| Background and Objective:Pancreatic cancer(PC)is a highly malignant tumor with the characteristics of occult onset,difficult early diagnosis,rapid development and poor prognosis.PC can escape immune surveillance and has a high degree of immunosuppression.This leaves patients with PC with limited options for effective treatment.Programmed Cell Death Protein 1(PD-1)blockers have provided new approaches and strategies for the treatment of many malignant tumors.The immunotherapy of PC has shown new hope,but the high immunosuppression of PC makes the efficacy of PD-1 blocker monotherapy little.In this experiment,the difference of PD-1 expression level in CD8~+T lymphocytes of peripheral blood from PC patients and healthy volunteers was studied,and the relationship between PD-1 expression rate in CD8~+T lymphocytes of peripheral blood from PC patients and clinical pathological characteristics was studied to verify the relationship between PD-1 expression and the occurrence and development of PC.Through further study on the combination of interferon-α(IFN-α),thymosinα1(Tα1)and tislelizumab,we analyzed the expression changes of PD-1 in CD8~+T lymphocytes of patients with PC and studied the activity,proliferation ability and cytokine secretion level of CD8~+T lymphocytes cultured with different treatments.The effect of CD8~+T lymphocyte on that cytotoxic activity of the PC cell line was then analyzed.To study the effect of combined medication on growth of PC xenografts.In order to provide a new meaningful program for immunotherapy of patients with PC.Methods:1.The CD8~+T lymphocytes in peripheral blood were sorted by magnetic bead sorting method,and PD-1 expression in CD8~+T lymphocytes of PC patients and healthy volunteers was detected by flow cytometry.The clinical data of PC patients were analyzed,and the relationship between PD-1 expression rate and clinicopathological features was analyzed.2.Peripheral blood CD8~+T lymphocytes and PC cell line were cultured,and the effects of different treatments with IFN-α,Tα1 and/or Tislelizumab on the expression of PD-1 in CD8~+T lymphocytes of PC patients in different groups were analyzed by flow cytometry.3.The proliferation and activity of sorted CD8~+T lymphocytes treated with different drugs were detected by trypan blue staining and blood count method.Then the IL-2,TNF-α,and IFN-γcytokine secretion levels of CD8~+T lymphocytes treated with different drugs and the effect of each drug treatment group on the cytotoxic activity of PC cell lines such as PANC-1,BXPC-3 and MIAPa Ca-2 were analyzed by Elisa and CCK-8experiments.4.The PC subcutaneous xenografts model was established in nude mice,and CD8~+T lymphocytes were injected into the tail vein to detect the effects of different drug treatments on the growth of PC tumors.Results:1.The PD-1 expression in CD8~+T lymphocytes of PC patients was significantly higher than that of healthy donors.The expression rate of PD-1 in peripheral blood CD8~+T lymphocytes of patients with PC has nothing to do with age,sex and tumor location.It was significantly correlated with tumor differentiation,lymph node metastasis and TNM stage,negatively correlated with tumor differentiation and positively correlated with TNM stage.2.When PD-1 blocker is used alone,the PD-1 expression in CD8~+T lymphocytes decreases with the increase of Tislelizumab monoclonal antibody concentration.The combination of IFN-αand Tα1 can decrease the expression of PD-1 on CD8~+T lymphocytes,but its inhibitory effect on PD-1 is less than that of Tislelizumab monoclonal antibody.While the combination of IFN-α,Tα1 and Tislelizumab can inhibit the expression of PD-1 to the maximum extent.After combined administration,we further increased the dose of Tislelizumab,but there was no statistical difference between the 1ug/ml mAb+IFN-α+Tα1 group and the 10ug/ml mAb+IFN-α+Tα1 group.3.In trypan blue staining and blood cell count experiments,compared with Ctrl group,IFN-α+Tα1 group and mAb group,the number of CD8~+T lymphocytes in 1ug/ml mAb+IFN-α+Tα1 group was significantly more and the proliferation ability was stronger.In Elisa experiment,the supernatant of cultured CD8~+T lymphocytes was taken,and the secretion of IL-2,TNF-αand IFN-γof CD8~+T lymphocytes increased after PD-1blocking.The secretion of the above three cytokines was the highest in the group of 1ug/ml mAb+IFN-α+Tα1.There was significant difference in cytokine secretion among the groups.4.In CCK-8 experiment,when CD8~+T lymphocytes were co-cultured with PC cell line in a certain proportion,it was found that compared with Ctrl group,IFN-α+Tα1 group and mAb group,1ug/ml mAb+IFN-α+Tα1group showed the highest cytotoxic activity.5.In the nude mouse experiment,the volume of BXPC-3subcutaneous transplanted tumor can be observed after the cultured CD8~+T lymphocytes are injected into the tail vein with different drugs.1ug/ml mAb+IFN-α+Tα1 group has the best inhibitory effect on the growth of nude mice.Conclusions:1.The difference of PD-1 expression in CD8~+T lymphocytes is related to the occurrence and development of PC.2.IFN-αand Tα1 can directly down-regulate the expression of PD-1,and the combination of IFN-α,Tα1 and Tislelizumab can enhance the therapeutic effect of PD-1 blockers and play a synergistic inhibitory role.Combination of IFN-α,Tα1 and Tislelizumab can effectively improve the proliferation and activity of CD8~+T lymphocytes in vitro.3.IFN-α,Tα1 combined with Tislelizumab can inhibit the growth of PC tumor to the maximum extent. |
PDF Full Text Request