Flow cytometry analysis of the numbers of circulating TFR and TFH cells. PBMCs were isolated from individual subjects and were stained in duplicate with Alexa Fluor 647-anti-CXCR5, PE-Cy7-anti-CD4, Alexa Fluor-488-anti-Bcl-6 for 30 min. After being washed, the cells were fixed and permeabilized cells via Cytofix/Cytoperm. The cells were stained with PE-anti-Foxp3 and were characterized by flow cytometry analysis by gating initially on living lymphocytes, and then on CD4+ T cells. The isotype-matched antibodies were used as controls. Data are representative FACS charts and expressed as the mean values of individual subjects and the difference between groups was analyzed by the Kruskal–Wallis test. (A) Flow cytometry analysis; (B) The numbers of CD4+CXCR5+ T cells, CD4+CXCR5+Foxp3− (TFH) cells, and CD4+CXCR5+Foxp3+ (TFR) cells. The horizontal lines indicate the median values for individual groups.