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

自然杀伤 (NK) 细胞中裂解颗粒胞吐作用的调节

基本信息

批准号：
8745425
负责人：
JOHN COLIGAN
金额：
$ 22.51万
依托单位：
NATIONAL INSTITUTE OF ALLERGY AND INFECTIOUS DISEASES
依托单位国家：
美国
项目类别：
财政年份：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8745425
关键词：
Abnormal Cell Adaptor Signaling Protein Address Adherence Affect Apoptosis Binding Biogenesis Caspase Cell Degranulation Cell Fractionation Cell Line Cell membrane Cell physiology Cell surface Cell-Mediated Cytolysis Cells Cellular biology Characteristics Complex Cytolysis Cytoplasmic Granules Cytoskeleton Data Defect Disease Dynein ATPase Epidermal Growth Factor Receptor Excision Exocytosis FCGR3B gene Flow Cytometry Fluorescence Goals Golgi Apparatus Granzyme Griscelli Syndrome Half-Life Hour Human Image Impairment Interleukin-2 K-562 Knowledge Laser Scanning Confocal Microscopy Lead Left Location Lysosomes LytA enzyme Lytic Mediating Membrane Membrane Proteins Messenger RNA Microtubule-Organizing Center Microtubules Motor Movement Natural Killer Cells Pathway interactions Play Proteins RNA Interference Regulation Role Site Small Interfering RNA Sorting - Cell Movement Surface Syndrome Time Transcription Factor AP-1 Transport Vesicles Travel Tumor Cell Line Vesicle YARS gene analytical ultracentrifugation cellular transduction cytokine cytotoxic cytotoxicity dynactin familial hemophagocytic lymphohistiocytosis granule cell granzyme B immunological synapse killings knock-down neoplastic cell perforin prevent protein complex research study trafficking

