NK-cell–mediated cytotoxicity of autologous T-cell blasts is dependent on NKG2D and calcium. (A) Purified CD4⁺and CD8⁺ T lymphocytes were cultured with medium alone or stimulated with PMA/ionomycin for 48 hours, and then stained with anti-MICA mAb (thick line) or control Ig (filled histogram). (B) Cytotoxicity assay was performed using untreated or PMA/ionomycin-activated CD4⁺or CD8⁺ T lymphocytes as targets, and IL-2–activated autologous NK cells as effectors, at the indicated effector-target ratios. The assay was performed in the presence of an anti-NKG2D neutralizing mAb, of an anti-CD56 mAb used as isotype control, or of the Ca^+‖+ chelator EGTA (final concentration of 4 mM). CD4⁺ T cells from 4 donors and CD8⁺ T cells from 3 donors were analyzed. Data shown derive from the same donor. (C) PBMCs were stimulated with SEB and after 3 days cells were stained with anti-CD3 and mAbs specific for NKG2D ligands. MFI values relative to MICA (thick line) and evaluated on CD3⁺ T cells are shown. Control IgG, filled histogram. MFI values of ULBP molecules were as follows: ULBP1 = 0, ULBP2 = 0, ULBP3 = 2 for donor 1; ULBP1 = 1, ULBP2 = 3, ULBP3 = 4 for donor 2 (data not shown). CD3⁺ T cells in both donors were approximately 80%. (D) The same cells described in panel C were used as targets in a cytotoxicity assay, together with IL-2-activated autologous NK cells as effectors. Data shown derive from 2 of 3 donors.