Analyses Of Immune Cellular Compositions Of Peripheral Blood In Patients With Systemic Lupus Erythematosus Based On Multicolor Flow Cytometry

Background:Systemic lupus erythematosus(SLE)is an autoimmune disease involved many organs.Immune cells of innate immune system and adaptive immune system take part in the development of SLE disease.With the rapid development of multicolor flow cytometry,there are more and more applications in autoimmune diseases.However,to date,there have not been a flow cytometry panel to study alterations of all the immune cells in SLE patients at once.Therefore,firstly,we aim to establish a stable multicolor flow cytometry method to detect all immune cells in human peripheral blood;secondly we apply this method to explore the immune cells alterations of patients with SLE compared to healthy controls;finally we aim to identify some cellular biomarkers related with disease activities et al.Objecyive:1.To establish a 14-color panel to measure lymphoid cells and myeloid cells in human peripheral blood.2.To visualize differences in the immune cellular compositions in the peripheral blood of patients with systemic lupus erythematosus(SLE)and healthy controls;and the correlation between cellular compositions and SLE disease activity as well as laboratory variables.3.To visualize differences in frequencies of CD127+ILCs(innate lymphoid cells)and their subsets in peripheral blood mononuclear cells(PBMCs)of patients with SLE and healthy controls;and the correlation between cellular compositions and SLE disease activity as well as laboratory variables.Methods:1.Use CD45,CD3,CD19,CD14,CD4,CD8,CD16,CD56,CD25,CD127,CD11c,CD 127 and HLA-DR fluorescent antibodies and 4’,6-diamidino-2-phenylindole(DAPI)to test peripheral lymphoid cells and myeloid cells by multi-parameters flow cytometry.2.Peripheral Immune cells were obtained from thirty-two SLE patients and sixteen healthy controls.Circulating granulocytes,basophils,dendritic cells(DCs),monocytes,T cells,B cells,natural killer(NK)cells,CD127+ILCs and their subpopulations were identified by 14-color flow cytometry.The association between disease activity,laboratory variables and the immune cells was examined by univariable and multivariable analysis.3.PBMCs was obtained from twenty-five SLE patients and twelve healthy controls Circulating total ILCs,its subsets(ILC1,ILC2,ILC3,CD336+ILC3 and CD336-ILC3)and the expression of CD4 and CD8 were identified by 8-color flow cytometry.The association between disease activity,laboratory variables and the immune cells was examined by univariable and multivariable analysis.Results:1.We successfully established a 14-color panel through optimization of the combination of antibody and fluorescein,titration of antibody concentration,detection voltage optimization and fluorescence compensation adjustment,to measure circulating immune cells,including granulocytes,basophils,dendritic cells,monocytes,T cells,B cells,NK(natural killer,NK)cells,CD127+ILCs and their subsets in one tube 1mL peripheral blood.And also we established an 8-color panel to measure circulating CD127+ILCs,ILC1,ILC2,ILC3 and their subpopulations from PBMCs.2.Increased frequencies of granulocytes,HLA-DR+CD4+T cells,CD8+T cells,CD25+DN T cells,HLA-DR+DN T cells,HLA-DR+DP T cells,CD16-NK cells,CD16-CD56dimNK cells,HLA-DR+CD56dim NK cells,CD 11 c+CD56dimNK cells,CD25+CD56dimNK cells,HLA-DR+CD56brightNK cells,CD25+CD56brightNK cells and CD4+CD127+ILCs were observed significantly in patients compared to healthy controls,while decreased frequencies of CD25hiB cells,T cells,CD4+T cells,Tregs(T regulatory cells),CD25+CD8+T cells,CD127+CD8+T cells,CD56+CD8+T cells,NK cells,CD16+NK cells,CD16brightCD56dim NK cells and CD127+ILCs were observed significantly in patients compared to healthy controls.Significantly increased frequencies of CD 123-positive granulocytes,monocytes,T cells,NK cells were found in SLE patients compared to healthy controls.3.Increased frequencies of ILC1,CD4-CD8-ILC2,CD4+CD8-ILC2 and CD4-CD8-CD336-ILC3 were observed in patients compared to controls.While decreased frequencies of ILC2,ILC3,CD4+CD8+ILCs,CD4+CD8-ILC1,CD4+CD8+ILC2 and CD4+CD8-CD336-ILC3 were found in patients compared with controls.4.There were many correlations between immune cells and disease activity,serum IgA,IgM,IgE levels and ESR(erythrocyte sedimentation rate),CRP(C-reactive protein)by univariate analyses.After multivariate analyses:frequencies of B cells,CD25+CD56bright NK cells,CD16+NK cells and CD 16-NK cells were correlated with disease activity.Frequency of MyDCs was negatively related with serum IgA level and frequency of CD25hiB cells was positively related with ESR.5.There were significance differences in frequencies of B cells,CD127+CD8+T cells,basophils,DCs,HLA-DR+CD56bright NK cells,intermediate monocytes,CD25+CD56dim NK cells,CD16+CD4+T cells,CD25+CD56bright NK cells,CD11c+CD56bright NK cells,HLA-DR+CD4+T cells,HLA-DR+Tregs,CD16+CD8+T cells,CD25+CD8+T cells,CD11c+CD4+T cells,CD25+DP T cells and CD 16-CD56briht NK cells between SLE patients with autoantibodies positive groups and SLE patients with autoantibodies negative groups.Conclusion:We successfully established a 14-color panel to measure circulating immune cells,including myeloid cells(granulocytes,basophils,dendritic cells and monocytes)and lymphoid cells(T cells,B cells,NK cells and innate lymphoid cells)and their subsets in one tube 1mL peripheral blood.We observed diverse alterations in the peripheral immune cells in SLE patients and some cell subsets were related with SLE disease activity and autoantibodies.This was a first study to analyze a comprehensive peripheral immuneophenotypes including myeloid cells,lymphoid cells and their subsets in patients with SLE.This 14-color flow cytometry panel could be easy and fast to understand the immune disorders in patients with SLE,and guide diagnosis and treatment of disease.However,the total sample size of our study was small,large number and multi-center studies were necessary to Validate.