Foxp3 nrrf 30

Tumor and surrounding regions were delineated macroscopically. Tissues were dissociated through 100 µm cell strainers in PBS with 3% bovine serum albumin (BSA). Hepatocytes were removed by centrifugation on a 35% Percoll gradient at 700 × g at 21°C for 12 min. Leukocytes present in the pellet were resuspended in red blood cell lysis buffer (0.15 M NH₄Cl, 0.1 mM KHCO₃, 0.1 mM Na₂-EDTA in water; pH 7.2) for 1 min, washed, and then resuspended in PBS with 3% BSA. Cell suspensions were incubated with anti-mouse CD16/CD32 (Becton Dickinson, BD, USA) to block Fc receptors and Fixable Viability Dye eFluor 506 (eBioscience, San Diego, CA, USA). Cells were then stained with a cocktail of directly conjugated mAbs [CD3 (17A2) BD, CD4 (RM4-5) eBioscience, CD8 (53-6.7) BioLegend, CD45 (30-F11) BioLegend and Foxp3 (NRRF-30) eBioscience] for 30 min at 4°C. Intracellular staining was performed with a transcription factor staining buffer set from (eBioscience). The relevant fluorescence-minus-one labeling conditions including the appropriate isotype-matched mAb were used as controls. All samples were acquired on an LSR Fortessa flow cytometer (BD) and analyzed with FlowJo version 9.3.1 or above (Tree Star, Ashland, OR, USA).

Mouse splenocytes and thymocytes were prepared from pooled thymus or spleen. Thymic and splenic cells were pre-incubated with Fc-block before staining with other antibodies. The mouse antibodies used included anti-CD4 (RM4–5, eBioscience); anti-CD8 (53–6.7, eBioscience); anti-CD45.1 (A20, BioLegend), anti-CD45.2 (104, eBioscience); anti-CD25 (PC61.5, eBioscience); anti-CD69 (H1.2F3, eBioscience); anti-CD24 (M1/69, eBioscience); anti-Helios (22F6, eBioscience) before flow cytometry analysis. For intracellular staining of Ki-67 (B56, BD), pSTAT5 (47/Stat5(pY694), BD) and Foxp3 (NRRF-30, eBioscience), cells were fixed and permeabilized with BD Cytofix/Cytoperm™ Fixation / Permeabilization Solution Kit (554,714, BD) and stained according to the manufacturer’s protocols. The CD4⁺ T cells from the thymus and spleen of WT or RelB^−/− mice were analyzed by flow cytometry and gated as shown in the Supplementary Figure 1. The samples were analyzed using a BD LSRFortessa flow cytometer and FlowJo software (Tree Star Inc). All single-cell suspensions from the tissues were stained with Abs diluted in PBS containing 2% FCS for 30 min on ice.