Figure 2.

Flow cytometry for quantitation of subsets of TFH cells. Fluorescence-activated cell sorting analysis of the numbers of different subsets of TFH cells in individual subjects. Peripheral blood mononuclear cells were isolated from individual subjects and were stained in duplicate with anti-CD4, anti-CXCR5, anti-ICOS, anti-PD-1 and intracellular anti-IL-21 or isotype-matched IgG antibodies, respectively. The cells were characterized using flow cytometry with gating, initially on living lymphocytes, and then on CD4+CXCR5+TFH cells. Subsequently, the frequency of ICOS+, PD-1+ and IL-21+TFH in total TFH was analyzed, and a minimum of 30,000 events were analyzed for each sample. Data are expressed as the mean values of individual participants from two separate experiments. (A) Flow cytometry analysis. (B-D) The numbers of (B) CD4+CXCR5+PD-1+, (C) CD4+CXCR5+ICOS+ and (D) CD4+CXCR5+IL-21+TFH cells. The horizontal lines indicate the median values for each group. OA: All OA patients (n=40), HC: healthy control group (n=13). TFH, follicular helper T; IL-21, interleukin-21; OA, osteoarthritis; (ICOS)+, inducible costimulator; (PD-1), programmed death 1; CXCR5, chemokine (C-X-C motif) receptor 5.