ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.
ILT4 inhibition prevents TAM- and dysfunctional T cell-mediated immunosuppression and enhances the efficacy of anti-PD-L1 therapy in NSCLC with EGFR activation.
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ILT 4抑制可预防TAM和功能失调的T细胞介导的免疫抑制，并增强抗PD-L1治疗在EGFR活化NSCLC中的疗效。
作者：
Chen X;Gao A;Zhang F;Yang Z;Wang S;Fang Y;Li J;Wang J;Shi W;Wang L;Zheng Y;Sun Y
基本原理：针对PD-1/PD-L1通路的免疫检查点抑制剂（ICI）在非小细胞肺癌（NSCLC）患者中显示出有限的成功，尤其是在具有活化表皮生长因子受体（EGFR）突变的患者中。目前迫切需要阐明EGFR介导的肿瘤免疫逃逸的机制，并开发有效的免疫治疗方法。免疫球蛋白样转录物（ILT）4是一种最初在骨髓细胞中发现的重要免疫抑制分子，在实体瘤细胞中富集并促进NSCLC的恶性行为。然而，ILT 4过表达的上游调控及其在EGFR激活的NSCLC肿瘤免疫中的作用尚不清楚。研究方法：使用免疫组织化学（IHC）、实时PCR、Western印迹、免疫荧光和流式细胞术分析人NSCLC组织和细胞系中ILT 4表达和EGFR磷酸化。使用mRNA微阵列和癌症基因组图谱（TCGA）数据库分析研究EGFR调节的ILT 4表达的分子信号传导，然后通过蛋白质印迹法证实。用CCK-8增殖和凋亡实验检测ILT 4对肿瘤细胞增殖和凋亡的调节作用。采用Transwell迁移试验、流式细胞术、酶联免疫吸附试验（ELISA）和实时荧光PCR检测ILT 4和PD-L1对肿瘤相关巨噬细胞（TAM）募集和极化的影响，采用CFSE增殖试验、凋亡试验、流式细胞术、ELISA和细胞溶解试验检测ILT 4和PD-L1对T细胞存活和细胞毒性的影响。在接种稳定的EGFR过表达刘易斯肺癌（LLC）细胞的C57 BL/6小鼠和接种EGFR突变体、吉非替尼抗性PC 9（PC 9-GR）或EGFR过表达野生型H1299细胞的人源化NSG小鼠中建立靶向成对Ig样受体B（PIR-B，小鼠中ILT 4的直系同源物）/ILT 4和/或PD-L1的肿瘤免疫治疗模型。PIR-B和ILT 4抑制通过特异性敲减慢病毒的感染实施，并且使用人/小鼠中和抗体阻断PD-L1。在注射PIR-B下调的LLC细胞的NSG小鼠中建立肿瘤生长模型以评估PIR-B对肿瘤增殖的影响。采用流式细胞术检测小鼠脾脏和血液中巨噬细胞和T细胞的频率和表型，采用免疫组化和免疫荧光法检测肿瘤组织中巨噬细胞和T细胞的频率和表型。结果：NSCLC组织中ILT 4的表达与EGFR的磷酸化水平呈正相关。使用NSCLC细胞系，我们证明了ILT 4通过酪氨酸激酶突变诱导的和表皮生长因子（EGF）依赖的EGFR激活以及随后的AKT/ERK 1/2磷酸化上调。在EGFR激活的肿瘤细胞中过表达ILT 4诱导TAM募集和M2样极化，这损害了T细胞功能。ILT 4还直接抑制T细胞增殖、细胞毒性和IFN-γ表达和分泌。在体外EGFR激活的细胞系和体内野生型EGFR激活的C57 BL/6和人源化NSG免疫治疗模型中，ILT 4（PIR-B）或PD-L1抑制通过抵消TAM和功能失调的T细胞诱导的免疫抑制性TME来增强抗肿瘤免疫并抑制肿瘤进展;两种分子的组合抑制显示出最显著的肿瘤收缩。令人惊讶的是，在EGFR突变体、TKI抗性人源化NSG免疫治疗模型中，单独的ILT 4抑制而不是与PD-L1抑制剂组合抑制肿瘤生长和免疫逃避。结论：ILT 4在NSCLC细胞中通过激活EGFR-AKT和ERK 1/2信号而被诱导表达。过表达的ILT 4通过募集M2样TAM和损害T细胞应答来抑制肿瘤免疫，而ILT 4抑制则防止免疫抑制和肿瘤促进。此外，ILT 4抑制可增强PD-L1抑制剂在EGFR野生型NSCLC中的疗效，但在EGFR突变型NSCLC中则无此作用。我们的研究确定了EGFR介导的肿瘤免疫逃逸的新机制，并为EGFR激活的NSCLC患者提供了有希望的免疫治疗策略。
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.
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DOI：
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