Circulating Regulatory T Cells And Relative Soluble Molecules In Myasthenia Gravis

Myasthenia gravis (MG) is an autoimmune disease affecting the neuromuscular junction of skeletal muscles. Acetylcholine receptor (AChR) antibodies are present in sera from 80% to 90% of patients with generalized MG. Both AChR-specific Th1, Th2 cells and cyokines are documented in MG patients, and the involvement of the complement system should cause an increased consumption of complement components such as C3 and C4. But it is not yet clear what are essential for driving the pathogenesis of MG patients. The pathogenesis of MG is involved by many of the relative immune factors. The imbalance of immune system plays an important role in it. As impaired regulatory T cell activity can result in autoimmune diseases, regulatory T cells play a central role in the maintenance of peripheral tolerance. Current evidence proves the disfunction of regulatory T cells (Tregs), especially CD4⁺CD25⁺Tregs in both circulation and thymus maybe contribute to the development of MG. We used flowcytometry to detect the percentage of circulating CD4⁺CD25^high Treg and the expression of some molecules which are important for the development and function of the cell, such as Foxp3 and so on; We also detected the level of other regulatory T cells and the activation of B cell in peripheral circulation of MG patients; And we used MACS and ³H-TdR to isolate the circulating CD4⁺CD25⁺Treg and CD4⁺CD25^-T cell for co-culture and to analyse the function of CD4⁺CD25⁺Treg in MG patients. Because of the important role of humoral immunity in MG, we used ELISA to detecte the soluble molecules in sera and PBMC cutured supernatants of MG patients and the healthy controls. We also aimed to find the relationship between the CD4⁺CD25^high Treg and the other regulatory T cells, BAFFR⁺ B cells, the soluble molecules in MG. A: Dynamic study of circulating CD4⁺CD25^high regulatory T cells inmyasthenia gravisObjective The aim of this study is to observe the difference of blood CD₄⁺CD₂₅^high T cells between the MG patients and healthy controls and to investigate the dynamic changes of the population in MG patients with various treatments. Methods The peripheral blood CD₄⁺CD₂₅^high T cells of 55 MG patients and 33 healthy controls were detected by four-color flow cytometry. And 26 MG patients were detected before and after various treatments. Results There was no significant difference in the percentages of CD₄⁺CD₂₅^high T cells between MG patients (6.2%±3.4) and the healthy control (5.2%±1.9, p=0.061). After short-term non-operated treatment, the percentages of CD₄⁺CD₂₅^high T cells were elevated, and had a negative correlation with the decrease of MGFA in 21 MG patients (r=-0.563, P=0.008). Conclusions Our results indicated that changes of the percentage of CD₄⁺CD₂₅^high T cells in MG patients after short-term treatment might be related to the disease. Except thymectomy, other treatments contributed to the increase of peripheral CD₄⁺CD₂₅^high regulatory T cells in MG patients after short term treatment.B: Circulating regulatory T cells and BAFF receptor on B cells in patients withmyasthenia gravisObjectives To investigate the levels of circulating regulatory T cells and B cell-activating factor receptor (BAFF-R) on B cells from patients with myasthenia gravis (MG). Methods Using flow cytometry, we tested the levels of circulating regulatory T cells ( CD4⁺CD25^highFoxp3⁺, CD8⁺CD28^-, CD8⁺CD122⁺ ) and CD19⁺BAFF-R⁺ B cells in 61 MG patients and 23 healthy controls. Results The percentages of the circulating CD4⁺CD25^highFoxp3⁺ T cells in MG patients ( 32.1%±16.1 ) were significantly declined compared with healthy controls (65.2%±14.7, p<0.01). The expression of BAFF-R on CD19⁺ B cells in MG patients (10.6%±5.6) was higher than that in healthy controls (5.4%±3.9, p<0.01). There were no significant differences in the subpopulations of CD8⁺CD28^- and CD8⁺CD122⁺T cells between MG patients and healthy controls (p>0.05) . Treatment of high-dose corticosteroid with or without additional intravenous IgG elevated the percentages of CD4⁺CD25^highFoxp3⁺ T cells in 20 MG patients (p<0.05). Conclusions These results revealed that decrease of circulating CD4⁺CD25^highFoxp3⁺ regulatory T cells in MG patients might be related to the pathogenesis of MG. An elevated BAFF-R on CD19⁺ B cells demonstrated that B cells were more active in MG patients.C: The function of circulating CD4⁺CD25⁺ regulatory T cells in myasthenia gravisObjectives To investigate the function of circulating CD4⁺CD25⁺ Treg