Facs symphony

Approximately 5 × 10⁶ cells were aliquoted into tubes, washed with FACS buffer (PBS containing 2% FCS and 0.05% sodium azide), and resuspended in 100 μl of FACS buffer containing appropriate conjugated antibodies. Surface staining was done for 30 minutes on ice in the dark. Cells were washed, fixed, and permeabilized (using the eBioscience Fixation/Permeabilization Transcription Factor Staining Kit), and intracellular staining (where indicated) was performed overnight at 4°C. Cells were washed in permeabilization buffer and resuspended in FACS buffer, and data were acquired on a BD FACSsymphony or LSRFortessa. Data were analyzed using FlowJo (TreeStar).

Biotinylated RBD protein (Acro Biosystems, cat# SPD-C82E9) was complexed with fluorochrome-conjugated streptavidin APC (BioLegend, cat# 405207) to yield RBD-Tetramers. Lymph node cells from FNA were incubated with the RBD-fluorochrome complex and subsequently stained with aqua live/dead dye (Invitrogen, L34957), anti-human IgM (FITC, clone: G20–127, BD cat# 555782, 1:5), anti-human IgG (PE-Cy7, clone: G18–145, BD cat# 561298, 1:20), anti-human CD3 (AF700, clone: SP34-2, BD cat# 557917, 1:20), anti-human PD-1 (BV650, clone: EH12.1, BD cat# 564104, 1:20), anti-human CD20 (PE/Dazzle 594, clone: 2H7, BioLegend cat# 302348, 1:20), anti-human CD4 (APC-Cy7, clone: OKT4, BioLegend cat# 317418, 1:20) and anti-human CXCR5 (PerCP-eF710, clone: MU5UBEE, ThermoFisher cat# 46-9185-42, 1:20). Cells were fixed/permeabilized using Transcription Factor Staining Buffer Set (ThermoFisher, cat# 00-5521-00) and further stained with anti-human Bcl-6 (PE, clone: 7D1, BioLegend cat# 358504, 1:20) and anti-human Ki-67 (BV421, clone: 11F6, BioLegend cat# 151208, 1:20). Sample acquisition was performed on a BD FACS Symphony and data were analyzed with BD FlowJo V10 software.

Cells were stimulated for 4 h with PMA (phorbol 12-myristate13-acetate; 50 ng/mL), ionomycin (1.0 µg/mL), and a protein-transport inhibitor containing monensin. PMA and ionomycin stimulate the immune cells in a non-antigen specific manner, and monensin is used to trap the cytokine within the cytosol. After stimulation, surface markers were stained for 15–20 min at room temperature in PBS with 1% FBS. Cells were then fixed in Cytofix and permeabilized with Perm/Wash Buffer using the BD Fixation Permeabilization solution kit and stained with anti-IL-17A (TC11-18H10.1, Biolegend, San Diego, CA, USA); anti-IFN-γ (XMG1.2, Biolegend); and anti-IL-4 (11B11, Biolegend) antibodies diluted in Perm/Wash buffer. Permeabilization was undertaken in order to make the intracellular cytokines accessible to the FACS antibodies. All antibodies were used in 1:500 dilutions. Flow cytometry was undertaken using BD FACS Symphony, and the data were analyzed with FlowJo software version 10.9.0.

Cells to be stained with lymphocyte antibodies were stimulated with a cell activation cocktail (2 mg/mL) containing phorbol myristate acetate/ionomycin/Brefeldin A (Biolegend-423303, San Diego, CA) for 4 h at 37°C. Cells to be stained with dendritic cell antibodies were stimulated with R848 (1ug/mL) (Invivogen- tlrl-r848, San Diego, CA) and Brefeldin A (1mg/mL) (Biolegend-42060) for 5 h at 37°C. Detailed staining protocol is included in Supplementary methods. Subsequent to staining, single-cell suspensions underwent flow cytometric analysis on a BD FACS Canto (5 samples) or BD FACS Symphony (2 samples) (BD Biosciences, San Jose, CA) and were analyzed with the FlowJo software (BD Biosciences). The intensity of the staining was measured by percent of parent population, and gating was determined by the mean fluorescence intensity (MFI) values. To distinguish between specifically stained cells and background fluorescence, we used appropriate controls, including unstimulated samples and fluorescence-minus-one controls [48 (link)]. We defined this MFI value as a cut-off and considered a cell as positive for a given marker if their MFI exceeded this cut-off value and cells with MFI values below this cut-off value were referred to as cytokine-negative cells. Further data regarding gating process can be found in the Supplementary methods.