PD-1 Controls Phospholipid Metabolic Reprogramming And Function Of CD8~+T Cell In Tumor Microenvironment

BackgroundMalignant tumors have become a major threat to human health.Epidemiological studies show that the incidence and mortality of malignant tumors in China have been rising in recent years.Clinically,tumor treatment is based primarily on surgery,chemotherapy,radiotherapy or a combination of these methods.However,the prognosis of tumor patients,especially advanced patients,has not improved significantly.Therefore,more effective tumor treatment methods need to be developed.After more than a century of development,immunotherapy has shown robust anti-tumor potential and has shown good results in clinical tumor treatment.For example,chimeric antigen receptor T(CAR-T)cell therapy and immune checkpoint blockade(ICB)has been successfully treated in some types of blood and solid tumor patients,greatly improving the patient's 5-year survival rate.However,these T cell-based immunotherapy technologies are only effective in a small number of patients,especially in solid tumor patients.Some studies have demonstrated that the interaction between the solid tumor microenvironment and T cells is a key factor affecting immunotherapy efficiency.There are various immunosuppressive molecules in the microenvironment of solid tumors,such as PDL1,CTLA-4,IDO,etc.and PDL1 is considered to be a key molecule that inhibits T cell activity.PDL1 can activate PD-1 on T cells and thereby inhibit the tumor-killing activity of T cells.In theory,blocking antibody therapy targeting PDL1 or PD-1 can effectively improve tumor treatment.Only a small number of patients benefit from blocking therapy,and in addition to PDL1,therapies that target CTLA-4,IDO,or other T cell inhibitors have also not achieved an effective response in most patients.Preclinical studies have shown that environmental factors can lead to changes in the T cell's metabolism,which in turn affects the T cell's response to block therapy and the effect of tumor treatment.Therefore,the premise of improving T cell immunotherapy is to understand the correlation between immunosuppressive factors and T cell metabolism and further to explore on the effect on blocking therapy,to discover pivotal metabolic molecules to develop more effective combined therapy strategies.Based on the above considerations,this topic mainly studies the changes of T cell lipid metabolism in the tumor microenvironment,the effect of lipid metabolism on T cell function,and the relationship between the PDL1/PD-1 pathway and T cell lipid metabolism.The effects of T cell metabolism on its function and the mechanism of action were discussed in depth using cell models,animal models,and clinical specimens.Part 1.Phospholipid metabolism of CD8+T cell decreased in the tumor microenvironmentObjectiveTo investigate the changes of CD8+T cell metabolic molecules in the solid tumor microenvironment and the correlation between metabolism and cell function.Methods(1)The expression of CD8+ T cell surface co-inhibitory molecules PD-1,CTLA4,Tim3,and killer molecules GranzymeB,IFN-y,as well as the expression of cell proliferation-related molecule Ki67 in CD8+T cells in peripheral blood and tumor tissue,were detected by flow cytometry;(2)The distributions of lipid molecules in peripheral blood and tumor tissues getting from tumor patients were detected by ultra-efficient liquid chromatography technology;and RT-PCR,flow cytometry,immunofluorescence staining technique was used to detect the expression of phospholipid metabolism-related enzyme PLPP1 in T cells;(3)RNA sequencing data of CD8+T cells in 36 lung cancer patients(GSE90728)was downloaded,which was used for analyzing significant signaling pathway associated with PLPP1 expression by GO and GSEA;(4)Analysis of the infiltration of TIL and the effects of PLPP1 expression on the prognosis of different tumor patients through the tumor immune dysfunction and exclusion website.Results(1)Compared with peripheral blood,the active CD8+T cells of tumor tissue in patients with lung cancer were highly expressed PD-1,CTLA4,and Tim3,while the expressions of Granzyme B,IFN-y and Ki67 were decreased;(2)The distribution of lipid molecules on activated CD8+T cells in peripheral blood and tumor tissue of patients with lung cancer was different.Compared with peripheral blood,the PA molecule of CD8-T cells in tumor tissue was higher,while the content of DG,PC and PE molecules was lower;(3)The expression of phospholipid metabolism pathway-related enzyme PLPP1 in activated CD8+T cells was significantly lower