Role Of Tfh In The Formation Of De Novo DSA After Renal Transplantation

Objective: To Analysis the risk factors of de novo DSA formation after renal transplantation.To investigate the role of Tfh and the imbalance of Tfh/Treg in the formation of de novo DSA after renal transplantation.Methods: A total of 218 renal transplant recipients were enrolled in our hospital from January 1,2013 to December 31,2013,and followed up for 3 year.The serum DSA and C1q+DSA were measured at 3,6,12,24 and 36 months after transplantation by Luminex.To Analysis of the incidence rate,time of onset,the characteristics of DSA and C1q+DSA,and survival of graft.The clinical data of DSA positive and negative group were compared by single and multivariate factor method.According to the de novo DSA,the patients were divided into DSA positive group(n=30),DSA negative group(n=30)and healthy control group(n=30).The percentage of lymphocyte subsets Tfh cells,Treg cells,CD19+CD38+B cells in peripheral blood were measured by flow cytometry.The expression levels of transcription factors Bcl-6 m RNA,Foxp3 m RNA and ROR-γt m RNA in peripheral blood mononuclear cells were detected by real-time quantitative polymerase chain reaction(RT-PCR).The levels of IL-2,IL-6,IL-10,IL-17,IL-21 and BAFF were detected by ELISA in the three groups of patients.To compare the differences between Tfh cells,Treg cells and transcription factors and cytokines in the three groups,and to analyze their role in the development of DSA.Result: 1.The average incidence of DSA was 18.7±10.9 months.The cumulative incidence of postoperative 1,2 and 3 years were 9.2%,12.9% and 14.2%.2.There were 39 DSA positive loci in the 31 DSA-positive patients,including 4 classⅠantibodies.There were 35 class Ⅱ antibodies,accounting for 92.2%,among which there was 30 HLA DQ(76.9%).3.The mean fluorescence intensity(MFI)of C1q+DSA was higher than C1q-DSA(8478 ± 3676 vs 4943 ± 2114,p = 0.003).71.4% C1q+DSA patients developed AMR.There was a significant difference between the two groups(P = 0.007 χ2 = 7.235).4.Binary Logistic regression analysis show that DQ mismatch(OR: 3.870),cyclosporine A(OR: 3.330)and low CNI drug concentration(OR: 2.580)were independent risk factors of de novo DSA.5.The proportion of Tfh in DSA positive group was higher than DSA negative group(8.95 ± 2.01% vs 5.02 ± 1.43% p = 0.000).The proportion of Treg in DSA positive group was significantly lower than DSA negative group(2.69 ± 0.77% vs 5.95 ± 1.27% P <0.01).The percentage of CD19+CD38+B cells in the DSA positive group was significantly higher than DSA negative group(48.9 ± 7.1% vs 34.5 ± 6.47% P <0.01).6.Tfh / Treg ratio was significantly higher in DSA positive group than that in DSA negative group(3.59 ± 1.41 vs 0.909 ± 0.414 P <0.001).7.The expression of BCL-6 m RNA in DSA positive group was significantly higher than DSA negative group(2.23 ± 0.40 vs 1.90 ± 0.47,P = 0.017).The expression of Foxp3 m RNA in DSA negative group was significantly higher than DSA positive group(1.12 ± 0.17 vs 0.94 ± 1.90 P = 0.003).The expression of Ro Rγt m RNA in DSA positive group was significantly higher than DSA negative group(2.98 ± 0.30 vs 2.65 ± 0.26 P = 0.000).8.The levels of IL-6,IL-17,IL-21 and BAFF in DSA-positive group were significantly higher than those in DSA-negative group,while the expression of IL-10 in DSA-positive group was lower than that in DSA-negative group.Conclusion: 1.DQ mismatch,cyclosporine A and low CNI drug concentration were independent risk factors of de novo DSA.2.The study shows that the peripheral blood transcription factor Bcl-6 increased,the number of Tfh cells increased,and increased IL-21 expression may be the cause of de novo DSA formation after renal transplantation.3.The imbalance of Tfh/Treg may also be the cause of DSA formation.4.IL-6,IL-17,IL-21,BAFF may be involved in the production of DSA.