Immune Tolerance Induced By Treg-exosomes In Transplanted Liver

[Objective]The optimized extraction method was used to isolate and activate Treg cells with high quality and high proliferation rate.It is confirmed that Treg cells can inhibitor CD8+T cell proliferation through non-contact as well as direct contact.It has also shown that the inhibitorion of CD8+T by Treg cells can be realized by exosomes.Treg and its exosomes can target LAT1(Slc7a5)via microRNA-194-1-3p to regulate mTOR signaling pathway and affect amino acid transport,thus inhibitoring CD8+T cell proliferation.The SD rat donor and Wistar rat orthotopic liver transplantation model was established.The protective effect of Treg-exosomes on allogeneic liver transplantation was confirmed by in vivo experiments.[Methods]1.Effects of Treg cells on the function and proliferation of CD8+T cell:The Spleens of BALB/C mice were taken and Treg cells were separated by magnetic beads adsorption and flow separation respectively.The isolated Treg cells were activated and amplified by the combined action of CD3,CD28,IL-2 monoclonal antibody and Rapamycin.CD8+T cells were adsorbed and sorted by magnetic beads,and CD8+T cells were activated by CD3 and IL-2 monoclonal antibodies.Treg cells and CD8+T cells were co-cultured by Transwell system and directly.CCK-8 was used to detect the proliferation of CD8+T cells at various time points,and PI staining was used to analyze the changes of CD8+T cell cycle.2.Effects of Treg-exosomes on the function and proliferation of CD8+T cell:Treg cell supernatants were collected,exosomes were extracted by exosomes extraction kit and ultra-high speed differential centrifugation respectively,and the expression of CD63 was detected by western blot to determine the content of Exosomes in Treg cell supernatants of different incubation time and different extraction methods.Exosome was identified by electron microscope analysis,particle size analysis and western blot to detect the expression of CD63 and LAPMP-1 proteins.CD63 lentivirus labeled with green fluorescent protein(GFP)was constructed to infect Treg cells,and the infection efficiency was detected under fluorescence microscope.Treg-exosomes with fluorescence was extracted to infect activated CD8+T cells,and the infection efficiency was detected under confocal fluorescence microscope.Intervention cells were divided into the following groups:Control Group A:normally-cultured CD8+T cells;Co-culture Group B:Treg cells and CD8+T cells group;High-dose Exosomes Group C:40 ug Treg cell-derived exosomes were added into CD8+T cells;Low-dose Exosomes Group D:adding 10 ug Treg cell-derived exosomes into CD8+T cells;inhibitoror Treg Group E:activated Treg cells were co-cultured with CD8+T cells after adding GW4869 inhibitoror for 2 hours;inhibitoror Exosomes Group F:40 ug Treg cell-derived exosomes were added into CD8+T cells while GW4869 was being added.CCK8 was used to detect the proliferation of CD8+T cells and the changes of CD8+T cell cycle in the above 6 groups at different time points.Meanwhile,the expression of CD8+T cell marker cytokines IFNy and Perforin were detected by QPCR,Western blotting and flow cytometry.3.Treg and exosomes can regulate mTOR signaling pathway through microRNA-194-1-3p targeting LAT1(Slc7a5):Treg cells and CD8+T cells after exosomes intervention are collected,microRNA chips are used to detect and screen differential microRNA,and QPCR verification,target gene prediction,target gene GO and KEGG analysis are performed.Bioinformatics prediction website predicts the targeted relationship between microRNA-194-1-3p and LAT1(Slc7a5),which is confirmed by double luciferase reporter gene experiment.mimics and inhibitor transfected CD8+T cells QPCR detected changes of mmu-miR-194-1-3p and Slc7a5 Genes mmu-miR-194-1-3p mimics and inhibitor interfered CD8+T cells detected CD8+T cells proliferation.The expression of target genes Slc7a5 and p-mTOR protein was detected by western blot after mimics and inhibitor of mmu-miR-194-1-3p intervened CD8+T cells.4.Protective effects of Treg-exosomes on allogeneic liver transplantation:In this experiment,Wistar rat allogeneic liver transplantation model was established by Kamada's "two cuff" method.The model was divided into two groups,Group A and Group B.Rapamycin was given on the 4th day after the operation,and Group A was injected with exosomes extracted from Treg cells on the 1st,3rd and 5th days.Plasma was collected from tail vein on the 3rd,11th and 18th 3day after the operation to examine ALT and TBIL levels and observe survival time.