Role Of ILT4 In Immunomodulation Of Tumor Microenvironment And Immunotherapy For EGFR Actived Non-small Cell Lung Cancer

BackgroundImmune checkpoint inhibitors(ICIs)targeting the PD-1/PD-L1 axis are the milestone of anti-tumor therapy in recent years.These agents have achieved promising results in multiple solid tumors and have been established as the standard care of front-line therapy in advanced non-small cell lung cancer(NSCLC).However,the efficacy of ICIs is still limited and no more than 20%in NSCLC patients with wild-type epidermal growth factor receptor(EGFR).Clinical trials of ICIs in EGFR-driven NSCLC have been largely disappointing thus far.It has been reported that activated EGFR signaling in NSCLC cells can utilize multiple strategies to create an immunosuppressive tumor microenvironment(TME).These include impeding T cell infiltration and cytotoxicity,recruitment of suppressive tumor-associated macrophages(TAMs)and regulatory T cells(Tregs),and secretion of inhibitory cytokines and metabolites,which represent major hurdles to effective anti-tumor immunity and immunotherapy.Therefore,exploration of novel mechanisms for EGFR-mediated immune escape and tumor promotion and reversal of the suppressive TME is essential to improve the efficacy of ICIs in NSCLC patients with EGFR activation.Immunoglobulin-like transcript(ILT)4 is an inhibitory receptor of the immunoglobulin superfamily.It is mainly expressed in myeloid cells,including dendritic cells(DCs),granulocytes,monocytes,macrophages and platelets[7,8],ILT4 in these cells represents their immunosuppressive phenotypes and negatively regulates antigen presentation of DCs,phagocytosis of neutrophils,maturation of macrophages,and platelet aggregation.In recent years,we and others have shown that ILT4 is enriched in many solid tumor cells and directly induces their proliferation,invasion,and migration.In addition,ILT4 directs immunosuppressive T cell subset infiltration in lung adenocarcinoma patients and predicts poor patient outcome.These findings identify ILT4 as a potential target for tumor treatment.However,the regulatory mechanism of ILT4 expression in NSCLC cells and its functional role in anti-tumor immunity and immunotherapy remain undetermined.TME is an integral part of cancer fundamentally orchestrating tumorigenesis,disease progression,and treatment resistance.Among the complex cellular components in TME,immunocytes,including TAMs and dysfunctional T cells,play central roles in ICI resistance.TAMs are the most abundant immune cells in the TME.There is substantial evidence revealing that TAMs acquire a pro-tumoral phenotype at the time of tumor initiation,and promote tumor progression by inducing T cell dysfunction,angiogenesis,and tumor cell invasion and motility.Immunologically,the pro-tumoral TAMs can either directly inhibit cytotoxic T lymphocyte(CTL)responses or indirectly regulate immunosuppression by reshaping the immune microenvironment,ultimately impairing ICI activity.Besides TAMs,T cells including CD4+T helper cells and CD8+CTLs have a crucial role in tumor rejection.While CD4+T cells kill tumor cells and recruit tumor-specific CTLs by producing IFN-y and IL-2,CTLs mediate tumoricidal activity directly through the release of cytotoxic granules(perforin and granzyme)or indirectly through secretion of cytokines(IFN-y and tumor necrosis factors).However,T cells in the TME are usually hyporesponsive to tumors due to their exhausted or senescent status,hindering effective anti-tumor immunotherapy.Although ICIs,targeting PD-1/PD-L1 signaling,partially reverse the exhaustion of T cells,the mechanisms for T cell dysfunction are far more complex than previously expected.Further exploring tumor-orchestrated immunosuppression and rescuing the suppressive TME by combination immunotherapy would provide potent approaches to improve ICI efficacy.In this context,whether TAMs and dysfunctional T cells participate in ILT4-mediated tumor promotion is still unclear.AimsIn this study,we will first clarify the expression mechanism and function of ILT4 in EGFR-activated NSCLC,and find that ILT4 can be up-regulated by EGFR-AKT/-ERK1/2 signal pathway.Secondly,the co-culture system of tumor-TAM/T cells in vitro and the model of transplanted tumor in mice were constructed to confirm that ILT4 in tumor cells induced TAMs recruitment and M2-like polarization,and inhibited T cell infiltration and cytotoxicity.Using the homologous mouse immunotherapy model,it was confirmed that blocking ILT4 could reverse tumor immunosuppressive TME and tumor growth activated by EGFR activated NSCLC in vivo and in vitro.In addition,we