项目摘要

The current emphasis is to understand first the role of lysosomal membrane protein 1, LAMP1, in cytotoxicity of human NK cells. To address this issue, RNAi was used to disrupt LAMP1 expression and assess its effects on lytic granule exocytosis and NK cell cytotoxic potential. We generated YTS and NK92 NK cell lines with a stable LAMP1 knockdown (LAMP1 KD) (80% decrease at protein level). With siRNA-mediated silencing of LAMP1 in ex vivo isolated IL-2 cultured human NK cells, we achieved a 40% to 50% decrease of LAMP1 protein. The lower LAMP1 knockdown level chieved in the transiently siRNA-transduced ex vivo NK cells was at least partially due to the stability of LAMP1; LAMP1 has a half-life of at least 30 hours, and the time point at which NK cells were analyzed (72 hours) was likely too short for stronger or full silencing of LAMP1. To investigate the role of LAMP1 in NK-cell function, we evaluated the effect of LAMP1 silencing on NK-cell cytotoxicity. We found that compared with untransduced YTS cells, or cells transduced with non-targeting control RNAi, LAMP1 RNAi cells had a 70% to 76% decrease in cytotoxicity against tumor cell lines (721.221 or K562). We obtained similar results using ex vivo isolated NK cells, where a transient knockdown of LAMP1 reduced cytotoxicity by 40% to 60%. We also used Ab-dependent cell-mediated cytotoxicity to evaluate the killing ability of LAMP1 RNAi NK cells. Although CD16-mediated recognition of anti-Human Epidermal Growth Factor Receptor 2 (HER2) Ab, bound to the cell surface of SK-OV3 cells, resulted in pronounced lysis of target cells by control-transduced NK cells, cytotoxicity by LAMP1 RNAi NK cells was decreased by 35% to 40%. Our results demonstrate for the first time that LAMP1 plays a critical role in NK cell cytolytic activity. Furthermore, we found that, in contrast to control RNAi-transduced cells, LAMP1 RNAi cells failed to deliver granzyme B to 721.221 target cells. Similarly, siRNA-mediated KD of LAMP1 in ex vivo NK cells also blocked the transfer of granzyme B from NK to tumor cells. Importantly, LAMP1 RNAi cells (both YTS and ex vivo NK cells) had normal level of granzyme B mRNA and protein, and the activity of granzyme B from LAMP1 RNAi cells was undistinguishable from the activity in control cells. LAMP1 RNAi cells had normal conjugation, and were able to externalize CD107b on the cell surface, excluding the possibility that the block of granzyme B delivery and, subsequently, cytotoxicity resulted from improper cell-cell adherence or inhibition of fusion of lytic granules with the plasma membrane. Our results suggested that the defect of granzyme B delivery could be due to problems with granule transport of lytic granules to the cell to cell contact site. Laser scanning confocal microscopy experiments showed that although LAMP1 RNAi cells were able to translocate lytic granules toward the immunological synapse, the granules were more dispersed around the MTOC and did not cluster efficiently at the immunological synapse, suggesting a possible defect in granule transport in LAMP1 RNAi cells. Indeed, we discovered that compared to control RNAi cells, granules in LAMP1 RNAi cells traveled shorter distances, had smaller displacement and moved slower, indicating that LAMP1 is important for the proper granule movement along cytoskeletal tracks. Using cell fractionation and analytical ultracentrifugation, we found that, when compared to control RNAi cells, lytic granules isolated from LAMP1 KD cells had less p150glued, an important component of the microtubule motor protein complex-dynein/dynactin. The decreased recruitment of motor proteins to lytic granules provides an explanation for the defects in the movement of the granules caused by the disruption of LAMP1 expression. More importantly, we also found that compared to control RNAi cells, silencing of LAMP1 resulted in 30-40% decrease of perforin fluorescence, despite normal level of perforin mRNA. Analysis of intracellular perforin level by flow cytometry confirmed the specific decrease of perforin in ex vivo NK and YTS cells with LAMP1 KD. Imaging and lytic granule isolation studies led us to discovery that LAMP1 RNAi cells had less perforin associated with lytic granules. Consequently, we found that the decreased level of perforin in lytic granules was due to the defect in perforin trafficking, and retention of perforin in the trans-Golgi (TGN)-derived transport vesicles. The adaptor protein complex AP-1, important for sorting proteins leaving the TGN, is known to directly interact with LAMP1. Therefore, we analyzed the effect of silencing the AP-1 complex subunit, adaptin gamma, on perforin location in YTS cells. Adaptin gamma silencing resulted in increased association of perforin with the transport vesicles. Importantly, adaptin gamma knock-down produced effects similar to LAMP1 silencing, indicating that these two binding partners are important for proper trafﬁcking of perforin to lytic granules and NK-cell cytotoxicity. Our data suggest that the direct interaction between LAMP1 and AP-1 could mediate transport of LAMP1-positive vesicles, containing perforin, to lytic granules. The lack of LAMP1 could prevent binding of AP-1 to perforin-positive vesicles and result in retention of perforin in the transport vesicles instead of its delivery to lytic granules. Similarly, the lack of AP-1 complex would prevent proper perforin transfer from the TGN to lytic granules because there would be no adaptor to interact with LAMP1 and enable the transport. 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.

