Aiolos promotes TH17 differentiation by directly silencing Il2 expression

Abstract

CD4(+) interleukin 17 (IL-17)-producing helper T cells (T(H)17 cells) are instrumental in the immune response to pathogens. However, an overactive T(H)17 response results in tissue inflammation and autoimmunity, and therefore it is important to identify the molecular mechanisms that control the development of T(H)17 cells. IL-2 suppresses such development, but how IL-2 production is actively suppressed during T(H)7 differentiation is not understood. Here we report that under T(H)17-polarizing conditions, the transcription factors STAT3 and AhR upregulated the expression of Aiolos, a member of the Ikaros family of transcription factors. Using Aiolos-deficient mice, we demonstrated that Aiolos silenced the Il2 locus, promoting T(H)17 differentiation in vitro and in vivo. Thus, we have identified a module in the transcriptional program of T(H)17 cells that actively limits IL-2 production and promotes their differentiation.

Figure 1

Expression of Aiolos by T_H17 cells. (a) Quantitative real-time PCR analysis of Ikzf3, Tbx21, Gata3 and Rorc mRNA in naive CD4⁺CD44^LoCD62L^hiCD25⁻ T cells differentiated for 48 h in T_H0, T_H1, T_H2 or T_H17 conditions, presented relative to the expression of Gapdh mRNA (encoding glyceraldehyde phosphate dehydrogenase). ND, not detected. (b) Quantitative real-time PCR analysis of mRNA encoding Ikaros (Ikzf1), Helios (Ikzf2), Aiolos (Ikzf3), Eos (Ikzf4) and Pegasus (Ikzf5) in naive CD4⁺ T cells differentiated for 48 h in T_H0 or T_H17 conditions (presented as in a). (c) Quantitative real-time PCR analysis of the time course of the expression of Ikzf3 and Il17a mRNA in naive CD4⁺ T cells polarized into T_H0 or T_H17 cells (presented as in a). Data are from one of three independent experiments with similar results (error bars, s.e.m.).

Figure 2

Aiolos controls the development of T_H17 cells. (a) Quantitative real-time PCR analysis of Il17a, Ifng and Ikzf3 mRNA in wild-type (WT) and Aiolos-deficient (KO) naive CD4⁺ T cells differentiated for 48 h in T_H0 or T_H17 conditions (presented as in Fig. 1a). (b) Enzyme-linked immunosorbent assay (ELISA) of IL-17 and IFN-γ in supernatants of wild-type and Aiolos-deficient naive T cells differentiated for 48 h in T_H0, T_H1 or T_H17 conditions. (c) Quantitative real-time PCR analysis of Rorc, Rora, Maf, Ahr, Tbx21, Il17a, Il17f and Il21 mRNA in wild-type and Aiolos-deficient naive CD4⁺ T cells differentiated for 48 h in T_H17 conditions (presented as in Fig. 1a). (d,e) Proliferative recall response to MOG(35–55) (d) and expression of IFN-γ and IL-17 by T cells in the CD3⁺CD4⁺ fraction (e, left) 7 d after immunization of wild-type and Aiolos-deficient mice with MOG(35–55) in complete Freund’s adjuvant. Right (e), ratio of IFN-γ⁺ cells to IL-17⁺ cells (IFN-γ⁺/IL-17⁺) among the cells at left. Numbers in quadrants (e, left) indicate percent cells in each throughout. *P < 0.01, compared with wild-type T cells (Student’s t-test). (f,g) EAE clinical scores of Rag2^−/− mice given wild-type or Aiolos-deficient CD4⁺ T cells reactivated with MOG(35–55) in the presence of IL-23 (f) or IL-12 (g). *P < 0.01 and **P < 0.001, compared with mice given wild-type donor cells (two-way analysis of variance (ANOVA)). Data are representative of three experiments (a–c) or two experiments (f,g) or are from one of three independent experiments with similar results (d,e; error bars, s.e.m.).

