Natural killer cells and immune control: Deciphering the transcription factors that regulate expression of the NKG2D-ligand MICA

NKG2D（由KLRK1基因编码，杀伤细胞凝集素样受体亚家族K，成员1）是一种c型凝集素受体，是参与肿瘤监测的重要激活免疫受体。NKG2D表达于大多数细胞毒性淋巴细胞表面，如NK细胞、CD8+ T细胞、一些γδ T细胞，也可能是一些CD4+ T细胞，被称为受损或危险细胞的传感器。NKG2D的配体（NKG2D- l）通常不在健康细胞上表达，而是在恶性细胞表面诱导表达。逃避先天nkg2d介导的免疫监视是许多实体和造血肿瘤的标志，并且依赖于可溶性NKG2D-L的蛋白水解脱落和释放。这使得靶细胞对依赖nkg2d的NK细胞攻击不可见，并且导致效应细胞上受体表面表达的下调。靶向NKG2D-NKG2D-L轴刺激抗肿瘤免疫是一种很有前景的治疗方法。在这里，我们的目标是确定特定的转录因子，调节MHC I类多肽相关序列A （NKG2D-L， MICA）的诱导表达。直到今天，调控MICA上调的转录因子网络还没有被定义。更好地了解影响NKG2D-L表达的因素将有助于开发新的治疗策略，旨在提高肿瘤细胞表面NKG2D-L的表达，从而更有效地杀死NK细胞。在我们之前的工作中，我们证明了CBP/p300乙酰转移酶和cAMP响应元件结合蛋白（CREB）对MICA的诱导表达起关键作用。使用enChIP方法，我们确定了几个转录因子，这些转录因子响应与活性MICA启动子相关的组蛋白去乙酰化酶抑制，包括kr<s:1> ppel样因子4 (KLF4)，阴阳1 （YY1）和ccctc结合因子（CTCF）。该项目的目的是评估这些候选蛋白，特别是转录因子，在MICA表达调控中的作用。为此，我们计划在启动子报告基因试验中分析这些因子调节MICA转录的程度。计划进一步的过表达和耗尽方法来研究对MICA表面蛋白表达和NK细胞介导的靶细胞消除的影响。最后，将在急性骨髓瘤白血病患者样本中评估所确定因素的临床相关性。为此，我们将进行Rhapsodytm （Beckman Coulter）单细胞测序，以关联MICA和候选因子表达，并测量这些因子是否调节MICA表达（功能的获得/丧失）和对NK细胞的易感性。我们期望我们将确定MICA的积极调节因子，从长远来看，这可能代表新的治疗靶点。

NKG2D (encoded by the KLRK1 gene, killer cell lectin-like receptor subfamily K, member 1) is a C-type lectin receptor and an important activating immunoreceptor involved in tumor surveillance. NKG2D is expressed on the surface of most cytotoxic lymphocytes such as NK cells, CD8+ T cells, some γδ T cells, and possibly also some CD4+ T cells and known as a sensor for damaged or dangerous cells. Ligands for NKG2D (NKG2D-L) are generally not expressed on healthy cells but are induced on the surface of malignant cells. Evasion from the innate NKG2D-mediated immune surveillance is a hallmark of many solid and hematopoietic tumors and depends among other strategies on proteolytic shedding and release of soluble NKG2D-L. This renders target cells invisible to an NKG2D-dependent NK cell attack and moreover causes a downregulation of receptor surface expression on effector cells. Targeting the NKG2D-NKG2D-L axis to stimulate anti-tumor immunity is a promising therapeutic approach. Here, we aim to identify specific transcription factors, which regulate the inducible expression of MHC Class I Polypeptide-Related Sequence A (NKG2D-L, MICA). Until today the network of transcription factors regulating the up-regulation of MICA is not defined. A better understanding of factors that direct NKG2D-L expression will contribute to the development of novel therapeutic strategies aiming at an increased NKG2D-L expression on the tumor cell surface and thus more efficient killing by NK cells. In our previous work, we demonstrated that CBP/p300 acetyltransferases and the cAMP responsive element binding protein (CREB) contribute critically to the inducible expression of MICA. Using an enChIP approach we identified several transcription factors which are in response to histone deacetylase inhibition associated with the active MICA promoter including Krüppel-like factor 4 (KLF4), Yin Yang 1 (YY1), and CCCTC-Binding Factor (CTCF). The aim of this project is to assess the involvement of these candidate proteins, in particular transcription factors, in the regulation of MICA expression. For this purpose, we plan to analyze to what extend these factors regulate the transcription of MICA in promoter-reporter assays. Further overexpression and depletion approaches are planned to investigate the impact on MICA surface protein expression and on NK cell-mediated target cell elimination. Finally the clinical relevance of the identified factors will be assessed in acute myeloma leukemia patient samples. To this end we will perform Rhapsodytm (Beckman Coulter) single-cell sequencing to correlate MICA and candidate factor expression and to measure whether these factors regulate (gain/loss of function) MICA expression and susceptibility against NK cells. We expect that we will identify positive regulators of MICA which may on the long run represent novel therapeutic targets.