Anti foxp3 clone mf 14

Single-cell suspensions from spleen of mice were stained with anti-CD4 (clone GK1.5; BioLegend), anti-CD25 (clone 3C7; BioLegend), and anti-FoxP3 (clone MF-14; BioLegend). In the case of intracellular cytokine staining, splenocytes were cultured in RPMI 1640 medium containing 10% fetal calf serum, 2 mM L-glutamine, 100 U/ml penicillin, and 100 U/ml streptomycin in presence of phorbol-12-myristate-13-acetate (PMA) (50 ng/ml; Sigma), ionomycin (1 μg/ml; Sigma), and monensin (BioLegend) for 5 h. Cells were washed, fixed, permeabilized, and stained with anti-IL-17 (clone TC11-18H10.1; BioLegend). Viable single cells were analyzed based on forward and side light scatter properties with a CyFlow Cube 6 flow cytometer (Sysmex) or MACSQuant Analyzer 10 flow cytometer (Miltenyi Biotec).

Antibodies used in this study: anti-hamster IgG, whole molecule (Sigma-Aldrich, St. Louis, MO), NA/LE hamster anti-mouse CD3ε, anti-mouse CD28 (BD Biosciences), anti-PRMT5 (A-11, Santa Cruz Biotechnology, Dallas, Tx), anti-H3R2me2s polyclonal (Invitrogen, Waltham, MA), alpha-tubulin mouse (DM1A, Cell Signaling Technologies (CST), Danvers, MA), anti-mouse secondary-IgG HRP linked F(ab’)₂ fragment (clone NA9310V Amersham Biosciences, Piscataway, NJ), anti-rabbit secondary – IgG HRP linked whole antibody (clone NA934V, Amersham Biosciences), anti-PRMT5 rabbit monoclonal (clone ST51-06, Invitrogen), anti-H3R2me2s ChIP-seq grade (EpigenTek, Farmingdale, NY), anti-CD4 FITC (Clone H129.19, BD Biosciences), anti-CD25 (clone PC61, BioLegend, San Diego, CA), anti-Foxp3 (clone MF-14, BioLegend), anti-IFNγ (BD Biosciences), anti-Tbet (BioLegend), nuclear stain Draq5 (ThermoFisher Scientific), anti-rabbit IgG Fab₂ Alexafluor 488 (CST), zombie violet fixable viability kit (BioLegend), CytoTell UltraGreen and CytoTell Red650 (AAT Bioquest, Pleasanton, CA).

Tumor and pleural immune cells were fixed, permeabilized, and stained with anti-CD45 (clone 30-F11), anti-CD11b (clone M1/70), anti-F4/80 (clone BM/8), anti-CD206 (clone C068C2), anti-Ly6C (clone HK1.4), anti-Ly6G (clone RB6-8C5), anti-CD11c (clone HL3), anti-MHCII (clone M5/114.15.2), anti–IL-10 (clone JES5-16E3), anti–IL-12 (clone C15.6), anti-CSF1R (clone AFS98), anti-CD3 (clone 145-2C11), anti-CD4 (clone GK1.5), anti-CD8 (clone YTS1567.7), anti-Foxp3 (clone MF14), and anti–granzyme B (clone QA18A28) (all purchased from BioLegend). Inflammatory cells were selected based on their forward- and side-scatter profiles and their CD45⁺ staining. Specifically, macrophage populations were characterized as CD11b⁺Ly6G^–Ly6C^lo F4/80⁺, Mo-MDSCs as CD11b⁺Ly6G^–Ly6C, polymorphonuclear MDSCs as CD11b⁺Ly6G⁺Ly6C^lo, and activated DCs as CD11c⁺MHCII⁺ cells. M2 macrophage phenotype was determined as F4/80⁺CD206⁺. In addition, M1/M2 phenotypes were evaluated according to the macrophage IL-12/IL-10 expression ratio. Total numbers of CD3⁺CD4⁺ and CD3⁺CD8⁺ lymphocytes were also enumerated. Tregs were determined as CD4⁺Foxp3⁺, as well as activated CD8⁺ T cells by granzyme B expression. The flow cytometry data were acquired using BD FACSCanto II flow cytometer and analyzed by FlowJo software.