Anti ifn γ pe

Mouse splenocytes were blocked with anti-CD16/CD32 mAbs and stained with eFluor 450 viability dye (93: catalog 101320, BioLegend). Antibodies used for cell surface proteins were Alexa Fluor 700–anti-CD3 (17A2: catalog 100215), PerCP-Cy5.5–anti-CD8a (57-6.7: catalog 100734), APC-Cy7–anti-CD4 (GKq.5: catalog 100412), PE-Cy7–anti-CD19 (6D5: catalog 115520), PE–anti-CD62L (W18021D: catalog 161203), PE-Cy7–anti-CD44 (IM4: catalog 103024) (all BioLegend), and APC–anti-CD11c (3.9: catalog 17-0116-42, Thermo Fisher Scientific). The cells were then stained with fluorescence antibodies or H-2D(b) SARS-CoV-2 N_219-227 LALLLLDRL (Alexa Fluor 647–Labeled Tetramer) or H-2K(b) SARS-CoV-2 S_539-546 VNFNFNGL (Alexa Fluor 647–Labeled Tetramer) (NIH Tetramer Facility, Emory University). For intracellular staining, the cells were permeabilized in PBS containing 0.1% saponin and fixed for 10 minutes in 4% paraformaldehyde. The cells were then stained with PE–anti–IFN-γ (XMG1.2: catalog 505808), APC-cy7–anti–TNF-α (MP6-XT22: catalog 506343), PE–anti-perforin (S16009A: catalog 154305), and PE-cy7–anti-granzyme (QA16A02: catalog 372213) (BioLegend). The cells were analyzed on an LSR II flow cytometer (BD) and the data were analyzed with FlowJo software.

Before detection of intracellular cytokine production, human PBMC or isolated mice splenocytes were restimulated with PMA (50 ng/ml) plus ionomycin (500 ng/ml) for 4h in the presence of Brefeldin A (GolgiStop; BD Biosciences). Staining with LIVE/DEAD Fixable Dead Cell Stain Kit (Molecular Probes) was performed before fixation to allow gating on viable cells. Cells were then blocked for 15 mins and stained with antibodies targeting specific surface markers for different lymphocytes (APC anti-CD3, PerCP/Cy5.5 anti-CD4/ PerCP/Cy5.5 anti-CD19/ FITC anti-CD16, APC anti-CD56/ FITC anti-HLA-DR, PerCP/Cy5.5 anti-CD11c/ FITC anti-CD14) at 4°C for 30 mins. After fixation and permeabilization with Fixation and Permeabilization solution (BD Biosciences) for 20 mins, cells were stained with PE anti-IFN-γ, PE anti-IL-4, PE anti-IL-17, PE anti-IL-10 or PE Rat IgG1, κ (BioLegend) 4°C for 30 mins respectively and then submitted to flow cytometry analysis (FACS Calibur; BD Biosciences).

In order to evaluate the antigen-specific T cells to the RBD protein, a single cell suspension was prepared from the spleen of the mice and stimulated in a 96-well plate, using eBioscience Cell Stimulation Cocktail (Invitrogen, Waltham, MA, USA) at 37 °C with 5% CO₂ for 8–12 h. The cells were processed by a fixation/permeabilization kit (BD Biosciences, Franklin Lakes, NJ, USA), followed by staining with IL-4 (BD), IFN-γ, and TNF-α (BioLegend, San Diego, CA, USA). The following cytokine antibodies were used: PE-Cy7-anti IL-4 (BD), PE-anti IFN-γ (BioLegend), and APC-anti TNF-α (BioLegend). The proportion of IFN-γ⁺ and TNF-α⁺ CD8⁺ T cells and memory T cells were analyzed by flow cytometry (BD Biosciences, Franklin Lakes, NJ, USA) and FlowJo software (version 10.6.2).