Single-cell suspensions were prepared from cultured cells or fresh tumor tissues. Red cells were removed using ammonium chloride lysis buffer when necessary. 1 × 106 cells were incubated at 4°C for 30 min with different combinations of fluorescent-conjugated antibodies for γδ T cells (anti-Vγ9 TCR-allophycocyanin [BioLegend, San Diego, CA, USA], anti-CD3-PECy7 [BioLegend], anti-Vδ2 TCR-peridinin chlorophyll [PerCP]-Cy5.5 [BioLegend], and anti-Vδ1 TCR-FITC [Thermo Fisher Scientific]), T cells (FITC-anti-CD4 [BD Biosciences, San Jose, CA, USA], PE-anti-CD8 [BD Biosciences], and PE/Cy7-anti-CD3 [BioLegend]). For intracellular staining of IFN-γ, cells were incubated with staphylococcal enterotoxin B for 6 hr in the presence of 19 brefeldin A as described by the manufacturer (BD Biosciences, San Jose, CA, USA). Cells were then stained with IFN-γ PerCP-Cy5.5 (BD Biosciences).
For γδTDE-mediated cell proliferation experiments, CD8+ T cells were stained with CFSE (4.5 mM) at 37°C for 20 min. The labeled cells were cultured in AIM V Medium with IL-2 and zoledronate at the presence of γδTDE derived from different conditions for 6 days. Cells were harvested, and CFSE was measured by a flow cytometer with 488 nm excitation and emission filters.
For γδTDE-mediated cell proliferation experiments, CD8+ T cells were stained with CFSE (4.5 mM) at 37°C for 20 min. The labeled cells were cultured in AIM V Medium with IL-2 and zoledronate at the presence of γδTDE derived from different conditions for 6 days. Cells were harvested, and CFSE was measured by a flow cytometer with 488 nm excitation and emission filters.