Impacts of BET bromdomain inhibition on DCs and T cells. (A) RelA was acetylated in LPS-stimulated DCs. Whole-cell lysates were prepared from CD11c microbead–purified DCs treated with or without LPS (250 ng/mL) and I-BET151 (500 nm) for 6 hours as indicated. Western blot was performed to detect expression of BRD4, RelA, and Acetyl-310 RelA. Left panel: Data are representative of 3 experiments with similar results. Right panel: Data represent a summary of 3 independent experiments (mean ± standard error of the mean [SEM]; *P < .001 for comparison of Acetyl-310 RelA between untreated and I-BET151–treated DCs. P values were obtained by two-way analysis of variance [ANOVA]). (B) RelA was acetylated in CD3/CD28-activated T cells. Whole-cell lysates were prepared from T cells after stimulation with CD3/CD28 antibodies and I-BET 151 (250 nm) for 24 hours. Western blot was performed to detect expression of BRD4, RelA, and Acetyl-310 RelA. Left Panel: Data are representative of 3 experiments with similar results. Right panel: Data are a combination of densitometric analyses from 3 independent experiments (mean ± SEM; *P < .001 for the comparison of Acetyl-310 RelA between untreated and I-BET151-treated T cells; P values were obtained by two-way ANOVA). (C-E) Impact of I-BET151 and JQ1 on cytokine expression in DCs. Data shown are the combined results of 3 independent experiments (mean ± SEM; *P < .01). (D) Quantitation of the indicated cytokine messenger RNAs was determined by quantitative real-time PCR. Data are combined from 3 independent experiments (mean ± SEM; *P < .001). (F-G) Impact of I-BET151 in mixed leukocyte reaction. Data were representative of 2 similar experiments (mean ± SEM). (F) Purified BALB/C T cells were labeled with CFSE and cocultured with B6 DCs treated as in (A) and analyzed with flow cytometry. (G) Data are representative of 2 similar experiments. (H) Impact of I-BET151 on surface costimulatory molecules on BM DCs. Data are representative of 3 similar experiments (mean ± SEM) (** P < .01; *** P < .001, one-way ANOVA Dunnett multiple comparison test). (I) Impact of I-BET151 on T-cell proliferation. CFSE-labeled BALB/C T cells were treated as in (B) for 2 days and analyzed by CFSE dye dilution. Data are shown as (left panel) representative or (right panel) combined results from 3 similar experiments (mean ± SEM; **P < .01, Student t test). (J) Impact of I-BET151 on cytokine productions in T cells treated the same as in (B). Cytokines in supernatants were analyzed by enzyme-linked immunosorbent assay. Data were combined from 2 similar experiments (mean ± SEM; *P < .01, Student t test). (K) Impact of I-BET151 and JQ1 on T-cell receptor and CD28 signaling. Purified T cells were stimulated with CD3/CD28 antibodies and treated with I-BET151 (250 nm) or JQ1 (100 nm) for 24 hours and analyzed for expression of CD90.2⁺TCRβ⁺, CD90.2⁺CD28⁺, and CD90.2⁺pZAP70⁺ with flow cytometry. Data are combined from 3 similar experiments (mean ± SEM). (L) Impact of I-BET151 on BM DC viability. Apoptosis analysis after incubation with I-BET151 as described in “Study design.” (M) Impact of I-BET151 on T-cell viability. BALB/C T-cell apoptosis was determined after the cells were stimulated with anti-CD3/CD28 antibodies and treated with I-BET151 for 2 days. Data are from 1 of 2 representative experiments. Abs, antibodies; 7-AAD, 7-aminoactinomycin D.