Icos pe

PBMCs from UC patients and healthy controls were harvested and stained in duplicate for CD45-PC7, CD3-ECD, CD8-PE, CD4-FITC, CD19-PC5, CD38-FITC, CD27-PC7, CD86-PE (Beckman Coulter, Brea, CA), CXCR5-PC5 and ICOS-PE (eBioscience, San Diego, CA) at room temperature for 30 min. After being washed with PBS, the cells were characterized by flowcytometry analysis using the Beckman Coulter Cytomics FC500 (Beckman Coulter). Cells stained with separate antibodies were defined as certain T_FH cells (CD4⁺ CXCR5⁺ T_H cells and CD4⁺CXCR5⁺ICOS⁺ T_H cells) and subsets of B cells (CD38⁺CD19⁺ B cells, CD86⁺CD19⁺ B cells and CD27⁺CD19⁺ B cells). Data were analysed with a CXP Analysis Cytometer (Beckman Coulter).

To determine the percentage of T cell subsets, heparin-anticoagulated whole blood were collected and stained with CD3-PerCP (BD Bioscience, New Jersey, US), CD4-FITC (BD Bioscience, New Jersey, US), CXCR5-APC (Biolegand, California, US), PD-1-PE (eBioscience, California, US), ICOS-PE (eBioscience, California, US) and CD25-APC (BD Bioscience, New Jersey, US). After fixed and permeabilized, samples were stained with FoxP3-PE (BD Bioscience, New Jersey, US), p-STAT3-PE (BD Bioscience, New Jersey, US), p-STAT5-PE (BD Bioscience, New Jersey, US) and p-STAT4-PE (BD Bioscience, New Jersey, US). After stimulation with phorbol 12-myristate 13-acetate (PMA) (50 ng/ml) (Sigma-Aldrich, US), ionomycin (1 μg/ml) (Sigma-Aldrich, US), and Golgi stop (BD Bioscience, New Jersey, US) for 5 h, the fixation and permeablication were performed. Then samples were stained with IL-21-PE (BD Bioscience, New Jersey, US). Samples were measured with FACS Canto II (BD Biosciences, New Jersey, US). Gating strategy used for the analysis of all immune parameters was shown in Additional file 1.