ILT4 functions as a potential checkpoint molecule for tumor immunotherapy.

Immune checkpoint blockade therapy targeting CTLA4 and PD-1/PD-L1 is a promising strategy in the treatment of different types of cancers. However, the clinical success rates of these therapies are still moderate and varied among cancer types. Therefore, identification of alternative and novel checkpoint molecules or interrupting tolerogenic pathways is urgently needed for successful tumor immunotherapy. Immunoglobulin-like transcript 4 (ILT4) is as an immunosuppressive molecule predominantly expressed in myeloid cells, including monocytes, macrophages, dendritic cells and granulocytes. Recent studies revealed that ILT4 is also enriched in tumor cells and stroma cells in the tumor microenvironment of various malignancies, modulating the biological behaviors of tumor cells and promoting their immune escape. However, the underlying mechanisms responsible for ILT4-mediated tumor development and progression are still poorly understood. In this review, we explore the functional role of ILT4 as a novel checkpoint molecule in cancers. We specifically discuss the mechanisms mediated by ILT4 for controlling tumor malignant behaviors, impairing effector anti-tumor immune responses, and sustaining the tumor suppressive microenvironment. We also highlight the potential role of ILT4 as a novel immune checkpoint target for tumor immunotherapy. Improved understanding of these issues is critical for elucidation of the role of ILT4 in tumor pathogenesis and should open new avenues for cancer immunotherapy specifically targeting this novel and alternative checkpoint molecule.

Gao A ，Sun Y ，Peng G 《BIOCHIMICA ET BIOPHYSICA ACTA-REVIEWS ON CANCER》

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》

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》

PD-1/PD-L1 immune checkpoint: Potential target for cancer therapy.

Recent studies show that cancer cells are sometimes able to evade the host immunity in the tumor microenvironment. Cancer cells can express high levels of immune inhibitory signaling proteins. One of the most critical checkpoint pathways in this system is a tumor-induced immune suppression (immune checkpoint) mediated by the programmed cell death protein 1 (PD-1) and its ligand, programmed death ligand 1 (PD-L1). PD-1 is highly expressed by activated T cells, B cells, dendritic cells, and natural killer cells, whereas PD-L1 is expressed on several types of tumor cells. Many studies have shown that blocking the interaction between PD-1 and PD-L1 enhances the T-cell response and mediates antitumor activity. In this review, we highlight a brief overview of the molecular and biochemical events that are regulated by the PD-1 and PD-L1 interaction in various cancers.

Dermani FK ，Samadi P ，Rahmani G ，Kohlan AK ，Najafi R ... -  《-》

Bladder cancer, a unique model to understand cancer immunity and develop immunotherapy approaches.

With the mechanistic understanding of immune checkpoints and success in checkpoint blockade using antibodies for the treatment of certain cancers, immunotherapy has become one of the hottest areas in cancer research, with promise of long-lasting therapeutic effect. Currently, however, only a proportion of cancers have a good response to checkpoint inhibition immunotherapy. Better understanding of the cancer response and resistance mechanisms is essential to fully explore the potential of immunotherapy to cure the majority of cancers. Bladder cancer, one of the most common and aggressive malignant diseases, has been successfully treated both at early and advanced stages by different immunotherapeutic approaches, bacillus Calmette-Guérin (BCG) intravesical instillation and anti-PD-1/PD-L1 immune checkpoint blockade, respectively. Therefore, it provides a good model to investigate cancer immune response mechanisms and to improve the efficiency of immunotherapy. Here, we review bladder cancer immunotherapy with equal weight on BCG and anti-PD-1/PD-L1 therapies and demonstrate why and how bladder cancer can be used as a model to study the predictors and mechanisms of cancer immune response and shine light on further development of immunotherapy approaches and response predictive biomarkers to improve immunotherapy of bladder cancer and other malignancies. We review the success of BCG and anti-PD-1/PD-L1 treatment of bladder cancer, the underlying mechanisms and the therapeutic response predictors, including the limits to our knowledge. We then highlight briefly the adaptation of immunotherapy approaches and predictors developed in other cancers for bladder cancer therapy. Finally, we explore the potential of using bladder cancer as a model to investigate cancer immune response mechanisms and new therapeutic approaches, which may be translated into immunotherapy of other human cancers. © 2019 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland.

Song D ，Powles T ，Shi L ，Zhang L ，Ingersoll MA ，Lu YJ ... -  《-》