Immunology 1991 73 433-437
ADONIS
001928059100177H
Does staphylococcal enterotoxin B bind directly to murine T cells? W. QASIM,t M. A. KEHOEt & J. H. ROBINSONt Departments of *Immunology and tMicrobiology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne Acceptedfor publication I May 1991
SUMMARY We have investigated the binding potential of staphylococcal enterotoxin B (SEB) to murine T cells using the induction of early activation events in Th I and Th2 T-cell clones in the absence of antigenpresenting cells (APC) as indicators of direct interactions between SEB and the T-cell receptor (TcR). We consistently found that concanavalin A (Con A) induced rises in intracellular free calcium as well as inostitol phosphate accumulation in APC-free T-cell clones. However, SEB uniformly failed to induce either calcium fluxes or inositol phosphate turnover in Th I and Th2 T-cell clones in the absence of APC. In addition, we have used proliferation assays to show that (i) T-cell clones prepulsed with SEB did not respond when APC were added, (ii) APC-independent T-cell clones responded to soluble anti-TcR antibodies but not to SEB in the absence of APC, and (iii) SEB coupled to Sepharose beads did not stimulate T-cell clones in the absence ofAPC. Taken together our results argue against SEB binding to the TcR without the participation of MHC class II molecules.
INTRODUCTION Staphylococcal enterotoxins (SE) are a group of bacterial exotoxins which polyclonally activate T cells in a major histocompatibility complex (MHC) class II dependent, but haplotype unrestricted, manner.",2 SE bind specifically to MHC class II molecules,34 selectively activating T cells bearing particular T-cell receptor (TcR) Vfl segments,',5 and MHC class II-dependent binding of SE to purified TcR Vf chains has been reported recently.6 Mitogenic activity thus depends on functional bivalency, as SE must interact with both MHC class II and TcR molecules to stimulate a T-cell response. However, radiolabelled SE bind MHC class IT-bearing antigen-presenting cells (APC), but not to pure T-cell populations,3 arguing against direct binding of SE to the TcR. MHC class IT-independent Tcell binding can be inferred from the demonstration that prepulsing of murine T-cell clones with large doses of staphylococcal enterotoxin B (SEB) followed by thorough washing and the addition of APC, leads to T-cell proliferation.7 Furthermore, these workers reported that one T-cell clone, which responded by interleukin-2 (IL-2) production to soluble anti-V#8 antibody, also responded to SEB in the absence of APC,7 suggesting that MHC class II was not necessary for SEB binding to the TcR in this particular clone. T-cell proliferation normally requires accessory cell-derived co-stimulatory signals in addition to
occupancy of the TcR,8 so that MHC class II dependence of proliferation to SE is a complex issue. However, it has been shown that under conditions which exclude APC contamination, staphylococcal enterotoxin A (SEA) does not induce proliferation in human T cells in combination with phorbol esters, which are known to bypass co-stimulatory factor requirements.9 The induction of early activation events in homogeneous Tcell populations offers a more direct means of assessing the ability of SE to interact with the TcR. It has been reported that SE interact directly with human T cells and trigger rises in intracellular free calcium, representative of early T-cell activation.9'10 However, human T cells express MHC class II molecules so that the question of whether SE bind to the TcR in the absence of MHC class II could not be addressed. There are no similar reports in the literature utilizing murine T cells, which do not express MHC class II molecules, apart from the demonstration that toxic shock syndrome toxin (TSST- 1) induces inositol phosphate accumulation in T cells in the presence of APC." Direct binding of SE to T cells is an issue central to the understanding of the mechanism of action of bacterial superantigens. We examine here the binding potential of SEB to murine Th 1 and Th2 T-cell clones in the absence of APC using the detection of intracellular calcium transients and inositol phosphate accumulation as indicators of direct interactions between SEB and T cells. In addition, proliferation assays were used to re-evaluate whether T-cell clones pre-pulsed with SEB responded when APC were added, as well as to determine whether soluble SEB or SEB-coated Sepharose beads activated APC-independent T-cell clones.
Abbreviations: SE, staphylococcal enterotoxins; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B. Correspondence: Dr J. H. Robinson, Dept. of Immunology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
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MATERIALS AND METHODS Mice and T-cell clones BALB/c, CBA.Ca and C57BL/6 mice were obtained from the Comparative Biology Centre, University of Newcastle Upon Tyne, U.K., and used at 6-10 weeks of age. The SEB-reactive Tcell clones investigated were as follows: DIO.G4, Vf8+, specific for conalbumin/Ak (a gift from Dr G. G. B. Klaus, National Institute for Medical Research, London, U.K.); Ni 1, Vf3 +, specific for ovalbumin/Ab; 6D, Vf8+, specific for ovalbumin/ Ab; R28, Vfl8+, specific for group A streptococcal type 5 M protein (M5)/Ad; QI, Vfi8+, specific for MS/Ak; and Q3, VB8+, specific for M5/Ak. All clones except D1O.G4 were generated by us (J. H. Robinson et al., manuscript submitted for publication). Clones DlO.G4 and Nil secreted IL-4 but not IL-2 on stimulation and were considered to be of the Th2 type, whereas clones R28, Q1, Q3 and 6D were designated as ThI type based upon secretion of IL-2 but not IL-4. During maintenance of Tcell clones, T-cell blasts were purified by one-step density centrifugation using Histopaque (Sigma Chemical Co., Poole, Dorset, U.K.) 3 days after every cycle of restimulation with antigen and irradiated spleen cells.
Stimulants SEB (Sigma Chemical Co.) was purified by isoelectric focusing to greater than 95% purity as judged by SDS-PAGE and stored at 10-4 M in phosphate-buffered saline (PBS) as aliquots at -80°. Concanavalin A (Con A; Sigma Chemical Co.) was dissolved in PBS and stored as aliquots at -80°. The monoclonal antibodies F23. 1 (anti-VP8), KJ.25 (anti-Vf3), and 145.2C II (anti-CD3) were concentrated by saturated ammonium sulphate precipitation of culture supernatants from antibody-secreting hybridomas, obtained from the American Type Culture Collection, Rockville, MA, and stored at -80°.
