A Study Of The Subsets Of Cells In The Human B Cell Subsets That Produce Anti-A Antibody

Objective Anti-ABO antibody-mediated rejection is the main hurdle in ABO-incompatible transplantation.However,cellular immune mechanisms of anti-ABO responses remain unclear.The present study aimed to identify the main human B cell subsets that produce anti-ABO antibodies by analyzing association of B cell subsets with anti-ABO titer.Methods The study protocol was approved by Seoul National University's Institutional Review Board(H-1411-020-623).Human peritoneal fluid and peripheral blood samples were collected from patients that initiated peritoneal dialysis and healthy volunteers,respectively.Peritoneal cells were obtained by centrifugation of the whole peritoneal fluid that was taken from peritoneal dialysis patients when the peritoneum was washed,2 weeks after catheter insertion.Peripheral blood mononuclear cells(PBMCs)were isolated by gradient centrifugation using Ficoll(GE Healthcare,Cleveland,OH,USA).PBMCs and peritoneal cells were subjected to red blood cell-lysis using BD Pharm LyseTM(BD Biosciences,San Diegeo,CA,USA).PBMC and peritoneal cells were stained with the following anti-human antibodies:blood type(tri)-PAA-fluor(Glycotech,Gaithersburg,MD,USA),anti-CD20(L27),anti-CD27(M-T271),anti-CD43(MEM-59),anti-CD5(L17F12),anti-CDllb(ICRF44),anti-CDlc(L161)(BioLegend,San Diego,CA,USA).Flow cytometric analyses were performed using a BD FACS Canto ? flow cytometer(BD Biosciences)and data analysis was performed using FlowJo(Tree Star,Ashland,OR,USA).A gel card method(ID-System DiaMed,Bio-Rad,Hercules,CA,USA)was used for the measurement of human blood group A-specific IgM and IgG.Briefly,50?L of 0.8%RBCs and 25 ?L of serially diluted serum samples were added to the gel card and incubated at room temperature(IgM)or 37?(IgG)for 15 minutes.After centrifugation for 10 minutes,the titer was determined based on the presence of the trapped agglutinates.The data were presented as the mean ± standard error of the mean.Continuous variables were analyzed using Student's t-tests and correlations were analyzed using Pearson's correlation analysis.A P value less than 0.05 was considered statistically significant.All analyses were performed using SPSS(version 20.0,IBM Corporation,Armonk,NY,USA).Results Overall,these data confirmed Bl,MZB,and MZ-B1 cells are present in the human peripheral blood and demonstrated that blood group A antigen-specific B cells were enriched in the B1 and MZ,B1 cells.Taken together,B1 cells,particularly CD11b+ B1 cells had a higher proportion of blood group A-specific B cells and showed a positive correlation with anti-A IgM titer,suggesting that these cells could be mainly involved in the anti-A antibody responses.Our previous studies have shown that CD49dhigh CD4+T cells can help B1 cells secrete IgM and IgG.Therefore,with the help of T cells,CD11b+B1 cells are likely to secrete anti-A antibody.Conclusion In this experiment,B cells were classified by two methods,and both methods confirmed that B1 cells produced anti-a antibodies.Furthermore,A good correlation between the frequency of human B1 cells and the titer of anti-a antibodies was demonstrated.Therefore,human B1 cells are potential therapeutic target for desensitization in ABO-incompatible transplantation.