T. Int. Immunol. 1994; 6: 1245-1255Google Scholar). 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 (39Groves T. Smiley P. Cooke M.P. Forbush K. Perlmutter R.M. Guidos C.J. Immunity. 1996; 5: 417-428Google Scholar, 40van Oers N.S.C. Lowin-Kropf B. Finlay D. Connolly K. Weiss A. Immunity. 1996; 5: 429-436Google Scholar). 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 β chain of TCR and staphylococcal enterotoxin B was reported (41Field B. Malchiodi E.L. Li H. Ysern X. Stauffacher C.V. Schlievert P.M. Karjalainen K. Mariuzza R.A. Nature. 1996; 384: 188-192Google Scholar). The complex formed by SAg with TCR is distinctive in structure from the complex formed by TCR with the MHC+ peptides (42Garboczi D.N. Ghosh P. Utz U. Fan Q.R. Biddison W.E. Wiley D.C. Nature. 1996; 384: 134-141Google Scholar). In addition, antibody treatment of the CD3 ε chain induced Syk activation and a Ca2+ increase in J.CaM1.6 cells (26Goldsmith M.A. Weiss A. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 6879-6883Google Scholar, 43Couture C. Baier G. Altman A. Mustelin T. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 5301-5305Google Scholar). 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 (13Yamasaki S. Takamatsu M. Iwashima M. Mol. Cell. Biol. 1996; 16: 7151-7160Google Scholar). 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 (44Pawson T. Nature. 1994; 373: 573-580Google Scholar). 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. 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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