Anti cd16 pe cy7 clone 3g8

NK cell degranulation upon co-incubation with target cells was determined by the expression of CD107a on the cell surface, which serves as a surrogate marker for NK cell degranulation [58 (link)]. In brief, overnight cultured NK cells enriched with the EasySep human NK cell enrichment kit (StemCell Technologies) from PBMCs from KIR2DL5A*001-positive donors or donors lacking KIR2DL5 genetically were co-cultured with CD155-expressing (CD155-transduced 721.221) or CD155-nonexpressing (721.221) target cells at an effector to target ratio of 1:2 in 200 μl complete R10 for 4 h at 37°C. During co-incubation, each well contained 2 μl anti-CD107a (clone LAMP-1, Biolegend) and 25μl/ml Brefeldin A. Cells were subsequently stained with LIVE/DEAD Fixable Near-IR and with the following antibodies: anti-CD3-BUV395 (clone UCHT1, BD), anti-CD16-PE-Cy7 (clone 3G8, Biolegend), anti-CD56-BV785 (clone NCAM16.1, BD), anti-KIR2DL5-PE (clone UP-R1, Biolegend) for 15 min at RT and fixed with CellFix (BD) before flow cytometric acquisition.

The cells obtained from the digestion of the biopsies were washed and resuspended in PBS, then collected and transferred into 5 mL tubes for flow cytometry, and then incubated for 15 min in the dark with the Zombie Violet™ fixable viability stain (Biolegend, San Diego, CA, USA). The cells were then washed and resuspended in FACS buffer (PBS, 2% FBS, 2 mM FBS EDTA) to proceed with the labeling with antibodies (and their isotopic controls) for the recognition of surface markers. For immunophenotyping of T lymphocytes and TRM, the cells were labeled with anti-CD3 FITC clone UCHT1, anti-CD103 PE clone Ber-ACT8, anti-CD69 PerCP-Cy5.5 clone FN50 (Biolegend, San Diego, CA, USA), anti-CD8 PE-Cy7 clone HIT8a, anti-CD45 APC-Cy7 clone 2D1. In addition, the following antibodies were used for immunophenotyping NK and γδ T cells: anti-CD56 PE clone B159, anti-CD16 PECy7 clone 3G8, anti-Vδ2 APC clone B6 (Biolegend, San Diego, CA, USA). All the antibodies used, except for anti-CD69 and anti-Vδ2, were obtained from the BD Biosciences Company, (Franklin Lakes, NJ, USA). The samples were acquired using a BD FACSAria flow cytometer at least 50,000 viable cells were acquired for each sample. The gating strategy for the detection of T lymphocytes and TRM T cells is shown in supplemental Figure S1. The data obtained were analyzed using the FlowJo software (BD Biosciences).