Foxp3 staining buffer set

We then thawed our cryopreserved PBMCs and washed them twice in PBS supplemented with 10% FBS. Single-cell suspensions were then processed for surface staining with an antibody cocktail (panel described in Table S2) for 20 min at 4°C and then washed with PBS containing 2% FBS, fixed, and permeabilized using the Foxp3 Staining Buffer Set (BD Biosciences, Franklin Lakes, NJ, USA) according to the manufacturer’s instructions. Cells were then stained with an intracellular antibody cocktail (panel described in Table S2) for 30 min at 4°C before being washed in Foxp3 permeabilization buffer and resuspended in CellFix (BD Biosciences). The stained cells were then evaluated using an LSR II Fortessa with FACS Diva software (BD Biosciences) and all analyses were completed using FlowJo software (BD Biosciences, Franklin Lakes, NJ, USA).

CD4⁺CD25⁺ Tregs isolated from the spleen of mice (10⁵ cells) were subjected to the staining solution with 2 μg/mL PE-conjugated anti-CTLA-4 Ab (BD Biosciences, USA) or anti-mouse IgG isotype control to characterize surface CTLA-4. Stained cells were washed twice with PBS and then analyzed by flow cytometry. Foxp3 expression was analyzed by intracellular staining. Tregs were fixed in 1 mL fresh fixation/permeabilization working solution (FoxP3 Staining Buffer Set, BD Biosciences). Fixed cells were washed with PBS and then re-suspended in the staining solution containing 5 μg/mL FITC-labeled anti-Foxp3 antibody or IgG isotype control (eBioscience, USA). The stained cells were washed twice with PBS and then analyzed using FACSCalibur cytometer and the CellQust software (BD Biosciences).

Intracellular staining of FoxP3 was performed using phycoerythrin-conjugated anti-FoxP3 mAb and the FoxP3 staining buffer set (Pharmingen, BD Biosciences) according to the manufacturer’s protocol. PerCP-Cy5.5-conjugated anti-CD4 mAb (clone RM4-5) from Pharmingen, BD was used. Cells were acquired on a FACScan cytometer (Becton Dickinson, Mountain View, CA). Data were analyzed by using FlowJo software (Tree Star, CA, USA)

Spleens of mice were dissected under sterile conditions and passed through a 40 μm cell strainer followed by ammonium chloride based erythrocyte lysis (BD Biosciences, Germany). Derived splenocytes were cultured in flat bottom 96-well plates in standard T cell medium (IMDM with 5% FCS, 2 mM L-glutamine and 50 μM 2-ME, life technologies). To restimulate cells, MOG_35-55 was added during the culture period with increasing concentration from 0 to 100 μg/ml. T cell proliferation was measured via [³H] thymidine incorporation during the last 24 h of a four day incubation. Counts per minute (cpm) of quadruplicate test cultures ± SEM were determined using liquid scintillation counting (BetaPlate1205, Perkin Elmer, Boston, US). Stimulation index was calculated as ratio of the cpm at the indicated MOG_35-55 concentrations to the proliferation of cells in the absence of MOG_35-55. Flow cytometry was performed from homogenized lymphoid organs. Cells were stained for cell surface CD4 (L3T4, APC or Pacific Blue labelled), CD25 (3C7, PerCP labelled) and intracellular FoxP3 (MF23, Pacific Blue labelled) using the FoxP3 staining buffer set (all from BD Biosciences). Flow cytometry was performed using a FACSCanto II flow cytometer (BD Biosciences).

Cell surface expression was assessed on freshly isolated TILs or PBMCs by staining with the following monoclonal antibodies: CD3-Pacific Orange (Clone UCHT1, Cat. 561416, BD Biosciences, San Jose, CA, USA), CD4-APC-Cy7 (Clone OKT4, Cat. 317418, Biolegend, San Diego, CA), CD8-FITC (Clone RPA-T8, Cat. 555366, BD Biosciences, San Jose, CA, USA), CD25-Phycoerythrin (Clone M-A251, Cat. 555432, BD Biosciences, San Jose, CA, USA) and PD-1-Pacific Blue (Clone EH12.2H7, Cat. 329916, Biolegend, San Diego, CA). For detection of intracellular expression of FOXP3, cells were permeabilized and fixed with the ‘FOXP3 Staining Buffer Set’ (BD Biosciences, San Jose, CA, USA) following the manufacturer’s instructions, and then they were incubated with the anti-FOXP3-PE-Cy7 monoclonal antibody (Clone PCH101, Ref: 25-4776-42, BD Biosciences, San Jose, CA, USA). Cell analysis was performed with a FACSCanto flow cytometer (BD Biosciences, San Jose, CA, USA).