Analysis of NK Cell Function in Lysosome Storage Disorders

溶酶体储存障碍中 NK 细胞功能分析

基本信息

批准号：
9566746
负责人：
JOHN COLIGAN
金额：
$ 22.02万
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/9566746
关键词：
Affect Anabolism Biogenesis Blood Platelets Blood specimen CHS1 gene Cell Degranulation Cell membrane Cell physiology Cells Cellular biology Ceroid Clinical Colitis Cytoplasmic Granules Defect Deposition Diagnosis Disease Disease susceptibility Early Endosome Exocytosis Genes Genetic screening method Goals Granzyme Griscelli Syndrome Growth Hemorrhage Hermanski-Pudlak Syndrome Host Defense Human Hypopigmentation Immune Immunologic Deficiency Syndromes Immunologics Impairment Inborn Genetic Diseases Infection Interferons Investigation Life Lymphocyte Lymphocyte Subset Lysosomal Storage Diseases Lysosomes Lytic Melanosomes Microscopic Missense Mutation Morphology Mutation Natural Immunity Natural Killer Cells Oculocutaneous Albinism Organelles Pathway interactions Patients Phenotype Pigments Play Process Protein Sortings Proteins Pulmonary Fibrosis Recording of previous events Regulation Reporting Site Skin Syndrome TNF gene Translating Transport Vesicles Vesicle Transport Pathway adaptive immunity arm cell motility chediak-higashi syndrome chemokine cytokine cytotoxic cytotoxicity familial hemophagocytic lymphohistiocytosis histiocyte immunological synapse killings late endosome macrophage perforin protein transport response trafficking tumor tumorigenesis

项目摘要

Several lysosomal storage disorders, such as CHS or HPS, display similarities in terms of their clinical manifestations and immunologic mechanisms, one of them being the decreased or absent cytotoxic activity of NK cells resulting in immune deficiency. Thus, we are investigating whether impaired NK cell function in those disorders is associated with defects in secretory lysosome (lytic granule) morphology, biogenesis, motility, exocytosis, or combination of these factors. We analyzed NK cells from atypical CHS patients with missense mutations in the LYST ARM/HEAT or BEACH domains. CHS NK cells displayed severely reduced cytotoxicity. Mutations in the ARM/HEAT domain led to a reduced number of perforin-containing granules, which were significantly increased in size, but still able to polarize to the immunological synapse (IS); however, they were unable to properly fuse with the plasma membrane. Mutations in the BEACH domain resulted in the formation of normal or slightly enlarged granules that had markedly impaired polarization to the immunological synapse, but could be exocytosed upon reaching the IS. Perforin-containing granules in CHS NK cells did not acquire certain lysosomal markers (LAMP1/2), but were positive for markers of transport vesicles (CI-MPR), late endosomes (Rab27a), and to some extent, early endosomes (EEA-1), indicating a lack of integrity in the endo-lysosomal compartments. CHS NK cells had normal cytokine compartments and cytokine secretion. Our study revealed that LYST is involved in regulation of multiple aspects of NK cell lytic activity ranging from governance of lytic granule size to control of their polarization and exocytosis, as well as the regulation of endo-lysosomal compartment identity. LYST functions in the regulated exocytosis, but not in the constitutive secretion pathway. Furthermore, we analyzed NK cells from patients diagnosed with HPS-1, HPS-2, HPS-4, and an unknown HPS subtype. The latter patient had clinical features of HPS, but genetic testing did not reveal any mutations in genes associated with HPS, suggesting a new HPS subtype; we classified this patient as HPS-new. All patients had oculocutaneous albinism; 7 of 10 patients with HPS-1 and the HPS-2 patient had pulmonary fibrosis. None of the patients had a history of unusual infections or tumorigenesis except the subject with HPS-2, who was previously reported to have a history of severe infections. NK cells from HPS-2 and HPS-new patients had markedly reduced cytotoxic capabilities that correlated with defective NK cell degranulation, while NK cells from HPS type 1 and HPS type 4 patients had only slightly decreased capacity to kill the target cells. Microscopic analysis revealed that NK cells from patients with HPS-2 and HPS-new had enlarged, granzyme A- and perforin-positive lytic granules that failed to translocate to the immunological synapse in response to target cell recognition; this translated to severely reduced granule exocytosis and deficient NK cell cytotoxicity. While both HPS-2 and HPS-new NK cells shared a similar cellular phenotype in regard to the impairment of regulated exocytosis, they differed in the regulation of the constitutive secretion pathway. HPS-new NK cells secreted normal amounts of TNF and IFN, whereas HPS-2 NK cells displayed defective cytokine exocytosis, and accumulated cytokines inside of the cell. Thus, HPS-new affects the cytolytic function of NK cells, while HPS-2 affects both lytic granule and cytokine secretion.

