The role of TSPO in T-cell immune control of glioblastoma

胶质母细胞瘤(GB)是最恶性、最常见的原发脑肿瘤，中位生存期不到21个月。除了分子水平的改变外，GB的发病机制在很大程度上依赖于免疫逃逸机制。转运蛋白18 kDa(TSPO)与正常脑组织相比表达上调，与肿瘤的恶性程度呈正相关，已被用作判断胆囊癌预后的生物标志物。根据初步发现，我们在第一个资助期假设，TSPO通过保护脑瘤起始细胞(BTIC)免受免疫系统的排斥而促进了GB的进展。我们证明了在BTICs与肿瘤反应性T细胞和T细胞衍生因子接触时可以诱导TSPO。T细胞来源的干扰素γ和肿瘤坏死因子α是BTICs发生TSPO的主要诱因。我们还可以通过分析原始GB样本的单细胞RNASeq数据集，证实干扰素γ和肿瘤坏死因子α介导的途径是原代人GB原位表达TSPO的主要原因。关于TSPO在GB免疫抵抗中的作用，我们证明TSPO的下调可使GB细胞株和BTICs对T细胞介导的细胞毒作用致敏，并削弱BTICs的迁移能力。我们还发现，TSPO选择性地保护BTICs免受TRAIL诱导的细胞凋亡。相应地，合成的TSPO配体对TSPO活性的实验调节一致地增加了TRAIL的促凋亡作用，并损害了线粒体的活性。对TSPO+/-BTICs的转录组表达分析显示，TSPO调控的差异表达基因与BTICs的抗凋亡、干细胞特性和诱导耐药间充质特性相关。在第二个资助期内，我们的第一个目标是揭示TSPO如何协调GB的干性和对免疫细胞诱导的凋亡的抗性，从而诱导形成耐药的GB表型。在这里，我们将特别研究TSPO调控的茎和抗凋亡驱动基因与TSPO对线粒体和外在细胞凋亡调控的直接影响的相对影响。为此，我们还将调查TSPO如何影响DNA中调控元件的可及性。其次，我们将通过在两个同基因小鼠模型中研究肿瘤排斥反应、免疫浸润以及对CAR T细胞实验性免疫治疗的抵抗力，来研究TSPO在GB体内免疫抵抗中的相关性。最后，我们将使用在我们的体外和体内模型中产生的结果来开发翻译策略来逆转TSPO在GB中施加的免疫抵抗。总体而言，我们将继续将抑制TSPO作为提高针对GB的免疫治疗效率的新手段。

Glioblastoma (GB) is the most malignant and common type of primary brain tumor with a median survival of less than 21 months. Apart from molecular alterations, the pathogenesis of GB is largely dependent on immune escape mechanisms. Translocator protein 18 kDa (TSPO) has been used as a prognostic biomarker for GB, due to its up-regulated expression compared to normal brain and positive correlation with malignancy. Based on preliminary findings we hypothesized in the first funding period that TSPO contributes to GB progression by protecting brain tumor initiating cells (BTICs) against rejection by the immune system. We demonstrated that TSPO is induced in BTICs upon their contact with tumor-reactive T-cells and T-cell derived factors. We could identify T-cell derived IFNγ and TNFα as the main inducers of TSPO in BTICs. We could also confirm IFNγ- and TNFα-mediated pathways as major causes of TSPO expression in primary human GB situ by analyzing single-cell RNA-Seq data set from primary GB samples. In regard to its` role in immune resistance of GB we demonstrated that downregulation of TSPO sensitized GB cell lines and BTICs against T cell-mediated cytotoxicity and impaired the migratory capacity of BTICs. We also found that TSPO protects BTICs selectively against TRAIL-induced apoptosis. In accordance, experimental regulation of TSPO activity by synthetic TSPO ligands consistently increased the pro-apoptotic effects of TRAIL and impaired mitochondrial activity. Transcriptome expression analysis on TSPO+/- BTICs revealed differentially expressed genes controlled by TSPO that were associated with apoptosis-resistance, stemness features, and induction of therapy-resistant mesenchymal characteristics in BTICs. Within the second funding period, our first aim is to unravel the mechanisms how TSPO orchestrates stemness and resistance against immune cell induced apoptosis in GB and thereby induces the formation of a treatment-resistant GB phenotype. Here, we will particularly study the relative impact of stemness- and anti-apoptotic driving genes regulated by TSPO compared to direct effects of TSPO on mitochondrial and extrinsic apoptosis regulation. To this end, we will also investigate how TSPO affects the accessibility of regulatory elements within the DNA. Second, we will study the relevance of TSPO in immune resistance of GB in vivo by investigating tumor rejection and immune infiltration as well as resistance against experimental immunotherapy with CAR T-cells in two syngeneic mouse models. Finally, we will use the results generated in our in vitro and in vivo models to develop translational strategies to revert immune resistance exerted by TSPO in GB. Overall, we will continue to establish TSPO inhibition as a new means to improve efficiency of immunotherapy against GB.