Selective inhibition of CD4+ T-cell cytokine production and autoimmunity by BET protein and c-Myc inhibitors

Abstract

Bromodomain-containing proteins bind acetylated lysine residues on histone tails and are involved in the recruitment of additional factors that mediate histone modifications and enable transcription. A compound, I-BET-762, that inhibits binding of an acetylated histone peptide to proteins of the bromodomain and extra-terminal domain (BET) family, was previously shown to suppress the production of proinflammatory proteins by macrophages and block acute inflammation in mice. Here, we investigated the effect of short-term treatment with I-BET-762 on T-cell function. Treatment of naïve CD4(+) T cells with I-BET-762 during the first 2 d of differentiation had long-lasting effects on subsequent gene expression and cytokine production. Gene expression analysis revealed up-regulated expression of several antiinflammatory gene products, including IL-10, Lag3, and Egr2, and down-regulated expression of several proinflammatory cytokines including GM-CSF and IL-17. The short 2-d treatment with I-BET-762 inhibited the ability of antigen-specific T cells, differentiated under Th1 but not Th17 conditions in vitro, to induce pathogenesis in an adoptive transfer model of experimental autoimmune encephalomyelitis. The suppressive effects of I-BET-762 on T-cell mediated inflammation in vivo were accompanied by decreased recruitment of macrophages, consistent with decreased GM-CSF production by CNS-infiltrating T cells. These effects were mimicked by an inhibitor of c-myc function, implicating reduced expression of c-myc and GM-CSF as one avenue by which I-BET-762 suppresses the inflammatory functions of T cells. Our study demonstrates that inhibiting the functions of BET-family proteins during early T-cell differentiation causes long-lasting suppression of the proinflammatory functions of Th1 cells.

Fig. 1.

I-BET-762 treatment differentially alters CD4⁺ T-cell cytokine production. (A) Antigen-inexperienced CD4⁺ T cells were stimulated with plate bound anti-CD3/anti-CD28 antibodies in the presence or absence of an active compound (I-BET-762) or an inactive stereoisomer (Control-768) for the initial 72 h of priming in the presence of indicated differentiation conditions. The cells were subsequently harvested and expanded for an additional 48 h without addition of compound. (B) The cells were restimulated with PMA/ionomycin for 6 h in the presence of Brefeldin-A, fixed with PFA, and cytokine production measured by intracellular staining. (C) CD4⁺ T cells were stimulated as described above in the presence of TGF-β (Upper) or TGF-β + RA (Lower) in the presence of indicated concentration of I-BET-762 or Control-768. (D) CD4⁺ T cells were stimulated under Th-17 biasing condition TGF-β + IL-6 or IL-1β + IL-6 + IL-23.

Fig. 2.

I-BET-762 treatment does not block the ability of Th17 cells to induce CNS inflammation. (A) CD4⁺ T cells isolated for 2D2 TCR-transgenic mice specific for MOG_(35-55) were stimulated under Th17-23 biasing condition in the presence of I-BET-762 (500 nM) or Control-768 compounds. Subsequently, the cells were expanded, restimulated, and transferred into irradiated recipients. (B) Changes in body weights of recipients transferred with Control-768 (n = 7) or I-BET-762 (n = 4) compounds. These data were obtained from two independent experiments. (C) Cytokine production from CNS infiltrating 2D2-transgenic Vβ11+ CD4+ T cells and (D) composition of inflammatory cells in the CNS was assessed. Results obtained in two individual recipients are depicted. (At indicated times, the differences in weight loss between the two groups were evaluated for statistical significance. P values in B was obtained by using Student’s t test; *P < 0.05.)

Fig. 3.

I-BET-762 treatment suppresses ability of autoreactive Th1 T cells to induce pathology in vivo. (A) CD4⁺ T cells isolated for 2D2 TCR-transgenic mice specific for MOG_35–55 were stimulated under Th1 biasing condition in the presence of I-BET-762 (500 nM) or Control-768 compounds. The cells were subsequently expanded without addition of compounds, restimulated, and transferred into B6 recipients. The recipients were monitored for changes in body weight and clinical manifestation of disease as described in Materials and Methods. (B) Peak clinical scores and changes in body weights of recipients transferred with Control-768 (n = 44) or I-BET-762 (n = 40) compounds. These data were obtained from nine independent experiments. In three of these experiments, none of the I-BET-762 recipients exhibited any clinical signs of disease (score = 0). (C) The time course of one representative experiment is depicted [Control-768 (n = 7); I-BET-762 (n = 10)]. Cytokine production from CNS infiltrating 2D2-transgenic Vβ11⁺ CD4⁺ T cells (D) and composition of inflammatory cells in the CNS (E) was assessed. Bar graphs in D and E represent variation between individual recipients, and these data are representative of two independent experiments. (P values in B, D, and E were obtained by using Student t test; **P < 0.01, ***P < 0.001. The P values obtained in C were obtained by using two-way ANOVA analyses).

Fig. 4.

I-BET-762 does not affect the ability of previously differentiated Th1 cells to induce neuroinflammation. (A) CD4+ T cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence of indicated differentiation conditions. After in vitro culture for 5 d, the cells were harvested, washed, and restimulated with PMA/ionomycin in the presence of indicated compounds for 6 h. (B) Cytokine production measured by intracellular staining. (C) TCR-transgenic 2D2 T cells were primed under Th1 conditions and expanded in the absence of any compounds. Five days after initial activation, the cells were restimulated with immobilized anti-CD3 and anti-CD28 for 24 h in the presence of control or I-BET-762 compounds. Subsequently, cells were harvested and transferred into irradiated B6 recipients. Clinical manifestation of neuroinflammation was assessed. These data are obtained from two independent experiments (Control-768, n = 16; I-BET-762, n = 17).

Fig. 5.

I-BET-762 treatment inhibits Myc expression during T-cell priming. (A) CD4+ T cells isolated for 2D2 TCR-transgenic mice were stimulated under Th1 biasing condition in the presence of Control-768 or I-BET-762 compounds. The cells were harvested at indicated times after activation and expression of indicated genes was assessed by real-time PCR analyses. The error bars represent the variation (SEM) among three independent experiments. (B) CD4+ T cells were stimulated with plate-bound anti-CD3/anti-CD28 antibodies in the presence or absence of an inhibitor of c-myc function (10058-F4) or solvent (DMSO) for the initial 48 h of priming in the presence of indicated differentiation conditions. The cells were subsequently harvested and expanded for an additional 72 h without the addition of compound. The cells were restimulated with PMA/ionomycin for 6 h in the presence of Brefeldin-A, fixed with PFA, and cytokine production was measured by intracellular staining. CD4+ T cells isolated from 2D2 TCR-transgenic mice were stimulated under Th1 biasing conditions in the presence of indicated compounds. Subsequently the cells were expanded, restimulated, and transferred into irradiated recipients. (C) GM-CSF production was assessed before transfer. (D) Changes in body weights of recipients transferred with DMSO (n = 5) or 10058-F4 (n = 5) treated cells. The data in B–D are representative of three independent experiments. (P values were obtained by two-way ANOVA analyses).