ILT4 Impairs Anti-tumor Immunity Through Reprogramming Suppressive T Cell Subsets In Lung Adenocarcinoma

Lung cancer is the leading cause of cancer morbidity and mortality worldwide.Traditional chemoradiotherapy showed mild improvement in patient prognosis with a 5-year survival rate of less than 21%.Lung adenocarcinoma(LUAD)has now become the most frequent subtype of lung cancer with the increase of their incidence.The application of targeted therapy has greatly changed the paradigm of LUAD treatment in patients with activated driver genes.However,most patients have no drugable targets.Furthermore,drug resistance and disease progression is inevitable for targeted therapy.Therefore,novel strategies are needed to improve patient outomes.Immune checkpoint blockade(ICB)against PD-1/PD-L1 or CTLA-4 are the latest revolution in lung cancer treatment.However,the objective response rate(ORR)of single PD-1/PD-L 1 inhibitors in lung cancer is only 20%.In addition to the inadequate patient selection and tumor intrinsic hypoimmunogenicity,the complex immunosuppressive microenvironment,which contains inhibitory immunocytes,cytokines and metabolites as well as decreased TIL number and functionality,presents a major hurdle to T cell immunity and effective ICB therapy.Therefore,development of novel immunotargets and treatment are urgently needed to break the suppressive barrier in anti-tumor immunotherapy.Tumor infiltrating lymphocytes(TILs)is an important component of the tumor microenvironment(TME).TIL infiltration in the TME predicted benefit patient prognosis and superior efficacy of tumor immunotherapy.As the most frequent and critical subset in TILs,T lymphocytes play central roles in anti-tumor immunity.Mature T lymphocytes are characterized by CD3 expression on the cell surface,and can be divided into two subgroups:CD3+CD4+T cells and CD3+CD8+T cells.CD4 is a classic marker for helper T cells.It binds to exogenous antigen-peptid-MHC-II molecules and secrets multiple cytokines to activate immune response.While CD8,the marker of cytotoxic T cells,mainly binds the complex of endogenous antigens and MHC-I molecules and directly eradicates tumor cells.FOXP3 is a lineage-specific transcript factor for regulatory T cells(Tregs).It not only controls their phenotype,but also maintains their immunosuppressive function,and is the most widely used Treg marker.Treg usually mediates immunosuppressive function through contact-dependent inhibition of Teffertor,NK or APC activation,or otherwise through secretion of inhibitory factors such as IL-10,TGF-?.CD4+TILs and CD8+TILs are considered as positive prognostic indicator for cancer patients.In contrast,Treg infiltration in the TME indicates poor patient outcomes.More importantly,the infiltrates of different T cell subsets determines ICB efficacy.Therefore,identifying novel immune targets and reprogramming the tumor eradicating TME is particularly important to improve the clinical benefit of ICB treatment.Immunoglobulin like transcript(ILT)4,a classic immunosuppressive receptor,is mainly expressed in myeloid innate cells and plays an important role in the maintenance of maternal and infant immune tolerance,organ transplantation tolerance and inflammatory response.ILT4 in these myeloid cells effectively inhibited their activation,and consequently suppressed the immune response of CD4+T cells,CD8+cytotoxic T cells,and also induced different types of Treg cells.In recent years,ILT4 has been found to be expressed in multiple tumor cells such as NSCLC,leukemia,breast cancer,esophageal cancer and pancreatic cancer.ILT4 in tumor cells promoted their malignant biologies including proliferation,invasion and metastasis.Meanwhile,ILT4 is also enriched in myeloid-derived suppressor cell(MDSC)and tumor-associated macrophages(TAMs),which promotes their M2 polarization and creates the immunosuppressive TME.However,the regulation of tumor-derived ILT4 on T lymphocyte subsets and the potential mechanisms have not been reported yet.Objectives1.We aimed to clarify the correlation of ILT4 in tumor cells with T cell subset distribution in the TME of lung adenocarcinoma,and probe into ILT4-regulated T subset infiltrates.2.To detect the effect of tumor cell-derived ILT4 on proliferation and apoptosis of T lymphocyte subsets and explore the mechanisms underlying ILT4-regulated T cell subset infiltration.Methods1.We collected the tissue samples and corresponding clinicopathological data from 216 primary LUAD patients in Yantaishan hospital.The correlation of ILT4 expression with different T cell subset density,clinicopathological features and patient survival were determined by immunohistochemistry and survival analysis.2.The online tool of KM-plotter(http://kmplot.com/)database was used to analyze ILT4-based survival in LUAD patients.A total of 720 and 461 LUAD patients from Gene Expression Omnibus(GEO)database were analyzed for progression free survival(PFS)and OS respectively.The correlation of ILT4 levels with Treg infiltration was analyzed in both GEO dataset(GSE50081)and the Cancer Genome Atlas(TCGA)TCGA database.3.Using R language 3.0.1and glmnet package for LASSO Cox regression model analysis,a nomogram was established to predict the 2-year and 3-year survival rate of LUAD patients based on the clinicopathologic variates and T cell subset infiltrates.4.ILT4-incuded T cell subset proliferation and apoptosis were evaluated using CCK-8 and apoptotic assay after coculturing different T cells with ILT4 up-/down-regulated tumor cells.The expression of ILT4 in LUAD cell line H1975 was up-/down-regulated,and the transfection efficiency of ILT4 was detected by Western Blot and RT-PCR.Human CD3+,CD4+and CD8+T lymphocytes were purified from health volunteers and pre-activated with CD3 antibody before co-culture.Results1.ILT4 was highly expressed in tumor cells of LUAD tissues.Enriched ILT4 was correlated with reduced T cell infiltration in the TME,advanced diseases and poor patient overall survival(OS).Further T cell subset analyses revealed that ILT4 expression was correlated with decreased CD8+T cell and increased Treg frequency in both cancer nest and stroma,but not with altered CD4+T cell frequency.High ILT4 level combined with low CD8+T cell/high Treg density predicted markedly poorer clinical outcomes compared with any of these biomarkers alone.2.Based on the immune indexes obtained by Lasso regression screening as well as clinical parameters,the nomogram can effectively predict the 2-and 3-year survival rate of LUAD patients,providing a quantitative method to predict patient prognosis.3.ILT4 overexpression inhibited the proliferation of CD3+and CD8+T cells and promoted their apoptosis,while ILT4 knockdown increased T cell proliferation and decreased their apoptosis.However,ILT4 overexpression/knockdown showed no effect on CD4+T subset proliferation and apoptosis.Conclusion1.Enriched ILT4 in LUAD cells prodiceted immunosuppressive T cell subset infiltration and poor clinical outcomes.Combined ILT4 expression and CD8+T cell/Treg frequency in tumor infiltrating lymphocytes(TILs)are stronger predictor for patient outcomes.2.A clinical useful nomogram tool was established to predict the 2-and 3-year survival rate for LUAD patients.3.Tumor cell-derived ILT4 directed T cell subset distribution and tumor immune escape through regulating their proliferation and apoptosis.