Coupling SEB to Sepharose beads CnBr-activated Sepharose-4B gel filtration medium (Sigma Chemical Co.) was washed and swollen for 15 min at 240 in I M HCI in a sintered glass funnel, and then washed in coupling buffer (0.1 M NaHCO3, 0.5 M NaCI, pH 8.3). Five milligrams of SEB in I ml of gel were mixed for 2 hr at 240, and washed twice by centrifugation. One mole of ethanolamine (Sigma Chemical Co.) was added and mixed for I hr at 240 and then 30 min at 40 to block remaining activated groups. The gel was then washed three times alternately in acetate buffer (pH 4-0) and Tris buffer (pH 8-0), and stored in Tris buffer (pH 7-4) at 4°. Measurement of intracellular calcium T cells were loaded for I hr at 370 with the calcium-specific dualwavelength fluorescent dye, Indo-I AM (8 Mm; Molecular Probes Inc., Eugene, OR).'2 107 loaded cells were resuspended in 800 p1 of phenol red-free HBSS (Gibco Ltd, Paisley, Renfrewshire, U.K.) containing 10 mm HEPES buffer (Gibco Ltd) in a quartz cuvette at 370 for analysis using a MPF-3 Perkin-Elmer fluorescence spectrophotometer. Excitation was at 356 nm and emissions were measured at 396 nm (calcium-bound Indo-1 AM) and 460 nm (calcium-free Indo- I AM) to allow calculation of flourescence ratio (R). Rmax was obtained by saturation of the intracellular compartment with calcium using the calcium ionophore Ionomycin (1 Mg/ml; Calbiochem Corp., San Diego, CA) and Rmin was calculated after cellular lysis and chelation of free calcium in a final concentration of 12 mm EGTA (BDH
8
g 460
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Figure 1. Measurement of intracellular free calcium. Fluorescence trace measured at 396 nm against time of 107 T-cell clone NIl cells, loaded with 8 jM Indo-l AM. The trace was interrupted during addition of Ig/ sequential doses of SEB (1 nM = 28 ng/ml) or 100 yM Con A (=10 ml), and on each plateau the emission wavelength was momentarily changed to 460 nm to allow subsequent calculation of fluorescence ratio (R).
Chemicals Ltd, Poole, Dorset, U.K.). Stimulants were added in
4-pl volumes. Absolute calcium concentrations ([Ca2+]i) were calculated from the observed fluorescence (F) by: Ca2+ = Kd x [F- Fmin)/((Fmax- F), where Kd=250 nM.1213 Measurement of inositol phosphates T-cell clones were saturated with myo [3H]inositol (Amersham International plc, Amersham, Bucks, U.K.) by incubation for 18 hr at 107 T cells/ml with 40 ,Ci/ml of label in HBSS buffered with 10 mm HEPES and supplemented with 10% vol/vol dialysed FCS (Sigma Chemical Co.). Analysis in triplicate, using 3 x 106 cells per sample, was carried out in RPMI-1640 (Gibco Ltd) supplemented with 5% vol/vol FCS and 10 mm lithium chloride to prevent recycling of the inositol phosphates. Stimulation was terminated after I hr by the addition of a 2: 1 vol/vol mixture of methanol and chloroform and the aqueous phase was separated after addition of chloroform and distilled water. Total inositol phosphates were measured by combined phase scintillation counting after extraction by anion-exchange chromatography (Dowex-l, AGI x 8; Sigma Chemical Co.).'4 Results are expressed as c.p.m. + SEM. The B-lymphoblastoid cell line A20 (American Type Culture Collection), untreated or pre-pulsed with 10-6 M SEB I hr at 37°, was used as APC where indicated.
Proliferation assays 2 x 104 cloned T cells were cultured for 48 hr in RPMI-1640 (Gibco Ltd) supplemented to 3 mM L-glutamine and 10% FCS vol/vol in flat-bottomed 96-well microtitre plates in the presence of stimuli, with or without 7-5 x 105 irridiated (20 Gy) syngeneic
spleen cells as APC. Proliferation was assessed by uptake of [3H]thymidine (Amersham International) during the final 4 hr. Cells were harvested and radioactivity was quantified by liquid scintillation spectroscopy. Results are expressed as mean d.p.m. of triplicates + SEM. In pre-pulsing experiments, 106 cloned T cells were incubated with 10-6 M SEB in I ml of culture medium for I hr at 370 before washing in 4 x 10 ml of culture medium prior to the proliferation assay. RESULTS Calcium transients and inositol phosphate accumulation in Thl and Th2 T-cell clones In preliminary experiments all T-cell clones used in this study were shown to be SEB responsive in proliferation assays. In the
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SEB and activation signals in murine T cells None SEB
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EGTA 0
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Figure 2. Fluorescence ratio stimulated with SEB. The mean fluorescence ratio+ SEM for five experiments is shown. In each cuvette, 107 clone N 1 cells were sequentially stimulated with 1000 nm SEB, 100 nM Con A, 1 ug/ml Ionomycin (Iono) and 12 mm EGTA. Mean [Ca]2+]I +SEM: no stimulus (None), 113 + 10 nM; SEB, 130+9 nm; and Con A 686 + 115 nm. See the Materials and Methods for calculation of R and
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Figure 3. Measurement of inositol phosphate accumulation. 3 x 106 clone DI O.G4 cells, labelled with myo [3HJinositol, were incubated for I hr with (a) Con A in the absence of APC, (b) dilutions of F23. 1 antibody or (c) SEB in the presence (-) or absence (0) of 106 A20 cells as APC. Results are expressed as mean c.p.m. of triplicate cultures + SEM.
Clone 6D Sol SEB/APC APC Clone alone Clone D1O.G4 Sol SEB/APC Antigen/APC APC Clone alone
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D.p.m. Figure 4. Proliferation response of SEB pre-pulsed T-cell clones. T-cell clones 6D and DlO.G4 were pre-pulsed with 10-6 M SEB for 1 hr and washed four times in 10-ml volumes. Proliferation responses of prepulsed (-) and untreated cloned T cells (0) were compared. Mean d.p.m. +SEM of 104 cloned cells, in the presence (APC) or absence (Clone alone) of 7-5 x 105 non-pulsed irradiated spleen cells are shown. Proliferation of cloned T cells with 1O-7 M soluble SEB and APC (Sol SEB/APC) was included to show that the clone remained SEBresponsive after treatment.
