Serum cytokines ELISA assay
The serum levels of IL-6, IL-21, IL-23, and TGF-β were quantified using commercially available ELISA kits according to the manufacturer’s instructions (Proteintach Group Inc, Chicago, USA).
Immunohistochemical double staining analysis of Th17 and Tregs in thymoma
A two-color immunohistochemical double stain with CD4 and IL-17A primary antibodies (Th17 cells) and CD4 and FOXP3 primary antibodies (Tregs) was performed according to the manufacturer’s protocol (KIT-9999: Fuzhou Maixin Biotechnology Development Co., Ltd. China). Paraffin-embedded thymoma tissue sections (4 µm) were de-waxed in xylene, dehydrated in ethanol, and subjected to antigen retrieval with Tris-EDTA under high temperature and high pressure for 3 min. Endogenous peroxidase activity was blocked by incubation with 3% H2O2for 10 min. Non-specific reactions were blocked by incubation with serum-free medium for 10 min at room temperature. Two sections from the same sample were incubated with anti-IL-17A antibody (1:100 dilution; ab136668, Abcam, Cambridge, UK) or anti-FOXP3 antibody (1:200 dilution; ab54501, Abcam) overnight at 4 °C. The following morning, the sections were incubated with goat anti-rabbit secondary antibody, and then were stained with BCIP/NBT (mandarin blue indicated positive staining). The slides were rinsed in PBS and incubated with double staining intensifier and serum-free medium for 10 min. The slides were then incubated with the second primary antibody, anti-CD4 (1:100 dilution; ab846, Abcam), overnight at 4 °C. Then, the sections were incubated with goat anti-rabbit secondary antibody and stained with AEC (3-amino-9-ethylcarbazole chromogen) solution and hematoxylin. Thus, the two primary antibodies could be easily differentiated by the various chromogens (IL-17A, mandarin blue immunolabeling in cytoplasm; FOXP3, mandarin blue immunolabeling in cytoplasm or nuclei; CD4, red immunolabeling in cell membrane), as well as by cellular stain localization. Double-positive cells were counted on five adjacent high-power fields (×400) of each section.
Analysis of RORγt and FOXP3 in thymoma
The immunohistochemical procedure of RORγt and FOXP3 staining as described above was used for staining of thymoma, with the exception of the second primary antibody. Sections were incubated with anti-human RORγt antibody (1:40 dilution; ab219496, Abcam) and anti-human FOXP3 antibody (1:50 dilution; ab54501, Abcam). The sections were then incubated with goat anti-rabbit secondary antibody and stained with DAB and hematoxylin. Brown granules in the nuclei were considered to represent a positive signal. The frequency of positive cells in five high-power fields (magnification, ×400, Leica DMIL, Germany) of each section was determined. More than 10% positive cells in each high-power field were found to have positive expression.
Real-time quantitative PCR (RT-qPCR) analysis of RORC (RORγt) and FOXP3 mRNA