Mg2+ regulates cytotoxic functions of NK and CD8 T cells in chronic EBV infection through NKG2D

Abstract

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium (Mg(2+)) concentrations. Individuals with genetic deficiencies in MAGT1 have high levels of Epstein-Barr virus (EBV) and a predisposition to lymphoma. We show that decreased intracellular free Mg(2+) causes defective expression of the natural killer activating receptor NKG2D in natural killer (NK) and CD8(+) T cells and impairs cytolytic responses against EBV. Notably, magnesium supplementation in MAGT1-deficient patients restores intracellular free Mg(2+) and NKG2D while concurrently reducing EBV-infected cells in vivo, demonstrating a link between NKG2D cytolytic activity and EBV antiviral immunity in humans. Moreover, these findings reveal a specific molecular function of free basal intracellular Mg(2+) in eukaryotic cells.

Figure 1. XMEN patients exhibit high EBV levels, lymphoma development, and defective cytotoxicity

Levels of EBV DNA in XMEN patient blood (n=7) expressed as copies/mL blood (†), copies/10⁶ cells (*), or copies/ug DNA (§) depending on the specific clinical laboratory. Each point represents an independent blood draw measurements at different times. Chronic active EBV (CAEBV) patient values are shown for comparison. Shaded area indicates normal range, EBV DNA is typically undetectable in EBV⁺ subjects with less than 10% of normal individuals usually showing no more than 20 – 80 copies/mL in the blood (37). (B) Histopathology of the various EBV⁺ lymphoproliferative disorders (LPD) in XMEN patients. The upper panel shows the hematoxylin and eosin (H&E) staining and the lower panel shows the EBV–encoded small RNA (EBER) staining by in situ hybridization. Yellow arrowheads show Reed-Sternberg cells. (Magnification: 400X for patients B.1 and E.1 (bottom panel) and 1000X for patient F.1 and E.1 (top panel)). (C) Cytotoxicity of EBV-specific CTLs from father (), mother (), and XMEN patients A.1 () and A.2 () on autologous LCLs. (D) Cytotoxicity of IL-2-expanded NK cells from normal control () or XMEN patients A.1 () and A.2 () against K562 cells. Results shown in C and D are representative of results of all XMEN patients tested (n=4) in at least three independent experiments (Two-Way ANOVA, ***: p<0.001; ****: p<0.0001).

Figure 2. Mg²⁺ supplementation restores intracellular Mg²⁺ and enhances cytotoxicity but does not restore TCR activation

(A) Flow cytometry profile of the ratio of the mean fluorescent intensity (MFI) of Mg²⁺-specific fluorescent probe MagFluo4 to the MFI of the Ca²⁺-sensitive probe Fura Red (MF4/FR) using PBMC from normal control () and XMEN patient A.1 (). The ratio was arbitrarily set at 1 for normal control (dotted line). (B) Dot plot of the MF4/FR ratios from PBMCs of normal controls (, n=5) and XMEN patients (, n=6), non-XMEN CAEBV patients (, n=5) or XLP patients (, n=4). (C) Flow cytometry profiles of MF4/FR ratio on normal control and XMEN patient CTLs cultured in media supplemented with indicated amounts of MgSO₄ for 5 days. (D) MF4/FR ratio of CTLs from normal control (, n=4) or XMEN patients (, n=4). (E) Percentage of CD69⁺ in CD8⁺ cells after stimulation with anti-CD3 (1 µg/ml) for 24h in presence of indicated amount of MgSO₄ or unstimulated. (F) Cytotoxicity of normal or XMEN patient EBV-specific CTLs, supplemented with or without 2 mM of MgSO₄ for 5 days, on autologous LCLs. (G) Cytotoxicity of normal or XMEN patient NK cells, supplemented with or without 2 mM of MgSO₄ for 5 days, on K562 targets. Results shown are representative of all XMEN patient tested (n=4) in at least three independent experiments (Two-Way ANOVA, *:p=0.01, **:p=0.0038, ****: p<0.0001).

Figure 3. Effect of Mg²⁺ on NKG2D expression and function in vitro

(A) Flow cytometry profiles of the surface expression of NK receptors on gated NK cells (CD3^-CD56⁺) from normal controls ( and ) and XMEN patient A.1 (). Isotype antibody staining () is shown as a reference for background fluorescence. Results are representative of all XMEN patients tested (n=5). (B) Normalized MFI of NKG2D surface staining on gated CTLs from whole blood of normal controls (, n=5) and XMEN patients (, n=6), non-XMEN CAEBV patients (, n=5) or XLP patients (, n=4). (C) Immunoblots of CTLs and NK cells from normal control and XMEN patient with indicated antibodies. (D) NKG2D-specific redirected lysis of normal or XMEN patient IL-2-expanded NK cells on P815 cells expressing the NKG2D ligand, ULBP1. (E) NKG2D-specific redirected lysis of normal or XMEN patient CTLs on P815 expressing the NKG2D ligand ULBP1 or not in presence of anti-CD3 antibodies. (F) Flow cytometry profiles of MF4/FR ratio in PBMCs (left) and NKG2D expression on CTLs (middle) and NK cells (right) from normal control or XMEN patient supplemented with the indicated concentrations of MgSO₄ for five days. (G) Immunoblot of cells lysates from CTLs from a normal control or an XMEN patient supplemented with or without 5 mM of MgSO₄ for 5 days. (H) Cytotoxicity of normal control or XMEN patient EBV-specific CTLs, supplemented with or without 2 mM of MgSO₄ for 5 days, on autologous EBV-transformed LCLs pre-treated with or without soluble NKG2D-Fc. In C and G, black arrows heads show the various forms of the indicated protein. “nNKG2D” indicates the normal form of NKG2D, “pNKG2D” the patient’s forms and * indicates a nonspecific band detected by the anti-NKG2D antibody. Results shown are representative of all XMEN patient tested (n=4) in at least three independent experiments (Student t-test (B), Two-Way ANOVA (D-E-H), ****: p<0.0001).

Figure 4. In vivo Mg²⁺ supplementation of XMEN patient restored NKG2D expression and decreased EBV⁺ PBMCs

Flow cytometry profiles of MF4/FR ratio in PBMC (A) and NKG2D expression on gated CTLs (CD3⁺CD8⁺) (B) from a normal control (Mother, untreated), patient A.1 (treated) and patient A.2 (treated) at the indicated times of magnesium supplementation in days (D). Mother was not available for sample on D14. (C) Quantification of the MF4/FR ratio (top), NKG2D MFI (middle) and percentage of EBV⁺ B cells measured by EBV–encoded small RNA (EBER) fluorescence in situ hybridization (FISH) (bottom) during the magnesium supplementation. The arrowhead and dotted line indicates the starting point of the magnesium supplementation. NKG2D-Fc staining on gated B cells (CD19⁺) from fleshly isolated PBMC of a normal control, XMEN patients (before D-63 and after D175 Mg²⁺ supplementation) alone by flow cytometry (D) or co-stained with EBER FISH in confocal microscopy (E). (F) Quantification of the EBER⁺/NKG2D-Fc⁺ cells shown as percentage of EBV⁺ cells that are NKG2D-Fc⁺ or NKG2D-Fc^- in the patient’s PBMCs during in vivo Mg²⁺ supplementation as measured by EBV-specific FISH. (scale bar: 5 µm).