The Effect And Mechanism Of PD-L1 Regulation In Mice Pulmonary Chronic GVHD

Objective:To establish a mouse lung cGVHD model and investigate the preventive effect and mechanism of PD-Ll on chronic lung cGVHD in mice.Methods:1.Mice:C57BL/6(H2b),B10.BR(H2k)and BALB/C mice were purchased from the National Cancer Institute(NCI).All mice were kept in sterile animal houses.2.In vitro test:B7H1-fc expression plasmid was constructed and amplified by escherichia coli,and then transfected into eukaryotic CHO cells.After isolation and purification,Westemblot was used to identify the expression of PD-L1.Through molecular exclusion chromatography(size exclusion chromatography)and combined with the test(Binding assay)to determine the purified protein activity and function.Mouse spleen CD4+T cells and CD8+T cells were isolated and purified.The proliferation and apoptosis of PD-L1 were studied in vitro.Annexin V and CD4+Foxp3+T cells were detected by flow cytometry.3.Build the donor C57BL/6 to B10.BR(H2k)lung chronic GVHD transplantation model in mice.Mice in eight hours before transplantation,given a lethal dose Cs137 total body irradiation(750 cGy TBI)and-3.-2d CTX120mg/kg I.P,and received T cells depleted BM,(TCD-BM)5×106+spleen cells(0.25-2.0×106).After transplantation,GVHD indicators such as hair loss,diarrhea,weight loss,survival,proteinuria,etc.After transplantation,Flexivent system(SCIREQ)instrument was used to detect changes in lung function,as well as histopathology of salivary glands,skin,lungs and liver.4.The mouse model of lung cGVHD-induced transplantation was established for in vivo experiments.The experimental group was injected with isolated and purified PD-L1 Ig,while the control group was injected with Rat Ig,with a dose of 400ug intraperitoneal injection(starting from the first day of transplantation to+5 days of daily pulse injection,and then injected 3 times a week until+28 days).Survival,proteinuria and other GVHD indicators were observed.Lymphocytes from spleen,peripheral lymph nodes,thymus,skin,liver and lung were collected and analyzed.1)Annexin V,BrdU,H2b,CD4,CD8,annexin V,and H2b,CD4,CD8 were used for staining of monocytes from recipient spleen and target organs.The number and proportion of Tcon,Thl,Th2 and Th17 cells in organ tissues were observed.The proliferation and apoptosis of infiltrated T cells and the expression of CD4+Foxp3+T cells were observed.In addition,T cell failure indicators such as PD-1,Tim-3 and IL7Ra of donor T cells were detected.2)Flexivent system was used to monitor and compare the changes and outcomes of lung function after transplantation.3)Mice were intraperitoneally injected with BCL/Luc+leukemia cells,and tumor cell amplification was detected by BLI to observe the effect of PD-L1 on GVL effect.5.Statistical analysis:all data were analyzed by GraphPad Software.The rank sum test or log-rank test was used to compare the clinical GVHD score and survival of different groups.The comparison of the two means was tested by t test.P<0.05 was considered statistically significant.Results:1.In vitro experiments showed that PD-L1 Ig significantly inhibited the proliferation of CD4+and CD8+T cells.PD-L1 Ig had no significant effect on apoptosis of CD4+T cells.In contrast,apoptosis of CD8+T cells was significantly inhibited in the PD-L1 Ig group.Meanwhile,PD-L1 can induce Foxp3+T cell proliferation.2.Our project successfully established a stable C57BL/6 to B10.BR(H2k)chronic lung GVHD transplantation model in mice.Mice in the TCD-BM 5 ×106+spleen cell(0.75×106)group showed chronic GVHD damage in skin,salivary glands,liver and lungs at about 40 days.Masson's trichrome staining suggested typical collagen deposition in lung tissues.The results of lung function test indicated that GVHD mice had significantly increased airway resistance and elasticity and significantly decreased compliance.Compared with the tcd-bm group,the proportion and yield of CD4+CD8+double-positive thymus cells in the TCD-BM+SPL 0.75m group were significantly reduced.However,immunofluorescence in the germinal center of the spleen showed that GC of the receptors in the TCD-BM+SPL group 0.75M/1.0M was seriously destroyed.3.PD-L1 Ig can alleviate the occurrence and severity of cGVHD through multiple mechanisms.1)Mice in the PD-L1 Ig group had better survival than those in the Control Ig group.Moreover,the severity of cGVHD in the target tissue of the PD-L1 Ig injection group was significantly lower than that of the control group.2)In the skin tissue,the expression level of IL-17 and IL-4 in the PD-L1 Ig injection group was close to that in the control group.However,the production of IFN-?+CD4+T,IL-17+CD4+T,and IL-4+CD4+T cells was significantly reduced compared with the control group.And,the expression level and production of IFN--? and IL-17 were significantly decreased in the PD-L1 Ig group.3)Compared with the control group,the yield of CD4+T cells in the donor group of PD-L1 Ig increased relatively in lymphoid tissues at 40 days and 60 days after transplantation,while the cGVHD target tissues in the skin and lungs decreased significantly.4)At 40 days after transplantation,the amplification of donor Tcon cells in the spleen and lung tissues of the injected group of PD-L1 Ig was significantly inhibited,and there was no difference with apoptosis.The proliferation and apoptosis of Tcon cells in liver were not affected.5)At 40 and 60 days after transplantation,there was no significant difference in the expression level of Tim-3 in CD4+T cells in the PD-L1 Ig group and the control group.However,the expression level of IL7R? in the donor CD4+T cells was significantly decreased in the PD-L1 Ig group,while the expression level of PD1 was significantly higher than that in the control group.6)At 60 days after transplantation,the production of Foxp3+CD4+T cells from the spleen,lung and skin of the mice injected with PD-L1 Ig was more than twice that of the control group(p<0.05),while there was no significant difference between the two groups in liver tissue.4.The results of mouse leukemia transplantation model showed that compared with the control group,the tumor cells in the PD-L1 injection group were also cleared within 20 days,and the long-term survival was up to 80 days.PD-L1 protein alleviates GVHD while retaining GVL effect.Conclusion1.C57BL/6 to B10.BR transplant model which was pretreated with TBI+CTX,and TCD BM combined with low-dose splenocyte infusion could stably induce the generation of pulmonary cGVHD.2.Early application of PD-L1 protein in transplantation can reduce the proliferation of Thl and Th17 cells in target organs.3.PD-L1 can induce Foxp3+CD4+T cell proliferation in lung tissues.thereby alleviating cGVHD.