TBI increases nuclear RelB translocation in CD11c^hi DCs and is necessary for the full development of GVHD. (A) Mice were subjected to TBI, and 24 hours later APCs were enriched from spleens of control or irradiated mice. Cytospins were prepared with 10⁵ cells/slide, fixed, and stained for RelB using an immunoperoxidase technique. RelB is stained with diaminobenzidine (brown), and the nucleus is counterstained with hematoxylin (blue) at magnification ×1000. (B) Nuclear RelB-positive cells were enumerated (arrows in panel A), with a minimum of 10 high-power fields counted per slide. Results are expressed as the mean ± SEM. (C) Spleens were harvested from B6 mice before or 4 hours after TBI, and CD19⁺, CD11c^neg, and CD11c^hi populations were sort purified. Nuclear protein was extracted, and RelB DNA binding was detected by ELISA as described in “Materials and methods.” Results are expressed as the mean ± SEM of duplicate samples and representative of 2 similar experiments. (D) Survival by Kaplan-Meier analysis. Irradiated WT or RelB^−/− BMCs were transplanted with BM and T cells from Balb/c (allo; WT n = 20, RelB^−/− n = 18) or B6 donors (syn; WT n = 11, RelB^−/− n = 10) as described in “Materials and methods.” ***P < .0001, WT allo versus RelB^−/− allo. (E) GVHD clinical scores were determined weekly as described in “Materials and methods.” Results are expressed as the mean ± SEM. **P < .01 and *P < .05, WT allo versus RelB^−/− allo. Data were combined from 2 replicate experiments.