absence of APC, SEB failed to induce rises in intracellular free calcium over the dose range of SEB which stimulated APCdependent proliferation in T-cell clones (Fig. 1). However, subsequent addition of Con A induced calcium signals in the same cells. The fluorescence ratio, measured at 396 nm and 460 nm, for five experiments is plotted for clone Nl 1 in Fig. 2. In addition, the anti-V#3 antibody KJ.25 stimulated a rise in calcium in clone Nl 1 without APC (R = 0-87 compared to a basal ratio of 0-61). Similar results were obtained for the Th2 clone DIO.G4 and the ThI clone 6D. The small rises seen after SEB treatment were not greater than changes induced by the addition of medium alone followed by agitation of the sample. It is possible that if SEB bound T cells directly without reaching the threshold necessary for inducing calcium signals, then steric hindrance could prevent binding by other stimulants. However, pretreatment with SEB failed to inhibit subsequent induction of calcium signals in cloned Ni 1 cells by the antibodies KJ.25, F23. 1 or 2CI I (data not shown). Attempts to measure calcium responses in the presence of APC were unsuccessful due to the effects of dye leakage over the extended period of incubation required for cell-cell interactions to occur. However, SEB was shown to induce inositol phosphate accumulation in the presence of APC, but failed to do so when APC were omitted. Figure 3a illustrates the dose-dependent increase in inositol phosphate accumulation in the Th2 clone DIO.G4 stimulated with Con A. Antibodies against Vfl8 (F23.1) also induced significant inositol phosphate turnover in the absence of APC (Fig. 3b), whereas SEB only elicited a response in the presence of APC and at higher doses of toxin (Fig. 3c). Similar results were obtained for the Thl clones, QI and Q3. Proliferation of SEB pre-pulsed T-cell clones and APC-independent T-cell clones
Preliminary experiments showed that spleen cells pre-pulsed with 10-6 M SEB for 1 hr at 370 induced proliferation in SEBreactive T-cell clones (data not shown). Pre-pulsing of Thl or Th2 T-cell clones with SEB at this dose in the absence of APC
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W. Qasim, M. A. Kehoe & J. H. Robinson
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Figure 5. Proliferation responses of APC-independent T-cell clone. 2 x 104 cloned QI cells were cultured for 48 hr in the presence (a) or absence (b) of 7 5 x 105 irradiated syngeneic spleen cells as APC, with the doses of SEB shown, 40 nm Con A or a 1:200 dilution of the antibodies F23.1 (anti-Vfl8), 2C 1I (anti-CD3) or KJ.25 (anti-Vfl3). Results are plotted as mean d.p.m. of triplicate cultures+ SEM.
followed by extensive washing did not lead to stimulation of proliferation upon subsequent culture with non-pulsed APC (Fig. 4). Extending the pre-pulse time to 3 hr or increasing the SEB dose to 10-5 M gave essentially the same results (data not shown). APC-independent T-cell clones were also used to investigate the direct binding potential of SEB to T cells uncomplicated by the requirement for APC-derived co-stimulatory factors. The Tcell clones Qi, Q3 and D1O.G4 proliferated in response to soluble antibodies directed against the Vf8 domain of the TcR (F23.1) or CD3 (2C1 1) in the absence of APC (results for the V/8+ clone Q1 are shown in Fig. 5b). Responses were considerably enhanced by the addition of irradiated syngeneic spleen cells (Fig. 5a). However, SEB failed to induce proliferation unless APC were present (Fig. 5). Similar results were obtained for clones Q3 and D10.G4. To evaluate whether the role of MHC class II on the APC surface was to present an array of SEB molecules to TcR on the T-cell surface, SEB was crosslinked by coupling to Sepharose beads. SEB-Sepharose, washed well before addition to the proliferation assay to remove any residual soluble SEB, stimulated the SEB-responsive T-cell clone R28 in the presence of APC, but not in their absence (Fig. 6). Similar results were obtained for the APC-independent T-cell clones Q1 and Q3. DISCUSSION Stimulation of proliferation in most T cells by specific antigens, superantigens, such as SE, and monoclonal antibodies against T-cell surface molecules, including CD3 and the TcR, requires APC or accessory cells to present the stimulus and/or to provide
40,0001 (b)
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Figure 6. Proliferation response of T-cell clone to SEB-Sepharose. 2 x 104 cloned R28 cells were cultured for 48 hr in the presence (A) or absence (A) of 7 5 x I0O irradiated syngeneic spleen cells as APC, and (a) concentrations of soluble SEB or (b) dilutions of SEB-Sepharose shown. Results are plotted as mean d.p.m. of triplicate cultures + SEM.
co-stimulatory activity. Specific antigens are processed to peptide epitopes and presented to T cells in association with selfMHC class II molecules on the surface of APC.'5 Superantigens do not require processing, but bind directly to MHC class II molecules, and activate T cells expressing selected TcR VB segments, independent of their MHC restriction phenotype.' However, it is not clear if SE bind to MHC class II and TcR independently, or alternatively whether MHC class II binding of SE is a pre-requisite step to T-cell triggering. Direct binding to the TcR or CD3 by monoclonal antibodies, in the absence of APC, is normally unable to stimulate proliferation in T cells,'6 but induces early activation signals such as increases in free intracellular calcium or accumulation of inositol phosphates.8 Thus, we measured early activation events in murine T-cell clones, as a more direct method than assessment of proliferation, to investigate whether SEB binds to the TcR in the absence of MHC class II. It has been reported that SE trigger rises in intracellular free calcium in human T cells,9"0 but as human T cells express MHC class II molecules, the question of whether SE bind to the TcR in the absence of MHC class II could not be addressed. The present report is the first studying induction of early activation signals in murine T cells by SE. The results show that Con A or anti-TcR antibodies induced calcium signals and/or inositol phosphate turnover in murine Th I and Th2 cell clones, but that SEB failed to do so, in the absence of APC. However, higher doses of SEB stimulated inositol phosphate turnover in the presence of APC, indicating that SEB was unable to initiate early activation events in the absence of MHC class II bearing APC. Both Th I and Th2 T-cell clones were used because of reported differences in their signalling requirements.'7 Our results are consistent with the demonstration that high doses of a related toxin, TSST-1, induces inositol phosphate accumulation in the presence of APC," although the
