IFN-γ Induces PD-L1 Expression And Promotes Immune Evasion Of Human Chronic Myeloid Leukemia

OBJECTIVES:(1)Expression of PD-L1 was tested on the surface of newly diagnosed CML and non-tumor patients cells in order to analyse the role of PD-L1 in immune evasion of CML.(2)A prognosis evaluation method was provided by analysing the correlation of PD-L1 and clinical stage 、 risk stratification and efficacy evaluation of newly diagnosed CML.(3)CML primary cells were stimulated with IFN-g to analyze the association of PD-L1 and IFN-g in CML clinical samples.(4)A possible new therapy is proposed by establishing CML cell specific CTL,whose killing ability was examined after treated with IFN-g or PD-L1 blocking antibody.METHODS:(1)White blood cells were isolated from bone marrow of 27 newly diagnosed CML patients and 17 non-tumor patients,and the expression of PD-L1 on CML and normal cell surface was analyzed by flow cytometry.(2)According to the CML staging standard and Sokal prognosis scoring system,patients were divided into groups to analyze the relationship between PD-L1 expression level and clinical staging and risk stratification.(3)The relationship between PD-L1 expression level and efficacy was analyzed by grouping patients according to whether they obtained EMR after 3 months of treatment,CCy R after 6 months of treatment and MMR after 12 months of treatment.(4)The expression of PD-L1 on the surface of primary CML cells was examined by flow cytometry after stimulation with 20ng/m L IFN-g for 24 h by flow cytometry.(5)Mononuclear cells were exacted from peripheral blood of healthy donors through Ficoll density gradient centrifuge.CD3+ T cells were isolated by magnetic cell sorting(MACS).CML reactive CTL was established by co-culture of the CD3+ T cells and CML,and its capability of specifically killing CML cells was tested with Cyto Tox 96 nonradioactive cytotoxity kits.(6)The killing ability of CML reactive CTL treated with IFN-g or PD-L1 blocking antibody was tested.(7)Data analysis was performed with SPSS 22.0 and Graphpadprism 8.0.The Student’s t-test was used to compare various experimental groups with various control groups.Mann-Whitney U test was used for comparison between two groups,and Kruskal-Wallis H test was used for comparison among multiple groups.P < 0.05 was considered statistically significant.RESULTS:(1)PD-L1 was highly expressed in CML patients,but low in non-tumor patients.(2)while there was no significant correlation between the expression of PD-L1 and the clinical stages,higher Sokal score was correlated with higher PD-L1 expression.(3)there was no significant difference in the expression level of PD-L1 between patients in the EMR group and those in the non-EMR group at 3 months,patients in the CCy R group and those in the non-CCy R group at 6 months,and patients in the MMR group and those in the non-MMR group at 12 months.(4)IFN-g unregulated the expression of PD-L1 on the surface of CML cells.(5)IFN-g treated CML cells inhibited the killing ability of CML reactive CTL,but the inhibition can be reversed by PD-L1 blocking antibody.CONCLUSIONS:(1)PD-L1 is highly expressed in CML patients,and its expression increases with uplifted risk stratification.(2)IFN-g induced the surface expression of PD-L1 in patients with CML.(3)IFN-g induced PD-L1 expression promoted immune evasion of CML via T cells.This escape mechanism may provide new therapeutic strategies for CML patients.