(A–C) GVHD in BALB/c mice was induced as described in Figure 1A. BM cells were isolated on day 15 after transplantation from these GVHD mice (n = 3–4) and activated with cytokines (i.e., Flt3L plus SCF plus GM-CSF) for 2 hours, followed by measuring p-S6K1 (A) and ROS production (B). (C) Rapamycin (1.5 mg/kg, daily, i.p.) was administered into GVHD mice from days 0 to 14, with solvent treatment as control. MPPs were examined in these mice on day 15 after transplant. (D–F) MPPs were sorted from GVHD mice on day 15 after transplantation and labeled with CellTrace and cultured with Flt3L and SCF, with normal MPPs as controls. (D) Plots show CellTrace dilution and cell death on day 3 of culture. Graphs show the recovery rates and dead cell percentage of cultured MPPs. (E and F) On day 9 of culture, cell viability and recovery rate of pDCs were examined without (E) or with (F) NAC (500 μM) for 9 days. (G) Normal c-kit⁺ HSPCs were cultured in the upper chamber of a Transwell plate in the presence of Flt3L plus SCF. Naive T cells and GVHD T cells from normal mice and GVHD mice, respectively, were activated with anti-CD3 Ab plus anti-CD28 Ab for 3 hours and transferred into the lower chamber. Nine days later, cells were collected for pDC measurement. (H) Neutralizing Abs specific for each cytokine were added to the plate that contained c-kit⁺ HSPCs and activated GVHD T cells as mentioned above in G. pDC production in culture was examined 9 days later. (I and J) GM-CSF (5.0 ng/mL) was added to MPP cultures stimulated with Flt3L plus SCF. (I) The frequencies of pDCs and mo-DCs were measured on day 9. (J) Gene expression in GM-CSF–stimulated MPPs on day 3 of culture. Results shown are representative of at least 2 independent experiments. Two-group comparisons were evaluated by unpaired t test (E, F, I, and J), and multiple comparisons by 1-way ANOVA with Turkey’s multiple-comparison test (G) or 1-way ANOVA with Dunnett’s multiple-comparison test (H). *P < 0.05; **P < 0.01; ***P < 0.001.