Proc. Nati. Acad. Sci. USA

Vol. 89, pp. 7727-7731, August 1992 Medical Sciences

T-cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A CAROL H. PONTZER*, MICHAEL J. IRWINt, NICHOLAS R. J. GASCOIGNEt, AND HOWARD M. JOHNSON* *Department of Microbiology and Cell Science, 3103 McCarty Hall, University of Florida, Gainesville, FL 32611; and tDepartment of Immunology, The Scripps Research Institute, La Jolla, CA 92037

Communicated by George K. Davis, May 12, 1992

ABSTRACT We have examined the interaction of the microbial superantigen staphylococcal enterotoxin A (SEA) with peptides corresponding to overlapping regions of the T-cell antigen receptor ( chain variable region V(33. SEA is known to stimulate murine T cells bearing certain VP3 elements, among them V(33. Five peptides were synthesized representing amino acids 1-24,20-44,39-60,57-77, and 74-95 of V.33. We demonstrate here that soluble VI33-bearing .8 chains can bind to a complex of SEA and major histocompatibility complex class II and that the synthetic peptide V(t33-(57-77) blocked this interaction. The peptide V.83-(57-77) also inhibited SEAinduced interferon-V production and SEA-induced proliferation of B1O.BR spleen cells. Conversely, the peptide corresponding to amino acids 57-77 of V.88.2, a VP3 element that is not recognized by SEA, decreased staphylococcal enterotoxin C-2-induced proliferation but did not affect SEA-induced proliferation. The peptide inhibition of SEA-induced function was due at least in part to inhibition of V(83-bearing T-cell activity, since the percentage of T cells reactive with an anti-V.83 monoclonal antibody was significantly reduced by V(33-(57-77). These data suggest that the region of V(3 encompassing amino acids 57-77 is an area that displays the appropriate sequence and conformation for binding of the SEA molecule and blocking of the resultant interaction with the T-ceil antigen receptor.

MATERIALS AND METHODS SEA. SEA was obtained from Toxin Technology, Madison, WI. It was homogeneous by SDS/gel electrophoresis (1). Synthetic Peptides. Overlapping peptides encompassing the V/3 of the TCR, V(33-(1-24), Vf33-(20-44), V,33-(39-60), V(33-(57-77), Vf33-(74-95), and Vj38.2-(57-77), were synthesized on a Biosearch 9500AT automated peptide synthesizer using fluoren-9-ylmethyloxycarbonyl (Fmoc) chemistry (13). Peptides were cleaved from the resins by using trifluoroacetic acid/ethanedithiol/thioanisole/anisole, 90:3:5:2 (vol/vol). The cleaved peptides were then extracted in ether and ethyl acetate and subsequently dissolved in water and lyophilized. Dimer formation is prevented with the use of acetamidomethyl as the protecting group on the cysteine, because acetamidomethyl is not removed by trifluoroacetic acid. Reversephase HPLC analysis of crude peptides indicated one major peak in each profile. Hence, further purification was not warranted. Amino acid analysis of these peptides showed that the amino acid composition corresponded closely to the

theoretical composition. Polyclonal antisera to peptides was produced by hyperimmunization of rabbits. For immunization, peptides were conjugated to keyhole limpet hemocyanin using gluteraldehyde as the coupling reagent. Soluble (3-Chain Binding. A gene encoding a truncated form of the P chain from the pigeon cytochrome c I-Ek-specific T-cell 2B4 was expressed in baculovirus (M.J.I. and N.R.J.G., unpublished results). The j3chain protein was purified by affinity chromatography with the anti-(3 chain constant region antibody H57-597, as described (14). For biotinylation, 100 1.l of purified soluble (3chain (2 mg/ml) and an equal volume of 50 mM bicarbonate (pH 9.6) containing 0.02 mg of NHS-LC-biotin (Pierce) were mixed and incubated on ice for 2 h. Unreacted biotin was removed by dialysis. One million cells of the human Burkitt lymphoma line Raji were preincubated with either 50 1.l of SEA (250 pg/ml) or FACS buffer (phosphate-buffered saline containing 10 mM sodium azide and 0.5% bovine serum albumin) for 1 h at 370C. The cells were washed and then incubated with 50 1ul of biotinylated soluble (3 chain (1 mg/ml) for 3 h at 370C. For peptide competition, cells were preincubated with 300 A&M peptides for 1 h followed by addition of 20 ,ld of biotinylated soluble P chain (1 mg/ml) and a further 3-h incubation. The cells were washed and incubated with 50 1.l of streptavidinphycoerythrin at 1 ,ug/ml for 15 min at 40C. Fluorescence was analyzed on a FACStar (Becton Dickinson) using logarithmic

