The percentage of CD4+ and CD8+ T lymphocyte from the mouse spleen was analyzed by flow cytometry, as described in previous studies with minor modifications [26 (link),27 (link)]. Briefly, spleen lymphocytes were isolated as described above and fixed with 1% formaldehyde solution. Subsequently, the lymphocytes were treated with FITC-conjugated anti-mouse CD4 antibody, phycoerythrin (PE)-conjugated anti-mouse CD8 antibody, FITC mouse IgG2b κ isotype control antibody, or PE mouse IgG1 κ isotype control antibody (BioLegend, San Diego, CA, USA) at 4°C for approximately 2 h following the instructions of manufacturer. The labeled cells were washed with cold PBS solution three times and analyzed by a flow cytometer (BD CantoII, Franklin Lakes, NJ, USA). All treatments were performed in triplicate.