Cd69 brilliantviolet510

Mixed cell suspensions were stained for surface markers with combinations of the following antibodies: CD45-vioblue450, CD8-FITC, CD19-APC (Tonbo, San Diego, CA), HLA-DR-FITC, CD3-viogreen, CD103-PE (Miltenyi Biotec, Auburn, CA), CD11c-PerCp-Cy5.5, CD103-PE-Cy7, CD4-PE (eBioscience, San Diego, CA), CD56-APC (BD Pharmingen, San Diego, CA), CD69-BrilliantViolet510 (BioLegend, San Diego, CA). Dead cells were excluded with 7AAD (Southern Biotech, Birmingham, AL) or zombie dye yellow staining (BioLegend, San Diego, CA). For spectral flow cytometry, the following antibodies were used: CX3CR1-PE eFluor610 (Thermo Fisher, Waltham, MA), CD3-viogreen (Miltenyi Biotec), CD4-BrilliantUV805, CD10-BrilliantViolet650, CCR5-PE-Cy5, CD45-BrilliantUV395 (BD Biosciences, Franklin Lakes, NJ), HLA-DR-AlexaFluor700, CCR7-BrilliantViolet750, CD8-SparkBlue550, CD62L-BrilliantViolet605 (BioLegend). Analysis was performed on Gallios (Beckman Coulter, Brea, CA) or Cytek Aurora 5 lasers configuration (Cytek, Fremont, CA) flow cytometers and data analyzed with FlowJo (Tree Star, Inc., Ashland, OR) or OMIQ software (www.omiq.ai). Expression of surface markers is shown as percentage of positive cells. Fluorescence minus one (FMO) strategy was used to establish appropriate gates.

Cells from animals were stained with fluorescently conjugated antibodies: CD69-Brilliant Violet 510 (clone FN50) or CD14-Brilliant Violet 510 (clone M5E2), CD3-Pacific Blue (clone Hit3a), CD8a-FITC (clone Hit8a), CD4-PE (clone RPA-T4), CD19-PE-Cy5 (clone SJ25C1), CD45-APC (clone 2D1), CD56-PE-Cy7 (clone MEM-188) (all from Biolegend) and Ghost Dye Red 780 (Tonbo Biosciences). All flow cytometry samples were run using a MACSQuant Analyzer 10 flow cytometer (Miltenyi) or Attune NxT. (Beckman Coulter). All data was analyzed using FlowJo v.10 (TreeStart, Inc) (Supplementary Fig. 10d).

PBMC were isolated from CPT tubes (BD Biosciences) according to the manufacturer’s instructions and cryopreserved in CTL-CryoTM ABC Media Kit (ImmunoSpot). Before T cell stimulation the PBMC vials were thawed, washed, counted, and suspended in an X-Vivo15 serum-free medium (Lonza). 1-2 million of PBMCs were used per condition. Overlapping peptide pools of SARS-CoV2 spike, nucleocapsid, and membrane proteins were purchased from Miltenyi Biotec and used at a final concentration of 1ug/ml of each peptide. The stimulations (mock, S, M, and N peptide pools) were carried out for 20 hours in the presence of anti-CD28 and anti-CD49d. CEFX peptide pool (JPT Peptides) was used as a positive control. After the stimulation T cells were stained with monoclonal antibodies: CD3 Brilliant Violet 650, CD4 Alexa Fluor 700, CD8 Brilliant Violet 605, CCR7 PE-Dazzle, CD45RA APC, CD69 Brilliant Violet 510 (all from Biolegend), and CD137 PE (from Miltenyi Biotech). Before the acquisition with LSRFortessa (BD Biosciences), 7AAD was added for the discrimination of dead cells. Data were analyzed using FCS Express 7 (DeNovo Software).