Staphylococcal enterotoxin A (SEA) is the most potent T-cell mitogen known. It is capable of stimulating DNA synthesis, interferon-y (IFN-y) production, and interleukin 2 production at concentrations as low as 10-16 M (1-3). In fact SEA has the characteristics of a superantigen because of its ability to stimulate all T cells bearing particular T-cell antigen receptor (TCR) (3 chain variable regions (V,() (4-6). The sequence and structural characteristics that enable certain VB families to interact with SEA have not yet been elucidated. Although particular V(3 residues have been shown to be associated with the functional interaction of the TCR (3 chain with various superantigens (7-10), direct binding studies of superantigens, class II major histocompatibility complex (MHC) molecules, and TCRs have not been carried out to directly identify a binding site for superantigen on the (3 chain of the TCR. Peptide blocking studies using soluble (3-chain binding to superantigen-class II complexes allow us to investigate the V,( binding site. We have examined the entire sequence of a V,(3 molecule by using overlapping synthetic peptides to identify a region of the molecule responsible for its interaction with SEA. Amino acids 57-77, which contain the predicted external TCR loop (complementarity-determining region 4, CDR4) (11, 12), represent a TCR binding site for SEA.

amplifiers. Abbreviations: SEA, staphylococcal enterotoxin A; SEC2, staphylococcal enterotoxin C-2; TCR, T-cell antigen receptor; IFNy, interferon-y; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2Htetrazolium bromide; MHC, major histocompatibility complex; CDR, complementarity-determining region; V,8, (3 chain variable region.

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Medical Sciences: Pontzer et al.





30 40 50 60 Residue




FIG. 1. Predicted composite surface profile for the VP3 region of the TCR by using the parameters of HPLC hydrophilicity, accessibility, and segmental mobility. Residues with composite profile values >50%o are predicted to be more accessible to the surface ofthe molecule.

SEA Induction of IFN-y. B10.BR spleen cells were suspended in RPMI 1640 medium containing 10%o (vol/vol) fetal bovine serum at 5 x 106 cells per well and incubated with 0.02 puM SEA in the presence or absence of 100 ,&M peptides for 72 h. Supernatant fluids were harvested, and IFN-'y production was assessed by ELISA as described (15). SEA-Induced Proliferation. Murine spleen cells were collected from B1O.BR/SgSnJ mice. They were washed and resuspended in RPMI 1640 medium supplemented with 10%6 fetal bovine serum, 0.1 mM 2-mercaptoethanol, and antibiotics (100 units of penicillin and 100 pg of streptomycin per ml). All tissue culture media and sera used in this study were negative for endotoxin, as determined by assay with Limulus amebocyte lysate (Associates of Cape Cod, Woods Hole, MA) at a sensitivity level of 0.07 ng/ml. Cell viability was assessed by trypan blue dye exclusion. SEA at 20 nM or concanavalin A (Con A) at 3 jg/ml was mixed with various concentrations of peptides in triplicate. Spleen cells were added to 96-well microtiter plates at 5 x 105 cells per well and incubated at 370C in an atmosphere of 5% C02/95% air for 3 days. The cells were pulse-labeled with 1 ,uCi of [3H]thymidine (specific activity, 21 mCi/mg; 1 Ci = 37 GBq) for 24 h before harvesting. Cells were harvested on a cell harvester (PHD, Cambridge, MA) and washed with distilled H20, and [3H]thymidine incorporation was determined in a (3-scintillation counter. Alternatively, cells were pulse-labeled with 10 jul of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT; 5 mg/ml) for 16 h prior to the termination of culture (16). Crystals were solubilized with 0.1 M HCl in either 10%6 (wt/vol) SDS or isopropanol, and the OD was read using a 570-nm filter. Significant differences were assessed by analysis of variance. Analysis of TCR Vf3 Expression. T cells were purified from B1O.BR/SgSnJ spleens by using ammonium chloride lysis followed by removal of B cells on goat anti-mouse IgG whole molecule (Sigma)-coated plates. Nonadherent cells were >90%o T cells (TCR+) as assessed by flow cytometry. SynTable 1. Overlapping

geneic spleen cells were treated with mitomycin C at 100 pg/ml for 20 min at 370C, washed three times, and added as antigen-presenting cells. Purified T cells (6 x 104 cells) were cocultured with 2 x 104 mitomycin C-treated cells and 20 nM SEA in the presence of various concentrations of VP peptides in a total volume of 1 ml for 4 days. Viable cells were recovered over Ficoll (1.083 g/ml), washed, and labeled with the appropriate biotinylated hamster anti-mouse VPe TCR monoclonal antibodies (Pharmingen, San Diego), followed by streptavidin-phycoerythrin. Cells were analyzed by flow cytometry. Forward and 900 light scatter were used to gate on blast cells.