当前的重点是首先了解溶酶体膜蛋白1，LAMP1在人NK细胞的细胞毒性中的作用。为了解决这个问题，RNAi被用来破坏LAMP1的表达，并评估其对裂解颗粒胞外增生和NK细胞细胞毒性潜力的影响。我们产生了具有稳定的LAMP1敲低（LAMP1 KD）（蛋白质水平下降80％）的YTS和NK92 NK细胞系。在离体分离的IL-2培养的人NK细胞中，siRNA介导的LAMP1沉默，我们达到了40％至50％的LAMP1蛋白。在短暂的siRNA转导的离体NK细胞中，较低的LAMP1敲低水平至少部分归因于LAMP1的稳定性。 LAMP1的半衰期至少为30小时，并且分析NK细胞的时间点（72小时）可能太短了，无法对LAMP1进行更强或完全沉默。为了研究LAMP1在NK细胞功能中的作用，我们评估了LAMP1沉默对NK细胞细胞毒性的影响。我们发现，与未转导的YTS细胞或用非靶向对照RNAi转导的细胞相比，Lamp1 RNAi细胞对肿瘤细胞系的细胞毒性降低了70％至76％（721.221或K562）。我们使用过体内分离的NK细胞获得了相似的结果，其中LAMP1的短暂敲低可将细胞毒性降低40％至60％。我们还使用依赖性细胞介导的细胞毒性来评估LAMP1 RNAi NK细胞的杀伤能力。尽管CD16介导的对抗人表皮生长因子受体2（HER2）AB的识别与SK-OV3细胞的细胞表面结合，但通过对照转导的NK细胞对靶细胞产生了明显的裂解，但通过LAMP1 RNAI NK细胞的细胞毒性降低了35％至40％。我们的结果表明，LAMP1在NK细胞溶解活性中起着至关重要的作用。此外，我们发现，与控制RNAi转导的细胞相比，LAMP1 RNAi细胞未能将颗粒酶B递送至721.221靶细胞。同样，siRNA介导的LAMP1的lamp1在体内NK细胞中也阻塞了颗粒酶B从NK转移到肿瘤细胞。重要的是，LAMP1 RNAi细胞（YTS和EX VIVO NK细胞）具有正常的颗粒酶B mRNA和蛋白质水平，并且来自LAMP1 RNAi细胞的颗粒酶B活性与对照细胞中的活性不可分割。 LAMP1 RNAi细胞具有正常的结合，并且能够在细胞表面上外部化CD107B，排除了颗粒酶B递送的块以及随后的细胞毒性的可能性，是由于细胞细胞细胞细胞粘附或抑制lytic颗粒与血浆膜的融合而产生的。我们的结果表明，颗粒酶B递送的缺陷可能是由于裂解颗粒向细胞到细胞接触部位的颗粒运输的问题。激光扫描共聚焦显微镜实验表明，尽管LAMP1 RNAI细胞能够将裂解颗粒旋转为免疫学突触，但颗粒在MTOC周围更具分散，并未在免疫突触上有效地聚集，表明在Lamp1 RNAi细胞中可能存在的缺陷。确实，我们发现与对照RNAi细胞相比，LAMP1 RNAi细胞中的颗粒移动较短，位移较小且移动较慢，这表明LAMP1对于沿细胞骨架轨道的适当颗粒运动很重要。使用细胞分馏和分析性超速离心，我们发现，与对照RNAi细胞相比，从LAMP1 KD细胞中分离出的裂解颗粒的P150GLED较少，P150GLED是微管运动蛋白蛋白质复合型 - 二氧蛋白/dynactin的重要组成部分。运动蛋白募集到裂解颗粒的募集减少为解释了由LAMP1表达的破坏引起的颗粒运动的缺陷。更重要的是，我们还发现，与对照RNAi细胞相比，LAMP1的沉默导致穿孔蛋白mRNA水平正常，导致perforin荧光降低30-40％。通过流式细胞术对细胞内的细胞素水平进行分析证实了用LAMP1 KD的ex Vivo NK和YTS细胞中穿孔蛋白的特异性降低。成像和裂解颗粒分离研究使我们发现Lamp1 RNAi细胞与裂解颗粒相关的穿孔蛋白较少。因此，我们发现裂解颗粒中的穿孔蛋白水平降低是由于穿孔蛋白运输的缺陷以及在反式高尔基体（TGN）衍生的转运囊泡中保留穿孔蛋白。已知适配器蛋白复合物AP-1对于分类离开TGN的蛋白质很重要，它是直接与LAMP1相互作用的。因此，我们分析了沉默的AP-1复合物亚基Adaptin伽马对YTS细胞中的perforin位置的影响。 Adaptin Gamma沉默导致穿孔蛋白与运输囊泡的缔合增加。重要的是，Adaptin Gamma敲低产生的作用类似于Lamp1沉默，这表明这两个结合伴侣对于适当的穿孔蛋白与裂解颗粒和NK细胞细胞毒性非常重要。我们的数据表明，LAMP1和AP-1之间的直接相互作用可以介导含有穿孔蛋白的LAMP1阳性囊泡的转运，向裂解颗粒。缺乏LAMP1可以防止AP-1与穿孔蛋白阳性囊泡的结合，并导致在运输囊泡中保留pert蛋白，而不是将其递送到裂解颗粒中。同样，缺乏AP-1复合物将防止适当的穿孔素从TGN转移到裂解颗粒，因为没有适配器与LAMP1相互作用并启用运输。我们已经表明，LAMP1表达的破坏对NK细胞具有多效性作用，从而导致临界裂解颗粒蛋白，穿孔蛋白和裂解颗粒运动较慢的流通损伤。这导致NK细胞无法将颗粒酶B输送到靶细胞并抑制NK细胞细胞毒性。因此，LAMP1不仅是NK细胞脱粒的标记，而且是NK细胞细胞毒性的关键成分。