For analysis of surface markers, cells were stained in phosphate-buffered saline containing 2% (w/v) BSA. The following fluorescent-labeled antibodies (purchased from Thermo Fisher Scientific, Tonbo, BD Biosciences, Cell Signaling Technology, and Sony Biotechnology) were used: anti-CD4 (RM4-5), anti-CD8 (53-6.7), anti-CD25 (PC61.5), anti-B220 (RA3-6B2), anti-CD62L (MEL-14), anti-CD44 (IM7), anti-Fas (Jo2), anti-GL7 (GL-7), anti–PD-1 (J43), anti-ICOS (C398.4A), anti–Nrp-1 (3DS304M), anti-CD98 (RL388), anti-CD45.1 (A20), anti-CD45.2 (104), anti-KLRG1 (2F1), and anti-TCRβ (H57–597). Biotin-conjugated anti-CXCR5 (2G8) antibody and phycoerythrin (PE)–labeled streptavidin from BD Biosciences were used for TFH staining. Active caspase-3 or annexin V staining was performed according to the manufacturer’s instructions (BD Biosciences). For intracellular staining, cells were fixed using the Foxp3 fixation buffer (Thermo Fisher Scientific) as per the manufacturer’s instructions. The following antibodies were used: anti-Foxp3 (FJK-16 s), anti-CTLA4 (UC10-4B9), anti–Ki-67 (SolA15), anti–c-Myc (D84C12), anti–interferon-γ (IFN-γ) (XMG1.2), anti–IL-17A (TC11-18H10.1), and anti–IL-4 (11B11). For intracellular cytokine staining, cells were stimulated for 4 to 5 hours with phorbol 12-myristate 13-acetate and ionomycin in the presence of monensin (BD Biosciences). Flow cytometry data were collected using LSRII or Fortessa (BD Biosciences) cytometers and analyzed with FlowJo v10 software (TreeStar). Fluorescence-activated cell sorting was performed using Synergy or Reflection instruments (Sony Biotechnology).

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