Study On The Activation Of Intracellular Oncogene Signaling By PD-1/PD-L1 Immunosuppressive Axis And Their Effect On The Prognosis In Peripheral T-cell Lymphoma Patients

Objective:The main purpose of this study was to explore the PD-1/PD-L1immunosuppressive axis directly stimulated by the PD-1/PD-L1 immunosuppressive axis to activate the intracellular oncogene signaling pathway of peripheral T-cell lymphoma(PTCL)and verify it in vitro.The correlation between the expression of key proteins in PD-L1 and oncogene signaling pathways and the relationship between clinical features and prognosis of patients was explored in PTCL tumor tissues.In this study,the PD-1/PD-L1 immunosuppressive axis in PTCL can regulate the T cell function through PD-1,and can also regulate the novel immune escape mechanism of oncogene signaling pathway in tumor cells.Methods:At the cell level,Three PTCL cell lines were selected to detect the total expression of PD-L1 by western blotting,and PD-1 stimulates PD-L1 high expression PTCL cell line for phosphorylation.And the expression of PD-L1 on the membrane of PTCL cell line was detected by flow cytometry.The recombinant PD-1 peptide was used to stimulate the PTCL cell line.The total protein of AKT and the level of activated AKT in the cell line were detected from 0h to 2h by western blotting.The clinical data of patients with peripheral T-cell lymphoma were collected.At the same time,the Formalin-fixed paraffin-embedded(FFPE)of these patients was collected and the expression of PD-L1 and p-AKT were detected by Opal stainingor multi-color immunofluorescence.The relationship between the expression of PD-L1and p-AKT and the clinical characteristics was analyzed by X~2 test.The patients were followed up,the survival time was recorded,the therapeutic effect was evaluated,and the effects of PD-L1 and p-AKT expression on the survival of patients with PTCL were analyzed by Graphpadprism5.0 software,respectively.The relationship between the expression of PD-L1 and p-AKT was detected by X~2 analyses.And the survival of patients was further analyzed by Kaplan-Meier analyses.Results:Cytology,three TCL cell lines and western-blotting were used to display the high expression of the total PD-L1 in the cells and PD-1/PD-L1 binding stimulates multiple oncogene signaling pathways in PTCL cell lines.The results of flow cytometry showed the high expression of membrane PD-L1(mPD-L1).The activated state of the AKT in each cell line was upregulated after the stimulation by the human recombinant PD-l for 0 h-2 h.That is,the level of phosphorylat was increased to a different extent and decreased with time.In the paraffin-tissue study of 75 patients with PTCL in Tianjin Cancer Hospital,Tianjin Medical University from January to May,2016,the results showed that the expression of PD-L1 in the PTCL was a typical cell membrane stainingand p-AKT was core staining.The positive rate of the expression of PD-L1 and p-AKT was 26.7%.The expression between PD-L1 and p-AKT showed a significant positive correlation(R=0.280,P=0.015).In total,40patients(53.3%)were PD-L1-positive cases,which correlated with stage and IPI score(R=0.278,P=0.016;R=0.280,P=0.015).The positive rate of PD-L1 in stage III-IV patients was higher than that in stage I-II patients(62.3%vs 31.8%).In total,28 patients(37.3%)were positive for p-AKT,which was associated with stage(R=0.255,P=0.027).The survival analysis of kaplan-meier(log-rank)showed that the prognosis of patients with positive expression of PD-L1 and p-AKT was worse and the difference was statistically significant(P=0.001,P=0.006).More interestingly,the survival of the PD-L1 and p-AKT bi-positive expression patients was poorest(P<0.001).Conclusions:1.PTCL cells highly express PD-L1 and PD-1/PD-L1 binds to activate multiple oncogene signaling pathways in PTCL cells.2.PD-1/PD-L1 pathway in PTCL cells may directly activate the AKT/ m TOR oncogene signaling pathway in PTCL cell lines 3.The expression of PD-L1 and p-AKT was higher than 1/3 in PTCL patients.PD-L1 was membraneexpression and p-AKT was nuclear.The expression of PD-L1 and p-AKT was correlated with clinical stage and the expression of PD-L1 was also correlated with IPI score.4.The co-expression rate of PD-L1 and p-AKT in PTCL tissues was 26.7%,and there was a significant positive correlation between the two biomarkers 5.The prognosis of patients with positive expression of PD-L1 or p-AKT was worse than patients with negative expression of PD-L1 or p-AKT,and the overall survival was shorter than that of patients with negative expression of PD-L1 or p-AKT.