IL-15–mediated acute GVHD requires expression of T-bet by donor splenocytes. (A) Highly purified wt B6 splenic T cells were cultured in the presence of rmIL-15 (10 μg/mL) for the times indicated. Mean fold induction of T-bet relative to time 0 plus or minus SEM is shown. The experiment was performed 3 times with similar results. (B) Peripheral blood mononuclear cells were harvested from 4- to 6-week-old wt B6 or IL-15 tg B6 mice. Expression of T-bet was measured by quantitative real-time PCR. N ≥ 6 mice per group. Data represent mean fold induction of T-bet relative to wt B6 mice plus or minus SEM. (C, D) Splenocytes were harvested from wt B6 or IL-15 tg B6 mice (n = 4 per group) and stained for cell-surface expression of CXCR3 (C) or stimulated for 6 hours using immobilized anti-CD3/28 antibodies followed by fixation and staining for intracellular IFN-γ (D). Data represent mean percent positive cells plus or minus SEM. (E) Lethally irradiated wt B6D2F1 mice underwent transplantation with 1 × 10⁷ wt B6 TCD BM cells and 5 × 10⁶ wt B6 unpurified splenic T cells (•, n = 9), 1 × 10⁷ IL-15 tg B6 TCD BM cells and 5 × 10⁶ wt B6 unpurified splenic T cells (□, n = 10), or 1 × 10⁷ IL-15 tg B6 TCD BM cells and 5 × 10⁶ T-bet^–/– B6 unpurified splenic T cells (⋄, n = 7). Survival was monitored daily and was compared using the logrank test. Data are combined from 2 similar experiments. (F) Body weights from animals in panel E were measured, normalized to day 0, and plotted as mean plus or minus SEM. Groups were compared as described in “Statistics.”