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(Journal of Leukocyte Biology. 2001;69:921-927.)
© 2001 by Society for Leukocyte Biology

Superantigen antagonist blocks Th1 cytokine gene induction and lethal shock

Gila Arad, Dalia Hillman, Revital Levy and Raymond Kaempfer

Department of Molecular Virology, The Hebrew University-Hadassah Medical School, Jerusalem, Israel

Correspondence: Dr. Raymond Kaempfer, Department of Molecular Virology, The Hebrew University-Hadassah Medical School, 91120 Jerusalem, Israel. E-mail: kaempfer{at}cc.huji.ac.il


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial superantigens trigger an excessive, Th1-cytokine response leading to toxic shock. We designed a peptide antagonist that inhibits SEB-induced expression of human genes for IL-2, IFN-{gamma}, and TNF-ß, cytokines that mediate shock. The peptide antagonist shows homology to a ß-strand-hinge-{alpha}-helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus and Streptococcus pyogenes yet remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. For Th1-cell activation, superantigens depend on this domain. The peptide protected mice against lethal challenge with SEB or SEA. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed broad-spectrum, protective immunity, which rendered them resistant to further lethal challenges with different staphylococcal and streptococcal superantigens. Thus, the lethal effect of superantigens, mediated by Th1 cytokines, can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade, before activation of T cells takes place.

Key Words: toxic shock • T-cell activation • antagonist peptide • protective immunity


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Bacterial superantigens are among the most potent activators of T cells known. Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes trigger an excessive, cellular, immune response that can lead to lethal, toxic shock [1 2 3 ]. The family of superantigens includes staphylococcal enterotoxins SEA-E, toxic shock syndrome toxin 1 (TSST-1), as well as the streptococcal pyrogenic exotoxins SPEA and SPEC [4 , 5 ]. Bypassing the restricted presentation of conventional antigens, superantigens bind directly to most major histocompatibility complex (MHC) class II molecules [6 ] and stimulate virtually all T cells bearing particular domains in the variable portion of the ß chain (Vß) of the T-cell receptor (TCR), without need for processing by antigen-presenting cells (APC) [1 , 7 8 9 10 ]. Superantigens interact with the TCR via the outer face of its Vß domain, a region not involved in conventional antigen recognition [11 ]. Staphylococcal enterotoxin B (SEB) can activate 30–40% of murine T cells to divide and produce cytokines [5 ]. Lethal toxicity results from massive induction of Th1 cytokines that include interleukin-2 (IL-2), interferon-gamma (IFN-{gamma}), and tumor necrosis factor ß (TNF-ß or LT-{alpha}) [12 13 14 ], with large amounts of TNF-{alpha} being produced by APC. Administration of recombinant human TNF-{alpha} to animals induced death within minutes to hours, as a result of respiratory arrest [15 , 16 ]. Human T cells are at least two orders of magnitude more sensitive to staphylococcal superantigens than murine ones [17 ], and although humans are sensitive to developing TSS, mice are resistant, apparently because cells that display the most highly reactive Vß chains of the TCR were deleted from the murine, T-cell repertoire, or the relevant Vß genes were eliminated [5 ].

Efforts to block downstream phenomena in the toxicity cascade set off by a pyrogenic toxin (for example, by inhibiting the action of TNF with monoclonal antibodies) did not succeed, most likely owing to the excessive, Th1-cytokine levels produced in response to superantigens. Although exogenous IL-10 inhibited murine IFN-{gamma} induction by SEB in vitro [18 ] and protected mice against SEB-induced lethal shock [19 ], it also induced long-term anergy in human CD4 cells [20 ].

We have explored the possibility of blocking the action of superantigens before activation of T cells takes place. We designed a peptide antagonist that inhibits the induction of human, Th1 cytokine gene expression by superantigens and protects mice from the lethal effects of these toxins, allowing rapid development of immunity against toxic shock. The antagonist peptide shows homology with a domain that is conserved structurally and spatially within the family of superantigens yet remote from known binding sites for the MHC class II molecule and TCR and not hitherto thought to be essential for superantigenicity.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and induction of cytokine gene expression
Peripheral blood mononuclear cells (PBMC) from healthy human donors were separated on Ficoll Paque (Pharmacia, Upsala, Sweden), washed twice with 50 ml RPMI 1640 medium, resuspended at a density of 4 x 106/ml, and cultured in this medium supplemented with 2% fetal calf serum, 2 mM glutamine, 10 mM modified Eagle’s medium (MEM), nonspecific amino acids, 100 mM Na-pyruvate, 10 mM HEPES, pH 7.2, 5 x 10-5 M 2-mercaptoethanol (2-ME), 100 units/ml penicillin, 100 µg/ml streptomycin, and 5 µg/ml nystatin. After overnight resting, PBMC were induced with 100 ng/ml SEB, lot 14–30, from the Department of Toxicology, United States Army Medical Research Institute of Infectious Diseases (USAMRIID; Frederick, MD), SPEA (Toxin Technologies, Sarasota, FL), SEA, or TSST-1 (both from Sigma Chemical Co., St. Louis, MO). When present, peptides were used at 1 µg/ml.

