Aria fusion cytometer

PBMCs from were thawed in R10 supplemented with DNaseI, washed twice by centrifugation (500 g) and rested for 2 hours at 37°C, 5% CO₂, followed by centrifugation and resuspension of the pellet in 50 μL antibody mixture for 20 minutes at room temperature. Cells were washed twice (PBS) and fixed with 2% PFA. Cells then acquired using an Aria Fusion Cytometer (BD) and analyzed with FlowJo Software, version 9.9.6 (Tree Star). The antibody cocktail consisted of LIVE/DEAD Fixable Near-IR Dead Cell Marker (Invitrogen, Thermo Fisher Scientific); anti–CD3 Brilliant Violet 785, clone OKT3; anti–γδ TCR PE, clone B1; anti–Vδ2 TCR PerCpCy5.5, clone B6; anti–Vγ9 TCR APC, clone B3; anti–Vα7.2 BV711, clone 3C10; anti–CD26 PE-Cy5 clone BA5b; anti–CD161 Brilliant Violet 605, clone HP3G10 (all from BioLegend); anti–CD45 V500, clone HI30 (BD); and anti–Vδ1 TCR FITC clone TS8.2 (Thermo Fisher Scientific).

Binding of IFN-γ to Mtb was assessed by mixing varying concentrations of IFN-γ with whole bacterial cells fixed in 4% PFA and incubating overnight at 4 °C. Bacteria were pelleted and resuspended in 0.1% Tween in PBS before seeding onto microtiter wells for 2 h at 37 °C. After washing, Ultra-LEAF anti-human IFN-γ antibody (Biolegend, clone B27) at 1 μg/mL was added to the wells which were incubated for a further 2 h at room temperature. Anti-mouse HRP at 1:5000 was then added for 1 h at room temperature. TMB solution (Sigma Aldrich) was used as a substrate and 1 M sulfuric acid as stop solution. Optical density was read at 450 nm measured with GloMax Discover microplate reader (Promega). For flow cytometry, after overnight incubation with IFN-γ, bacterial pellets were stained with human anti-IFN-γ Brilliant Violet 421 (Biolegend, clone 4 S.B3) or anti-TNF-α Alexa Fluor 700 (BD, clone MAb11) at 1:20 for 1 h at room temperature. Data was acquired using BD Aria Fusion cytometer and analyzed using FlowJo Software v.10.