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

在哺乳动物细胞中，MAGT 1转运蛋白在细胞内游离Mg 2+水平的选择性调节中起关键作用。游离Mg ~（2+）在真核细胞中的分子功能尚未完全确定。我们发现，MAGT 1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。在研究来自这些患者的淋巴细胞时，我们发现MAGT 1的缺乏引起基础细胞内游离Mg 2+减少，导致NK和CD 8 + T细胞中自然杀伤激活受体NKG 2D的表达缺陷。在没有NKG 2D的情况下，针对EBV的细胞溶解反应减弱，从而揭示了真核细胞中细胞内基础游离Mg 2+的第一个特异性分子功能。细胞内游离Mg 2+、NKG 2D表达和功能可通过孵育患者细胞和升高的Mg 2+水平在体外进行挽救。此外，在MAGT 1缺陷患者中，镁给药可改善NKG 2D表达和细胞溶解功能，并减少体内EBV感染的细胞。因此，我们的数据表明游离基础Mg 2+在免疫中具有重要的分子功能，并证明在人体的基本EBV抗病毒应答中需要NKG 2D细胞溶解功能。 我们特别感兴趣的是探讨与Mg 2+在EBV控制中的作用相关的其他问题。尽管与上皮（鼻咽和胃）和淋巴（伯基特和霍奇金淋巴瘤）恶性肿瘤有关，但目前还没有已知的方法用于慢性EBV感染或相关恶性肿瘤的一级或二级预防。我们发现，Mg 2+离子转运蛋白的遗传缺陷导致选择性免疫缺陷，导致受影响儿童中不受控制的EBV感染和极高的EBV+淋巴瘤发生率，（一种广泛使用且价格低廉的营养品）通过增加一种称为NKG 2D的特异性抗病毒受体来纠正免疫缺陷，从而显著减少或消除EBV，这提供了一个新的假设关于非洲的慢性EB病毒。我们正在与美国国家癌症研究所研究员Sam Mbulaiteye合作，他研究非洲的EB病毒/淋巴瘤风险。我们已经进行了一项初步研究，以前收集的病例对照血液样本来自非洲，并表明，有一个统计学上显着的缺乏血清Mg 2+伯基特淋巴瘤（BL）高EBV患者。这项初步研究无法回答这些患者的细胞内Mg 2+和NKG 2D表达是否缺乏，这需要在非洲现场进行流式细胞术分析，或者是否可以通过向细胞中添加更多的Mg 2+来恢复。然而，这些结果有希望，地方性EBV和随之而来的淋巴瘤可以通过简单的饮食补充Mg 2+来预防。 虽然我们最初发现XMEN患者的细胞内水平和Mg 2+转运异常，但我们扩大的患者队列显示总血清镁和离子血清镁浓度均正常，因此不能诊断XMEN。细胞内Mg 2+测定或T细胞受体诱导的Mg 2+通量已被广泛测试，但由于不可靠而未被证明可用于临床诊断测试。因此，我们仍然推荐我们先前描述的通过流式细胞术进行的NKG 2D表面表达评估作为一种优秀的诊断工具，现在可以使用标准临床诊断测试进行增强。 与XMEN患者一样，自身免疫性淋巴细胞增生综合征（ALPS）患者也经常表现出双阴性幼稚T细胞升高、细胞凋亡缺陷和次级淋巴细胞扩增。在我们的研究队列中包括ALPS患者，使我们能够使用飞行时间质谱细胞术对32种免疫标志物进行深度免疫表型分析，并将其与XMEN患者进行比较，以寻求两种疾病之间的可区分特征。我们的分析显示，两个群体的幼稚B细胞的丰度可以区分XMEN，ALPS和健康个体。我们还对T淋巴细胞进行了糖蛋白组学分析，结果表明XMEN病是一种先天性糖基化障碍，影响糖蛋白的一个有限的子集。MAGT 1 mRNA的转染使我们能够拯救糖基化缺陷的蛋白质。总之，这些数据提供了新的临床和病理生理学基础，为XMEN疾病的诊断和治疗提供了重要的分支。

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.