Ifn γ fitc

The CD8⁺ T cells were incubated with IFN‐γ‐FITC (11‐7311‐41, Invitrogen), anti‐human CD8‐APC (#344721, BioLegend), or anti‐mouse CD8a‐APC (17‐0081‐82, Invitrogen), and TAMs were incubated with F4/80‐FITC (#23107, BioLegend) and Arg‐1‐APC (IC5868A, R&D Systems) in the dark for 30 min. The phenotype of CD8⁺ T cells and TAMs was then analyzed by flow cytometry.

Cell suspensions were made and cells stained as described (25 (link), 37 (link)) using the following directly conjugated antibodies from BioLegend unless otherwise stated: CD11b APC (clone M1/70), Siglec-F PE (BD Bioscience, clone E50-2440), CD4 FITC, AF700 (clone GK1.5), ST2 APC (clone DIH9), CD117 APC (ACK2), FcεRI PerCP-eFluor 710 (eBioscience, clone MAR1), CD49b PE (clone DX5), IL4rα PE (clone I015F8), and CD25 (clone 3C7). A minimum of 1 × 10⁵ cells were acquired, and doublets were excluded by gating cells on FSC-H/FSCA, as illustrated in Supplementary Figure 1. For intracellular cytokine staining, mLN cells were stimulated with Cell Activation Cocktail (with Brefeldin A, BioLegend) at 1 × 10⁶ cells/ml, incubated in complete RPMI (10% FBS and 5% Pen/Strep) at 37°C for 6 h. Then, cells were fixed using Ic Fixation buffer (Invitrogen) and permeabilized using Permeabilization Buffer (Invitrogen) to carry out the intracellular staining for IL-4 PE (clone 11B11), IL-13 Pecy7 (Invitrogen, clone eBio13A), and IFN-γ FITC (clone XMG1.2). Cells (2 × 10⁶) were used for transcription factor staining. Staining against Foxp3 PE and Gata3 PE was performed using the Transcription Factor Staining Buffer Set according to the manufacturer’s instructions. Data were acquired on a C6 Accuri flow cytometer (BD Biosciences) or Cytoflex (Beckman) and analyzed using FlowJo v10.6.

Cells were stained with specific antibodies or control isotypes using conventional protocols. The following anti-mouse antibodies were used for FACS analysis of MDSCs: CD11b/PECy5, Gr1/PE, Gr1/APC, CD62L/FITC, F4/80/FITC, CD11c/PE, and MHC CI OVA peptide/Biotin from eBiosciences (San Diego, CA); as well as CD124/PE, CD40/FITC, CD86/FITC, I^A-I^E/FITC, Ly6C/FITC and Ly6G/PE that were purchased from BD Biosciences (San Jose, CA). Lymphocytes characterization was performed with the next specific antibodies: CD8/PE and CD8/Biotin, CD69/PECy5, CD62L/FITC, CD247/PE, CD4/PE, CD4/PECy5, CD25/PECy5, Foxp3/Biotin, IFN-γ/FITC, IL-4/PE and IL-17/PE, all from eBiosciences. FITC-conjugated Streptavidin was purchased also from eBiosciences. In all cases, cells were previously incubated with anti-mouse FcγR 2.4G2 ascites (HB-197; ATCC) to reduce the non-specific binding. For intracellular staining, cells were fixed and permeabilized, according to the manufacturer’s protocol, with either Foxp3 Staining Buffer Set or IC Fixation and 10X Permeabilization Buffers from eBiosciences. Cells were acquired using both FACScan (BD Biosciences) and Gallios (Beckman Coulter, Miami, FL) flow cytometers and analyzed with Kaluza 1.2 (Beckman Coulter) and FlowJo 5.7.2 (Tree Star Inc., USA) softwares.