IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+ T cells - PubMed (original) (raw)

IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+ T cells

Gopal Murugaiyan et al. J Immunol. 2009.

Abstract

Although the physiologic pathways that control regulatory T cells (Foxp3-expressing regulatory T cells, IL-10-secreting Tr1 cells) and Th17 cells in rodents have been defined, the factors that control these differentiation pathways in humans are not well understood. In this study, we show that IL-27 promotes the differentiation of IL-10-secreting Tr1 cells while inhibiting Th17 generation and molecules associated with Th17 function. Furthermore, IL-27 inhibits IL-17-polarizing cytokines on dendritic cells, which in turn decrease IL-17 secretion from T cells. Our results demonstrate that IL-27 plays a key role in human T cells by promoting IL-10-secreting Tr1 cells and inhibiting Th17 cells and thus provides a dual regulatory mechanism to control autoimmunity and tissue inflammation.

PubMed Disclaimer

Figures

FIGURE 1

IL-27 stimulation induces IL-10 production from human peripheral blood CD4+ T cells. ELISA of total CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of recombinant human IL-27 (100 ng/ml). A and B, IL-27 stimulation increased anti-CD3- and anti-CD28-induced IL-10 and IFN-γ production. C and D, IL-27 inhibited anti-CD3- and anti-CD28-induced IL-17 without affecting TGF-β production. E–H, Quantitative PCR of the expression of mRNA encoding T-bet, GATA-3, RORC, and Foxp3 in total CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 (100 ng/ml), presented relative to the expression of mRNA encoding GAPDH. Data are from 11 randomly selected healthy donors. Horizontal bars indicate the median.

FIGURE 2

IL-27 stimulation induces Tr1 like cells. A, Purity of naive and memory CD4+ T cells. Flow cytometry of naive CD4+ T cells after purification and staining with anti-CD4 PE, anti-CD45RA allophycocyanin, and anti-CD45RO FITC. B, Analysis of the cytokine expression of naive and memory CD4+ T cells stimulated with anti-CD3 and anti-CD28 in the presence or absence of recombinant human IL-27 (100 ng/ml). C, Naive CD4+ T cells expressed more IL-27 receptor on their surface compared with memory CD4+ T cells as measured by FACS. D and E, Analysis of the cytokine expression of secondary stimulated T cells. CD4+ T cells, first activated with CD3/CD28/IL-27, produced large amounts of IL-10 on secondary stimulation. Cells were stimulated for 3 days with anti-CD3 and anti-CD28 in the presence or absence of IL-27 and 3 days after primary stimulation cells were expanded and subjected to secondary stimulation. IL-10 secretion was measured at day 3. F, IL-27 induces the generation of IFN-γ+ and IL-10+CD4+ T cells. Flow cytometry of naive CD4+ T cells activated with anti-CD3 and anti-CD28 in the presence or absence of IL-27 stained for intracellular IL-10 and IFN-γ. G and H, IL-27-induced IL-10 production is IL-2 dependent. CD4+ T cells were incubated with the plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 and IL-10 secretion was measured at day 3. rIL-2 (100 U/ml) or neutralizing Ab to IL-2 (20 _μ_g/ml) was added as indicated. I, IL-27-stimulated T cells inhibit the proliferation of bystander CD4+ T cells in an IL-10-dependent manner. Naive CD4+ T cells were incubated with the plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 for 3 days. Cell-free culture supernatants were transferred to freshly purified CD4+ T cells cultured with plate-bound anti-CD3 alone or with anti-CD3 and CD28. Neutralizing anti-IL-10 Ab (20 _μ_g/ml) was added to the indicated condition and proliferation was measured at day 5. Data represent one of four (A–C) experiments or one of three (D and E) or one of two (F–I) experiments involving nine randomly selected donors, and the error bars represent the mean ± SD.

