Systemic Analysis Of B Cells And The Specific Mechanisms Of CD20^hiB Cells In The Pathogenesis Of Systemic Lupus Erythematosus

Objective:Distinct abnormalities of B cells are considered to be an important cause of Systemic Lupus Erythematosus.This study aims to investigate the disturbed peripheral B cell phenotypes of SLE patients and explore the specific mechanisms of CD20^hiB cells in the pathogenesis of SLE,thus providing a new strategy for targeted therapy.Methods:1.Peripheral blood samples were collected from 72 na(?)ve SLE patients as well as 40 cases of healthy donors from Renji Hospital.The whole picture of immune cell phenotypes was studied by flow cytometry.2.The distribution of B cell subsets in 54 SLE patients was focused,and the correlation between B cell subsets and clinical informations was analyzed.3.23 SLE patients were followed up and the peripheral blood samples were collected at baseline and at 4,12,24 weeks.The changes of pheripheral B cell subsets were observed and the B cell subset which was most relevant to clinical characters was found out.4.B cell-derived cytokines were detected by flow cytometry,serum cytokines were detected by ELISA and luminex.Correlation analysis was further perfomed with clinical informations and B cell subsets.5.Phenotypes and functions of CD20^hiB cells were analyzed by flow cytometry,and the specific autoantibody spectrum of CD20^hiB cells was detected by ELISA.6.RNA-seq technologies were used to detect the different gene expression profiles between CD20^hiB cells and classical Bm,and the different signal pathway between CD20^hiB cells and classical Bm was further studied by q PCR and flow cytometry.7.Immunofluorescence technologies were used to analyse the infiltration of CD20^hiB cell in renal tissues of lupus nephritis and in paracancer renal tissues.Correlation analysis with clinical informations was further studied.Results:We identified elevated percentage of B cells in na(?)ve SLE patients.CD20^hiB cells were significantly expanded,and exhibited good relationship with disease activity indexes and lupus nephritis.After 24-week treatment,the percentage of CD20^hiB cells in flare/resistant group was much higher than in remission group,indicating it may have the same value as plasma cells in predicting disease recurrence.BCR crosslinking induced markedly reduced Ca²⁺influx in CD20^hiB cells.CD20^hiB cells also exhibited increased apoptosis rate and decreased ability of total antibody secretion,but showed a strong capacity in secreting anti-nucleosome antibodies and relatively potent ability to promote the proliferation and IFN?secretion of CD4⁺T cells.In our study,we first time pointed out that the m TOR pathway was highly activated in CD20^hiB cells,and participated in the differentiation of this B cell subset.Rapamycin could specifically block the m TOR pathway,thus inhibiting the differentiation of CD20^hi(T-bet⁺)B cells induced by IFN?and R848.Besides,rapamycin could effectively inhibit the secretion of antibodies of CD20^hiB cells.For the first time CD20^hiB cells were found to be infiltrated in renal tissues of LN patients,which also showed a good relationship with 24h urinary protein,renal functions and activity indexes.The infiltration rate of CD20^hiB cell was highest in type IV LN and in highest renal-SLEDAI score group.Conclusion:CD20^hiB cells were significantly expanded in peripheral blood of SLE patients,showed a good relationship with disease activity indexes and lupus nephritis,and exhibited to be a sensitive index in evaluating disease recurrence.CD20^hiB cells were found to infiltrate in renal tissues of LN patients.It may participate in the development of SLE and LN by secreting anti-nucleosome antibodies and playing a role in promoting the proliferation and IFN?secretion of CD4⁺T cells.m TOR pathway was highly activated in CD20^hiB cells,which participated in the differentiation of this B cell subset.Rapamycin could specifically block the m TOR pathway,thus inhibiting the differentiation of CD20^hi(T-bet⁺)B cells mediated by IFN?and R848 and the secretion of antibodies.It may improve the symptoms of lupus and prevent the progression of SLE by this way.