Anti cd107a pe cy5

NK-cell responses to target cell lines were determined as previously described [18 (link)]. PBMC were incubated with culture media alone or with K562 cells, 221 cells or antibody-coated p815 cells at an effector to target ratio of 10:1 for 5 hours in the presence of anti-CD107a PE-Cy5 (BD Biosciences, 10 ml/mL), brefeldin A (2.5 mg/mL) and Golgistop (2.5 mg/mL, BD Bioscience). Cells were then washed, stained with the Fixable Blue Dead Cell Stain prior to staining for surface receptors, then fixed, permeabilized (BD Fix/perm, BD Biosciences) and stained with anti-IFNγ-FITC, anti-MIP1β-PE and anti-TNF-Alexafluor700 (all BD Biosciences). Gates were set on media-only controls, which were also used for background subtraction prior to analysis.

PBMCs were cultured overnight with 10 μg therapeutic antibody per milliliter of culture medium. Alternatively, plates were coated as described above. Anti-CD107a PE-Cy5 (BD Biosciences, San Jose, CA, USA) was added at the beginning of the stimulation. The next morning, PBMCs were washed with PBS, incubated in PBS supplemented with Zombie Aqua™ Fixable Viability Kit (final conc. 1/500; BioLegend, San Diego, CA, USA) and incubated for 20 min on ice. Subsequently, PBMCs were washed in 2% FCS in PBS and incubated for 30 min on 4 °C in 2% FCS in PBS supplemented with anti-CD56 Brilliant Violet 421 (Clone NCAM16.2; BD), anti-CD16 FITC (Immunotech, Marseille, FRA), anti-CD3 APC (UCHT1), anti-CD19 APC-Cy7 (HIB19), and anti-CD69 PE (all from BioLegend). After a final washing step, PBMCs were directly analyzed on a three-laser flow cytometer (LSRII, BD). Data were processed using FlowJo® software (FlowJo LCC, Ashland, OR, USA). The gating strategy is shown in Additional file 1.

Therapeutic antibodies and anti-CD107a PE-Cy5 (BD Biosciences, San Jose, CA, USA) were added at the same time point to freshly isolated PBMCs (1 × 10⁶ cells/ml). CD107a surface expression on NK cells was measured after culture overnight by flow cytometry as described.

In brief, plates were coated with AVI-tag gp120, blocked, and patient plasma added before incubating for 2 h at 37 °C. NK cells isolated from a healthy donor's whole blood were added simultaneously with anti-CD107a PE-Cy5 (BD Biosciences), Brefeldin A (Sigma) and Golgi Stop (BD Biosciences) and incubated for 5 h at 37 °C as previously reported [37 ]. NK cells were then stained with anti-CD56 PE-Cy7, anti-CD16 allophycocyanin (APC)-Cy7 and anti-CD3 Alexa Fluoro 700 (BD Biosciences), fixed (FIX&PERM cell fixation and permeabilization kit, Thermo Fisher Scientific, Waltham, Massachusetts, USA), and stained intracellularly with anti-IFNγ-APC and anti-MIP-1β-PE (BD Biosciences). Surface expression of CD107a and intracellular production of IFNγ and MIP1β by NK cells (CD16+/56+CD3−) were then analysed by flow cytometry. For each Fc-assay, a negative control (human purified IgG, Sigma) was included. Mean of signal in the negative controls wells was subtracted as background signal. The gating strategy for the Fc-mediated functions are illustrated in Supplementary Fig. 1.

ELISA plates were coated with 2 ug/mL of spike, incubated for 2 hours at 37°C, washed three times with PBS and blocked overnight at 4°C in 5% BSA in PBS. Human NK cells were isolated from peripheral blood (MGH Blood Bank) using RosetteSep kit (Stem Cell Technologies) followed by Ficoll separation to isolate cells. NK cells were maintained overnight at 37°C in RPMI media with 10% fetal bovine serum, L-glutamine, HEPES, penicillin/streptomycin and IL-15. Blocked plates were washed three times with PBS, and diluted serum (1:50) and diluted breastmilk (1:5) were added to the coated ELISA plates. Plates were incubated for 2 hours at 37°C. After the incubation, plates were washed three times with PBS, and NK cells were added at a concentration of 2.5 × 10ˆ5 cells/mL in media supplemented with GolgiStop (BD), Brefeldin A (BFA, Sigma Aldrich) and anti-CD107a PE-Cy5 (BD) and were incubated for 5 hours at 37°C. Following the incubation, NK cells were stained for surface markers with anti-CD3 PacBlue (BD), anti-CD16 APC-Cy5 (BD), and anti-CD56 PE-Cy7 (BD). After staining, cells were fixed using the FIX&PERM A/B kit (Life Tech) and stained for MIP-1b (anti-MIP-1b PE, BD). Fluorescence was acquired using an iQue (Intellicyt). NK cells were gated as CD56+/CD16+/CD3- and NK cells activity was determined as the percentage of NK cells that were positive for CD107a and MIP-1b.