Golgistop monensin

To assess innate and adaptive immune cell function, Tonsillar (1 x10⁶ cells) were resuspended in RPMI 1640(Gibco) containing 10% FBS and either stimulated with PMA (50 ng/ml) and ionomycin (1 μg/ml) or cultured in medium alone. Anti-CD107a was added to each tube at a concentration of 20 μl/ml and, Golgiplug (brefeldin A) [Cat No: 555029, BD biosciences] and Golgistop (monensin) [Cat No: 554724, BD biosciences] were added at final concentrations of 6 μg/ml; then all samples were cultured for 12 h at 37°C in 5% CO2. After culture, samples were surface stained using markers to delineate live cells (LIVE/DEAD Aqua dye), leukocytes (CD45), and innate and adaptive immune cell populations (complete list of antibodies in the Supplemental Table 1). Cells were then permeabilized using Caltag Fix & Perm, and intracellular cytokine staining was performed for combinations of antibodies to measure IFN-γ, TNF-α, IL-22.

Virus-specific CD8⁺ T-cell frequencies were measured by flow cytometric analysis of gamma interferon (IFN-γ) induction after specific stimulation as described previously [42 (link)]. PBMCs were prepared from whole blood by density gradient centrifugation using Ficoll-Paque PLUS (Cytiva). Lymph node-derived lymphocytes were prepared from minced lymph nodes by density gradient centrifugation using Ficoll-Paque PLUS. Cells were pulsed and cocultured with peptide pools (at a final concentration of more than 0.1 μM for each peptide) using panels of overlapping peptides spanning the SARS-CoV-2 S, N, M, and E amino acid sequences (PM-WCPV-S-1, PM-WCPV-NCAP-1, PM-WCPV-VME-1, and PM-WCPV-VEMP-1; JPT Peptide Technologies) in the presence of GolgiStop (monensin, BD), 1 μg/ml of anti-CD28 (CD28.2, BD) and 1 μg/ml anti-CD49d (9F10, BD) for 6 hours. Intracellular IFN-γ staining was performed with a CytofixCytoperm kit (BD) and anti-CD3 APC-Cy7, anti-CD4 FITC, anti-CD8 PerCP, and anti-IFN-γ PE (4S.B3; BioLegend). Stained cells were analyzed by BD FACS Lyric. A representative gating schema for flow cytometric analysis is shown in Fig 5A. Specific T-cell frequencies were calculated by subtracting nonspecific IFN-γ⁺ T-cell frequencies from those after peptide-specific stimulation. Specific T-cell frequencies less than 0.03% of CD8⁺ T cells were considered negative.

Detection of antigen specific CD4 T cells detection was quantified by activation induced marker (AIM) and intracellular cytokine staining (ICS). PBMCs/LN cells were stimulated with overlapping peptide pools of HIV consensus C and HIV-1 C.1086 Env gp140C protein (NIH AIDS Reagent Program) in AIM/R10 media in the presence of 0.2 μg CD28/49d co-stimulatory antibodies (BD) per test. As a positive control, cells were stimulated with 1 X Cell Stimulation Cocktail (PMA and ionomycin) (eBioscience, USA). Unstimulated controls were treated with volume-controlled DMSO (Sigma-Aldrich). Tubes were incubated in 5% CO₂ at 37 °C overnight for AIM assay. For ICS assay, after 1 hr of stimulations, protein transport inhibitors 2 μl/mL GolgiPlug (Brefeldin A) and 1.3 μl/mL GolgiStop (Monensin) (BD, Biosciences, USA) were added to the tubes for 8 hr at 37 °C, 5% CO₂. Following stimulation, the cells were stained for AIM and ICS surface markers (see Table 1). Cells were then fixed with cytofix/cytoperm for 10 min at 4 °C, permeabilized with 1 X Perm wash buffer (BD, Biosciences, USA), and stained for intracellular markers (see Table 1) for 45 min. Cells were then washed and acquired the same day on a BD FACS Symphony.

Isolated NK cells were cocultured (1:1 ratio, 25,000 each) with ovarian cancer cells in a flat-bottom 96-well plate with or without different patient ascites or ascites-isolated antibody samples (25% addition), bovine serum albumin [fraction V (pH 7.0), PanReac], or Rituximab (MabThera, 10 mg/mL, Roche). The human anti-EGFR-antibody Cetuximab 1 μg/mL [Erbitux, 5 mg/mL, Merck (Serono)] was added to the coculture to induce NK cell-mediated ADCC. For induction of unspecific degranulation, PMA (Sigma-Aldrich, 50 ng/mL) and Ionomycin (Sigma-Aldrich, 1 µg/mL) were added. NK cells were labeled with anti-CD107a-FITC (25 µg/mL, clone H4A3, Mouse IgG1, k, BD Biosciences) or isotype control and incubated for 1 h at 37°C and 5% CO₂. Afterwards, Golgistop Monensin (BD Biosciences) was added (1:600). Following 5 h of incubation, NK cells were stained with CD56-BV421 (12 µg/mL, NCAM 16.2, IgG2b, k, BD Biosciences). CD107a and CD56 expressions were analyzed by flow cytometry.

Fresh blood was obtained from healthy individuals recruited from the Montreal Neurological Institute and Hospital (MNI/H), McGill University and University of Pennsylvania. All subjects provided an informed consent as approved by the corresponding institutional ethics review boards. The study was approved by and carried out in accordance with the guidelines and regulations of the Institutional Review Board of the Montreal Neurological Institute (McGill University) and University of Pennsylvania and in accordance with the Declaration of Helsinki. Peripheral blood mononuclear cells (PBMC) were isolated from whole blood using Ficoll-Paque density gradient centrifugation (GE Healthcare) and a strict standardized protocol^{53 (link)}. B cells were selected from PBMC using CD19 microbeads (Miltenyi Biotec) according to manufacturer’s recommendations. Typical purities routinely assessed by flow cytometry were > 98%. B cells were cultured in serum-free X-Vivo medium (Lonza) and plated in flat-bottom 24 well plate at 2 × 10⁶/well in a total volume of 1500 μl of medium. Cells were activated with phorbol 12-myristate 13-acetate (PMA; 20 ng/ml, Sigma-Aldrich) and Ionomycin (500 ng/ml, Sigma-Aldrich) for 4 h. In the case of intracellular cytokine staining (ICS), Golgi stop (Monensin, BD Biosciences) was added to cells at the start of stimulation.