cells in patients with Myasthenia Gravis (MG). Methods By using flow cytometry, we detected the fluorescence intensity of function-relative molecules on circulating CD4⁺CD25^highT cells (GITR/Neuropilin-l/CTLA-4/CD103) and appotosis-relative molecule (CD95) in 20 MG patients and 16 healthy controls. And we sorted the CD4⁺CD25⁺T and CD4⁺CD25^-T cells from 10 MG patients and 8 healthy controls and cultured in differents experimental groups: Group A: CD4⁺CD25⁺ T cell alone; Group B: CD4⁺CD25⁺T plus CD4⁺CD25^-T cell; Group C: CD4⁺CD25^-T cell alone). The proliferation was detected by ³H-TdR incorperation and the cytokines were detected by ELISA. Results The pecentages of CD4⁺CD25^highNeuropilin-1⁺ and CD4⁺CD25^highCD103⁺T cells were lower in MG patients than in the healthy controls. The proliferation of the group B was higher than group A and C in MG patients, while there was no difference in healthy controls. The secretion of sICAM-1 in group A and B were higher in MG patients, and the secretion of IL-10 in group C was lower in MG patients. Conclusions Some function-relative molecules in circulating CD4⁺CD25⁺ T cells in MG patients were decreased. The immunosuppression and the secretion of IL-10 of CD4⁺CD25⁺ T cells in MG patients were abnormal as well. A: The level of different kinds of cytokines, sCTLA-4 and sICAM-1from serum and cultured-cell supernatant in myasthenia gravisObjectives Myasthenia gravis (MG) is caused by T-cell dependent autoantibodies against muscle acetylcholine receptors (AChR) at the neuromuscular junction. Methods Here, we adopted ELISA and flow cytometry techniques to measure the levels of Th1, Th2, Th3 cytokines, inflammatory cytokines, sCTLA-4 and sICAM-1 from 75 MG patients and 50 healthy controls. Results There were no differences in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 between MG patients and healthy controls in both sera and culture supernatants. The level of IL-12 was decreased in culture supernatants from MG patients, and the level of sICAM-1 was increased in both sera and culture supernatants from MG patients. Conclusions It revealed that there were no significant differences in the levels of different soluble factors between MG patients and healthy controls. While the function of sICAM-1 should be studied further.B: An effective method of detection sICAM-1 for patients withmyasthenia gravisObjectives ICAM-1 is a member of the immunoglobulin supergene family and a reliable marker of disease, especially soluble ICAM-1. Methods In this study, we used ELISA to detect the level of sICAM-1 in the serum and PBMC culture supernatant from MG patients and healthy controls. Results There was no significant (p=0.20) differences of the level of sICAM-1 in serum between MG patients and healthy controls. The level of the sICAM-1 in culture supernatant was increased in MG patients (10.4±1.4) compared with healthy controls (4.5±0.1) (after cultured with FBS (p=0.0048) or with serum of themselves (p=0.024) for 48 hours). The level of IL-10, IL-12 and TNF-αwere all elevated cultured with FBS compared with the serum of themselves, except for IL-4 (no difference). The supernatant level of sICAM-1 from seven MG patients co-cultured with corticosteroid was decreased (p=0.0136) after 48 hours, while there was no significant difference of the level of sICAM-1 in serum before and after treated with high dosage of corticosteroid in fourteen MG patients (p=0.329). Conclusions So we drew the conclusion that PBMC secreted more sICAM-1 in MG patients than normal. It was a better method to analyze the supernatant level of sICAM-1 in PBMC cultured with FBS in MG patients. Corticosteroid can down-regulate the secretion of sICAM-1 but it was not seen in the level in serum after treated with high dosage of corticosteroid in MG.Conclusions:1. The disfunction of circulating CD4⁺CD25^high regulatory T cells and more active B cell in MG patients reveal the imbalance of T cells and the activation of B cell might be related to the pathogenesis of MG.2. There was no difference in the levels of IL-2, IL-4, IL-10, IL-13, IFN-γ, TNF-α, TGF-βand sCTLA-4 in sera and culture supernatants between MG patients and healthy controls. But the level of sICAM-1 was increased in culture supernatants from MG patients and most of the increase of sICAM-1 was likely caused by effctive CD4⁺CD25^-T cells in MG patients.3. Dynamic observation of the circulating CD4⁺CD25^high T cells and the secretion of sICAM-1 in cultured supernatant of PBMC in MG patients would be helpful for understanding the change of the disease.