Kaluza version 2

Whole-blood aliquots (60 µl), at T3, were withdrawn from Nil (negative control) and mitogen (positive control) tubes and from the three Ag tubes (20 µl each, mixed together) of the QuantiFERON SARS-CoV-2 kit (Qiagen, Hilden, Germany) before centrifugation, and stained with the following combination of anti-human fluorescent monoclonal antibodies: CD3BV786, CD4FITC, CD8PE, CD137APC, CD69BV711 (Becton Dickinson, San Jose, CA, USA), CD154(CD40L)APC-VIO770, and CD134(OX-40)PE-VIO770 (Miltenyi Biotec, Auburn, CA, USA). Pharm Lyse solution (Becton Dickinson, San Jose, CA, USA) was used to remove red blood cells. Then T cells were analyzed by using a 16-color FACS Celesta SORP flow cytometer (Becton Dickinson, San Jose, CA, USA) with the same instrument setting. At least 10⁴ cells were analyzed using the Kaluza Version 2.1.1 software (Beckman Coulter, CA, USA). Cells were gated on the forward scatter/side scatter cell gate and then on the CD3+CD4+ gate for the quantification of CD40L+CD69+ and CD137+OX-40+ SARS-CoV-2-specific CD4 T cells, and on the CD3+CD8+ gate for the quantification of CD137+CD69+ SARS-CoV-2-specific CD8 T cells.

M1-like macrophages were harvested, washed with 1X PBS and then stained with the following anti-human monoclonal antibodies: CD209-FITC, CD64-PE-Vio770, CD80-APC, CD86-PE (all from Miltenyi Biotec, Auburn, CA, USA). For each antibody, isotype controls were used according to fluorescence and antibody specificity (REA293 Isotype control antibody, human IgG1—APC, FITC and PE-Vio770, Miltenyi Biotec, Auburn, CA, USA). Cells were incubated for 20 min at RT in the dark. Cells acquisition has been performed with a 16-colors BD FACS Celesta SORP Cell Analyzer (BD Biosciences, San Jose, CA, USA) with the same instrument setting. At least 10⁴ cells were analyzed using Kaluza Version 2.1.1 software (Beckman Coulter, Carlsbad, CA, USA).

The venous blood of ICs was collected in K3EDTA tubes (Greiner Bio-One GmbH, Kremsmünster, Austria). Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation on Lympholyte Cell Separation Media (Cedarlane Laboratories Limited, Burlington, ON, Canada). Afterward, CD19+ B cells were separated from PBMCs by immune-magnetic sorting using anti-CD19 magnetic microbeads (REAlease CD19 MicroBeads Kit, Miltenyi Biotec, Auburn, CA, USA). The CD19+ B cells obtained from immune-magnetic sorting displayed a purity yield higher than 98%, which was determined by flow cytometry analysis. The isolated fraction was stained with SARS-CoV-2 spike B Cell Analysis Kit (ref. 130-128-022, Miltenyi Biotec, Auburn, CA, USA) to quantify the SARS-CoV-2-specific B cells at T2 and T3 following the manufacturer’s instructions. Samples were acquired by a MACSQuant Cytometer (Miltenyi Biotec, Auburn, CA, USA) and analyzed with the Kaluza Version 2.1.1 software (Beckman Coulter, CA, USA).

Samples were collected pre-challenge and 7, 14 and 21 days post challenge (dpc). Whole blood was mixed 1:1 with medium [RPMI (Gibco, Waltham, MA, USA) plus 1% penicillin-streptomycin (Sigma-Aldrich, St Louis, MO, USA)] referred to as the unstimulated control or with LSDV Neethling strain, BEFV virus or with BHK-21 cells at a 1:10 dilution in medium. Blood was seeded in 96-well plates in triplicate with a total volume of 200 µL/well. Blood was incubated for 24 h in a humidified, 5% CO₂ incubator at 37 °C and brefeldin A was added during the last 5 h of incubation. Red blood cells were removed by lysis using BD Pharm Lyse™ lysing solution (BD Biosciences, Franklin Lakes, NJ, USA), cells were fixed with 4% paraformaldehyde, perforated (0.05% Saponin) and then stained with anti-CD4-FITC (1:50 dilution; BioRad, Hercules, CA, USA); anti-CD8-PE (BioRad, 1:20 dilution) and Mouse anti-Bovine Interferon Gamma:Alexa Fluor^®647 (1:100 dilution; BioRad, Hercules, CA, USA). Samples were assayed on a FC 500 Beckman Coulter flow cytometer and data analysed using Kaluza version 2.1 (Beckman Coulter, Brea, CA, USA). Values significantly higher than unstimulated control (p ≤ 0.05) were considered positive. The 21 dpc analyses data only included anti-CD8 antibodies and IFNγ due to a shortage of anti-CD4 antibody.

