New Roles of Magnesium as a Regulatory Ion in Immune Responses and Cell Behavior

MAGT1转运蛋白在哺乳动物细胞内游离镁离子水平的选择性调节中起重要作用。真核细胞中游离镁离子的分子功能尚未完全确定。我们发现MAGT1基因缺陷的患者具有高水平的EB病毒(EBV)和淋巴瘤的易感性。在研究这些患者的淋巴细胞时，我们发现MAGT1的缺陷导致基础细胞内游离镁离子减少，导致NK和CD8+T细胞中自然杀伤分子激活受体NKG2D的表达缺陷。没有NKG2D，对EBV的细胞溶解反应被减弱，从而揭示了真核细胞中细胞内基础游离镁离子的第一个特定的分子功能。细胞内游离镁离子、NKG2D的表达和功能可以通过孵育患者细胞和升高镁离子水平在体外得到挽救。此外，在MAGT1缺陷患者体内，给予镁可以改善NKG2D的表达和细胞溶解功能，减少EBV感染细胞。因此，我们的数据表明游离基础镁离子在免疫中具有重要的分子功能，并表明在人类基本的EBV抗病毒反应中需要NKG2D细胞溶解功能。 我们特别感兴趣的是寻求与镁在EB病毒控制中的作用有关的其他问题。尽管与上皮性(鼻咽部和胃部)和淋巴系(Burkitt和Hodgkin淋巴瘤)的恶性肿瘤有关，但目前还没有已知的方法来一级或二级预防慢性EBV感染或相关的恶性肿瘤。我们发现，镁离子转运体的遗传缺陷会导致选择性免疫缺陷，从而导致不受控制的EBV感染和受影响儿童中极高的EBV+淋巴瘤发生率，而膳食中补充镁(一种广泛获得且廉价的营养食品)可以通过增加称为NKG2D的特定抗病毒受体来纠正免疫缺陷，从而显著减少或消除EBV，这为非洲慢性EBV提供了一个新的假说。我们正在与国家癌症研究所研究员Sam MBulaitye合作，他研究非洲的EBV/淋巴瘤风险。我们对以前从非洲收集的病例对照血液样本进行了初步研究，结果表明，EBV高的Burkitt淋巴瘤(BL)患者血清镁离子明显缺乏。这项初步研究不能回答这些患者是否细胞内镁离子和NKG2D表达不足，这需要在非洲进行现场流式细胞仪分析，或者是否可以通过向细胞中添加更多镁离子来恢复这些表达。然而，这些结果表明，地方性EB病毒和随后的淋巴瘤可以通过简单的饮食补充镁离子来预防。 虽然我们最初在XMEN患者中发现了细胞内镁离子水平和镁离子转运的异常，但我们扩大的患者队列显示总镁和电离血清镁浓度均正常，因此不能诊断XMEN。细胞内镁离子测定或T细胞受体诱导的镁离子通量已被广泛测试，但由于不可靠而不能用于临床诊断测试。因此，我们仍然推荐我们之前描述的通过流式细胞术评估NKG2D表面表达作为一种优秀的诊断工具，现在可以通过标准的临床诊断测试来增强这一点。 与XMEN患者一样，自身免疫性淋巴增殖性综合征(ALPS)患者也经常表现为双阴性幼稚T细胞升高、细胞凋亡缺陷和继发性淋巴样扩张。将阿尔卑斯患者纳入我们的研究队列，使我们能够使用飞行时间质谱仪对32个免疫标记物进行深入的免疫表型分析，并将其与XMEN患者进行比较，以寻找这两种疾病之间的区别特征。我们的分析表明，两个群体的幼稚B细胞的丰度可以区分XMEN、阿尔卑斯和健康个体。我们还对T淋巴细胞进行了糖蛋白质组学分析，结果表明XMEN病是一种先天性糖基化障碍，影响有限的糖蛋白亚群。MAGT1基因的转导使我们能够挽救糖基化缺陷的蛋白质。总之，这些数据为XMEN病的诊断和治疗提供了新的临床和病理生理基础，具有重要的影响。

The MAGT1 transporter is critically involved in the selective regulation of intracellular free Mg2+ levels in mammalian cells. The molecular functions of free Mg2+ in eukaryotic cells have not been fully established. We found that patients with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. In studying lymphocytes from these patients, we found that a deficiency of MAGT1 caused decreased basal intracellular free Mg2+ leading to defective expression of the natural killer activating receptor NKG2D in NK and CD8+ T cells. Without NKG2D, cytolytic responses against EBV are diminished, thereby revealing the first specific molecular function of intracellular basal free Mg2+ in eukaryotic cells. Intracellular free Mg2+, NKG2D expression and function can be rescued in vitro by incubating patient cells and elevated levels of Mg2+. Moreover, NKG2D expression and cytolytic function can be improved and EBV-infected cells reduced in vivo, in MAGT1-deficient patients by magnesium administration. Thus, our data indicate an important molecular function for free basal Mg2+ in immunity and demonstrate a requirement for NKG2D cytolytic function in an essential EBV antiviral response in humans. We are especially interested in pursuing additional questions related to the role of Mg2+ in the control of EBV. Despite being linked to both epithelial (nasopharyngeal and gastric) and lymphoid (Burkitt and Hodgkin lymphoma) malignancies, there are currently no known methods for primary or secondary