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).