Golgiplug containing brefeldin a

PBMCs from healthy donors’ fresh blood were cultured in RPMI 1640 medium (Gibco) containing 10% of FBS and stimulated 50% (v/v) with supernatant from macrophages treated with 1 mM of SB and PBA. Stimulation was performed for 6 h at 37 °C 5% CO₂ in the presence of Golgi-Plug containing Brefeldin A (BD Biosciences, San Jose, CA, USA) and Golgi-Stop containing Monensin (BD Biosciences) added after 1 h of the stimulation according to the manufacturer’s instructions. After that, PBMCs were washed and stained with the Live/Dead fixable Blue (Invitrogen) and surface markers (listed in Supplementary Table S1) for 30 min at room temperature, twice washed, fixed and permeabilized using the eBioscience™ Transcription Factor Fixation/Permeabilization (Invitrogen) according to the manufacturer’s instructions. Subsequently, the fixed and permeabilized PBMCs were staining using fluorochrome-conjugated antibodies against intracellular makers listed in Supplementary Table S1. Labeled cells were acquired on a Cytek Aurora Spectral Cytometer (Cytek Biosciences, Fremont, CA, USA). Data were analyzed using FlowJo (TreeStar, Ashland, OR, USA) v10.6.2 software.

For the intracellular cytokine staining, PBMC were cultured in the presence of SARS-CoV-2-specific MPs [1 ug/mL] for 9 h at 37°C. Golgi-Plug containing brefeldin A (BD Biosciences, San Diego, CA) and monensin (Biolegend, San Diego, CA) were added 3 h into the culture. Prior to addition of peptide MPs, cells were blocked at 37°C for 15 min with 0.5ug/mL anti-CD40 mAb (Miltenyi Biotec). Cells were then washed and surface stained for 30 min on ice, fixed with 1% of paraformaldehyde (Sigma-Aldrich, St. Louis, MO) and kept at 4°C overnight. Antibodies used in the ICS assay are listed in Table S4. The gates applied for the identification of CD40L⁺, CD40L⁺IFNg⁺, CD40L⁺IL-2⁺, CD40L⁺IL-10⁺, CD40L⁺IL-17⁺ or CD40L⁺TNFa⁺ production on non-CD45RA⁺CCR7⁺ CD4⁺ T cells were defined according to the cells cultured with DMSO for each individual. The gates applied for the identification of IFNg⁺, IFNg⁺TNFa⁺ or IFNg⁺GzmB⁺ production on non-CD45RA⁺CCR7⁺ CD8⁺ T cells were defined according to the cells cultured with DMSO for each individual. Antibodies used in the ICS assay are listed in Table S5.

Single-cell suspensions of digested tumors from OPC and OPC-API mice (1 × 10⁶ cells) were plated onto a 96 well plate coated with purified anti-mouse CD3 (10µg/mL) (Biolegend). Soluble purified anti-mouse CD28 (2µg/mL) (Biolegend) in RPMI 1640 media (10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin) was added to the assay wells and then incubated at 37 °C in 5% CO₂. After two days of incubation, Golgi Plug, containing brefeldin A, (1 ug/mL) (BD Bioscience) was added to each assay well and incubated for 4 hrs. After incubation, cells were surface stained with fluorescent antibodies including anti-mouse CD3-FITC (Biolegend) and anti-mouse CD8-PercP/Cy5.5 (Biolegend). Samples were then intracellularly stained with anti-mouse IFN-γ-PE (eBioscience), along with isotype CTRL, using a Cytofix/Cytoperm kit (BD Bioscience), according to the manufacturer’s protocol. Acquisition of activated CD8⁺ T cell samples were performed using a flow cytometer BD LSRII (BD Biosciences Immunocytometry Systems). FlowJo software (BD Bioscience) was used for data analysis.

Fresh blood collected from donors was subjected to centrifugation over a Ficoll-Paque Plus (GE Healthcare Life Sciences) density gradient, washed twice with PBS + 2% FBS and resuspended in R10. Isolated PBMCs were cryopreserved in Recovery Cell Culture Freezing Medium (Life Technologies). IFN-γ expression in NK cells was detected by intracellular cytokine flow cytometry. Briefly, frozen PBMCs (2 × 10⁵ cells/well) were incubated with K562 cells expressing or lacking HLA-B molecules at 1:1 (PBMC:K562) ratio in 200 μL complete media in 96-well U-bottom plates. GolgiPlug (containing brefeldin A, BD Biosciences) was added at 1:1000 1h later. After incubation for an additional five hours, cells were stained with Pacific Blue-conjugated anti-CD3, PE-Cy7-conjugated anti-CD56 and FITC-conjugated anti-KIR3DL1 mAbs for 30 minutes at 4°C, fixed in 4% paraformaldehyde for 10 minutes at room temperature, and permeabilized with 0.2% saponin for 10 minutes. Cells were then stained with Alexa Fluor 700-conjugated anti-IFN-γ for 30 minutes at 4°C and analyzed by flow cytometry.

Single-cell suspensions of the lung were prepared and cells were incubated with PMA (50 ng/ml; Sigma), ionomycin (1000 ng/ml; Sigma), and GolgiPlug-containing brefeldin A (BD Biosciences) for 4–6 h as described previously [17 (link)]. The cells were harvested and blocked with antibody to mouse CD16/CD32 (Fc Block, BD). Samples were immunostained with antibody to mouse CD3, CD4, CD8, or isotype control conjugated with PerCP-cy5.5, FITC or PeCy7 for 30 min on ice and then fixed with 1% Formaldehyde in FACS Staining Buffer. The indicated antibodies were obtained from eBioscience (San Diego, CA). For intracellular IL-17A detection, cells were fixed and permeabilized with CytoFix/CytoPerm solution (554722; BD) and Perm/Wash buffer (554723; BD) and then stained with APC-conjugated anti-IL-17 mAb (BD Biosciences). Stained samples were measured on a flow cytometer, FACSCalibur (BD Biosciences). The data were analyzed using CellQuest software (BD Biosciences).

Single-cell suspensions of lung were prepared and cells were incubated with PMA (50 ng/ml; Sigma), ionomycin (1000 ng/ml; Sigma) and GolgiPlug-containing brefeldin A (BD Biosciences) in 1 ml complete RPMI (RPMI 1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100 U/ml penicillin and 100 μg/ml streptomycin). After 4–6 h incubation, the cells were harvested and blocked with antibody to mouse CD16/CD32 (Fc Block, BD). Samples were immunostained with antibody to mouse CD3, CD4, CD8 or isotype control conjugated with PerCP-cy5.5, FITC or PeCy7 for 30 minutes on ice and then fixed with 1% Formaldehyde in FACS Staining Buffer. The indicated antibodies were obtained from eBioscience (San Diego, CA). For intracellular IFN-γ detection, cells were fixed and permeabilized with CytoFix/CytoPerm solution (554722; BD) and Perm/Wash buffer (554723; BD) and then stained with APC-conjugated anti-IFN-γ mAb (BD Biosciences). Stained samples were measured on a flow cytometer, FACSCalibur (BD Biosciences). The data were analyzed using CellQuest software (BD Biosciences).