Zombie aqua zombie aqua fixable viability kit

Endometrial cancer digests (n = 12) or frozen peripheral blood mononuclear cells were thawed, washed, and centrifuged. Pellets were resuspended in RPMI with 10% FCS. Half of each sample was incubated with PMA/ionomycin (eBioscience 1× Cell Stimulation Cocktail) for 4 h at room temperature, and all samples were incubated with a protein transport inhibitor to enable intracellular cytokine staining (1:1000, BD GolgiPlug™ Protein Transport Inhibitor, Thermo Fisher Scientific, BD555029, Waltham, MA, USA). Cells were stained with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, #423101, BioLegend) for 15 min, fixed, and permeabilized according to the manufacturer’s protocol (FIX & PERM™ Cell Permeabilization Kit, #GAS003, ThermoFisher Scientific), followed by incubation with antibodies for CD3, CD8α, TNF-α, IFN-γ, IL-2 with either IL-21 and IL-13 or IL-10, and GM-CSF (Supplementary Table S6). Samples were incubated at room temperature for 15 min, washed and suspended in PBS + 2% FCS. Samples were analyzed on FacsVerse (BD Biosciences) and results were analyzed with Cytobank premium software (cytobank.org).

Multiparameter flow cytometry was performed in accordance to BD Pharmingen™ Protocol. In brief, splenocytes were harvested at day 14 post second boost; 5 × 10⁶ cells were stimulated in a Nunc™ 96-well conical-bottom plate (Thermo Scientific™, USA) for 6 h at 37 °C with 10 μg of M2eh peptide and 1 μg of influenza virus A/Aichi/2/68 (H3N2) in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis and subsequently stained with antibodies (CD3e-FITC, CD8a-APC-Cy7, CD4-PerCP, CD62L-PE-Cy7, CD44-APC) at 4 °C for 30 min (antibodies from BD Pharmingen, USA). Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocol and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza, version 1.5 (Beckman Coulter, USA).

Multi-parameter flow cytometry was performed in accordance with the BD Pharmingen^TM protocol. In brief, mouse lungs were harvested at day 14 post second boost; lung lymphocytes (5x10⁶) were stimulated in Nunc^TM 96-well conical bottom plates (Thermo Scientific™, USA) for 6 h at 37°C with 10 μg of M2eh peptide, in combination with 1 μg of either the A/Aichi/2/68 (H3N2) or the A/California/07/09 (H1N1pdm09) influenza virus, in the presence of 1 μg/ml of Brefeldin A (BD Bioscience, USA) and purified hamster anti-mouse CD28. The cells were then washed, and Fc receptors were blocked using CD16/CD32 antibodies (Mouse BD Fc Block, BD Pharmingen, USA) for 30 min. Next, cells were incubated with Zombie Aqua (Zombie Aqua™ Fixable Viability Kit, Biolegend, USA) to enable gating of live cells during analysis, and they were subsequently stained with CD3e-FITC, CD8a- APC-Cy™7, CD4- PerCP, CD62L-PE-Cy™7, or CD44-APC (BD Pharmingen, USA) antibodies at 4⁰ C for 30 min. Cells were permeabilized using the BD Cytofix/Cytoperm Plus (BD Bioscience, USA) protocols and stained with anti-TNF-α-BV421 or anti-IFN-γ-PE antibodies (BD Pharmingen, USA). Sample acquisition (50,000 live CD3+ T-cells were collected) was performed with a BD FACS Canto II flow cytometer (Becton Dickinson, USA) and analyzed using Kaluza version 1.5 (Beckman Coulter, USA).