SEB and activation signals in murine T cells requirement for MHC class II bearing APC was not addressed in this report. In the absence of any data to show binding of radiolabelled SE to T cells,3'0 the concept of direct binding of SE to murine T cells is based on pre-pulsing experiments.2'7 We were unable to confirm these findings, as we have shown that pre-pulsing T-cell clones with high doses of SEB, followed by extensive washing, did not stimulate proliferation upon addition of APC. The prepulsing protocols employed similar doses of SEB but different T-cell clones were used. It is also possible that the discrepancy in the results is due to residual APC in the cloned T-cell populations of Yagi et al.,7 as we purified T-cell blasts by density centrifugation after every cycle restimulation with antigen and spleen cells. In addition, we have shown that SEB, in contrast to anti-Vfi8 antibodies, failed to stimulate three APC-independent T-cell clones to proliferate in the absence of APC, whereas these clones proliferated to SEB in the presence of syngeneic irradiated splenocytes as APC, indicating that SEB does not bind to the TcR of these clones with sufficient affinity to induce proliferation. Yagi et al.7 reported one clone which responded to SEB in the absence of APC, suggesting that the TcR of rare clones could bind SEB, but that this was not a general phenomenon. Finally, we evaluated whether the role of MHC class II on the APC surface was to present an array of SEB molecules to TcR, by attempting to mimic the array by coupling SEB to Sepharose beads. This approach has been used successfully to induce proliferation in T-cell clones by antibodies against CD3 or the TcR, when soluble antibodies were ineffective.'6 However, we have shown that SEB-Sepharose failed to activate T-cell clones, including APC-independent clones, in the absence of MHC class II bearing APC, indicating that MHC class II plays a more complex role than simply presenting SEB in a way which cross-links TcR on the T-cell surface. It was surprising that SEB coupled to Sepharose could mediate the interaction between TcR and MHC class II, given the large size of the beads, and it is possible that APC remove SEB from the beads prior to MHC class II binding, or that coupling to beads allows SEB to deliver a signal directly through the TcR but that APC are required for the delivery of an additional signal independent of surface MHC class II. We are further investigating the role of SEB coupled to beads in T-cell triggering, and have preliminary data to show that SEB-Sepharose does not induce inositol phosphate turnover in T-cell clones, including APC-independent clones, in the absence of APC. In conclusion, five separate lines of investigation in this study have failed to provide any evidence to suggest that SEB binds the TcR of murine T cells in the absence of MHC class II. We cannot, however, exclude the possibility that binding occurred, but that the affinity was too low to have any functional consequences. Our results are consistent with the model of T-cell activation by SE proposed by Janeway et al.,2 in which intact superantigens cross-link MHC class II and the TcR. However, the results described here argue against SEB binding independently to the TcR, and we postulate that SEB first interacts with MHC class II, which induces a conformational change in SEB necessary for subsequent TcR binding. ACKNOWLEDGMENTS The authors wish to thank Marian Atherton and Gwyn Pyle for skillful assistance with cell culture, Dr G. G. B. Klaus, National Institute for Medical Research, Mill Hill, U.K. for valuable guidance on assays for
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measuring intracellular calcium and inositol phosphates as well as for the gift of DlO.G4 cells, and Dr Richard Virden, Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K., for access to the fluorescence spectrophotometer. This research was funded by U.K. MOD and a Nuffield Foundation Student Bursary to W. Qasim.
REFERENCES 1. MARRACK P. & KAPPLER J.W. (1990) The staphylococcal enterotoxins and their relatives. Science, 2A4, 705. 2. JANEWAY C.A., YAGI J., CONRAD P.J., KATZ M.E., JONES B., VROEGOP S. & BUXSER S. (1989) T cell responses to Mls and to bacterial proteins that mimic its behavior. Immunol. Rev. 107, 61. 3. FRASER J.D. (1989) High affinity binding of staphylococcal enterotoxin A and B to HLA-DR. Nature, 339,221. 4. SCHOLL P., DIEZ A., KARR R., SEKALY R., TROWSDALE J. & GEHA, R.S. (1990) Effect of isotypes and allelic polymorphism on the binding of staphylococcal exotoxins to MHC class II molecules. J. Immunol. 144, 226. 5. WHITE J., HERMAN A., PULLEN A.M., KuBo R., KAPPLER J. W. & MARRACK P. (1989) The VPi-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell, 56, 27. 6. GASCOIGNE N.R.J. & AMEs K.T. (1991) Direct binding of secreted T-cell receptor P chain to superantigen associated with class II major histocompatibility complex protein. Proc. natl. Acad. Sci. U.S.A. 88, 613. 7. YAGI J., BARON J., BUXSER S. & JANEWAY C.A. (1990) Bacterial proteins that mediate the association of a defined subset of T cell receptor: CD4 complexes with class II MHC. J. Immunol. 144, 892. 8. MUELLER D.L., JENKINS M.K. & SCHWARTZ R.H. (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol. 7,445. 9. FLEISCHER B. & SCHREZENEMEIER H. (1988) T cell stimulation by staphylococcal enterotoxins. J. exp. Med. 167, 1697. 10. FLEISCHER B., SCHREZENEMEIER H. & CONRADT P. (1989) T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures. Cell. Immunol. 120, 92. 11. NORTON S.D., SCHLIEVERT P.M., NOVICK R.P. & JENKINS M.J. (1990) Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-I. J. Immunol. 144, 2089. 12. GRYNKIEWICZ G., POENIE M. & TsIEN R.T. (1985) A new generation of Ca2 + indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440. 13. BISTERBOSCH M.K., RIGLEY K.P. & KLAUS G.G.B. (1985) Cross linking of surface immunoglobulin on B lymphocytes induces both intra-cellular Ca2+ release and Ca2+ influx: analysis with Indo-l. Biochem. Biophys. Res. Comm. 137, 500. 14. BERRIDGE M.J., DAWSON R.M.C., DOWNES C.P., HESLOP J.P. & IRVINE R.F. (1983) Changes in the levels ofinositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem. J. 212, 473. 15. SCHWARTZ R.H. (1985) T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann. Rev. Immunol. 3, 237. 16. MEUER S.C., HODGDON J.C., HUSSEY R.E., PROTENTIS J.P., SCHLOSSMAN S.F. & REINHERZ E.L. (1983) Antigen like effects of monoclonal antibodies directed at receptors on human T cell clones. J. exp. Med. 158,988. 17. GAJEWSKY T.F., SCHELL R.S., NAU G. & FITCH F.W. (1989) Regulation of T cell activation: differences among T cell subsets. Immunol. Rev. 111, 79.
