Loss of RIPK3 in IECs reduces both local and systemic GVHD. (A) Survival of the lethally irradiated WT, Ripk3^−/−, and Ripk3^−/−caspase-8^−/− B6 recipients (BALB/c→B6) that received BALB/c TCD BM cells (BM) with or without CD4⁺ T cells (BM+CD4⁺ T). (B) Survival of the lethally irradiated WT and Ripk3^−/− BALB/c recipients (B6→BALB/c) that received B6 TCD BM cells with CD4⁺ T cells. (C) Survival of the lethally irradiated Ripk3^fl/fl and Vil-cre Ripk3^fl/fl B6 recipients (BALB/c→B6) that received BALB/c TCD BM cells with CD4⁺ T cells. (D-E) Histology analysis of small intestine, colon, and liver obtained from WT, Ripk3^−/− and Vil-cre Ripk3^fl/fl B6 recipients (BALB/c→B6) on day 17 after allo-HCT. Representative hematoxylin and eosin (H&E) images (D) and quantification of pathology scores (E). Bars in colon and small intestine represent 100 μm. Bar in liver represents 50 μm. Black arrows indicate areas of inflammatory cell infiltration or cell damage. (F) Representative 2D and 3D confocal images of OLFM4⁺ ISCs and lysozyme⁺ Paneth cells in the small intestine from the indicated B6 recipients on day 17 after allo-HCT. Bar represents 100 μm. (G) Quantification of IHC staining of OLFM4 in the small intestine from the indicated B6 recipients on day 17 after allo-HCT. (H) Quantification of infiltrated CD3⁺ T cells in the small intestine from the indicated B6 recipients (BALB/c→B6) on day 17 after allo-HCT. Three mice were examined in each group. (I) TNF-α and IFN-γ levels in serum from the indicated B6 recipients on day 17 after allo-HCT. Data shown are representative of 2 or 3 independent experiments. Data are shown as the mean ± SD (G-H), histology scores, and concentrations of cytokines are shown as the mean ± SEM (E,I). ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. Survival comparisons were evaluated by log-rank test (A-C). Multiple comparisons were evaluated by one-way ANOVA (E,G-I). ANOVA, analysis of variance; IHC, immunohistochemistry; SD, standard deviation; SEM, standard error of the mean.