in tumor tissue than peripheral blood;(4)According to the expression of PLPP1 in infiltrating CD8-T cells in tumor tissue,36 patients with non-small cell lung cancers were divided into PLPP1loW and PLPP1high groups.DNA replication,cell division,oxidation phosphorylation,fatty acid metabolism,and IFN-y reaction-related signaling pathways were more active in the tissue-infiltrating CD8+T cells of tumor patients from the PLPP1high group;(5)The expression of PLPP1 in 36 non-cellular lung cancer patients had a positive correlation with the number,activation,function,and tissue-resident memory cells of infiltrating CD8+T cells in tumor tissue;(6)The expression of PLPP1 in activated CD8+T cells was significantly lower in tumor tissue in patients with rectal and esophageal cancer than peripheral blood;(7)Patients with high expression of PLPP1 in rectal cancer,malignant melanoma,kidney cancer,and lung cancer had a positive correlation with infiltrating lymphocytes in tumor tissue.ConclusionCD8+ T cell phospholipid metabolism is abnormal in patients with different solid tumor tumors,in which phospholipid metabolism-related enzyme PLPP1 is reduced,and PLPP1 is positively associated with T-cell infiltration,activation,and metabolism.Part 2.PLPP1 controls the function and metabolism of CD8+T cellObjectiveTo investigate the direct effect of the metabolism and function of activated CD8+T cells after PLPP1 is knocked down.Methods(1)The expression of IFN-y and TNF-? in CD8+ T cells from PLPP1low and PLPP1high groups in lung cancer tumor tissue were detected by flow cytometry;(2)The malignant melanoma mouse model was constructed to detect the expression of IFN-y and TNF-? in CD8+T cells which isolating from PLPP1lowand PLPP1high groups of melanoma tumor tissue by flow cytometry;(3)By molecular cloning technology to build PLPP1-sh plasmids,transfect T cells,then through flow cytometry and RT-PCR method to detect the knockdown efficiency;(4)The distributions of lipid molecules in the activated CD8+T cells and PLPP1-sh1 and PLPP1-sh2 CD8+T cells were detected by ultra-efficient liquid chromatography technology;(5)The expression of metabolic pathway-related enzymes in the activated CD8+ T cells and PLPP1-shl and PLPP1-sh2 CD8+ T were detected by RT-PCR method;(6)The ECAR and OCR metabolic strengths of the activated CD8+T cells and PLPP1-shl and PLPP1-sh2 CD8+T cells were detected by the hippocampus energy detection technology;(7)The proliferation and apoptosis of activated CD8+ T cells and PLPP1-shl and PLPP1-sh2 CD8+T cells were detected by flow cytometry;(8)The function and intracellular Ca2+concentration of the activated CD8+T cells and PLPP1-shl and PLPP1-sh2 CD8+T cells were detected by flow cytometry.Results(1)The function and proliferation ability of CD8+T cells in the tumor microenvironment in PLPP1high group is higher;(2)An elevated expression of PLPP1 was detected after CD8+T cell activation;(3)The overall metabolic level of CD8+ T cells was lower after PLPP1 was knocked down,such as phospholipid metabolism,glycolysis,and fatty acid anabolic metabolism;(4)CD8+T cell apoptosis is not affected after PLPP1 knockdown,but T cells proliferation is reduced;(5)The concentration of Ca2+in the CD8+T cell cytoplasm was decreased after PLPP1 knockdown,meanwhile the ability of T cells to secrete killer cytokines IFN-y and TNF-? was also reduced.ConclusionPLPP1 is a key regulatory molecule for regulating the metabolism and function of CD8+T cells.Part 3.PD-1 binding to PDL1 downregulates PLPP1 expression in CD8+ T cellObjectiveTo investigate the key molecules which regulate the expression of PLPP1 in CD8+ T cells in the tumor microenvironment.Methods(1)Co-incubated CD8+ T cells and tumor cells,subsequently detected the expression of PLPP1,PD1 in T cells and PDL1 on the surface of tumor cells by RT-PCR and flow cytometry technology;(2)Activated CD8+T cells from lung cancer tumor tissues were divided into PLPP1low and PLPP1high groups,then detected the surface PD-1,CTLA4 and Tim3 expression by flow cytometry;(3)Activated the normal CD8+ T cells and CRISPR/Cas9 knockout PD-1 CD8+ T cells with PDL1 Fc fragment and detected PLPP1 expression by imaging flow cytometry and flow cytometry;(4)Activated the normal CD8+T cells and PLPPl-shl and PLPP1-sh2 CD8+T cells with PDL1 Fc fragment,then detected OCR changes and intracellular mitochondrial funct1ion by hippocampal energy detection technology and flow cytometry;(5)Normal CD8+ T cells and PLPP1-shl and PLPPl-sh2 CD8+ T cells were activated by PDL1 Fc fragment and detected their function and Ca2+ concentration