egc Superantigens Impair Monocytes/Macrophages Inducing Cell Death and Inefficient Activation.

Bacterial superantigens (SAgs) are enterotoxins that bind to MHC-II and TCR molecules, activating as much as 20% of the T cell population and promoting a cytokine storm which enhances susceptibility to endotoxic shock, causing immunosuppression, and hindering the immune response against bacterial infection. Since monocytes/macrophages are one of the first cells SAgs find in infected host and considering the effect these cells have on directing the immune response, here, we investigated the effect of four non-classical SAgs of the staphylococcal egc operon, namely, SEG, SEI, SEO, and SEM on monocytic-macrophagic cells, in the absence of T cells. We also analyzed the molecular targets on APCs which could mediate SAg effects. We found that egc SAgs depleted the pool of innate immune effector cells and induced an inefficient activation of monocytic-macrophagic cells, driving the immune response to an impaired proinflammatory profile, which could be mediated directly or indirectly by interactions with MHC class II. In addition, performing surface plasmon resonance assays, we demonstrated that non-classical SAgs bind the gp130 molecule, which is also present in the monocytic cell surface, among other cells.

Kinetic and thermodynamic studies of the interaction between activating and inhibitory Ly49 natural killer receptors and MHC class I molecules.

Natural killer (NK) cells are lymphocytes of the innate immune system that eliminate virally infected or malignantly transformed cells. NK cell function is regulated by diverse surface receptors that are both activating and inhibitory. Among them, the homodimeric Ly49 receptors control NK cell cytotoxicity by sensing major histocompatibility complex class I molecules (MHC-I) on target cells. Although crystal structures have been reported for Ly49/MHC-I complexes, the underlying binding mechanism has not been elucidated. Accordingly, we carried out thermodynamic and kinetic experiments on the interaction of four NK Ly49 receptors (Ly49G, Ly49H, Ly49I and Ly49P) with two MHC-I ligands (H-2Dd and H-2Dk). These Ly49s embrace the structural and functional diversity of the highly polymorphic Ly49 family. Combining surface plasmon resonance, fluorescence anisotropy and far-UV circular dichroism (CD), we determined that the best model to describe both inhibitory and activating Ly49/MHC-I interactions is one in which the two MHC-I binding sites of the Ly49 homodimer present similar binding constants for the two sites (∼106 M-1) with a slightly positive co-operativity in some cases, and without far-UV CD observable conformational changes. Furthermore, Ly49/MHC-I interactions are diffusion-controlled and enthalpy-driven. These features stand in marked contrast with the activation-controlled and entropy-driven interaction of Ly49s with the viral immunoevasin m157, which is characterized by strong positive co-operativity and conformational selection. These differences are explained by the distinct structures of Ly49/MHC-I and Ly49/m157 complexes. Moreover, they reflect the opposing roles of NK cells to rapidly scan for virally infected cells and of viruses to escape detection using immunoevasins such as m157.

Uptake and intracellular trafficking of superantigens in dendritic cells.

Bacterial superantigens (SAgs) are exotoxins produced mainly by Staphylococcus aureus and Streptococcus pyogenes that can cause toxic shock syndrome (TSS). According to current paradigm, SAgs interact directly and simultaneously with T cell receptor (TCR) on the T cell and MHC class II (MHC-II) on the antigen-presenting cell (APC), thereby circumventing intracellular processing to trigger T cell activation. Dendritic cells (DCs) are professional APCs that coat nearly all body surfaces and are the most probable candidate to interact with SAgs. We demonstrate that SAgs are taken up by mouse DCs without triggering DC maturation. SAgs were found in intracellular acidic compartment of DCs as biologically active molecules. Moreover, SAgs co-localized with EEA1, RAB-7 and LAMP-2, at different times, and were then recycled to the cell membrane. DCs loaded with SAgs are capable of triggering in vitro lymphocyte proliferation and, injected into mice, stimulate T cells bearing the proper TCR in draining lymph nodes. Transportation and trafficking of SAgs in DCs might increase the local concentration of these exotoxins where they will produce the highest effect by promoting their encounter with both MHC-II and TCR in lymph nodes, and may explain how just a few SAg molecules can induce the severe pathology associated with TSS.