RESULTS TCR VjJ3 Peptides. Those regions of a molecule expected to be antigenic or important in function are likely to be located on or exposed to the surface of the molecule. Using the amino acid sequence of the TCR V,83 clone 2B4 (17), a surface profile was generated from a computer program that uses a composite of three parameters: HPLC hydrophilicity, flexibility, and accessibility (18) (Fig. 1). As a result of this profile as well as the primary amino acid sequence, five overlapping peptides were synthesized corresponding to sequential regions of V(33 (Table 1). A V.88.2 peptide was also synthesized as a control. Each peptide was designed to contain some predicted surface accessible residues, and all were approximately the same length. The peptide sequences were hydrophilic with the exception of V,(3-(74-95). Of interest, the C-terminal region of TCR VPe families exhibit the greatest degree of sequence homology (19). Binding of Soluble P Chain. A soluble i chain of the VP3 family bound to SEA-coated Raji cells as assessed by flow cytometry (Fig. 2). The mean fluorescence for Raji cells increased from 17.80 for cells alone to 43.23 for cells in the presence of SEA, and the mode shifted from 7.84 to 14.48. Preliminary incubations showed that binding required a 3-h incubation with soluble (3chain (data not shown). The data are direct evidence for binding of the SEA-MHC complex to a TCR and are consistent with the superantigen requirement for interaction of soluble P chain with MHC observed in a cell binding assay (20). We have examined the ability of the synthetic peptides corresponding to V(33 to compete with the soluble P chain sequence for binding to the complex of SEA and MHC (Fig. 3). The peptide encompassing amino acids 57-77 of V.83 significantly reduced soluble chain binding. V(33-(57-77) was a much better competitor of soluble chain binding than was V(38.2-(57-77), which is consistent with the specificity of V(33 but not V(38.2 for SEA (21). The minor effects ofthe V(38.2 peptide may be due to the five homologous amino acids of V.83 and V(88.2 found between residues 67 and 77 (Table 1). These data suggest that (-chain residues 57-77 are required for binding of the TCR to the superantigen and MHC, but additional regions may be involved in this binding and contribute to the specificity of the V(3 interaction. Vj3 Peptide Inhibition of SEA-Induced IFN-y Production. V(33 peptide effects on T-cell function were investigated as a

V183 peptides

Net Molecular Hydropathic Sequence index charge weight +7 NSKVIQTPRYLVKGQGQKAKMRCI -0.7 2744 V,83-(1-24) +4 KMRCIPEKGHPVVFWYQQNKNNEFK 3118 -1.2 25 VP3-(20-44) KNNEFKFLINFQNQEVLQQIDM 0 -0.7 22 2738 VB3-(39-60) QIDMTEKRFSAECPSNSPCSL 0 2340 -0.7 21 Vj3-(57-77) -2 0.2 2228 PCSLEIQSSEAGDSALYLCASS 22 VP3-(74-95) +1 KGDIPDGYKASRPSQENFSL -1.3 20 2206 V,88.2-(57-77) Translated VP3 protein sequence of the 2B4 clone, the V.8.2 sequence of the TB2 clone, and amino acid position number assignments so as to maximize homology were obtained from Kabat et aL (19). A space has been included in the numbering of amino acids 57-77 of VB8.2 to optimize alignment with amino acids 57-77 of V,33.



no. 24

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Pontzer et al.




