Interleukin-27 priming of T cells controls IL-17 production in trans via induction of the ligand PD-L1

Abstract

Interleukin-27 (IL-27) is a key immunosuppressive cytokine that counters T helper 17 (Th17) cell-mediated pathology. To identify mechanisms by which IL-27 might exert its immunosuppressive effect, we analyzed genes in T cells rapidly induced by IL-27. We found that IL-27 priming of naive T cells upregulated expression of programmed death ligand 1 (PD-L1) in a signal transducer and activator of transcription 1 (STAT1)-dependent manner. When cocultured with naive CD4(+) T cells, IL-27-primed T cells inhibited the differentiation of Th17 cells in trans through a PD-1-PD-L1 interaction. In vivo, coadministration of naive TCR transgenic T cells (2D2 T cells) with IL-27-primed T cells expressing PD-L1 inhibited the development of Th17 cells and protected from severe autoimmune encephalomyelitis. Thus, these data identify a suppressive activity of IL-27, by which CD4(+) T cells can restrict differentiation of Th17 cells in trans.

Figure 1. IL-27-primed cells inhibit Th17 cell differentiation in trans

Briefly, naïve CD45.2⁺CD4⁺ T cells were primed with the indicated cytokines. After washing, cells were mixed with naïve CD45.1⁺CD4⁺ T cells and cultured with IL-6, TGF-β and anti-IFN-γ for 3 days. (A, B) IL-17A and IFN-γ production in CD45.1⁺CD4⁺ T cells was assessed by intracellular staining. Representative intracellular staining is shown for Th17 cells (A). Pooled data from six independent experiments are provided in B (mean values, NS, not significant). (C–F) Production of IFN-γ and IL-17A in CD45.1⁺OT-II CD4⁺ T cells was analyzed by flow cytometry. Total numbers of CD45.1⁺OT-II CD4⁺ T cells are provided in C. Representative results from 2 individual mice are shown in D and pooled data from 2 independent experiments are provided in E and F (mean values, NS, not significant).

Figure 2. IL-27-primed naïve CD4⁺ T cells inhibit pathogenicity of T cells in trans

Briefly, naïve CD4⁺ T cells were primed with or without IL-27 and transferred with or without sorted naïve TCR transgenic 2D2 CD4⁺ T cells into Rag2^−/− mice. To induce EAE, which were immunized with MOG peptide in CFA. (A) Data provided represent mean ± s.e.m. of the EAE clinical score of 40 mice pooled from 3 independent experiments (***P<0.001, *P<0.05). (B–D) CNS-infiltrating CD4⁺ T cells from recipient mice of 2 groups from A were analyzed on day 20 after immunization. Absolute numbers (B), representative flow cytometric analysis of IL-17A and IFN-γ (C) and absolute numbers of subsets of CD4⁺ T cells (D) are provided from 2 independent experiments (mean values, NS, not significant).

Figure 3. IL-27 priming rapidly induces PD-L1 on naïve CD4⁺ T cells

(A–C) Naïve CD4⁺ T cells were cultured in medium alone (control), or stimulated with IL-6 or IL-27 for 3 hours before analyzing differential gene expression by microarray. The arrow indicates Cd274 expression. Volcano plots depict differential gene expression induced by IL-27 or IL-6 compared to control (A). Comparison of IL-27- and IL-6-dependent gene expression is shown as a scatter plot (B). (C and D) Naïve CD4⁺ T cells (C), naïve CD8⁺ T cells or freshly isolated CD11c⁺ DCs (D) were stimulated as in A and expression of PD-L1 and PD-L2 was determined by flow cytometry. (E and F) Wild type or Ebi3^−/− mice were orally administered T. gondii, and sacrificed on day 9. Mesenteric lymph node (mLN) and Peyers patch (PP) cells were gated on CD4⁺, TCR-β ⁺, CD8⁻, and Foxp3⁻ cells. PD-L1 expression is shown as percentages of CD4⁺ T cells, and by geometric mean channel fluorescence (E). DCs were gated on CD3⁻, NK1.1⁻, CD19⁻ and CD11c^high. B cells are gated on CD3⁻, NK1.1⁻ and CD19⁺ (F). The experiment shown is representative of 2 separate experiments, (mean values, NS, not significant).