Ribonuclease protection analysis
Total RNA, extracted from 30-ml cultures of PBMC with guanidinium isothiocyanate, was subjected to ribonuclease protection analysis [21 ] using antisense RNA probes transcribed with [{alpha}-32P]UTP in vitro from DNA inserted into pBS (Promega, Madison, WI). The IL-2 probe [600 nucleotides (nt)], transcribed from the T7 promoter, is complementary to the third exon and a portion of the third intron; in 8-M urea-polyacrylamide gels, it yields an RNA fragment of 117 nt protected by mRNA [21 ]. The IFN-{gamma} probe (274 nt), transcribed from the T3 promoter, is complementary to the third exon and a portion of the third intron and yields an RNA fragment of 183 nt protected by mRNA [22 ]. The TNF-ß probe (700 nt), transcribed from the T3 promoter, is complementary to part of exon 1, exon 2, and exon 3, and portions of intron 3 and exon 4; mRNA protects two fragments of 274 and 263 nt [23 ]. Sense RNA transcripts yielded no detectable hybridization signal. Analysis with an antisense RNA probe for 18S rRNA, yielding a protected fragment of 90 nt, served as loading control.

Quantitative dot-blot hybridization of RNA
RNA was isolated from 1-ml cultures of PBMC by collecting the cells and lysing them in 7.5-M guanidinium-HCl. RNA, precipitated overnight in ethanol at -20°C, was dissolved into formaldehyde and incubated for 15 min at 60°C. Four serial, twofold dilutions, made in 10x saline sodium citrate, were applied in duplicate to nitrocellulose sheets using a 96-well, dot-blot apparatus. To detect expression of human IL-2 and IFN-{gamma} genes, sheets were hybridized with the 32P-labeled, antisense RNA probes. Exposed autoradiograms were scanned at 630 nm in an enzyme-linked immunosorbent assay (ELISA) reader. RNA levels were expressed in units of A630. Hybridization intensity is linear with amount of RNA and number of cells and can be quantitated accurately over a 200-fold range [22 , 24 , 25 ]. As analyzed by Northern blotting, over 85% of the IL-2 and IFN-{gamma} hybridization signals thus detected consists of mature mRNA [22 ].

Structures of superantigens
Molecular modeling was based on the atomic coordinates derived by X-ray diffraction for SEB [Protein Data Bank (PDB) code 1SEB; 26–30], SEA (PDB code 1SEA) [31 ], and TSST-1 (PDB code 1TSS) [32 ], and on the predicted atomic coordinates for SPEA (PDB code SPEA_STRPY).

Peptide synthesis
Peptides were synthesized using fluoronyl-methoxycarbonyl chemistry. They were cleaved, and the side chain was deprotected with triflouroacetic acid. In culture medium, triflouroacetic acid-peptide salts were soluble. D-Ala was linked using the same procedure. High-pressure liquid chromatography showed that peptides were >95% pure.

Protection of mice against toxic shock
Groups of 10 female, BALB/c mice (Harlan, Jerusalem, Israel), aged 10–12 weeks, were sensitized by intraperitoneal (i.p.) injection with 20 mg D-galactosamine (Sigma) at the time of challenge with a toxin, injected i.p. Except for SEB (Sigma), sources of superantigens for murine trials were as detailed for induction of cytokine-gene expression. When present, antagonist peptide was injected i.p. Survival was monitored. All experiments involving the use of mice were in accordance with protocols approved by the Animal Care and Use Committee of the Hebrew University-Hadassah Medical School (Jerusalem, Israel).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Induction of Th1 cytokine gene expression by superantigen
Exposure of human PBMC to the superantigen SEB led to induction of IL-2 and IFN-{gamma} mRNA, shown by quantitative dot-blot hybridization (Fig. 1A 1B 1C ) and RNase protection analysis (Fig. 1D) . Both methods yielded similar patterns of induction, characterized by a transient wave of IL-2 mRNA and a more prolonged expression of IFN-{gamma} mRNA. A wave of TNF-ß mRNA was induced more gradually (Fig. 1E) .



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  		Figure 1. Induction of IL-2, IFN-{gamma}, and TNF-ß gene expression by SEB. Aliquots of 4 x 106 human PBMC were induced with SEB. Total RNA was extracted at times indicated, and serial twofold dilutions (vertical rows) were subjected to dot-blot hybridization analysis with 32P-labeled IL-2 and IFN-{gamma} antisense RNA probes; autoradiograms (B and C) were quantitated by densitometry at 630 nm, plotted in A. In separate experiments, aliquots of 3 x 107 human PBMC were induced with SEB, and total RNA was extracted at times indicated. IL-2, IFN-{gamma} (D), and TNF-ß mRNA (E) were quantitated by RNase protection analysis. IL-2 mRNA protects a fragment of 117 nt; IFN-{gamma} mRNA protects a fragment of 183 nt; TNF-ß mRNA protects two fragments of 274 and 263 nt. ß-Actin RNA served as loading control.

