Dalia Hillman

Superantigen antagonist protects against lethal shock and defines a newdomain for T-cell activation

Superantigens trigger an excessive cellular immune response, leading to toxic shock. We have designed a peptide antagonist that inhibits superantigen-induced expression of human genes for interleukin-2, gamma interferon and tumor necrosis factor-b, which are cytokines that mediate shock. The peptide shows homology to a b-strand–hinge–a-helix domain that is structurally conserved in superantigens, yet is remote from known binding sites for the major histocompatibility class II molecule and T-cell receptor. Superantigens depend on this domain for T-cell activation. The peptide protected mice against lethal challenge with staphylococcal and streptococcal superantigens. Moreover, it rescued mice undergoing toxic shock. Surviving mice rapidly developed protective antibodies against superantigen that rendered them resistant to further lethal challenges, even with different superantigens. Thus, the lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the beginning of the toxicity cascade, before activation of T cells takes place.

Binding of superantigen toxins into the CD28 homodimer interface is essential for induction of cytokine genes that mediate lethal shock.

Bacterial superantigen toxins bind directly to the dimer interface of CD28, the principal co-stimulatory receptor, to induce a lethal cytokine storm, and peptides that prevent this binding can suppress superantigen lethality.

Superantigen antagonist blocks Th1 cytokine gene induction and lethal shock

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‐γ, and TNF‐β, cytokines that mediate shock. The peptide antagonist shows homology to a β‐strand‐hinge‐α‐helix domain that is conserved structurally in superantigens produced by Staphylococcus aureus andStreptococcus 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.

CD28: Direct and Critical Receptor for Superantigen Toxins

Every adaptive immune response requires costimulation through the B7/CD28 axis, with CD28 on T-cells functioning as principal costimulatory receptor. Staphylococcal and streptococcal superantigen toxins hyperstimulate the T-cell-mediated immune response by orders of magnitude, inducing a lethal cytokine storm. We show that to elicit an inflammatory cytokine storm and lethality, superantigens must bind directly to CD28. Blocking access of the superantigen to its CD28 receptor with peptides mimicking the contact domains in either toxin or CD28 suffices to protect mice effectively from lethal shock. Our finding that CD28 is a direct receptor of superantigen toxins broadens the scope of microbial pathogen recognition mechanisms.

Superantigens hyperinduce inflammatory cytokines by enhancing the B7-2/CD28 costimulatory receptor interaction

Significance Superantigens—bacterial virulence factors—cause toxic shock by hyperinducing inflammatory cytokines. T-cell activation is mediated both by antigen and by interaction between principal costimulatory receptors B7-2 and CD28. Superantigens must bind CD28 to elicit cytokine overexpression through a hitherto unknown mechanism. We show that, by binding not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the B7-2/CD28 interaction, thereby inducing T-cell hyperactivation. Superantigens engage B7-2 and CD28 at their homodimer interfaces, far from where the receptors interact, demonstrating the regulatory properties of these interfaces. B7-2 dimer interface peptides attenuate cytokine overexpression and prevent superantigen lethality by blocking costimulatory receptor engagement by superantigen. Thus, bacterial superantigens induce a pathogenic “cytokine storm” by strongly enhancing formation of the B7-2/CD28 costimulatory axis. Full T-cell activation requires interaction between the costimulatory receptors B7-2 and CD28. By binding CD28, bacterial superantigens elicit harmful inflammatory cytokine overexpression through an unknown mechanism. We show that, by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the avidity between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using the same 12-aa β-strand-hinge-α-helix domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, areas remote from where these coreceptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm not only by mediating the interaction between MHC-II molecule and T-cell receptor but also, critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our results reveal a role for B7-2 as obligatory receptor for superantigens. B7-2 homodimer interface mimotopes prevent superantigen lethality by blocking the superantigen–host costimulatory receptor interaction.

CD28 Homodimer Interface Mimetic Peptide Acts as a Preventive and Therapeutic Agent in Models of Severe Bacterial Sepsis and Gram-Negative Bacterial Peritonitis

BACKGROUND Severe gram-negative bacterial infections and sepsis are major causes of morbidity and mortality. Dysregulated, excessive proinflammatory cytokine expression contributes to the pathogenesis of sepsis. A CD28 mimetic peptide (AB103; previously known as p2TA) that attenuates CD28 signaling and T-helper type 1 cytokine responses was tested for its ability to increase survival in models of polymicrobial infection and gram-negative sepsis. METHODS Mice received AB103, followed by an injection of Escherichia coli 0111:B4 lipopolysaccharide (LPS); underwent induction E. coli 018:K1 peritonitis induction, followed by treatment with AB103; or underwent cecal ligation and puncture (CLP), followed by treatment with AB103. The effects of AB103 on factors associated with and the lethality of challenge infections were analyzed. RESULTS AB103 strongly attenuated induction of tumor necrosis factor α and interleukin 6 (IL-6) by LPS in human peripheral blood mononuclear cells. Receipt of AB103 following intraperitoneal injection of LPS resulted in survival among 73% of CD1 mice (11 of 15), compared with 20% of controls (3 of 15). Suboptimal doses of antibiotic alone protected 20% of mice (1 of 5) from E. coli peritonitis, whereas 100% (15 of 15) survived when AB103 was added 4 hours following infection. Survival among mice treated with AB103 12 hours after CLP was 100% (8 of 8), compared with 17% among untreated mice (1 of 6). In addition, receipt of AB103 12 hours after CLP attenuated inflammatory cytokine responses and neutrophil influx into tissues and promoted bacterial clearance. Receipt of AB103 24 hours after CLP still protected 63% of mice (5 of 8). CONCLUSIONS Single-dose AB103 reduces mortality in experimental models of polymicrobial and gram-negative bacterial infection and sepsis, warranting further studies of this agent in clinical trials.

Bacterial superantigen toxins induce a lethal cytokine storm by enhancing B7-2/CD28 costimulatory receptor engagement, a critical immune checkpoint

Formation of the costimulatory axis between the B7-2 and CD28 coreceptors is critical for T-cell activation. Superantigens, Gram-positive bacterial virulence factors, cause toxic shock and sepsis by hyperinducing inflammatory cytokines. We report a novel role for costimulatory receptors CD28 and B7-2 as obligatory receptors for superantigens, rendering them therapeutic targets. We show that by engaging not only CD28 but also its coligand B7-2 directly, superantigens potently enhance the interaction between B7-2 and CD28, inducing thereby T-cell hyperactivation. Using a conserved twelve amino-acid domain, superantigens engage both B7-2 and CD28 at their homodimer interfaces, sites far removed from where these receptors interact, implying that inflammatory signaling can be controlled through the receptor homodimer interfaces. Short B7-2 and CD28 dimer interface mimetic peptides bind diverse superantigens, prevent superantigen binding to cell-surface B7-2 or CD28, attenuate inflammatory cytokine overexpression, and protect mice from lethal superantigen challenge. Thus, superantigens induce a cytokine storm by mediating not only the interaction between MHC-II molecule and T-cell receptor but critically, by promoting B7-2/CD28 coreceptor engagement, forcing the principal costimulatory axis to signal excessively. Our findings highlight the B7/CD28 interaction as a bottleneck in signaling for expression of inflammatory cytokines. B7-2 and CD28 homodimer interface mimetic peptides prevent superantigen lethality by blocking the superantigen-host costimulatory receptor interaction.