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。含有穿孔素和颗粒酶的NK细胞裂解颗粒是NK细胞杀伤所必需的，因为它们的释放会导致靶细胞的凋亡。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。 溶解颗粒具有溶酶体的特征，被称为分泌型溶酶体。溶酶体膜中存在许多蛋白质，它们在溶酶体腔的酸化、蛋白质输入、膜融合和降解产物向细胞质的运输中发挥作用。令人惊讶的是，最丰富的蛋白质，溶酶体相关膜蛋白(LAMP)1和LAMP2(分别为CD107a和CD107b)的作用尚不明确。在人类中，LAMP2的突变会导致达农病，据信是由异常的自噬引起的。LAMP2基因缺陷的小鼠也会在几个组织中积累自噬空泡。LAMP1缺陷小鼠的表型几乎正常，但LAMP2水平升高，这表明LAMP2可以补偿LAMP1的丢失。最近使用耗尽LAMP的成纤维细胞的研究表明，这两种蛋白都是正确的溶酶体运输和有效的(自动)吞噬小体-溶酶体融合所必需的。LAMP1也被广泛用作鉴定NK细胞脱颗粒的标志，因为它出现在溶酶体与质膜融合后的细胞表面，但LAMP1在NK细胞生物学中的作用尚不完全清楚。由于LAMP1是溶酶体膜上含量最丰富的蛋白之一，而NK细胞利用其溶酶体诱导靶细胞死亡，因此我们试图确定LAMP1在NK细胞杀伤活性中的重要性。 因此，本项目的重点是了解溶酶体膜蛋白1在调节人类NK细胞杀伤活性中的作用。为解决这一问题，利用RNA干扰技术(RNAi)干扰LAMP1在人NK细胞系和原代NK细胞中的表达，并研究了LAMP1沉默对裂解颗粒组成、运动、胞吐和NK细胞杀伤活性的影响。 我们已经证明，LAMP1沉默导致抑制NK细胞的细胞毒作用，因为LAMP1 RNAi细胞无法将颗粒酶B递送到靶细胞。LAMP1表达的降低影响溶解颗粒的运动，导致颗粒中穿孔素水平的降低，但不影响颗粒酶B的水平。在LAMP1 RNAi细胞中，更多的穿孔素被保留在跨高尔基网络衍生的运输小泡的溶酶体室外。LAMP1结合伙伴--适配蛋白1(AP-1)分选复合体的表达中断也会导致穿孔素滞留在运输小泡中并抑制细胞毒性，表明AP-1分选复合体与运输小泡表面的LAMP1之间的相互作用对于穿孔素运输到溶解颗粒是重要的。我们的结论是，LAMP1缺陷细胞裂解颗粒中穿孔素水平的降低，加上裂解颗粒的运动障碍，导致无法将诱导凋亡的颗粒酶B递送到靶细胞，从而抑制NK细胞的细胞毒作用。 因此，我们已经证明，LAMP1的表达中断对NK细胞具有多效性，导致关键的溶解颗粒蛋白穿孔素的功能受损和溶解颗粒运动减慢。这导致NK细胞不能将颗粒酶B运送到靶细胞，从而抑制了NK细胞的细胞毒作用。因此，LAMP1不仅是NK细胞脱颗粒的标志，也是NK细胞细胞毒作用的重要组成部分。

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 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. Lytic granules have the characteristics of lysosomes and are referred to as secretory lysosomes. There are many proteins residing in lysosomal membranes that function in acidification of the lysosomal lumen, protein import, membrane fusion, and transport of degradation products to the cytoplasm. Surprisingly, the role of the most abundant proteins, lysosome-associated membrane protein (LAMP) 1 and LAMP2 (CD107a and CD107b, respectively), is ill-defined. In humans, mutations in LAMP2 cause Danon disease, believed to arise from aberrant autophagy. LAMP2-deficient mice also accumulate autophagic vacuoles in several tissues. LAMP1-deficient mice have an almost normal phenotype but have elevated levels of LAMP2, suggesting that LAMP2 can compensate for the loss of LAMP1. Recent studies using LAMP-depleted fibroblasts demonstrate that both proteins are required for proper lysosomal transport and efficient (auto)phagosome-lysosome fusion. LAMP1 is also widely used as a marker for the identification of NK-cell degranulation, as it appears on the cell surface following the fusion of lysosomes with the plasma membrane, but the role of LAMP1 in NK-cell biology is completely unknown. Since LAMP1 is one of the most abundant proteins in the lysosomal membrane and NK cells use their lysosomes to induce the death of target cells, we sought to determine the importance of LAMP1 in NK-cell cytolytic activity. Therefore, this project emphasis has been to understand the role of lysosomal membrane protein 1, LAMP1, in regulation of human NK cell cytotoxicity. To address this issue, RNA interference (RNAi) was used to disrupt LAMP1 expression in human NK cell lines, as well as primary NK cells, and the effects of LAMP1 silencing on lytic granule composition, movement, exocytosis and NK cell cytotoxic potential were investigated. We have demonstrated that LAMP1 silencing causes inhibition of NK-cell cytotoxicity, as LAMP1 RNAi cells fail to deliver granzyme B to target cells. Reduction of LAMP1 expression affects the movement of lytic granules and results in decreased levels of perforin, but not granzyme B, in the granules. In LAMP1 RNAi cells, more perforin is retained outside of lysosomal compartments in trans-Golgi networkderived transport vesicles. Disruption of expression of LAMP1 binding partner, adaptor protein 1 (AP-1) sorting complex, also causes retention of perforin in the transport vesicles and inhibits cytotoxicity, indicating that the interaction between AP-1 sorting complex and LAMP1 on the surface of the transport vesicles is important for perforin trafficking to lytic granules. We conclude that the decreased level of perforin in lytic granules of LAMP1-deficient cells, combined with disturbed motility of the lytic granules, leads to the inability to deliver apoptosis-inducing granzyme B to target cells and to inhibition of NK-cell cytotoxicity. Thus, we have shown that disruption of LAMP1 expression has a pleiotropic effect on NK cells, causing impairment of trafficking of a critical lytic granule protein, perforin, and slower movement of lytic granules. This results in the inability of NK cells to deliver granzyme B to target cells and inhibition of NK-cell cytotoxicity. Thus, LAMP1 is not only a marker of NK-cell degranulation, but also a crucial component of NK-cell cytotoxicity.