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. 2013 Oct;14(10):1054-63.
doi: 10.1038/ni.2695. Epub 2013 Sep 1.

IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39

Ivan D Mascanfroni  1 , Ada Yeste, Silvio M Vieira, Evan J Burns, Bonny Patel, Ido Sloma, Yan Wu, Lior Mayo, Rotem Ben-Hamo, Sol Efroni, Vijay K Kuchroo, Simon C Robson, Francisco J Quintana
Affiliations

IL-27 acts on DCs to suppress the T cell response and autoimmunity by inducing expression of the immunoregulatory molecule CD39

Ivan D Mascanfroni et al. Nat Immunol. 2013 Oct.

Abstract

Dendritic cells (DCs) control the balance between effector T cells and regulatory T cells in vivo. Hence, the study of DCs might identify mechanisms of disease pathogenesis and guide new therapeutic approaches for disorders mediated by the immune system. We found that interleukin 27 (IL-27) signaling in mouse DCs limited the generation of effector cells of the TH1 and TH17 subsets of helper T cells and the development of experimental autoimmune encephalomyelitis (EAE). The effects of IL-27 were mediated at least in part through induction of the immunoregulatory molecule CD39 in DCs. IL-27-induced CD39 decreased the extracellular concentration of ATP and downregulated nucleotide-dependent activation of the NLRP3 inflammasome. Finally, therapeutic vaccination with IL-27-conditioned DCs suppressed established relapsing-remitting EAE. Thus, IL-27 signaling in DCs limited pathogenic T cell responses and the development of autoimmunity.

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Conflict of interest statement