ADONIS
001928059100177H
Does staphylococcal enterotoxin B bind directly to murine T cells? W. QASIM,t M. A. KEHOEt & J. H. ROBINSONt Departments of *Immunology and tMicrobiology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne Acceptedfor publication I May 1991
SUMMARY We have investigated the binding potential of staphylococcal enterotoxin B (SEB) to murine T cells using the induction of early activation events in Th I and Th2 T-cell clones in the absence of antigenpresenting cells (APC) as indicators of direct interactions between SEB and the T-cell receptor (TcR). We consistently found that concanavalin A (Con A) induced rises in intracellular free calcium as well as inostitol phosphate accumulation in APC-free T-cell clones. However, SEB uniformly failed to induce either calcium fluxes or inositol phosphate turnover in Th I and Th2 T-cell clones in the absence of APC. In addition, we have used proliferation assays to show that (i) T-cell clones prepulsed with SEB did not respond when APC were added, (ii) APC-independent T-cell clones responded to soluble anti-TcR antibodies but not to SEB in the absence of APC, and (iii) SEB coupled to Sepharose beads did not stimulate T-cell clones in the absence ofAPC. Taken together our results argue against SEB binding to the TcR without the participation of MHC class II molecules.
INTRODUCTION Staphylococcal enterotoxins (SE) are a group of bacterial exotoxins which polyclonally activate T cells in a major histocompatibility complex (MHC) class II dependent, but haplotype unrestricted, manner.",2 SE bind specifically to MHC class II molecules,34 selectively activating T cells bearing particular T-cell receptor (TcR) Vfl segments,',5 and MHC class II-dependent binding of SE to purified TcR Vf chains has been reported recently.6 Mitogenic activity thus depends on functional bivalency, as SE must interact with both MHC class II and TcR molecules to stimulate a T-cell response. However, radiolabelled SE bind MHC class IT-bearing antigen-presenting cells (APC), but not to pure T-cell populations,3 arguing against direct binding of SE to the TcR. MHC class IT-independent Tcell binding can be inferred from the demonstration that prepulsing of murine T-cell clones with large doses of staphylococcal enterotoxin B (SEB) followed by thorough washing and the addition of APC, leads to T-cell proliferation.7 Furthermore, these workers reported that one T-cell clone, which responded by interleukin-2 (IL-2) production to soluble anti-V#8 antibody, also responded to SEB in the absence of APC,7 suggesting that MHC class II was not necessary for SEB binding to the TcR in this particular clone. T-cell proliferation normally requires accessory cell-derived co-stimulatory signals in addition to
occupancy of the TcR,8 so that MHC class II dependence of proliferation to SE is a complex issue. However, it has been shown that under conditions which exclude APC contamination, staphylococcal enterotoxin A (SEA) does not induce proliferation in human T cells in combination with phorbol esters, which are known to bypass co-stimulatory factor requirements.9 The induction of early activation events in homogeneous Tcell populations offers a more direct means of assessing the ability of SE to interact with the TcR. It has been reported that SE interact directly with human T cells and trigger rises in intracellular free calcium, representative of early T-cell activation.9'10 However, human T cells express MHC class II molecules so that the question of whether SE bind to the TcR in the absence of MHC class II could not be addressed. There are no similar reports in the literature utilizing murine T cells, which do not express MHC class II molecules, apart from the demonstration that toxic shock syndrome toxin (TSST- 1) induces inositol phosphate accumulation in T cells in the presence of APC." Direct binding of SE to T cells is an issue central to the understanding of the mechanism of action of bacterial superantigens. We examine here the binding potential of SEB to murine Th 1 and Th2 T-cell clones in the absence of APC using the detection of intracellular calcium transients and inositol phosphate accumulation as indicators of direct interactions between SEB and T cells. In addition, proliferation assays were used to re-evaluate whether T-cell clones pre-pulsed with SEB responded when APC were added, as well as to determine whether soluble SEB or SEB-coated Sepharose beads activated APC-independent T-cell clones.
Abbreviations: SE, staphylococcal enterotoxins; SEA, staphylococcal enterotoxin A; SEB, staphylococcal enterotoxin B. Correspondence: Dr J. H. Robinson, Dept. of Immunology, The Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH, U.K.
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MATERIALS AND METHODS Mice and T-cell clones BALB/c, CBA.Ca and C57BL/6 mice were obtained from the Comparative Biology Centre, University of Newcastle Upon Tyne, U.K., and used at 6-10 weeks of age. The SEB-reactive Tcell clones investigated were as follows: DIO.G4, Vf8+, specific for conalbumin/Ak (a gift from Dr G. G. B. Klaus, National Institute for Medical Research, London, U.K.); Ni 1, Vf3 +, specific for ovalbumin/Ab; 6D, Vf8+, specific for ovalbumin/ Ab; R28, Vfl8+, specific for group A streptococcal type 5 M protein (M5)/Ad; QI, Vfi8+, specific for MS/Ak; and Q3, VB8+, specific for M5/Ak. All clones except D1O.G4 were generated by us (J. H. Robinson et al., manuscript submitted for publication). Clones DlO.G4 and Nil secreted IL-4 but not IL-2 on stimulation and were considered to be of the Th2 type, whereas clones R28, Q1, Q3 and 6D were designated as ThI type based upon secretion of IL-2 but not IL-4. During maintenance of Tcell clones, T-cell blasts were purified by one-step density centrifugation using Histopaque (Sigma Chemical Co., Poole, Dorset, U.K.) 3 days after every cycle of restimulation with antigen and irradiated spleen cells.
Stimulants SEB (Sigma Chemical Co.) was purified by isoelectric focusing to greater than 95% purity as judged by SDS-PAGE and stored at 10-4 M in phosphate-buffered saline (PBS) as aliquots at -80°. Concanavalin A (Con A; Sigma Chemical Co.) was dissolved in PBS and stored as aliquots at -80°. The monoclonal antibodies F23. 1 (anti-VP8), KJ.25 (anti-Vf3), and 145.2C II (anti-CD3) were concentrated by saturated ammonium sulphate precipitation of culture supernatants from antibody-secreting hybridomas, obtained from the American Type Culture Collection, Rockville, MA, and stored at -80°.
