γδ tcr apc

After 14 days of γδ T cell expansion, IFN-γ and TNF-α production was measured using a flow cytometric-based intracellular staining assay. Measurements were also performed after an additional hour of stimulation with Daudi or U266 cells (E:T ratio = 5:1). Brefeldin A (Golgi-Plug 1 μL/mL; BD) was added to the different conditions (1 × 10⁶ cells/mL) and incubated for 3 h at 37 °C/5 % CO₂. γδ T cells were then washed and incubated with Live/Dead^® Fixable Aqua Stain, CD3-PerCP-Cy5.5 (BD) and γδ TCR-APC (Miltenyi) for 30 min at 4 °C. Subsequently, cells were fixed and permeabilized, using the Foxp3/Transcription Factor Staining Buffer Set (eBioscience), according to the manufacturer’s instructions. Intracellular staining antibodies (IFN-γ-FITC and TNF-α-PE-Cy7, BD) or the corresponding isotype control were added and allowed to bind for 1 h at 4 °C.

γδ T cells were phenotyped after 48-hour co-culture using the following monoclonal antibodies (mAbs): γδ TCR-FITC (Miltenyi), CD86-FITC, CD80-PE, PD-1-PE, NKG2D-PE, CD16-PB, BTLA-BV421, γδ TCR-APC (Miltenyi), NKp30-AF647, and CD3-APC-H7. Unless specified otherwise, all mAbs were purchased from BD (Erembodegem, Belgium). Live/Dead^® Fixable Aqua Stain (Invitrogen, Merelbeke, Belgium) was used to exclude dead cells from analysis. Corresponding species- and isotype-matched antibodies were used as controls. The corresponding gating strategy can be retrieved from Figure S1 in Supplementary Material.

Freshly isolated and 5-day proliferated γδ T cells were membrane-stained with the following monoclonal antibodies; γδ TCR-FITC (Miltenyi), CD56-PE (BD), CD69-PE (BD), and HLA-DR-PE (BD). Propidium iodide (PI; Life Technologies) was added to exclude dead cells from phenotypic analysis. Data acquisition was performed on a FACScan multiparametric flow cytometer (BD). Phenotypic characterization of γδ T cells was examined pre- and post-expansion, using CD27-FITC (BD), CD69-FITC (BD), CD56-PE (BD), CD80-PE (BD), CD45RA-PE-Cy7 (BD), CD28-PerCP-Cy5.5 (BD), CD16-PB (BD), CD86-V450 (BD), γδ TCR-APC (Miltenyi), and HLA-DR-APC-H7 (BD). Live/Dead^® Fixable Aqua Stain was used to distinguish viable from non-viable cells. Data were acquired on a FACSAria II flow cytometer (BD). Corresponding species- and isotype-matched antibodies were used as controls.