Figure 4. IL-27-induced expression of PD-L1 on naïve CD4⁺ T cells inhibits IL-17 production in trans

(A and B) Naïve CD45.2⁺CD4⁺ T cells were primed with IL-6 or IL-27 or cultured in medium alone. After 3 hours, cells were washed and co-cultured with naïve CD45.1⁺CD4⁺ T cells under Th17 cell-polarizing conditions with an isotype control antibody (upper panel) or anti-PD-L1 antibody (lower panel). After 3 days IL-17A protein expression of CD45.1⁺CD4⁺ T cells was analyzed by intracellular staining. Representative intracellular staining is depicted in A and pooled data from four separate experiments with mean values are shown in B (NS, not significant). (C) Data provided represent mean ± s.e.m. of the EAE clinical score of 32 mice pooled from 2 independent experiments (***P<0.001, **P<0.01, *P<0.05). (D–F) CNS-infiltrating CD4⁺ T cells from recipient mice of 2 groups from C were analyzed on day 19 after immunization. Absolute numbers (D), representative flow cytometric analysis of IL-17A and IFN-γ (E) and absolute numbers of subsets of CD4⁺ T cells (F) are provided (mean values, NS, not significant).

Figure 5. Soluble PD-L1 affects Th17 cell differentiation

(A and B) Naïve CD4⁺ T cells were cultured with IL-6, TGF-β and anti-IFN-γ (Th17 cell-polarizing conditions, upper panel) or IL-12 and anti-IL-4 (Th1 cell-polarizing conditions, lower panel) for 3 days with PD-L1 or IgG1-Fc as a control. IL-27 and IL-6 were added as additional controls. IL-17A, IFN-γ and FoxP3 protein expression were analyzed by intracellular staining. Representative flow cytometry plots are depicted in A and pooled data from 3 (Th1 cell-polarizing condition) or 4 (Th17 cell-polarizing condition) individual experiments with mean values are shown in B (NS, not significant). (C and D) Naïve CD4⁺ T cells were cultured under Th17 cell-polarizing conditions with PD-L1 or IgG1-Fc in the presence of anti-PD-L1 antibody (lower panel) or isotype control antibody (upper panel) for 3 days. IL-17 and FoxP3 protein expression were analyzed by intracellular staining. Representative intracellular staining is depicted in C and pooled data from 3 individual experiments with mean values are shown in D (NS, not significant).

Figure 6. Rapid induction of PD-L1 by IL-27 is directly mediated by STAT1

(A) Naïve CD4⁺ T cells from Stat1^−/−, Stat3^−/− or wild type control mice were stimulated with the indicated cytokines for 3 hours. Surface expression of PD-L1 is shown. (B) Naïve CD4⁺ T cells, freshly isolated CD8⁺ T cells, B220⁺ cells, CD11c⁺ cells and CD11b⁺ cells were stimulated with indicated cytokines for 3 hours and expression of PD-L1 was determined by flow cytometry. Representative data are provided from 2 separate experiments. (C) Naïve CD4⁺ T cells were stimulated with IL-27 for 30 minutes. Fixed cells were immunoprecipitated with anti-STAT1 antibody. Eluted DNA was analyzed by quantitative PCR (mean ± s.d.). Representative data are provided from 2 separate experiments. (D, E) Briefly, naïve CD45.2⁺CD4⁺ T cells were primed with the indicated cytokines. After washing, cells were mixed with naïve CD45.1⁺CD4⁺ T cells and cultured with IL-6, TGF-β and anti-IFN-γ for 3 days. IL-17A and IFN-γ production in CD45.1⁺CD4⁺ T cells was assessed by intracellular staining. Representative intracellular staining is shown for Th17 cells (D). Pooled data from three independent experiments are provided in E (mean values). (F) Data show mean ± s.e.m. of the EAE clinical score of 20 mice pooled from 2 independent experiments (***P<0.001, **P<0.01, *P<0.05).

Figure 7. STAT1 is critical for induction of PD-L1 and inhibition of Th17 cell differentiation in vivo

(A, B) Wild type or Stat1^−/− mice received an intraperitoneal injection of LPS with PBS. One day after injection, spleens were harvested and CD4⁺ T cells were analyzed by flow cytometry for PD-L1 expression. Representative results are shown in A and pooled data from 2 independent experiments are provided in B (mean values). (C) Data show mean ± s.e.m. of the EAE clinical score of 32 mice pooled from 2 independent experiments (***P<0.001, **P<0.01, *P<0.05).