 
Antagonist peptide blocks Th1 cytokine gene induction by SEB
The powerful ability of superantigens to activate T cells involves their tight binding to the TCR and MHC class II molecule, stabilized by interactions at multiple sites [26 27 28 29 30 ]. A short, unstructured peptide would not be expected to exhibit that property, yet we conjectured that should it compete with SEB for an essential site, it might prevent cooperative interactions. Thus, we synthesized peptides that contain all or part of amino acid residues 13–33, 41–61, 81–93, and 208–218 in SEB, essential for binding to the TCR and MHC class II molecule; residues 21–29 and 48–61, essential for binding to the TCR; and residues 13–17 and 44–52, essential for binding to the MHC class II molecule [26 27 28 29 30 ]. We also synthesized a peptide, pSEB(150–161), containing residues 150–161 of SEB (TNKKKVTAQELD), which are relatively well-conserved among superantigens yet not known to be involved in binding to TCR or MHC class II molecule [26 27 28 29 30 ], as well as a variant of this sequence, p12(150–161) (YNKKKATVQELD), and pSEB(152–161) and p10(152–161), which lack two N-terminal residues of the corresponding dodecamers.

When present in 100- to 200-fold higher molar amounts than SEB, none of these peptides had significant SEB-agonist activity, as judged by a lack of the ability to induce IL-2 or IFN-{gamma} mRNA expression in human PBMC [33 ]. The peptides were then assayed for the ability to inhibit SEB-mediated induction of human IL-2, IFN-{gamma}, and TNF-ß gene expression by direct quantitation of mRNA, using RNase protection analysis. At 100- to 200-fold molar excess over SEB, none of the peptides chosen to target SEB domains interacting with the TCR and/or MHC class II molecule was inhibitory (cf. [33 ]), but antagonist activity was exhibited by dodecapeptide p12(150–161) and, to a lesser extent, pSEB(150–161) (Fig. 2 ).



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  		Figure 2. Inhibition of SEB-mediated induction of IL-2, IFN-{gamma}, and TNF-ß mRNA by p12(150–161) and pSEB(150–161). Aliquots of 3 x 107 PBMC were induced with SEB in the absence (No peptide) or presence of 1 µg/ml of the indicated peptide (10x: 10 µg/ml). At times shown, total RNA was extracted and subjected to RNase protection analysis, using a 32P-labeled, IL-2, IFN-{gamma}, TNF-ß, or ribosomal RNA (rRNA), antisense RNA probe. Autoradiograms show levels of mRNA; rRNA served as loading control (from [33]).

 
p12(150–161) blocked the induction of IL-2 and IFN-{gamma} mRNA almost completely, whereas the peptide having the natural SEB sequence, pSEB(150–161), inhibited no more than twofold, even at a tenfold-higher concentration. Likewise, expression of TNF-ß mRNA was inhibited strongly by p12(150–161) but only weakly by pSEB(150–161) (Fig. 2) . Peptides p10(152–161) and pSEB(152–161), which lack two N-terminal residues of the corresponding dodecamers, showed lower antagonist activity [33 ]. Peptides exhibiting antagonist activity were not cytotoxic because the viability of cells was unaffected, as judged by trypan blue exclusion analysis and recovery of total cellular RNA [33 ]. Thus, p12(150–161) is a potent antagonist of SEB in vitro.

Antagonist activity of p12(150–161) was improved by abutting D-Ala residues to its N- and C-termini to render it more resistant to proteolysis, and this peptide was termed p12A [33 ]. The related man-made peptide, p14A, having two additional amino acids from the SEB sequence at its N-terminus (VQTNKKKVTAQELD) and also abutted by D-Ala residues, showed antagonist activity comparable to that of p12A in vitro (unpublished results).

Protection and rescue of mice from lethal shock
The ability of p12A and p14A to antagonize the SEB-mediated induction of Th1 cytokine gene expression in human PBMC prompted us to examine their ability to protect mice from lethal challenge with this toxin. Using the D-galactosamine-sensitized mouse, an accepted animal model for studying lethality of the superantigens [13 , 34 , 35 ], we investigated the protective activity of p12A [33 ] and p14A.

In the experiment of Figure 3 , 90% of control mice were killed within less than 20 h when exposed to SEB. However, when p14A was administered just before lethal challenge with SEB, 90% of these mice survived (Fig. 3) . A protective effect of p14A was observed reproducibly in over 15 experiments. Surviving animals showed no signs of distress and remained indistinguishable from normal controls in behavior; they survived for as long as monitored, 2 weeks. Mice exposed to p14A alone stayed fully viable and showed no detectable side effects.



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  		Figure 3. Antagonist peptide protects mice from lethal shock induced by SEB. Ten BALB/c mice were sensitized with 20 mg D-galactosamine and challenged simultaneously with 10 µg SEB ({circ}). Another 10 mice received 25 µg p14A 30 min before SEB challenge (•). Survival was monitored.

