Pathogenesis Of Th17 And Treg Imbalance In Thymoma Associated Myasthenia Gravis

Objective: Thymoma is the most common anterior mediastinal tumor,often associated with autoimmune diseases such as myasthenia gravis(MG).However,the pathogenesis of MG caused by thymoma is not yet clear.The present study suggests that Th17 cells and Treg cells play an important role in central and peripheral immune tolerance.Through the detection of peripheral blood and thymoma tissue in patients with thymoma with or without MG,this study hopes to proof that the imbalance of Th17/Treg exist in peripheral blood and thymoma of MG patients,and IL-6/TGF-β increased in peripheral blood,RORγt/Foxp3 m RNA and protein increased in thymoma of MG patients.In vitro experiments,thymoma cell line can change the Th17/Treg percentage in lymphocyte subsets,the mechanism is thymoma cell line can secrete large amount of IL-6 and inhibit of TGF-β.Methods:(1)60 thymoma patients were divided into two groups according to combine with or without MG,and the clinical data and immune indexes were analyzed.Peripheral blood mononuclear cells were separated by Ficoll density gradient centrifugation.The contents of Th17 and Treg cells were detected by flow cytometry.The expression of IL-6 and TGF-β in serum was detected by ELISA.(2)Thymoma specimens were paraffin embedded,Th17 cells(CD4+,IL17+)and Treg cells(CD4+,Fox P3+)were detected in thymoma by immunohistochemical double staining method.Protein or m RNA of RORγt and Foxp3 were detected in thymoma by immunohistochemistry or RT-q PCR.(3)The expression of IL-1,IL-4,IL-6,IL-12,IL-21,IL-23 and TGF-β in the culture supernatant of thymoma cell line Thy0517 were detected by ELISA.CD4+T lymphocytes were isolate from healthy human peripheral blood by MACS microspheres,and co cultured with thymoma cell line Thy0517 for 3 days.The proportion of Th17 cells and Treg cells was detected by flow cytometry.Results:(1)Ig E increased,C3 and CRP decreased significantly in thymoma with MG patients,and there was no significant difference in other immune indexes and CD4+T cells,CD8+T cells and CD4+T/CD8+T between the two groups.(2)Compared with thymoma patients,the proportion of Th17 cells elevated(2.02 +3.80 + 1.22% vs 2%,P= 0.006),the proportion of Treg cells decreased(5.90 + 2.21% vs 4.14 + 1.28%,P=0.015),the ratio increased significantly(P=0.023)in thymoma with MG patients.IL-6 in serum increased significantly(20.32 + 7.40 pg/ml vs 101.10 + 20.3 pg/m,P=0.01),TGF-β did not change significantly(P=0.07),and the ratio of two increased significantly(P<0.001)in thymoma with MG patients.(3)The number of Th17 cells in thymoma tissues of MG patients was significantly higher than that in thymoma without MG(9.77 + 3.86 vs 4.93 + 2.13,P<0.0001),while the number of Tregs cells was significantly lower(6.77 + 2.94 vs,9.93 4.40,P =0.002).The ratio of Th17/Tregs was significantly higher in thymoma tissues of MG patients(P<0.001).(4)The m RNA and protein expression of RORγt in thymoma tissues of MG patients were significantly higher than those without MG(P<0.05),and the m RNA and protein expression of Fox P3 was significantly lower(P<0.05).(5)Thymoma cell line Thy0517 has exocrine function.IL-6 and IL-21 expression is increased and TGF-β expression is decreased in cell culture supernatant,while IL-1 beta,IL-4 and IL-23 are not changed significantly.(6)After co culture of Thy0517 and human CD4+T lymphocytes,the proportion of Th17 cells were increased(0.66 + 0.07 vs 1.14 + 0.08,P<0.01),Treg cells were inhibited(3.10 + 0.22 vs,2.41 + 0.39,P<0.01).Conclutions: The Th17 cells increased and Treg cells decreased in the peripheral blood and thymoma tissue of thymoma with MG patients,at the same time,increased serum IL-6/TGF-β and RORγt/Fox P3 gene and protein expression also present in thymoma with MG patients.Thymoma cell can promote the differentiation of Th17 cells and inhibit the Treg cells.The above results indicate that thymoma can cause abnormal expression of cytokines IL-6,TGF-β and RORγt and Foxp3,and then affect the differentiation of Th cells,resulting in the imbalance of Th17/Treg cells and the occurrence of MG.