Characteristics Of B Cell Subsets And B Cell Receptor Repertoire Analysis In Systemic Lupus Erythematosus

Systemic lupus erythematosus?SLE?is a chronic systemic autoimmune disease of unknown etiology,characterized by the destruction of B cell immune tolerance and the production of a large number of autoantibodies,resulting in the deposition of immune complexes in tissues and organs.The prevalence of systemic lupus erythematosus in China is about 0.7¹/1000,and the incidence of systemic lupus erythematosus in women is much higher than that in men.Although all kinds of innate and adaptive immune cells are involved in the pathogenesis of SLE,according to current studies in mice and humans,the destruction of B cell immune tolerance and the abnormal activation of autoreactive B cells are still the core pathogenic factors of SLE.The proportion of B cell subsets in SLE is disordered and so is the immune homeostasis.The detection of the proportion and functional status of B cell subsets in peripheral blood can be used to explore the role of different B cell subsets in the pathogenesis of SLE.There are a large number of autoantibody secreting cells?ASC?and high levels of autoantibodies in SLE patients,and the expansion of autoantibody secreting cells will lead to the increase of SLE activity.However,the origin and production process of autoreactive ASC cells are still unclear.Human anergic B cells(B_ND for short)are a subset of autoreactive cells in Mature Na?ve Cells in human peripheral blood.They reach the state of immune"inactivity"by down-regulating surface Immunoglobulin?Ig?M to avoid antigen stimulation.In addition,the intracellular protein phosphorylation responses and intracellular calcium flux activity were prohibited.A number of studies have shown that anergic B cells are missing in autoimmune diseases,suggesting that these cells may be activated and further differentiated.Anergic B cells are likely to be a potentially dangerous source of ASC cells.Adaptive immune receptor repertoire sequencing?AIRR-seq?is an important method to study the characteristics of B cell activation and antibody spectrum.the analysis of B cell receptor?BCR?sequence by high-throughput sequencing can explore the understanding of the diversity,clonal overlap,and maturation of immune repertoires.Further understanding of the abnormal immune response in SLE is also helpful to find pathogenic B cell subsets.However,there is still a lack of research on the BCR repertoire in patients with SLE,and the characteristics of Anergic B cells?IgM⁺Conventional Na?ve B cell?CN B??Switch-memory B cells?SM B?and ASC cells and the relationships among them are still unknown.How autoreactive B cells are activated and converted into ASC cells is still an important question to be solved.In our study,the proportion of B cell subsets in SLE patients was detected by flow cytometry and their functional status was evaluated.The significance of B cell subsets in the pathogenesis of SLE was further analyzed.Moreover,IgM⁺CN B cells?B_ND cells,SM B cells and ASC cells were sorted and further analyzed their BCR repertoire,which helped to elucidate the transformation relationship between the four B cell subsets in SLE and find the pathogenic cell subsets in SLE.Chapter 1:Study on the proportion and function of B cell subsets in peripheral blood of patients with SLEObjective:Study on the proportion and Activation level of different subsets of B cells in patients with SLEMethod:1.Peripheral blood mononuclear cells?PBMC?from 61 patients with SLE and 61 healthycontrols were isolated by density gradient centrifugation.the surface markers of differentB cell subsets were labeled by flow cytometry,and the proportion of each cell group wasstatistically analyzed.And the clinical correlation analysis was further analysized.2.The expression of CD21?CD40?CD80?CD24?CD95?CD183?Interleukin 4 receptor?IL-4R?and B-cell activating factor?BAFF-R?on different B cell subsets was detectedby flow cytometry.3.B cell activation mitochondria were labeled with MitoTracker?Green probe todetermine the mitochondrial activation status of different B cell subsets.4.Intracellular calcium flux activities were detected in CN B cells and B_ND cells under thestimulation by BCR crosslink.Results:1.The proportion of B cell subsets in SLE patients was disordered.The proportion of DNB,ASC,Tr B cells was significantly higher than that of healthy controls;while theproportion of UM B and B_ND cells was significantly lower than that of healthy controls,while the proportion of Na?ve,CN