 
The protective effect was not specific to the BALB/c mouse strain. A/J mice were also protected by i.p. p12A against killing by SEB [33 ]. This indicates that protection is not narrowly MHC-restricted, in agreement with the fact that the antagonist peptide targets a superantigen domain not known to interact with the MHC class II molecule.

Remarkably, p14A was not only protective when given before SEB challenge but was able to rescue mice undergoing lethal shock even when injected as late as 7 h after the toxin (Fig. 4 ). Here, only 30% of the control mice were still alive at 24 h after exposure to SEB and 20% at later times. All of the SEB-challenged mice were protected when p14A was given at 30 min before SEB. When administration of the antagonist peptide was delayed to 3, 5, or 7 h after lethal challenge, a significant, although partial, protection was obtained. A progressively decreasing, protective effect was seen between 20 and 40 h, yielding 70%, 60%, and 50% survival, respectively.



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  		Figure 4. Antagonist peptide protects and rescues mice from SEB-induced lethal shock. Groups of 10 BALB/c mice were sensitized with 20 mg D-galactosamine and challenged simultaneously with 10 µg SEB. An aliquot of 25 µg p14A was injected at 30 min before SEB ({blacktriangleup}) or at 3 ({blacksquare}), 5 ({circ}), or 7 (•) h after SEB. Ten sensitized, naive mice that did not receive p14A served as SEB controls ({square}).

 
Protection or rescue was observed when the antagonist peptide was in 20- to 40-fold molar-excess over SEB, showing that the peptide is a potent superantigen antagonist in vivo.

Protected mice develop broad-spectrum resistance to lethal shock rapidly
To examine whether the antagonist peptide exhibits broad-spectrum, protective activity, we studied its effect during lethal challenge with SEA, a superantigen showing only 27% overall amino-acid sequence homology with SEB [36 ]. As seen in Figure 5A , p14A also protected mice from lethal challenge with SEA. We have shown [33 ] that the antagonist peptide p12A protected mice from death induced by SEB, the streptococcal toxin SPEA (having 30% homology with SEA and 48% with SEB), or TSST-1, which exhibits only 7% and 6% overall sequence homology with SEA and SEB, respectively [36 ].



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  		Figure 5. Mice protected by antagonist develop rapidly broad-spectrum immunity to lethal shock. (A) Ten BALB/c mice received p14A 30 min before sensitization with 20 mg D-galactosamine and simultaneous challenge with 10 µg SEA (•). Without further injection of p14A, the seven survivors were re-challenged after 2 weeks with 10 µg SEB ({blacktriangleup}; B) and 2 weeks later, with 10 µg TSST-1 ({blacksquare}; C) and then, 2 weeks later, with 15 µg SPEA ({blacktriangleup}; D). At each challenge, 10 sensitized, naive mice served as toxin controls ({circ}, {triangleup}, {square}).

 
In the experiment of Figure 5 , seven of the mice that had survived a lethal challenge with SEA owing to protection by p14A (Fig. 5A) were rechallenged 2 weeks later with SEB, this time in the absence of peptide antagonist (Fig. 5B) . All of these mice survived, whereas in the control group of 10 mice, none survived SEB exposure. Two weeks later, mice that had survived this second challenge with SEB were exposed once more to yet another superantigen toxin, this time TSST-1, again in the absence of peptide antagonist. Whereas all 10 mice died in the control group, only 1 out of 7 mice died in the group that had been challenged with TSST-1 (Fig. 5C) . These six survivors were challenged 2 weeks later for a fourth time, now with SPEA. Four out of six of these mice survived, whereas in the control group, only 1 out of 10 mice survived SPEA exposure. Thus, mice that were protected by p14A against SEA-mediated lethal shock had acquired resistance to subsequent toxin challenges rapidly with different superantigens.

The antagonist targets a structurally conserved, superantigen domain essential for the induction of Th1 cytokine gene expression
The finding that a peptide derived from the SEB(150–161) domain exhibits SEB antagonist activity (Fig. 2) , whereas peptides from regions known to be essential for the interaction of SEB with the TCR and/or MHC class II molecule lacked such activity [33 ] drew our attention to the location and conformation of the 12-amino acid 150–161 domain within the structure of SEB. This domain is well-removed from functionally important regions in SEB shown to participate in binding of TCR and/or MHC II [26 27 28 29 30 ], which map into the left half of the molecule (Fig. 6A ). Moreover, this domain lies outside the region sufficient for mitogenic activity, the N-terminal 138 amino acids [37 , 38 ]. Thus, the ability of p12(150–161) to act as a SEB antagonist is surprising. The SEB(150–161) domain forms a central turn starting within a ß-strand and connecting it, via another short ß-strand, to an {alpha}-helix (Fig. 6A) . The sequence in this domain, TNKKKVTAQELD, is conserved among pyrogenic toxins, with 10/12 identities for SEA, SEC1, SEC2, and SPEA, 9/12 for SEE, and 4/12 for the most remotely related member of the staphylococcal superantigen family, TSST-1 [39 ]. Although highly homologous with SEB in the 150–161 domain, SEA shows, as stated, only 27% overall sequence homology with SEB; SPEA has 48% homology with SEB and 30% with SEA, and TSST-1 has merely 6% homology with SEB [36 ]. Notwithstanding these differences in overall sequence, the 3-dimensional structures of these superantigens, resolved for SEB, SEA, and TSST-1 by X-ray diffraction and predicted for SPEA, are remarkably similar, especially in their right halves (Fig. 6A) . A structurally conserved ß-strand-hinge-{alpha}-helix domain corresponding to residues 150–161 in SEB is found in each, including TSST-1 (Fig. 6A) . Indeed, antagonist peptide p12A inhibits the induction of IL-2 and IFN-{gamma} mRNA by each of SEB, SEA, SPEA, and TSST-1 [33 ]. Our results show that the induction of Th1 cytokine gene expression by a superantigen depends critically on this domain.



