The role of inhibitory receptors in human NK cell development and function.

NK细胞自然杀伤(Natural Killer，NK)细胞是先天免疫系统的效应淋巴细胞，能够对抗病毒感染和癌症。NK细胞通过释放可直接溶解靶细胞的细胞毒性颗粒(如Perforin和Granzyme B)，或通过产生可激活免疫系统其他手臂的各种细胞因子(如IFNy和TNFa)来调节其功能(Caligiuri，2008)。NK细胞的功能是一系列激活的和抑制的细胞表面受体之间平衡的结果，这些受体传递信号以响应环境。特别是，NK细胞的激活被特定的抑制性受体与HLA配体的结合所抑制，这些受体通常在健康细胞中高表达，但在许多癌细胞中表达下调，以逃避细胞毒CD8+T细胞的检测(Long Etal)。ILT2和KIR2DL4已有研究表明，某些实体癌类型(例如卵巢癌、肝细胞癌、非小细胞肺癌、胶质瘤和肾细胞癌)已经进化到高水平表达HLA-G配体，这是一种非经典的HLAI类分子，导致疾病预后不良(Khan等人，2020)。人类NK细胞上的抑制性受体ILT2(Fille等人，2010)和KIR2DL4(Rajagopalan等人，2006)识别HL-G，它们与免疫受体结合，抑制NK细胞的激活和增殖。ILT2阻断已被证明可以恢复NK细胞对抗实体肿瘤和血液病的功能(Godal等人，2010；Roberti等人，2015；Villa-álvarez等人，2018；Chen等人，2020)，但ILT2和HLA-G相互作用对NK细胞的具体机制还需要进一步研究。KIR2DL4已被证明同时具有激活和抑制作用(Rajagopalan和Long，2012)。KIR2DL4介导NK细胞功能激活的机制已被研究(Rajagopalan等人，2006年)，但对其在NK细胞功能抑制中的作用知之甚少。IL-1R8与HLA特异性抑制受体相似，也参与了NK细胞介导的免疫监视。其中一种受体是IL-1R8，它最近作为NK细胞上的一种新的免疫检查点出现(Molgora等人，2017年)。与IL-1R8充足的细胞相比，缺乏IL-1R8的巨噬细胞和小鼠NK细胞采用了增强的成熟和细胞毒性表型(Molgora等人，2017年)。最近的其他证据表明，与常规NK细胞不同，来自巨细胞病毒感染者的适应性NK细胞对肿瘤诱导的免疫抑制具有高度抵抗力，这归因于IL-1R8表达减少(Sarhan等人，2018年)。IL-1R8能够与IL-37结合，IL-37的相互作用已被证明可以抑制炎症反应(Nold-Peter等人，2015年)。与这些发现一致的是，由于抗肿瘤免疫受损，IL1R8的高表达被证明促进了乳腺肿瘤的生长(Campeato等人，2017年)，而血清IL-37水平的升高与上皮性卵巢癌患者的不良预后有关(霍等人，2017年)。这些发现表明，研究IL-1R8/IL-37信号在实体瘤背景下对NK细胞发育和功能的潜在意义。目的利用慢病毒和/或核重组介导的CRISPRCas9基因组编辑方法，从脐带血造血干细胞中产生缺乏ILT2、KIR2DL4和IL-1R8的人NK细胞。这一结果不仅将导致NK细胞被迅速激活，甚至在抑制性肿瘤微环境中也可能具有更强的细胞毒性，而且还将揭示这些基因在NK细胞发育、激活和功能中的作用。最终，它可能为实体癌症更有效的基于NK细胞的免疫疗法铺平道路。

NK cellsNatural Killer (NK) cells are effector lymphocytes of the innate immune system, capable of fightingviral infection and cancer. NK cells mediate their function by releasing cytotoxic granules (e.g., Perforinand granzyme B) that can directly lyse target cells, or by producing a variety of cytokines (e.g., IFNyand TNFa) that can activate other arms of the immune system (Caligiuri, 2008). NK cell function istuned as a result of a balance between an array of activatory and inhibitory cell surface receptors thattransduce signalling in response to their environment. In particular, NK cell activation is suppressed bythe binding of specific inhibitory receptors to HLA ligands, which are usually well expressed in healthycells, but downregulated in many cancer cells to escape detection by cytotoxic CD8+ T cells (Long etal. 2013).ILT2 and KIR2DL4It has been shown that certain solid cancer types (e.g., ovarian cancer, hepatocellular carcinoma, nonsmall cell lung carcinoma, glioma, and renal cell carcinoma) have evolved to express high levels ofHLA-G ligand, a non-classical HLA class I molecule, leading to poor disease prognosis (Khan et al.,2020). HLA-G is recognised by the inhibitory receptors ILT2 (Favier et al., 2010) and KIR2DL4(Rajagopalan et al., 2006) on human NK cells, which upon immunoreceptor engagement, suppressesNK cell activation and proliferation. ILT2 blockade has been shown to restore NK cell function againstsolid and haematological cancers (Godal et al., 2010; Roberti et al., 2015; Villa-Álvarez et al., 2018;Chen et al., 2020), however the specific mechanism of ILT2 and HLA-G interaction on NK cell requiresfurther investigation. KIR2DL4 has been shown to possess both activating and inhibitory roles(Rajagopalan and Long, 2012). The mechanism by which KIR2DL4 mediate activation of NK cellfunction has been investigated (Rajagopalan et al., 2006), however little is known about its role in thesuppression of NK cell function.IL-1R8Similarly to HLA-specific inhibitory receptors, non-HLA-specific inhibitory receptors have also beenimplicated in NK cell-mediated immunosurveillance. One such receptor is the IL-1R8, which hasrecently emerged as a novel immune checkpoint on NK cells (Molgora et al., 2017). IL-1R8 deficienthuman and mouse NK cells have shown to adopt enhanced mature and cytotoxic phenotypes compared to IL-1R8 sufficient cells (Molgora et al., 2017). Additional recent evidence has shown that, unlikecanonical NK cells, adaptive NK cells derived from cytomegalovirus-infected individuals are highlyresistant to tumour-induced immune suppression, attributed to reduced IL-1R8 expression (Sarhan etal., 2018). IL-1R8 is capable of binding to IL-37, the interaction of which has been shown to dampeninflammatory responses (Nold-Petry et al., 2015). Consistent with such findings, high expression of IL1R8 has been shown to promote breast tumour growth as a result of impaired antitumor immunity(Campesato et al., 2017), while elevated serum IL-37 level has been associated with poor prognosisin epithelial ovarian cancer patients (Huo et al., 2017). These findings indicate the potential significanceof investigation of IL-1R8/IL-37 signalling on NK cell development and function in the context of solidcancers.AIMSThis PhD project seeks to generate ILT2, KIR2DL4 and IL-1R8 deficient human NK cells derived fromumbilical cord blood hematopoietic stem cells, using lentivirus- and/or nucleofection-mediated CRISPRCas9 genome editing methods. The outcome of this would not only result in NK cells that are morereadily activated and potentially more cytotoxic even in a suppressive tumour microenvironment, butwould also uncover the roles of these genes in NK cell development, activation and function. Ultimately,it could pave the way for a more effective NK cell-based immunotherapy for solid cancers.