Chimeric Anti-Staphylococcal Enterotoxin B Antibodies and Lovastatin Act Synergistically to Provide In Vivo Protection against Lethal Doses of SEB

Staphylococcal enterotoxin B (SEB) is one of a family of toxins secreted by Staphylococcus aureus that act as superantigens, activating a large fraction of the T-cell population and inducing production of high levels of inflammatory cytokines that can cause toxic shock syndrome (TSS) and death. Extracellular engagement of the TCR of T-cells and class II MHC of antigen presenting cells by SEB triggers the activation of many intracellular signaling processes. We engineered chimeric antibodies to block the extracellular engagement of cellular receptors by SEB and used a statin to inhibit intracellular signaling. Chimeric human-mouse antibodies directed against different neutralizing epitopes of SEB synergistically inhibited its activation of human T-cells in vitro. In the in vivo model of lethal toxic shock syndrome (TSS) in HLA-DR3 transgenic mice, two of these antibodies conferred significant partial protection when administered individually, but offered complete protection in a synergistic manner when given together. Similarly, in vivo, lovastatin alone conferred only partial protection from TSS similar to single anti-SEB antibodies. However, used in combination with one chimeric neutralizing anti-SEB antibody, lovastatin provided complete protection against lethal TSS in HLA-DR3 transgenic mice. These experiments demonstrate that in vivo protection against lethal doses of SEB can be achieved by a statin of proven clinical safety and chimeric human-mouse antibodies, agents now widely used and known to be of low immunogenicity in human hosts

NOTCH1 can initiate NF-κB activation via cytosolic interactions with components of the T cell Signalosome.

T cell stimulation requires the input and integration of external signals. Signaling through the T cell receptor (TCR) is known to induce formation of the membrane-tethered CBM complex, comprising CARMA1, BCL10, and MALT1, which is required for TCR-mediated NF-κB activation. TCR signaling has been shown to activate NOTCH proteins, transmembrane receptors also implicated in NF-κB activation. However, the link between TCR-mediated NOTCH signaling and early events leading to induction of NF-κB activity remains unclear. In this report, we demonstrate a novel cytosolic function for NOTCH1 and show that it is essential to CBM complex formation. Using a model of skin allograft rejection, we show in vivo that NOTCH1 acts in the same functional pathway as PKCθ, a T cell-specific kinase important for CBM assembly and classical NF-κB activation. We further demonstrate in vitro NOTCH1 associates physically with PKCθ and CARMA1 in the cytosol. Unexpectedly, when NOTCH1 expression was abrogated using RNAi approaches, interactions between CARMA1, BCL10, and MALT1 were lost. This failure in CBM assembly reduced inhibitor of kappa B alpha phosphorylation and diminished NF-κB-DNA binding. Finally, using a luciferase gene reporter assay, we show the intracellular domain of NOTCH1 can initiate robust NF-κB activity in stimulated T cells, even when NOTCH1 is excluded from the nucleus through modifications that restrict it to the cytoplasm or hold it tethered to the membrane. Collectively, these observations provide evidence that NOTCH1 may facilitate early events during T cell activation by nucleating the CBM complex and initiating NF-κB signaling

Potent Neutralization of Staphylococcal Enterotoxin B by Synergistic Action of Chimeric Antibodies▿

Staphylococcal enterotoxin B (SEB), a shock-inducing exotoxin synthesized by Staphylococcus aureus, is an important cause of food poisoning and is a class B bioterrorism agent. SEB mediates antigen-independent activation of a major subset of the T-cell population by cross-linking T-cell receptors (TCRs) with class II major histocompatibility complex (MHC-II) molecules of antigen-presenting cells, resulting in the induction of antigen independent proliferation and cytokine secretion by a significant fraction of the T-cell population. Neutralizing antibodies inhibit SEB-mediated T-cell activation by blocking the toxin's interaction with the TCR or MHC-II and provide protection against the debilitating effects of this superantigen. We derived and searched a set of monoclonal mouse anti-SEB antibodies to identify neutralizing anti-SEB antibodies that bind to different sites on the toxin. A pair of non-cross-reactive, neutralizing anti-SEB monoclonal antibodies (MAbs) was found, and a combination of these antibodies inhibited SEB-induced T-cell proliferation in a synergistic rather than merely additive manner. In order to engineer antibodies more suitable than mouse MAbs for use in humans, the genes encoding the VL and VH gene segments of a synergistically acting pair of mouse MAbs were grafted, respectively, onto genes encoding the constant regions of human Igκ and human IgG1, transfected into mammalian cells, and used to generate chimeric versions of these antibodies that had affinity and neutralization profiles essentially identical to their mouse counterparts. When tested in cultures of human peripheral blood mononuclear cells or splenocytes derived from HLA-DR3 transgenic mice, the chimeric human-mouse antibodies synergistically neutralized SEB-induced T-cell activation and cytokine production

Synergistic neutralization /inhibition of SEB-mediated T-cell activation by a combination of anti-SEB antibody and lovastatin.

