MAGT1-mediated disturbance of Mg2+ homeostasis lead to exhausted of HBV-infected NK and CD8+ T cells

The magnesium transporter 1 (MAGT1) is a critical regulator of basal intracellular free magnesium ([Mg2+]i) levels. It has been shown that MAGT1 was involved in the disorder in Mg2+ homeostasis after Epstein-Barr virus (EBV) infection. Here, we identified the effects of MAGT1-mediated disturbance of Mg2+ homeostasis on chronic hepatitis B virus (HBV)-infected natural killer (NK) and CD8+ T cells. The expression of MAGT1 was gradually decreased with the increase of infected time in CD8+ T cells, but not with that in NK cells, of the patients. Decreased level of intracellular free Mg2+ ([Mg2+]i) leads to defective expression of programmed cell death 1 (PD-1) and the NK activating receptor (NKG2D) in NK and CD8+ T cells. Our data illustrate that [Mg2+]i plays a key role in control of HBV infection.

groups was at normal levels ( Fig. 1B and D). Moreover, the levels of both Mg 2+ and Ca 2+ were not significantly different in NK cells ( Fig. 1E and F).

MAGT1 expression is defective in CD8 + T cells of patients. MAGT1 is a critical regulator of basal
[Mg 2+ ]i concentrations. To investigate whether defective Mg 2+ influx is the cause of defective MAGT1 expression, MAGT1 expression was detected in different cells. As shown in Fig. 2A, MAGT1 expression was gradually decreased with the increase of infected time in CD8 + T cells, but not with that in NK cells, of the patients. However, the mRNA levels of MAGT1 in CD8 + T cells of the patients did not change as compared to those in healthy controls (Fig. 2B). These results indicated that MAGT1 expression was defective in CD8 + T cells of the patients possibly through post-transcriptional regulation.
To primarily confirm whether MAGT1 was regulated at the post-transcriptional level, such as through non-coding RNA, including microRNAs (miRNAs), the expression of miRNAs related to MAGT1 was detected. The detected miRNAs were selected from TargetScan (http://www.targetscan.org/vert_71/) prediction result, among which miR-199a-5p and miR-199b5p were predicted as the most conserved potential target regulators for MAGT1. As shown in Figure S1, the expression levels of miR-199a-5p and miR-199b5p in CD8 + T cells of the patients infected with HBV for more than 3 years were significantly increased as compared to those in normal patients.
Expression of NKG2D and programmed cell death 1 is abnormal in CD8 + T cells. As mentioned above, MAGT1 expression was defective after HBV infection. Lack of MAGT1 leads to decreased plasma Mg 2+ levels in infected CD8 + T cells. NKG2D expression was continuously regulated by the free [Mg 2+ ]i. Notably, NKG2D expression is induced by infection, cellular transformation, and cell stress in humans 11,12 . Here, the expression of NKG2D and programmed cell death 1 (PD-1) in CD8 + T and NK cells was detected by flow  2+ and Ca 2+ was measured in the serum by spectrophotometry with XB-1 and indirect potentiometric determination, respectively, using a Dxc800 automatic biochemical analyzer. (C-F) The concentration of Ca 2+ and Mg 2+ was measured in CD8 + T and NK cells by the fluorescence probe Mag-Fura4-AM and Fura-2AM, respectively, using flow cytometry. Data are expressed as mean ± SD from at least 3 independent experiments, *P < 0.05 vs. Healthy controls, **P < 0.01 vs. Healthy controls.
cytometry. As shown in Figs 3 and 4, the expression of NKG2D and PD-1 is abnormal in CD8 + T cells. NKG2D expression in CD8 + T cells of the patients decreased significantly in the late infection stage. On the contrary, expression of PD-1 increased significantly in CD8 + T cells of the patients.

Mg 2+ supplementation in clinical studies.
To confirm the exact role of Mg 2+ , clinical studies were conducted. Forty patients were included in the clinical trials, which were divided into two groups-one was treated with Mg 2+ and entecavir, while the other was treated with placebo and entecavir as the control. There was no significant difference in gender and age between the two groups. The clinical studies continued for over nine months. Consequently, the Mg 2+ level in the serum of patients with Mg 2+ supplementation significantly increased to an affordably normal level, and the protein level of MAGT1 also recovered to normal. In contrast, the Mg 2+ level in the serum and the protein level of MAGT1 remained in an aberrant situation in the patients with no Mg 2+ supplementation ( Fig. 5A and B).

