Proposed model of granzyme A-mediated suppression of intracellular mycobacterial growth by γ₉δ₂ T cells.

<p>Mycobacteria-infected macrophages present antigens to γ₉δ₂ T cells (1) which secrete granzyme A upon activation (2). Granzyme A in turn induces TNF-α production by infected and/or bystander macrophages (3) apparently independent of perforin. TNF-α activates intracellular mechanisms which alone or in concert with other unknown granzyme A-induced intracellular mechanisms suppress mycobacterial growth (4).</p

TNF-α is critical for γ₉δ₂ T cell-mediated inhibition of intracellular mycobacterial growth.

<p><i>A</i>, γ₉δ₂ T cell lines significantly suppressed intracellular growth of mycobacteria (n = 5; *p<0.05 by Wilcoxon matched-pairs test). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages for 3 d. Surviving bacteria were quantified by H³-uridine incorporation and compared with cultures absent of γ₉δ₂ T cells. <i>B</i>, The effector mechanism responsible for γ₉δ₂ T cell-mediated mycobacterial growth inhibition involves soluble factors. γ₉δ₂ T cells were stimulated in the top chambers of semi-permeable transwell membrane (0.4 µm pores) systems with BCG-infected macrophages and/or soluble HMB-PP. The levels of intracellular growth inhibition were assayed in the bottom chambers and compared to the levels of inhibition achieved by direct co-culture of γ₉δ₂ T cells with the bottom layer of BCG-infected macrophages (*p<0.05, n = 5 by Wilcoxon matched pairs tests compared with unstimulated γ₉δ₂ T cells separated by transwells; **p<0.02, n = 7 by Wilcoxon matched pairs tests compared with HMB-PP stimulated γδ T cells separated by transwells; ***p<0.003, n = 15 by Wilcoxon matched pairs tests compared with HMB-PP stimulated γδ T cells separated by transwells). <i>C</i>, Neutralization of TNF-α alone prevents γ₉δ₂ T cell-mediated inhibition while neutralization of IFN-γ alone did not (*p<0.02, n = 7 by Wilcoxon matched pairs tests compared with no antibody control; **p<0.02, n = 6 by Wilcoxon matched pairs tests compared with no antibody control; n.s. – not significantly different). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages in the presence or absence of the indicated neutralizing antibody and the surviving bacteria quantified by H³-uridine incorporation. <i>D</i>, shRNA-mediated knockdown of TNF-α in macrophages, but not γ₉δ₂ T cells, eliminates γ₉δ₂ T cell-mediated mycobacterial growth inhibition. (*p<0.02, n = 7 by Wilcoxon matched pairs tests compared with adenovirus expressing the empty vector). γ₉δ₂ T cells were co-cultured with BCG-infected macrophages. Prior to co-culture, either the T cells or uninfected macrophages were infected with shRNA expressing Adenovirus. Surviving bacteria were quantified by H³-uridine incorporation.</p

Granzyme A secretion by γ₉δ₂ T cells mediates inhibition of intracellular mycobacteria by induction of inflammatory responses in mycobacteria-infected macrophages.

<p><i>A</i>, The levels of granzyme A produced by γ₉δ₂ T cells are directly and highly correlated with inhibition of intracellular mycobacterial growth (r = 0.7564; p<0.0001). Supernatant levels of granzyme A, measured by CBA, were correlated with the level of mycobacterial growth inhibition observed in γ₉δ₂ T cell co-cultures with BCG-infected macrophages. <i>B</i>, Purified granzyme A induces the production of pro-inflammatory cytokines TNF-α and IL-1β by BCG-infected macrophages (*p<0.05, n = 4; statistical comparisons performed using Friedman's test). The indicated concentrations of purified granzyme A were added to BCG-infected monocytes for 3 days and the levels of TNF-α and IL-1β in the culture supernatants determined by CBA. <i>C-D</i>, Purified granzyme A alone can induce inhibition of intracellular mycobacterial growth in the absence of perforin. The indicated concentrations of granzyme A were added to BCG-infected (<i>C</i>) or <i>M. tuberculosis</i> H37Rv-infected (<i>D</i>) co-cultures for 3 days and the surviving bacteria quantified by H³-uridine incorporation and CFU counting. (*p<0.03; n = 6–12 by Wilcoxon matched pairs test comparing levels of mycobacterial intracellular growth in cultures with and without added purified granzyme A). Purified granzyme A had no direct effects on extracellular mycobacterial growth (data not shown). <i>E</i>, siRNA knockdown of granzyme A in γ₉δ₂ T cells reduces the inhibitory activity of γ₉δ₂ T cells for mycobacterial growth (*p<0.05; n = 3 by one-way ANOVA comparing levels of intracellular mycobacterial growth inhibition in cultures with or without γ₉δ₂ T cells producing granzyme A). γ₉δ₂ T cells transduced with the indicated siRNA-lentivirus targeting GzmA or a negative control (NC) were co-cultured with BCG-infected macrophages for 3 days and surviving bacteria quantified by H³-uridine incorporation.</p

Classical effector mechanisms involving direct contact of γ₉δ₂ T cells with BCG-infected macrophages are not required for mycobacterial suppression.

<p>γ₉δ₂ T cells were co-cultured with BCG-infected macrophages in the presence or absence of the indicated treatment and surviving bacteria quantified by H³-uridine incorporation. Blocking of Fas/FasL interactions did not prevent γ₉δ₂ T cell-mediated inhibition (p = 0.23, n = 7). In contrast, γ₉δ₂ T cells pre-treated for 16 hours with 25 mM strontium chloride were unable to mediate mycobacterial inhibition (*p<0.05, n = 5 by Wilcoxon matched pairs test compared with untreated γ₉δ₂ T cells). Despite strontium depletion indicating the importance of γ₉δ₂ T cell cytolytic granules for mycobacterial inhibitory effects, antibody neutralization of perforin had no effect (p = 0.28, n = 3).</p