α foxp3

Single cell suspensions were prepared from the spleens of recipient mice. Cells were labeled for surface and intracellular markers using fluorescence-labeled anti-mouse α-CD4, α-CXCR5, α-ICOS, α-PD1, α-FOXP3, α-GL7, and α-Fas antibodies (eBioscience, CA, USA). Intracellular FOXP3 staining was performed using a commercially available staining kit that included permeabilization solution and buffer (eBioscience). Flow cytometric measurements were performed on a FACSAria III (BD Bioscience, CA, USA) and data were analyzed using FlowJo (FlowJo Software, OR, USA). For proper gating, apoptotic cells were excluded, and the samples were compared with isotype controls, fluorescence minus one (FMO)-stained, permeabilized, and unpermeabilized unstained cells. For the carboxyfluorescein diacetate succinimidyl ester (CFSE; Invitrogen, CA, USA) cell proliferation assay, cells were labeled with 1 µM CFSE. The CFSE dilution was measured by flow cytometry.

Cells were stained with LIVE/DEAD Violet (Life Technologies) before antibody staining. The following fluorophore-conjugated antibodies were used (Biolegend): α-CD62L, CD44, CD8, CD3, CD4, CD8, CD137, CD45.1, PD-1, OX40, GITR, TIM-3, LAG-3, Granzyme B, TNFα, IFNγ, CD25, and CD69. α-FOXP3 was from eBiosciences. For TNFα and IFNγ intracellular staining, cells were restimulated with SIINFEKL peptide (Sigma) for 4 hours in the presence of Golgi-stop (BD) or cell stimulation cocktail (eBiosciences) and then fixed and permeabilized with Fix/perm buffer (eBiosciences) before intracellular staining and acquisition on a Fortessa (BD). Data was analyzed by FlowJo (TreeStar).
For flow cytometric analyses of TILs, excised tumors were minced finely with a scalpel blade in a Petri dish, and incubated for 25 minutes at 37°C in Collagenase-D and DNAse-I (Roche) in RPMI before passage through a 70-μm cell strainer (BD) to obtain single-cell suspensions and perform antibody staining.