几种溶酶体储存障碍（例如CHS或HPS）在其临床表现和免疫学机制方面表现出相似性，其中一种是NK细胞的降低或没有细胞毒性活性，导致免疫缺陷。因此，我们正在研究这些疾病中的NK细胞功能受损与分泌溶酶体（裂解颗粒）形态，生物发生，运动，胞吐作用或这些因素组合的缺陷有关。我们分析了来自非典型CHS患者的NK细胞，该患者在LYST ARM/HEAT或海滩域中具有错义突变。 CHS NK细胞显示出严重降低的细胞毒性。手臂/热结构域中的突变导致含状颗粒的数量减少，大小显着增加，但仍然能够偏振与免疫突触（IS）；但是，他们无法与质膜正确融合。海滩结构域中的突变导致形成正常或略微扩大的颗粒，这些颗粒显着损害了对免疫突触的极化，但在达到IS时可能会胞外。 CHS NK细胞中的含状颗粒没有获得某些溶酶体标记（LAMP1/2），但对传输囊泡（CI-MPR），晚期内体（RAB27A）的标记呈阳性，并在某种程度上，早期内体（EEA-1）呈阳性，表明内摩层的综合性缺乏完整性。 CHS NK细胞具有正常的细胞因子室和细胞因子分泌。我们的研究表明，LYST参与了NK细胞裂解活性的多个方面的调节，从裂解颗粒大小的治理到控制其极化和胞吐作用，以及调节内溶剂体腔室认同。 LYST在受调节的胞吐作用中起作用，但在本构分泌途径中不作用。此外，我们分析了诊断为HPS-1，HPS-2，HPS-4和未知HPS亚型的患者的NK细胞。后一患者具有HPS的临床特征，但是基因检测并未揭示与HPS相关的基因突变，这表明新的HPS亚型。我们将该患者归类为HPS-NEW。所有患者均具有眼形白化病。 10例HPS-1和HPS-2患者中有7例患有肺纤维化。除了患有HPS-2的受试者外，没有患者有异常感染或肿瘤发生病史。来自HPS-2和HPS NEW患者的NK细胞显着降低了与缺陷的NK细胞脱粒相关的细胞毒性能力，而来自HPS 1型和HPS 4型HPS的NK细胞仅略微降低了杀死靶细胞的能力。显微镜分析表明，来自HPS-2和HPS-NEW患者的NK细胞增大，颗粒酶A-和穿孔蛋白阳性裂解颗粒，未能响应靶细胞识别而无法转移到免疫突触；这转化为严重降低的颗粒胞吐作用和缺乏NK细胞毒性。尽管HPS-2和HPS-NEW NK细胞都在受调节胞吐作用的损害方面具有相似的细胞表型，但它们在组成型分泌途径的调节方面有所不同。 HPS-NEW NK细胞分泌了正常量的TNF和IFN，而HPS-2 NK细胞显示出缺陷的细胞因子胞吐作用，并在细胞内部积聚细胞因子。因此，HPS-NEW会影响NK细胞的细胞溶解功能，而HPS-2则影响裂解颗粒和细胞因子分泌。