Figure 3

Aiolos limits IL-2 production in T_H17 cells. (a) Quantitative real-time PCR analysis of Il2, Il17a and Ifng mRNA in naive CD4⁺ T cells differentiated for 0, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 42, 48, 50, 52, 60 or 72 h in T_H17 conditions (presented as in Fig. 1a). (b) Quantitative real-time PCR analysis of Il2 mRNA in naive wild-type and Aiolos-deficient CD4⁺ T cells differentiated into T_H17 cells with IL-6 and TGF-β1 (presented as in Fig. 1a). (c) ELISA of IL-2 in supernatants of wild-type and Aiolos-deficient naive T cells differentiated for 48 h in T_H0 or T_H17 conditions. *P < 0.05 and **P < 0.01, compared with wild-type T cells (two-way ANOVA). (d) Flow cytometry analysis of the expression of IL-2 and IL-17 in naive wild-type or Aiolos-deficient CD4⁺ T cells differentiated for 48 h or 72 h in T_H17 conditions. (e) Flow cytometry analysis of IL-17 expression in naive wild-type and Aiolos-deficient CD4⁺ T-cells differentiated for 72 h in T_H17 conditions without antibodies (None) or with the addition of blocking antibody to IL-2 (Anti-IL-2) or isotype-matched control antibody (IC). Numbers adjacent to outlined areas indicate percent IL-17⁺CD4⁺ (T_H17) cells. Data are from one of two independent experiments with similar results (error bars (b,c), s.e.m.).

Figure 4

Aiolos silences the Il2 promoter in T_H17 cells. (a) Il2 promoter; red indicates the Aiolos-binding site. (b) EMSA of the interaction of nuclear extracts of T_H0, T_H1 and T_H17 cells with a radiolabeled oligonucleotide containing the putative Aiolos-binding site in Il2 promoter, and competition of that interaction with an antibody to Aiolos that disrupts the oligomeric form of Aiolos responsible for DNA binding in EMSA (top). Anti-tubulin serves as a control. Below, SDS-PAGE and silver staining of the nuclear extracts normalized for total protein content, to demonstrate equal loading of samples. M, molecular size markers in kilodaltons (kDa). (c) ChIP analysis of the interaction of Aiolos, or of proteins precipitated with isotype-matched control antibody (Ctrl), with the binding site in the Il2 promoter in T_H17, T_H1 or T_H0 cells differentiated in vitro. (d,e) ChIP analysis of the abundance of H3K4me3, H3K9me3 and H3K27me3 (d) and of H3ac and H4ac (e) in the Il2 promoter of wild-type and Aiolos-deficient T_H0, T_H1 and T_H17 cells, presented relative to results obtained with isotype-matched control antibody. (f) Luciferase activity in Jurkat T cells transfected with various concentrations (horizontal axis) of an IL2 luciferase reporter (pIKZF3) plus a construct encoding Aiolos, then activated for 24 h with the phorbol ester PMA and ionomycin; results are presented relative to renilla luciferase activity (cotransfected control). *P < 0.001 (one-way ANOVA). Data are from one of two to three independent experiments with similar results (error bars (c–f), s.e.m.).

