Figure 3. ST2–IL-33 signaling enhances AREG expression on T9 cells, leading to protection of gut epithelial cells from alloreactivity. (A) B6 T1, WT T9IL-33, or ST2−/− T9IL-33 cells differentiated in MLR conditions were co-cultured with BALB-5047 colonic epithelial cells together (left) or through Transwells (right) for 6 h. The percentage of residual live BALB-5047 cells was measured by viability dye staining negative and flow cytometry (n = 4 from two independent experiments, unpaired t test; data are shown as mean ± SEM). (B) Representative plots of AREG and Foxp3 expression on CD4+ cells from in vitro differentiated T cell subsets from Foxp3-GFP reporter mice (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) AREG expression in in vitro differentiated and sorted CD4 subsets or T reg isolated freshly form spleen isolated magnetically using regulatory T cell isolation kit (Miltenyi). Gene expression was measured by real-time PCR and protein level by flow cytometry (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; ***, P < 0.001). (D) Percentage of residual live cells of BALB-5047 cells after co-culture with T1, T9, or T9IL-33 cells for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; **, P < 0.01). (E) Percentage of residual live cells of BALB-5047 cells after co-culture with T1, WT T9IL-33, or ST2−/− T9IL-33 cells for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; **, P < 0.01; ***, P < 0.001). (F) Percentage of residual live cells among BALB-5047 cells after co-culture with T1 cells, T9IL-33 cells, T1 + T9IL-33 cells, or T1 + in vitro polarized T reg cells (purified magnetically using the regulatory T cell isolation kit) for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, mean ± SEM). (G) AREG expression in sorted CD4 subsets from intestine of GVHD mice collected on day 14 after allo-HCT with T1, WT T9IL-33, or ST2−/− T9IL-33 cells (n = 4, unpaired t test; data are shown as mean ± SEM; ***, P < 0.001). (H) Representative plots of ex vivo expression of AREG and Foxp3 in gut CD4 T cells collected on day 14 after allo-HCT with allogeneic T9IL-33 cells from Foxp3-GFP reporter mice depleted or nondepleted of Foxp3 T reg cells (n = 3, unpaired t test; data are shown as mean ± SEM). (I) Clinical scores of GVHD and survival curves for C3H.SW mice receiving B6 BM cells and in vitro differentiated T9IL-33 cells treated with five doses of 100 µg anti-AREG or isotype control every other day from day −1 to day 7 (n = 7 per group, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; ***, P < 0.001. For survival by log-rank test (**, P < 0.01; ***, P < 0.001.). (J) Representative plots of ex vivo IFN-γ and IL-17 expression by gut-infiltrating T cells 10 d after HCT in mice treated with αAREG or isotype control antibodies, and bar graphs showing frequencies of IFN-γ–positive T cells. B6 WT T9IL-33 cells were cultured for 5 d, after which these cells were injected into lethally irradiated C3H.SW mice along with B6 WT BM cells. Mice were treated with a total of five doses of anti-AREG or isotype control (100 µg each) every other day from day −1 to day 7 (n = 4, unpaired t test; data are shown as mean ± SEM).
Figure 3.

ST2–IL-33 signaling enhances AREG expression on T9 cells, leading to protection of gut epithelial cells from alloreactivity. (A) B6 T1, WT T9IL-33, or ST2−/− T9IL-33 cells differentiated in MLR conditions were co-cultured with BALB-5047 colonic epithelial cells together (left) or through Transwells (right) for 6 h. The percentage of residual live BALB-5047 cells was measured by viability dye staining negative and flow cytometry (n = 4 from two independent experiments, unpaired t test; data are shown as mean ± SEM). (B) Representative plots of AREG and Foxp3 expression on CD4+ cells from in vitro differentiated T cell subsets from Foxp3-GFP reporter mice (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; **, P < 0.01; ***, P < 0.001). (C) AREG expression in in vitro differentiated and sorted CD4 subsets or T reg isolated freshly form spleen isolated magnetically using regulatory T cell isolation kit (Miltenyi). Gene expression was measured by real-time PCR and protein level by flow cytometry (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; ***, P < 0.001). (D) Percentage of residual live cells of BALB-5047 cells after co-culture with T1, T9, or T9IL-33 cells for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; **, P < 0.01). (E) Percentage of residual live cells of BALB-5047 cells after co-culture with T1, WT T9IL-33, or ST2−/− T9IL-33 cells for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, from three independent experiments, unpaired t test; data are shown as mean ± SEM; **, P < 0.01; ***, P < 0.001). (F) Percentage of residual live cells among BALB-5047 cells after co-culture with T1 cells, T9IL-33 cells, T1 + T9IL-33 cells, or T1 + in vitro polarized T reg cells (purified magnetically using the regulatory T cell isolation kit) for 6 h in the presence of anti-AREG blocking antibody or isotype control (n = 3, mean ± SEM). (G) AREG expression in sorted CD4 subsets from intestine of GVHD mice collected on day 14 after allo-HCT with T1, WT T9IL-33, or ST2−/− T9IL-33 cells (n = 4, unpaired t test; data are shown as mean ± SEM; ***, P < 0.001). (H) Representative plots of ex vivo expression of AREG and Foxp3 in gut CD4 T cells collected on day 14 after allo-HCT with allogeneic T9IL-33 cells from Foxp3-GFP reporter mice depleted or nondepleted of Foxp3 T reg cells (n = 3, unpaired t test; data are shown as mean ± SEM). (I) Clinical scores of GVHD and survival curves for C3H.SW mice receiving B6 BM cells and in vitro differentiated T9IL-33 cells treated with five doses of 100 µg anti-AREG or isotype control every other day from day −1 to day 7 (n = 7 per group, unpaired t test; data are shown as mean ± SEM; *, P < 0.05; ***, P < 0.001. For survival by log-rank test (**, P < 0.01; ***, P < 0.001.). (J) Representative plots of ex vivo IFN-γ and IL-17 expression by gut-infiltrating T cells 10 d after HCT in mice treated with αAREG or isotype control antibodies, and bar graphs showing frequencies of IFN-γ–positive T cells. B6 WT T9IL-33 cells were cultured for 5 d, after which these cells were injected into lethally irradiated C3H.SW mice along with B6 WT BM cells. Mice were treated with a total of five doses of anti-AREG or isotype control (100 µg each) every other day from day −1 to day 7 (n = 4, unpaired t test; data are shown as mean ± SEM).

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