<p>(<b>A</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in BALB/c splenocytes. BALB/c splenocytes were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 100 ng/ml SEB; Hu IgG1κ+SEB, 10 µg/ml Human IgG1κ+100 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+100 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+100 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg /ml chimeric anti-SEB 82 M+2.5 µM lovastatin+100 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M+lovastatin was significantly more inhibitory than either agent alone (P<0.01). (<b>B</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in HLA-DR3 transgenic mice splenocytes. HLA-DR3 transgenic mice splenocytes were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 10 ng/ml SEB; HuIgG1κ+SEB, 10 µg/ml Human IgG1κ+10 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+10 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+10 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg/ml chimeric anti-SEB 82 M+2.5 µM lovastatin+10 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M and lovastatin was significantly more inhibitory than either agent alone (P<0.01). (<b>C</b>) Anti-SEB Ch 82 M and lovastatin inhibit SEB action in human PBMCs. PBMCs were cultured as outlined in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0027203#s4" target="_blank">materials and methods</a> with the following additives: Medium alone, SEB alone, 1 ng/ml SEB; HuIgG1κ+SEB, 10 µg/ml Human IgG1κ+1 ng/ml of SEB; Ch 82 M+SEB, 10 µg/ml chimeric anti-SEB 82 M+1 ng/ml SEB; Lova+SEB, 2.5 µM lovastatin+1 ng/ml SEB; Ch 82 M+Lova+SEB, 10 µg/ml chimeric anti-SEB 82 M+2.5 µM lovastatin+1 ng/ml SEB. Each bar represents the means ± s.d. of triplicate measurements, and the data shown are representative of two or more independent experiments. The combination of Ch 82 M and lovastatin was significantly more inhibitory than either agent alone (P<0.01).</p

Chimeric anti-SEB 82M and lovastain provide <i>in vivo</i> protection against SEB toxicity in HLA-DR3 transgenic mice.

<p>(<b>A</b>) Inhibition of SEB-mediated hypothermic effect. Age-matched HLA-DR3 transgenic mice were injected with the following: Ch 82 M+Lova+SEB (♦), 1 mg chimeric 82 M+1 mg lovastatin+50 µg SEB; Ch 82 M+SEB (▪), 1 mg chimeric 82 M+50 µg SEB; Lova+SEB (▴), 1 mg lovastatin +50 µg SEB; SEB alone (×), 50 µg SEB in PBS. Rectal temperatures were recorded at the indicated time points. Error bars are the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>B</b>–<b>D</b>) Inhibition of SEB-induced cytokine production. Serum levels of interleukin-2 (<b>B</b>), interferon-γ (<b>C</b>) and interleukin-6 (<b>D</b>) were determined at 6 hours post-SEB injection in groups of age-matched HLA-DR3 transgenic mice treated with the following: naïve, PBS only; Ch 82 M + SEB, 1 mg Ch 82 M+50 µg SEB; Lova + SEB, 1 mg lovastatin +50 µg SEB; Ch 82 M + Lova + SEB, 1 mg Ch 82 M+1 mg lovastatin+50 µg SEB; SEB alone, 50 µg SEB in PBS. Differences between the combination of Ch 82 M and lovastatin and either drug or antibody alone were significant (P<0.05) in inhibiting SEB-induced cytokine production. Each bar represents the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>E</b>) Protection against SEB-mediated death. Survival was monitored within groups of age-matched HLA-DR3 transgenic mice receiving the following: SEB alone, 50 µg SEB in PBS; Ch 82+SEB, 1 mg of Ch 82 M+50 µg SEB; Lova+SEB, 1 mg of lovastatin+50 µg SEB; Ch 82 M+Lova + SEB, 1 mg Ch 82 M+1 mg lovastatin+50 µg SEB. Survival was monitored for 7 days. Combination of Ch 82M and lovastatin provided statistically significant protection (P<0.01) against SEB-induced death compared with mice treated with SEB alone. The data shown are representative of two or more independent experiments.</p

Chimeric anti-SEB 82 M and 63 synergistically protect HLA-DR3 mice from the toxic effects of SEB.