B and SM B cells was not significantly different fromthat of healthy controls.2.The proportion of ASC cells was positively correlated with Systemic lupuserythematosus activity index?SLEDAI?and anti double-strand DNA?dsDNA?antibodytiters,the proportion of B_ND cells was negatively correlated with SLEDAI and anti-dsDNA antibody titers,and the proportion of Tr cells was negatively correlated with anti-dsDNA antibody titers.3.The expression of CD21,CD80,CD95 and CD183 in B_ND cells was lower than that inB cells;the expression of CD40 and BAFF-R was similar;while the expression of IL-4R in B_ND cells was higher than that in CN B cells.4.The mitochondrial activity of Na?ve B cells(CN B,B_ND)in SLE patients wassignificantly higher than that in healthy controls.5.The intracellular calcium flux activities of B_ND cells in SLE patients was significantlyhigher than that in healthy controls.Conclusion:the proportion of B_ND cells in SLE patients is lower than that in healthy controls,and is negatively correlated with SLEDAI and anti-dsDNA antibody titers.it is a group of cells with important clinical significance.In addition,the activity of mitochondria and the intracellular calcium flux activities were increased in SLE B_ND cells compared with healthy controls,which indicated that B_ND cells in SLE patients were functionally activated and increased in response to antigen stimulation.It is suggested that abnormal B_ND cells in patients with SLE may be a potential cause of the disease.Chapter 2:BCR repertoire analysis of CN B?BND?SM B?and ASC cell subsets in patients with SLE.Objective:Study on the BCR repertoire characterstics of different subsets of B cells in patients with SLEMethod:1.PBMC from 7 patients with SLE and 7 healthy controls were isolated by density gradientcentrifugation and labeled with flow antibodies.2.CN B cells,B_ND cells,SM B cells and ASC cells were sorted by flow cytometry in SLEpatients and healthy controls,according to the surface markers.3.RNA was extracted from different cell populations by Trizol and reverse transcriptionwas performed.The heavy chain was amplified by Polymerase Chain Reaction?PCR?and the PCR products were identified.The target fragment band was recovered and thetarget fragment was amplified by PCR again.4.The PCR products were sequenced in the second generation.The sequencing data werecompared and statistically analyzed by MIXCR and VDJtool software.Results:1.In healthy population,CN B and B_ND cells have similar V genes;The IGHV4-34 geneusages in CN B and B_ND cells were both significantly higher than that in SM B and ASCcells.When compared with SLE patients and healthy controls,the IGHV4-34 gene usagein SM B and ASC cells of SLE patients was significantly higher than that of healthycontrols.2.In healthy population,The IGHJ6 gene usage in CN B was significantly lower than thatin B_ND cells.The IGHJ6 gene usages in CN B and B_ND cells were both significantlyhigher than that in SM B and ASC cells.When compared with SLE patients and healthycontrols,the IGHJ6 gene usage of B_ND cells in SLE patients was significantly lower thanthat in healthy controls,while the IGHJ6 gene usage of ASC cells was significantlyhigher than that in healthy controls.3.In healthy population,the Complementary decision region?CDR3?length of CN B11cells was significantly shorter than that of B_ND cells.The CDR3 lengths of CN B andB_ND cells were both longer than that of SM B and ASC cells.When compared with SLEpatients and healthy controls,the length of CDR3 length of B_ND cells in SLE patientswas lower than that of healthy controls,while that of SM B cells was longer than that ofhealthy controls.Conclusion:In healthy population,the autoreactivity of B_ND cells was higher than that of CN B cells,which is mainly manifested in higher IGHJ6 utilization and longer CDR3 length.In the conversion of Naive B cells(CN B,B_ND)to SM B and ASC cell subsets,the immune tolerance checkpoint will screen out the autoreactive B cells,resulting in the decrease of IGHV4-34 and IGHJ6 gene usage and shorter CDR3 length.Our results suggest that immune tolerance checkpoints are deficient in patients with SLE.The IGHJ6 gene usage of B_ND cells was lower than that of healthy controls,and the length of CDR3 was shorter.While the IGHJ6 gene usage of SM B or ASC cells was higher than that of healthy controls,and the length of CDR3 was longer.These results suggested that some autoreactive B_ND cells are activated in SLE patients,resulting in high autoreactivity of SM B and ASC cell subsets in SLE patients.