Analysis of NK Cell Function in Lysosome Storage Disorders

目前的重点是描述和了解一组Chediak-Higashi和Hermansky-Pudlak(1、2、4和10型)综合征患者的NK细胞功能缺陷。 我们分析了四名CHS活动期患者、他们的健康直系亲属以及一名接受骨髓移植的CHS患者的PBMCs中的NK细胞。我们发现，两名CHS患者的NK细胞百分比略低，但接近正常(患者1和2分别为2%和3.3%，而父母分别为4.39%和4.06%)。两组受试者NK细胞表面受体活性和抑制性水平均正常，与其家族成员或无血缘关系的健康人比较，差异无统计学意义，提示本病并未影响NK细胞表面受体的转运。此外，裂解颗粒的关键成分穿孔素和颗粒酶B的细胞内水平在家族成员之间非常相似。然而，与健康家庭成员相比，CHS患者的NK细胞由于脱颗粒能力受损而严重降低了细胞毒活性。令人惊讶的是，虽然所有NK细胞都损害了脱颗粒，但这种缺陷背后的原因取决于Lyst突变的位置。蛋白质N端的突变导致形成巨大的溶酶体，这些溶酶体能够极化到细胞-细胞接触区，但太大而不能释放；LYST的C端部分的突变导致颗粒略大，流动性降低，从而阻碍胞吐作用。有趣的是，在细胞因子的产生和分泌的情况下，没有观察到溶解颗粒的释放缺陷。与父母相比，CHS患者对细胞因子刺激的反应中，MIP-1a和TNFa的上调水平相似。此外，细胞因子刺激导致两个患者的NK细胞产生更多的IFNG(患者的NK细胞的67-69%，而父母为40 53%)。 相比之下，接受骨髓移植的CHS患者的NK细胞似乎是正常的，NK细胞激活和抑制受体的细胞表面表达水平也与健康人相似。移植患者的NK细胞与靶细胞结合正常，对两种不同的靶细胞株的杀伤作用与健康人的NK细胞相当。此外，移植患者的NK细胞很容易将穿孔素和颗粒酶A极化到细胞-细胞接触部位。有趣的是，与健康供者相比，移植患者的NK细胞对CD16的参与表现出更多的脱颗粒(27%对健康供者的10%)，因此，在移植患者中ADCC增加。在细胞因子刺激下，移植的CHS患者NK细胞产生MIP-1a、IFNG和TNFa的能力与健康供者的NK细胞相当。因此，骨髓移植完全恢复了患者的NK细胞功能。 我们还分析了8名HPS-1、1名HPS-2、2名HPS-4和1名HPS-10患者的NK细胞。HPS 2型和10型患者的NK细胞在自然杀伤和ADCC检测中均不能杀伤靶细胞，而HPS 1型和4型患者的NK细胞对靶细胞的杀伤能力仅略有下降。HPS NK细胞与靶细胞的结合似乎没有受到明显影响；F-肌动蛋白聚集在细胞-细胞接触部位，表明这些NK细胞的突触形成可能没有受到影响。然而，来自HPS 2型和10型的NK细胞未能脱颗粒，这与受损的细胞毒作用一致。此外，我们发现HPS-2 NK细胞中的裂解颗粒没有有效地聚集在MTOC周围，没有极化到IS，并且看起来略有增大。HPS-10 NK细胞含有较大的溶解颗粒，不能极化到IS。大的溶酶体使人联想到在CHS中观察到的巨大的溶酶体。在一些病例中，大颗粒仅为穿孔素或颗粒酶A阳性；结合增大的大小，这些数据表明蛋白质分类不当和囊泡融合。所有HPS NK细胞均能在刺激后产生细胞因子(TNFa和IFNG)。当HPS-1和HPS-10NK细胞分泌正常水平的细胞因子时，HPS-2NK细胞在细胞刺激后不能释放细胞因子。因此，HPS-10影响NK细胞的细胞溶解功能，而HPS-2同时影响裂解颗粒和细胞因子的分泌(调节和构成胞吐途径)。

The current emphasis is to describe and understand the defects in NK cell function in a group of patients with Chediak-Higashi and Hermansky-Pudlak (type 1, 2, 4 and 10) syndromes. We analyzed NK cells in PBMCs of four CHS patients with the active disease, their healthy immediate family members, as well as one CHS patient that underwent bone marrow transplantation. We found that the two CHS patients have slightly lower, but close to normal percentage of NK cells (2% and 3.3% for patient 1 and 2, vs. 4.39% and 4.06% for parents). The levels of the investigated activating and inhibitory NK cell surface receptors were normal and there was no difference between receptor levels in the two patients when compared to their family members or unrelated healthy individual, indicating that the disease is not affecting the trafficking of NK cell surface receptors. In addition, the intracellular levels of critical components of lytic granules, perforin and granzyme B, were very similar between family members. When compared to the healthy family members, however, NK cells from CHS patients had severely decreased cytotoxic potential, due to impaired degranulation ability. Surprisingly, while all NK cells had impaired degranulation, the cause underlying this defect depended on the position of LYST mutation. Mutations in the N-terminal part of the protein resulted in formation of giant lysosomes that are able to polarize to the cell-cell contact area, but are too big to be released; mutations in C-terminal part of LYST result in slightly enlarged granules that have decreased mobility, and