constructed a humanized mouse immunotherapy model and confirmed that ILT4 blocking and PD-L1 inhibitors showed synergistic anti-tumor effect in EGFR wild type,but not with EGFR mutant NSCLC.Our study identified a new mechanism of tumor immune escape mediated by ILT4 in EGFR-activated NSCLC and provided a promising immunotherapy and combined therapy strategy for patients with EGFR-activated NSCLC.Methods1.Immunohistochemical method was used to detect the expression of ILT4 and EGFR phosphorylation in tissue samples of patients with NSCLC,and the correlation between the two expressions and the clinicopathological parameters of the patients were statistically analyzed.2.EGFR-TKIs/EGF was used to stimulate EGFR mutant/wild type NSCLC cells to inhibit/activate EGFR activation,or knockout/overexpression of EGFR mutation/wild type NSCLC in EGFR,real-time PCR,western blot,immunofluorescence and flow cytometry to analyze the regulation of ILT4 expression by EGFR activation in human NSCLC cell line.3.mRNA microarray and TCGA database were used to analyze and screen the signal pathways regulated by EGFR,and the specific molecular signal pathways of ILT4 expression induced by EGFR in tumor cells were determined by western blot through corresponding signal pathway inhibitors.4.The co-culture system of tumor cells with TAM or T cells was constructed by knocking out ILT4,from EGFR-activated NSCLC cells.The effect of tumor cell ILT4 on TAMs recruitment and polarization was detected by Transwell migration test,flow cytometry and real-time PCR,and the effect of tumor cell ILT4 on T cell survival and cytotoxicity was analyzed by CFSE proliferation test,apoptosis test,flow cytometry,ELISA and cytotoxicity test.5.The co-culture system of tumor cells with TAM or T cells was constructed by blocking ILT4 and PD-L1,in NSCLC cells activated by EGFR,respectively.As mentioned above,TAMs recruitment and M2-like polarization and T cell survival and killing levels were detected.It was verified that ILT4 combined with PD-L1 blockade synergistically reversed TAMs/T cell-mediated immunosuppression.6.C57BL/6 mice were subcutaneously injected with lung cancer cell line LLC,which knocked out PIR-B(the homologue of mouse ILT4)and regularly treated with PD-L1 inhibitors to establish tumor immunotherapy model.To study the effect of blocking PIR-B and PD-L1 monoclonal antibody on tumor growth.The number and phenotype of TAMs and T cells in spleen and peripheral blood of mice were detected by flow cytometry,and the number and phenotype of TAMs and T cells in tumor tissue were detected by immunohistochemistry and immunofluorescence.To determine the dual effects of PIR-B on tumor growth and immune escape.7.The tumor growth model was established by subcutaneous injection of PIR-B knockout lung cancer cell line LLC in NSG mice,and the effect of PIR-B on tumor growth in immunodeficient mice was observed.8.Human PBMC was injected into NSG mice via tail vein to construct humanized immune system,and the tumor immunotherapy model was established by inoculating gefitinib resistant PC9(PC9-GR)and EGFR wild type H1299 cells with ILT4 knockout,respectively,and regularly treated with PD-L1 antibody or control antibody.The number and phenotype of TAMs and T cells in spleen and peripheral blood of mice were detected by flow cytometry,and the number and phenotype of TAMs and T cells in tumor tissue were detected by immunohistochemical methodResults1.EGFR activation induces ILT4 expression in NSCLC cellsIn the NSCLC tissues of patients,we found that the expression of ILT4 in tumor cells was positively correlated with the level of EGFR phosphorylation.In NSCLC cell line,two ways of EGFR activation,EGFR activation induced by tyrosine kinase mutation and EGFR activation dependent on ligand(EGF)binding,were proved to up-regulate the expression of ILT4.2.EGFR mediates ILT4 expression by activating ERK and AKT signaling pathwaysMRNA gene chip and TCGA big data analysis showed that EGFR was closely related to MAPK,NF-?B and AKT signal pathway;using specific inhibitors of related pathways and WB verification,it was confirmed that EGFR activation induced ILT4 expression in NSCLC cells through ERK and AKT signal pathways.3.ILT4 in EGFR activated NSCLC promotes TAM recruitment and M2-like polarization,and inhibits T cell proliferation and cytotoxicityUsing clinical tumor tissue samples and TCGA big data,it was found that the expression of ILT4 in lung cancer tissue was correlated with TAMs recruitment polarization and T cell quantitative function in TME.In vitro mechanism studies have confirmed that EGFR-activated ILT4 in NSCLC promotes TAM