The Role And Mechanism Of Follicular Helper T Cell And Monocyte/macrophage-derived Exosomes In Antibody-mediated Rejection After Renal Transplantation

ObjectiveAntibody-mediated rejection(ABMR)is a key factor leading to loss of renal function in the kidney.Currently,the molecular mechanism of ABMR is not sufficient.Clinically,the treatment of ABMR remains in non-specific inhibition.Clearing the antibody-producing cells or blocking the layer of antibody produced,the targeting is poor,the side effects are large,and the efficacy is poor.Therefore,finding new therapeutic targets has important clinical implications.Follicular helper T cells(Tfh)are T cell subsets of helper B cells that promote the diferentiation of B cells into plasma cells and produce high affinity antibodies.Exosomes are nanoscale vesicles actively assembled and released by cells,containing components such as proteins,nucleic acids and lipids,which mediate communication functions between cells.At present,the relationship between Tfh cell-derived exosomes for B cell proliferation and differentiation and ABMR is unclear.After renal transplantation,the donor glomerular endothelial cells first contact the recipient's immune cells,and the study of exocytosis between the two is essential for the study of transplant rejection.The transplanted kidney enters the body and immediately activates the innate immunity of the receptor,and a large number of mononuclear/macrophage homing and infiltrating into the graft.Mononuclear/macrophage surface ligands can bind to VCAM-1,ICAM-l,and E-selectin on endothelial cells,mediating pro-inflammatory function,so it can be used as an effective model to study exocrine transit between immune cells and transplanted kidney after kidney transplantation.Based on the previous research on the mechanism of action of Tfh cells in antibody-mediated rejection,this study further studied the role of derived exosomes in ABMR by detecting Tfh cells in peripheral blood of patients with chronic renal allograft dysfunction.Its subpopulation-derived exosomes were analyzed for their relevance to ABMR.Tfh cell-derived exosomes were obtained by further isolation of Tfh cells in vitro,and they were co-cultured with B cells under the stimulation of SEB to analyze their effects on B cell proliferation and differentiation.It not only provides a new perspective for elucidating the mechanism of Tfh cell-assisted B cell proliferation and differentiation,but also provides a new approach for the prevention and monitoring of antibody-mediated rejection.Netrin-1 is used as a molecular model to discuss the effects of neural molecules on the interaction between monocytes/macrophages and glomerular endothelial cells,in order to understand the relationship between nerves,immunity and blood vessels after organ allografts and provide new insightsof the delivery of exosomes.First partMethods1.Selection of study subjects:Patients with chronic renal allograft dysfunction(CRAD)after renal transplantation at the eighth medical center of Chinese PLA General Hospital,were selected as subjects,and renal function after renal transplantation was selected.Normal patients were used as a control group.2.Collection of specimens:The subjects in the group were collected in the early morning fasting liquid samples,and the specimens of the antibody-mediated rejection group were clinically diagnosed.ABMR was collected in time for treatment.3.Study grouping:The subjects were divided into two groups according to the Banff diagnostic criteria for antibody-mediated rejection in 2013,including antibody-mediated rejection(ABMR)and non-ABMR groups.4.Exosomes isolation method:The exosomes in the serum of the study subject were separated by polymer precipitation method,and were operated using SBI ExoQuickTM Exosome Exosomes Isolation Kit.5,identification of exosomes:transmission electron microscopy(TEM)to identify the morphology of exosomes;nanoparticle tracking analysis(NTA)technology to identify the size and concentration of exosomes;Western blot to identify exosome surface specific molecular markers.6.Detection of exosome biomarkers by flow cytometry:using magnetic beads coated with anti-CD63 antibody to bind exosomes,labeling CD4,CXCR5 and other surface markers by flow cytometry:using Invitrogen Exosome-Human CD63/Detecting reagents,combining exosomes with magnetic beads,using flow cytometry to detect the proportion of exosomes derived from different Tfh cell subsets and the expression of CTLA-4 and HLA-G on exosomes,comparing patients with ABMR group Differences in Tfh-derived exosomes between patients with non-ABMR groups were analyzed for their association with ABMR.7.Collection of other clinical data:general clinical conditions(including gender,age,time of donor kidney transplantation,etc.)and laboratory indicators(key records of transplanted kidney function indicators(serum creatinine,urea nitrogen,uric acid),blood routine,urine routine and other test indicators).8.Numerical data are calculated in the form of mean±standard deviation.The data was analyzed and calculated using SPSS 19.0 software.Clinical data were compared using a two-sided Wilcoxon rank sum test.Spearman rank test correlation and paired t test were used to compare experimental results.P<0.05 was set to have statistical differences.Results1.Basic characteristics of exosomes:Transmission electron microscopy(TEM)results show that the isolated exosomes are tea disc-shaped or hemispherical with concave surfaces with a diameter of 50-100 nm;nanoparticle tracking analysis(NTA)results show that the capsules The diameter of the bubble is about 100nm,which is consistent with the diameter of the exosomes;Western Blot is used to detect the expression of exosome protein;the three marker proteins CD9,CD81 and TSG101 are expressed in the vesicles separated from the serum.2.Flow cytometry was used to compare the exosomes and their subtypes separated by serum from each group of recipients.The results of flow cytometry showed that the percentage of Tfh cell-derived exosomes in the serum of