Fig. 1

FACS analysis of circulating CD4⁺ T cells. PBMCs from individual ESRF patients and HC subjects were stained with APC-anti-CD4, or PerCP-anti-CD4 and FITC-anti-CD25, or isotype controls, fixed and permeabilized, followed by intracellular staining with FITC-anti–IL-17 and PE-Cy7-anti-IFN-γ and PE-anti-Foxp3. The frequency of CD4⁺, CD4⁺CD25⁺Foxp3⁺ Treg, CD4⁺IFN-γ⁻IL-17⁺ Th17, CD4⁺IFN-γ⁺IL-17⁻ Th1 and CD4⁺IFN-γ⁺IL-17⁺ Th1/17 cells were determined by flow cytometry analysis. The cells were gated on living lymphocytes and then gated on CD4⁺ cells, and at least about 30,000 events were analyzed for each sample. The numbers of each type of CD4⁺ T cells were calculated, according to the total numbers of PBMCs, the frequency of total CD4⁺, and different types of CD4⁺ T cells. a The representative charts of flow cytometry analysis. b-g Quantitative analysis. Data shown are representative FACS charts or the mean numbers of each type of cells per ml of peripheral blood in individual subjects from two separate experiments. The horizontal lines indicate the median values for each group