rLIF administration inhibits radiation-induced IL-12 production in DCs through the STAT1 signaling to protect against GVHD. (A) TBI (11 Gy) induced IL-12 production from DCs in MLNs, which was greatly reduced by rLIF administration as examined by using the cytokine panel at 24 hours after TBI in C57BL/6 mice. n = 8 mice/group. (B) TBI (11 Gy) increased percentage (left) and number (right) of IL-12⁺ cells in DCs in MLNs at 24 hours after TBI, which were largely decreased by rLIF administration in IL-12–p40–YFP C57BL/6 reporter mice as examined by flow cytometric assays. n ≥ 4 mice/group. The gating strategy and representative flow images are shown in supplemental Figure 8D. (C) The significantly decreased expression of T-bet in donor CD4⁺ T cells in MLNs from B6C3F1 mice at 7 days after allo-BMT with rLIF administration compared with mice without rLIF administration as determined by flow cytometric assays. n = 8 mice/group. (D) The induction of the expression of Th1 cytokine IFNγ by BM + T in B6C3F1 mice was largely decreased by rLIF administration as determined at 7 days after allo-BMT. Relative mRNA expression levels of IFNγ in the SI were determined by quantitative real-time PCR assays (left). Protein levels of IFNγ in the SI (middle) and serum (right) were determined by ELISAs. n ≥ 4 mice/group. (E-F) Administering rIL-12 largely abolished the protective effect of rLIF on GVHD. Lethally irradiated B6C3F1 mice that received allo-BMT from C57BL/6 mice along with or without rLIF administration were treated with rIL-12 (500 ng/d for 7 days from D-4 to D3) or PBS. (E) The spleen weight (left) and length of SI (right) in B6C3F1 mice measured at 7 days after allo-BMT. (F) MFI of MHC-II on IECs of B6C3F1 mice were determined at 7 days after allo-BMT. n ≥ 3 mice/group. (G-H) Administering rIL-12 largely abolished the inhibitory effect of rLIF on donor immune cell infiltration after allo-BMT. (G) The relative numbers of infiltrating donor cells in the spleen, MLN, and LP tissues from B6C3F1 mice at 7 days after allo-BMT. (H) The relative numbers of infiltrating donor immune cells in the spleen (top), MLN (middle), and LP (bottom) tissues from B6C3F1 mice at 7 days after allo-BMT. n ≥ 6 mice/group. (I) BMDCs were activated by IFNγ (10 ng/ml) and LPS (100 ng/ml) with or without LIF treatment (100 ng/ml) for 6 hours. The mRNA levels of IL12b in BMDCs were determined by quantitative real-time PCR assays and normalized with β-actin. n = 7/group. (J) rLIF treatment increased the phosphorylation levels of STAT1 at Tyr-701 (p-STAT1) in activated BMDC as determined by Western blot assays. (K) rLIF treatment increased the binding of STAT1 to a putative STAT1 binding site in the intron 1 of IL12b gene as determined in BMDCs by chromatin immunoprecipitation assays. (Top) The sequence and location of the putative STAT1 binding site in IL12b gene. A region containing no STAT1 binding site was included as a negative control. n = 5/group. n.d.: non-detectable. (L) Blocking the STAT1 signaling by fludarabine (2 μM) and pravastatin (2 μM), 2 small-molecule STAT1 inhibitors, largely abolished the inhibitory effect of rLIF on IL12b production in activated BMDCs. The mRNA levels of IL12b in BMDCs were determined by quantitative real-time PCR assays and normalized with β-actin. n = 3/group. Data are presented as mean ± standard deviation from 3 independent experiments. ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, n.s.: not significant; unpaired t test with Welch’s correction.