<p>(<b>A</b>) Protection against SEB-induced hypothermia. Age-matched HLA-DR3 transgenic mice were injected with the following: Ch 82 M+Ch 63+SEB (♦), 500 µg chimeric 82 M+500 µg chimeric 63+50 µg SEB; Ch 63+SEB (▪), 1 mg chimeric 63+50 µg SEB; Ch 82 M+SEB (▴),1 mg chimeric 82 M+50 µg SEB; Hu IgG1κ+SEB (×), 1 mg human IgG1κ+50 µg SEB; SEB alone (*); 50 µg SEB in PBS. Rectal temperatures were recorded at the indicated time points. Error bars are the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. <b>(B</b>–<b>D)</b> Inhibition of SEB-induced cytokine production. Serum levels of interleukin-2 (<b>B</b>), interferon-γ (<b>C</b>), and interleukin-6 (<b>D</b>) were determined at 6 hours post-SEB injection in groups of age-matched HLA-DR3 transgenic mice treated with the following: naïve, PBS only; Ch 82 M+SEB, 1 mg Ch 82 M+50 µg SEB; Ch 63+SEB, 1 mg Ch 63+50 µg SEB; Ch 82 M+Ch 63+SEB, 500 µg Ch 82 M+500 µg Ch 63+50 µg SEB; SEB alone, 50 µg of SEB in PBS. Differences between the combination of anti-SEBs and either antibody used alone were significant (P<0.05) in inhibiting SEB-induced cytokine production. Each bar represents the means ± s.d. for each group, and the data shown are representative of two or more independent experiments. (<b>E</b>) Appearance of mice protected with anti-SEB and unprotected mice 6 hours after a 50 µg dose of SEB. The mouse on the left received no protective antibody, suffered hyperthermia, shivered and displayed hunched posture and a rough coat. The mice on the right, which were treated with 50 µg SEB+500 µg Ch 82 M+500 µg Ch 63 appeared normal, were sleek of coat and animated. (<b>F</b>) Protection against SEB-mediated death. Survival was monitored within groups of age-matched HLA-DR3 transgenic mice receiving the following: SEB alone, 50 µg SEB in PBS; Hu IgG1κ+SEB, 1 mg human IgG1κ+50 µg SEB; Ch 82 M+SEB, 1 mg Ch 82 M+50 µg SEB; Ch 63+SEB, 1 mg Ch 63+50 µg SEB; Ch 82 M+Ch 63+SEB, 500 µg Ch 82 M+500 µg Ch 63+50 µg SEB. Combination of Ch 82 M and Ch 63 provided statistically significant protection (P<0.001) against SEB-induced death compared with untreated controls. The data shown are representative of two or more independent experiments.</p

An allosteric site in the T-cell receptor Cβ domain plays a critical signalling role.

The molecular mechanism through which the interaction of a clonotypic αβ T-cell receptor (TCR) with a peptide-loaded major histocompatibility complex (p/MHC) leads to T-cell activation is not yet fully understood. Here we exploit a high-affinity TCR (B4.2.3) to examine the structural changes that accompany binding to its p/MHC ligand (P18-I10/H2-Dd). In addition to conformational changes in complementarity-determining regions (CDRs) of the TCR seen in comparison of unliganded and bound X-ray structures, NMR characterization of the TCR β-chain dynamics reveals significant chemical shift effects in sites removed from the MHC-binding site. Remodelling of electrostatic interactions near the Cβ H3 helix at the membrane-proximal face of the TCR, a region implicated in interactions with the CD3 co-receptor, suggests a possible role for an allosteric mechanism in TCR signalling. The contribution of these TCR residues to signal transduction is supported by mutagenesis and T-cell functional assays

Mouse Cytomegalovirus m153 Protein Stabilizes Expression of the Inhibitory NKR-P1B Ligand Clr-b

ABSTRACT Natural killer (NK) cells are a subset of innate lymphoid cells (ILC) capable of recognizing stressed and infected cells through multiple germ line-encoded receptor-ligand interactions. Missing-self recognition involves NK cell sensing of the loss of host-encoded inhibitory ligands on target cells, including MHC class I (MHC-I) molecules and other MHC-I-independent ligands. Mouse cytomegalovirus (MCMV) infection promotes a rapid host-mediated loss of the inhibitory NKR-P1B ligand Clr-b (encoded by ) on infected cells. Here we provide evidence that an MCMV m145 family member, m153, functions to stabilize cell surface Clr-b during MCMV infection. Ectopic expression of m153 in fibroblasts augments Clr-b cell surface levels. Moreover, infections using -deficient MCMV mutants (Δm144-m158 and Δm153) show an accelerated and exacerbated Clr-b downregulation. Importantly, enhanced loss of Clr-b during Δm153 mutant infection reverts to wild-type levels upon exogenous m153 complementation in fibroblasts. While the effects of m153 on Clr-b levels are independent of transcription, imaging experiments revealed that the m153 and Clr-b proteins only minimally colocalize within the same subcellular compartments, and tagged versions of the proteins were refractory to coimmunoprecipitation under mild-detergent conditions. Surprisingly, the Δm153 mutant possesses enhanced virulence , independent of both Clr-b and NKR-P1B, suggesting that m153 potentially targets additional host factors. Nevertheless, the present data highlight a unique mechanism by which MCMV modulates NK ligand expression. Cytomegaloviruses are betaherpesviruses that in immunocompromised individuals can lead to severe pathologies. These viruses encode various gene products that serve to evade innate immune recognition. NK cells are among the first immune cells that respond to CMV infection and use germ line-encoded NK cell receptors (NKR) to distinguish healthy from virus-infected cells. One such axis that plays a critical role in NK recognition involves the inhibitory NKR-P1B receptor, which engages the host ligand Clr-b, a molecule commonly lost on stressed cells (“missing-self”). In this study, we discovered that mouse CMV utilizes the m153 glycoprotein to circumvent host-mediated Clr-b downregulation, in order to evade NK recognition. These results highlight a novel MCMV-mediated immune evasion strategy