Cd8a pecy7

Before immunofluorescence staining of cells, Fc receptors were blocked using FcR Blocking Reagent (Miltenyi Biotec). Subsequently, the cells were stained with fluorochrome-labeled mouse antibodies and analyzed using a MACSQuant^® Analyzer (Miltenyi Biotec). Dead cells identified by propidium iodide (Miltenyi Biotec) staining were excluded from the analysis. All antibodies were purchased from Miltenyi Biotec except where noted. The following antibodies were used to stain tumor infiltrating CD8⁺ T cells: CD45-VioBlue, CD4-FITC, CD8a PE-Cy7 (Thermo Fisher Scientific, MA, U.S.A.), MHC class II-APC and APC/Cy7-TCRβ (BioLegend, CA, U.S.A.). The following antibodies were used for detailed analysis of CD8⁺ T cells: CD45-VioBlue, CD4-VioGreen, CD44-FITC, CD8a PE-Cy7 (Thermo Fisher Scientific), CD25-APC, CD69-APC-Vio770 and CD62L-PE. The following antibodies were used in the anti-CD8 antibody experiment: CD45-VioBlue, CD8a-PE-Vio770 and APC/Cy7-TCRβ (BioLegend).

Spleen and MLN single cell suspensions (0.5–1 × 10⁶ cells/well) were incubated with anti-mouse CD16/CD32 (Mouse BD Fc Block, BD Biosciences, San Jose, CA, USA) in PBS supplemented with 1% BSA and 5% FCS for 15 min on ice to block non-specific binding sites. Subsequently, cells were incubated for 30 min with the following antibodies: CD4-PerCP Cy5.5, CCR6-PE (both from Biolegend, San Diego, CA, USA) CD8a-PECy7, CD69-PE, CD25-Alexa Fluor 488, CD3-PerCP Cy5.5, CD27-PE, CD19-APC and B220-FITC (all from Thermo Fisher). For intracellular staining, cells were first fixated and permeabilized with Foxp3 Staining buffer set (Thermo Fisher) according to manufacturer’s protocol, followed by incubation with Foxp3-PECy7 (Thermo Fisher), RORγT-Alexa Fluor 647 (BD) or Tbet-eFluor 660 (Biolegend). Dead cells were excluded using Fixable Viability Dye eFluor^® 780 (Thermo Fisher). Stained cells were measured by FACS Canto II (BD Biosciences) and analyzed using Flowlogic software version 7 (Inivai Technologies, Mentone, VIC, Australia).

Tumor tissue was digested in a 0.3% collagenase/0.1% hyaluronidase solution, pressed through a nylon mesh filter to obtain a single cell suspension and incubated in red cell lysis buffer (0.17 M Tris-HCl, 0.16 M NH₄Cl) for 3 min, spun down, and resuspended in FACS buffer (PBS + 1.5% FBS). Equal numbers of cells were stained with a viability dye and combinations of the following antibodies: CD8a-PECy7 (eBioscience, San Diego, CA, USA, 25-0081-82), F4/80-PECy7(eBioscience, 25-4801-82), CD206-FITC (Biolegend, 141704), Ly6G-APC (eBioscience, 17-5931-81), CD11b-PE (eBioscience, 12-0112-82), CD45-PE-Cy5 (eBioscience, 15-0451-83), IFN-γ-APC (eBioscience, 17-7311-82), and SIINFEKL pentamer-PE (ProImmune, Oxford, UK). For autophagy analysis using Cyto-ID staining of bone marrow derived macrophages, cells were first stained for surface markers (CD80-PE-Cy5 (eBioscience, 15-0801-81) and CD206-APC (eBioscience, 17-2061-82)) followed by staining with Cyto-ID for 30 min at 37 °C, per the manufacturer’s protocol (Enzo Life Sciences, Farmingdale, NY, USA, ENZ-51031). Flow cytometric data were acquired on a BD FACSCanto II cytometer and analyzed using FACSDiva software version 9.2 (BD Biosciences, San Jose, CA, USA).