FIG 2.

Initial unbiased evaluation of nature of MtbWL. (A) Mycobacterial lysates were treated in order to remove large classes of molecules. The initial biochemical treatments consisted of (1) reduction and alkylation to disrupt disulfide bonds, (2) delipidation via organic extraction with chloroform-methanol (2:1) to remove nonpolar lipids, (3) RNase and DNase treatment to degrade nucleotide polymers, (4) base hydrolysis to cleave base-labile bonds, (5) acid hydrolysis to cleave acid-labile bonds, (6) pronase digestion to extensively degrade proteins, and (7) trypsin digestion to more selectively cleave proteins to complement pronase digestion. All the treated mycobacterial lysates were tested for the ability to expand γ₉δ₂ T cells capable of inhibiting intracellular mycobacteria. The expansion index was calculated by dividing the absolute number of γ₉δ₂ T cells after stimulation with treated lysates by the absolute number of γ₉δ₂ T cells after rest in medium. Percent inhibition was calculated as described previously (35). Specific activity (SA) was defined as (expansion index × percent inhibition of intracellular mycobacterial growth)/dry weight of sample. The expansion index and SA are shown in panel B. Mycobacterial antigens stimulating inhibitory γ₉δ₂ T cells were highly sensitive to acid hydrolysis.