Study On B Cell Receptor Repertoire In Different Disease States Of Rheumatoid Arthritis Based On Single Cell Sequencing

Background and objective:Rheumatoid arthritis(RA)is a common chronic,systemic,inflammatory and autoimmune disease.Although the application of new drugs such as biologics and small-molecule targeted drugs has brought good news to RA patients,RA is still difficult to cure and often flares.Patients often relapse between different disease states such as active and remission.Insufficient research on the pathogenesis of the disease and the mechanism of disease state changes have brought great challenges to the goals of individualized treatment and standard treatment.The pathogenesis of RA is still unknown.It is currently believed that the dysfunction of B cells is involved in important links,such as a large number of autoreactive B cells,immune tolerance disorders,high activation and secretion of a large number of autoantibodies,and regulatory B cell ratio disorders that aggravate immune intolerance,but at present,the specific mechanism of B cells involved in RA is still unclear.B cell receptor(BCR)is the most important part of B cells to recognize antigens and perform functions.On a certain time section,the sum of all BCRs in an individual constitutes the B cell immune repertoire.By studying the BCR repertoire It is of great significance for understanding the characteristics of pathogenic B cells,the mechanism of breaking immune tolerance,and the mechanism of participating in pathogenicity.At the same time,the development of single-cell immune repertoire sequencing technology has realized the joint analysis of the transcriptome and immune repertoire at the level of single cells,which can analyze richer biological information and find disease-specific BCR clones more accurately.And locate the corresponding self-reactive B cells,providing new ideas for revealing the pathogenesis of RA.Based on this,we used single-cell immune repertoire sequencing technology to perform a combined analysis of the single-cell transcriptome and BCR repertoire of peripheral blood B cells in two RA patients in the active and remission phases.Explore the commonalities and differences of the B cell immune repertoire in different disease states by analyzing the changes in the number of B cell subgroups under different disease states,as well as the shared clones and differential clones of the BCR repertoire in the active and remission period Pathogenesis and potential therapeutic targets.Methods:1.Determine the inclusion and exclusion criteria,and conduct regular follow-up of 8patients with RA who meet the criteria,and use the Disease Activity Score in 28 joints(DAS28)as the main indicator for evaluating the disease activity of the patients.Collect peripheral blood samples of all patients in the active phase(DAS28>3.2)and remission phase(DAS28<2.6).Finally,2 patients(RA01,RA05)were selected from 8 follow-up patients to enter the study.2.The density gradient centrifugation method was used to extract peripheral blood mononuclear cells(PBMC)from two patients in remission and active phases respectively,and CD19+ B cells were obtained by Easyseq magnetic bead sorting.3.Perform single-cell 5-terminal transcriptome and immune repertoire combined sequencing on the sorted B cells,and obtain transcriptome data and BCR immune repertoire data after data filtering and quality control.4.Perform cell cluster analysis and label annotation on single-cell transcription data,and analyze the changes in the number of major subgroups of B cells in different disease states.The analysis of the B cell immune repertoire is carried out from three dimensions and three directions: the three dimensions refer to the three dimensions of shared clones,differential clones and high-frequency differential clones in the active and remission phases of two patients;three directions;It refers to the discussion from the three directions of clonotype,VDJ usage preference,and CDR3 region characteristics,to study the commonalities and differences of the B cell immune repertoire in different disease states.Results:1.Through clustering and annotation of single-cell transcriptome data,we identified11 cell clusters,including 3 types of Naive B cells,5 types of Switch Memory B(SM B)cells,2 types of Unswitch Memory B(UM B)cells and 1 type of Double Negative B(DN B)cells.By comparing the changes in the number of cells in each group under different disease states,it is found that UM B Cells and DN B cells showed an increase in the number of remission cells in both patients.2.Through the analysis of the single-cell immune repertoire data,it is found that compared with the active period,the BCR repertoire of RA patients showed an increase in the total number of clones,a decrease in the degree of clonal expansion,a decrease in the number of high-frequency clones,and a more scattered clone abundance in the remission period.,B cell immune repertoire is more diverse,and the length of amino acids in the CDR3 region of the heavy chain is reduced.3.By analyzing shared clones under different disease states,we found 21 light and heavy chain gene fragments are prefer to be used in both patients,including IGHV1-18,IGHV3-21,IGHV3-23 D,IGHV3-30-3,IGHD6-13,IGHD3-9,IGHD1-7,IGHD2-21,IGHD3-22,IGHD3-3,IGHD5-12;IGKV3-20,IGLV1-47,IGLV2-23,IGLV2-8,IGLV3-1and IGKJ1,IGLJ2,IGKJ4,IGLJ3,IGLJ1.4.Through the analysis of differential clones in different disease states,8 heavy chain gene fragments which are high-frequency used and the frequency reduced in the remission period were found,including IGHV3-23 D,IGHV4-34,IGHV3-9,IGHV3-30,IGHV1-3,IGHJ6,IGHD2-2,and IGHD1-26.Meanwhile,the frequency of IGHV3-23 D,IGHV4-34,IGHV3-15,IGHJ6,IGHD3-22,IGHD3-16 are lower during the remission period in high-frequency differential clones.Last,through comprehensive comparison,IGHV3-23 D,IGHV4-34,IGHJ6 are strongly suggested be related to the pathogenicity and disease activity of RA.Conclusion:1.Through the analysis of single-cell transcriptome data,it was found that both patients presenting increasing in the proportion of UM B cells and DN B cell in the remission period,suggesting that it may be related to the remission of the disease.2.Through the analysis of the single-cell immune repertoire data,it is found that compared with the active period,the BCR repertoire of RA patients showed an increase in the total number of clones,a decrease in the degree of clonal expansion,a decrease in the number of high-frequency clones,and a more scattered clone abundance in the remission,B cell immune repertoire is more diverse,and the length of amino acids in the heavy chain CDR3 region is reduced,which may be related to the remission of the disease and the decline of autoimmunity.3.At last,we identified several gene fragments related to the pathogenesis and progression of RA,including IGHV3-23 D,IGHV4-34,IGHJ6,etc.It may provide new clues for exploring the involvement of B cells in the pathogenesis of RA and potential therapeutic targets and biomarkers.