Tumor-derived ILT4 Induces T Cell Senescence And Immune Escape Via Upregulated Fatty Acid Synthesis In Tumor Cells

BackgroundImmune system is a significant barrier to tumor development and progression,in which adaptive T cell immunity plays a dominant role.However,the infiltrated T cell frequency is typically low in the tumor microenvironment(TME).Furthermore,tumor cells can utilize multiple strategies to restrict T cell migration and induce their dysfunction.Therefore,improving the infiltration and killing ability of tumor specific T cells represents promising anti-tumor immunotherapeutic strategies in recent years.Immunotherapy mainly contains immune checkpoint blockade(ICB)and adoptive T cell transfer(ACT).While ICB releases negative regulator(CTLA-4,PD1/PD-l pathway)of anti-tumor T cell immunity using specific antibodies and was designed to modulate the existed T cell activity in TME,ACT aimed to generate a robust anti-tumor immune response through adoptive transfer of ex vivo manipulated tumor antigen-specific T cells.However,these treatments are still clinically challenged.The clinical objective response rate for ICB is limited due to the primary and secondary resistance,which indicated that ICB alone is insufficient to break the complex immunosuppressive TME.Further more,the clinical efficancy of ACT(such as CAR-T)in solid tumors have not paralleled that in hematopoietic malignancies.Except for more heterogeneitic target selection in solid tumors,the immunosuppressive factors such as immune checkpoint molecules,inflammatory cytokines and suppressive metabolites also set a great hurdle to the killing ability of transferred T cells.Therefore,identification of novel checkpoint molecules and designation of combination strategies are urgently needed to overcome the immune resistance and maximize the clinical benefit.Immunoglobulin-like transcript 4(ILT4)is an inhibitory molecule of immunoglobulin superfamily.It is mainly expressed in myeloid cells including monocytes,macrophages,dendritic cells(DCs)and granulocytes.With three immunoreceptor tyrosine inhibitory motifs(ITIMs)in the cytoplasm,ILT4 functions to suppress their activation,antigen presentation and immune response.The ortholog of ILT4 in mouse is paired Ig-like receptor-B(PIR-B).In rescent years,ILT4 and PIR-B were found to be enriched in tumor cells and stroma cells(such as HSCs,MDSCs and TAMs)of the TME.They were reported to regulate malignant behaviors of tumor cells,DC maturation,HSC renewal and M2 macrophage polarization,which might in turn induce effector T cell dysfunction and regulatory T cell differentiation,and amplify the immunosuppressive microenvironment.Based on these findings,we for the first time proposed the concept that ILT4 functions as a novel checkpoint for tumor immunotherapy.However,the direct effect of tumor-derived ILT4 on the fate and function of tumor infiltrating T cells is still uncleanT cell senescence is a newly defined mechanism for T cell dysfunction in the TME.The core feature of senescent T cells is durable cell-cycle arrest.Phenotypically,it is characterized by loss of co-stimulatory molecules(CD27,CD28),expression of anti-proliferation molecules(pr.6,p53,p21)and secretion of pro-inflammatory and inhibitory cytokines(IL-6,IL-8,IFN-γ,TNF-α).More importantly,senescent T cells have impaired killing capacity and even direct suppressive activity.Accumulating evidence suggests that tumor cells and stroma components such as regulatory T cells(Tregs)and bone marrow mesenchymal stromal cells(BMSCs)can induce tumor infiltrating T cell senescence and create an immunosuppressive microenvironment.Mechanically,tumor cell-derived cAMP directly tranferred into T cells through gap junction,which consequently activated LCK/cAMP/PKA/CREB in T cells and induced their senescence.However,the functional role of ILT4 in tumor-induced T cell senescence is undetermined.AimsIn this study,we first want to detect enriched ILT4 expression in multiple tumor cell lines and patient tissues,analyze the correlation of ILT4 expression with clinicopathological features and patient OS,and raise ILT4 