γδ T Cells In Kidney Transplantation Recipients And Immunity Rejection Mechanism Of Mouse Skin Transplantation

BackgroundThe function of the immune system of kidney transplantation recipient(KTR)lacks effective monitoring methods.Either too low or too high doses of immunosuppressive agents can lead to graft loss.Therefore,it is very important to explore the immune status of KTR.Cytometry by Time-of-Flight(CyTOF)uses metal isotope labeled antibodies to overcome the limitation of overlapping emission spectrum signals in the traditional flow chamber,detected more than 40 parameters in a cell simultaneously to accurately analyze cell subsets.In this study,CyTOF was used to identify specific immune cell subsets in KTR,and then an animal model of immune rejection was constructed to further study its mechanism.Immunosuppressive therapy after organ transplantation may lead to various complications.Regulating gut microbiota to induce the immune tolerance of the host may provide a new approach.We constructed an animal model of skin transplantation in mice.The gut microbiota and immune microenvironment of skin-transplanted mice were influenced further by sodium acetate solution.We analyzed the changes of intestinal immune cells in the mice transplantation model before and after the intervention,and further studied the immune regulation mechanism of gut microbiota.ObjectivesCyTOF was used to analyze the immune cell subsets and status of KTR before and after immunosuppressive agents,and screen the target immune cells subsets for the further research.Investigate the mechanism of gut microbiota regulate the immune rejection of skin transplantation through γδ T cells in intestine.Methods1.Peripheral blood samples were collected from patients before kidney transplantation and after 2 weeks after receiving immunosuppressant.2.CyTOF analysis was performed on peripheral blood samples.3.Use Cytobank(https://www.cytobank.org)to analyze.fcs data.4.Use t-SNE algorithm to generate vi-SNE graph of immune cell subsets.SPADE algorithm was used to re-cluster the above immune cells and identify cell subsets.5.Cell subsets that could be used to evaluate the immune status of patients were screened by ROC analysis.6.Mice skin transplantation model was constructed.7.Skin graft survival was evaluated by skin graft quantitative score,HE and Mason pathological analysis.8.16S rRNA gene sequencing and bioinformation analysis were used to analyze the status of gut microbiota in mice.9.Intraperitoneal injection of SCFAs sodium acetate was used to intervene the gut microbiota of skin transplantation mice.10.Alpha diversity analysis,community composition analysis,PCoA analysis,and LEfSe analysis were conducted to analyzed the differences in the species structure of each level of microbial classification based on the results of bacterial microbiota sequencing.11.RNA-seq sequencing was used to analyze the transcriptome expression of intestinal tissues of mice,and CIBERSORT was used to analyze the sequencing data of intestinal lymphocytes.12.Similar mice RNA-seq sequencing data were downloaded from the GEO,and the intestinal lymphatic infiltration was analyzed by CIBERSORT and compared with our data.13.The contents of γδ T cells and Treg cells in peripheral blood of mice were analyze by flow cytometry.14.Hamster anti-mice TCR-γδ monoclonal antibody was injected intraperitoneally to deplete γδ T cells in mice.15.The concentration of IL-17,IL-4,IL10,TNFα and IFN-γ in peripheral blood of mice 14 days after operation were tested by ELISA.16.Statistical analysis.ResultsThe results of CyTOF showed that the proportions of seven types of immune cells were different in each sample.We detected a total of 363,342 live single immune cells.Re-clustered NK cells/γδ T cells showed that CD57+NK cells were significantly down-regulated and tolerance NK cells were significantly increased in the post-treatment group.Heat-maps showed that the expression patterns of molecular markers in peripheral blood NK/γδ T cell subsets were different in pre-treatment and post-treatment group,such as CCR6、CXCR5、IL-2R.ROC analysis showed that CD57+NK cells and γδ T cells were the best predictors of immunosuppressant efficacy pre-treatment and post-treatment(AUC 0.88 and 0.75).Re-cluster analysis of CD4+ T cells showed that the central memory CD4+T cells and auxiliary follicular CD4+T cells were significantly reduced in the post-treatment group.Effector CD8+T cells,effector memory CD8+T cells and naive CD8+ T cells were significantly down-regulated in the post-treatment group.The B cells were re-clustered into 5 subsets.After treatment,the content of memory B cells was significantly up-regulated and the expression pattern of B cell subsets was significantly changed.DCs cells were re-cluster into 2 subsets.The relative abundance of DC subsets in peripheral blood of patients before and after treatment not change significantly.Skin graft rejection was slight in SA group.HE and Masson’s trichrome staining pathological analysis showed that the morphology of grafts in SA group was normal and the structure was clear.Masson showed that collagen fibrosis infiltration in SA group was lighter than that in Allo group.After skin transplantation,the abundance of SA group,Allo group and Auto group changed at phylum level and genus level.The relative abundance of lactobacillus in SA group was lower than that in NC group,Allo group and Auto group.Further analysis of the overall structure of gut microbiota in SA group and NC group showed that there was significant difference in the overall species composition of gut microbiota between groups.At the phylum level,the differences between groups showed that the Cyanobacteria had obvious differences.Compared between the two groups,Proteobacteria in SA group was lower than that in Allo and Auto groups,suggesting that SA interferes with the abundance of Proteobacteria.The abundance of Cyanobacteria in SA group was lower than that in NC group,and in Allo group was lower than that in NC group.There was no difference in species between Auto and NC,suggesting that Cyanobacteria may be affected by transplantation related immune factors.The small intestine tissues of mice were sequenced by RNA-sequencing,and CIBERSOT was used to evaluate the infiltration of immune cells in the small intestine of mice.The results showed that γδ T cells in sodium acetate group were higher than those in other groups.The percentage of IL17+/TCR-γδ+cells in SA group was lower than that in Allo group.The content of IL17+/TCR-γδ+cells in Allo group was significantly higher than that in Auto group,indicating that the composition ratio of IL17+/TCR-γδ+cells increased after skin transplantation,and SA could reduce the content of IL17+γδ T cells in peripheral blood to a certain extent.After intraperitoneal injection of hamster anti-mice TCR-γδ monoclonal antibody,the protective effect of sodium acetate on skin grafts was absent.The expression of IL-17A in peripheral blood of mice was detected by ELISA,and the level of IL-17A in SA group was lower than that in Allo group and Auto group.ConclusionBefore and after immunosuppressive agents in KTR,γδ T cells and other immune cell subsets were significantly changed.In the early stage of receiving immunosuppressive agents,the proportion of activated immune cell subsets(such as central memory CD4+T cells)decreased and the proportion of tolerant immune cells(such as tolerant NK cells)increased.The structure and abundance of gut microbiota changed after skin transplantation.Short chain fatty acid sodium acetate can regulate the abundance and composition of intestinal microorganisms after transplantation,inhibit the differentiation of intestinal γδ T cells into IL-17+γδ T subsets,reduce the number of peripheral blood IL-17+γδ T cells and reduce the secretion of IL-17A,and then inhibit the occurrence of skin rejection.