Anti cd16 cd32 antibody clone 2.4g2

To minimize nonspecific binding, 2 million cells of lung suspension, were pre-incubated with an anti-CD16/CD32 antibody (Clone 2.4G2, BD Biosciences, Erembodegem, Belgium). After viability staining, using the Zombie Aqua™ Fixable Viability Kit, cells were labelled with combinations of anti-mouse fluorochrome-conjugated mAbs against CD45 (clone 30-F11), CD11c (clone N418), MHCII (clone M5/114.15.2), CD11b (clone M1/70), CD103 (clone 2E7), CD64 (clone X54-5/7.1) and Siglec-H (clone 551) (all from Biolegend, San Diego, California). Sample acquisition was performed on a LSR Fortessa SORP flow cytometer running DIVA software (BD Biosciences, Erembodegem, Belgium). FlowJo software (BD Bioscience, Ashland, Oregon) was used for data analysis. Gating strategy is available in Appendix A Fig. S3. Flow cytometry configuration are available in Appendix B.

Lung MNCs were resuspended in the FACS buffer (1–2 × 10⁶ cells/mL). Cells were first blocked with anti-CD16/CD32 antibody (clone 2.4G2; BD Biosciences, San Diego, CA) and then labeled with isotype controls or the following antibodies: FITC-IL-4, FITC-IFN-γ, PeCy5-TCR-β (eBioscience), PE-PBS-57 (α-GalCer analog)/CD1d tetramer (NIH tetramer core facility). Dead cells were excluded by forward scatter and side scatter. iNKT cells were measured as PBS-57/CD1d tetramer and TCR-β double-positive cells. For intracellular cytokine staining, lung MNCs were cultured for 4 to 6 h with 500 ng/mL ionomycin and 50 ng/mL phorbol 12-myristate 13-acetate (PMA) in the presence of monension (1 μL/mL). Cells were harvested and washed, and intracellular staining was performed according to the manufacturer’s protocol (eBioscience). Cells were analyzed by flow cytometry (Epics Altra; Beckman, Seattle, WA).

ICS assays were performed using 10⁶ live splenocytes per well in 96-well U-bottom plates. Splenocytes in RPMI media supplemented with 10% FBS were stimulated by the addition of pools of overlapping peptide for S or N antigens at 2 μg/mL/peptide for 6 h at 37 °C in 5% CO₂, with protein transport inhibitor, GolgiStop (BD) added two hours after initiation of incubation. The S peptide pool (JPT: Cat #PM-WCPV-S-1) is a total of 315 spike peptides split into two pools comprised of 158 and 157 peptides each. The N peptide pool (JPT; Cat # PM-WCPV-NCAP-1) was also used to stimulate cells. A SIV-Nef peptide pool (BEI Resources) was used as an off-target negative control. Stimulated splenocytes were then stained for a fixable cell viability stain followed by the lymphocyte surface markers CD8β and CD4, fixed with CytoFix (BD), permeabilized, and stained for intracellular accumulation of IFN-γ, TNF-α and IL-2 (in studies 2 and 3). Fluorescent-conjugated antibodies against mouse CD8β antibody (clone H35-17.2, ThermoFisher), CD4 (clone RM4-5, BD), IFN-γ (clone XMG1.2, BD), TNF-α (clone MP6-XT22, BD) and IL-2 (clone JES6-5H4; BD), and staining was performed in the presence of unlabeled anti-CD16/CD32 antibody (clone 2.4G2; BD). Flow cytometry was performed using a Beckman-Coulter Cytoflex S flow cytometer and analyzed using Flowjo Software.