as a common feature and indicator of poor patient outcome in malignancies.Then we aimed to establish tumor cell-T cell co-culture system in vitro and tumor transplantation models in vivo to investigate ILT4-induced responder T cell senescence and tumor immune escape.Furthermore,we will clarify the metabolic and signaling mechanisms for ILT4-induced T cell senescence by detecting ILT4-regulated alterations in tumor lipid metabolism,signaling pathways and the reverse of T cell senescence upon treatment with corresponding inhibitors.Finally,we want to verify that PIR-B bolckade could reverse adoptively transferred tumor specific cytotoxic T lymphocytes(CTLs)and enhance their anti-tumor activity in tumor immunotherapeutic mouse models,and propose ILT4 as a novel target for tumor immunotherapy and ACT-based combination strategy.Methods1.Part 1:Tumor-derived ILT4 mediates tumor immune escape via induction of T cell senescence.1)Compare ILT4 expression in human tumor cells and cancer tissues with that in normal epithelial cells using real-time PCR,flowcytometry and immunohistochemistry,and analyze the correlation between tumor ILT4 expression and patient outcome using corresponding clinical patient data and GEO database.2)Compare PIR-B expression in mouse tumor cells with that in epithelial cell line using real-time PCR and flowcytometry.Detect the effect of PIR-B overexpression/knockdown on malignant behaviors of mouse tumor cells including proliferation,adhesion and invasion using tumor growth,adhesive and wound healing assays.3)Establish tumor-T cell co-culture system using ILT4-overexpressed/downregulated tumor cells,and illustrate ILT4-induced CD4+/CD8 T cell senescence.4)Establish mouse tumor transplantation model in C57BL/6J mice using PIR-B-overexpressed/downregulated E0771,investigate the effect of PIR-B on tumor growth and development.Isolate T cells from blood,spleen,lymph node and tumor tissues of these mice and identify T cell subset distribution,senescence and function using SA-β-Gal staining and flowcytometry.5)Establish mouse tumor transplantation model in immunocompromised NSG mice using PIR-B-overexpressed/-downregulated E0771,observe how PIR-B impact tumor growth.2.Part 2:The mechanisms for ILT4-induced T cell senescence.1)Detect the gene expression level of key enzymes involved in lipid metabolism and lipid droplets formation in ILT4-overexpressed/downregulated tumor cells,and delineate ILT4-regulated tumor lipid metabolism using real-time PCR and oil red O staining.2)Verify that lipid metabolism and its products are responsible for ILT4-induced T cell senescence using corresponding lipid metabolism inhibitors in tumor-T cell co-culture system.3)Screen the altered signaling proteins in tumor cells upon ILT4 overexpression/knockdown and explore the molecular signaling involved in ILT4-induced fatty acid synthesis and T cell senescence using corresponding signaling pathway inhibitors.3.Part 3:ILT4 blockade enhanced the efficacy of ACT therapy.Separate gp100 specific CD8 T cells from Pmel TCR transgene mice,inject B16F0 subcutaneously and gp100-CD8 T cells intravenously into B6 mice to establish adoptively transferred T cell immunotherapy model.Block PIR-B expression in B16F0 and investigate how PIR-B control the immunosenescence and eradication of adoptively transferred CTLs using SA-β-Gal staining and flowcytometry analysis.Results1.Part 1:Tumor-derived ILT4 mediates tumor immune escape via induction of T cell senescence.1)ILT4 is highly expressed in multiple tumor cells and predicts poor patient survival.Human NSCLC,breast cancer,melanoma and prostate cancer cells showed significantly higher ILT4 gene and protein expression compared with breast epithelial cell line.In human NSCLC and breast cancer tissues,ILT4 expression is significantly higher in tumor cells compared with that in adjacent normal epithelical cells,and high ILT4 expression is positively correlated with lymph node metastasis,advanced TNM stage and poor patient OS.Analysis result from GEO database showed that high ILT4 expression in NSCLC,breast cancer and prostate cancer patients