prevention of chronic EBV infection or the associated malignancies. Our discovery that a genetic deficiency of a Mg2+ ion transporter caused a selective immunodeficiency that led to uncontrolled EBV infection and an extremely high rate of EBV+ lymphoma in affected children and that dietary supplementation with Mg2+ (a widely available and inexpensive nutraceutical) could correct the immune defect by increasing a specific antiviral receptor called NKG2D which markedly decreased or eliminated EBV offered a new hypothesis about chronic EBV in Africa. We are collaborating with Sam Mbulaiteye, a National Cancer Institute investigator, who studies EBV/lymphoma risk in Africa. We have carried out a preliminary study of previously collected case-control blood samples from Africa and showed that there was a statistically significant deficiency of serum Mg2+ in Burkitt lymphoma (BL) patients with high EBV. This preliminary study could not answer whether intracellular Mg2+ and NKG2D expression were deficient in these patients which requires flow cytometric analysis on site in Africa or whether these could be restored by adding more Mg2+ to the cells. However, these results held promise that endemic EBV and the consequent lymphomas could be prevented by simple dietary supplementation with Mg2+. While we initially found abnormalities of intracellular levels and transport of Mg2+ in patients with XMEN, our expanded patient cohort showed both total and ionized serum magnesium concentrations were normal and therefore not diagnostic of XMEN. Intracellular Mg2+ determination or T cell receptor-induced Mg2+ fluxes have been extensively tested and not proven useful for clinical diagnostic tests due to unreliability. Therefore, we still recommend our previously described NKG2D surface expression evaluation by flow cytometry as an excellent diagnostic tool, and this can now be augmented with the standard clinical diagnostic test. Like XMEN patients, Autoimmune Lymphoproliferative Syndrome (ALPS) patients also often present with elevated double negative naive T cells, defects in apoptosis, and secondary lymphoid expansion. Including ALPS patients in our study cohort allowed us to conduct deep immunophenotyping of 32 immune markers using time-of-flight mass cytometry and compare those to XMEN patients to seek distinguishable characteristics between the two diseases. Our analysis revealed that the abundance of two populations of naive B cells could differentially classify XMEN, ALPS, and healthy individuals. We also performed glycoproteomics analysis on T lymphocytes and showed that XMEN disease is a congenital disorder of glycosylation that affects a restricted subset of glycoproteins. Transfection of MAGT1 mRNA enabled us to rescue proteins with defective glycosylation. Together, these data provide new clinical and pathophysiological foundations with important ramifications for the diagnosis and treatment of XMEN disease.