Coupling SEB to Sepharose beads CnBr-activated Sepharose-4B gel filtration medium (Sigma Chemical Co.) was washed and swollen for 15 min at 240 in I M HCI in a sintered glass funnel, and then washed in coupling buffer (0.1 M NaHCO3, 0.5 M NaCI, pH 8.3). Five milligrams of SEB in I ml of gel were mixed for 2 hr at 240, and washed twice by centrifugation. One mole of ethanolamine (Sigma Chemical Co.) was added and mixed for I hr at 240 and then 30 min at 40 to block remaining activated groups. The gel was then washed three times alternately in acetate buffer (pH 4-0) and Tris buffer (pH 8-0), and stored in Tris buffer (pH 7-4) at 4°. Measurement of intracellular calcium T cells were loaded for I hr at 370 with the calcium-specific dualwavelength fluorescent dye, Indo-I AM (8 Mm; Molecular Probes Inc., Eugene, OR).'2 107 loaded cells were resuspended in 800 p1 of phenol red-free HBSS (Gibco Ltd, Paisley, Renfrewshire, U.K.) containing 10 mm HEPES buffer (Gibco Ltd) in a quartz cuvette at 370 for analysis using a MPF-3 Perkin-Elmer fluorescence spectrophotometer. Excitation was at 356 nm and emissions were measured at 396 nm (calcium-bound Indo-1 AM) and 460 nm (calcium-free Indo- I AM) to allow calculation of flourescence ratio (R). Rmax was obtained by saturation of the intracellular compartment with calcium using the calcium ionophore Ionomycin (1 Mg/ml; Calbiochem Corp., San Diego, CA) and Rmin was calculated after cellular lysis and chelation of free calcium in a final concentration of 12 mm EGTA (BDH
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Figure 1. Measurement of intracellular free calcium. Fluorescence trace measured at 396 nm against time of 107 T-cell clone NIl cells, loaded with 8 jM Indo-l AM. The trace was interrupted during addition of Ig/ sequential doses of SEB (1 nM = 28 ng/ml) or 100 yM Con A (=10 ml), and on each plateau the emission wavelength was momentarily changed to 460 nm to allow subsequent calculation of fluorescence ratio (R).
Chemicals Ltd, Poole, Dorset, U.K.). Stimulants were added in
4-pl volumes. Absolute calcium concentrations ([Ca2+]i) were calculated from the observed fluorescence (F) by: Ca2+ = Kd x [F- Fmin)/((Fmax- F), where Kd=250 nM.1213 Measurement of inositol phosphates T-cell clones were saturated with myo [3H]inositol (Amersham International plc, Amersham, Bucks, U.K.) by incubation for 18 hr at 107 T cells/ml with 40 ,Ci/ml of label in HBSS buffered with 10 mm HEPES and supplemented with 10% vol/vol dialysed FCS (Sigma Chemical Co.). Analysis in triplicate, using 3 x 106 cells per sample, was carried out in RPMI-1640 (Gibco Ltd) supplemented with 5% vol/vol FCS and 10 mm lithium chloride to prevent recycling of the inositol phosphates. Stimulation was terminated after I hr by the addition of a 2: 1 vol/vol mixture of methanol and chloroform and the aqueous phase was separated after addition of chloroform and distilled water. Total inositol phosphates were measured by combined phase scintillation counting after extraction by anion-exchange chromatography (Dowex-l, AGI x 8; Sigma Chemical Co.).'4 Results are expressed as c.p.m. + SEM. The B-lymphoblastoid cell line A20 (American Type Culture Collection), untreated or pre-pulsed with 10-6 M SEB I hr at 37°, was used as APC where indicated.
Proliferation assays 2 x 104 cloned T cells were cultured for 48 hr in RPMI-1640 (Gibco Ltd) supplemented to 3 mM L-glutamine and 10% FCS vol/vol in flat-bottomed 96-well microtitre plates in the presence of stimuli, with or without 7-5 x 105 irridiated (20 Gy) syngeneic
spleen cells as APC. Proliferation was assessed by uptake of [3H]thymidine (Amersham International) during the final 4 hr. Cells were harvested and radioactivity was quantified by liquid scintillation spectroscopy. Results are expressed as mean d.p.m. of triplicates + SEM. In pre-pulsing experiments, 106 cloned T cells were incubated with 10-6 M SEB in I ml of culture medium for I hr at 370 before washing in 4 x 10 ml of culture medium prior to the proliferation assay. RESULTS Calcium transients and inositol phosphate accumulation in Thl and Th2 T-cell clones In preliminary experiments all T-cell clones used in this study were shown to be SEB responsive in proliferation assays. In the
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Figure 2. Fluorescence ratio stimulated with SEB. The mean fluorescence ratio+ SEM for five experiments is shown. In each cuvette, 107 clone N 1 cells were sequentially stimulated with 1000 nm SEB, 100 nM Con A, 1 ug/ml Ionomycin (Iono) and 12 mm EGTA. Mean [Ca]2+]I +SEM: no stimulus (None), 113 + 10 nM; SEB, 130+9 nm; and Con A 686 + 115 nm. See the Materials and Methods for calculation of R and
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Figure 3. Measurement of inositol phosphate accumulation. 3 x 106 clone DI O.G4 cells, labelled with myo [3HJinositol, were incubated for I hr with (a) Con A in the absence of APC, (b) dilutions of F23. 1 antibody or (c) SEB in the presence (-) or absence (0) of 106 A20 cells as APC. Results are expressed as mean c.p.m. of triplicate cultures + SEM.
Clone 6D Sol SEB/APC APC Clone alone Clone D1O.G4 Sol SEB/APC Antigen/APC APC Clone alone
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D.p.m. Figure 4. Proliferation response of SEB pre-pulsed T-cell clones. T-cell clones 6D and DlO.G4 were pre-pulsed with 10-6 M SEB for 1 hr and washed four times in 10-ml volumes. Proliferation responses of prepulsed (-) and untreated cloned T cells (0) were compared. Mean d.p.m. +SEM of 104 cloned cells, in the presence (APC) or absence (Clone alone) of 7-5 x 105 non-pulsed irradiated spleen cells are shown. Proliferation of cloned T cells with 1O-7 M soluble SEB and APC (Sol SEB/APC) was included to show that the clone remained SEBresponsive after treatment.
