Monensin

The cell phenotypes were analyzed by FACS Aria II flow cytometer (BD Bioscience), and data were analyzed with the FlowJo V10.0.7 (FlowJo, OR, USA). Cells were collected and washed in PBS, then the single-cell suspensions were labeled with various fluorochrome-conjugated antibodies for 30 minutes within PBS containing 0.5% BSA on ice. The gating strategies for CD4+ central memory T cells, CD8+ central memory T cells, CD4+ effector memory T cells, Tfh cells were CD4+CD62L+CD44+, CD8+CD62L+CD44+, CD4+CD62L-CD44+, CD4+CXCR5+PD-1+, respectively. The following antibodies were used: anti-mouse CD4-AF700 (clone RM4-5, eBioscience), anti-mouse CD8a-BV510 (clone 53-6.7, BD Horizon), anti-mouse CD62L-PE (clone MEL-14, Biolegend), anti-mouse CD44-Percp/Cy5.5 (clone 1M7, Biolegend), anti-mouse CXCR5-APC (clone SPRCL5, eBioscience) and anti-mouse PD-1-PE/Cy7 (clone J43, eBioscience). For ICCS, mice splenocytes were collected and co-stimulated with anti-CD3 antibody and anti-CD28 antibody (Biolegend) for 1 hour at 37 °C, then Cells were incubated with 5 mg/mL brefeldin A (Topscience), 2 mM monensin (Topscience). Cells were then harvested, stained with indicated antibodies with a Fixation/Permeabilization Solution Kit (BD Bioscience). We used these antibodies to detect cytokine secretion of T cells: anti-IFN-γ (clone XMG1.2, Biolegend) and anti-IL-2 (clone JES6-5H4, Biolegend).