Commercial preparations of bovine tuberculin (PPD-B) and avian tuberculin (PPD-A, Thermo Fisher Scientific) were used to stimulate PBMCs at a final concentration of 300 and 250 IU/ml, respectively, as per kit instructions. Two sets of peptides [PCL, 40-mer length with a 20-residue overlap; PCS (peptide cocktail–short), 16 to 20 mers with an 8- to 12-residue overlap] were chemically synthesized to a minimal purity of 98 and 85%, respectively. The identity was confirmed by mass spectrometry (see table S1 for amino acid sequences). A histidine-tagged fusion recombinant protein of ESAT-6, CFP-10, and Rv3615c, expressed in Escherichia coli, was purified by nickel affinity chromatography (Lionex Ltd., Braunschweig, Germany).

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