Zhou Siqi, Ye Shiqi, Chen Liang, Ge Qintao, Lu Jiahe, Anwaier Aihetaimujiang, Tian Xi, Wang Zhongyuan, Zhu Shuxuan, Chang Kun, Yang Jianfeng, Li Tian, Zhang Hailiang, Ye Dingwei, Xiang Jianfeng, Xu Wenhao
Department of Urology, Fudan University Shanghai Cancer Center; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai, 200032, China.
Shanghai Genitourinary Cancer Institute, Shanghai, 200032, China.
Int J Biol Sci. 2025 Jun 9;21(9):3827-3851. doi: 10.7150/ijbs.113737. eCollection 2025.
Tertiary lymphoid structures (TLSs), organized clusters of immune cells within non-lymphoid tissues, significantly influence tumor progression and therapeutic response. However, their prognostic relevance and underlying regulatory mechanisms in clear cell renal cell carcinoma (ccRCC) remain insufficiently characterized. We integrated transcriptomic and clinical data from 928 ccRCC patients to construct a TLS-related prognostic RiskScore using machine learning algorithms. TLS maturation heterogeneity was characterized via immunohistochemistry and multiplex immunofluorescence analyses. The functional role of interferon regulatory factor 4 (IRF4), a key regulator within the TLS gene network, was investigated using assays. Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics were employed to dissect the involvement of IRF4 in TLS formation and maturation. The derived TLS-associated signature RiskScore, comprising effectively stratified patients into distinct prognostic groups and showed robust associations with clinical parameters, tumor microenvironment (TME) features, and predicted immunotherapy responses. Functional assays demonstrated that IRF4 significantly enhanced the malignant phenotype of 786-O and 769-P ccRCC cells. Clinically, elevated IRF4 expression independently predicted worse patient outcomes, characterized by a predominance of immature TLS phenotypes, reduced TLS density, and diminished CD8⁺ T cell infiltration. Mechanistically, scRNA-seq analyses revealed that active IRF4 signaling was predominantly confined to immature B cell states and was inversely associated with TLS maturation trajectories. Spatial transcriptomics further confirmed IRF4 enrichment within TLS regions, notably spatially segregated from high endothelial venules (HEVs) and mature TLS compartments. In conclusion, this study establishes a robust TLS-related prognostic signature for ccRCC and elucidates the mechanistic role of IRF4 in promoting TLS immaturity and immune dysfunction. By potentially recruiting immature B cells while impairing their maturation, IRF4 contributes to an ineffective anti-tumor immune landscape, offering a promising target for therapeutic intervention.
三级淋巴结构（TLSs）是非淋巴组织内有组织的免疫细胞簇，对肿瘤进展和治疗反应有显著影响。然而，它们在透明细胞肾细胞癌（ccRCC）中的预后相关性及潜在调控机制仍未得到充分表征。我们整合了928例ccRCC患者的转录组和临床数据，使用机器学习算法构建了一个与TLS相关的预后风险评分。通过免疫组织化学和多重免疫荧光分析来表征TLS成熟异质性。使用相关检测方法研究了TLS基因网络中的关键调节因子干扰素调节因子4（IRF4）的功能作用。采用单细胞RNA测序（scRNA-seq）和空间转录组学来剖析IRF4在TLS形成和成熟中的作用。所推导的与TLS相关的特征风险评分有效地将患者分为不同的预后组，并与临床参数、肿瘤微环境（TME）特征以及预测的免疫治疗反应显示出强烈关联。功能检测表明，IRF4显著增强了786-O和769-P ccRCC细胞的恶性表型。在临床上，IRF4表达升高独立预测患者预后较差，其特征为不成熟TLS表型占主导、TLS密度降低以及CD8⁺T细胞浸润减少。从机制上讲，scRNA-seq分析显示活跃的IRF4信号主要局限于未成熟B细胞状态，并且与TLS成熟轨迹呈负相关。空间转录组学进一步证实了IRF4在TLS区域内的富集，特别是在空间上与高内皮小静脉（HEVs）和成熟TLS区室分隔开。总之，本研究为ccRCC建立了一个强大的与TLS相关的预后特征，并阐明了IRF4在促进TLS不成熟和免疫功能障碍中的机制作用。通过潜在地招募未成熟B细胞同时损害其成熟，IRF4导致抗肿瘤免疫格局无效，为治疗干预提供了一个有前景的靶点。
