A novel mycobacterial in vitro infection assay identifies differences of induced macrophage apoptosis between CD4 and CD8 T cells

Macrophages are natural host cells for pathogenic mycobacteria, like Mycobacterium tuberculosis (M.tb). Immune surveillance by T cells and interaction with M.tb infected macrophages is crucial for protection against M.tb reactivation and development of active tuberculosis. Several factors play a role in the control of M.tb infection but reliable biomarkers remain elusive. One major obstacle is the absence of functional in vitro 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 in vitro assay based on flow cytometry analysis of monocyte-derived macrophages (MDM) infected with a Mycobacterium bovis BCG strain containing a tetracycline inducible live/dead reporter plasmid (LD-BCG). MDM of healthy human donors were generated in vitro 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. Coculture with pre-activated effector T cells reduced viability of both, LD-BCG and MDM, in a concentration-dependent manner. M.tb 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 in vitro assay has the potential to contribute to the identification of biomarkers for protective T-cell responses against tuberculosis.