A Potential Prime Culprit, The Role Of T Follicular Helper Cells In The Pathogenesis Of Myasthenia Gravis

Myasthenia gravis(MG) is a typical autoimmune disease caused by antibodies against the acetylcholine receptor(ACh R) at neuromuscular junction. It is now widely accepted that the thymus played key role in development and maintenance of MG. Ectopic germinal centers(GCs) were found in thymus of MG patients with thymic hyperplasia or thymoma. T follicular helper(Tfh) cell, as one of the most important subsets of CD4+ T cells, has been recently identified responsible for B cell maturation and autoantibody production. Some studies showed that frequency of Tfh cells or its subsets is increasing in the peripheral blood of many autoimmune diseases including MG. However, little is known about Tfh cells frequency in the thymus of MG patient and Tfh cells interactions with B cells within thymic GCs. In this study, our major focus is to detect peripheral and intrathymic Tfh cells frequency and Tfh-B cells interactions in triggering occurrence and maintaining development of MG.MethodsWe also collected peripheral blood of 113 MG(34 OMG and 79 GMG) patients, 20 healthy controls(HCs), and 9 non-MG-associated thymomas(NMG). Meanwhile, thymi were obtained from 28 MG patients(9 OMG and 19 GMG) with abnormal thymus and 8 non-MG-associated thymomas(NMG), and all patients underwent thymectomy at Department of Thoracic Surgery of Tangdu Hospital. Normal thymi were obtained from 9 young adults(aged 7 months-21 years) after cardiac surgery as healthy controls(HCs). Peripheral blood mononuclear cells(PBMCs) were isolated and thymocytes were mechanically isolated from fresh thymic tissue. Flow cytometry was used to analyze the frequency of Tfh cells and B cells in PBMCs and thymocytes of MG patients and HCs. Radioimmunoassay was used to detect the anti-ACh R Ab titers in serum and tissue homogenate from thymi. The expression of transcription factors m RNA in the PBMCs and thymi were quantificationally analyzed by Real-time PCR(RT-PCR). Cytokines production were detected by RT-PCR, Western Blotting and Enzyme-linked immunosorbent assay(ELISA) at both m RNA and protein levels. Next, to further prove the intrathymic Tfh-B cells interactions in the pathogenesis of MG, Tfh and B cells were isolated from MG patients PBMCs by immuno-magnetic microbeads and co-cultured for a period time, and then anti-ACh R Ab titers and cytokine IL-21 level were detected in co-culture supernatant. Furthermore, the dynamic interaction between Tfh and B cells was visualized through live-cell image analysis. Additionally, the co-localization of Tfh/B cells in thymus was observed by confocal fluorescence microscopy. ACh R-specific long-lived plasma cells were detected in the bone marrow of recurrent MG patients with thymectomy by flow cytometry。ResultsIncreased frequencies of CD4+CXCR5+ICOS+T(Tfh) cells and B cells were detected in thymocytes and PBMCs of GMG patients, and meanwhile anti-ACh R antibody titers in thymus tissue homogenate or serum of GMG patients were also increased remarkably. A positive correlation between the percentages of Tfh cells and anti-ACh R antibody levels/MG clinical scores was confirmed. Meanwhile, Tfh-associated transcription factors Bcl-6 and cytokine IL-21 expression were also increased in peripheral blood and thymus of MG patients, but other Th-associated molecules such as transcription factors T-bet, GATA-3 and cytokine IFN-γ, IL-4 expression were not increased. In vitro cell culture proved Tfh-B cells interaction could induce anti-ACh R antibody production, and live-cell image analysis showed dynamical processes of Tfh-B cells interaction. Immunofluorescence microscopy results showed Tfh cells and B cells co-localized within ectopic GCs of MG patients’ thymus. To further search the source of autoantibody in patients with thymectomy, we detected the CD138+ACh R+ cells viewed as ACh R specific long-lived plasma cells in the bone marrow of these patients.ConclusionThe current study proved peripheral and intrathymic Tfh cells were implicated in the pathogenesis of MG, and intrathymic Tfh-B cells interactions could promote anti-ACh R antibody production and ectopic GCs formation, which may be a potential prime culprit triggering occurrence and maintaining development of MG. Additionally, the identification of ACh R specific long-lived plasma cells suggested bone marrow may be another source of autoantibody for recurrent MG patients with thymectomy and a new therapeutic target.