Analysis of NK Cell Function in Lysosome Storage Disorders

目前的重点是描述和了解一组Chediak-Higashi综合征(CHS)患者的自然杀伤(NK)细胞功能缺陷。我们分析了两名CHS活动性疾病患者、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。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。与他们的父母相比，两名CHS患者对细胞因子刺激的反应中，MIPb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细胞产生更强劲的IFNG-1(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细胞活化和抑制受体的细胞表面表达水平与健康人相当。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CHS患者NK细胞产生MIP-1b、IFNG和TNFa的能力与健康供者NK细胞相当。因此，骨髓移植完全恢复了患者的NK细胞功能。

The current emphasis is to describe and understand the defects in natural killer (NK) cell function in a group of patients with Chediak-Higashi syndrome (CHS). We analyzed NK cells in PBMCs of two 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. 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 two CHS patients have similar level of up-regulation of MIPb; 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 comparable to those of 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-1b, 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.