Figure 5

AhR and STAT3 control Aiolos expression in T_H17 cells. (a) Quantitative real-time PCR analysis of Rorc, Ahr, Stat3 and Ikzf3 mRNA in naive CD4⁺ T cells differentiated for 0, 1, 2, 4, 6, 8, 10, 12, 16, 20, 24, 30, 42, 48, 50, 52, 60 or 72 h in T_H17 conditions (presented as in Fig. 1a). (b) STAT3-responsive elements (blue; SRE-1, SRE-2 and SRE-3) and AhR-binding sites (green; xenobiotic response elements XRE-1, XRE-2 and XRE-3) in the Ikzf3 promoter. (c,d) ChIP analysis of the interaction of AhR (c) or STAT3 (d), or proteins precipitated with an isotype-matched control antibody (ctrl), with AhR-binding sites (c) or STAT3-responsive elements (d) in the Ikzf3 promoter in T_H17 or T_H0 cells differentiated in vitro. (e) Luciferase activity in Jurkat T cells transfected with an IKZF3 luciferase reporter, alone (−) or with a construct encoding constitutively activated STAT3 (STAT3ca) or AhR, separately (middle) or together (STAT3ca + AhR; far right), then activated for 24 h with PMA and ionomycin (presented as in Fig. 4f). (f) Quantitative real-time PCR analysis of Ikzf3 mRNA in naive CD4⁺ T cells cultured for 48 h in T_H0 conditions (T_H0) or with IL-6 and TGF-β1 alone (T_H17) or in the presence of the AhR agonist FICZ (T_H17 + FICZ) or the AhR antagonist CH-223191 (T_H17 + Inh; presented as in Fig. 1a). *P < 0.05 (one-way ANOVA). Data are from one of two independent experiments with similar results (error bars (c–f), s.e.m.).

Figure 6

Aiolos controls the generation of IL-17-producing CD4⁺ memory T cells. (a) Flow cytometry of splenic CD4⁺ T cells from wild-type and Aiolos-deficient mice. Numbers adjacent to outlined areas indicate CD62L^hiCD44^lo cells (top left) or CD62L^loCD44^hi cells (bottom right). (b) Frequency of CD62L^loCD44^hi T cells in the CD3⁺CD4⁺ T cell fraction of wild-type and Aiolos-deficient mice. *P < 0.01 (Student’s t-test). (c) Total CD62L^loCD44^hi CD4⁺ T cells in wild-type and Aiolos-deficient mice. *P < 0.05 (Student’s t-test). (d,e) Flow cytometry analysis of the expression of IL-17 and IFN-γ on splenic CD4⁺CD44^hiCD62L^lo T cells (d) and ratio of IFN-γ⁺ cells to IL-17⁺ cells (e) among CD3⁺ CD4⁺ T cells stimulated in vitro for 4 h with PMA and ionomycin. *P < 0.05 (Student’s t-test). (f) Quantitative real-time PCR analysis of Rorc and Tbx21 mRNA in splenic CD4⁺CD44^hi T cells sorted by flow cytometry (presented as in Fig. 1a). Data are from one of two to three independent experiments with similar results (error bars (b,c,e,f), s.e.m.).

Figure 7

Aiolos-deficient CD4⁺CD25⁻CD45RB^hi T cells induce an aggressive wasting disease in Rag2^−/− recipient mice. (a) ELISA of IL-17, IFN-γ and IL-2 in supernatants of CD4⁺ T cells isolated from Rag2^−/− mice reconstituted with wild-type or Aiolos-deficient CD4⁺ T cells, followed by activation of the cells in vitro for 48 h. (b) Ratio of IFN-γ⁺ cells to IL-17⁺ cells among CD3⁺CD4⁺ T cells from the lamina propria of Rag2^−/− mice reconstituted as in a. (c) Change in body weight of Rag2^−/− mice that received wild-type or Aiolos-deficient CD4⁺CD25⁻CD45RB^hi T cells. Dashed line indicates baseline (set as 0%). (d) Linear-regression curves of results in c. Dashed lines indicate the 95% confidence intervals. (e) Colon lengths of Rag2^−/− mice that received wild-type or Aiolos-deficient cells, measured from the colocecal junction to the anal verge. (f) Quantification of pathological changes in the mice in c. (g) ELISA of IFN-γ and IL-17 in supernatants of CD4⁺ T cells isolated from Rag2^−/− mice reconstituted as in c, followed by activation for 48 h. (h) Ratio of IFN-γ⁺ cells to IL-17⁺ cells among CD3⁺CD4⁺ T cells from the lamina propria of Rag2^−/− mice reconstituted as in c. *P < 0.05 and **P < 0.01, compared with recipients of wild-type T cells (Student’s t-test). Data are representative of three experiments with similar results, with four to five mice per group (error bars, (a–c),(e–h) s.e.m.).