The homodimer interfaces of costimulatory receptors B7 and CD28 control their engagement and pro-inflammatory signaling.

BACKGROUND: The inflammatory response is indispensable for protective immunity, yet microbial pathogens often trigger an excessive response, 'cytokine storm', harmful to the host. Full T-cell activation requires interaction of costimulatory receptors B7-1(CD80) and B7-2(CD86) expressed on antigen-presenting cells with CD28 expressed on the T cells. We created short peptide mimetics of the homodimer interfaces of the B7 and CD28 receptors and examined their ability to attenuate B7/CD28 coligand engagement and signaling through CD28 for inflammatory cytokine induction in human immune cells, and to protect from lethal toxic shock in vivo. METHODS: Short B7 and CD28 receptor dimer interface mimetic peptides were synthesized and tested for their ability to attenuate the inflammatory cytokine response of human peripheral blood mononuclear cells, as well as for their ability to attenuate B7/CD28 intercellular receptor engagement. Mice were used to test the ability of such peptides to protect from lethal superantigen toxin challenge when administered in molar doses far below the toxin dose. RESULTS: B7 and CD28 homodimer interfaces are remote from the coligand binding sites, yet our finding is that by binding back into the receptor dimer interfaces, short dimer interface mimetic peptides inhibit intercellular B7-2/CD28 as well as the tighter B7-1/CD28 engagement, attenuating thereby pro-inflammatory signaling. B7 mimetic peptides exhibit tight selectivity for the cognate receptor in inhibiting intercellular receptor engagement with CD28, yet each diminishes signaling through CD28. In a prominent example of inflammatory cytokine storm, by attenuating formation of the B7/CD28 costimulatory axis, B7-1 and CD28 dimer interface mimetic peptides protect mice from lethal toxic shock induced by a bacterial superantigen even when administered in doses far submolar to the superantigen. CONCLUSIONS: Our results reveal that the B7 and CD28 homodimer interfaces each control B7/CD28 costimulatory receptor engagement and highlight the protective potential against cytokine storm of attenuating, yet not ablating, pro-inflammatory signaling via these receptor domains.

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

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 ß-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.

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

Bacterial superantigens, a diverse family of toxins, induce an inflammatory cytokine storm that can lead to lethal shock. CD28 is a homodimer expressed on T cells that functions as the principal costimulatory ligand in the immune response through an interaction with its B7 coligands, yet we show here that to elicit inflammatory cytokine gene expression and toxicity, superantigens must bind directly into the dimer interface of CD28. Preventing access of the superantigen to CD28 suffices to block its lethality. Mice were protected from lethal superantigen challenge by short peptide mimetics of the CD28 dimer interface and by peptides selected to compete with the superantigen for its binding site in CD28. Superantigens use a conserved ß-strand/hinge/α-helix domain of hitherto unknown function to engage CD28. Mutation of this superantigen domain abolished inflammatory cytokine gene induction and lethality. Structural analysis showed that when a superantigen binds to the T cell receptor on the T cell and major histocompatibility class II molecule on the antigen-presenting cell, CD28 can be accommodated readily as third superantigen receptor in the quaternary complex, with the CD28 dimer interface oriented towards the ß-strand/hinge/α-helix domain in the superantigen. Our findings identify the CD28 homodimer interface as a critical receptor target for superantigens. The novel role of CD28 as receptor for a class of microbial pathogens, the superantigen toxins, broadens the scope of pathogen recognition mechanisms.

A peptide antagonist of CD28 signaling attenuates toxic shock and necrotizing soft-tissue infection induced by Streptococcus pyogenes.

Staphylococcus aureus and group A Streptococcus pyogenes (GAS) express superantigen (SAg) exotoxin proteins capable of inducing lethal shock. To induce toxicity, SAgs must bind not only to the major histocompatibility complex II molecule of antigen-presenting cells and the variable ß chain of the T-cell receptor but also to the dimer interface of the T-cell costimulatory receptor CD28. Here, we show that the CD28-mimetic peptide AB103 (originally designated "p2TA") protects mice from lethal challenge with streptococcal exotoxin A, as well as from lethal GAS bacterial infection in a murine model of necrotizing soft-tissue infection. Administration of a single dose of AB103 increased survival when given up to 5 hours after infection, reduced inflammatory cytokine expression and bacterial burden at the site of infection, and improved muscle inflammation in a dose-dependent manner, without compromising cellular and humoral immunity. Thus, AB103 merits further investigation as a potential therapeutic in SAg-mediated necrotizing soft-tissue infection.

