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Spencer C.T.,Saint Louis University | Spencer C.T.,University of Texas at El Paso | Abate G.,Saint Louis University | Sakala I.G.,Saint Louis University | And 10 more authors.
PLoS Pathogens

Human γ9δ2 T cells potently inhibit pathogenic microbes, including intracellular mycobacteria, but the key inhibitory mechanism(s) involved have not been identified. We report a novel mechanism involving the inhibition of intracellular mycobacteria by soluble granzyme A. γ9δ2 T cells produced soluble factors that could pass through 0.45 μm membranes and inhibit intracellular mycobacteria in human monocytes cultured below transwell inserts. Neutralization of TNF-α in co-cultures of infected monocytes and γ9δ2 T cells prevented inhibition, suggesting that TNF-α was the critical inhibitory factor produced by γ9δ2 T cells. However, only siRNA- mediated knockdown of TNF-α in infected monocytes, but not in γ9δ2 T cells, prevented mycobacterial growth inhibition. Investigations of other soluble factors produced by γ9δ2 T cells identified a highly significant correlation between the levels of granzyme A produced and intracellular mycobacterial growth inhibition. Furthermore, purified granzyme A alone induced inhibition of intracellular mycobacteria, while knockdown of granzyme A in γ9δ2 T cell clones blocked their inhibitory effects. The inhibitory mechanism was independent of autophagy, apoptosis, nitric oxide production, type I interferons, Fas/FasL and perforin. These results demonstrate a novel microbial defense mechanism involving granzyme A-mediated triggering of TNF-α production by monocytes leading to intracellular mycobacterial growth suppression. This pathway may provide a protective mechanism relevant for the development of new vaccines and/or immunotherapies for macrophage-resident chronic microbial infections. © 2013 Spencer et al. Source

Metkar S.S.,NorthShore University HealthSystems Research Institute | Wang B.,NorthShore University HealthSystems Research Institute | Catalan E.,University of Zaragoza | Anderluh G.,University of Ljubljana | And 3 more authors.

The cytotoxic cell granule secretory pathway is essential for host defense. This pathway is fundamentally a form of intracellular protein delivery where granule proteases (granzymes) from cytotoxic lymphocytes are thought to diffuse through barrel stave pores generated in the plasma membrane of the target cell by the pore forming protein perforin (PFN) and mediate apoptotic as well as additional biological effects. While recent electron microscopy and structural analyses indicate that recombinant PFN oligomerizes to form pores containing 20 monomers (20 nm) when applied to liposomal membranes, these pores are not observed by propidium iodide uptake in target cells. Instead, concentrations of human PFN that encourage granzyme-mediated apoptosis are associated with pore structures that unexpectedly favor phosphatidylserine flip-flop measured by Annexin-V and Lactadherin. Efforts that reduce PFN mediated Ca influx in targets did not reduce Annexin-V reactivity. Antigen specific mouse CD8 cells initiate a similar rapid flip-flop in target cells. A lipid that augments plasma membrane curvature as well as cholesterol depletion in target cells enhance flip-flop. Annexin-V staining highly correlated with apoptosis after Granzyme B (GzmB) treatment. We propose the structures that PFN oligomers form in the membrane bilayer may include arcs previously observed by electron microscopy and that these unusual structures represent an incomplete mixture of plasma membrane lipid and PFN oligomers that may act as a flexible gateway for GzmB to translocate across the bilayer to the cytosolic leaflet of target cells. © 2011 Metkar et al. Source

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