Granzyme A Produced by γ₉δ₂ T Cells Induces Human Macrophages to Inhibit Growth of an Intracellular Pathogen

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.