thus impaired exocytosis. Intriguingly, the defect of release of the lytic granules was not observed in case of cytokine production and secretion. When compared to their parents, the CHS patients have similar level of up-regulation of MIP-1a and TNFa in response to cytokine stimulation. Moreover, the cytokine stimulation resulted in more robust IFNg production by NK cells of both patients (67-69% of NK cells in patients vs. 40 53% in parents). In comparison, NK cells from the CHS patient that received a bone marrow transplant appeared to be normal and the levels of the cell surface expression of NK cell activating and inhibitory receptors were also just like in healthy individuals. NK cells of the transplanted patient conjugated with target cells normally and the killing of two different target cell lines was comparable to NK cells from a healthy individual. Furthermore, NK cells of the transplanted patient readily polarized perforin and granzyme A to the cell-cell contact site. Interestingly, when compared to a healthy donor, NK cells of the transplanted patient showed increased degranulation in response to engagement of CD16 (27% vs 10% of the healthy donor) and, consequently, ADCC was increased in case of the transplanted patient. In response to cytokine stimulation, the production of MIP-1a, IFNg and TNFa by NK cells from the transplanted CHS patient was comparable to that of NK cells from a healthy donor. Thus, the bone marrow transplant fully restored NK cell functionality in this patient. We also analyzed NK cells from eight HPS-1, one HPS-2, two HPS-4, and one HPS-10 patient. NK cells from HPS type 2 and type 10 patients failed to kill target cells in natural cytotoxicity and ADCC assays, while NK cells from HPS type 1 and HPS type 4 patients had only slightly decreased capacity to kill the target cells. Conjugation of HPS NK cells to target cells did not appear to be significantly affected; F-actin accumulated at the cell-cell contact site, suggesting that the synapse formation was likely unaffected in those NK cells. However, NK cells from HPS type 2 and 10 failed to degranulate, in line with impaired cytotoxicity. Furthermore, we found that lytic granules in HPS-2 NK cells do not cluster efficiently around the MTOC, do not polarize to the IS, and appear to be slightly enlarged. HPS-10 NK cells contained large lytic granules that failed to polarize to the IS. The large lysosomes were reminiscent of the giant lysosomes observed in CHS. In several cases the large granules were positive only for perforin or granzyme A; combined with the increased size, these data suggest improper protein sorting and vesicular fusion. All HPS NK cells were able to produce cytokines (TNFa and IFNg) in response to stimulation. While HPS-1 and HPS-10 NK cells secreted normal levels cytokines, HPS-2 NK cells failed to release the cytokines following the cell stimulation. Thus, HPS-10 affects the cytolytic function of NK cells, while HPS-2 affects both lytic granule and cytokine secretion (regulated and constitutive exocytosis pathways).