Analysis of NK Cell Function in Lysosome Storage Disorders

几种溶酶体贮积症，如CHS或HPS，在其临床表现和免疫机制方面显示出相似性，其中之一是NK细胞的细胞毒性活性降低或缺失，导致免疫缺陷。因此，我们正在研究这些疾病中NK细胞功能受损是否与分泌性溶酶体（溶解颗粒）形态、生物发生、运动性、胞吐或这些因素的组合缺陷相关。 我们分析了来自非典型CHS患者的NK细胞，这些患者在LYST ARM/HEAT或海滩结构域中具有错义突变。CHS NK细胞显示出严重降低的细胞毒性。ARM/HEAT结构域中的突变导致含有穿孔素的颗粒数量减少，其尺寸显著增加，但仍然能够粘附到免疫突触（IS）;然而，它们无法与质膜适当融合。海滩结构域中的突变导致形成正常或轻微增大的颗粒，其具有明显受损的免疫突触极化，但在到达IS时可以被胞吐。CHS NK细胞中含有穿孔蛋白的颗粒没有获得某些溶酶体标志物（LAMP 1/2），但对于转运囊泡（CI-MPR）、晚期内体（Rab 27 a）和在一定程度上的早期内体（EEA-1）的标志物是阳性的，表明内-溶酶体区室中缺乏完整性。CHS NK细胞具有正常的细胞因子区室和细胞因子分泌。我们的研究表明，LYST参与调节NK细胞裂解活性的多个方面，从裂解颗粒大小的管理到控制其极化和胞吐作用，以及内-溶酶体区室身份的调节。LYST在受调节的胞吐作用中起作用，但不在组成性分泌途径中起作用。 此外，我们分析了来自诊断为HPS-1、HPS-2、HPS-4和未知HPS亚型的患者的NK细胞。后一名患者具有HPS的临床特征，但基因检测未发现任何与HPS相关的基因突变，提示为新的HPS亚型;我们将该患者归类为HPS-新。所有患者都有眼皮肤白化病; 10例HPS-1和HPS-2患者中有7例有肺纤维化。除HPS-2受试者既往报告有重度感染史外，所有患者均无异常感染或肿瘤发生史。来自HPS-2和HPS-新患者的NK细胞具有与缺陷性NK细胞脱粒相关的显著降低的细胞毒性能力，而来自HPS 1型和HPS 4型患者的NK细胞仅具有略微降低的杀死靶细胞的能力。显微镜分析显示，来自HPS-2和HPS-new患者的NK细胞具有增大的颗粒酶A和穿孔素阳性溶解颗粒，这些颗粒未能移位至免疫突触以响应靶细胞识别;这转化为严重减少的颗粒胞吐作用和NK细胞细胞毒性缺陷。虽然HPS-2和HPS-新NK细胞在调节胞吐的损伤方面具有相似的细胞表型，但它们在组成性分泌途径的调节方面不同。HPS-新NK细胞分泌正常量的TNF和IFN，而HPS-2 NK细胞显示出缺陷的细胞因子胞吐作用，并在细胞内积累细胞因子。因此，HPS-新影响NK细胞的细胞溶解功能，而HPS-2影响溶解颗粒和细胞因子分泌。

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.