00 4




FL2 0

FIG. 2. Binding of soluble , chain to SEA and MHC on cells of the human Burkitt lymphoma line Raji. One million Raji cells were preincubated with either 50 jil of SEA (250 pg/ml; dotted line) or FACS buffer alone (solid line) for 1 h at 37°C. The cells were washed and then incubated with 50 id of biotinylated soluble chain (1 mg/ml) for 3 h at 3TC. They were further labeled with streptavidinphycoerythrin and analyzed by flow cytometry. Results are expressed as a one-parameter fluorescence histogram. FL2, fluorescence intensity.

correlate to binding. Spleen cells from B10.BR mice (H-2k, I-E+) were stimulated with SEA to produce IFN-y. SEA induction of IFN-'y was inhibited by 60%o by 100 ,uM VB3(57-77) (Fig. 4), which is consistent with inhibition of binding to soluble chain. Other peptides, including V(38.2-(57-77), were much less effective in blocking function. V83 Peptide Inhibition of SEA-Induced T-Cefl Proliferation. As observed for IFN-y production, V/33-(57-77) also significantly decreased SEA-induced proliferation of B10.BR spleen cells (Table 2). V(33-(39-60) also reduced SEA function, but this reduction was not consistently observed. In agreement with the binding results, V(38.2-(57-77) did not affect SEA-induced proliferation. Cell viability was not affected by addition of the peptides alone, thus the inhibitory effects were not due to peptide toxicity (data not shown). The peptides did not alter Con A-induced T-cell proliferation. 20




M 10

VBE1-24) VPE20-44) VC39SO) V"537-77) V"74-95) V6C57-77)

POptd (100 IAM) FIG. 4. V(33 peptide inhibition ofIFN-'yproduction. B1O.BR spleen cells at 5 x 106 cells per well were incubated with 0.02 pM SEA with or without 100 pLM peptides for 72 h. Supernatants were harvested, and IFN-Y production was assessed by ELISA. Results are expressed as mean percent reduction of four experiments. SEA induced IFN-yat 92 ± 35 units/ml (mean ± SEM) in the absence of peptide.

Thus, peptide inhibition of SEA function is consistent with peptide inhibition of SEA binding by the TCR (3 chain. SEA-induced spleen-cell proliferation could also be inhibited by antisera. As expected, an anti-V(33 monoclonal antibody significantly decreased proliferation, whereas an antiVf38.2 monoclonal antibody did not (Fig. 5). An anti-Vf3(57-77) antiserum significantly decreased proliferation, whereas an anti-V(38.2-(57-77) antiserum did not. Significant inhibition of SEA-induced proliferation was not observed using rabbit polyclonal antisera to the other V,33 peptides (data not shown). The observation that the inhibition by anti-V,83-(57-77) antiserum was not complete suggests that although the anti-V(83-(57-77) antiserum cross-reacts with the corresponding region on the TCR, which is involved in SEA-induced function, there may be additional functional sites on the TCR. The specificity of peptide inhibition of toxin-induced proliferation was examined by comparing the effect of V,33-(5777) and V,38.2-(57-77) on both SEA and staphylococcal enterotoxin C-2 (SEC2) function in a dose-response assay. At a concentration as low as 30 1uM, V,83-(57-77) significantly inhibited SEA-induced proliferation (Fig. 6A). V38.2-(57-77) was without effect. Conversely, spleen-cell proliferation induced by SEC2, which stimulates T cells bearing V(38.2 and Vp610, could be inhibited by low concentrations of Vp8.2-(5777), but not by V,33-(57-77) (Fig. 6B). Thus, this region of the ,( chain of the TCR appears to control superantigen specificity. Table 2. V(33 peptide inhibition of SEA-induced


spleen-cell proliferation



Treatment SEA or Con A



1-24) VE20-44) VE39-40)

VM57-77) V374-95) V3657-77)

Peptido FIG. 3. Peptide inhibition of soluble t3-chain binding. The experiments were performed as described in Fig. 2, except that, prior to addition of soluble chain (20-60 01 at 1 mg/ml), cells were incubated with 300 AM peptides for 1 h. Results of three replicate experiments are normalized on the basis of mean fluorescence in the absence of SEA and presented as mean percent reduction relative to binding of soluble ,B chain in the absence of peptides. Significant differences were assessed by least significant difference with P < 0.05.


Con A

277,044 ± 13,418 335,295 ± 26,035 + Vj63-(1-24) 253,535 + 12,999 492,850 ± 23,243 + V(3-(20-44) 299,882 ± 12,403 323,306 ± 19,967 + Vf33-(39-60) 310,011 ± 23,414 192,080 ± 11,728* + VP3-(57-77) 328,404 + 40,718 193,588 ± 7,420* + Vf33-(74-95) 297,880 ± 25,404 346,921 ± 47,916 + Vp8.2-(57-77) 432,177 ± 63,659 385,633 ± 11,483 x were incubated with B1O.BR spleen cells (1.5 107 cells per ml) either 0.02 ,uM SEA or Con A (3 Atg/ml) in the presence or absence of 300 j&M of the indicated VB peptides for 72 h. Results are expressed as cpm of triplicate determinations (mean ± SD). We repeated the experiments once with [3H]thymidine and three times with MTT as the indicator system. Unstimulated cells had 26,068 _ 2782 cpm. *, P < 0.01.