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  		Figure 6. Structures of superantigens. (A) Backbone structures of SEB, SEA, SPEA, and TSST-1. Domains corresponding to residues SEB150–161 are shown as magenta ribbons. Domains in SEB that interact with TCR, MHC II, or both are shown as orange and yellow ribbons. N-terminal 138 residues in SEB are shown as orange ribbons and cyan strands; corresponding regions in SEA, SPEA, and TSST-1 are cyan. (B) Detail showing domains homologous to p12(150–161). Magenta domains as in A are SEB150–161, TNKKKVTAQELD; SEA145–156, TNKKNVTVQELD; SPEA119–130, TDKKMVTAQELD; TSST-1135–146, FDKKQLAISTLD. Boldfaced residues are shown as blue or green side chains in B. RasMol 2.6 software was used in A and Insight II software (Molecular Simulations, San Diego, CA) in B (from [33])._art>

 
The SEB(150–161) domain and its equivalents in SEA, SPEA, and TSST-1 show not only overall spatial conservation (Fig. 6A) but also a remarkable structural similarity (Fig. 6B) . In SEB, SEA, and SPEA, this domain forms a pocket in which Thr, Lys, and Glu residues are oriented to allow hydrogen bonding. In TSST-1, the core of this pocket is hydrophobic in character, containing proximal Phe, Leu, and Thr residues, with the Lys residue extending away from the pocket.

The sequence of p12(150–161) differs in several positions from the corresponding sequence in SEB. The KKK and QELD motifs are spaced equally in both, but p12(150–161) contains the hydrophobic residue Tyr where SEB contains Thr, resembling Phe more closely at this position in TSST-1 (Fig. 6B) . Thus, p12(150–161) combines features of the four superantigens in their corresponding domains. We have shown elsewhere that this peptide indeed has broad-spectrum, antagonist activity against superantigens, including TSST-1, on human PBMC and in mice [33 ].


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
The activation of Th1 cells by superantigens elicits a powerful wave of IL-2, IFN-{gamma}, and TNF-ß gene expression, as well as expression of TNF-{alpha} in the APC. Excessive expression of these cytokines leads to shock and death. Our finding is that a short peptide composed of 12 or 14 amino acids, having limited homology to a domain in superantigens not hitherto thought to be essential for their ability to induce T-cell activation and proliferation, can block Th1 cytokine gene induction and protect mice against death evoked by superantigens. The observation that the antagonist peptide inhibits not only the superantigen-mediated induction of Th1 cytokine genes but also its lethal effect supports the view that it blocks superantigenicity itself.

The action of superantigens depends on their ability to bind to the TCR and MHC class II molecule at multiple binding sites, rendering it likely that the antagonist peptide acts by competing with the superantigen domain to which it has homology to block this concerted interaction and thus to prevent the activation of Th1 cells at the top of the toxicity cascade. Most likely, the antagonist peptide interferes with the binding of a bacterial superantigen to a receptor that is required for Th1-cell activation. This receptor has yet to be identified. Interaction of a short, unstructured peptide such as p12(150–161) with this receptor may induce the peptide to fold into a conformation mimicking the corresponding domain within the intact superantigen molecule, allowing it to compete with the superantigen.

The fact that a molar excess of only 20- or 40-fold over the challenge superantigen will protect mice against lethal shock and that mice can be rescued from superantigen-induced killing that is virtually complete within 24 h even when the antagonist is given as late as 7 h after the onset of shock (Fig. 4) argue in favor of the concept that the antagonist peptide must possess a high affinity for this receptor. The antagonist peptide blocks induction of Th1-cytokine mRNAs by a superantigen yet fails to inhibit their induction by a conventional antigen such as tuberculin PPD [33 ]. Hence, the antagonist peptide exhibits specificity for superantigens and is not a general TCR or MHC class II antagonist.

