Figure 5.

Mg²⁺ regulates glycosylation and plays a unique role in T-cell–mediated immunity, especially in immune protection against EBV. A, flow cytometry histograms of surface expression of NKG2D, CD70, HLA-DR, and CD5 in cycling T cells from HCs cultured either in cRPMI for 5 days, dRPMI for 5 days, or cells that were cultured in dRPMI for 3 days followed by the addition of Mg²⁺ (0.5 mm) back into the dRPMI for 2 days. B, quantification of the MFI in A. Error bars represent the standard error of the mean of eight independent experiments, and p values were calculated with a paired t test. C, representative Western blot analysis of NKG2D, CD70, TCR-β, and β-actin in cycling T cells from a HC as described in A. Numbers at left indicate kDa standards. Glycosylation patterns are shown at right: fully-glycosylated (2); partially-glycosylated (1); and unglycosylated (0). Data are mean of eight (A and B) or are representative of three (C) independent replicates. D, schematic diagram for killing pathway with NK cells (top) and EBV-721.221(target; bottom) cells. E, flow cytometry analysis of surface expression of NKG2D in EBV-specific NK cells from a HC with cRPMI and dRPMI. F, quantification of the MFI in F. G, percent lysis of autologous EBV-LCLs by EBV-specific NK cells from HC with a different dose of the Mg²⁺ with significance determined by one-way ANOVA. n indicates the number of independent samples. H, quantification from G. Data are representative of three independent biological replicates. NS, nonsignificant.