A novel mycobacterial <i>In Vitro</i> infection assay identifies differences of induced macrophage apoptosis between CD4⁺ and CD8⁺ T cells

<div><p>Macrophages are natural host cells for pathogenic mycobacteria, like <i>Mycobacterium tuberculosis (M</i>.<i>tb)</i>. Immune surveillance by T cells and interaction with <i>M</i>.<i>tb</i> infected macrophages is crucial for protection against <i>M</i>.<i>tb</i> reactivation and development of active tuberculosis. Several factors play a role in the control of <i>M</i>.<i>tb</i> infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional <i>in vitro</i> assays which allow concomitant determination of i) mycobacterial eradication; ii) cytotoxic effects on host macrophages; and iii) effector T-cell functions. We established a novel functional <i>in vitro</i> assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a <i>Mycobacterium bovis</i> BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated <i>in vitro</i> and infected with defined LD-BCG numbers. After short-term MDM/LD-BCG co-incubation with autologous effector T cells or in the presence of antibiotics, proportions of MDM containing live or dead LD-BCG were determined by flow cytometry. Concomitant measure of defined numbers of added beads allowed comparison of absolute MDM numbers between samples. Differential effects of T-cell subpopulations on anti-mycobacterial cytotoxicity and on MDM apoptosis were determined. Flow cytometry measure of MDM/LD-BCG treated with rifampicin correlated well with mycobacterial colony forming units and fluorescence microscopy results. Co-culture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. <i>M</i>.<i>tb</i> protein specific CD4⁺ and CD8⁺ T-cells contributed similarly to anti-mycobacterial cytotoxicity but CD4⁺ T cells induced higher levels of apoptosis in infected MDMs. This novel assay enables rapid quantification of anti-mycobacterial cytotoxicity and characterization of effector functions. Our functional <i>in vitro</i> assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.</p>