Function Of The Lesional Dsg-specific B Cell In The Pathogenesis Of Pemphigus

Object:Pemphigus is a severe skin and mucosal membrane-targeting autoimmune bullous disease.It is generally believed that the circulating anti-Desmoglein1(Dsg1)and Desmoglein3(Dsg3)autoantibodies lead to acantholysis,and intraepidermis blister formation by destructing the desmosome between keratinocytes.Scientists consider that the pathogenic antibodies come from circulating B cells rather than the skin,which is only believed as the target of the disease,so most of the studies focus on the peripheral lymphocytes.In order to demonstrate the lesional immune mechanism of pemphigus,we investigate the phenotype and function of the infiltrated lymphocytes in pemphigus lesion in this project.Methods:Measurement of the frequencies of lesional CD19~+B cells,Dsg-specific B cells,lesional and peripheral follicular helper T cells by flow cytometry,and compared them with the percentages of healthy controls;ELISPOT assay was used to detect the percentage of Dsg-specific antibody secreting cells in PV lesion;ELISA was used for detecting the titer of autoantibodies in supernatant from in vitro culture of PV lesional lymphocytes and sera;Western blotting assay was used for verifying if the antibodies produced by in vitro culture of PV lesional lymphocytes could bind to the Dsg1 or Dsg3antigens of keratinocyte extracts;data were analyzed by GraphPad Prism version 5.0software and presented as x~-±SEM;to analyze the correlation between titers of antibodies secreted by lesional lymphocytes after in vitro culture and Pemphigus Disease Area Index(PDAI),Pearson product moment correlation coefficient was used;Spearman rank correlation was used to study the correlation between the titer of anti-Dsg autoantibody in PV patients'sera and the percentage of follicular helper T cell in PBMC;ROC analysis was used for exporting paired sensitivity and specificity values and calculating cut-off values to discriminate positive and negative results of the titer of antibodies from lesional lymphocytes in vitro culture.Results:The proportion of CD19+B cells was found much higher in PV lesion than that in healthy skin(4.27±1.13%vs.0.61±0.31%;P=0.0002),and CD19~+B cells from the PV lesions could specifically bind Dsg1 and Dsg3;the short-term ELISPOT assay revealed that the frequencies of Dsg1 and Dsg3-specific IgG secreting lymphocytes isolated from lesional skin of PV patients were 0.22±0.031%and 0.54±0.095%respectively;antibodies produced by PV lesional lymphocytes after in vitro culture specifically bond to a 130kDa protein by Western blotting assay;PV lesion contained a significantly higher proportion of CD3~+T cells than healthy skin(71.18±3.95%vs.57.18±3.56%;P=0.0127),but no difference in cell percentage was found between CD4~+T cells and Tfh cells(59.04±3.57%vs.62.06±3.85%,P=0.5783 for CD4+T cells and 2.12±0.32%vs.2.47±0.48%,P=0.5861,Unpaired t test for Tfh cells);there were significantly higher proportion of CD4~+IL-21~+T cells in PV lesions than that in healthy skin(3.98±0.87%vs.1.21±0.06%;P=0.0195),and 7.87±7.803%of IL-21 secreting CD4~+T cells could also produce IL-17a;the frequencies of Tfh cells and CD45RA~-Tfh cells in pemphigus patients were remarkably higher than those in healthy human controls(P<0.0001);after treated with glucocorticoid,the frequencies of first-onset patients'Tfh cells(0.16±0.11)%and CD45RA~-Tfh cells(0.11±0.09)%were lower than before;pemphigus patients'Tfh cells in PBMC showed positive correlation with the titer of anti-Dsg1 and anti-Dsg3 autoantibodies in patients'sera(R=0.631,P=0.017 and R=0.285,P=0.004).Conclusion:Dsg1 and Dsg3-specific B cells and Dsg-specific antibody secreting cells are present in pemphigus lesion;after in vitro culture for PV lesional lymphocytes,anti-Dsg1and Dsg3 antibodies were produced;IL-21 was secreted by Th17 cells in PV lesional skin,assisting B cells maturation and producing specific pathogenic antibodies,leading to the formation of lesion and severity of the disease.