Flow cytometry.

The antibodies used for flow cytometry are as follows: PerCP-Cy5.5 anti-mouse CD3 (BioLegend, clone 17A2, catalog 100218), APC anti-mouse/human CD45R/B220 (BioLegend, clone RA3-6B2, catalog 103212), FITC anti-mouse/human CD45R/B220 (BioLegend, clone RA3-6B2, catalog 103206), APC anti-mouse CD8a (BioLegend, clone 53-6.7, catalog100712), PE-Cy7 anti-mouse CD8 (BioLegend, clone 53-6.7, catalog 100722), Alex Fluor 700 anti-mouse CD4 (BioLegend, clone GK1.5, catalog 100430), Alex Fluor 488 anti-mouse CD69 (BioLegend, clone H1.2F3, catalog 104516), PE-Cy7 anti-mouse CD25 (BioLegend, clone 3C7, catalog 101916), FITC anti-mouse CD25 (BioLegend, clone 3C7, catalog 101908), FITC anti-mouse CD62L (BioLegend, clone MEL-14, catalog 104406), APC anti-mouse/human CD44 (BioLegend, clone IM7, catalog 103012), APC anti-mouse IFN-γ (BioLegend, clone XMG1.2, catalog 505810), APC Granzyme B (eBioscience, clone NGZB, catalog 17-8898-82), and PE anti-RPS6 Phospho (Ser235/Ser236) (BioLegend, clone A17020B, catalog 608604). For cell surface markers, cells were stained in 1% BSA/PBS with indicated antibodies for 30 minutes at 4°C. For intracellular staining following surface marker staining, cells were fixed and permeabilized using Cytofix/Cytoperm Fixation/Permeabilization Solution Kit (BD Biosciences, catalog 554714), then stained with antibodies for intracellular markers. Dead cells were excluded by using Zombie Aqua Fixable Viability Kit (BioLegend, catalog 423102) during surface marker staining.

For BrdU incorporation assay, BrdU was injected intraperitoneally (2 mg/mouse) and splenocytes were collected 20 hours later for analysis. BrdU staining was performed following BD Pharmingen BrdU Flow Kits (BD Biosciences, catalog 559619) instruction. Phospho-flow cytometry of p-S6 was performed following BD Phosflow Protocols for Mouse Splenocytes or Thymocytes. Data were collected on LSRII flow cytometer (BD Biosciences) or CytoFLEX (Beckman Coulter) and was analyzed using FlowJo software.