Competing financial interests

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Il27ra expression in DCs
(a–c) IL-27RA expression was analyzed by flow cytometry (a), q-PCR (b) and immunoblot (c) in sorted cDCs and pDCs. The numbers within the histogram show the percentage of positive cells. *P < 0.05 (Student’s t-test). Data are representative of more than three independent experiments with similar results.
Figure 2
Figure 2. IL-27 modulates the APC function of cDCs
(a) Flow cytometry of IL-27- (20 ng/ml) treated cDC in the presence or absence of ecLPS (100 ng/ml). Mean fluorescence intensity (MFI) +/− SD is shown. (b) Cytokine secretion by IL-27-treated cDCs, determined in culture supernatants by ELISA. (c) q-PCR analysis of Il27 expression in IL-27-treated cDCs. (d–f) Naive 2D2+ CD4+ T cells were stimulated with MOG35–55 and cDCs pre-treated with IL-27 and ecLPS, and proliferation (d), cytokine secretion to culture supernatants (e) and frequency of CD4+ IFN-γ+, IL-17+, IL-10+ and FoxP3+ (f) were analyzed. (g–h) Naive 2D2+ CD4+ T cells were stimulated with MOG35–55 and cDCs pre-treated with IL-27 and ecLPS in the presence of exogenous cytokines to promote the differentiation of TH1, TH2, Tr1 or FoxP3+ T cells, and cytokine secretion (g) and the frequency of CD4+ FoxP3+ T cells (h) were analyzed. Data are representative (h, left panel) or are the mean ± SEM (a–h) of three independent experiments. *P < 0.05; **P < 0.01 (One-way ANOVA).
Figure 3
Figure 3. IL-27RA signaling in cDCs controls T cell differentiation and EAE development
(a) Flow cytometry of IL-27RA expression in splenic cDCs sorted from naive DC (WT) or DC (IL-27RA-KO) mice. (b) Development of EAE in DC (WT) and DC (IL-27RA-KO) mice, clinical score (left panel) and linear-regression curves of disease for each group (dashed lines indicate 95% confidence interval). (c) CNS-infiltrating CD4+ T cells analyzed for the expression of IFN-γ, IL-17, IL-10 and FoxP3 by flow cytometry. (d) Recall response to MOG (35–55) in splenocytes from DC (WT) and DC (IL-27RA-KO) mice isolated 21 days after EAE induction. (e) Frequency of CD4+CD44+CD40Lhi splenic IFN-γ+, IL-17+, IFN-γ+ IL-17+ (DP), IL-10+ and FoxP3+ CD4+ T cells in DC (WT) and DC (IL-27RA-KO) mice 21 days after EAE induction. (f) Expression of Il27ra, Il6, Il12a, Il23a, Il27, Ifnb, Il10 and Tgfb1 in cDCs sorted from DC (WT) and DC (IL-27RA-KO) mice 21 days after immunization. (g) Quantitative nCounter expression profiling of cDCs isolated from DC (WT) and DC (IL-27RA-KO) mice 21 days after immunization. (h–i) Naive CFSE labeled 2D2+ CD4+ T cells were stimulated with MOG35–55 and cDCs sorted from DC (WT) and DC (IL-27RA-KO) mice 21 days after immunization, and proliferation (h) and cytokines secretion (i) were analyzed. The frequency of proliferated cells is shown in the histogram and the proliferation index is shown in the right (h). Numbers within histograms show the percentage of positive cells. Shown is a representative experiment (of three) with n ≥ 5 mice/group. *P < 0.05, **P < 0.01 and ***P < 0.001 (Student’s t-test) compared with DC (WT).
Figure 4
Figure 4. ENTPD1 is required for the inhibitory effects of IL-27 on DCs
(a–c) Naive CD4+ T cells were stimulated with anti-CD3 and ecLPS- or ecLPS+IL-27-treated cDCs in the presence of either isotype control (IC), anti-IL-27, anti-IL-10, anti-IFN-β or anti-TGF-β blocking antibodies (a) or in the presence of the IDO inhibitor 1-D-MT (b) and proliferation was analyzed. (c) T cells were stimulated with anti-CD3 and ecLPS- or ecLPS+IL-27-treated cDCs from ENTPD1-deficient mice (DC (CD39-KO)) and T-cell proliferation was analyzed. (d) Expression of Entpd1 in cDCs sorted from naive DC (WT) and DC (IL-27RA-KO) mice analyzed by qPCR (left panel) and flow cytometry. The numbers within the histogram show the percentage of positive cells. (e–f) Splenic cDCs were exposed to IL-27 (20 ng/ml) and after indicated time periods, cells were harvested and analyzed by immunoblot (e) and flow cytometry (f) for phosphorylated or unphosphorylated STAT 1 and STAT3. (g) STAT1 and STAT3 binding sites in the Entpd1 promoter. Schematic representation of the Entpd1 promoter with STAT1- (green; IRF-1), STAT3- (blue; SRE-1 and SRE-2) and STAT1 and STAT3-binding sites. (h) ChIP analysis of the interaction of STAT3 in the Entpd1 promoter of cDCs treated with IL-27 and ecLPS. (i) ChIP analysis of the interaction of STAT1 in the Entpd1 promoter of cDCs treated with IL-27 and ecLPS. (j) Luciferase activity in HEK293 cells transfected with a Entpd1 (CD39) luciferase reporter, alone (Control) or with a construct encoding constitutively activated STAT1 or STAT3, separately or together (STAT1c + STAT3c). *P < 0.05; **P < 0.01 (One-way ANOVA). Data are representative of more than three independent experiments with similar results.
Figure 5
Figure 5. IL-27-induced ENTPD1 controls extracellular ATP and NLRP3 inflammasome activation
(a) Extracellular ATP levels in culture supernatants of ecLPS- or ecLPS+IL-27-treated cDCs. (b) Residual extracellular ATP in culture supernatants of ecLPS- or ecLPS+IL-27-treated cDCs after incubation with 500 μM of exogenous ATP. Please note different scale used in comparison with the data showed in (a). (c) TLC assays to assess CD39 enzyme activity in ecLPS- or ecLPS+IL-27-treated cDCs. (d) Quantification of AMP band intensity, expressed relative to ADP in CD39 deficient DCs. (e) Immunoblot blot (left) and densitometric analysis of caspase-1 and IL-1β in ecLPS- or ecLPS+IL-27-treated cDCs. (f) Quantification of IL-1β in culture supernatants of ecLPS- or ecLPS+IL-27-treated cDCs. *P < 0.05; **P < 0.01; ***P < 0.001 (One-way ANOVA). Data are representative of two independent experiments with similar results. AU, arbitrary units.
Figure 6
Figure 6. ENTPD1 in DCs controls T cell differentiation and EAE development
(a) flow cytometry of ENTPD1 (CD39) expression of splenic DC sorted from naive WT (DC (WT))- or CD39-KO (DC (CD39-KO))-reconstituted mice. (b) Development of EAE in WT DC (WT) and DC (CD39-KO) mice, clinical score (left panel) and linear-regression curves of disease for each group (dashed lines indicate 95% confidence intervals). (c) CNS-infiltrating CD4+ T cells analyzed for the expression of IFN-γ, IL-17, IL-10 and FoxP3 by flow cytometry. (d) Recall response to MOG (35–55) in splenocytes from DC (WT) and DC (CD39-KO) mice isolated 21 days after EAE induction. (e) Frequency of CD4+CD44+ CD40Lhi splenic IFN-γ+, IL-17+, IFN-γ+ IL-17+ (DP), IL-10+ and FoxP3+ CD4+ T cells in DC (WT) and DC (CD39-KO) mice 21 days after EAE induction. (f) Expression of Entpd1, Il6, Il12a, Il23a, Il27, Ifnb, Il10 and Tgfb1 in cDCs sorted from DC (WT) and DC (CD39-KO) mice 21 days after immunization. (g–h) Naive CFSE labeled 2D2+ CD4+ T cells were stimulated with MOG35–55 and cDCs sorted from DC (WT) and DC (CD39-KO) mice 21 days after immunization, and proliferation (g) and cytokine secretion (h) were analyzed. The frequency of proliferated cells is shown in the histogram and the proliferation index is shown in the right (g). Numbers within histograms show the percentage of positive cells. Shown is a representative experiment (of three) with n ≥ 5 mice/group. *P < 0.05 and **P < 0.01 (Student’s t-test) compared with DC (WT).
Figure 7
Figure 7. Vaccination with IL-27 conditioned DCs suppresses EAE
EAE was induced by immunization of naive SJL mice with PLP131–159, and DCs were administered i.v. 4 times, once every 4 days, starting at day 20. (a) The course of EAE is shown as the mean EAE score ± SEM (n = 5 mice per group) for the whole observation period (left panel), and also as the linear regression curves (right panel) of the disease for each group from day 20 until the termination of the experiment. Arrows indicate treatment with DC vaccines. (b,c) Recall proliferative (b) and cytokine (c) response to PLP131–159 in splenocytes taken from DCs-treated mice 55 days after EAE induction. (d,e) Recall proliferative (d) and cytokine (e) response to PLP178–191 in splenocytes taken from DCs-treated mice 55 days after EAE induction. (f) Heatmap depicting the antibody response to myelin antigens on day 55 after EAE induction as measured on antigen microarrays. Each column represents the mean IgG serum reactivity in each treatment group, according to the colorimetric scale shown on the bottom. Data are representative of at least three independent experiments. NS, not significant. *P < 0.05, **P < 0.01 and **P < 0.001 (One-way ANOVA)versus control mice.
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