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

Abstract

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.

Effector Vγ9Vδ2 T cells dominate the human fetal γδ T-cell repertoire

“…Perforin was enriched in Vγ9Vδ2 T cells at the RNA level but not at the protein level, in contrast to adult Vγ9Vδ2 T cells, which are known to express perforin, granzyme B, and granulysin protein (3). Interestingly, it has been shown recently that granzyme A produced by adult Vγ9Vδ2 T cells, independently of perforin and granzyme B, induces macrophages to inhibit the growth of intracellular mycobacteria (77). Fetal Vγ9Vδ2 T-cell effectors expressed high levels of the inflammatory chemokine receptors CCR5 and CXCR3, which have been described as being particularly highly expressed on adult blood Vγ9Vδ2 T cells (78).…”

Granzymes Regulate Proinflammatory Cytokine Responses

“…We are currently investigating the molecular mechanism underlying this GrA effect. Interestingly, hGrA blocks growth of intracellular mycobacteria in human macrophages (49). Human gd T cells efficiently inhibit the growth of these bacteria inside macrophages.…”

“…Human gd T cells efficiently inhibit the growth of these bacteria inside macrophages. Recombinant GrA, as well as GrA produced by gd T cells, induces TNF-a production in macrophages, which in turn inhibits the growth of the pathogen (49). The mechanism by which TNF-a exerts this effect remains unknown.…”

“…Overview of effects of Grs on cytokine secretion by different cell types. GrA, GrB, and GrK from human or mouse induce the release of cytokines from several cell types, including fibroblasts (45, 48, 53), monocytes (11,46,48), and macrophages (10,11,49). Grs cause this release on their own or potentiate LPSinduced cytokine responses.…”

Mycobacterium-Specific γ9δ2T Cells Mediate Both Pathogen-Inhibitory and CD40 Ligand-Dependent Antigen Presentation Effects Important for Tuberculosis Immunity

“…In addition, memory CD8 ϩ T cells can recognize and destroy M. tuberculosis-infected macrophages by the secretion of perforin, granzyme, and granulysin (8). ␥ 9 ␦ 2 T cells also have been shown to produce Th1 cytokines and exhibit cytolytic activity, as well as inhibit intracellular mycobacteria (9)(10)(11)(12). ␥ 9 ␦ 2 T cells comprise only 3% to 5% of total human peripheral blood lymphocytes (13,14), and yet, they expand to large numbers after exposure to antigen (15).…”