Study On The Expression And Mechanism Of TIGIT On CD8+T Cells In Non-small Cell Lung Cancer

Under the continuous stimulation of tumor antigens in the tumor microenvironment,CD8+ T cells will enter a state of dysfunction or exhaustion,and thus cannot effectively prevent the progression of cancer.The interaction between the inhibitory ligand PD-L1 on the surface of tumor cells and PD-1 on CD8+ T cells is one of the mechanisms that mediate T cell failure;drugs that block the PD-1/PD-L1 pathway are effective in many cancers.However,in most cancer types,only a small proportion of patients(20-30%)respond to antiPD-1/PD-L1 immunotherapy.Non-responders generally experience short-term activation of T cell function in the body and the disease continues to progress.This may be because blocking the PD-1 pathway is difficult to reverse the fate of the exhausted T cells,suggesting that there are other mechanisms that cooperate with the PD-1 pathway to regulate T cell exhaustion.Since immune checkpoint molecules can cooperatively mediate immune suppression and promote T cell failure,it is necessary to find immune checkpoint molecules that coordinate with the PD-1 pathway to regulate T cell failure and try to use combination drugs(while blocking different pathways)to further enhance the activity of effector cells to kill cancer cells and improve the efficacy of tumor immunotherapy.TIGIT(T cell immune receptor with Ig and ITIM domains)is one of the immune checkpoint molecules that negatively regulate T cell function.It is overexpressed in many cancers,so it may be used as a potential targets for immunotherapy.In this project,we studied the expression of TIGIT in the circulating blood and tumor microenvironment of patients with non-small cell lung cancer(NSCLC),and its regulation of anti-tumor immune response.We further explored the anti-TIGIT+anti-PD-1 combination treatment in the improvement of tumor progression.This study can provide theoretical basis for the clinical treatment of non-small cell lung cancer.We found that the levels of TIGIT and the co-expression of TIGIT and PD-1 on CD8+ T cells in the peripheral blood of NSCLC patients were higher than those of healthy controls.In the tumors of NSCLC patients,TIGIT and PD-1 are highly expressed on CD8+ T cells’ surface.These results indicate that in the tumor microenvironment of NSCLC patients,TIGIT and PD-1 signaling pathways may work together to regulate T cell-mediated antitumor immunity by regulating CD8+ T cells.Further studies confirmed that TIGIT deficiency(gene knockout or anti-TIGIT antibody therapy)could inhibit lung tumors’ progression in mice.Then,we used the LLC mouse model to study the immunosuppressive mechanism of TIGIT molecules: TIGIT was up-regulated as CD8+ T cells infiltrated or expanded into the tumor,and then decreased after reaching the highest point on the 12 th day,and co-expressed with PD-1.By comparing the phenotypes of wild-type mice and TIGIT knockout mice,we found that TIGIT deficiency can significantly increase the number and effector functions of tumor-infiltrating CD8+ T cells and inhibit the process of T cell exhaustion.In the LLC mouse tumor model,the combination of anti-TIGIT and anti-PD-1 therapy can inhibit tumor growth more significantly than monotherapy.Our comparative analysis found that the level of tumor antigen-specific CD8+ TIL infiltration in mice treated with anti-TIGIT+anti-PD-1 increased and had a stronger level of cytokine secretion while significantly down-regulating the functional phenotype of Treg cells in the tumor microenvironment,Indicating that TIGIT and PD-1 co-expressed in CD8+ TIL may synergistically inhibit the function of anti-tumor CD8+ T cells by acting on a common downstream signaling molecule.The combination of anti-TIGIT and anti-PD-1 can further increase the effect function and proliferation,and survival ability of CD8+ TIL.Part one TIGIT expression in patients with non-small cell lung cancerObjective: To explore the expression of TIGIT in the