Study On The Mechanism Of Plasma Exosomal PD-L1-mediated Cancer Cell Immune Escape In Patients With Gastric Cancer

Objective:1.To determine whether a positive correlation can be found between the level of exosomal PD-L1（ePD-L1）in plasma of gastric cancer patients and the TNM stage of gastric cancer.2.To determine whether T cell activation can be suppressed by ePD-L1 in plasma of gastric cancer patients.Methods:1.Exosome purification was carried out using the plasma of patients diagnosed with gastric cancer.2.The number and size of plasma exosomes were analyzed by performing nanoparticle tracking analysis（NTA）;the structure of plasma exosomes was characterized by electron microscopy;multiple antigens on exosomes were labeled and observed by confocal microscopy.3.The level of ePD-L1 in plasma of gastric cancer patients was determined by performing ELISA and the correlation between ePD-L1 level and the TNM stage of gastric cancer was analyzed.4.CD8⁺T cells were incubated with exosomes with different levels of PD-L1,and the levels of intracellular TNFαand IFNγand surface CD69 and PD-1 were detected by performing flow cytometry.Results:1.The electron microscope results showed that exosomes we isolated have a double membrane structure.2.The NTA results showed that exosomes purified from plasma of gastric cancer patients are remarkably larger in size,compared to exosomes purified from the plasma of healthy people.3.The confocal imaging showed that CD63 and PD-L1 on exosomes can be fluorescently labelled.The patient’s plasma has significantly morePD-L1⁺exosomes than the healthy control’s plasma.4.The ELISA results showed that ePD-L1 is expressed at a low level in plasma of30 healthy people.However,it is apparently expressed at a high level in plasma of gastric cancer patients（P<0.01）.30 gastric cancer patients were classified into stage3/4（late stage）patients and stage 1/2（early stage）patients.These two groups of patients have different levels of ePD-L1 in their plasma,with ePD-L1 level being relatively higher in plasma of stage 3/4 patients.30 gastric cancer patients were classified into male patients and female patients.These two groups of patients do not differ in their plasma ePD-L1 levels.5.The flow cytometry results showed that surface CD69 and intracellular TNFαand IFNγare greatly reduced in CD8⁺T cells treated with PD-L1⁺exosomes,while the accumulation of surfacePD-1 remain unaltered（P>0.05）.Furthermore,the accumulation of surface CD69 and intracellular TNFαand IFNγis reduced to a greater extent in CD8⁺T cells treated with PD-L1^highexosomes than in CD8⁺T cells treated with PD-L1^lowexosomes（P<0.05）,although the accumulation of surfacePD-1remain unaltered regardless of being treated with PD-L1^highexosomes or PD-L1^lowexosomes（P>0.05）.Conclusion:1.ePD-L1 is expressed at a much higher level in the plasma of gastric cancer patients.A positive correlation indeed exists between the level of ePD-L1 in plasma of gastric cancer patients and the TNM stage of gastric cancer.2.ePD-L1 in plasma of gastric cancer patients can exert a suppressive effect on the activation of CD8⁺T cells.Cancer cells may achieve immune escape by secreting PD-L1⁺exosomes to suppress T cell activation.