Figure 2
Figure 2. I-BET151 mitigates GVHD, preserves GVT, and regulates NF-kB. (A) I-BET151 decreases GVHD severity. Lethally irradiated B6 recipients (10 Gy) were transplanted from either syngeneic B6 or allogeneic BALB/C donors and injected with I-BET151 or diluent control as in “Study design.” Survival and GVHD clinical score were monitored over time. Data shown are the combined results of 2 independent experiments (mean ± SEM). Mann-Whitney U test was used for the statistical analysis of clinical scores (*P < .05) and log-rank test was used to compare survival curves (**P < .01). (B) I-BET151 decreases GVHD severity in a second BMT model. BALB/C mice were irradiated (8.5 Gy) and transplanted with 5 × 106 T-cell depleted BM cells and 0.6 × 106 CD90+ T cells from either syngeneic BALB/C or allogeneic B6 donors and treated with I-BET151 as in “Study design.” Survival was monitored over time. Data are combined from 2 independent experiments (n = 10 mice in the allogeneic groups). Log-rank test was used to compare survival curves (** P < .01). (C) Histopathologic analysis of bowel and liver after BMT. The GVHD model and I-BET were treated as in (B). Total GVHD scores are the mean ± SE of the sum of scores (*P < .01). (D) Sera cytokine concentration. Mice were transplanted as in “Study design.” Sera were collected on day +7 from allogeneic animals treated with or without I-BET151 and analyzed for TNF-α, IL-6, IFN-γ, and IL-17. Data shown are combined results in each group (mean ± SE; *P < .01). (E) Impact of I-BET151 on cytokine expression in donor T cells. Spleens were collected on day 7 after BMT. Donor-derived T cells (H2b + CD3+) were stained for intracellular IFN-γ, IL-17, T-BET, and ROR-γ by flow cytometry. Data are from 5 mice per group (mean ± SEM). P values were obtained by Student t test (*P < .01). (F) Impact of I-BET151 on donor T cell (H2b+, CD3+, CD4+, or CD8+) proliferation. Data were obtained by combined numbers from 4 to 5 mice per group (mean ± SEM; *P < .01). (G) Induction of leukemia and lymphoma: Mice were transplanted and injected with MBL-2 tumors and analyzed for tumor-free survival as in “Sudy design.” Data from 2 similar but independent experiments were combined. Survival curve was plotted by using Kaplan-Meier estimates.21-23 (H) Disruption of interaction between Acetyl-K310 RelA and BRD4 in DCs by I-BET151. DCs were treated as in Figure 1A, and whole-cell lysates were processed for IP by RelA antibody. Interaction between acetyl-K310 RelA and BRD4 was analyzed by immunoblotting (IB). Data are representative of 3 similar experiments. (I-J) Disruption of interaction between acetyl-K310 RelA and BRD4 in T cells by (I) I-BET151 and (J) JQ1. Purified T cells were treated with CD3/CD28 antibodies and I-BET151 (250 nm) or JQ1 (100 nm) for 24 hours as indicated. IP was performed as in Figure 2H. Data are representative of 3 similar experiments. Tx, treatment.

I-BET151 mitigates GVHD, preserves GVT, and regulates NF-kB. (A) I-BET151 decreases GVHD severity. Lethally irradiated B6 recipients (10 Gy) were transplanted from either syngeneic B6 or allogeneic BALB/C donors and injected with I-BET151 or diluent control as in “Study design.” Survival and GVHD clinical score were monitored over time. Data shown are the combined results of 2 independent experiments (mean ± SEM). Mann-Whitney U test was used for the statistical analysis of clinical scores (*P < .05) and log-rank test was used to compare survival curves (**P < .01). (B) I-BET151 decreases GVHD severity in a second BMT model. BALB/C mice were irradiated (8.5 Gy) and transplanted with 5 × 106 T-cell depleted BM cells and 0.6 × 106 CD90+ T cells from either syngeneic BALB/C or allogeneic B6 donors and treated with I-BET151 as in “Study design.” Survival was monitored over time. Data are combined from 2 independent experiments (n = 10 mice in the allogeneic groups). Log-rank test was used to compare survival curves (** P < .01). (C) Histopathologic analysis of bowel and liver after BMT. The GVHD model and I-BET were treated as in (B). Total GVHD scores are the mean ± SE of the sum of scores (*P < .01). (D) Sera cytokine concentration. Mice were transplanted as in “Study design.” Sera were collected on day +7 from allogeneic animals treated with or without I-BET151 and analyzed for TNF-α, IL-6, IFN-γ, and IL-17. Data shown are combined results in each group (mean ± SE; *P < .01). (E) Impact of I-BET151 on cytokine expression in donor T cells. Spleens were collected on day 7 after BMT. Donor-derived T cells (H2b + CD3+) were stained for intracellular IFN-γ, IL-17, T-BET, and ROR-γ by flow cytometry. Data are from 5 mice per group (mean ± SEM). P values were obtained by Student t test (*P < .01). (F) Impact of I-BET151 on donor T cell (H2b+, CD3+, CD4+, or CD8+) proliferation. Data were obtained by combined numbers from 4 to 5 mice per group (mean ± SEM; *P < .01). (G) Induction of leukemia and lymphoma: Mice were transplanted and injected with MBL-2 tumors and analyzed for tumor-free survival as in “Sudy design.” Data from 2 similar but independent experiments were combined. Survival curve was plotted by using Kaplan-Meier estimates.21-23  (H) Disruption of interaction between Acetyl-K310 RelA and BRD4 in DCs by I-BET151. DCs were treated as in Figure 1A, and whole-cell lysates were processed for IP by RelA antibody. Interaction between acetyl-K310 RelA and BRD4 was analyzed by immunoblotting (IB). Data are representative of 3 similar experiments. (I-J) Disruption of interaction between acetyl-K310 RelA and BRD4 in T cells by (I) I-BET151 and (J) JQ1. Purified T cells were treated with CD3/CD28 antibodies and I-BET151 (250 nm) or JQ1 (100 nm) for 24 hours as indicated. IP was performed as in Figure 2H. Data are representative of 3 similar experiments. Tx, treatment.

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