Decidual cells and PBMCs were stained for flow cytometric analysis using standard staining procedures. See Supplementary Table S1 for antibodies used. Before staining with antibodies, all samples were stained with Live/Dead Fixable Aqua Dead cell stain (Molecular Probes, Thermo Fisher) for the exclusion of dead cells.
Cells were incubated with antibodies for surface markers, followed by fixation and permeabilization where indicated. For decidual samples and PBMCs from the same donors, the eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher) was used for fixation and permeabilization prior to staining for intracellular markers. Stimulated PBMCs from women pregnant in the 3^rd trimester and from non-pregnant controls were fixed and permeabilized with Cytofix/Cytoperm (Becton Dickinson), followed by staining for intracellular proteins. Staining with the MR1 5-OP-RU and 6-FP tetramers was carried out at room temperature, before staining with antibodies for surface markers. Samples were acquired on an Aria III flow cytometer (Becton Dickinson) and all data was analyzed in Kaluza version 2.1 (Beckman Coulter). Gates were set either based on clear negative/positive populations or based on staining with isotype antibodies.

For GrB and IL-10 detection, cells were harvested at the end of the culture period and stained with anti-CD19 Pacific Blue (clone J3-119, Beckman Coulter, Krefeld, Germany) and 7AAD (BioLegend, Eching, Deutschland) followed by fixation/permeabilization (Cytofix/Cytoperm kit, BD Biosciences, Heidelberg, Germany). Cells were then stained intracellulary for GrB (anti-GrB, clone GB11, PE, eBioscience) or IL-10 (anti-human IL-10, APC, clone JES3-9D7, Biolegend). For the plasma blast formation assay, cells were harvested after culture and stained with anti-human CD19 (clone J3-119, FITC, Beckman Coulter), anti-human CD38 (clone HIT-2, PE, Biolegend), anti-human CD27 (clone O323, APC-H7, Biolegend). Appropriate isotype controls were used to confirm specificity of staining. Flow cytometric measurement was performed the same day with a fluorescence activated cell sorter (FACS) NAVIOS™ from Beckman Coulter. Kaluza Version 2.1 (Beckman Coulter) was used to analyze FACS data.

Cal 27 cells were trypsinised, washed, and resuspended in phosphate-buffered saline (PBS) upon reaching 70%–80% confluency. Whole blood (1 mL) was diluted in 1:2 dilution by PBS. Trypsinised cancer cells were spiked in diluted blood. Approximately 2 mL of diluted blood was carefully layered over 1 mL of HiSep™ LSM 1077 (#LS001, HiMedia, Mumbai, India) in a 15-mL conical tube and centrifuged at 400 g for 30 min at 22°C in a swinging bucket rotor. The upper layer was aspirated without disturbing the mononuclear cell layer at interphase. The mononuclear cell layer was carefully transferred to the other conical tube. Then, PBS was added to this tube, mixed, and centrifuged at 250 g for 10 min at 4°C. The supernatant was discarded.
For cell counting, 10 µL of cell suspension was mixed with an equal volume of trypan blue dye, and cells were placed on a haemocytometer and counted under an inverted microscope. Approximately 100,000 cells (Cal 27 and peripheral blood mononuclear cells (PBMCs) separately) were resuspended in PBS–ethylenediaminetetraacetic acid (EDTA) and subjected to staining for 1 h at 4°C using the following antibodies:
Cells were washed, resuspended with PBS–EDTA, and then analysed using a flow cytometer (Navios, Beckman Coulter; BD FACS Aria™ III) after employing a range of internal quality controls. Data were analysed using Kaluza version 2.1 (Beckman Coulter).