ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.

Rationale: Immune checkpoint inhibitors (ICIs) against the PD-1/PD-L1 pathway showed limited success in non-small cell lung cancer (NSCLC) patients, especially in those with activating epidermal growth factor receptor (EGFR) mutations. Elucidation of the mechanisms underlying EGFR-mediated tumor immune escape and the development of effective immune therapeutics are urgently needed. Immunoglobulin-like transcript (ILT) 4, a crucial immunosuppressive molecule initially identified in myeloid cells, is enriched in solid tumor cells and promotes the malignant behavior of NSCLC. However, the upstream regulation of ILT4 overexpression and its function in tumor immunity of NSCLC with EGFR activation remains unclear. Methods: ILT4 expression and EGFR phosphorylation in human NSCLC tissues and cell lines were analyzed using immunohistochemistry (IHC), real-time PCR, Western blotting, immunofluorescence, and flow cytometry. The molecular signaling for EGFR-regulated ILT4 expression was investigated using mRNA microarray and The Cancer Genome Atlas (TCGA) database analyses and then confirmed by Western blotting. The regulation of tumor cell proliferation and apoptosis by ILT4 was examined by CCK8 proliferation and apoptosis assays. The impact of ILT4 and PD-L1 on tumor-associated macrophage (TAM) recruitment and polarization was evaluated using Transwell migration assay, flow cytometry, enzyme linked immunosorbent assay (ELISA) and real-time PCR, while their impact on T cell survival and cytotoxicity was analyzed by CFSE proliferation assay, apoptotic assay, flow cytometry, ELISA and cytolytic assay. Tumor immunotherapy models targeting at paired Ig-like receptor B (PIR-B, an ortholog of ILT4 in mouse)/ILT4 and/or PD-L1 were established in C57BL/6 mice inoculated with stable EGFR- overexpressing Lewis lung carcinoma (LLC) cells and in humanized NSG mice inoculated with EGFR mutant, gefitinib-resistant PC9 (PC9-GR) or EGFR-overexpressing wild type H1299 cells. PIR-B and ILT4 inhibition was implemented by infection of specific knockdown lentivirus and PD-L1 was blocked using human/mouse neutralizing antibodies. The tumor growth model was established in NSG mice injected with PIR-B-downregulated LLC cells to evaluate the effect of PIR-B on tumor proliferation. The frequencies and phenotypes of macrophages and T cells in mouse spleens and blood were detected by flow cytometry while those in tumor tissues were determined by IHC and immunofluorescence. Results: We found that ILT4 expression in tumor cells was positively correlated with EGFR phosphorylation in human NSCLC tissues. Using NSCLC cell lines, we demonstrated that ILT4 was upregulated by both tyrosine kinase mutation-induced and epidermal growth factor (EGF)-dependent EGFR activation and subsequent AKT/ERK1/2 phosphorylation. Overexpressed ILT4 in EGFR-activated tumor cells induced TAM recruitment and M2-like polarization, which impaired T cell function. ILT4 also directly inhibited T cell proliferation, cytotoxicity, and IFN-γ expression and secretion. In EGFR-activated cell lines in vitro and in wild-type EGFR-activated C57BL/6 and humanized NSG immunotherapy models in vivo, either ILT4 (PIR-B) or PD-L1 inhibition enhanced anti-tumor immunity and suppressed tumor progression by counteracting TAM- and dysfunctional T cell- induced immuno-suppressive TME; the combined inhibition of both molecules showed the most dramatic tumor retraction. Surprisingly, in EGFR mutant, TKI resistant humanized NSG immunotherapy model, ILT4 inhibition alone rather than in combination with a PD-L1 inhibitor suppressed tumor growth and immune evasion. Conclusions: ILT4 was induced by activation of EGFR-AKT and ERK1/2 signaling in NSCLC cells. Overexpressed ILT4 suppressed tumor immunity by recruiting M2-like TAMs and impairing T cell response, while ILT4 inhibition prevented immunosuppression and tumor promotion. Furthermore, ILT4 inhibition enhanced the efficacy of PD-L1 inhibitor in EGFR wild-type but not in EGFR mutant NSCLC. Our study identified novel mechanisms for EGFR-mediated tumor immune escape, and provided promising immunotherapeutic strategies for patients with EGFR-activated NSCLC.