patients in the CRAD group and the control group.No significant difference.In the CRAD group,the proportion of exosome derived from IL-21-producing Tfh cells(Tfh2 and Tfh17 cells)was higher than that of the control group.CRAD patients are further divided into ABMR group and non-ABMR group.The results showed that there was no significant difference in the proportion of exosome derived from Tfh and Tfhl cells between the two groups,while the proportion of exosomes derived from Tfh cells(Tfh2 and Tfh 17 cells)producing IL-21 in the ABMR group was significantly higher than that in the non-ABMR group.3.Flow cytometry was used to detect the expression of HLA-G and CTLA-4 in Tfh cell-derived exosomes:Tfh cell-derived exosomes were not significantly expressed in CRAD patients and controls in HLA-G and CTLA-4.Variety.In CRAD patients,the expression of CTLA-4 on Tfh cell-derived exosomes in the ABMR group was significantly lower than that in the non-ABMR group,but there was no statistical difference in HLA-G expression between the two groups.ConclusionsIncreased expression of exosome derived from IL-21-producing Tfh cells(Tfh2 and Tfh17)in serum from antibody-mediated rejection(ABMR)after renal transplantation,suggesting that IL-21-producing Tfh cells(Tfh2 and Tfh17)are derived Exosomes are closely related to ABMR.The expression of CTLA-4 on the surface of Tfh cell-derived exosomes in the blood of ABMR patients was significantly reduced.Second partMethods1.Research objects and groupings:same as the first part.2.Collection of specimens:Obtain peripheral blood samples from subjects and store them in heparin anticoagulation tubes.3.Separation of PBMC:Peripheral blood PBMC were isolated using lymphocyte separation fluid.4,sorting follicular helper T cells(Tfh):first immunomagnetic beads negative sorting peripheral blood specimens CD4+T lymphocytes;further immunomagnetic beads positive sorting CXCR5+ follicular helper T cells(Tfh).5.Culture of follicular helper T cells(Tfh):Tfh cells were cultured for 48 hours in RPMI1640 medium containing 10%fetal exfoliated fetal bovine serum.6.Isolation of Tfh cell-derived exosomes:Exosomes were isolated using SBI's ExoQuickTM Exosome Exosomes Isolation Kit.7.Flow cytometry was used to detect the expression of HLA-G and CTLA-4 in exosomes.8.Exosomes tracer experiment:PKH67 was stained with exosomes,and then added to B cell culture medium to observe whether exosomes were engulfed by B cells.9.The exosomes were added to the lymphocyte medium depleted of Tfh cells for 48 hours,and the ratio of B cells(CD3-CD19+ cells)and plasma cells(CD3-CD19-CD38+cells)was measured by flow cytometry.The concentration of Ig was measured by ELISA.Results1.In the ABMR group,the expression of CTLA-4 on Tfh cell-derived exosomes was decreased,but there was no difference between HLA-G and non-ABMR groups;2.Tfh cell-derived exosomes can be engulfed by B cells;3,Tfh cell-derived exosomes in ABMR patients promote B cell proliferation and differentiation,compared with non-ABMR group,the proportion of B cells and plasma cells increased by 87.52%and 110.2%,respectively;4.Tfh cell-derived exosomes in ABMR patients promote plasma cell production of IgG and IgA,but have no significant effect on IgM production.Conclusions1.Tfh-derived exosomes can be engulfed by B cells;2.Tfh-derived exosomes from ABMR patients stimulate B cells to differentiate and mature,and can promote the production of IgG and IgA by plasma cells;3.The expression of CTLA-4 on Tfh cell-derived exosomes was decreased in ABMR group,but the expression of HLA-G was not different from that in non-ABMR group.It is speculated that exosomes may be an important carrier for CTLA-4 to exert immunosuppressive effects..Third partMethods1.Obtain peripheral blood from healthy volunteers,isolate PBMC,and sort mononuclear/macrophage cells using CD 14+magnetic beads;2.Mixed lymphocyte culture(MLC)with mitomycin C-treated glomerular endothelial cells and lymphocytes from the same volunteers;3.Replace the medium and continue to culture mononuclear/macrophage cells,add Netrin-1 and exosome inhibitor GW4869,and analyze the phenotype of mononuclear/macrophage and the expression of HLA-G protein by flow cytometry;4.After 48 hours of MLC,the stimulating factor was removed from the mononuclear/macrophage cells,and then the cells were added to the upper portion of the chamber,and glomerular endothelial cells were cultured in advance in the lower portion of the chamber.The lower endothelial cells were collected after 48 hours of co-cultivation;5.Flow cytometry was used to detect the expression of HLA-G protein and its receptors(CD152d,CD85j and CD85d),ICAM-1,VCAM-1 and E-selectin in endothelial cells;6.The polymer precipitation method kit separates the mononuclear/macrophage-derived exosomes,and the tracer test detects whether it is engulfed by endothelial cells;7.The exosomes were cultured in endothelial cell culture medium,and the expression of HLA-G protein was detected by flow cytometry.ResultsNetrin-1 promotes phenotypic transformation of mononuclear/macrophages and expression of HLA-G,and Netrin-1 stimulated mononuclear/macrophage promotes HLA-G and its receptors in endothelial cells via exosomal delivery Expression,mononuclear/macrophage significantly increased exogenous HLA-G expression after Netrin-1 stimulation;using antibody to seal HLA-G on exosomes significantly inhibited HLA of endothelial cells in the Netrin-1 group-G expression.Netrin-1 promotes targeted delivery of monocyte/macrophage-derived exosomes HLA-G to endothelial cells.Netrin-1 stimulated monocytes/macrophages inhibit the expression of pro-inflammatory molecules in endothelial cells.Conclusions1.MLC induces monocyte/macrophage to polarize M1 and enhance its HLA-G expression.The neurite-directed factor Netrin-1 partially reverses this polarization and further promotes HLA-G expression in monocytes/macrophages.;2.Mononuclear/macrophage targeted delivery of HLA-G to endothelial cells via exosomes,and the neurite targeting factor Netrin-1 promotes this mode of delivery.