Study Of PD-1 In Regulating T Cell Infiltration To Kill Hepatocellular Carcinoma And Its Mechanism

BackgroundIn recent years,tumor immunotherapy has made significant progress in the field of cancer therapy.Immune checkpoint inhibitors(ICIs),such as programmed death-1receptor(PD-1)and its ligand(programmed death ligand,PD-L1),cytotoxic T lymphocytes-associated antigen-4(CTLA-4)and other related antibodies,have been massively investigated in the clinical research of liver cancer treatment.Although the median progression-free survival(PFS)reached 9.7 months,it is still difficult to sustainably inhibit the growth of liver cancer cells.For ICIs,the number of lymphocytes infiltrating into tumor is the basic condition to achieve effectiveness.Immune checkpoint related blocking therapy ultimately depends on T cells.However,no matter it is tumor induced T cells,adoptive activated T cells or chimeric antigen receptor T cells(CAR-T),the evolution in the tumor environment after contacting with tumor cells is not clear.PD-1 is a type I transmembrane protein,which is preferentially expressed in immune cells like T,B and NK cells.PD-L1 is a member of the B7 family.The costimulatory/co-inhibitory molecules for antigen presentation in this family are expressed by a variety of cell types(including cancer cells).Upon combining with PD-L1,PD-1 strongly interferes with T cell receptor(TCR)signal transduction through several poorly understood molecular mechanisms.In the tumor microenvironment,the role of PD-1/PD-L1 in regulating the infiltration and function of T cells is still not clear.In conventional T cell adoptive immunotherapy and CAR-T cell therapy targeting solid tumors,T cells are almost at the outer edge of tumor tissue.Chemokine receptor 3(CXCR 3)is an important factor for the chemotaxis of T cells.Inflammatory body nod-like receptor protein 3(NLRP 3)is the key regulatory protein of CXCR 3 expression on T cells.Moreover,both NLRP 3activation and CXCR3 expression on T cells are closely related to phosphatidylinositol-3-kinase/protein kinase B(PI3K/Akt)and mitogen-activated protein kinase(MAPK)signal pathways.There are currently 12 types of MAPKs that are further divided into three types,for example extracellular signal regulated kinase 1/2(ERK 1/2).Studies have shown that the combination of PD-1 and PD-L1 can regulate the metabolism of T cells from glycolysis to β-oxidation of fatty acids by inhibiting the activity of PI3K/Akt and ERK pathways.We aimed to explore the influence of different cytokines on the expression of CXCR3 on activated T cells,as well as the underlying mechanisms,to help understanding the migration characteristics of adoptively administrated T cells and CAR-T cells in the tumor microenvironment,which could provide beneficial support for tumor immunotherapy.Objective To clarify whether PD-1/PD-L1 can affect the activation of NLRP 3 via the PI3 K or MAPK pathway and subsequently affect the expression of CXCR 3,which finally can regulate the infiltration of T cells into the tumor.The mechanisms behind may provide a more effective strategy for the immunotherapy of hepatocellular carcinoma.Methods In this study,CRISPR/Cas 9 was used to knock out PD-1 expression in the activated T cells and chimeric glypican 3 T cells(CAR-GPC 3-T)to establish PD-1 knock-out T cells.To investigate the effects of activated T cells and human liver cancer cell lines on the expression of PD-1 and PD-L1,and the effects of different cytokines on the expression of CXCR 3 on T cells,the co-culture system was established containing the activated T cells or CAR-T cells induced by multiple cytokines and liver cancer cell lines with different genetic backgrounds.Flow cytometry was performed to determine the knockdown efficiency,changes of T cell phenotyping,expression of PD-1 and CXCR 3 on T cells,and PD-L1 expression on tumor cells.ELISA was used to detect the level of IFN-γ secreted by T cells.CCK-8 assay was used to determine the proliferation and the killing effect of T cells against hepatoma cells.Cell