recruitment by up-regulating the expression and secretion of chemokine CCL2 and CCL5.Knockout of ILT4 expression in NSCLC cell lines activated by EGFR significantly decreased the migration ability of TAMs induced by EGFR,inhibited the expression of M2-like markers such as CD206,CD209,IL-10 and Argl in TAMs,increased the expression of Ml-like markers such as IL-12,TNFa and IL-6,and weakened the immunosuppressive effect induced by TAMs.Knockout of ILT4 expression in EGFR activated NSCLC cell line significantly enhanced the proliferation,IFN-y expression and secretion level and killing ability of co-cultured T cells.4.ILT4 blockers can act synergistically with PD-L1 inhibitors in EGFR activated NSCLC cells to reverse tumor immunosuppression mediated by TAM and disabled T cellsEGFR mutant,EGFR mutant TKIs and EGFR wild type NSCLC tumor cells were pretreated with ILT monoclonal antibody or/and PD-L1 monoclonal antibody,respectively,and then co-cultured with TAMs or T cells.The results showed that ILT4 or PD-L1 in NSCLC cells blocking the above three EGFR activation states inhibited the migration of induced TAM,and the combination of the two antibodies had the most significant inhibitory effect on the migration of TAM.At the same time,blocking ILT4 or PD-L1 could reduce the levels of CD 163 and CD206 in TAMs,and the expression of CD 163 and CD206 was the lowest in the combined antibody group.Co-culture with anti-ILT4-or anti-PD-L1-pretreated tumor cells could increase the level of IFN-y,tumor killing ability and proliferation of T cells,while the combined blocking of these two molecules showed the most significant increase.5.Combination of PIR-B and PD-L1 blockers can synergistically inhibit tumor growth and immune escapePIR-B knockout/control LLC was subcutaneously injected into whole-gene C57BL/6J mice,followed by intraperitoneal injection of PD-L1 monoclonal antibody/control antibody.The results showed that both PIR-B knockout and PD-L1 blocking could inhibit the growth of tumor in mice and improve the synergistic therapeutic effect of T cells and TAMs-mediated immunosuppressive TME;.The knockout of PIR-B in the tumor growth model of NSG mice also inhibited the growth of tumor in mice,but the change trend was significantly smaller than that of full-gene C57BL/6J mice,suggesting that the immune system is involved in PIR-B-induced tumorigenesis and development.6.ILT4 alone,rather than in combination with PD-L1 inhibitors,can inhibit tumor progression and immune escape of TKI-resistant EGFR mutant NSCLCThe immunotherapy model of immune reconstruction NSG mice was established by using TKI-resistant EGFR mutant NSCLC cell line PC9-GR.It was confirmed that ILT4 gene knockout significantly inhibited tumor growth and enhanced T cell infiltration and function in spleen,peripheral blood and tumor tissues;however,PD-L1 monoclonal antibody promoted but not inhibited the tumor growth of PC9-GR,and PD-L1 alone or combined with ILT4 inhibition could not improve the infiltration of T cells in the above-mentioned organs.It is suggested that blocking ILT4 alone rather than combining with ICIs may be an effective treatment strategy for patients with EGFR-TKI-resistant EGFR mutations.7.ILT4 blockade enhances the efficacy of PD-L1 inhibitors in the treatment of EGFR wild-type NSCLC in vivoThe tumor immunotherapy model of humanized NSG mice was established by knocking out ILT4,from EGFR-H1299 cells.It was confirmed that both ILT4 and PD-L1 blockers could inhibit tumor growth in vivo,and the combination therapy had the most significant inhibitory effect on tumor growth,and they synergistically enhanced T cell infiltration and IFN-y expression in spleen,peripheral blood and tumor tissues.Conclusions1.The expression of ILT4 in 1.NSCLC cells is induced by the activation of EGFR-AKT/-ERK1/2 signal pathway.2.On the one hand,2.EGFR-activated ILT4 in NSCLC can induce TAM recruitment and M2-like polarization,enhance the tumor immunosuppressive effect of TAMs;on the other hand,it can directly inhibit T cell proliferation,cytotoxicity and interferon-y expression and secretion.ILT4 blocking and PD-L1 blocking synergistically reversed tumor immunosuppression in vitro.3.3.In vivo studies have confirmed that PIR-B not only promotes tumor malignant biological behavior,but also induces TAM/T cell-mediated immunosuppressive microenvironment,thus promoting tumor progression.PIR-B and PD-L1 blockade showed a synergistic effect in EGFR-activated NSCLC therapy.4.In the humanized mouse treatment model,ILT4 blockade could enhance the efficacy of PD-L1 inhibitors on EGFR wild-type NSCLC,but had no effect on EGFR mutant NSCLC.