in the cytoplasm by flow cytometry;(6)Establish a mouse model of malignant melanoma and adoptively transferred OT1 T cells.RT-PCR and flow cytometry were utilized to detect the function of CD8+T cells and PLPP1 expression of PD-1 high T cells in mouse tumor tissues after treatment with PDL1-blocking monoclonal antibodies.Results(1)In the tumor microenvironment of lung cancer patients,PD-1 expression on the surface of CD8+T cells with high expression of PLPP1 was lower,while PD-1 expression was high on CD8+ T cells with low expression of PLPP1,indicating that the PD-1 molecule has May regulate the expression of PLPP1 in CD8 T cells;(2)Activated CD8+T cells derived from peripheral blood of healthy people are directly or indirectly co-incubated with lung cancer tumor cell lines H460 and A549,The expression of PD-1 on the surface of CD8+ T cells and the expression of PDL1 on the surface of tumor cell lines were significantly increased in a direct co-incubation system,and the expression of PLPP1 in CD8+T cells in this system was significantly lower than that of unincubated or indirectly co-incubated CD8+T cells.This result further proves that PD-1 can regulate the expression of PLPP1 in CD8+T cells;(3)Activated CD8+T cells and PLPPl-shl and PLPPl-sh2 CD8+T cells were treated with PDL1 Fc fragment in vitro.It was found that the OCR metabolism rate,intracellular mitochondrial function,and cell function and intracellular Ca2+concentration was reduced in activated CD8+T cells,but there was no significant change in OCR metabolism rate,intracellular mitochondrial function,cell function,and intracellular Ca2+ concentration in PLPP1-shl and PLPP1-sh2 CD8+T cells,indicating that PDL1/PD-1 regulates CD8+ T cells metabolism and function mainly depend on the expression of PLPP1 in T cells;(4)PDL1 blocking monoclonal antibody can reduce the growth rate of tumor cells in mouse malignant melanoma tumor models,and further found that it can increase the expression of PLPP1 and improve the function of antigen-specific T cells.ConclusionCD8+ T cell surface molecule PD-1 binds to its ligand PDL1 can reduce PLPP1 expression in the tumor microenvironment,which further decreases the metabolism and function of T-cells.Part 4.The mechanism of PDL1/PD-1 regulates PLPP1 expression in CD8+T cellObjectiveThe main signaling pathways of PDL1/PD-1 regulating PLPP1 expression in CD8+T cells in the tumor microenvironment.were discussed.Methods(1)Activated the normal CD8+T cells,jurkat and jurkat-PD1 cells with PDL1 Fc fragment and detect the activation of AKT/mTOR signaling pathway by flow cytometry and Western blot;(2)Used PCR array and website to predict and analyze the changes in transcription factors of CD8+T cells which were treated by PDL1 Fc fragment,and detected PLPP1 expression after knocking down relevant transcription factors in CD8+T cells in vitro;(3)Detected the binding relationship between transcription factor GATA1 and the promoter region of the target gene PLPP1 by Chip technology;(4)The experiment of double fluorescein proves that GATA1 can bind to the promoter region of PLPP1,then regulate the expression of the purpose gene;(5)Detection of nuclear translocation of GATA1 in CD8+T cells after treated with PDL1 Fc fragment by imaging flow cytometry and Western blot;(6)Establish a mouse model of malignant melanoma and adoptively transferred OT1 T cells.The tumor growth was detected by mouse imaging technology,and through H&E staining to prove the tumorigenesis.Tissue immunofluorescence was used to detect the infiltration of CD8 T cells and the expression of PLPP1 in tumor tissues of mice treated with PDL1-blocking monoclonal antibodies.Flow cytometry was used to detect AKT/mTOR signaling pathway in antigen-specific T cells after treated with PDL1-blocking monoclonal antibodies.Results(1)PDL1/PD-1 binding can inhibit the AKT/mTOR signaling pathway in CD8+T cells,and the pathway has a positive correlation with the expression of PLPP1;(2)PDL1/PD-1 binding can promote the expression of GATA1 in CD8+ T cells,and promote the nuclear translocation of GATA1;(3)GATA1 in CD8+ T cells can up-regulate PLPP1 expression by binding to the PLPP1 promoter region,and is regulated by the AKT/mTOR signaling pathway;(4)PDL1 blocking monoclonal antibody therapy can reactivate the AKT/mTOR signaling pathway and improve the infiltration of PLPP1+CD8+ T cellsin the tumor microenvironment and further to reduce the growth rate of tumor cells.ConclusionPDL1/PD-1 reduces the expression of PLPP1 in CD8+ T cells mainly by inhibiting the AKT/mTOR/GATA1 signaling pathway.