| BackgroundAllograft kidney transplantation is still an effective method to treat end-stage renal disease.Since 1954 the American Murray has successfully performed kidney transplants between a pair of identical twins,It has been more than 60 years of development,and millions of uremia patients around the world have gained new life.In the early stages of renal transplantation,research on the rejection of transplanted kidneys has focused on T cell rejection.With the development of microsurgical technology and the wide application of immunosuppressive drugs in clinical practice,the incidence of T cell mediated rejection has been significantly reduced,and the early survival rate of transplanted kidneys has also been greatly improved.However,the long-term survival of transplanted kidney is still not ideal.In recent years,Antibody mediated rejection(AMR)is considered to be the key reason for long-term survival of transplanted kidney.At present,proteasome inhibitors or monoclonal antibodies are used clinically to inhibit or eliminate antibody-producing cellular functions,but the side effects are large,and the effect is not very satisfactory.Donor-Specific Antibody(DSA)is the main antibody involved in mediating AMR,and follicular helper T cells(Tfh)play a key role in antibody production.It is a class with strong adjuvants.Tfh cells are a class of CD4~+T cells with a strong auxiliary capacity,they can produce somatic high-frequency mutations,antibody class switching,and affinity maturation by secreting IL-21 and other helper B cells,and ultimately produce high-affinity plasma cells and long-term protective memory B cells.Recent studies have found that Tfh cells and their subtypes play an important role in the occurrence and development of AMR after renal transplantation.Targeting Tfh cells may be the main research direction of AMR therapy in the future.Mesenchymal stem cells(MSC),a precursor stem cell derived from bone marrow,umbilical cord or fat,is immunomodulatory,and has now entered clinical or clinical experiments to induce immune tolerance after transplantation.In recent years,it has been found that MSC can inhibit the differentiation of Tfh cells and thus achieve the purpose of treating autoimmune diseases mediated by B cells.However,whether MSC has the function of AMR after renal transplantation and which source of MSC has not been reported at home and abroad.ObjectiveTo determine the changes in the proportion of Tfh and its subtypes in peripheral blood of AMR patients after renal transplantation.To study the immunomodulatory effects and mechanisms of different sources of MSC.To explore the effect and effect of MSC on Tfh and subtypes of different sources,and to provide a theoretical basis for the treatment of AMR after renal transplantation.Methods1.66 cases of renal transplant patients in our hospital were included in this study.According to the 2013 edition of Banff conference,the diagnostic criteria of AMR will be divided into normal group(n=20),AMR group(n=20)and non AMR CRAD group(n=26).Peripheral blood mononuclear cells(PBMCs)were extracted from the above patients and the corresponding CD4,CXCR5,CXCR3,CCR6 and Foxp3 were labeled.Flow cytometry were used to detect Tfh(CD4~+C XCR5~+),Tfh1(CD4~+CXCR5~+CXCR3~+CCR6~-),Tfh2(CD4~+CXCR5~+CXCR3~-CCR6~-),Tfh17(CD4~+CXCR5~+CXCR3~-CCR6~+)andTfr(CD4~+CXCR5~+Foxp3~+)cell.2.Bone marrow-derived mesenchymal stem cells(BM-MSCs),umbilical cord mesenchymal stem cells(UC-MSCs),and fat-derived MSCs(Fat mesenchymal s tem cells,Fat-MSCs)were recovered,passage cultured,phenotype Identified.The expression of CD34,CD44 surface markers and HLA-G was detected by flow cytometry.The Enzyme-Linked Immunosorbent Assays(ELISA)method was used to detect the secretion of the immunotolerant molecule 2,3-dioxygenase(IDO) and interleukin from different sources of MSCs.10(Interleukin-10,IL-10)and transforming growth factor-?(TGF-?)ability.3.Different sources of MSC were co cultured with PBMC in peripheral blood of AMR patients after renal transplantation,respectively.Negative controls were set up.After 48 hours,centrifugation was performed.Tfh cells stimulated by MSCs and supernatants were collected,and labeled with flow cytometry.The proportion of Tfh cells was detected by flow cytometry,and the concentration of in terleukin-21(IL-21)in the supernatant was detected by ELISA.To further investig ate the mechanism of differentiation of Tfh by MSCs,1-methyltryptophan(1-MT),IL-10 antibody,and HLA-G antibody were added to the co-culture system.After 48 hours of culture,the flow cytometry was used to detect the change of Tfh ratio.Results1.The proportion of Tfh cells in peripheral blood of CRAD patients was not significantly different from that in normal group after renal transplantation.The proportion of Tfh2 cells and Tfh17 cells in lymphocytes of CRAD patients was significantly higher than that in normal group after renal transplantation(P<0.05).CRAD patients were further divided into AMR group and non AMR group.There was no significant difference in Tfh cells and Tfh1 cells between the two groups,but the proportion of IL-21 Tfh(Tfh2+Tfh17cell)cell subtype in AMR group was significantly higher than that in non AMR group.2.MSC from different sources all overexpressed CD44 but did not express CD34 of hematopoietic cells,indicating that MSC used in this experiment had high purity and could be used for subsequent experiments.The average fluorescenc e intensity of BM-MSC expressed in HLA-G was 96,which was 39.13%and 31.51%higher than that of UC-MSC and Fat-MSC,respectively.The results of ELISA showed that the ability of BM-MSC to secrete IDO,IL-10 and TGF-?cytokine was higher than that of UC-MSC and Fat-MSC.3.In the co-culture system,the percentage of Tfh cells in the peripheral blood of AMR patients after renal transplantation decreased by 43.4%?15.4% and 14.3%,respectively,compared with the control group(P<0.05).Compared with UC-MSC and Fat-MSC groups,the proportion of Tfh cells in BM-MSCs decreased by 33.11%and 34.0%,respectively,with statistically significant differences(P<0.05).The results of ELISA assay for the concentration of IL-21 in the supernatant of the culture system showed that the concentrations of IL-21 in the BM-MSC,UC-MSC and Fat-MSC groups were reduced by 56.9%?39.7%and 34.5%,resp ectively,compared to the control group(P<0.05).Compared with UC-MSC and Fat-MSC,the concentration of IL-21 in BM-MSC group decreased by 28.6%and34.2%respectively(P<0.05).The blockade of IDO inhibitor 1-MT,IL-10 anti body and HLA-G antibody can partially weaken the inhibitory effect of BM MSCs on Tfh cells.Conclusions1.Compared with non AMR CRAD patients,the proportion of Tfh cells and Tfh1 subtypes in AMR patients did not change significantly,while the proportion of Tfh cell subtypes(Tfh2+Tfh17 cells)increased significantly,so Tfh and its subtypes were correlated with the occurrence of AMR after kidney transplantation.2.The BM-MSC,UC-MSC and Fat-MSC used in our experiments are highly expressed CD44,but do not express CD34 of hematopoietic cells.BM-MSC has the strongest ability to induce immune tolerance.3.BM-MSC,UC-MSC and Fat-MSC can inhibit Tfh cell differentiation and IL-21 secretion,BM-MSC inhibition is the most significant.IDO,IL-10 and HL A-G may be the main mechanisms of this inhibition. |
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