Anti cd107a fitc clone h4a3

After 16 days in culture, CBMC were resuspended at 2×10⁶ cells/ml in fresh complete medium and re-stimulated in 96-well plates pre-coated with anti-γδ TCR (clone B1.1, eBioscience, San Diego, CA). Plates were coated overnight at 4°C with anti-γδ TCR (diluted 1:250 in PBS, 50 µl/well) and with different concentrations (0.4, 2 and 10 µg/ml) of human recombinant PDL1-Fc (Sino biological, China) or, for a subset of specimens, with human recombinant CD19-Fc, 2 µg/ml (ACROBiosystems, Newark, DE). CMBC were plated in triplicate (100 µl/well) with anti-CD107a FITC (clone H4A3, BD Biosciences, 5 µl/well) and GolgiPlug (brefeldin A, 1 µg/ml, BD Biosciences). After a 6-hour incubation, cells were collected, washed once with cold PBS, and stained for membrane markers. After staining of surface markers, cells were permeabilized by incubating for 20 minutes at 4°C with fixation/permeabilization solution (BD Biosciences). Cells were then washed twice with 1× Perm/wash buffer (BD Biosciences). Anti-human TNF-α APC (clone MAb11, BD Biosciences) was added for 40 minutes at room temperature. Finally, cells were washed once with Perm/wash buffer. At least 10⁵ lymphocytes were collected for each sample.

T cell functions were assessed using flow cytometry as described previously (14 (link)). Briefly, 6C5 T cells were incubated with T2 cells ± peptide (10 μg/ml) in the presence of GolgiStop (0.7 μl/ml; BD Biosciences) and anti-CD107a–FITC (clone H4A3; BD Biosciences). Negative control tubes contained equivalent DMSO or no T2 cells, and positive control tubes contained PMA (10 ng/ml; Sigma-Aldrich) and ionomycin (1.5 μg/ml; Sigma-Aldrich). Cells were incubated for 6 hr at 37°C, washed with FACS buffer (Thermo Fisher Scientific), and stained for 15 min at 4°C with anti-CD8a–PerCP-Cy5.5 (clone SK1), anti-CD19–BV510 (clone HIB19), and Zombie NIR (all from BioLegend). After a further wash, cells were fixed/permeabilized using a FIX & PERM Cell Fixation & Permeabilization Kit (Thermo Fisher Scientific), washed again, and stained for 20 min at 4°C with anti-IFN-γ–V450 (clone B27; BD Biosciences), anti-IL-2–APC (clone MQ1-17H12; BioLegend), anti-MIP-1β–PE (clone D21-1351; BD Biosciences), and anti-TNF-α–PE-Cy7 (clone MAb11; BioLegend). Data were acquired immediately using a FACSCanto flow cytometer (BD Biosciences) and analyzed using FlowJo software version 9.9.4 (FlowJo LLC). The gating strategy is shown in Supplemental Fig. 1.