BLI of gastrointestinal tissues and spleen combined with histology and immunofluorescence microscopy of Peyer patches during induction of acute GVHD. (A-D) Until day 3 after allogeneic HCT, BLI signals increase over Peyer patches (PPs), mesenteric lymph nodes (mLNs) and the spleen, while staying confined to these organs. Day 4 represents a transition, showing spread of BLI signals within the small bowel, preferentially in areas adjacent to PPs. Day 6 shows a diffuse BLI signal, covering the entire GIT. BLI signal increased over the spleen until day 3 and remained highly positive until day 6. (E-H) In H&E staining, the PPs of day 1 show irradiation-induced necrosis of B-cell follicles surrounded by large macrophages loaded with nuclear debris, which showed a high degree of green autofluorescence (compare panels I-L). Between days 3 and 4 the B-cell follicles disappear completely and are replaced by histiocytic infiltrates, while the T-cell zones appear to be conserved. On day 6 the architecture of PPs is further disturbed by a collapse of dome regions, leaving behind a flat structure, diffusely infiltrated by lymphocytes. (I-L) Triple-color staining performed with CD4-PE (red), CD8-FITC (green), and Thy1.1-APC as donor-specific marker (blue). CD4⁺ donor T cells specifically home to parafollicular T-cell areas of PPs within 1 day after transfer of splenocytes, while completely sparing subepithelial dome regions and B-cell follicles. Donor-derived CD4⁺ T cells clearly dominate in PPs over CD8⁺ T cells until day 3 and stay confined to the T-cell areas. The transition on day 4 shows a diffuse emigration of donor T cells, which become visible in the small bowel mucosa and submu-cosa adjacent to the PPs. On day 6 the PPs and other parts of the intestinum are diffusely infiltrated by donor T cells, now predominantly CD8⁺.