Discussion
T cells are majorly acquired immune cells in humans. Depending on the CD molecules on the cell surface, T cells are divided into CD8 + and CD4 + T cells. As the major effector cells in the process of HBV infection, CD8 + T cells mainly exert their effects in the chronic phase and mediate immune responses (cytotoxic T lymphocyte, CTL). In this study, we found that serum Mg 2+ /Ca 2+ concentrations were at normal levels in patients with chronic hepatitis B, and were similar to those in healthy controls. In the case of CD8 + T cells, the Mg 2+ level decreased significantly in infected patients after 6 months as compared to those in non-infected patients. At the end of a 3-year investigation, Mg 2+ levels in CD8 + T cells of the infected patients decreased to the lowest. Consistent with a previous study, Ca 2+ concentration in both the groups was at normal levels 5 . The levels of both Mg 2+ and Ca 2+ were not significantly different in NK cells. Expression of PD-1 increased and that of NKG2D decreased in CD8 + T cells of the patients.
It has been reported that MAGT1 acts not only as a TCR-gated transporter, but also as a basal free [Mg 2+ ] regulator 13,14 . In this study, MAGT1 expression was gradually decreased with the increase of infected time in CD8 + T cells, but not with that in NK cells, of the patients. The mRNA levels of MAGT1 in CD8 + T cells of the patients did not change as compared to those in healthy controls. The Mg 2+ levels in CD8 + T cells in the plasma of patients also decreased significantly. The expression of the potential target regulators, miR-199a-5p and miR-199b-59, increased in patients with HBV, implying that decreased MAGT1 expression might be regulated at the post-transcriptional level ( Figure S1). MAGT1 expression may be defective after HBV infection. Lack of MAGT1 leads to decreased plasma Mg 2+ levels in infected CD8 + T cells. NKG2D expression in CD8 + T cells of the patient also decreased significantly in the late infection stage. These observations are consistent with those in the previous studies [15][16][17] . NKG2D expression was continuously regulated by free [Mg 2+ ]i. Notably, NKG2D expression is induced by infection, cellular transformation, and cell stress in humans 11,12 . Consistently, it could be explained as follows: in the late stage of chronic infection, viral replication is not well controlled and decreased Mg 2+ level caused defective NKG2D expression. On the contrary, PD-1 expression increased significantly in CD8 + T cells of the patients. In the T cells, the inhibitory effect of PD-1 is based on the TCR conduction. Therefore, it might play a role as a negative regulator in CD8 + T cells. The clinical utility of Mg 2+ supplementation strongly supports the data. There was no significant difference in gender and age between each experimental group and the control groups ( Table 1). All human subjects in this study provided written informed consent in accordance with Chinese legal principles and were approved by the Medical Ethics Committee of Wuhan General Hospital of the Chinese People's Liberation Army.

Materials and Methods
Cell purification and culture. PBMCs were isolated using whole blood from the normal control and patients by Ficoll-Paque PLUS (GE Healthcare, USA) and density-gradient centrifugation. The cells were washed twice in phosphate buffered saline (PBS), resuspended at a density of 10 6 cells/mL in complete RPMI 1640 medium (Lonza, Switzerland) supplemented with 10% fetal bovine serum (FBS), 2 mM glutamine, and penicillin and streptomycin (100 U/mL each; Invitrogen, USA), and incubated in a humidified incubator at 37 °C and 5% CO 2 .

Isolation and identification of CD8 + T and NK cells.
To prepare NK cells, PBMCs were incubated with a cocktail of anti-CD3, anti-CD19, and anti-CD14 monoclonal antibody (mAb)-coated microbeads, and NK cells were isolated by passing the PBMCs through a magnetic cell separation system (MZSC; Miltenyi Biotec, Germany) with column type VR. More than 95% of the cells were confirmed to be CD56 + (Miltenyi Biotech, Germany) NK cells by flow cytometry. Simultaneously, to isolate CD8 + T cells, PBMCs were suspended in labeling buffer and incubated with anti-CD8 mAb-coated microbeads. CD8 + T cells were isolated using a magnetic cell separation system with column type VR. More than 95% of the cells were confirmed to be CD8 + (Miltenyi Biotech, Germany) T cells by flow cytometry. 2+ . The concentration of Ca 2+ and Mg 2+ in the serum was measured by spectrophotometry with XB-1 and indirect potentiometric determination, respectively, using a Dxc800 automatic biochemical analyzer (Beckman, USA). The cell density was adjusted to 1.5 × 10 6 cell/mL in assay buffer without Mg 2+ and Ca 2+ , and cells were respectively loaded with 3 μM Mag-Fura4-AM (ThermoFisher, USA) or 2 μM Fura-2AM (ThermoFisher, USA) for 20 min at room temperature (RT) in the dark. Ten milliliters PBS was added, centrifuged at 300 × g for 5 min, and the cells were resuspended in assay buffer. The measurement of [Mg 2+ ]i and [Ca 2+ ]i were conducted at RT using flow cytometry. Flow cytometry data were analyzed using FlowJo software 3.3.