The antagonist peptide shows homology to a ß-strand-hinge-{alpha}-helix domain that is conserved structurally in superantigens yet remote from known binding sites for the TCR and MHC class II molecule in each of the four superantigens studied here (Fig. 6) . For Th1-cell activation, superantigens depend on this domain because the induction in human PBMC of IL-2 and IFN-{gamma} gene expression is sensitive to inhibition by the antagonist peptide (Fig. 2) as well as to antibodies raised against it [33 ]. Conservation of domains among superantigens of the pyrogenic toxin family is, however, not unique to SEB residues 150–161, which are targeted by the antagonist peptide. SEB domains consisting of residues 76–86, 113–124, and 213–226 also show extensive sequence homology [39 ] as well as spatial conservation (unpublished results) within this family. Structural and mutational analysis (26–30) has shown that these domains function in the binding of TCR and/or MHC class II molecule. By contrast, SEB domain 150–161 was not implicated in the binding of either ligand [26 27 28 29 30 ].

Individual superantigens differ in their mode of interaction with TCR or MHC class II molecule. Thus, in TSST-1, binding of TCR requires residues on the top back of the molecule [40 ] rather than on the top front as for SEB (Fig. 6A) ; unlike SEB, SEA binds the MHC class II molecule also via residues in its beta-grasp [31 , 41 , 42 ]. Thus, it is remarkable that an antagonist peptide of man-made amino acid sequence p12(150–161) is a more effective SEB antagonist than pSEB(150–161), which has full homology with the corresponding domain in this superantigen (Fig. 2) . Apparently, by unifying features of this domain in SEB, SEA, SPEA, and TSST-1 (Fig. 6B) , p12(150–161) is a more effective competitor for the receptor.

Of note, once they have been protected by the antagonist peptide against superantigen-mediated toxic shock and death, mice develop, within less than 2 weeks, a broad-spectrum resistance to further lethal challenges by superantigens of both staphylococcal and streptococcal origin. We have shown that mice protected against SEA lethal shock by a single dose of antagonist p14A will survive, in the absence of further antagonist administration, successive lethal challenges with SEB, TSST-1, and SPEA (Fig. 5) . Apparently, when the antagonist peptide blocks induction of a cellular immune response leading to lethal toxic shock, it thereby allows the superantigen to induce a vigorous, humoral immune response directed against itself, leading to protective immunity. This immunity is broad-spectrum in nature (Fig. 5) . By adoptive transfer, we have shown that the immunity is based on protective antibodies [33 ]. We did not detect antibodies against p12A peptide in the sera of surviving mice (unpublished results). Instead, immunoglobulin (Ig)M and IgG were elicited against the challenge toxin [33 ]. Whether Th1 and Th2 responses are affected differentially by the antagonist peptide will require further study.


   ACKNOWLEDGEMENTS
 
The authors thank L. Zisu of the Bletterman Macromolecular Research Laboratory of the Hebrew University-Hadassah Medical School for synthesis of peptides, M. Katzenellenbogen for help in determination of TNF-ß mRNA, and Y. Banai for assistance.

