Gastric Stem Cells Are Targeted in Upper Gastrointestinal GVHD

Introduction: Graft-versus-host disease (GVHD) is the major obstacle to perform allogeneic hematopoietic stem cell transplantation (SCT). We previously demonstrated that intestinal stem cells (ISCs) were targeted in lower gastrointestinal (GI) GVHD (Takashima et al, J Exp Med. 2011). However, it remains to be investigated whether gastric stem cells (GSCs) could be targeted in upper GI GVHD. We evaluated if leucine-rich repeat-containing G protein-coupled receptor (Lgr) 5⁺ GSCs, which replenish antrum and pyloric epithelium, could be damaged in GVHD. Wnt agonist R-Spondin 1 (R-Spo1), which binds to Lgr5, protects ISCs and ameliorates GVHD. We also evaluated if R-Spo1 could protect Lgr5⁺GSCs against GVHD.

Methods: B6D2F1 mice (H-2^b/d) were transplanted with 5 × 10⁶ splenocytes plus 5 × 10⁶ bone marrow cells from allogeneic B6 (H-2^b) or syngeneic B6D2F1 donors following lethal X-ray total body irradiation on day 0 and histological evaluation of the pylorus and antrum of stomach and flow cytometric analysis of infiltrating donor T cells in the stomach were performed on day +7 after SCT. Recipients of SCT were intravenously injected with 100 μg/day of R-Spo1 from days -3 to -1 and days +1 to +3 after SCT, or naïve mice were injected with 200 µg/day of R-Spo1 for six days.

Results: We investigated the fate of GSCs after SCT in Lgr5-creERT2 / eGFP B6D2F1 recipients. Immunofluorescent studies demonstrated development of gastric GVHD with significant donor T-cell infiltration in the lamina propria of gastric mucosa and cleaved-caspase-3⁺ apoptotic epithelial cells in the stomach of allogeneic animals, but not in syngeneic controls (Figure A). Numbers of Lgr5⁺ GSCs were significantly decreased in allogeneic recipients compared to those in syngeneic controls on day +7, suggesting that gastric GVHD targets Lgr5⁺ GSCs (Figure B). We then evaluated effects of R-Spo1 on GSCs. When injected to naïve B6D2F1 mice for 6 days, R-Spo1 significantly prolonged the crypt depth with an increase in numbers of Ki-67⁺ proliferating epithelial cells in the stomach. Fate mapping analysis using Lgr5-creERT2/eGFP × Rosa26^Tomato mice, in which Lgr5⁺ GSCs and their progenies are labeled with tdTomato and Lgr5⁺ GSCs are labeled with eGFP, showed that R-Spo1 did not increase eGFP⁺ GSCs but significantly increased Ki67⁺ cells and tdTomato⁺ epithelial cells. These data suggested that R-Spo1 stimulated differentiation of Lgr5⁺ GSCs into mucosal epithelial cells. We then tested if GVHD prophylaxis with R-Spo1 protects GSCs and ameliorates gastric GVHD after allogeneic SCT. Immunofluorescent analysis showed that brief administration of R-Spo1 significantly suppressed infiltration of donor T-cells and epithelial apoptosis, and protected GSCs from GVHD 7 days after allogeneic SCT (Figure A, B).

Conclusion: Lgr5⁺ GSCs are targeted in upper GI GVHD and R-Spo1 protects GSCs against GVHD after allogeneic SCT.

Figure: After allogeneic SCT, cleaved-caspase-3⁺ apoptotic epithelium was significantly increased (A), and number of Lgr5⁺ GSCs was significantly decreased on day +7 (B). R-Spo1 inhibited epithelial apoptosis (A) and Lgr5⁺ GSC loss (B).

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