CD8+ T cells were enriched from PBMCs by negative selection using magnetic bead separation according to the manufacturer’s instructions (STEMCELL Technologies), added to uninfected or infected A549 cells at a ratio of 3:1, and incubated for 16 h in the presence of α-TNF (Beckman Coulter) and TAPI-O (EMD Millipore) to prevent TNF cleavage from the cell surface (Haney et al., 2011 (link)). The following day, cells were stained with: (a) α-CD4 and α-CD8 (BioLegend) and (b) α–TRAV1-2 and α–TCRγδ (Thermo Fisher Scientific). Dead cells were excluded from the analysis using propidium iodide (BioLegend). Viable CD4−TCRγδ−CD8+TRAV1-2+TNF+ cells were sorted at high purity using a FACSAria flow cytometer (BD) directly into RNAlater (Applied Biosystems). The corresponding nonfunctional populations were sorted in parallel for control purposes. In all cases, cell numbers were standardized (5,000 per aliquot) to minimize sampling bias.