Two-Hit Assay for Mycobacterial Antigen Response

The two-hit assay has been performed [as described previously (19 (link))] with minor modifications. In brief, 10⁵ PBMCs were cultured in 200 μl RPMI supplemented with Penicillin/Streptomycin (100 U/ml), L-glutamine (2 mM), 10 mM HEPES buffer (all Gibco), 7.5% human serum (Sigma Aldrich) and 5 ng/ml of recombinant IL-7 (Sigma-Aldrich) in 96-U bottom plate. Cells were stimulated or left unstimulated with two Mtb antigens PPD (1μg/ml), ESAT6/CFP10 fusion protein (E6/C10; 1 μg/ml) and four latency antigens (i.e., Rv1733, Rv2628, Rv2031, and Rv3407; 1μg/ml). E6/C10 as well as the Mtb latency antigens are recombinant proteins produced in the laboratory of K. Franken and have been thoroughly used in previous studies (18 (link), 19 (link), 24 (link)). Samples were then incubated for 6 days at 37°C and 5% CO₂. On the sixth day, 100 μl of culture supernatants were discarded and samples were re-stimulated with the respective Mtb antigens (same concentrations as on d1) and Brefeldin A (3.75 μg/ml) (Sigma Aldrich) in reconstituted in fresh medium for 20 h. Afterwards, cells were then fixed, permeabilized and stained with the following panel of antibodies: CD3 APC (clone UCTH1), CD4 PerCP/Cy5.5 (clone RPA-T4), IFNγ PE (clone B27) and TNF-α FITC (clone Mab 11) (all BioLegend). Cells were measured as described above.