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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.