Fitc anti il17a

The intracellular production of IFNγ and of IL17A by T lymphocytes from PBMC and tissue infiltrates was analyzed as follows: the cells (resuspended in culture medium conditioned with 10% autologous plasma at a concentration of 1 × 10⁷/ml) were stimulated with phorbol-12-myristate-13-acetate (PMA 50 ng/ml, Sigma) and ionomycin (2 μg/ml, Sigma) for 5 hours at 37°C. Brefeldin A (BFA 10 μg/ml, Sigma) was added to the cells for the last 4 hours of incubation. After washings, the samples were stained with fluorochrome-conjugated mAbs specific for surface markers [PerCPCy5.5 anti-CD3 (BD), APCCy7 anti-CD8 (e-Biosciences) and Violet Live/Dead Fixable Dead Cell stain (Life Technologies, CA, USA)], before fixing and permeabilizing the lymphocytes with the Cytofix/Cytoperm kit (BD Bioscience) following the manufacturer's instructions. The cells were washed in Perm-Wash buffer (BD Bioscience) and incubated with Pe-Cy7 anti-IFNγ (BD) or FITC anti-IL17A (e-Biosciences) mAbs. Thereafter, the samples were washed in Perm-Wash buffer, fixed with FACS Lysing solution (BD Bioscience) and stored at 4°C. The cytokine profile was analyzed using a FACS Canto II flow cytometer (BD Bioscience) by the FACS DIVA software. The gating strategy for phenotypic analyses is shown in Supplementary Figure 3.

The frequencies of immune cells including Th1, Th2, Th17, regulatory T cell (Treg), Tfh, and Breg in the peripheral blood were measured by flow cytometry. Briefly, PBMCs were isolated and incubated with PMA (10 ng/ml, eBioscience) and BFA (10 μg/ml, eBioscience) for 4 h then harvested and washed twice for 30 min. Then, cells were stained with anti-CD4 and anti-CD25 for 30 min at 4°C. During the intracellular staining, antibodies against IFN-γ, IL-4, and IL-17A were according to stain Th1, Th2, and Th17, respectively. Intracellular FoxP3 was also stained, and CD4⁺FoxP3⁺ was used to determine Treg. CD19⁺CD24^HighCD38^High cells were determined as Breg. CD4⁺PD1⁺CXCR5⁺ cells were determined as Tfh. The following anti-human antibodies for surface staining or intracellular staining were used: PE-CY7-anti-CD4, PE-CY5.5-anti-CD25, FITC-anti-IL-17A, PE-anti-Foxp3, FITC-anti-IFN-γ, PE-anti-IL-4, FITC-anti-CD19, PE-anti-CD24, PE-CY7-anti-CD38, PE-anti-PD1, and FITC-anti-CXCR5 (all eBioscience).