Figure 4.
TRAIL contributes to enhanced GVL effects upon MDM2 inhibition. (A) Percentage survival of C57BL/6 recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) and treated with the MDM2-inhibitor RG-7112 and either anti-TRAIL antibody or IgG isotype, as indicated. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (B) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c), either WT T cells or TRAIL−/− T cells. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (C) Western blots showing the activation of caspase-3, caspase-8, and loading control (β-actin) in OCI-AML3 cells. Activated T cells were pretreated with 10 µg/mL anti-TRAIL neutralizing antibody or IgG control for 1 hour and were cocultured with OCI-AML3 cells exposed to DMSO or RG-7112 (1 µM) at an E:T ratio of 10:1 for 4 hours. (D) Quantification of the ratio of cleaved caspase-3/total caspase-3 normalized to isotype control. One representative experiment of 3 experiments with comparable results is shown. (E) Quantification of the ratio of cleaved caspase-8/total caspase-8 normalized to isotype control. One representative experiment of 3 experiments with comparable results is shown. A total of n = 3 independent experiments were performed. (F) Survival of Rag2−/−Il2rγ−/− mice receiving WT OCI-AML3 cells or CRISPR/Cas-mediated TRAIL-R2 knockout (TRAIL-R2−/−) OCI-AML3 cells. Mice were additionally injected with primary human T cells isolated from healthy donors and treated with vehicle or MDM2 inhibitor RG-7112. A total of n = 10 animals from 2 independent experiments are shown. P values were calculated using the 2-sided Mantel-Cox test. (G) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and BMT using allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) on day 2 after BMT. When indicated, CD8 T cells or NK cells were depleted from the allogeneic T-cell transplant. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (H) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) derived from previously challenged and treated (MDM2 inhibitor or vehicle) mice. Shown are n = 10 independent animals from 2 experiments, and P values were calculated using the 2-sided Mantel-Cox test.

TRAIL contributes to enhanced GVL effects upon MDM2 inhibition. (A) Percentage survival of C57BL/6 recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) and treated with the MDM2-inhibitor RG-7112 and either anti-TRAIL antibody or IgG isotype, as indicated. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (B) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c), either WT T cells or TRAIL−/− T cells. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (C) Western blots showing the activation of caspase-3, caspase-8, and loading control (β-actin) in OCI-AML3 cells. Activated T cells were pretreated with 10 µg/mL anti-TRAIL neutralizing antibody or IgG control for 1 hour and were cocultured with OCI-AML3 cells exposed to DMSO or RG-7112 (1 µM) at an E:T ratio of 10:1 for 4 hours. (D) Quantification of the ratio of cleaved caspase-3/total caspase-3 normalized to isotype control. One representative experiment of 3 experiments with comparable results is shown. (E) Quantification of the ratio of cleaved caspase-8/total caspase-8 normalized to isotype control. One representative experiment of 3 experiments with comparable results is shown. A total of n = 3 independent experiments were performed. (F) Survival of Rag2−/−Il2rγ−/− mice receiving WT OCI-AML3 cells or CRISPR/Cas-mediated TRAIL-R2 knockout (TRAIL-R2−/−) OCI-AML3 cells. Mice were additionally injected with primary human T cells isolated from healthy donors and treated with vehicle or MDM2 inhibitor RG-7112. A total of n = 10 animals from 2 independent experiments are shown. P values were calculated using the 2-sided Mantel-Cox test. (G) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and BMT using allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) on day 2 after BMT. When indicated, CD8 T cells or NK cells were depleted from the allogeneic T-cell transplant. A total of n = 10 independent animals from 2 experiments are shown, and P values were calculated using the 2-sided Mantel-Cox test. (H) Percentage survival of C57BL/6-recipient mice after transfer of AMLMLL-PTD FLT3-ITD cells (C57BL/6 background) and allogeneic BALB/c BM is shown. Mice were injected with additional allogeneic T cells (BALB/c) derived from previously challenged and treated (MDM2 inhibitor or vehicle) mice. Shown are n = 10 independent animals from 2 experiments, and P values were calculated using the 2-sided Mantel-Cox test.

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