Lymph node– and Peyer's patch–deficient LTα^−/− mice develop intestinal aGVHD, while splenectomized LTα^−/− mice are protected. Myeloablatively conditioned C57Bl/6 wild-type recipients (B6.WT) were compared with C57Bl/6-Lymphtoxin alpha^−/− mice (B6.LTα^−/−, deficient for PP and LNs) and splenectomized B6.LTα^−/− recipients (B6.LTα^−/−_SplEx) after transplantation with allogeneic luc⁺ FVB/N T cells plus WT BM cells. (A) Ex vivo BLI signals projected to the spleen, mLN, and PP in B6.WT mice, solely to the spleen in B6.LTα^−/− (B), but were not apparent in B6.LTα^−/−_SplEx recipients (C) on day 3 after aHCT. (D,E) On day 6 ex vivo BLI signals increased over the intestines in B6.WT and B6.LTα^−/− recipients in similar strength, in stark contrast to B6.LTα^−/−_SplEx recipients (F) Ex vivo imaging correlated well with H&E histopathology on day 6, by showing widespread signs of aGVHD (grade 2) in the small and large bowel of B6.WT (G) and B6.LTα^−/− recipients (H). In contrast, the intestinal mucosa of B6.LTα^−/−_SplEx recipients hardly showed any lymphocytes and no aGVHD (I).