TGFBR2-High mesenchymal glioma stem cells phenocopy regulatory T cells to suppress CD4+ and CD8+ T cell function

Attempts to activate an anti-tumor immune response in glioblastoma (GBM) have been met with many challenges due to its inherently immunosuppressive tumor microenvironment. The degree and mechanisms by which molecularly and phenotypically diverse tumor-propagating glioma stem cells (GSCs) contribute to this state are poorly defined. In this study, our multifaceted approach combining bioinformatics analyses of clinical and experimental datasets, single-cell sequencing, and molecular and pharmacologic manipulation of patient-derived cells identified GSCs expressing immunosuppressive effectors mimicking regulatory T cells (Tregs). We show that this Immunosuppressive Treg-Like (ITL) GSC state is specific to the mesenchymal GSC subset and is associated with and driven specifically by TGF-beta type II receptor (TGFBR2) in contrast to TGFBR1. Transgenic TGFBR2 expression in patient-derived GBM neurospheres promoted a mesenchymal transition and induced a 6-gene ITL signature consisting of CD274 (PD-L1), NT5E (CD73), ENTPD1 (CD39), LGALS1 (galectin-1), PDCD1LG2 (PD-L2), and TGFB1. This TGFBR2-driven ITL signature was identified in clinical GBM specimens, patient-derived GSCs and systemic mesenchymal malignancies. TGFBR2High GSCs inhibited CD4+ and CD8+ T cell viability and their capacity to kill GBM cells, effects reversed by pharmacologic and shRNA-based TGFBR2 inhibition. Collectively, our data identify an immunosuppressive GSC state that is TGFBR2-dependent and susceptible to TGFBR2-targeted therapeutics. GBM1A neurospheres derived from patient GBMs were transduced to transgenically express both POU5F1 (Oct4) and SOX2. Parental cells (GBM1A) and cells co-expressing transgenic Oct4 and Sox2 (GBM1A-OS) were analyzed using scRNA-seq.

针对胶质母细胞瘤（glioblastoma, GBM）激活抗肿瘤免疫应答的研究尝试，因其固有的免疫抑制性肿瘤微环境而面临诸多挑战。分子与表型异质性的肿瘤起始性胶质瘤干细胞（tumor-propagating glioma stem cells, GSCs）在促成该免疫抑制状态中的作用程度与具体机制，目前仍尚未明确。本研究整合临床与实验数据集的生物信息学分析、单细胞测序技术，以及对患者来源细胞的分子与药理学操控手段等多维度研究策略，成功鉴定出一类表达模拟调节性T细胞（regulatory T cells, Tregs）免疫抑制效应分子的GSCs。研究证实，这类免疫抑制性类调节性T细胞（Immunosuppressive Treg-Like, ITL）GSC状态仅特异性存在于间充质亚型GSCs中，且其发生与维持特异性依赖于转化生长因子βⅡ型受体（TGF-beta type II receptor, TGFBR2）的调控，而非转化生长因子βⅠ型受体（TGFBR1）。在患者来源的GBM神经球中转基因过表达TGFBR2，可促进细胞发生间充质转化，并诱导形成由CD274（PD-L1）、NT5E（CD73）、ENTPD1（CD39）、LGALS1（半乳凝素-1）、PDCD1LG2（PD-L2）及TGFB1这6个基因组成的ITL特征基因集。这种由TGFBR2驱动的ITL特征基因集，在临床GBM标本、患者来源的GSCs以及系统性间充质恶性肿瘤中均被检测到。高表达TGFBR2的GSCs可抑制CD4+与CD8+ T细胞的存活率及其杀伤GBM细胞的能力，而通过药理学干预或基于短发夹RNA（shRNA）的TGFBR2敲低手段可逆转这一免疫抑制效应。综上，本研究鉴定出一种依赖于TGFBR2的免疫抑制性GSC状态，该状态可被靶向TGFBR2的治疗手段有效干预。本研究将源自患者GBM的GBM1A神经球进行病毒转导，使其同时转基因表达POU5F1（Oct4）与SOX2。随后分别对亲本细胞（GBM1A）以及共表达转基因Oct4与Sox2的细胞（GBM1A-OS）进行单细胞RNA测序（scRNA-seq）分析。