migration and invasion ability was measured using trans-well assay.Western blotting was used to detect the binding of PD-L1 to PD-1 on T cells.Meanwhile,MAPK phosphorylation was also determined to explore the regulatory role of CXCR 3 on T cells.A Huh-7 nude mouse liver carcinoma model in situ was established.The activated T cells were injected via tail vein.89Zr-labeled T cells were scanned by PET-CT to observe the distribution of activated T cells in vivo.Hematoxylin-eosin(H&E)staining was used to observe the histopathological changes of hepatocellular carcinoma cells.Immunohistochemistry(IHC)and immunofluorescence(IF)were performed to analyze the correlation of PD-1/PD-L1 expression,the chemotactic ability,and the distribution of activated T cells in situ.This study aimed to explore the mechanism of PD-1 in regulating T cell infiltration and killing hepatocellular carcinoma,and the associated pathways using in vitro assays and in vivo models.Results1.In vitro assays1.1 Co-cultured liver cancer cell lines and activated T cells mutually affect expression of PD-L1 and PD-1All the liver cancer cell lines with different genetic backgrounds express PD-L1.When co-cultured with activated T cells,except for the MHCC 97 cell line having HBs Ag expression,the PD-L1 expression of other liver cancer cell lines was significantly increased.Among them,the poorly differentiated SMMC7721 cells showed the highest expression of PD-L1,suggesting that the interaction with T cells could upregulate PD-L1 to diverse extent on liver cancer cell lines with different genetic backgrounds.1.2 Activated T cells can be induced to express PD-1 after contacting with liver cancer cells After 24 hours of co-cultivation with liver cancer cell lines,the increased PD-1expression on activated T cells was observed,and the introduction of PD-1-sg RNA-Cas9 complex by electrotransfection could significantly inhibit the expression of PD-1 on activated T cells.1.3 Knock out or blocking PD-1 in the activates T cells can enhance the killing effect against liver cancer cells in vitro After co-cultured with liver cancer cells for 24 hours,the killing efficiency of PD-1knockout(KO)T cells against liver cancer cells was higher than that of the PD-1blocking group and the control group.In the 72 hours co-culture system,the killing efficiency of the blocking group and the knockout group was similar,and both were high than control.The killing efficiency of PD-1 KO T cells or T cells with PD-1mono-antibody blockage was positively correlated with the PD-L1 expression on liver cancer cell lines.the killing rate of normal activated T cells against liver cancer cells is negatively correlated with PD-L1 expression on liver cancer cells.After co-cultivation with liver cancer cells,an increase in IFN-γ secretion was observed in activated T cells,but PD-1 KO T cells exhibited a significant increase in IFN-γ after 24 hours of stimulation.However,activated T cells blocked with PD-1 monoclonal antibody did not show a significant increase in IFN-γ until 72 hours.Our results indicated that the killing effect of PD-1 KO T cells is more time-efficient than monoclonal antibody blockade.1.4 The combination of PD-1 on T cells and PD-L1 on liver cancer cells affects the expression of CXCR 3The study found that when co-cultured with T cells,different expression levels of PD-L1 on liver cancer cell lines were observed,and the expression of CXCR 3 on T cells was negatively correlated with the PD-L1 expression,suggesting that the combination of PD-L1 and PD-1 could directly affect the expression of CXCR 3 on T cells.1.5 PD-1 inhibits the chemotaxis and migration of T cells with high expression of CXCR 3The combination of CD3/CD28,IL-2 and GM-CSF successfully cultured activated T cells and CAR-T cells with high expression of CXCR 3 in vitro.Trans-well experiments found that activated T cells and CAR-T cells with high expression of CXCR 3 exhibited higher migration ability than T cells treated with other factors.The addition of PD-L1 in advance could prevent T cell migration.1.6 PD-1 regulates T cell infiltration and killing efficiency by PI3K/Akt-NLRP 3and ERK 1/2 signaling pathways(1)The combination of PD-1 and PD-L1 inhibited the phosphorylation of Akt induced by CXCL 9/CXCR 3It is known that the combination of CXCL 9 and CXCR 3 can activate downstream Akt signaling and induce actin reorganization and T cell chemotaxis.Our results showed that PD-L1 can block Akt phosphorylation,which is a necessary condition for T cell chemotaxis,suggesting that the combination of PD-1 and PD-L1 might inhibit T cells chemotaxis via inhibiting Akt phosphorylation.