FIGURE 3

IL-27 inhibits IL-17 production from total CD4+ T cells. A, Total CD4+ T cells were stimulated with anti-CD3 and anti-CD28 in the presence or absence of IL-27 for 3 days and cell-free culture supernatants were assayed for IL-17A by ELISA. B, IL-27 inhibits RORC in total CD4+ T cells. Quantitative PCR of the expression of mRNA total CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 (100 ng/ml). C–G, IL-27 inhibits molecules associated with Th17 effector molecules in total CD4+ T cells. Quantitative PCR of the expression of mRNA encoding IL-17F, IL-22, IL-23R, CCR6, and CCL20 from total CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 (100 ng/ml). Data are from five randomly selected healthy donors. Horizontal bars indicate the median. H and I, IL-27-mediated IL-17 suppression was mediated by IFN-γ. Total CD4+ T cells stimulated with plate-bound anti-CD3 and anti-CD28 in the presence or absence of IL-27 (100 ng/ml). To some conditions, neutralizing anti-IL-10 Ab (20 _μ_g/ml) or anti-IFN-γ (20 _μ_g/ml) was added as indicated. Data represent one of three independent experiments with cells from two randomly selected donors.

FIGURE 4

IL-27 inhibits IL-17 production from Th17-polarized cells. Naive CD4+ T cells were activated with plate-bound anti-CD3 and anti-CD28 in the presence of Th17-polarizing cytokines IL-1_β_ (50 ng/ml) and IL-23 (50 ng/ml) with or without IL-27 (100 ng/ml). A, ELISA of IL-17 in cell-free culture supernatants. B, Real-time quantitative RT-PCR of transcript expression of RORC at 24 h after activation. C, IL-27 inhibits TGF-β in combination with other proinflammatory cytokine-induced IL-17 secretion. ELISA of IL-17 secretion by Th17-polarized cells by TGF-β in combination with IL-1_β_ (50 ng/ml), IL-6 (50 ng/ml), IL-21 (12.5 ng/ml), and IL-23 (50 ng/ml) with or without IL-27. D, IL-27 inhibits RORC expression as determined by real-time quantitative RT-PCR. E, IL-27 induces IL-10 secretion from CD4+ T cells stimulated with TGF-β in combination with other proinflammatory cytokines. ELISA of IL-10 secretion from T cells stimulated with TGF-β in combination with IL-1_β_ (50 ng/ml), IL-6 (50 ng/ml), IL-21 (25.0 ng/ml), and IL-23 (50 ng/ml) with or without IL-27. Data represent one of three independent experiments with cells from three randomly selected donors.

FIGURE 5

IL-27 inhibits IL-17 polarizing cytokines from monocyte derived dendritic cells. A–C, Cytometric bead assay of IL-1_β_ and IL-6 and ELISA of IL-23 in monocyte-derived DCs stimulated with LPS or PGN alone or in the presence of IL-27 (100 ng/ml). D, DCs express IL-27R on their surface. Monocyte-derived DCs used in our experiments were >95% CD11c+ and these cells expressed IL-27R on their surface as determined by flow cytometry. E, Total CD4+ T cells were cultured with a 1: 3 ratio of DCs prestimulated with LPS or PGN alone or in the presence of IL-27. The DCs were washed three times before they were cultured with T cells. After 4 days of coculture, the cell-free culture supernatants were assayed for IL-17 by ELISA. Data are representative of three experiments with three donors.

References

1. Van Parijs L, Abbas AK. Homeostasis and self-tolerance in the immune system: turning lymphocytes off. Science. 1998;280:243–248. -PubMed
1. Kroemer G, Martinez C. Mechanisms of self tolerance. Immunol Today. 1992;13:401–404. -PubMed
1. Webb S, Morris C, Sprent J. Extrathymic tolerance of mature T cells: clonal elimination as a consequence of immunity. Cell. 1990;63:1249–1256. -PubMed
1. Groux H, O'Garra A, Bigler M, Rouleau M, Antonenko S, de Vries JE, Roncarolo MG. A CD4+ T-cell subset inhibits antigen-specific T-cell responses and prevents colitis. Nature. 1997;389:737–742. -PubMed
1. Roncarolo MG, Gregori S, Battaglia M, Bacchetta R, Fleischhauer K, Levings MK. Interleukin-10-secreting type 1 regulatory T cells in rodents and humans. Immunol Rev. 2006;212:28–50. -PubMed

IL-27 is a key regulator of IL-10 and IL-17 production by human CD4+ T cells - PubMed (original) (raw)