Chen X ，Gao A ，Zhang F ，Yang Z ，Wang S ，Fang Y ，Li J ，Wang J ，Shi W ，Wang L ，Zheng Y ，Sun Y ... -  《Theranostics》

The canonical TGF-β/Smad signalling pathway is involved in PD-L1-induced primary resistance to EGFR-TKIs in EGFR-mutant non-small-cell lung cancer.

Zhang Y ，Zeng Y ，Liu T ，Du W ，Zhu J ，Liu Z ，Huang JA ... -  《RESPIRATORY RESEARCH》

EGFR mutations induce the suppression of CD8(+) T cell and anti-PD-1 resistance via ERK1/2-p90RSK-TGF-β axis in non-small cell lung cancer.

Non-small cell lung cancer (NSCLC) patients with EGFR mutations exhibit an unfavorable response to immune checkpoint inhibitor (ICI) monotherapy, and their tumor microenvironment (TME) is usually immunosuppressed. TGF-β plays an important role in immunosuppression; however, the effects of TGF-β on the TME and the efficacy of anti-PD-1 immunotherapy against EGFR-mutated tumors remain unclear. Corresponding in vitro studies used the TCGA database, clinical specimens, and self-constructed mouse cell lines with EGFR mutations. We utilized C57BL/6N and humanized M-NSG mouse models bearing EGFR-mutated NSCLC to investigate the effects of TGF-β on the TME and the combined efficacy of TGF-β blockade and anti-PD-1 therapy. The changes in immune cells were monitored by flow cytometry. The correlation between TGF-β and immunotherapy outcomes of EGFR-mutated NSCLC was verified by clinical samples. We identified that TGF-β was upregulated in EGFR-mutated NSCLC by EGFR activation and subsequent ERK1/2-p90RSK phosphorylation. TGF-β directly inhibited CD8+ T cell infiltration, proliferation, and cytotoxicity both in vitro and in vivo, but blocking TGF-β did not suppress the growth of EGFR-mutated tumors in vivo. Anti-TGF-β antibody combined with anti-PD-1 antibody significantly inhibited the proliferation of recombinant EGFR-mutated tumors in C57BL/6N mice, which was superior to their monotherapy. Mechanistically, the combination of anti-TGF-β and anti-PD-1 antibodies significantly increased the infiltration of CD8+ T cells and enhanced the anti-tumor function of CD8+ T cells. Moreover, we found that the expression of TGF-β1 in EGFR-TKI resistant cell lines was significantly higher than that in parental cell lines. The combination of anti-TGF-β and nivolumab significantly inhibited the proliferation of EGFR-TKI resistant tumors in humanized M-NSG mice and prolonged their survival. Our results reveal that TGF-β expression is upregulated in NSCLC with EGFR mutations through the EGFR-ERK1/2-p90RSK signaling pathway. High TGF-β expression inhibits the infiltration and anti-tumor function of CD8+ T cells, contributing to the "cold" TME of EGFR-mutated tumors. Blocking TGF-β can reshape the TME and enhance the therapeutic efficacy of anti-PD-1 in EGFR-mutated tumors, which provides a potential combination immunotherapy strategy for advanced NSCLC patients with EGFR mutations.

Huang H ，Zhu X ，Yu Y ，Li Z ，Yang Y ，Xia L ，Lu S ... -  《Journal of Translational Medicine》

Tumor-derived ILT4 induces T cell senescence and suppresses tumor immunity.