Received December 7, 2000; revised February 5, 2001; accepted February 6, 2001.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Herman, A., Kappler, J. W., Marrack, P., Pullen, A. M. (1991) Superantigens: mechanisms of T-cell stimulation and role in immune responses Annu. Rev. Immunol. 9,745-772[Medline]
  2. Schlievert, P. M. (1993) Role of superantigens in human disease J. Infect. Dis. 167,997-1002[Medline]
  3. Murray, D. L., Ohlendorf, D. H., Schlievert, P. M. (1995) Staphylococcal and streptococcal superantigens: their role in human diseases ASM News 61,229-235
  4. Bohach, G. A., Fast, D. J., Nelson, R. D., Schlievert, P. M. (1990) Staphylococcal and streptococcal pyrogenic toxins involved in toxic shock syndrome and related illnesses Crit. Rev. Microbiol. 17,251-272[Medline]
  5. Marrack, P., Kappler, J. (1990) The staphylococcal enterotoxins and their relatives Science 248,705-711[Abstract/Free Full Text]
  6. Scholl, P., Diez, A., Mourad, W., Parsonnet, J., Geha, R. S., Chatila, T. (1989) Toxic shock syndrome toxin 1 binds to major histocompatibility complex class II molecules Proc. Natl. Acad. Sci. USA 86,4210-4214[Abstract/Free Full Text]
  7. Fraser, J. D. (1989) High-affinity binding of staphylococcal enterotoxins A and B to HLA-DR Nature 339,221-223[Medline]
  8. Choi, Y., Herman, A., DiGiusto, D., Wade, T., Marrack, P., Kappler, J. (1990) Residues of the variable region of the T-cell-receptor beta-chain that interact with S. aureus toxin superantigens Nature 346,471-473[Medline]
  9. Kappler, J., Kotzin, B., Herron, L., Gelfand, E. W., Bigler, R. D., Boylston, A., Carrel, S., Posneit, C. D., Choi, Y., Marrack, P. (1989) Vß-specific stimulation of human T cells by staphylococcal toxins Science 244,811-814[Abstract/Free Full Text]
  10. Janeway, C. A., Yagi, J., Katz, M. E., Jones, B., Vregop, S., Buxser, S. (1989) T cell responses to Mls and to bacterial proteins that mimic its behavior Immunol. Rev. 107,61-68[Medline]
  11. Choi, Y. W., Kotzin, B., Herron, L., Callahan, J., Marrack, P., Kappler, J. (1989) Interaction of Staphylococcus aureus toxin "superantigens" with human T cells Proc. Natl. Acad. Sci. USA 86,8941-8945[Abstract/Free Full Text]
  12. Marrack, P., Blackman, M., Kushnir, E., Kappler, J. (1990) The toxicity of staphylococcal enterotoxin B in mice is mediated by T cells J. Exp. Med. 171,455-464[Abstract/Free Full Text]
  13. Miethke, T., Wahl, C., Heeg, K., Echtenacher, B., Krammer, P. H., Wagner, H. (1992) T cell-mediated lethal shock triggered in mice by the superantigen staphylococcal enterotoxin B: critical role of tumor necrosis factor J. Exp. Med. 175,91-98[Abstract/Free Full Text]
  14. Hackett, S. P., Stevens, D. L. (1993) Superantigens associated with staphylococcal and streptococcal toxic shock syndrome are potent inducers of tumor necrosis factor-ß synthesis J. Infect. Dis. 168,232-235[Medline]
  15. Tracey, K. J., Beutler, B., Lowry, S. F., Merryweather, J., Wolpe, S., Milsark, I. W., Hariri, R. J., Fahey, T. J., Zentella, A., Albert, J. D., et al (1986) Shock and tissue injury induced by recombinant human cachectin Science 234,470-474[Abstract/Free Full Text]
  16. Tracey, K. J., Lowry, S. F., Fahey, T. J., Albert, J. D., Fong, Y., Hesse, D., Beutler, B., Manogue, K. R., Calvano, S., Wei, H., et al (1987) Cachectin/tumor necrosis factor induces lethal shock and stress hormone responses in the dog Surg. Gynecol. Obstet. 164,415-422[Medline]
  17. Uchiyama, T., Kamagata, Y., Yan, X. J., Kawachi, A., Fujikawa, H., Igarashi, H., Okubo, M. (1989) Relative strength of the mitogenic and interleukin-2-production-inducing activities of staphylococcal exotoxins presumed to be causative exotoxins of toxic shock syndrome: toxic shock syndrome toxin-1 and enterotoxins A, B and C to murine and human T cells Clin. Exp. Immunol. 75,239-244[Medline]
  18. Fiorentino, D. F., Zlotnik, A., Vieira, P., Mosmann, T. R., Howard, M., Moore, K. W., O’Garra, A. (1991) IL-10 acts on the antigen-presenting cell to inhibit cytokine production by Th1 cells J. Immunol. 146,3444-3451[Abstract]
  19. Bean, A. G., Freiberg, R. A., Andrade, S., Menon, S., Zlotnik, A. (1993) Interleukin 10 protects mice against staphylococcal enterotoxin B-induced lethal shock Infect. Immun. 61,4937-4939[Abstract/Free Full Text]
  20. Groux, H., Bigler, M., de Vries, J. E., Roncarolo, M. G. (1996) Interleukin-10 induces a long-term antigen-specific anergic state in human CD4+ T cells J. Exp. Med. 184,19-29[Abstract/Free Full Text]
  21. Gerez, L., Arad, G., Efrat, S., Ketzinel, M., Kaempfer, R. (1995) Post-transcriptional regulation of human interleukin-2 gene expression at processing of precursor transcripts J. Biol. Chem. 270,19569-19575[Abstract/Free Full Text]
  22. Arad, G., Ketzinel, M., Tal, C., Nussinovich, R., Deutsch, E., Schlesinger, M., Gerez, L., Kaempfer, R. (1995) Transient expression of human interleukin-2 and interferon-{gamma} genes is regulated by interaction between distinct cell subsets Cell. Immunol. 160,240-247[Medline]
