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

National Institute Of Allergy And Infectious Diseases

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Abstract

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. We coined the disease as XMEN, X-linked immunodeficiency with magnesium defect, EBV infection, and neoplasia. 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+ and 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+. Additionally, we became interested in using CRISPR technology as a therapeutic intervention. A major barrier to clinical translation of promising CRISPR-gene correction procedures is the low engraftment rates of edited hematopoietic stem/progenitor cells (HSPCs) with substantially reduced correction rates following transplant in mouse xenograft models, indicating poor targeting of engrafting human HSPCs. We addressed this by transient suppression of specific protein suppression to achieve highly efficient MAGT1 correction in engrafting XMEN patient HSPCs for potential gene therapy. This versatile approach was also readily applied to correct XMEN T cells. We were able to restore MAGT1 and NKG2D levels in CD8+ T cells and NKG2D receptor expression and cytotoxic activity in NK cells ex vivo. Freezing and thawing of the targeted cells maintained high levels of the MAGT1, NKG2D, and NKG2D receptor expression and cytotoxic activity. These results are exciting as a potential gene therapy treatment to control chronic high loads of EBV and may reduce the risk of EBV+ lymphoma in XMEN disease.