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

MAGT1转运体在哺乳动物细胞内游离Mg2+水平的选择性调节中起着关键作用。游离Mg2+在真核细胞中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）和淋巴瘤易感性。在研究这些患者的淋巴细胞时，我们发现MAGT1的缺乏导致基底细胞内游离Mg2+减少，导致NK和CD8+ T细胞中自然杀伤激活受体NKG2D的表达缺陷。没有NKG2D，抗EBV的细胞溶解反应减弱，从而揭示了真核细胞中细胞内基底游离Mg2+的第一个特异性分子功能。体外培养患者细胞并提高Mg2+水平，可恢复细胞内游离Mg2+、NKG2D的表达和功能。此外，在magt1缺乏的患者体内，镁可以改善NKG2D的表达和细胞溶解功能，减少ebv感染的细胞。因此，我们的数据表明游离基础Mg2+在免疫中具有重要的分子功能，并且在人类基本的EBV抗病毒应答中需要NKG2D的细胞溶解功能。

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.