Mechanism Of Helicobacter Pylori CagA Promotes Gastric Cancer Immune Escape Through P53 Regulating Exosomal PD-L1

Background and Objective:Gastric cancer(GC)was a common malignant tumor among residents in China and Hunan Province.It was a malignant tumor with high incidence rate and mortality,ranking the third in China.The pathogenesis of GC was unknown.Helicobacter pylori(HP)was a carcinogenic factor,which may play a causal role in the pathogenesis of GC and had a strong immune regulation function.p53tumor suppressor was closely related to the occurrence and development of GC.Immuno-therapy of tumors was currently emerging as an effective approach after surgery,chemotherapy,radiotherapy,and targeted therapy.Immune checkpoint inhibitor therapy such as blocking programmed death protein-1(PD-1)had been shown to have good clinical efficacy,but most patients were not sensitive to it.Recent studies had shown that programmed death protein ligand-1(PD-L1)in exosomes was involved in the immunesuppressive process of tumors.Therefore,it was crucial to search for the mechanism of HP regulating the level of GC exosomal PD-L1 and tumor immune escape,and to find potential new targets for GC immunotherapy.Methods:The clinical study in this study used a reagent method to extract exosomes from plasma,while an ultracentrifugation method to extract exosomes from cell culture supernatants.Characterization of exosomes was performed by transmission electron microscopy(TEM),nanoparticle tracking analysis(NTA)and Western Blot experiments.Plasma exosomal PD-L1 protein levels were detected by Western Blot.The expressions of Cytotoxin-Associated Gene A(Cag A),p53 and PD-L1in pathological specimens of HP infected GC were detected by immunehistochemistry.The clinic-pathological data were collected and the relationship between them and clinicopathological parameters was analyzed.On this basis,q PCR experiments and Western Blot experiments were performed on fresh tissue samples from HP-infected GC after surgery to study the relationship between Cag A,p53 and PD-L1.The regulation mechanism of HP on GC PD-L1 expression and exosomal PD-L1 level was studied by co-culture of HP and gastric cancer cells,transfection of Cag A eukaryotic expression plasmid into gastric cancer cells,overexpression and silencing of p53 gene in gastric cancer cells,and transfection of gastric cancer cells with mi R-34a mimics.The exosomes from the cell culture supernatant were extracted,and the protein level of exosomal PD-L1 was detected by Western Blot experiment,and the effect of HP on the level of exosomal PD-L1 in gastric cancer was studied.Using gastric cancer cell lines stably expressing Cag A,PD-L1-enriched exosomes were extracted.Through the co-culture experiment of exosomes and CD8~+T cells,CCK8 was used to detect the proliferation of T cells and ELISA was used to detect the ability of T cells to secrete cytokines,and the effect of exosomal PD-L1 on the proliferation and function of CD8~+T cells was studied.The effects of PD-L1-enriched exosomes injected through the tail vein on the growth of GC in mice were investigated by in vivo experiments in animals,so as to evaluate the effect of Cag A in HP on GC immune escape.Results:(1)NTA detection,TEM photography and the expression of exosomal marker proteins CD9,CD63 and TSG101 indicated that exosomes were successfully extracted.A total of 87 patients with HP positive gastric cancer were included in this study.The level of peripheral blood exosomal PD-L1 was higher in patients with poorly differentiated cancer,T3-T4 stage,lymph node metastasis and III-IV gastric cancer.The overall survival time of the exosomal PD-L1 low level group was better than that of the exosomal PD-L1 high level group and the difference was statistically significant(P<0.05).Immunohistochemical analysis of postoperative tissue samples from patients with HP-infected GC showed that the positive rate of Cag A,P53,and PD-L1was 71.3%,44.8%and54.0%,separately.The positive rate of Cag A was high in patients with intestinal gastric cancer,T3-T4 stage,lymph node metastasis and III-IV gastric cancer.And the positive rate of PD-L1 was high in undifferentiated cancer,T3-T4 stage,lymph node metastasis and III-IV gastric cancer.While,the positive rate of p53 was high in patients with diffuse gastric cancer,T1-T2 stage,no lymph node metastasis and I-II gastric cancer.(2)In additional,there was a positive correlation between the expression of HP-Cag A,PD-L1 and the level of exosome PD-L1,while a negative correlation between the expression of p53 and Cag A,PD-L1 and the level of exosome PD-L1.q PCR experiments showed that the m RNA expression levels of p53 gene and PD-L1 gene were negatively correlated.Western Blot experiments showed that the expression of p53 protein in the Cag A expression group was relatively low,and the expression of PD-L1 protein was relatively high;while the expression of p53 protein in the Cag A non-expression group was relatively high,and the expression of PD-L1 protein was relatively low.(3)The co-culture experiment of HP and GC showed that HP promoted the expression of PD-L1 and increased the level of exosomal PD-L1 in GC,which may be mediated by p53.In addition,the experiment of HP Cag A transfected gastric cancer cells showed that Cag A degraded p53and promoted the expression of PD-L1 and increased the level of exosomal PD-L1 in gastric cancer.Moreover,experiments of overexpression and interference of p53 expression and transfection of mi R-34a mimics into gastric cancer cells showed that p53 regulated the expression of PD-L1 by regulating mi R-34a.(4)The co-culture experiments of exosomes and CD8~+T cells showed that PD-L1 in exosomes inhibited the proliferation of CD8~+T cells and the ability to secrete cytokines IFN-γ,TNF-α,and IL-2.But PD-L1 antibody restored the ability of CD8~+T cells to proliferate and secreting cytokines IFN-γ,TNF-α,and IL-2 that were inhibited by PD-L1 in exosomes.(5)HP-Cag A promoted GC exosomal PD-L1 expression,which inhibit CD8~+T cells infiltrating into mice GC,and then caused tumor immune escape and promoted GC growth in mice.Conclusions:(1)The high level of PD-L1 in peripheral blood exosomes indicated a shorter overall survival time and was a new prognostic indicator for GC.(2)HP promoted the expression of GC PD-L1 and increased the level of exosomal PD-L1 through the Cag A/p53/mi R-34a/PD-L1 axis,which inhibited the proliferation and function of CD8~+T cells,inhibit CD8~+T cells infiltrating into mice GC,led to tumor immune escape,and promoted the growth of GC in mice.(3)PD-L1 antibody could relieve the inhibition of exosomal PD-L1 on CD8~+T,restored the anti-tumor ability of CD8~+T,and reversed tumor immune escape.(4)The mechanism of HP infection induced immune escape in GC may be related to HP promoting the expression of GC PD-L1 and increasing the level of exosomal PD-L1 and inhibiting CD8~+T cell function through the Cag A/p53/mi R-34a/PD-L1 axis.