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

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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.