[Results]1.inhibitorion of Treg Cells on the Function and Proliferation CD8+T Cell:Flow Cytometry Identified CD4+CD25+Treg Positive Ratio of Flow Sorting and Magnetic Bead Adsorption Sorting Treg Cells were 93.2%and 87%respectively;The ratio of CD8+T cells were 91.44%respectively.CCK-8 test results after co-culture showed that after Treg cells and CD8+T cells were co-cultured,the proliferation of CD8+T decreased significantly(P<0.05).The proliferation cycle test results after co-culturing showed that the CD8+T fine ratio in G0/G1 phase increased significantly,and there were fewer enriched cells in S phase and G2/M phase.2.Treg-exosomes inhibitor the function and proliferation of CD8+T cell:(1)Western Blot results show that the expression of Exosome CD63 extracted by polymerization precipitation method is significantly higher than that by ultracentrifugation method.Exosomes are round or quasi-round in diameter distribution in the range of 30-150 nm observed under transmission electron microscope.Western Blot results showed that CD63 and LAPMP-1 proteins in exosomes were all positive.Fluorescence microscope observation showed that lentivirus infected Treg cells showed that the fluorescence expression content reached more than 70%after 48 hours.Confocal fluorescence microscope can observe Treg-exosomes infected CD8+T cells.(2)After co-culture of Treg-exosomes and CD8+T cells,the proliferation of CD8+T cells detected by CCK-8 showed that after co-culturing of Treg-exosomes and activated CD8+T cells,the proliferation of CD8+T cells was significantly reduced,and the inhibitorory effect was more obvious in high-dose Exosomes group.However,GW4869 was added to Treg in advance to inhibitor the secretion of exosomes,and the results showed that Treg cells'inhibitorory effect on CD8+T cells was weakened.Flow cytometry was used to detect CD8+T cell cycle and similar results were obtained.(3)QPCR,Western blotting and flow cytometry were used to detect perforin and IFN-y.The results showed that the expression of IFN-y and Perforin protein was significantly down-regulated after Treg cells and high-dose exosomes intervened CD8+T cells(P<0.05).In contrast,the expression of IFN-y and Perforin protein didn't change significantly after Treg cells were added GW4869.3.Treg and exosomes target LAT1(Slc7a5)through microRNA-194-1-3p to regulate mTOR signaling pathway:Gene chip and QPCR confirm that there are 13 microRNA that Treg cells and exosomes have consistent changes.The double luciferase reporter gene experiment proves that mmu-miR-194-1-3p is combined with Slc7a5 gene 3'-UTR in a targeted way.Cell lines with high expression of miR-194-1-3p and low expression of miR-194-1-3p constructed by cell transfection were used to detect changes in mRNA and protein levels.The results showed that miR-194-1-3p was directly and negatively correlated with the expression of Slc7a5 and p-mTOR in CD8+T cells.High expression of miR-194-1-3p can significantly inhibitor the proliferation of CD8+T cells,with marked increase in G0/G1,G2/M phase and significant decrease in S phase.4.Protective effects of Treg-exosomes on allogeneic liver transplantation:on the 3rd day after the operation,ALT and TBIL levels in Group A were lower than those in Group B,but the difference was not significant.On the 11th day,ALT and TBIL levels in both groups were lower,but they were more obvious in Group A,with significant difference between the two groups(P<0.05).On the 18th day,the levels of ALT and TBIL in the two groups continued to decrease,but it was still more obvious in Group A(P<0.05),with significant difference between the two groups.The survival time of rats injected with Treg-exosomes was significantly prolonged.The Control Group(Group B)had a median survival time of 28 days after the transplantation,while the Treg-exosomes treatment group(Group A)had a significantly longer survival time of 90 days.[Conclusion]1.Treg cells obtained by magnetic beads adsorption and sorting can proliferate well under the stimulation of CD3/CD28 monoclonal antibody,rapamycin and IL-2.Treg has obvious inhibitorory effects on the proliferation of CD8+T cells.Treg can inhibitor the proliferation of CD8+T cells either through direct contact between cells or through non-contact.2.More Treg-exosomes can be extracted by ExoQuick precipitation method,and exosomes concentration obtained from Treg cell supernatant collected at 72h is higher.Treg cells can be transfected with pCT-CD63-GFP lentivirus and labeled with exosomes secreted by Treg cells,and the exosomes secreted by treg can be visually observed to be transferred and integrated into CD8+T cells.Treg cells can affect the distribution of cell cycle of CD8+T cells through secreted exosomes,inhibitor the proliferation of CD8+T cells,inhibitor their secretion of perforin and IFN-y.3.Treg cells and exosomes secreted by treg cells can exert their inhibitorory effect on through miR-194-1-3p.MiR-194-1-3p may inhibitor mTOR signaling pathway and affect amino acid transport through targeted down-regulation of LAT1(Slc7a5),thus inhibitoring CD8+T cell proliferation.4.After Treg cell-derived exosomes were injected into allogeneic liver transplantation rats,the survival time of transplanted rats was significantly prolonged.Treg-exosomes needs a certain reaction time to exert immunosuppressive effects in rats,and has a tendency to expand further with time.