Figure 7.
IL-6 signaling is dispensable for GI tract pathology and GVL responses. (A-C) Villin-Cre− × IL-6Rfl and Villin-Cre+ (Villin-CreERT2) × IL-6Rfl mice were treated with tamoxifen to induce Cre recombinase activity and cohoused. Two weeks after the first tamoxifen injection, recipient mice were lethally irradiated and received allografts containing 10 × 106 BM and 3 × 106 to 5 × 106 T cells or 10 × 106 TCD BM alone from BALB/c.45.1 donors. (A) Survival indices and weight loss percentage of recipients after transplantation (T-cell grafts, n = 14 mice per group from 3 experiments; TCD, n = 2-3 mice from 1 experiment). (B-C) Representative histology sections of ileum at day 6 to 7 after transplantation (200× magnification) (B) and semiquantitative GVHD histopathology (C) (n = 8-9 mice per group from 2 experiments). (D-F) The direct responsiveness of leukemic cells to IL-6 was first assessed. Lethally irradiated B6D2F1 mice received B6 wild-type (B6.WT) TCD BM grafts supplemented with either 1 × 106 B6D2F1-derived BCR-ABL/NUP98-HOXA9 GFP+ chronic myeloid leukemia blast crisis or MLL-AF9 GFP+ acute myeloid leukemia cells. (D-E) Peripheral blood was collected from mice after engraftment and expression of mIL-6R (D) and gp130 (E) determined on GFP+ cells. (F) The functional ability of the leukemia cells to respond to IL-6 was confirmed by whole-blood stimulation with IL-6 (to induce classical signaling) or hyper–IL-6 (H-IL-6) (to induce trans signaling) and upregulation of phosphorylated STAT3 (pSTAT3) measured by flow cytometry. (G-I) To assess the role of donor IL-6 classical signaling on GVL responses, lethally irradiated B6D2F1 mice received grafts containing 1 × 106 BCR-ABL/NUP98-HOXA9 GFP+ leukemic cells along with either TCD BM and T cells (0.1 × 106) or TCD BM alone (non-GVL control) from either VAVCre− × IL-6Rfl (control) or VAVCre+ × IL-6Rfl mice (donor leukocytes deficient in IL-6 classical signaling) (G); survival indices by Kaplan-Meier analyses (H) and peripheral blood leukemia burden (I) at the indicated day after transplantation (survival indices, n = 8-12 mice per group; percentage leukocyte GFP+, n = 4-12 mice per group; from 2 experiments). (J-K) To assess the role of IL-6 trans signaling on GVL responses, lethally irradiated B6D2F1 (control) or sgp130Fc.F1 (IL-6 trans signaling inhibited) mice received grafts containing MLL-AF9 GFP+ cells with TCD BM and T cells (0.5 × 106 T cells [T]) or TCD BM alone (non-GVL control) from B6.WT donor mice. (J) Survival indices by Kaplan-Meier analysis (n = 11 mice per group from 2 experiments). (K) Representative images of blood smears at day 21 after transplantation (Wright-Giemsa stain; 200× magnification). Data presented as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001. TBI, total-body irradiation.

IL-6 signaling is dispensable for GI tract pathology and GVL responses. (A-C) Villin-Cre− × IL-6Rfl and Villin-Cre+ (Villin-CreERT2) × IL-6Rfl mice were treated with tamoxifen to induce Cre recombinase activity and cohoused. Two weeks after the first tamoxifen injection, recipient mice were lethally irradiated and received allografts containing 10 × 106 BM and 3 × 106 to 5 × 106 T cells or 10 × 106 TCD BM alone from BALB/c.45.1 donors. (A) Survival indices and weight loss percentage of recipients after transplantation (T-cell grafts, n = 14 mice per group from 3 experiments; TCD, n = 2-3 mice from 1 experiment). (B-C) Representative histology sections of ileum at day 6 to 7 after transplantation (200× magnification) (B) and semiquantitative GVHD histopathology (C) (n = 8-9 mice per group from 2 experiments). (D-F) The direct responsiveness of leukemic cells to IL-6 was first assessed. Lethally irradiated B6D2F1 mice received B6 wild-type (B6.WT) TCD BM grafts supplemented with either 1 × 106 B6D2F1-derived BCR-ABL/NUP98-HOXA9 GFP+ chronic myeloid leukemia blast crisis or MLL-AF9 GFP+ acute myeloid leukemia cells. (D-E) Peripheral blood was collected from mice after engraftment and expression of mIL-6R (D) and gp130 (E) determined on GFP+ cells. (F) The functional ability of the leukemia cells to respond to IL-6 was confirmed by whole-blood stimulation with IL-6 (to induce classical signaling) or hyper–IL-6 (H-IL-6) (to induce trans signaling) and upregulation of phosphorylated STAT3 (pSTAT3) measured by flow cytometry. (G-I) To assess the role of donor IL-6 classical signaling on GVL responses, lethally irradiated B6D2F1 mice received grafts containing 1 × 106 BCR-ABL/NUP98-HOXA9 GFP+ leukemic cells along with either TCD BM and T cells (0.1 × 106) or TCD BM alone (non-GVL control) from either VAVCre− × IL-6Rfl (control) or VAVCre+ × IL-6Rfl mice (donor leukocytes deficient in IL-6 classical signaling) (G); survival indices by Kaplan-Meier analyses (H) and peripheral blood leukemia burden (I) at the indicated day after transplantation (survival indices, n = 8-12 mice per group; percentage leukocyte GFP+, n = 4-12 mice per group; from 2 experiments). (J-K) To assess the role of IL-6 trans signaling on GVL responses, lethally irradiated B6D2F1 (control) or sgp130Fc.F1 (IL-6 trans signaling inhibited) mice received grafts containing MLL-AF9 GFP+ cells with TCD BM and T cells (0.5 × 106 T cells [T]) or TCD BM alone (non-GVL control) from B6.WT donor mice. (J) Survival indices by Kaplan-Meier analysis (n = 11 mice per group from 2 experiments). (K) Representative images of blood smears at day 21 after transplantation (Wright-Giemsa stain; 200× magnification). Data presented as mean ± standard error of the mean. *P < .05, **P < .01, ***P < .001. TBI, total-body irradiation.

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