Regulation of lytic granule exocytosis in Natural Killer (NK) Cells

自然杀伤(NK)细胞由参与防御微生物病原体和肿瘤的淋巴细胞亚群组成。由于NK细胞具有细胞毒作用，因此能够直接清除异常细胞。NK细胞的裂解颗粒(分泌性溶酶体)含有穿孔素和颗粒酶，它们的释放会诱导靶细胞的凋亡，因此对NK细胞的细胞毒作用是必不可少的。溶血颗粒分泌缺陷与严重疾病有关，如噬血细胞性淋巴组织细胞增多症和Chediak-Higashi、Hermansky-Pudlak或Griscelli综合征。对参与调节溶解颗粒释放的蛋白质的了解非常有限，而参与NK细胞胞吐的颗粒特异性蛋白质机制也知之甚少。 Chediak-Higashi综合征(CHS)是一种罕见的溶酶体储存障碍，由溶酶体运输调节基因Lyst突变引起。Lyst编码一个429 kDa的蛋白，有几个不同的结构域与囊泡运输的不同方面有关：ARM/HEAT、PH、BASH和WD-40，但其确切功能仍有待阐明。研究表明，CHS患者的NK细胞存在形态和功能异常。然而，我们对CHS中NK细胞缺陷的了解有限，Lyst在细胞毒淋巴细胞生物学中的确切作用尚不清楚。 因此，本项目的重点是了解Lyst在调节人NK细胞杀伤活性中的作用。为了解决这个问题，我们在人NK细胞系中阻断了Lyst的表达，并研究了Lyst功能丧失对裂解颗粒组成、运动、胞吐和NK细胞杀伤潜力的影响。我们已经成功地建立了CHS的人类细胞模型，可以用来在细胞水平上研究Lyst缺陷的后果。

Natural killer (NK) cells comprise a subset of lymphocytes involved in protection against microbial pathogens and tumors. Because of their cytotoxic capacity, NK cells are able to directly eliminate abnormal cells. Lytic granules (secretory lysosomes) of NK cells, containing perforin and granzymes, are indispensable for NK-cell cytotoxicity because their release results in the induction of target-cell apoptosis. Defects in lytic granule secretion are associated with serious diseases, such as hemophagocytic lymphohistiocytosis and Chediak-Higashi, Hermansky-Pudlak, or Griscelli syndromes. Knowledge about the proteins involved in regulation of lytic granule release is very limited, and the granule-specific protein machinery involved in NK-cell exocytosis is poorly understood. Chediak-Higashi syndrome (CHS) is a rare lysosomal storage disorder caused by mutations in the lysosomal trafficking regulator gene, LYST. LYST encodes a 429 kDa protein with several distinct domains implicated in various aspects of vesicular trafficking: ARM/HEAT, PH, BEACH and WD-40, but its exact function remains to be elucidated. NK cells in CHS patients have been shown to have abnormal morphology and function. Nevertheless, our understanding of NK cell defects in CHS is limited and the exact role of LYST in cytotoxic lymphocyte biology is unknown. Therefore, this project emphasis has been to understand the role of LYST in regulation of human NK cell cytotoxicity. To address this issue, we disrupted LYST expression in human NK cell lines, and the effects of LYST loss-of-function on lytic granule composition, movement, exocytosis and NK cell cytotoxic potential were investigated. We have successfully generated a human cellular model of CHS that can be used to study consequences of LYST deficiency at a cellular level.