Correlation Analysis Of HLA Alleles And Functional Subsets Of Memory B Cells Between Myasthenia Gravis Patients

Objective The objective of this study was to analyze the distribution and function of T and B cell subsets in myasthenia gravis（MG）patients,and to find the correlation between MG and human leukocyte antigen（HLA）alleles,and to screen the advantageous epitopes of anti-acetylcholine receptor（ACh R）antibodies,so as to provide some reference for the diagnosis and treatment of MG patients.Methods MG patients meeting the inclusion and exclusion criteria and healthy controls were collected and divided into MG group and healthy control group.Flow cytometry was used to detect the classification and counting of T cells and B cell subsets in peripheral blood.In order to study the effect of treatment on the expression level of T and B cell subsets,MG group was further divided into conventional treatment group and immunosuppressive treatment group,and the differences in the expression level of T and B cell subsets among each group and their correlation with MG were analyzed and compared with statistical methods.HLA alleles were measured in MG group and healthy control group,and the association between MG and HLA alleles was analyzed by statistical method.Bioinformatics methods were used to screen possible dominant peptides of ACh R.The secretion of immunoglobulin 1（Ig G1）in peripheral blood mononuclear cells（PBMCs）of each group after stimulation by synthetic ACh R peptide was determined by enzyme-linked immunospot assay（ELISPOT）,and the difference of specific B cell count among the groups was statistically compared.Results 1.Flow cytometry analysis showed no significant differences in the proportion and activation rate of T cell subsets among all groups（all P>0.05）.The expression level of B cells in immunosuppressive treatment group was lower than that in conventional treatment group（P<0.01）and healthy control group（P<0.05）.The expression level of Memory B cells(B_MEM)in conventional treatment group was higher than that in immunosuppressive treatment group（P<0.01）and healthy control group（P<0.01）.The proportion of unswitched memory B cells,switched memory B cells（SM B cells）and double negative B cells in conventional treatment group and immunosuppressive treatment group were higher than those in healthy control group（all P<0.01）.The na（?）ve B cells expression level of the healthy control group was significantly higher than that of the conventional treatment group（P<0.01）and the immunosuppressive treatment group（P<0.01）,and na（?）ve B cells expression level of the conventional treatment group was lower than that of the immunosuppressive treatment group（P<0.01）.2.The proportions of B cells（r=0.845,P<0.01）,B_MEM（r=0.822,P<0.01）and SM B cells（r=0.866,P<0.05）in MG patients were positively correlated with the reduction of disease severity.The proportions of B cells（r=-0.834,P<0.01）,B_MEM（r=-0.875,P<0.01）and SM B cells（r=-0.903,P<0.01）in MG patients were negatively correlated with the change of ACh R titer.The na（?）ve B cells expression level in MG patients was negatively correlated with the reduction of disease severity（r=-0.798,P<0.01）,and positively correlated with the change of antibody titer（r=0.742,P<0.05）.3.No association was found between MG and HLA alleles in MG group and healthy control group（all P>0.05）.4.The antigenic peptide simulation showed that the ACh Rα88-96（NPDDYGGVK）peptide and ACh Rε88-98（SKDDFGGIETL）peptide were the dominant peptides.5.The number of specific B cells secreting Ig G1 in PBMCs in MG group was significantly higher than that in healthy control group（P<0.01）;The Ig G1-secreting specific B cells count of PBMCs in patients stimulated by ACh Rα88-96 peptide was higher than that in patients stimulated by ACh Rε88-98 peptide（P<0.05）.Conclusion 1.Immunosuppressants can reduce disease severity and ACh R antibody titers in MG patients by blocking the survival and differentiation of memory B cells and SM B cell subsets.2.Dynamic monitoring of the changes in the composition of memory B cell subsets in blood can partly reflect the therapeutic effect of MG patients.3.Linear ACh Rα88-96 peptide is the dominant epitope of anti-ACh R antibody compared with ACh Rε88-98 peptide.