Antibodies used for flow cytometry were as follows: CD4-Pacific Blue, CD25-PE, CD25-APC, CD127-FITC, Foxp3-PE, CD38-PE-Cy7, AnnexinV-PE, PD1-APC, CD8-FITC, CTLA4-PE-Cy7, CD44-FITC, CD62L-FITC, ICOS-FITC, GITR-PE, OX40-FITC, CD138-FITC, PD-L1-PE, and their isotype-matched mAbs (all from Biolegend). Intracellular staining of Foxp3, CTLA4, GITR, and OX40 were performed after fixation and permeabilization using cytofix/cytoperm kit (BD Biosciences), according to manufacturer’s protocol. Tregs were gated as CD4+CD25highFoxp3+ cells in CD4+ population and then sequential markers were assayed on Tregs, whereas CD4+CD25− cells were identified as Tcons. The remaining CD4+CD25low/intermediate subset was excluded in the current study because of their limited immunosuppressive activity compared with CD25high population (34 (link)). To avoid the effect of permeabilization when apoptosis assay was performed, CD4+CD25highCD127low/− cells were identified as Tregs (35 (link)). All flow cytometry was performed by BD FACS CantoII, and analyzed on FlowJo software version 10 (Treestar).