For flow cytometric analysis of splenocytes, single-cell suspensions were prepared. In general, 3 × 106 cells were surface stained in ice-cold PBS for 1 h at 4°C. CXCR5 staining was performed at room temperature for 1 h to optimize detection. After staining, cells were washed and analyzed on an LSRII Multilaser Cytometer (BD Biosciences). Intracellular staining for the indicated transcription factors was performed using the FoxP3/Transcription Factor Fixation/Permeabilization Kit (eBioscience) according to the manufacturer’s directions. Cell number was quantified using a reference number of microsphere beads (Spherotech ACBP70-10) added to specific samples. For intracellular cytokine staining, cells were plated in 96-well plates in T cell media. Cells were stimulated with PMA/ionomycin or media control. After 1 h, GolgiPlug (BD Biosciences) was added to each well. After 3 h, cells were harvested and surface stained as described above. Cells were then fixed with BD Cytofix (BD Biosciences) for 15 min followed by permeabilization with Permeabilization Buffer (Invitrogen) for 1 h. IL-21 staining was performed sequentially using an IL-21R-Fc chimeric protein (R&D Systems), followed by PE-labeled anti-human IgG (Jackson ImmunoResearch Laboratories) as previously described (Ray et al., 2015 (link)). IFN-γ antibody (Biolegend) was added at the initial step of IL-21 staining to simultaneously detect intracellular IFN-γ. For cell proliferation assays, CellTrace Yellow was purchased from ThermoFisher and used according to the manufacturer’s direction. For cell viability, Zombie Fixable Viability Kit was purchased from Biolegend and used according to the manufacturer’s directions.