Activated NK cells promote CD8⁺ cell responses. (A) NK cells stimulate CD8⁺ T-cell proliferation. Purified NK cells from Rag1^-/- mice were prestimulated with 0.5 μg/mL rLIGHT for 2 days and added to the culture of 2C T and irradiated Ag104 L^d cells at the indicated days for 7-day incubation. ³H-TdR incorporation and IFNγ assays were described previously. Significant stimulation of NK + 2C T cells to tumor can be detected by all 3 groups. (B) NK cells promote CD8⁺ cytolytic activity. 2C T cells with above treatment were cocultured with ⁵¹Cr-labeled Ag104 L^d at 50 of E/T ratio for specific lysis assay. (C) NK cells induce CD8⁺ T-cell maturation. Activated 2C T cells as above were stained with anti-CD8⁺ and anti-NKG2D (top) or anti–perforin through intracellular staining by permeabilization with 0.5% saponin (bottom). Data shown represent 1 of at least 3 independent experiments. Number in each quadrant indicates percentage of gated lymphocytes. (D) Perforin-positive CD8⁺ cells dynamically increased inside Ag104 L^d LIGHT tumor during the process of rejection. Single-cell suspension of tumor tissues from different times was stained with anti-CD8 and anti–perforin antibodies. Data shown represent 1 of at least 3 independent experiments. (E) Freshly isolated CD8⁺ T cells, not NK cells, from intratumor show marked killing activity. NK and CD8⁺ cells were isolated by NK or CD8⁺ purification kit from tumor tissues of day 22 Ag104 L^d LIGHT or Ag104 L^d tumor, followed by coculturing with ⁵¹Cr-labeled Ag104 L^d cells at the indicated E/T ratios for specific lysis assay. The results are representative of at least 3 independent experiments. Data analysis was performed by using the Student t test. Averaged results are expressed as mean plus or minus SD.