Quantification of T Cell Subsets

The numbers of circulating CD4⁺ T and CD8⁺ T lymphocytes were determined using BD Trucount absolute count tubes according to the manufacturer’s instructions (BD Biosciences). For detection of central memory CD4⁺ T (CD3 ⁺CD4 ⁺CD28 ⁺CD95 ⁺cells), effector memory CD4⁺ T (CD3 ⁺CD4 ⁺CD28^-CD95⁺ cells) and TFH cell (CD3 ⁺CD4 ⁺CXCR5 ⁺PD-1⁺) cells, PBMCs were stained with the following fluorescein-labeled antibodies: anti-CD3 (BD Bioscience), anti-CD4 (BD Bioscience), anti-CD8 (BD Bioscience), anti-CD28 (BD Bioscience), anti-CD95 (BD Bioscience), anti-CXCR5 (eBioscience), and anti-PD-1 (eBioscience) for 30 min and detected with a BD FACS LSR Fortessa flow cytometer (BD Biosciences, USA). Data were analyzed using FlowJo software (Tree Star, USA).