absence of APC, SEB failed to induce rises in intracellular free calcium over the dose range of SEB which stimulated APCdependent proliferation in T-cell clones (Fig. 1). However, subsequent addition of Con A induced calcium signals in the same cells. The fluorescence ratio, measured at 396 nm and 460 nm, for five experiments is plotted for clone Nl 1 in Fig. 2. In addition, the anti-V#3 antibody KJ.25 stimulated a rise in calcium in clone Nl 1 without APC (R = 0-87 compared to a basal ratio of 0-61). Similar results were obtained for the Th2 clone DIO.G4 and the ThI clone 6D. The small rises seen after SEB treatment were not greater than changes induced by the addition of medium alone followed by agitation of the sample. It is possible that if SEB bound T cells directly without reaching the threshold necessary for inducing calcium signals, then steric hindrance could prevent binding by other stimulants. However, pretreatment with SEB failed to inhibit subsequent induction of calcium signals in cloned Ni 1 cells by the antibodies KJ.25, F23. 1 or 2CI I (data not shown). Attempts to measure calcium responses in the presence of APC were unsuccessful due to the effects of dye leakage over the extended period of incubation required for cell-cell interactions to occur. However, SEB was shown to induce inositol phosphate accumulation in the presence of APC, but failed to do so when APC were omitted. Figure 3a illustrates the dose-dependent increase in inositol phosphate accumulation in the Th2 clone DIO.G4 stimulated with Con A. Antibodies against Vfl8 (F23.1) also induced significant inositol phosphate turnover in the absence of APC (Fig. 3b), whereas SEB only elicited a response in the presence of APC and at higher doses of toxin (Fig. 3c). Similar results were obtained for the Thl clones, QI and Q3. Proliferation of SEB pre-pulsed T-cell clones and APC-independent T-cell clones
Preliminary experiments showed that spleen cells pre-pulsed with 10-6 M SEB for 1 hr at 370 induced proliferation in SEBreactive T-cell clones (data not shown). Pre-pulsing of Thl or Th2 T-cell clones with SEB at this dose in the absence of APC
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Figure 5. Proliferation responses of APC-independent T-cell clone. 2 x 104 cloned QI cells were cultured for 48 hr in the presence (a) or absence (b) of 7 5 x 105 irradiated syngeneic spleen cells as APC, with the doses of SEB shown, 40 nm Con A or a 1:200 dilution of the antibodies F23.1 (anti-Vfl8), 2C 1I (anti-CD3) or KJ.25 (anti-Vfl3). Results are plotted as mean d.p.m. of triplicate cultures+ SEM.
followed by extensive washing did not lead to stimulation of proliferation upon subsequent culture with non-pulsed APC (Fig. 4). Extending the pre-pulse time to 3 hr or increasing the SEB dose to 10-5 M gave essentially the same results (data not shown). APC-independent T-cell clones were also used to investigate the direct binding potential of SEB to T cells uncomplicated by the requirement for APC-derived co-stimulatory factors. The Tcell clones Qi, Q3 and D1O.G4 proliferated in response to soluble antibodies directed against the Vf8 domain of the TcR (F23.1) or CD3 (2C1 1) in the absence of APC (results for the V/8+ clone Q1 are shown in Fig. 5b). Responses were considerably enhanced by the addition of irradiated syngeneic spleen cells (Fig. 5a). However, SEB failed to induce proliferation unless APC were present (Fig. 5). Similar results were obtained for clones Q3 and D10.G4. To evaluate whether the role of MHC class II on the APC surface was to present an array of SEB molecules to TcR on the T-cell surface, SEB was crosslinked by coupling to Sepharose beads. SEB-Sepharose, washed well before addition to the proliferation assay to remove any residual soluble SEB, stimulated the SEB-responsive T-cell clone R28 in the presence of APC, but not in their absence (Fig. 6). Similar results were obtained for the APC-independent T-cell clones Q1 and Q3. DISCUSSION Stimulation of proliferation in most T cells by specific antigens, superantigens, such as SE, and monoclonal antibodies against T-cell surface molecules, including CD3 and the TcR, requires APC or accessory cells to present the stimulus and/or to provide
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Figure 6. Proliferation response of T-cell clone to SEB-Sepharose. 2 x 104 cloned R28 cells were cultured for 48 hr in the presence (A) or absence (A) of 7 5 x I0O irradiated syngeneic spleen cells as APC, and (a) concentrations of soluble SEB or (b) dilutions of SEB-Sepharose shown. Results are plotted as mean d.p.m. of triplicate cultures + SEM.
co-stimulatory activity. Specific antigens are processed to peptide epitopes and presented to T cells in association with selfMHC class II molecules on the surface of APC.'5 Superantigens do not require processing, but bind directly to MHC class II molecules, and activate T cells expressing selected TcR VB segments, independent of their MHC restriction phenotype.' However, it is not clear if SE bind to MHC class II and TcR independently, or alternatively whether MHC class II binding of SE is a pre-requisite step to T-cell triggering. Direct binding to the TcR or CD3 by monoclonal antibodies, in the absence of APC, is normally unable to stimulate proliferation in T cells,'6 but induces early activation signals such as increases in free intracellular calcium or accumulation of inositol phosphates.8 Thus, we measured early activation events in murine T-cell clones, as a more direct method than assessment of proliferation, to investigate whether SEB binds to the TcR in the absence of MHC class II. It has been reported that SE trigger rises in intracellular free calcium in human T cells,9"0 but as human T cells express MHC class II molecules, the question of whether SE bind to the TcR in the absence of MHC class II could not be addressed. The present report is the first studying induction of early activation signals in murine T cells by SE. The results show that Con A or anti-TcR antibodies induced calcium signals and/or inositol phosphate turnover in murine Th I and Th2 cell clones, but that SEB failed to do so, in the absence of APC. However, higher doses of SEB stimulated inositol phosphate turnover in the presence of APC, indicating that SEB was unable to initiate early activation events in the absence of MHC class II bearing APC. Both Th I and Th2 T-cell clones were used because of reported differences in their signalling requirements.'7 Our results are consistent with the demonstration that high doses of a related toxin, TSST-1, induces inositol phosphate accumulation in the presence of APC," although the
