Figure 3.

Supplemental magnesium restores impaired degranulation and cytotoxicity associated with T cell signaling in vitro in cells from a patient with homozygous ITK mutations. (A) Cytotoxicity is impaired in CD8+ cells from the patient compared to CD8+ cells from a healthy control. Patient or healthy control CD8+ cells were incubated with L1210 murine cells with anti-CD3 and anti-Fas antibodies. Cytotoxicity was measured by flow cytometry as the percentage of L1210 cells positive for active caspase-3. E:T ratio is the effector cell to target cell ratio. Data represent mean ± standard deviation (SD). (B) Degranulation is impaired in CD8+ cells from the patient following CD3/CD28 stimulation. Prior to stimulation with anti-CD3/CD28 antibodies, anti-CD107a antibody (a marker of degranulation) was added to cells from the patient or healthy normal control (NC). Following stimulation, patient and control cells positive for CD107a were measured by flow cytometry. Degranulation was quantified as the fold change of the percent of CD107a positive cells from the stimulated (Stim) to non-stimulated (NS) samples and normalized to the healthy control. PMA/ionomycin was used as a positive control, which shows similar degranulation in patient and control cells. Data represent mean ± SD. (C) Cytotoxicity in CD8+ cells following culture with 1 mM supplemental magnesium (Mg) or no additional magnesium. Data represent mean ± SEM. (D) Degranulation when patient and healthy control cells were cultured in supplemental Mg for 5 days. Data represent mean ± SD. (A–D) Asterisk indicates p < 0.05. anti-CD3, anti-CD28, and anti-CD107a antibodies were from Biolegend, PE-anti-caspase 3 antibody was from BD Bioscience, and anti-Fas antibody was from Millipore. All experiments were repeated two to four times and pooled data are shown.