The role of inhibitory receptors in human NK cell function for immunotherapy

背景自然杀伤(Natural Killer，NK)细胞是一种先天效应淋巴细胞，在病毒感染和肿瘤免疫监控中发挥着重要作用。NK细胞通过释放细胞毒颗粒(如颗粒酶B)直接杀伤靶细胞，或通过产生各种细胞因子和趋化因子(如干扰素)激活免疫系统的其他手臂。NK细胞的激活是由多种激活的细胞表面受体驱动的，这些受体可以感知异常或应激的环境。激活也受多个抑制性受体的调节。PD-1和CTLA-4是两种抑制性受体，也被称为免疫检查点受体，以前在T细胞中发现了它们。一旦与配体结合，这些受体就会减少免疫细胞的激活和增殖，这一过程经常被癌细胞劫持以逃避免疫系统。因此，免疫检查点抑制剂在T细胞的背景下得到了广泛的研究，目前已被用于治疗几种癌症。最近的证据表明，PD-1阻断疗法即使对MHC缺陷的肿瘤仍然有效，这揭示了NK细胞上抑制受体的重要性。此外，卵巢癌患者NK细胞表面PD-1细胞高表达，与预后不良有关。CTLA-4在小鼠NK细胞上已被发现，但其在人NK细胞活性中的作用尚需进一步研究。目的首先，采用慢病毒介导的CRISPR/Cas9基因组编辑方法，制备缺乏PD-1和CTLA-4的人NK细胞，以研究其对NK细胞生物学的影响。这将包括NK细胞对各种靶细胞的细胞毒性研究，以及NK细胞激活时细胞因子的产生研究。进一步的特征将包括观察单个信号通路和转录组分。此外，这些修饰的NK细胞将被测试它们在癌症免疫治疗中的增强功能。

BACKGROUNDNatural Killer (NK) cells are innate effector lymphocytes that play a crucial role in viral infections and tumour immunosurveillance. NK cells directly kill target cells by releasing cytotoxic granules (e.g. granzyme B) or activating other arms of the immune system by producing a variety of cytokines and chemokines (e.g. IFN). NK cell activation is driven by multiple activatory cell surface receptors that sense an aberrant or stressful environment. Activation is also modulated by multiple inhibitory receptors.PD-1 and CTLA-4 are two inhibitory receptors, also known as immune checkpoint receptors, which have previously been identified in T cells. Upon ligand binding, these receptors reduce immune cell activation and proliferation, a process which is often hijacked by cancer cells to evade the immune system. Therefore, immune checkpoint inhibitors have been intensively researched in the context of T cells and are currently used to treat several cancers.Recent evidence suggests that PD-1 blockade therapies remain efficient even for MHC-deficient tumours, which sheds light on the importance of inhibitory receptors on NK cells. Moreover, high PD-1 cell surface expression on NK cells was observed in ovarian cancer patients, associated with poor disease prognosis. CTLA-4 has been identified on mice NK cells, but determination of its role in human NK cell activity requires further investigation. AIM OF PHD PROJECTFirstly, to generate human NK cells lacking PD-1 and CTLA-4, using lentiviral-delivered CRISPR/Cas9 genome editing methods, in order to study the effect on NK cell biology. This will then include NK cell cytotoxicity studies against a variety of target cells, and cytokine production studies upon NK cell activation. Further characterisation would involve looking at individual signalling pathways and transcriptomics. Furthermore, these modified NK cells will be tested for their enhanced function in cancer immunotherapy.