(2)CXCR 3 expression depends on PI3K/Akt-NLRP 3 and ERK 1/2 signaling pathways In the in vitro T cell culture system,glibenclamide(blocking NLRP 3)or p38 MAPK inhibitor,ERK 1/2 inhibitor was added 30 minutes in advance,followed by introducing GM-CSF 1000U/ml to stimulate for 36 hours.the expression of CXCR 3 in the control group,p38 blocking group,ERK blocking group,and glibenclamide group was85.32±5.63%,83.67±3.19%,63.21±3.34%,and 15.14±3.35%,respectively.The results suggested that the expression of CXCR 3 in T cells was dominated by the PI3K/Akt-NLRP 3 pathway.(3)The combination of PD-1 and PD-L1 inhibits phosphorylation of NLRP 3 and ERK.In order to clarify the mechanism of how PD-1/PD-L1 inhibit the expression of CXCR3 in T cells,we stimulated T cells with Kyn at a final concentration of 400 mmol/L for 48 hours.Subsequently,PD-L1(50 μg/ml)was introduced into the system for 24 hours,and then stimulated T cells with LPS for 24 hours.It was found that after PD-L1 treatment,the expression of NLRP 3 in T cells was inhibited,as well as the phosphorylation of ERK.In addition of PD-1 monoclonal antibody in advance or PD-1KO T cells,after treatment with PD-L1 and stimulated with LPS,high expression of NLRP 3 and phosphorylation of ERK can be observed,suggesting that PD-1 KO or PD-1 blockade can increase the expression of CXCR 3 by increasing the level of NLRP3 and phosphorylation of ERK.2.In vivo experiments2.1 Activated T cells possess the natural characteristics of homing to the liver,but are less distributed in liver cancer PET-CT was used to observe the distribution of adoptively re-infused activated T cells in the orthotopic liver cancer nude mouse model.It was found that after 89Zr-labeled activated T cells were injected via the tail vein of nude mice,the radioactive substances were mainly distributed in liver,spleen and lung.Among them,the radioactive substances in the lungs decreased rapidly within 4 hours,while increased rapidly in liver and spleen after 4 hours,and remained at a high level after 336 h,suggesting that activated T cells have natural homing property to liver.Immunohistochemistry showed that there was only a small amount of T cells infiltrated in the liver cancer in situ,and PD-1 and PD-L1 appeared rapidly.2.2 Knockout of PD-1 can enhance the infiltration and killing effect of T cells with high expression of CXCR3 against liver cancer in situ The knockout of PD-1 in T cells could increase the number of T cells around the sinusoids of hepatocellular carcinoma,and the number of T cells infiltrated into the tumor was also increased,which indicated that the PD-1 KO T cells have an enhanced ability to chemoattract into the tumor.Conclusions1.The co-culture of hepatoma cell lines and activated T cells could mutually affect the expression of PD-L1 and PD-1,and is related to the genetic background of hepatoma cell lines;2.The knockout or blocking PD-1 on T cells could significantly enhance the killing effect on hepatoma cells,which is positively correlated with the PD-L1 expression on tumor cells;3.Knockout of PD-1 in T cells can increase the expression of CXCR 3 and improve the infiltration and killing ability of T cells;4.PD-1 and PD-L1 decreased the expression of CXCR 3 in T cells by inhibiting PI3 K and ERK pathways;PD-1 can inhibit Akt phosphorylation of T cells and prevent the chemotaxis and migration of T cells with highly expressed CXCR 3 induced by CD3/CD28,IL-2 and GM-CSF stimulation.