  23. Osman, F., Jarrous, N., Ben-Asouli, Y., Kaempfer, R. (1999) A cis-acting element in the 3'-untranslated region of human TNF-{alpha} mRNA renders splicing dependent on the activation of protein kinase PKR Genes Dev 13,3280-3293[Abstract/Free Full Text]
  24. Gerez, L., Madar, L., Arad, G., Sharav, T., Reshef, A., Ketzinel, M., Sayar, D., Silberberg, C., Kaempfer, R. (1991) Aberrant regulation of interleukin-2 but not of interferon-{gamma} gene expression in Down Syndrome (Trisomy 21) Clin. Immunol. Immunopathol. 58,251-266[Medline]
  25. Kaempfer, R., Gerez, L., Farbstein, H., Madar, L., Hirschman, O., Nussinovich, R., Shapiro, A. (1996) Prediction of response to treatment in superficial bladder carcinoma through pattern of interleukin-2 gene expression J. Clin. Oncol. 14,1778-1786[Abstract/Free Full Text]
  26. Swaminathan, S., Furey, W., Pletcher, J., Sax, M. (1992) Crystal structure of staphylococcal enterotoxin B, a superantigen Nature 359,801-805[Medline]
  27. Jardetzky, T. S., Brown, J. H., Gorga, J. C., Stern, L. J., Urban, R. G., Chi, Y. I., Stauffacher, C., Strominger, J. L., Wiley, D. C. (1994) Three-dimensional structure of a human class II histocompatibility molecule complexed with superantigen Nature 368,711-718[Medline]
  28. Li, H., Llera, A., Tsuchiya, D., Leder, L., Ysern, X., Schlievert, P. M., Karjalainen, K., Mariuzza, R. A. (1998) Three-dimensional structure of the complex between a T cell receptor beta chain and the superantigen staphylococcal enterotoxin B Immunity 9,807-816[Medline]
  29. Papageorgiou, A. C., Tranter, H. S., Acharya, K. R. (1998) Crystal structure of microbial superantigen staphylococcal enterotoxin B at 1.5 A resolution: implications for superantigen recognition by MHC class II molecules and T-cell receptors J. Mol. Biol. 277,61-79[Medline]
  30. Leder, L., Llera, A., Lavoie, P. M., Lebedeva, M. I., Li, H., Sekaly, R. P., Bohach, G. A., Gahr, P. J., Schlievert, P. M., Karjalainen, K., Mariuzza, R. A. (1998) A mutational analysis of the binding of staphylococcal enterotoxins B and C3 to the T cell receptor ß chain and major histocompatibility complex class II J. Exp. Med. 187,823-833[Abstract/Free Full Text]
  31. Schad, E. M., Zaitseva, I., Zaitsev, V. N., Dohlsten, M., Kalland, T., Schlievert, P. M., Ohlendorf, D. H., Svensson, L. A. (1995) Crystal structure of the superantigen staphylococcal enterotoxin type A EMBO J 14,3292-3301[Medline]
  32. Prasad, G. S., Earhart, C. A., Murray, D. L., Novick, R. P., Schlievert, P. M., Ohlendorf, D. H. (1993) Structure of toxic shock syndrome toxin 1 Biochemistry 32,13761-13766[Medline]
  33. Arad, G., Levy, R., Hillman, D., Kaempfer, R. (2000) Superantigen antagonist protects against lethal shock and defines a new domain for T-cell activation Nat. Med. 6,414-421[Medline]
  34. Galanos, C., Freudenberg, M. A., Reutter, W. (1979) Galactosamine-induced sensitization to the lethal effects of endotoxin Proc. Natl. Acad. Sci. USA 76,5939-5943[Abstract/Free Full Text]
  35. Lowell, G. H., Kaminsky, R. W., Grate, S., Hunt, R. E., Charney, C., Zimmer, S., Colleton, C. (1996) Intranasal and intramuscular proteosome-staphylococcal enterotoxin B (SEB) toxoid vaccines: immunogenicity and efficacy against lethal SEB intoxication in mice Infect. Immun. 64,1706-1713[Abstract]
  36. Betley, M. J., Mekalanos, J. J. (1988) Nucleotide sequence of the type A staphylococcal enterotoxin gene J. Bacteriol. 170,34-41[Abstract/Free Full Text]
  37. Buelow, R., O’Hehir, R. E., Schreifels, R., Kummerehl, T. J., Riley, G., Lamb, J. R. (1992) Localization of the immunologic activity in the superantigen Staphylococcal enterotoxin B using truncated recombinant fusion proteins J. Immunol. 148,1-6[Abstract]
  38. Kappler, J. W., Herman, A., Clements, J., Marrack, P. (1992) Mutations defining functional regions of the superantigen staphylococcal enterotoxin B J. Exp. Med. 175,387-389[Abstract/Free Full Text]
  39. Hoffmann, M. L., Jablonski, L. M., Crum, K. K., Hackett, S. P., Chi, Y. I., Stauffacher, C. V., Stevens, D. L., Bohach, G. A. (1994) Predictions of T-cell receptor- and major histocompatibility complex-binding sites on staphylococcal enterotoxin C1 Infect. Immun. 62,3396-3407[Abstract/Free Full Text]
  40. Earhart, C. A., Mitchell, D. T., Murray, D. L., Pinheiro, D. M., Matsumura, M., Schlievert, P. M., Ohlendorf, D. H. (1998) Structures of five mutants of toxic shock syndrome toxin-1 with reduced biological activity Biochemistry 37,7194-7202[Medline]
  41. Hudson, K. R., Tiedemann, R. E., Urban, R. G., Lowe, S. C., Strominger, J. L., Fraser, J. D. (1995) Staphylococcal enterotoxin A has two cooperative binding sites on major histocompatibility complex class II J. Exp. Med. 182,711-720[Abstract/Free Full Text]
  42. Abrahmsen, L., Dohlsten, M., Segren, S., Bjork, P., Jonsson, E., Kalland, T. (1995) Characterization of two distinct MHC class II binding sites in the superantigen staphylococcal enterotoxin A EMBO J 14,2978-2986[Medline]



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