Spleens were collected and placed in complete RPMI media [RPMI, Gibco, 10% FBS, penicillin/streptomycin (100 U/ml), 0.4 mM 2-mercaptoethanol, 20 mM Hepes, 4 mM l-glutamine]. Single-cell suspension from spleens was obtained by passing the tissue through a 70-μm nylon strainer (Corning), and erythrocytes were eliminated with ACK Lysis buffer (0.15 M NH4Cl, 1 mM KHCO3, 0.1 mM EDTA). Single cells were washed and resuspend in 5 ml of complete RPMI (Gibco). Two-fifth of the spleen sample was used for the staining. Prior antibody staining mouse FC receptors were blocked using TruStain FCX (BioLegend, San Diego, California) at 1:200 dilution in FACS buffer (PBS, 2% FBS) for 15 min on ice. Surface markers were stained for 40 min using combinations of labeled monoclonal antibodies diluted in PBS: CD45.2-PE-dazzle (clone 104), CD11c-PE-Cy7 (clone N418), MHC-II-BV421 (clone M5/114.15.2), F4/80-APC-Cy7 (clone CI-A3–1), CD40-FITC (3/23), CD80-PE (clone 16-10A1), rat IgG2a, κ isotype Ctrl-FITC (clone RTK2758), Armenian hamster IgG isotype Ctrl-PE (clone HTK888), rat IgG2b, and κ isotype Ctrl-APC (RTK4530) (BioLegend). For Foxp3 intracellular staining, the Foxp3 staining buffer set kit (eBioscience) was used following the manufacturer’s recommendations. Before analysis, samples were washed twice with permeabilization buffer. All samples were resuspended in 200 μl of PBS before analysis, and 10-μl Beckman Coulter count beads were added for absolute number quantification. Stained samples were analyzed on Flow Cytometry BD LSRFortessa. Measurements of median were used to calculate the mean fluorescence intensity of cell populations using FlowJo software.

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