Foxp3+ Treg cells assay

A 200‐μl sample of peripheral blood was collected, and 10 μl EDTA (0·5 m, 15575‐038; Invitrogen) was added to prevent the blood from clotting. Erythrocytes were lyzed in red blood cell lysis buffer (420301; Biolegend, San Diego, CA) under the basic manufacturer's instructions. Then, cells were stained with Fc‐block anti‐antibody CD16/32. Next, cells were stained with the FITC‐conjugated CD4 (clone GK1.5; BD Pharmingen) and APC‐conjugated CD25 (clone PC61.5; eBioscience, San Diego, CA). Then, cells were fixed and permeabilized with the Foxp3/Transcription Factor Straining Buffer Set Kit (005523‐00; eBioscience) according to the manufacturer's instructions. The permeabilized cells were stained with PE‐conjugated Foxp3 (clone FJK‐16s; eBioscience). The CD4⁺ CD25⁺ Foxp3⁺ T cells in peripheral blood were detected with the BD Canto II flow cytometer and analyzed by flowjo V10.