Zombie aqua viability dye

Murine peritoneal cell suspensions were diluted to 5 × 10⁵ cells/ml in fluorescence-activated cell sorting (FACS) buffer (PBS with 2% FCS and 2 mM ethylenediaminetetraacetic acid). To exclude dead cells from the analysis, cells were incubated with Zombie Aqua viability dye (BioLegend, San Diego, CA, USA) for 15 min at room temperature (RT). Afterwards, the cells were washed with FACS buffer. To block the Fc receptors, cell suspensions were incubated with Fc receptor blocking agent (anti-CD16/CD32; Miltenyi Biotec) for 15 min at RT. Afterwards, the cells were washed and stained with the following anti-mouse antibodies for 30 min at 4°C: APC-labeled anti-CD11b (clone M1/70; eBioscience, Thermo Fisher Scientific) and BUV395-labeled anti-Ly-6G (clone 1A8; BD Biosciences, San Jose, CA, USA). The cells were subsequently washed and fixed with 0.4% formaldehyde in PBS. Acquisition was carried out using an LSRFortessa X-20 cell analyzer (BD Biosciences) and data analysis was carried out using FlowJo software (Tree Star, Ashland, OR, USA).

Mouse bladders bearing MB49 tumors were surgically excised followed by mechanical and enzymatic (Liberase, Sigma) dissociation. Fluorochrome-conjugated antibodies against Fc-receptor, CD3, NK1.1, CD4, CD8, CD11c, CD19, F4-80 and Gr-1 (eBioscience) were used. Stained cells were analyzed by flow cytometry on LSR Fortessa (BD Biosciences). For all the flow cytometry data analysis, dead cells were excluded from the analysis by using Zombie Aqua viability dye (BioLegend).

Cryopreserved samples containing 1-10 × 10⁷ PBMC from DRB1*07:01 (DR7)⁺ convalescent subjects or pre-pandemic negative controls were thawed, washed twice with PBS containing 2% FBS, and resuspended in either complete EHAA or RPMI 1640 with 5% FBS. Staining with a pre-mixed cocktail of DR7:p-streptavidin-PE, DR7:p-streptavidin-PE-Cy7, and DR7:p-streptavidin-APC tetramers at a final concentration of 10 nM for each reagent was for 1 hour at room temperature. Anti-PE and anti-APC magnetic beads (Miltenyi Biotec) were then added, and bead-bound cells were enriched as previously described (22). Cells in the bound and unbound fractions were stained with Zombie Aqua viability dye (BioLegend) and a surface marker antibody panel consisting of anti-CD20^BV510 (clone 2H7, BioLegend), anti-CD14^BV510 (clone M5E2, BioLegend), anti-CD3^AF700 (clone UCHT1, BioLegend), anti-CD4^BV605 (clone OKT4, BioLegend), anti-CD8^BUV395 (clone RPA-T8, BioLegend), anti-CD45RO^APC/Cy7 (clone UCHL1, BioLegend), anti-CXCR3^PE/Dazzle594 (clone G025H7, BioLegend), anti-CXCR5^BV421 (clone J252D4, BioLegend), anti-PD-1^BV785 (clone EH12.2H7, BioLegend), and anti-CCR4^PerCP/Cy5.5 (clone L291H4, BioLegend) for 30 min at room temperature. Data were acquired on an LSR Fortessa X-20 (BD) flow cytometer and analyzed with FlowJo software (BD).

Single cell suspensions generated as above or stimulated cells were stained with Zombie Aqua viability dye (Biolegend), Fc receptors blocked with TruStain FcX (Biolegend) and then stained with fluorophore conjugated antibodies against CD45 (Clone 30-F11, BD AB_2872789), CD3 (145-2C11, BD AB_2738278), CD4 (RM4.4, Biolegend AB_2563110), CD8a (53–6.7, Biolegend AB_493423 or AB_2562610), CD44 (IM7, Biolegend AB_493679), CD69 (H1.2F3, BD AB_2740186). For intracellular staining, cells were fixed and permeabilized with FOXP3 fix/perm kit (eBioscience) and then stained with antibodies against IFNγ (XMG1.2, Biolegend AB_315404), TNFα (MP6-XT22, Biolegend AB_2629800), Perforin (S16009A, Biolegend AB_2721463), IL-2 (JES6-5H4, Biolegend AB_2650897) or Ki67 (16A8, Biolegend AB_2564285). For all presented data cells were gated by forward and side-scatter to exclude debris, doublets and non-viable cells. For all studies, T-cells were defined as CD45⁺CD3⁺, CD8 T-cells were defined as CD45⁺CD3⁺CD4⁻CD8⁺ and CD4 T-cells were defined as CD45⁺CD3⁺CD4⁺CD8⁻. Gating strategy is provided in Supplemental Figure S1. Gating for activation markers and cytokines was set using cells which were stained with antibodies except those against activation markers or cytokines or stimulated in the absence of CD107a.