Expression Characteristics Of PD-1/PD-L1 And Its Preliminary Regulatory Mechanism Of Promoting Immune Escape Of Tumor Cell In PTCL

Objective:The purpose of this study was to reveal the relationship between sPD-L1 levels in peripheral blood or expression of PD-1,PD-L1 and p-Erk in tissues and clinical characteristics as well as prognosis,simultaneously further investigate the correlation between PD-L1 and PD-1/p-Erk,preliminary explore whether PD1/PD-L1 pathway can directly activate intratumoral Ras/Raf/Erk pathway in PTCL patients.Methods:The sPD-L1 levels of healthy people and PTCL patients were detected by ELISA.The relationship between sPD-L1 levels and clinical characteristics were analyzed.The response of all patients was evaluated,and their relationship with sPD-L1 was further analyzed using SPSS 23.0.In addition,expression of PD-1,PD-L1,and p-Erk in PTCL was detected by multicolor immunofluorescence,and the relationship between them and clinical features as well survival was also analyzed using GraphPad Prism.In vitro,five PTCL cell lines were selected,and the total and membrane expression of PD-L1 were detected by western blot and flow cytometry.The cells with high PD-L1 expression were stimulated with recombinant human PD-1 peptide.The expression of ERK and phosphorylated ERK was detected using western blot at different time.Results:Forty-one PTCL patients from the Tianjin Medical University Cancer Hospital from January 2010 to January 2015 were enrolled in this study.We found that sPD-L1 level in peripheral blood was significantly higher in PTCL patients than that in healthy people(P<0.0001).sPD-L1 level was significantly related to age,clinical stage,LDH and ?2 microglobulin levels.The therapeutic response of patients with sPD-Ll low level was significantly higher compared to those with sPD-L1 high level(P=0.022).In addition,FFPE from 76 newly diagnosed patients with PTCL were also collected from January 2008 to January 2015.Multi-color immunofluorescence results showed that PD-1,PD-L1,and p-Erk highly expressed in PTCL with respective positive rates of 48.7%,53.9%,and 53.9%.Co-expression rate of PD-1 and PD-L1 was 26.3%,and co-expression rate of PD-L1 and p-Erk was 35.50%.Spearman test showed that there was no significant correlation between PD-1 and PD-L1 expression(R=0.002,P=1.000),but a significant correlation between PD-L1 and p-Erk expression(R=0.259,P=0.037).PD-1 expression was correlated with clinical stage and ?2 microglobulin,but not gender,age,LDH and subtypes.The expression of PD-L1 and p-Erk was related to the clinical stage,but not gender,age,LDH,?2 microglobulin and subtypes.Kaplan-Meier analysis showed that patients with positive expression of PD-1,PD-L1,and p-Erk had poorer prognosis than those with negative expression(P<0.05).Patients with PD-1 and PD-L1 co-expression were worse than those with double-negative and single-positive expression(P=0.0021).Similarly,patients with co-expression of PD-L1 and p-Erk also had worse prognosis than those with double-negative and single-positive expression(P=0.0044).Western blot and flow cytometry results showed that all five PTCL cell lines expressed total and membrane PD-L1.Phosphorylated ERK in different cell lines was changed with different intensity after stimulated by human recombinant PD-1 peptide at 0,0.5,1,2 h.Conclusions:1.The sPD-L1 level in peripheral blood in PTCL was higher than that in healthy people.sPD-L1 level was related to age,clinical stage,LDH and ?2 microglobulin levels.The therapeutic response of patients with sPD-L1 low level was significantly higher compared to those with sPD-L1 high level.2.The expressions of PD-1,PD-L1 and p-Erk highly expressed in PTCL,which was related to clinical stage.In addition,PD-1 expression was also related to ?2 microglobulin.Patients with positive expression of PD-1 or PD-L1 had worse prognosis than patients with negative expression.However,there was no significant correlation between them.Further,patients with PD-1 and PD-L1 co-expression had the worst prognosis in three groups.There was a significant correlation between the expression of PD-L1 and p-Erk in PTCL patients.Patients with PD-L1 or p-Erk positive expression had worse prognosis than patients with negative expression.Furthermore,patients with PD-L1 and p-Erk co-expression experienced the worst prognosis in three groups.3.The PD-1/PD-L1 pathway might directly activate the Ras/Raf/Erk oncogene signaling pathway in PTCL.