LTBR acts as a novel immune checkpoint of tumor-associated macrophages for lung adenocarcinoma immunotherapy

Background: Tumor-associated macrophages (TAMs) has been reported to mediate the resistance of LUAD patients to ICI therapy. However, its underlying mechanisms and whether TAMs could be promising targets to overcome the non-response after ICI application remain to be unveiled. Methods: Immune multi-omics analysis was implemented with a large clinical cohort encompassing 1045 LUAD cases to screen out potential immune checkpoints. scRNA-seq analysis revealed their distribution in LUAD immune cells. The key molecule and related mechanisms were investigated in vitro and in vivo. Clinical verification in ICI therapy cohorts and TAMs-targeted delivery system had been performed for further clinical translation. Findings: LTBR, as a potential immune checkpoint, was screened out. Its high expression, duplication and low methylation were corelated with LUAD unfavorable survival. scRNA-seq and FACS analysis showed the highest expression of LTBR in TAMs. Moreover, TAM-targeted inhibition of LTΒR disrupted TISM following with CD8+ T cell boost as well as G-MDSC and M2-like TAM reduction, especially improved the therapeutic effect of ICI. Further studies showed that LTΒR maintained TAM immunosuppressive activity and M2 phenotype by non-canonical NF-kB signaling and Wnt/beta-catenin signaling. Clinical investigation indicated that LTΒR could predict LUAD stages, immunotherapy responses and clinical outcomes after ICI treatment. Conclusions: Through integrative analysis of immune Multi-Omics and ScRNA-seq data (iMOS), our study develops an immune-checkpoint discovery pipeline, and identifies LTBR as a novel immune checkpoint of TAMs that orchestrates TISM through non-canonical NF-kB and Wnt/beta-catenin signaling. Finally, we propose a promising strategy by targeting LTBR+ TAMs for improving the effect of ICI therapy.

背景：已有研究表明，肿瘤相关巨噬细胞（Tumor-associated macrophages, TAMs）可介导肺腺癌（Lung Adenocarcinoma, LUAD）患者对免疫检查点抑制剂（Immune Checkpoint Inhibitor, ICI）治疗的耐药性。然而，其背后的潜在机制，以及TAMs是否可作为克服ICI应用后治疗无应答的潜在靶点，仍有待阐明。方法：本研究针对包含1045例肺腺癌患者的大型临床队列开展免疫多组学分析，以筛选潜在的免疫检查点；通过单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）分析，明确目标分子在肺腺癌免疫细胞中的分布特征；在体外及体内实验中对关键分子及其相关机制进行探究；此外，在ICI治疗队列中开展临床验证，并构建TAMs靶向递送系统，以推进后续临床转化。结果：本研究筛选得到潜在免疫检查点淋巴毒素β受体（Lymphotoxin beta receptor, LTBR）。该分子高表达、基因拷贝数扩增及低甲基化水平与肺腺癌患者不良生存显著相关。单细胞RNA测序及流式细胞术（Fluorescence Activated Cell Sorting, FACS）分析显示，LTBR在TAMs中表达量最高。进一步研究发现，靶向TAMs抑制LTBR可破坏肿瘤免疫抑制微环境（Tumor Immune Suppressive Microenvironment, TISM），同时促进CD8+ T细胞浸润，并减少粒细胞系髓系来源抑制细胞（granulocytic myeloid-derived suppressor cells, G-MDSC）及M2型肿瘤相关巨噬细胞的数量，尤其可增强ICI的治疗效果。机制研究表明，LTBR可通过非经典NF-κB信号通路及Wnt/β-连环蛋白信号通路维持TAMs的免疫抑制活性与M2表型。临床调研显示，LTBR可用于预测肺腺癌分期、免疫治疗应答情况及ICI治疗后的临床结局。结论：本研究通过整合免疫多组学与单细胞RNA测序数据（iMOS），构建了一套免疫检查点筛选流程，并鉴定出LTBR是TAMs的新型免疫检查点，其可通过非经典NF-κB及Wnt/β-连环蛋白信号通路调控肿瘤免疫抑制微环境。最终，本研究提出了一种靶向LTBR阳性TAMs以增强ICI治疗效果的潜在临床转化策略。