Anti cd16 allophycocyanin apc cy7

An assay to determine NK cell activation (NKA) state based on the expression of surface CD107a and intracellular production of IFNγ and MIP1β was performed as previously described. NK cells were isolated from whole blood from healthy donors using negative selection with RosetteSep (STEMCELL Technologies). Following a pulse with SF162 gp120 (60 μg/ml), T lymphoblast CEM‐NKr cells and isolated primary NK cells were mixed at a ratio of 1:5, and purified IgG, anti‐CD107a‐phycoerythrin (PE)‐Cy5 (BD), brefeldin A (10 mg/ml) (Sigma), and GolgiStop (BD) were added. After a 5‐h incubation at 37°C, cells were first stained for surface markers using anti‐CD16‐allophycocyanin (APC)‐Cy7 (BD), anti‐CD56‐PE‐Cy7 (BD), and anti‐CD3‐Alexa Fluor 700 (BD) and then stained intracellularly with anti‐IFNγ‐APC (BD) and anti‐MIP1β‐PE (BD) after treatment with Fix and Perm A and B solutions (Invitrogen). Cells were then fixed in 4% paraformaldehyde and analyzed by flow cytometry. NK cells were defined as CD3‐negative and CD16‐positive and/or CD56‐positive, and the percent of NK cells positive for each marker was determined.

A modified ELISA-based assay for the detection of CD107a as a surrogate marker of NK-cell-mediated degranulation and cytolysis was performed as previously described (40 (link)). Briefly, a 96-well ELISA plate was coated overnight at 4°C with recombinant protein. Purified IgG was added to each well, and the plate was incubated at 37°C for 2 h. HIV-negative plasma samples or medium alone was used as a negative control, while HIVIG (pooled HIV immunoglobulin G) (NIH AIDS Reagents Program) was used as a positive control. A total of 5 × 10⁴ NK cells enriched via negative selection from healthy blood donors (RosetteSep; Stemcell Technologies) were added to each well in the presence of brefeldin A (BioLegend), Golgi stop, and anti-CD107a-PE-Cy5 (BD Biosciences). Plates were incubated for 5 h at 37°C with 5% CO₂. Cells were then stained with anti-CD3-AlexaFluor700, anti-CD56-PE-Cy7, and anti-CD16-allophycocyanin (APC)-Cy7 (BD); fixed with Perm A; permeabilized using Perm B (Invitrogen); and stained with anti-IFN-γ–APC and anti-MIP-1β–PE (BD). The cells were fixed with a 2% paraformaldehyde solution and analyzed by flow cytometry.

ELISA plates were coated with PPD (300 ng/well) or BSA at 4°C for 16 hours [33 (link)]. Purified IgG (25 µg) from study participants was added to each well. NK cells were isolated from whole blood from seronegative donors with RosetteSep. NK cells (5 × 10⁴/well) were incubated with anti-CD107a–phycoerythrin (PE)–Cy5 (BD), brefeldin A (10 mg/mL) (Sigma), and GolgiStop (BD) at 37°C for 5 hours. Cells were stained for surface markers using anti-CD16–allophycocyanin (APC)–Cy7 (BD), anti-CD56–PE–Cy7 (BD), and anti-CD3–AlexaFluor 700 (BD), and then intracellularly with anti-IFN-γ–APC (BD) and anti-MIP1β–PE (BD) using Fix and Perm A and B solutions (ThermoFisher). Frequency (%) of NK cells positive for CD107a, IFN-γ, and MIP1β were determined with NK cells defined as CD3⁻ and CD16/56⁺.

ELISA-based Ab-dependent NK cell activation assay was modified for use with ID93 antigen.^{24 (link)} Briefly, ELISA plates (Thermo Fisher NUNC MaxiSorp flat bottom) were coated with ID93 (3 ng/well) or BSA as a negative control at 4 °C for 16 h. Serum samples from Days 0 and 84 from all cohorts were diluted 1:100 and added to each well. NK cells were isolated from whole blood from HIV-seronegative donors with RosetteSep (STEMCELL Technologies) and cultured overnight with IL-15 (1 ng/mL). NK cells (5 × 10⁴ per well), anti-CD107a-phycoerythrin (PE)-Cy5 (catalog number 555801, BD), brefeldin A (10 mg/mL Sigma), and GolgiStop (BD) were added to each well, and the plates were incubated for 5 h at 37 °C. Cells were then stained for surface markers using anti-CD16–allophycocyanin (APC)-Cy7 (catalog number 557758, BD), anti-CD56-PE-Cy7 (catalog number 557747, BD), and anti-CD3-AlexaFluor 700 (catalog number 557943, BD), and then stained intracellularly with anti-IFNγ-APC (catalog number 554702, BD) and anti-MIP1β-PE (catalog number 550078, BD) using Fix and Perm A and B solutions (Thermo Fisher Scientific). Fixed cells were analyzed by flow cytometry. NK cells were defined as CD3− and CD16/56+.

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.