SEB and activation signals in murine T cells requirement for MHC class II bearing APC was not addressed in this report. In the absence of any data to show binding of radiolabelled SE to T cells,3'0 the concept of direct binding of SE to murine T cells is based on pre-pulsing experiments.2'7 We were unable to confirm these findings, as we have shown that pre-pulsing T-cell clones with high doses of SEB, followed by extensive washing, did not stimulate proliferation upon addition of APC. The prepulsing protocols employed similar doses of SEB but different T-cell clones were used. It is also possible that the discrepancy in the results is due to residual APC in the cloned T-cell populations of Yagi et al.,7 as we purified T-cell blasts by density centrifugation after every cycle restimulation with antigen and spleen cells. In addition, we have shown that SEB, in contrast to anti-Vfi8 antibodies, failed to stimulate three APC-independent T-cell clones to proliferate in the absence of APC, whereas these clones proliferated to SEB in the presence of syngeneic irradiated splenocytes as APC, indicating that SEB does not bind to the TcR of these clones with sufficient affinity to induce proliferation. Yagi et al.7 reported one clone which responded to SEB in the absence of APC, suggesting that the TcR of rare clones could bind SEB, but that this was not a general phenomenon. Finally, we evaluated whether the role of MHC class II on the APC surface was to present an array of SEB molecules to TcR, by attempting to mimic the array by coupling SEB to Sepharose beads. This approach has been used successfully to induce proliferation in T-cell clones by antibodies against CD3 or the TcR, when soluble antibodies were ineffective.'6 However, we have shown that SEB-Sepharose failed to activate T-cell clones, including APC-independent clones, in the absence of MHC class II bearing APC, indicating that MHC class II plays a more complex role than simply presenting SEB in a way which cross-links TcR on the T-cell surface. It was surprising that SEB coupled to Sepharose could mediate the interaction between TcR and MHC class II, given the large size of the beads, and it is possible that APC remove SEB from the beads prior to MHC class II binding, or that coupling to beads allows SEB to deliver a signal directly through the TcR but that APC are required for the delivery of an additional signal independent of surface MHC class II. We are further investigating the role of SEB coupled to beads in T-cell triggering, and have preliminary data to show that SEB-Sepharose does not induce inositol phosphate turnover in T-cell clones, including APC-independent clones, in the absence of APC. In conclusion, five separate lines of investigation in this study have failed to provide any evidence to suggest that SEB binds the TcR of murine T cells in the absence of MHC class II. We cannot, however, exclude the possibility that binding occurred, but that the affinity was too low to have any functional consequences. Our results are consistent with the model of T-cell activation by SE proposed by Janeway et al.,2 in which intact superantigens cross-link MHC class II and the TcR. However, the results described here argue against SEB binding independently to the TcR, and we postulate that SEB first interacts with MHC class II, which induces a conformational change in SEB necessary for subsequent TcR binding. ACKNOWLEDGMENTS The authors wish to thank Marian Atherton and Gwyn Pyle for skillful assistance with cell culture, Dr G. G. B. Klaus, National Institute for Medical Research, Mill Hill, U.K. for valuable guidance on assays for
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measuring intracellular calcium and inositol phosphates as well as for the gift of DlO.G4 cells, and Dr Richard Virden, Department of Biochemistry and Genetics, University of Newcastle upon Tyne, U.K., for access to the fluorescence spectrophotometer. This research was funded by U.K. MOD and a Nuffield Foundation Student Bursary to W. Qasim.
REFERENCES 1. MARRACK P. & KAPPLER J.W. (1990) The staphylococcal enterotoxins and their relatives. Science, 2A4, 705. 2. JANEWAY C.A., YAGI J., CONRAD P.J., KATZ M.E., JONES B., VROEGOP S. & BUXSER S. (1989) T cell responses to Mls and to bacterial proteins that mimic its behavior. Immunol. Rev. 107, 61. 3. FRASER J.D. (1989) High affinity binding of staphylococcal enterotoxin A and B to HLA-DR. Nature, 339,221. 4. SCHOLL P., DIEZ A., KARR R., SEKALY R., TROWSDALE J. & GEHA, R.S. (1990) Effect of isotypes and allelic polymorphism on the binding of staphylococcal exotoxins to MHC class II molecules. J. Immunol. 144, 226. 5. WHITE J., HERMAN A., PULLEN A.M., KuBo R., KAPPLER J. W. & MARRACK P. (1989) The VPi-specific superantigen staphylococcal enterotoxin B: stimulation of mature T cells and clonal deletion in neonatal mice. Cell, 56, 27. 6. GASCOIGNE N.R.J. & AMEs K.T. (1991) Direct binding of secreted T-cell receptor P chain to superantigen associated with class II major histocompatibility complex protein. Proc. natl. Acad. Sci. U.S.A. 88, 613. 7. YAGI J., BARON J., BUXSER S. & JANEWAY C.A. (1990) Bacterial proteins that mediate the association of a defined subset of T cell receptor: CD4 complexes with class II MHC. J. Immunol. 144, 892. 8. MUELLER D.L., JENKINS M.K. & SCHWARTZ R.H. (1989) Clonal expansion versus functional clonal inactivation: a costimulatory signalling pathway determines the outcome of T cell antigen receptor occupancy. Ann. Rev. Immunol. 7,445. 9. FLEISCHER B. & SCHREZENEMEIER H. (1988) T cell stimulation by staphylococcal enterotoxins. J. exp. Med. 167, 1697. 10. FLEISCHER B., SCHREZENEMEIER H. & CONRADT P. (1989) T lymphocyte activation by staphylococcal enterotoxins: role of class II molecules and T cell surface structures. Cell. Immunol. 120, 92. 11. NORTON S.D., SCHLIEVERT P.M., NOVICK R.P. & JENKINS M.J. (1990) Molecular requirements for T cell activation by the staphylococcal toxic shock syndrome toxin-I. J. Immunol. 144, 2089. 12. GRYNKIEWICZ G., POENIE M. & TsIEN R.T. (1985) A new generation of Ca2 + indicators with greatly improved fluorescence properties. J. biol. Chem. 260, 3440. 13. BISTERBOSCH M.K., RIGLEY K.P. & KLAUS G.G.B. (1985) Cross linking of surface immunoglobulin on B lymphocytes induces both intra-cellular Ca2+ release and Ca2+ influx: analysis with Indo-l. Biochem. Biophys. Res. Comm. 137, 500. 14. BERRIDGE M.J., DAWSON R.M.C., DOWNES C.P., HESLOP J.P. & IRVINE R.F. (1983) Changes in the levels ofinositol phosphates after agonist-dependent hydrolysis of membrane phosphoinositides. Biochem. J. 212, 473. 15. SCHWARTZ R.H. (1985) T-lymphocyte recognition of antigen in association with gene products of the major histocompatibility complex. Ann. Rev. Immunol. 3, 237. 16. MEUER S.C., HODGDON J.C., HUSSEY R.E., PROTENTIS J.P., SCHLOSSMAN S.F. & REINHERZ E.L. (1983) Antigen like effects of monoclonal antibodies directed at receptors on human T cell clones. J. exp. Med. 158,988. 17. GAJEWSKY T.F., SCHELL R.S., NAU G. & FITCH F.W. (1989) Regulation of T cell activation: differences among T cell subsets. Immunol. Rev. 111, 79.
