| ObjectiveMyeloid-derived suppressor cells(MDSCs)are important immunoregulatory cell populations that have a significant inhibitory effect on T cell-mediated immune responses.In addition to their development in the process of cancer has a negative regulatory role,but also on transplantation and autoimmune has a strong regulatory role.CD11 b + Gr1 + medullary inhibitory cells are capable of producing allograft resistance in mice and humans.However,MDSCs inducible factors,detailed phenotypes and functional molecules are significantly different in a variety of transplant models.Their therapeutic effect in chronic allograft rejection is unclear.In this study,we tried to establish the model of sepsis induced by lipopolysaccharide(LPS).Immunohistochemistry,flow cytometry and immunomagnetic sorting were used to isolate MDSCs.The number of cells was determined,and the Phenotype and cellular function were also analyzed.MDSCs were transferred to allogeneic corneal transplant recipients by tail vein injection to observe the survival of the grafts after corneal transplantation,and to explore the role of MDSCs in corneal transplantation.Methods1.A 6-8 week male C57 BL / 6(H-2b)mice were randomly divided into control group(PBS group)and experimental group(LPS treatment group).The experiment was repeated twice.2.5mg / kg LPS intraperitoneally injected into C57 BL / 6 mice once daily for 7 days.The same dose was given to the control group by intraperitoneal injection of the same dose of PBS.Daily observation of vital signs,drawing life curve,the he mice were sacrificed after 7 days.2.Preparation of mouse spleen,bone marrow and peripheral blood monocyte suspension,FITC-CD11 b antibody,PE-CY5-Gr-1 antibody was added to 50 μl of each group of single cell suspension,to seperate MDSCs by FITC,PE / CY5 access ring door,flow cytometry software analysis,mapping.0.4 ml of PBS and 0.1 ml of anti-Gr-1 biotin were added to the cell pellet of the above prepared single cell suspension by centrifugation to supernatant,and the reaction was carried out at 4 ° C for 10 minutes.Centrifuge,discard the supernatant,resuspend in 2ml PBS,shake well.CD11 b + Gr-1 MDSCs were sorted out by single cell suspension into MDSCs immunomagnetic beads sorting column.The purity of MDSCs was detected by flow cytometry.CD4 + T cell immuno-magnetic beads were sorted by the same method,and the purity of CD4 + T cells was detected by flow cytometry.3.Separated CD11 b + cells were added to PBS buffer to adjust the concentration to 1 x 106 / ml,10% FITC-labeled Ly6 C,CD40,CD80,CD86,CD273,CD275,TLR2,TLR4,MHC-II antibody were added to the prepared cells suspension,each group was provided with the same antibody type of control group,room temperature incubation for 15 minutes.The antibody was washed by centrifugation with PBS buffer and detected by flow cytometry.The experimental data were analyzed statistically.4.After isolation of the CD11 b + cells,the final concentration of 1 mg / ml FITC-bound OVA(OVA-FITC),or the same proportion of CD4 + T cells and CD11 b + T cells was added and then subjected to flow cytometry.CD11 b + cells isolated from LPS induced mouse bone marrow were co-cultured with CYSE-stained mouse spleen CD4 + T cells.T cells were seeded in 96-well plates at a density of 1×105 / CD4 + T cells and CD11 b + cells were inoculated at 1: 1,1: 2,1: 4 and 1: 8 ratio to detect the inhibitory effect of CD11 b + cells on CD4 + T lymphocyte proliferation.CD4 + T cells stained with CFSE were not positive for CD11 b + cells,and 1 μl of murine anti-CD3 / 28 proliferated beads was added to each well to stimulate cells with CD3 / 28 proliferation-free beads as negative control.Flow cytometry was used to detect the CFSE fluorescence intensity of each cell subgroup and its different proportion of CD4 + T cells to reflect its proliferation.Flowjo software was used to calculate the inhibition rate.5.The mice were randomly divided into experimental group and control group(n = 10).Immediately after penetrating keratoplasty,mice were injected with 1×106 LPS-induced mouse CD11 b + Gr1 + cells,and the control group was injected with the same amount of PBS.The corneal grafts were observed daily by slit lamp microscopy.The graft survival rate of each group was observed for more than 8 weeks.According to corneal transplantation flap,edema,neovascularization three indicators were scored.When the scoring score of 6 or 6 or more,or a plate opacity score reached 3,that is considered as the occurrence of immune rejection.Graft survival rate was recorded by mapping Kaplan-Meier survival curves,and log-rank analysis was used between groups.Results1.In this study,we successfully replicated the model of sepsis mice by intraperitoneal injection of the appropriate dose(2.5mg / kg)LPS continuously,Mice began to develop a morbid response after injection of LPS for 12 hours,with a peak of mortality rate in 3-4 days and a case fatality rate of about 30% at 7 days.PBS mice were not seen within 7 days of death.The expression of CD11 b + Gr1 + cells in the LPS-induced model was measured on the 8th day,and the CD11 b + Gr1 + cells in the spleen,blood and bone marrow were increased.The number of CD11 b + Gr1 + cells in the spleen of the control group increased by 2.9%,compared with 15.8% in the LPS-induced mice group.Similarly,LPS induced increased CD11 b + Gr1 + cells in mouse bone marrow from 5.4% to 29.4% and 0.3% in blood to 9.3%.These results showed a significant increase in CD11 b + Gr1 + cells in spleen,blood and bone marrow of sepsis mice.The largest increase is in bone marrow.2.CD11 b + Gr1 + cell phenotype: Flow cytometry was used to detect the expression of different cell surface markers.The expressions of CD80 / CD86,CD40 and PD1 L / PD-1 and TLR4 were similar to those of control group,but the expressions of Ly6 C,TLR2 and MHC-Ⅱ were significantly enhanced in LPS-induced CD11 b + Gr1 + cell surface.3.The co-culture of CD11 b + cells with CD4 + T cells and OVA showed that more than 98% of CD11 b + cells responded to soluble Ag-OVA after 6 hours,whereas 67% to 72% of CD4 + T cells were observed to have reaction after 12 hours and 24 hours.When with CD11 b + / T cells ratio of 1: 2,CD4 + T cell proliferation inhibition was 52%.When the ratio was 1: 8,the inhibition rate of LPS-induced mice was 30%,while the control group did not observe any inhibitory effect.4.The mice were injected with 1×106 LPS-induced CD11 b + Gr1 + cells in the tail vein of mice immediately after the operation of penetrating keratoplasty.The survival status of the mouse cornea graft was observed and the whole body condition of the mice was observed.No significant abnormal reactions were observed in the whole body of mice,indicating that LPS-induced CD11 b + cell adoptive transfer was safe.The graft survial rate of CD11 b + cell treatment group was significantly higher than the control group(P = 0.02).The mean graft survival time of the CD11 b + cell treated group was 21.4 days,compared with 10.9 days in the control group.ConclusionOur experimental results show that CD11 b + Gr1-1 + cells are significantly increased in the spleen,blood and bone marrow after LPS induction,and can inhibit activated CD4 + T cells.High expression of TLR2 in MDSCs seperated from bone marrow indicates phenotypic differences between endotoxin-infected and non-endotoxin-infected models.Lipopolysaccharide-induced CD11 b + cells can play a role as inhibitory APCs,suppress CD4+ T cells proliferation and improve corneal allograft survival.Predicting it can be used for clinical cell transfer therapy for transplant rejection in future. |
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