The Experimental Study Of PD-L1/PD-1 Pathway On MtDNA-Stimulated Macrophages And The Research Of It’s Inhibition Effect On T Cells

Objective:1.To observe the influence of PD-L1 phenotype mourse peritoneal macrophages stimulated by mt DNA, and to determine whether mt DNA can up-regulated the PD-L1 of macrophages expression.2.PD-L1 changes observed macrophages stimulated by mt DNA,to observe macrophages PD-L1 expression and TLR9-p38 signaling pathway.3.To investigate the effects of blockade of PD-L1/PD-1 pathways on the cocultivation of macrophages stimulated by mt DNA and T cells.Methods:1.Flow cytometry analysis of PD-L1 expression on macrophages after incubation for 24 h with medium only(control)、5ug/mlmt DNA and 10ug/ml mt DNA respectively;Additionally, PD-L1 expression on mouse peritoneal macrophages with 10ug/ml mt DNA-stimulated by flow cytometry after culturation for 0、6、12、24、48 and 72 h.. All data represent one typical result out of four independent experiments.2. FACS analysis of PD-L1 expression on mouse peritoneal macrophages after 24 h of incubation with 10ug/ml mt DNA with or without 1-hour pretreatment with signal inhibitors, 20 u MSN50(NF-k B) 、 20 u M UO126(MEK1/2), 10 u M SB203580(p38MAPK) and 25 u M LY294002(PI3K). Western blotting analysis of p38 phosphorylation in macrophages before and after activation with 10ug/ml mt DNA with or without 30 min pretreatment with TLR9 inhibitors chloroquine(CQ) for indicatedperiods of time.3. Allogeneic CD4+T cells were isolated and cocultured with mouse peritoneal macrophages(for 24 h with 10ug/ml mt DNA) in the presence or the absence of Ig G isotype control, anti-PD-L1 m Ab. The level of IFN-γin supernatants was determined by ELISA after 3d. After 48 h, the MTT proliferative response of CD4+T cell. All data represent one typical result out of experiments.and expressed as mean±SD.Result:1.Along with the increasing of concentration of mt DNA,PD-L1 upregulation on macrophages;The same concentration of mt DNA, expression of PD-L1 with time increased, and reached the peak at 24 hours for 48 hours, then 72 hours decreased.2.SB203580 inhibits the expression of PD-L1 on macrophages by mt DNA-stimilated; 10ug/mlmt DNA group increased expression of P-P38 protein,with inhibiting by CQ(TLR9 inhibition).3.Blockage of PD-L1 increased the amount of T-cells-derived IFN-g and the proliferation of T Cells in those cocultures.Conclusion:1.Upregulation of PD-L1 expression on macrophages by mt DNA;2.PD-L1 expression of stimulate-mt DNA in macrophages is TLR9- p38 MAPK dependent;3.mt DNA-triggered PD-L1 expression inhibits IFN-γ release and T cell proliferation in macrophages with CD4+T cells. Anti-PD-L1 acts to reverse inhibition of T cell and can induce T cell proliferation and restore cytokine production.