Open AccessA novel mycobacterial In Vitro infection assay identifies differences of induced macrophage apoptosis between CD4+ and CD8+ T cells
Author(s) -
Vanesa Nkwouano,
Sven Witkowski,
Nidja Rehberg,
Rainer Kalscheuer,
Norman Nausch,
Ertan Mayatepek,
Marc Jacobsen
Publication year - 2017
Publication title -
plos one
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.99
H-Index - 332
ISSN - 1932-6203
DOI - 10.1371/journal.pone.0171817
Subject(s) - flow cytometry , biology , cytotoxic t cell , mycobacterium tuberculosis , cd8 , immune system , effector , mycobacterium bovis , macrophage , in vitro , microbiology and biotechnology , immunology , tuberculosis , medicine , biochemistry , pathology
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. Co-culture 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.