predicts shortened patient PFS and OS.2)PIR-B is highly expressed in mouse tumor cells and controls their proliferation,adhesion and invasion.Mouse lung cancer,breast cancer and melanoma cells showed significantly higher PIR-B gene and protein expression compared with breast epithelial cell line.PIR-B overexpression/knockdown in tumor cells promoted/inhibited their proliferation,adhesion and invasion respectively.3)Tumor-derived ILT4 induced T cell senescence in tumor-T cell co-culture system.Compared with breast epithelial cell line,human NSCLC,breast cancer,melanoma and prostate cancer cells triggered markedly hightened CD4+/CD8 T cell senescence showed by SA-β-Gal staining,accompanied with restricted co-stimulating molecules CD27,CD28 expression and elevated p53,p21 expression.Anti-ILT4 dramatically reversed tumor induced CD4+/CD8 T cell senescence.We identified that tumor rather than T cell-derived ILT4 induced T cell senescence when pretreating tumor or T cells in co-culture system by anti-ILT4 respectively.Then we confirmed this result using ILT4-overexpressed/-downregulated tumor cells in co-culture system.4)PIR-B induced immunosenescence of tumor infiltrating lymphocytes in tumor tissues and promoted tumor growth in vivo.PIR-B-overexpressed/-downregulated E0771 were transplanted into wild type C57BL/6J mice to evaluate PIR-B-regulated tumor growth and T cell immunity.The results showed that overexpressed/downregulated PIR-B in E0771 markedly promoted/inhibited tumor growth and T cell senescence separated from blood and tumor tissues of B6 mouse.Meanwhile,CD4+/CD8 T cell function was concurrently suppressed/improved indicated by decreased/increased CD4+IN-γ+,CD8+IFN-γ+,CD8 GranzymeB+and CD8+Perforin+T cells.In NSG mice,overexpressed/downregulated PIR-B in E0771 promoted/inhibited tumor growth,but the difference was smaller than that in B6 mice,suggesting immune system participates in PIR-B-promoted tumor progress.2.Part 2:The mechanisms for ILT4-induced T cell senescence.1)ILT4-regulated fatty acid synthesis in tumor cells mediated T cell senescence.Anti-ILT4 inhibited ACC1 and FASN(key enzymes for fatty acid synthesis)gene expression as well as lipid droplets formation in multiple tumor cells.Meanwhile,ILT4 overexpression/knockdown in tumor cells remarkablely up-/down-regulated ACC1,FASN gene expression and lipid droplets formation.When ILT4-overexpressed tumor cells were pretreated with C75(fatty acid synthesis inhibitor)and then co-cultured with T cells,ILT4-induced CD4+/CD8+T cell senescence was significantly reversed.2)ILT4 induced fatty acid synthesis and T cell senescence via activated MAPK ERK1/2 signaling pathway.ILT4 overexpression/knockdown markedly up-/down-regulated ERK 1/2 phosphorylation in tumor cells.When ILT4-overexpressed tumor cells were pretreated with U0126(MEK inhibitor)and then co-cultured with T cells,ILT4-induced ACC1,FASN expression and CD4+/CD8+ T cell senescence was significantly reversed.3.Part 3:ILT4 blockade enhanced the efficacy of ACT therapy.In adoptive transfer immunotherapy model of B6 mouse,gp-100-CD8+ T cells specifically inhibited B16F0 tumor growth,while PIR-B knockdown further slowed down tumor growth.SA-β-Gal staining and flowcytometry results showed that PIR-B knockdown reversed gp100-CD8+T cell senescence,and enhanced their IFN-y expression and killing ability.Conclusions1.ILT4/PIR-B is highly expressed in human/mouse tumor cells and predicts poor patient survival.Tumor-derived ILT4/PIR-B induces the immunosenescence of CD4+and CD8+T cells,inhibits their killing ability and promotes tumor growth both in vitro and in vivo.2.ILT4-activated MAPK ERK1/2 signaling pathway elevated fatty acid synthesis and lipid accumulation in tumor cells,which contributes to T cell senescence.This finding provides molecular and metabolic target for anti-ILT4 therapy.3.In vivo PIR-B blockade reversed tumor-specific CTL senescence,enhanced their killing ability,and improved the efficancy of ACT.ILT4 blockade is a promising strategy to overcome ACT resistance and serves as an active treatment for ACT-based combined immunotherapy.