The Role Of PD-1/PD-L1 In The Immunological Mechanism Of Primary Biliary Cholangitis

Objective:To investigate the role of programmed death death-1(PD-1)and its ligand(PD-1 ligand 1,PD-L1)in the immunological mechanism of primary biliary cholangitis(PBC).Methods:The expression of PD-1 in CD8+ T cells and Treg cells of PBC patients was detected by flow cytometry.The expression of PD-L1 in biliary epithelial cells was analyzed by immunofluorescence assay.Cytokines related to PD-1/PD-L1 pathway were detected by ELISA.The relative gene expression of PD-1 and regulatory pathway in CD8+T lymphocytes were measured by RT-PCR.To confirm the effect of PD-1/PD-L1 pathway on the interaction between CD8+ cytoxic T lympghocytes(CTL)and human intrahepatic biliary epithelial cells(HiBEC)in PBC,futher co-culture of PD-1+/-CD8+ CTL and HiBEC were done in vitro.Results:We analyzed 69 patients diagnosed with PBC at Peking Union Medical College Hospital as well as 58 health controls(HC).The proportion of perifheral PD-1+ CD8+ T cell decreased in PBC(12.0 ± 8.8%)than HC(19.9 ±12.5%)(p<0.001).PBC patients had less Treg cells(5.6 ± 3.7%)than HC(7.4 ± 2.4%)(p = 0.005),with increased proportion of PD-1+ cells in FoxP3+ CD4+ T cells(29.9± 11.8%vs 25.8 ± 8.1%,p =0.044).The plasma concentration of IL-10,IFN-? and TGF-? in the PBC group were higher than that in HC(8.29±9.00 pg/ml v.s.4.43±5.08 pg/ml,p=0.0066;51.94 ± 52.94 vs 26.71±26.26 pg/ml,p= 0.0015;1302.0 ± 1972.8 pg/ml vs 205.8 ± 298.9 pg/ml,p=0.0018,resp.).Compared with HC(n=19),Tbet gene expression in CD8+ T lymphocyteswas increase in PBC group(n=21)(0.82 ± 0.76 vs 3.03 ± 4.23,p=0.028),with decreased trend of Pdcd1 gene expression(0.59 ± 0.73 vs 1.49±2.20,p=0.110).Immunofluorescence co-localization revealed that PD-L1 positive cells were less in PBC patients and without expression on HiBEC compared to HC.In the CTL and HiBEC co-culture,compared to PD-1-CD8+ T cells,the cytotoxicity of PD-1+ CD8+ T cells was weaker,with less proliferation and tendency of decreased production of IFN-? and TGF-?.Meawhile,HiBEC apoptosis was relatively more in PD-1-CD8+ T cells co-cluture group.These effects could be antagonized by anti-PD-1 antibody and enhanced by PD-L1.Conclusion:In PBC,the expression of Tbet is up-regulated,which leads to the down-regulated expression of PD-1 on CD8+ T cells.Meanwhile,the expression of PD-L1 in HiBEC may be down-regulated.The silenced PD-1/PD-L1 pathway,decreased Treg cells as well as the interaction and co-regulation of cytokines including IL-10,IFN-y and TGF-?,cause more CD8+ T cells proliferation,more related cytokines production and the enhanced CTL cytotoxic effects on HiBEC.