Second messenger role for Mg2+ revealed by human T-cell immunodeficiency

Abstract

The magnesium ion, Mg(2+), is essential for all life as a cofactor for ATP, polyphosphates such as DNA and RNA, and metabolic enzymes, but whether it plays a part in intracellular signalling (as Ca(2+) does) is unknown. Here we identify mutations in the magnesium transporter gene, MAGT1, in a novel X-linked human immunodeficiency characterized by CD4 lymphopenia, severe chronic viral infections, and defective T-lymphocyte activation. We demonstrate that a rapid transient Mg(2+) influx is induced by antigen receptor stimulation in normal T cells and by growth factor stimulation in non-lymphoid cells. MAGT1 deficiency abrogates the Mg(2+) influx, leading to impaired responses to antigen receptor engagement, including defective activation of phospholipase Cγ1 and a markedly impaired Ca(2+) influx in T cells but not B cells. These observations reveal a role for Mg(2+) as an intracellular second messenger coupling cell-surface receptor activation to intracellular effectors and identify MAGT1 as a possible target for novel therapeutics.

Figure 1. Patients have a proximal TCR activation defect

a, T cell CD4 and CD8 expression and ratio. b, CD31 expression in naïve CD4⁺ CD3⁺ T cells. c, CD69 expression in CD4⁺ T cells after 5 ug/ml anti-CD3 (αCD3) stimulation, PMA/Ionomycin (P/I) or unstimulated (Unstim). d, CD86 surface expression in purified B cells after stimulation with anti-IgM, SAC or unstimulated (Unstim). e, Confocal imaging of p65 nuclear translocation after αCD3 or P/I stimulation (scale bar: 10 μm). f, Percent cells with p65 (left) and NFAT (right) nuclear translocation. Numbers represent percent cells in indicated gates. Error bars represent s.e.m. (n=3), **** (P<0.0001).

Figure 2. Patients have MAGT1 null mutations and defective uptake of Mg²⁺

a, Pedigree of the families A (left) and B (right). b, X-chromosome inactivation assay. Peaks of PCR products from the different alleles are highlighted in yellow and pink. c, Schematic representation of the MAGT1 gene exons (boxes) and introns (lines), the mutations (*) and the probes used for RT-PCR. d, RT-PCR showing decreased expression of MAGT1 mRNA in T cells. e, Expression of MagT1 and actin control in T cells by immunoblot. f, Confocal images of T cells stained with anti-MagT1 antibody (scale bar: 5 μm).

Figure 3. TCR stimulation induces a MagT1-dependent Mg²⁺ influx

Mg²⁺ (upper panels) and Ca²⁺ (lower panels) flux: a, Fluxes in normal PBMC stimulated with ConA, PHA, αCD3, or αCD3/αCD28. b, Fluxes in healthy control T cells or Patient A.1 after 5 ug/ml αCD3 stimulation. c, Peak value of the fluxes in healthy control T cells and the 3 patients upon stimulation with indicated αCD3 concentrations. Error bars represent s.e.m. (n=3), **** (P<0.0001).

Figure 4. Requirement of receptor-stimulated Mg²⁺ flux for signaling

a, Mg²⁺ (MagFluo4, upper panel) and Ca²⁺ (Ratio F3/FR, lower panel) flux in healthy control T cells stimulated with αCD3 in buffers containing 1mM Mg²⁺ and Ca²⁺ (control) or lacking either ion. b, Ca²⁺ flux in B cells stimulated with αIgM and αCD40. Calcimycin (Cal) and EDTA addition display control influx and ion chelation, respectively. c, Graphs represent the fold change of the peak of Mg²⁺ (upper panels) and Ca²⁺ (lower panels) flux in A549 cells either unstimulated (Unstim) or stimulated with epidermal growth factor (EGF) or carbachol (Carb) as indicated. Error bars represent s.e.m. (n=3).

Figure 5. Knockdown and rescue of MagT1

Healthy T cells transfected with non-specific (NS) or MagT1 siRNAs. a, Mg²⁺ (MagFluo4, left) and Ca²⁺ (Fluo3, right) flux upon αCD3 stimulation. b, Percent nuclear p65 after MagT1 knockdown. Error bars represent s.e.m. (n=3). c, Time-lapse imaging (left, s = sec) or cytometry (right) of Mg²⁺ (upper) and Ca²⁺ (lower) flux in T cells transduced with lentiviruses expressing mCherry or mCherry + MagT1. d, Flow cytometry of CD69 expression on CD4+ T cells transduced with lentiviruses expressing MagT1 or not, either unstimulated (Unstim) or after anti-CD3 stimulation. Percent cells are shown for the indicated gates. Calcimycin (Cal) displays control influx.

Figure 6. MagT1 deficiency impairs PLCγ1 activation upon TCR stimulation

a, Confocal images of TCR clustering induced by αCD3. Cells were stained for LAT, PLCγ1 or phospho-PLCγ1. (Scale bar: 5 μm). Immunoblot of the indicated signaling proteins and phosphoproteins (b) and quantification of cellular IP3 level (c) in healthy control and patient T cells stimulated with αCD3 for indicated times. Error bars represent s.e.m. (n=3). ****(P<0.0001). d, Hypothetical schematic depicting how the MagT1 mediated Mg²⁺ influx participates in TCR signaling. Solid arrows indicate direct effects; dotted arrows indicate indirect effects.