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.

Superantigen concomitantly induces Th1 cytokine genes and the ability to shut off their expression on re-exposure to superantigen.

Superantigens, exemplified by staphylococcal enterotoxin B (SEB), are the strongest known inducers of a cellular immune response; they elicit the production of excessive amounts of Th1 cytokines, IL-2, IFN-gamma and TNF, leading to toxic shock. We show that increasing doses of SEB cause not only a greater induction but also a more rapid cessation of IL-2 gene expression. Remarkably, exposure of human PBMC to a second dose of SEB, even at concentrations 10- or 100-fold lower than the initial inducing dose and even within 2 h after the first exposure to SEB, resulted in an immediate and essentially complete shutoff of the induced IL-2 and IFN-gamma mRNA expression. The shutoff response was observed when primary induction of IL-2 and IFN-gamma gene expression was by SEB but not when it was by phytohemaggutinin-P. Signaling by a superantigen thus results not only in a vigorous induction of Th1 cytokine genes but concomitantly induces the ability to shut off their expression upon re-exposure to superantigen. Without induction of this negative control mechanism, the cellular immune response to a superantigen would be even more pronounced.

Broad-spectrum immunity against superantigens is elicited in mice protected from lethal shock by a superantigen antagonist peptide.

Bypassing the restricted presentation of conventional antigens, superantigens trigger an excessive cellular immune response leading to toxic shock. Antagonist peptides that inhibit the induction of human Th1 cytokine gene expression by a variety of bacterial superantigens protect mice from lethal toxic shock. We show that the surviving mice rapidly develop a broad-spectrum, protective immunity against further lethal toxin challenges with the same superantigen and even with superantigen toxins that they have not encountered before. By blocking the induction of a cellular immune response leading to toxic shock, the antagonist peptide allows the superantigen to induce a vigorous humoral immune response directed against itself, resulting in anti-toxin IgM and IgG that are broadly protective. Adoptive transfer of these antibodies to naïve mice rendered them resistant to lethal superantigen challenge. The appearance of these antibodies does not require immunization with an adjuvant and is not elicited by the antagonist peptide. Our results show that superantigens are potent immunogens when given the opportunity to induce a B cell response, in conditions where a deleterious Th1 response is prevented by the superantigen antagonist peptide.

Defense against biologic warfare with superantigen toxins.

BACKGROUND: Superantigens produced by Staphylococcus aureus and Streptococcus pyogenes are among the most lethal of toxins. Toxins in this family trigger an excessive cellular immune response leading to toxic shock. OBJECTIVES: To design an antagonist that is effective in vivo against a broad spectrum of superantigen toxins. METHODS: Short peptide antagonists were selected for their ability to inhibit superantigen-induced expression of human genes for cytokines that mediate shock. The ability of these peptides to protect mice against lethal toxin challenge was examined. RESULTS: Antagonist peptide protected mice against lethal challenge with staphylococcal enterotoxin B and toxic shock syndrome toxin-1, superantigens that share only 6% overall amino acid homology. Moreover, it rescued mice undergoing toxic shock. Antagonist peptides show homology to a beta-strand/hinge/alpha-helix domain that is structurally conserved among superantigens, yet remote from known binding sites for the major histocompatibility class II molecule and T cell receptor that function in toxic T cell hyperactivation. CONCLUSIONS: The lethal effect of superantigens can be blocked with a peptide antagonist that inhibits their action at the top of the toxicity cascade before activation of T cells occurs. Superantigenic toxin antagonists may serve not only as countermeasures to biologic warfare but may be useful in the treatment of staphylococcal and streptococcal toxic shock, as well as in some cases of septic shock.

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.