peripheral blood and tumor microenvironment of patients with non-small cell lung cancer,and to lay the foundation for subsequent research.Methods: Flow cytometry was used to detect the expression of immune checkpoint molecule TIGIT on the surface of CD8+ T cells in the peripheral blood and tumor microenvironment of NSCLC patients,and the level of PD-1 on TIGIT+CD8+ T cells was further examined.Immunofluorescence staining was used to determine the cell types expressing TIGIT in NSCLC patients.Results: The levels of TIGIT and the co-expression of TIGIT and PD-1 on CD8+ T cells in the peripheral blood of NSCLC patients were higher than those of healthy controls.In patient tumor tissues,TIGIT was highly expressed on the surface of CD8+ T cells,and PD-1 was highly expressed on TIGIT+CD8+ T cells.Conclusion: TIGIT and PD-1 are both highly expressed on CD8+ T cells in NSCLC patients,indicating that TIGIT and PD-1 signaling pathways may jointly regulate CD8+ T cell-mediated anti-tumor immunity.Part two: Mechanism study of TIGIT inhibits immune responses in lung cancerObjective: To explore the effect of TIGIT on lung tumor progression and the mechanism of TIGIT affecting tumor immunity by regulating the function of CD8+ T cells.Methods: The lung tumor models of LLC subcutaneous injection was used to establish lung tumor model in TIGIT knockout mice and wild-type mice,and the role of TIGIT in mouse anti-tumor immunity was determined.Flow cytometry and immunofluorescence staining were used to determine the cell types that express TIGIT molecules in the tumor microenvironment.At the same time,the role of PD-1+ TIGIT-CD8+ T cells in the LLC mouse model was determined,and the effect of TIGIT on the function of CD8+ T cells was examined.Results: TIGIT deletion or blocking TIGIT can inhibit the growth of lung tumors in mice.Flow cytometry analysis showed that PD-1+TIGIT-CD8+ T cells were the main activated cytotoxic lymphocyte population in the tumor microenvironment.Blocking the TIGIT signaling pathway can enhance the activity of CD8+ T cells.Conclusion: TIGIT may promote the transformation of activated CD8+ T cells to depleted T cells,thereby inhibiting the proliferation and effector function of CD8+ T cells in tumors,making them unable to effectively eliminate tumor cells in the body and causing tumor progression.Part three: The tumor suppressor effect of anti-TIGIT and anti-PD-1 combined treatmentObjective: To investigate the therapeutic effect of anti-TIGIT and anti-PD-1 combined therapy on lung tumor,and to provide new ideas for tumor immunotherapy.Methods: An LLC mice model was established,and the mice were treated with control antibodies,anti-PD-1,anti-TIGIT and anti-PD-1+anti-TIGIT,respectively.The tumor growth was observed.Flow cytometry was used to detect the proportion of CD8+ T and regulatory T cells infiltrating the tumor microenvironment in LLC tumor models.Functional differences of CD8+ T cells and the phenotypic changes of regulatory T cells in different treatment groups were then examined.Results: A single treatment of anti-TIGIT or anti-PD-1 can significantly inhibit the growth of mouse lung tumors,while the effect of anti-TIGIT and anti-PD-1 combination therapy was more obvious than that of single therapy.Also,the combined treatment of antiTIGIT and anti-PD-1 can effectively promote the infiltration of CD8+ T cells in tumor tissues and increase the secretion of IFN-γ in CD8+ T cells.In addition,anti-TIGIT and antiPD-1 combined therapy can reduce the expression of TIGIT on regulatory T cells.Conclusion: In the LLC mouse tumor model,the combination of anti-TIGIT and antiPD-1 therapy can inhibit tumor growth more significantly than monotherapy.Anti-TIGIT and anti-PD-1 combined therapy can further improve the tumor invasion and effector function of CD8+ T cells,and potentially inhibit the function of regulatory T cells.Therefore,the combination of anti-TIGIT and anti-PD-1 is expected to further improve the curative effect of current anti-PD-1 immunotherapy.