NKG2D is upregulated by AAT and is required for tumor cell kill. (A) NKG2D expression on NK cells from spleens of AAT-treated (blue) or albumin-treated (green) control mice; red = isotype control. Histograms are gated on NK1.1⁺CD3− populations (n = 5, P = .001). (B) Cytolytic function of NK cells obtained from spleens of AAT-treated donors (n = 7) and assayed at effector to target ratios of 1:1; 5:1, 10:1, and 50:1 against A-20 cells. NK 1.1 cells purified from AAT-treated donors were incubated with isotype control antibodies (Iso IgG2b), with anti-NKG2D antibody or with anti-Rae, the cognate receptor on A-20 cells. Lytic activity was measured after 18 hours of incubation with the effector cells (error bars indicate standard deviation of 5 independent experiments). (C) Recipient mice received 800 cGy TBI and were injected with 10⁴ A-20 cells IV 4 days before transplantation of allogeneic cells from AAT-treated C57BL/6 donors. Tumors were imaged at 6, 10, and 14 days after HCT using the luciferase reporter signal. Tumor growth as determined by bioimaging was more aggressive in mice treated with NKG2D blocking antibody (anti-CD314 [NKG2D] left panel), leading to death or requiring sacrifice. In mice not given the blocking antibody (control, right panel) the tumor regressed by day 14. (n = 12, each condition, transplant experiments were done in triplicates, results presented as the sum of transplantations). (D) Tumor size (luciferase activity) with NKG2D blockade (red), Fc Isotype control IgG2b (black); photon reading of luciferase activity; higher readings indicate greater tumor volume (values represent mean ± SEM of 12 mice, P = .0043). (E) Survival of Balb/c (H-2d Kd) recipients of C57BL/6 [H-2b] donors that had been treated with the above conditions, P values displayed in the figure, (n = 12 per arm).