Association of (tumor-) stem cell characteristics with failure of NKG2D-mediated immunosurveillance

现有数据表明，只有肿瘤细胞的一个子集，即所谓的肿瘤干细胞（TSC），负责癌症的启动、维持和常规治疗后的复发。然而，恶性疾病的发生发展过程在很大程度上还取决于转化细胞逃避肿瘤免疫监视的能力，其中NK细胞作为天然免疫的细胞毒性淋巴细胞起着重要作用。NKG2D是一种活化NK受体，它的发现导致了免疫监视假说的复兴，因为它的配体（NKG2DL）在恶性转化时被诱导，并有效地刺激抗肿瘤免疫。肿瘤细胞采用各种策略来逃避nkg2d介导的免疫监视，其中包括从细胞表面脱落NKG2DL以减少NK细胞的刺激信号量。其他机制包括直接调节nkg2dl的表达，例如通过表观遗传调控。目前对TSC与免疫系统之间的相互作用以及TSC与非TSC相比是否具有逃避免疫监测的特殊性质所知甚少。我们对急性髓系白血病（AML）患者白血病细胞的初步分析表明，AML-TSC（以CD34的表达、体外克隆生成能力和NSG小鼠异种移植时的体内白血病启动特性为特征）与Non-TSC相比，NKG2DL的表达显著降低，这也导致NK细胞特异性地对TSC部分的反应性功能受损。此外，缺乏NKG2DL表达能够识别出一个独立于CD34表达的AML细胞亚群，显示出增强的克隆生成能力，这表明缺乏NKG2DL表达可能被用作鉴定AML中TSC的新标记物。基于这些数据，在拟议的项目中，我们的目标是全面表征AML- tsc与非tsc中NKG2DL的表达及其对nk介导的AML免疫监测的影响。此外，我们计划阐明AML-TSC与非tsc中NKG2DL水平差异的分子机制，以及通过治疗诱导AML-TSC中NKG2DL表达以增强抗肿瘤免疫的可能性。最后，AML-TSC中NKG2DL的表达与患者的临床病程相关。综上所述，该项目旨在进一步阐明AML-TSC的特性，特别是它们逃避肿瘤免疫监测的能力，从而也有望识别与恶性细胞干细胞特性相关的新型免疫雕刻特性。

Available data suggest that only a subset of tumor cells, the so-called tumor stem cells (TSC), are responsible for initiation, maintenance and also relapse after conventional therapy of cancer. However, the development and course of malignant diseases also largely depends on the ability of the transformed cells to evade tumor immunesurveillance, for which NK cells as cytotoxic lymphocytes of innate immunity play an important role. NKG2D is an activating NK receptor, the discovery of which has led to the renaissance of the immune surveillance hypothesis, because its ligands (NKG2DL) are induced upon malignant transformation and potently stimulate anti-tumor immunity. Tumor cells employ various strategies to evade NKG2D-mediated immunesurveillance, which, among others, comprise shedding of NKG2DL from the cell surface to reduce the amount of stimulatory signals for NK cells. Other mechanisms involve direct modulation of NKG2DL-expression, for example via epigenetic regulation. Only little is currently known about the interactions between TSC and the immune system and on whether TSC, as compared to non-TSC, possess special properties to evade immunesurveillance. Our preliminary analyses with leukemia cells of patients with Acute Myeloid Leukemia (AML) reveals that AML-TSC (characterized by expression of CD34, their in vitro clonogenic ability and their in vivo leukaemia-initiating properties upon xenotransplantation in NSG mice) display substantially reduced NKG2DL expression as compared to Non-TSC, which also results in functionally impaired NK cell reactivity specifically against the TSC fraction. Moreover, lack of NKG2DL-expression is able to identify a subpopulation of AML cells displaying enhanced clonogenic capacity independently of CD34 expression, suggesting that lacking NKG2DL expression may be used as a novel marker for the identification of TSC in AML. Based on these data, in the proposed project we aim to comprehensively characterize NKG2DL expression in AML-TSC versus Non-TSC and the consequences for NK-mediated AML immunesurveillance. Moreover, we plan to elucidate the molecular mechanisms responsible for the differential NKG2DL levels in AML-TSC versus Non-TSC as well as the possibilities to therapeutically induce NKG2DL expression in AML-TSC to reinforce anti-tumor immunity. Finally the prevalence of NKG2DL expression on AML-TSC will be correlated with the clinical course of the disease in patients. Taken together, the proposed project serves to further elucidate properties of AML-TSC, especially with regard to their ability to evade tumor immunesurveillance and thereby also holds promise to identify novel immune-sculpting characteristics associated with stemness properties of malignant cells in general.