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Pontzer et al.



400,000 350000








* 200,000






z 20


100,000 upnon TWOuu 10






Antbody dion Vile

V03 o

VIW1-24) V3C20-44) VUM304) V5357-77) V374-95) V08C57-r)

VIM5f7-77) VOW57-77


FIG. 5. Antibody inhibition of toxin-induced spleen-cell proliferation. Approximately 1.5 x 106 spleen cells were incubated for 72 h with 0.02 uM SEA in the presence or absence of various dilutions of monoclonal antibodies to V/33 (KJ-25) (22), V,38.2 (F23.2) (23), or rabbit anti-V(3 peptide antisera. All anti-peptide antisera had titers greater than 1:30,000 to their corresponding peptide as assessed by ELISA. Unstimulated cells had 5546 + 157 cpm (mean ± SEM) and SEA-stimulated cells had 340,167 ± 24,569 cpm.

V.83 Peptide Reduction of SEA-Induced Clonal Expansion of V.83-Bearing T Cells. The percentage of SEA-stimulated T cells reactive with an anti-V,33 monoclonal antibody was significantly reduced by three peptides in the order of potency: V/33-(57-77) > Vf33-(1-24) > V,33-(39-60) (Fig. 7). Again, no reduction by V138.2-(57-77) was observed. No alteration in the percentage of V/38.2-bearing T cells was observed upon treatment with any of the peptides (data not shown). Thus, the peptide inhibition of SEA-induced function appears to be due to inhibition of the activity of V,83bearing T cells. The findings are consistent with binding and functional data indicating that SEA activates V,83 T cells via binding to the V,83-(57-77) region of the f3 chain.

DISCUSSION We have shown here binding of the soluble TCR P-chain molecule to the SEA and MHC on the cell surface. Addi-

FIG. 7. Inhibition of SEA-induced V(33-bearing T-cell blastogenesis by VP3 peptides. B10.BR spleen cells at 1 x 106 cells per ml were stimulated with 0.02 ILM SEA for % h with or without 300 pM (experiment 1) or 100 1AM (experiments 2 and 3) V,8 peptides. Cells were then treated with KJ-25, a biotinylated anti-V#3 monoclonal antibody, followed by streptavidin-phycoerythrin. Ten thousand events were analyzed by flow cytometry. Data are expressed as the percentage reduction (mean ± SD) of V,83-positive cells relative to SEA alone from three replicate experiments.

tionally, we have demonstrated that a peptide corresponding to amino acids 57-77 of Vf33 inhibited binding of the soluble molecule. Therefore, the region of V,33 necessary for formation of the trimolecular complex has been localized using synthetic peptides and direct binding. Stimulation of the TCR by superantigens such as the staphylococcal enterotoxins is associated with particular Vf3 families (4-6). Recently, SEA associated with MHC class II molecules on the cell surface was shown to bind to immobilized soluble TCR 8 chain (20). We have shown here that binding can also be demonstrated with biotinylated soluble /3 chain as assessed by flow cytometry. Consistent with binding studies, the V,33 peptide inhibited SEA induction of IFN-'y and T-cell proliferation. Thus, peptide binding and function data confirm the importance of 0.34 B

A 0.371-





0 to



c 0.35



0.28 A





0.331F 1




Peptide (,uM)

1I-0 100







Peptide (A&M)

FIG. 6. Specificity of peptide inhibition of toxin-induced proliferation. The experiment was performed as described in Table 2, except proliferation was assessed using MTT. Both SEA (A) and SEC2 (B) were used at 0.02 IzM. V,83-(57-77) (solid line) and V.88.2-(57-77) (dotted line) were used at the indicated concentrations. Results are expressed as the mean optical density (OD) of triplicate determinations. The OD in the absence of SEA was 0.189 + 0.003, which increased to 0.362 + 0.010 in the presence on SEA and to 0.281 0.005 in the presence of SEC2. The overall coefficient of variation was

T-cell antigen receptor binding sites for the microbial superantigen staphylococcal enterotoxin A.

We have examined the interaction of the microbial superantigen staphylococcal enterotoxin A (SEA) with peptides corresponding to overlapping regions o...
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