R-Spo1 mitigates GVHD-induced GSC injury. (A-D) Lethally irradiated B6 (Syn) or B6D2F1 (allo) mice received transplantation with bone marrow cells plus splenocytes from B6-Lck-cre donors on day 0. Allogeneic recipients were treated with R-Spo1 or PBS on days −3 to −1 and days +1 to +3. The stomach was harvested on day +7. (A) Macroscopic images of the gastric mucosa (top). The area in the black squares were magnified and shown in the bottom of the original images. Arrow heads indicate the erosions observed macroscopically. Bars, 5 mm (top) and 1 mm (bottom). (B) Immunofluorescent images of Lck^tdTomato+ T cells (red) in the gastric mucosa with DAPI nuclear staining (blue). (C-D) Numbers of CD4⁺ and CD8⁺ T cells infiltrated in the gastric mucosa. (E-J) Lethally irradiated B6D2F1-Lgr5-EGFP-creER mice received transplantation from B6D2F1 (syn) or B6 (allo) donors on day 0 and treated with R-Spo1 or PBS, as in panel A. On day +7, stomach was harvested. H&E staining (E), immunofluorescent images of cleaved caspase-3 (F) (red) with DAPI nuclear staining (blue), numbers of cleaved caspase-3 (G), PAS staining (H), immunofluorescent images of EGFP⁺ Lgr5⁺ GSCs (green) (I), and numbers of Lgr5⁺ GSCs (J) in the stomach. (H) Red arrow heads represent disrupted mucin layer. (B,E,F,H,I) Bars represent 100 μm. Data from 2 experiments were combined and shown as means ± standard error of the mean (n = 4-5 per group). ∗P < .05.