Disrupting usp14-mediated PARP1 dynamics reinstates mic-a/b-driven antigen-independent CD8⁺ t-cell killing in glioma

Antigen loss is a major mechanism of resistance to immunotherapy. MIC-A/B are stress-inducible ligands expressed by tumour cells that activate NKG2D on cytotoxic immune cells and mediate NKG2D-dependent, antigen-independent tumour cell killing, yet the mechanisms underlying their reduced expression in glioma remain unclear. Using single-cell RNA sequencing and spatial transcriptomics, we investigated ectopic MIC-A/B in mouse glioma and identified USP14 as a key regulator through deubiquitinase screening. Proteomic, coimmunoprecipitation, chromatin immunoprecipitation, immunofluorescence and ubiquitination assays characterized the interactions between USP14, PARP1 and NFIL3, while an intracranial tumour model combined USP14 inhibition and immunotherapy to evaluate effects on tumourigenesis and antitumour immunity. We found that MIC-A/B increased CD8⁺ T-cell infiltration and reversed exhaustion, and that USP14 stabilized PARP1 via K63-linked deubiquitination at lysine 653, reducing NFIL3 ..., , , # Disrupting usp14-mediated PARP1 dynamics reinstates mic-a/b-driven antigen-independent CD8⁺ t-cell killing in glioma Dataset DOI: [10.5061/dryad.vq83bk47n](https://doi.org/10.5061/dryad.vq83bk47n) This dataset contains processed data to reproduce key transcriptomic analyses testing the hypothesis that restoring stress-ligand signaling (MIC-A/B–NKG2D axis) and/or inhibiting the deubiquitinase USP14 (IU1) can reshape the immune microenvironment in the GL261 mouse intracranial glioma model, consistent with enhanced cytotoxic immune programs. Data are provided as per-sample normalized expression matrices to facilitate downstream reuse. ## Description of the data and file structure **The submission comprises three coordinated modules:** (1) CD45⁺ scRNA-seq from MICAB overexpression (MICAB-OE) and matched control tumors (per-sample normalized matrices), enabling immune-cell clustering/annotation and comparison of CD8⁺ T-cell state distributions (e.g., effector vs exhausted programs); ...抗原丢失是免疫治疗耐药的核心机制之一。主要组织相容性复合体I类链相关蛋白A/B（MIC-A/B）是肿瘤细胞表达的应激诱导配体，可激活细胞毒性免疫细胞表面的NKG2D受体，介导依赖NKG2D、不依赖抗原的肿瘤细胞杀伤作用，但胶质瘤中其表达下调的具体机制仍未明确。本研究借助单细胞RNA测序与空间转录组学技术，对小鼠胶质瘤中的异位MIC-A/B进行了分析，并通过去泛素化酶筛选鉴定出USP14作为其关键调控因子。本研究通过蛋白质组学、免疫共沉淀（coimmunoprecipitation）、染色质免疫共沉淀（chromatin immunoprecipitation）、免疫荧光及泛素化实验，阐明了USP14、PARP1与NFIL3之间的相互作用；同时构建颅内肿瘤模型，联合USP14抑制剂与免疫治疗，评估其对肿瘤发生及抗肿瘤免疫的影响。本研究发现，MIC-A/B可促进CD8⁺ T细胞浸润并逆转其耗竭状态；USP14通过在赖氨酸653位点进行K63连接型去泛素化修饰稳定PARP1，进而下调NFIL3……# 阻断USP14介导的PARP1动态调控可恢复胶质瘤中MIC-A/B驱动的抗原非依赖型CD8⁺ T细胞杀伤作用 数据集DOI：[10.5061/dryad.vq83bk47n](https://doi.org/10.5061/dryad.vq83bk47n) 本数据集包含可复现关键转录组学分析的处理后数据，用于验证以下假说：恢复应激配体信号通路（MIC-A/B–NKG2D轴）或/和抑制去泛素化酶USP14（IU1），可重塑GL261小鼠颅内胶质瘤模型中的免疫微环境，同时增强细胞毒性免疫程序。数据集以逐样本标准化表达矩阵的形式提供，便于后续复用。 ## 数据与文件结构说明 **本次提交的数据集包含三个协同模块：** (1) 过表达MICAB（MICAB-OE）及匹配对照肿瘤的CD45⁺ 单细胞RNA测序数据（逐样本标准化矩阵），可用于免疫细胞聚类注释及CD8⁺ T细胞状态分布的比较（如效应型与耗竭型程序的差异）；……