| Objective: To investigate the role of rapamycin target protein(m TOR)pathway in the regulation of regulatory T cells in the pathogenesis of IgG4 related disease(IgG4-RD);to explore the clinical characteristics and treatment efficacy of IgG4-related disease(IgG4-RD)with different levels of serum IgG4.Methods: Part I:The peripheral blood of newly diagnosed,remission IgG4-RD patients in Peking Union Medical College Hospital and sex,age matched healthy controls were collected.Peripheral blood mononuclear cells(PMBCs)were isolated and the percentages of Treg cells and their TGF β 1 and IL-10 expression were detected by flow cytometry.The plasma of HC and newly diagnosed IgG4-RD patients were collected and the expression of IL-10 in plasma was detected by ELISA.The CD4+T cells of HC and newly diagnosed IgG4-RD patients were isolated by magnetic bead sorting technique,RNA was extracted and transcribed into c DNA,lytic cell extract protein.The levels of m RNA PTEN,m TOR,TSC1,4EBP1 and P70S6 K were detected by RT-PCR technique.The protein expression levels of phosphorylated PTEN(p-PTEN),PTEN,p-m TOR,p-4EBP1,4EBP1,p-P70S6 K and P70S6 K in m TOR pathway were detected by Western Blot.Flow cytometry was used to detect the expression of p-PTEN and p-4EBP1 in CD4+T cells and Treg cells of HC and IgG4-RD.Part II:A total of 299 newly diagnosed patients with IgG4-RD were enrolled in this study.Patients were classified into four groups according to baseline serum IgG4 levels,Group A: normal serum IgG4 concentration,Group B: Serum IgG4 > normal but < 2× upper reference limit(URL),Group C: Serum IgG4 between 2× & 5× URL,Group D: Serum IgG4 > 5×URL.All patients were followed up for 12 months.Patients clinical characteristics,laboratory parameters,plasmablast/plasma cells and treatment efficacy were analysed.Results: Part I:The percentage of CD4+CD25+Treg cells and CD4+CD25+Foxp3-Treg cells in newly diagnosed IgG4-RD patients was significantly higher than that in HC(P ≥0.0356,P ≥ 0.023),and decreased after treatment(P < 0.0453),but there was no significant difference in CD4+CD25+Foxp3+Treg cells among the three groups.There was no significant difference in IL-10 expression among subtypes of Treg cells.The expressions of TGF β 1 of CD4+CD25+Treg cells and CD4+CD25+Foxp3-Treg cells in newly diagnosed IgG4-RD patients were significantly higher than that in HC(P < 0.0493),and decreased significantly after treatment(P < 0.0001,P ≤ 0.0013).The expressions of m RNA PI3 K,m RNA PTEN and m RNA TSC1 in CD4+T cells of newly diagnosed IgG4-RD patients were significantly lower than those of HC(P < 0.0001,P < 0.0001),and increased after treatment,while the expressions of m RNA P70S6 K and m RNA m TOR were lower than HC(P ≥ 0.0002,P ≤ 0.0003).The expressions of m RNA 4EBP1 increased significantly after treatment(P ≥ 0.0189).The protein expression of p-PTEN,p-m TOR and p-4EBP1 in CD4+T cells of newly diagnosed IgG4-RD patients was significantly higher than that of HC(P ≥ 0.0172,P ≤ 0.0290,P ≤ 0.0194),but there was no significant difference among the expression of PTEN,m TOR,4EBP1,p-P70S6 K and P70S6 K.The expression of p-PTEN in CD4+T cells of newly diagnosed IgG4-RD patients was significantly higher than that in HC(P ≤ 0.0381),and the expressions of p-PTEN and p-4EBP1 in CD4+T cells and Treg cells of newly diagnosed IgG4-RD patients were higher than those in HC(P ≤ 0.0016,P ≥ 0.0031).The MFI of p-PTEN in IgG4-RD was correlated with serum IgG4 level(r =0.6505,P=0.0465).Part II:Of organs affected,the prevalence of submandibular gland,lacrimal gland,parotid gland,pancreas,bile duct and lung involvement increased gradually from Group A to Group D.After 12 months’ follow-up,31(68.9%)patients in Group B,43(51.8%)patients in Group C and 20(12.7%)patients in Group D had achieved normal serum IgG4 successfully.Besides,serological relapse in Group D(19.4%)was higher than Group B(4.9%)and Group C(7.7%).The clinical relapse of patients whose serum IgG4 levels decreased ≥40% in Group B and Group D were lower than whose serum IgG4 levels decreased <40% and increased(P=0.009;P=0.043).Conclusion: 1.CD4+CD25+Treg cells and CD4+CD25+Foxp3-Treg cells in IgG4-RD patients were significantly higher than those in HC and after treatment.The expression of TGF β 1 in Treg cells of IgG4-RD patients was significantly higher than that in HC,treatment,TGF β 1 may promote the process of fibrosis,and serum IL-10 in IgG4-RD patients was significantly higher than that in HC patients.Serum IL-10 level related with laboratory parameter which reactived disease activity.2.m TOR pathway was highly activated in CD4+T cells and Treg cells of IgG4-RD patients,MFI of p-PTEN was correlated with serum IgG4 level,suggesting that m TOR pathway participated in the pathogenesis of IgG4-RD by regulating Treg cells.m TOR pathway is expected to be a new target for IgG4-RD therapy.3.Patients with higher serum IgG4 tend to had higher percentage of dacryoadenitis,sialadenitis,autoimmune pancreatitis,and higher prevalence of allergy history,whereas patients with retroperitoneum and mediastinum lesions usually had lower serum IgG4.Those with serum IgG4>5× URL were most prone to have serological relapse and were the most difficult to achieve normal level after treatment.Targeting decrease IgG4 levels ≥40% may be beneficial to reduce their clinical relapse rate. |
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