IFN-β Selectively Inhibits IL-2 Production through CREM-Mediated Chromatin Remodeling

Abstract

IFN-β is widely used in the treatment of multiple sclerosis, yet the mechanism facilitating its efficacy remains unclear. IL-2 production by activated T cells, including those mediating autoimmunity, and subsequent autocrine stimulation is vital for T cell expansion and function. In this study, we demonstrate that in mouse and human T cells, IFN-β specifically inhibits the production of IL-2 upon TCR engagement without affecting other cytokines or activation markers. Rather than disrupting TCR signaling, IFN-β alters histone modifications in the IL-2 promoter to retain the locus in an inaccessible configuration. This in turn is mediated through the upregulation of the transcriptional suppressor CREM by IFN-β and consequent recruitment of histone deacetylases to the IL-2 promoter. In accordance, ablation of CREM expression or inhibition of histone deacetylases activity eliminates the suppressive effects of IFN-β on IL-2 production. Collectively, these findings provide a molecular basis by which IFN-β limits T cell responses.

Figure 1. Specific inhibition of IL-2 expression by IFNβ

(A) Splenocytes from wild type 129/SvEv mice were treated with or without IFNβ or IFNγ for 16 h and then stimulated with 10 μg/mL immobilized anti-CD3 and 2 μg/mL anti-CD28 for 5 h followed by intracellular staining for IL-2. Upper plots were gated on live CD4⁺ cells and lower plots were gated on live CD8⁺ cells and are representative of at least five experiments. (B) Splenocytes from wild type 129/SvEv mice were treated for 16 h with IFNβ and stimulated with PMA/Ionomycin for 5 h followed by intracellular stain for IL-2. Plots are gated on live CD4⁺ cells. (C) Splenocytes were stimulated with the indicated mitogens and cultured for 48 h in the presence of increasing doses of IFNβ. IL-2 in the conditioned medium was quantified by ELISA (n=3). (D) CD4⁺ splenocytes were treated for 16 h with IFNβ prior to stimulation with anti-CD3/anti-CD28. 6 h later RNA was purified and quantitative PCR was used to measure IL-2 and IL-4 mRNA levels (n=3). (E) CD4+ splenocytes were treated as in (C), stained for CD25 and CD44 expression and analyzed by flow cytometry.

Figure 2. Inhibition of IL-2 expression requires pre-treatment with IFNβ

(A) Purified CD4⁺ splenic T cells were treated with IFNβ for 16 h (lower plots) before (middle plots) or after (right plots) 3 days in culture with anti-CD3 and anti-CD28 and then re-stimulated with PMA/Ionomycin for 5 h followed by intracellular stain for IL-2. (B) Splenocytes were cultured for a total of 4 days following stimulation with anti-CD3 and anti-CD28. IFNβ was added up to 12 h prior to or up to 12 h post stimulation. The amount of IL-2 in the conditioned medium was measured by ELISA (n=3).

Figure 3. IFNβ inhibition of IL-2 expression is independent of Treg cells and is reproduced in human peripheral blood leukocytes

(A) CD4⁺ T cells were depleted of CD25⁺ T_reg cells and then treated for 16 h with IFNβ followed by stimulation with anti-CD3/anti-CD28 for 4 h in the presence or absence of CD25⁺ Treg cells. IL-2 mRNA was measured by quantitative PCR. (n=3) (B) Human PBLs were stimulated with anti-CD3/anti-CD28 dynabeads with and without IFNβ pretreatment and RNA was collected after 3 h stimulation and analyzed for IL-2 message by quantitative PCR. (n=3) (C) Human PBLs were stimulated as in (b) except that cells were stained for intracellular IL-2 after 5 h. (representative of five experiments).

Figure 4. Inhibition of IL-2 by IFNβ in naive T cells is dependent on STAT1 but does not affect proximal signaling through the T cell receptor

(A) Splenocytes from wild type and STAT1^−/−, Tyk2^−/−, STAT3^−/− and STAT5^−/− mice were treated with IFNβ for 16 h and then stimulated with PMA/Ionomycin for 5 h followed by intracellular IL-2 stain. Plots are gated on CD4⁺ cells. Representative of 3-5 mice each. (B) Splenic T cells were treated for 16 h with IFNβ and then stimulated with anti-CD3/CD28 for the indicated time. Western blots of whole cell lysates were carried out and probed for the indicated phosphorylated proteins. (C) Splenic T cells, treated for 16 h with IFNβ and then loaded with Fluo-4 and Fura Red, were run on flow cytometer for 30 sec to establish baseline, and then stimulated with anti-CD3. After 1 min, a crosslinking antibody was added to cells to induce calcium flux (representative of at least five experiments).

Figure 5. IFNβ affects chromatin remodeling of the IL-2 promoter through histone deacetylase activity and CREM

(A) Diagram showing location of primers in the IL-2 promoter used in the subsequent figures. (B) Chromatin accessibility of the IL-2 promoter/enhancer. Purified CD4⁺ T cells were stimulated with anti-CD3/anti-CD28 for 4 h with and without IFNβ pretreatment. Chromatin accessibility was determined by quantitation of the qPCR products obtained with the indicated primer sets. (n=4) (C) Chromatin IPs using anti-acetylated H3 and anti-acetylated H4 antibodies and primers within the IL-2 promoter/enhancer following stimulation of CD4⁺ T cells with either anti-CD3/CD28 or PMA/Ionomycin. (D) IL-2 mRNA was quantitated from CD4⁺ T cells following 3 h anti-CD3/CD28 stimulation with or without IFNβ pre-treatment. Increasing doses of Trichostatin A were added to cells 1 h prior to stimulation. Left graph is a representative of three independent experiments the average % inhibition of which in shown on the right. (E) CD4⁺ T cells were treated with IFNβ for 16 h and Western blots of cell lysates were probed for CREM, ISG15, and GAPDH as a loading control. (F) mRNAs for CREM and ISG15 were measured in CD4⁺ T cells from 129WT or STAT1^−/− mice stimulated with IFNβ for 5 h as measured by quantitative PCR. (G) and (H) CD4⁺ T cells were transfected with control or CREM-specific siRNA prior to treatment with IFNβ for 16 h and subsequent stimulation with anti-CD3/CD28 for 3 h. (G) CREM mRNA and (H) IL-2 mRNA was measured by quantitative PCR. (H) The average % inhibition of IL-2 production was determined from 4 independent experiments.

Figure 6. Inhibition of IL-2 production in T cells from mice injected with IFNβ or infected with LCMV Cl13

(A) OTII TCR transgenic mice were injected with 100ug Ova_323-339 24 h after I.V. injection of 10,000 U IFNβ. Splenic T cells were collected 4 h following peptide injection and subjected to intracellular stain for IL-2. Cells were also analyzed for surface expression of Vα2/Vβ5 TCR chains as well as the activation marker CD69. (B) Graph represents average number of IL-2 positive OTII T cells from three independent experiments. (C) Single cell suspensions from spleens of Day 9 LCMV Cl13 infected mice were subjected to intracellular stain for CREM. CD4⁺ cells are shown. (D) Graph represents % CREM positive CD4 T cells from 5 uninfected and 7 LCMV Cl13 infected mice stained as in (b). (E) Single cell suspensions from spleens of Day 9 LCMV Cl13 infected mice were stimulated with anti-CD3/CD28 beads for 5 h and then intracellular stained for IL-2. (F) Graph represents % IL-2 positive CD4 T cells from 4 mice each uninfected and LCMV Cl13 infected mice stained as in (d).