Patients and samples
A total 60 patients with thymoma were recruited at the inpatient service of Tianjin Medical University General Hospital (Tianjin, China) between 2015 and 2017. All patients had undergone surgery but had not received chemotherapy or radiotherapy. Thirty patients had simple thymoma without other relative autoimmune diseases (Tm group), while 30 others were diagnosed with thymoma and AChR-MG (MG group). The ethics committee of Tianjin Medical University General Hospital approved this study.
Fasting venous blood samples were collected from individual patients before surgery and immunotherapy. Anticoagulated whole blood (3 mL) was examined by flow cytometry (FCM) within 4 h. Plasma samples were stored at −80 °C for enzyme-linked immunosorbent assay (ELISA). Thymoma tissue samples were collected from individual patients during surgery; one portion was fixed in 10% neutralized formalin overnight and was paraffin-embedded. The remaining tissues were frozen in liquid nitrogen.
Flow cytometry analysis of Th17 and Tregs in peripheral blood
PBMCs were processed by Ficoll density gradient centrifugation according to the manufacturer’s instructions (Hao Yang Biological Manufacture Co., Ltd., Tianjin, P.R.Chian). PBMCs were surface labeled with antibodies against each subpopulation for 20 min at room temperature. Following fixation and permeabilization, intracellular staining of FOXP3 and IL-17A was performed. For Th17 analysis, cells were stained with APC-conjugated anti-human CD4 and PE-conjugated anti-human IL-17A antibodies (Miltenyi Biotec Technology & Trading (Shanghai) Co., Ltd. Shanghai, P.R.China). For Treg analysis, the cells were stained with FITC-conjugated anti-human CD4, APC-conjugated anti-human CD25, and PE-conjugated anti-human FOXP3 antibodies (Miltenyi Biotec). Isotype controls were used to correct compensation and confirm antibody specificity. Stained cells were analyzed by FCM (FACSCanto Ⅱ; BD Biosciences, San Jose, CA, USA). Data analysis was performed using Cell Quest software (BD Biosciences).