Current immunotherapies including checkpoint blockade therapy have limited success rates in certain types of cancers. Identification of alternative checkpoint molecules for the development of effective strategies for tumor immunotherapy is urgently needed. Immunoglobulin-like transcript 4 (ILT4) is an immunosuppressive molecule expressed in both myeloid innate cells and malignant tumor cells. However, the role of tumor-derived ILT4 in regulating cancer biology and tumor immunity remains unclear. ILT4 expression in tumor cells and patient samples was determined by real-time PCR, flow cytometry, and immunohistochemistry. T cell senescence induced by tumor was evaluated using multiple markers and assays. Moreover, metabolic enzyme and signaling molecule expression and lipid droplets in tumor cells were determined using real-time PCR, western blot and oil red O staining, respectively. Loss-of-function and gain-of-function strategies were used to identify the causative role of ILT4 in tumor-induced T cell senescence. In addition, breast cancer and melanoma mouse tumor models were performed to demonstrate the role of ILT4 as a checkpoint molecule for tumor immunotherapy. We reported that ILT4 is highly expressed in human tumor cells and tissues, which is negatively associated with clinical outcomes. Furthermore, tumor-derived ILT4/PIR-B (ILT4 ortholog in mouse) is directly involved in induction of cell senescence in naïve/effector T cells mediated by tumor cells in vitro and in vivo. Mechanistically, ILT4/PIR-B increases fatty acid synthesis and lipid accumulation in tumor cells via activation of MAPK ERK1/2 signaling, resulting in promotion of tumor growth and progression, and induction of effector T cell senescence. In addition, blocking tumor-derived PIR-B can reprogram tumor metabolism, prevent senescence development in tumor-specific T cells, and enhance antitumor immunity in both breast cancer and melanoma mouse models. These studies identify a novel mechanism responsible for ILT4-mediated immune suppression in the tumor microenvironment, and prove a novel concept of ILT4 as a critical checkpoint molecule for tumor immunotherapy.

Gao A ，Liu X ，Lin W ，Wang J ，Wang S ，Si F ，Huang L ，Zhao Y ，Sun Y ，Peng G ... -  《Journal for ImmunoTherapy of Cancer》

Hypoxia-inducible factor-1α and nuclear factor-κB play important roles in regulating programmed cell death ligand 1 expression by epidermal growth factor receptor mutants in non-small-cell lung cancer cells.

Some driver gene mutations, including epidermal growth factor receptor (EGFR), have been reported to be involved in expression regulation of the immunosuppressive checkpoint protein programmed cell death ligand 1 (PD-L1), but the underlying mechanism remains obscure. We investigated the potential role and precise mechanism of EGFR mutants in PD-L1 expression regulation in non-small-cell lung cancer (NSCLC) cells. Examination of pivotal EGFR signaling effectors in 8 NSCLC cell lines indicated apparent associations between PD-L1 overexpression and phosphorylation of AKT and ERK, especially with increased protein levels of phospho-IκBα (p-IκBα) and hypoxia-inducible factor-1α (HIF-1α). Flow cytometry results showed stronger membrane co-expression of EGFR and PD-L1 in NSCLC cells with EGFR mutants compared with cells carrying WT EGFR. Additionally, ectopic expression or depletion of EGFR mutants and treatment with EGFR pathway inhibitors targeting MEK/ERK, PI3K/AKT, mTOR/S6, IκBα, and HIF-1α indicated strong accordance among protein levels of PD-L1, p-IκBα, and HIF-1α in NSCLC cells. Further treatment with pathway inhibitors significantly inhibited xenograft tumor growth and p-IκBα, HIF-1α, and PD-L1 expression of NSCLC cells carrying EGFR mutant in nude mice. Moreover, immunohistochemical analysis revealed obviously increased protein levels of p-IκBα, HIF-1α, and PD-L1 in NSCLC tissues with EGFR mutants compared with tissues carrying WT EGFR. Non-small-cell lung cancer tissues with either p-IκBα or HIF-1α positive staining were more likely to possess elevated PD-L1 expression compared with tissues scored negative for both p-IκBα and HIF-1α. Our findings showed important roles of phosphorylation activation of AKT and ERK and potential interplay and cooperation between NF-κB and HIF-1α in PD-L1 expression regulation by EGFR mutants in NSCLC.

Guo R ，Li Y ，Wang Z ，Bai H ，Duan J ，Wang S ，Wang L ，Wang J ... -  《-》