The differentiation of human T(H)-17 cells requires transforming growth factor-beta and induction of the nuclear receptor RORgammat

Abstract

T(H)-17 cells are interleukin 17 (IL-17)-secreting CD4+ T helper cells involved in autoimmune disease and mucosal immunity. In naive CD4+ T cells from mice, IL-17 is expressed in response to a combination of IL-6 or IL-21 and transforming growth factor-beta (TGF-beta) and requires induction of the nuclear receptor RORgammat. It has been suggested that the differentiation of human T(H)-17 cells is independent of TGF-beta and thus differs fundamentally from that in mice. We show here that TGF-beta, IL-1beta and IL-6, IL-21 or IL-23 in serum-free conditions were necessary and sufficient to induce IL-17 expression in naive human CD4+ T cells from cord blood. TGF-beta upregulated RORgammat expression but simultaneously inhibited its ability to induce IL-17 expression. Inflammatory cytokines relieved this inhibition and increased RORgammat-directed IL-17 expression. Other gene products detected in T(H)-17 cells after RORgammat induction included the chemokine receptor CCR6, the IL-23 receptor, IL-17F and IL-26. Our studies identify RORgammat as having a central function in the differentiation of human T(H)-17 cells from naive CD4+ T cells and suggest that similar cytokine pathways are involved in this process in mice and humans.

Figure 1. RORγT is necessary and sufficient for the expression of IL-17 in human CD4⁺ T cells

(a) Flow cytometry on sorted CD45RO⁻ and CD45RO⁺CCR6⁺ activated and expanded in the presence of IL-2 with or without IL-1β. IL-17 and IFN-γ production was analyzed at day 6. (b,c) RT-PCR for RORC and ACTB mRNA expression (b) and flow cytometry for intracellular IL-17 and IFN-γ (c) in sorted CD45RO⁺CCR6⁺ cells transduced with an empty vector or vector encoding for RORγT-specific shRNA (shRNA-1 and shRNA-2). Cells were selected in puromycin at day 2 and expression of mRNA and cytokines was analyzed at day 6. Data are representative of four independent experiments. (d) Flow cytometry of naive cord blood CD4⁺ T cells activated, transduced by vectors encoding IRES-HSA or RORγT-IRES-HSA and then expanded for 6 days in the presence of IL-2. Intracellular IL-17 and IFN-γ production was analyzed at day 6. (e) RT-PCR of ACTB, IL17, IL17F and IL26 in naive cord blood CD4⁺ T cells transduced with vectors encoding IRES-GFP or RORγT-IRES-GFP. GFP⁺ cells were sorted at day 6 and the levels of mRNAs were analyzed. (f) Flow cytometry of naive cord blood CD4⁺ T cells transduced with vectors encoding IRES-HSA, RORγT-IRES-HSA, GATA-3-IRES-HSA or T-bet-IRES-HSA. CCR6 cell surface expression was measured at day 12. Each panel is representative of three independent experiments unless noted otherwise.

Figure 2. TGF-β induces RORγT and inhibits its activity, but the inhibition is relieved by inflammatory cytokines

(a) Flow cytometry of naive cord blood CD4⁺ T cells transduced with a vector encoding RORγT-IRES-HSA alone or with combination of IL-1β, IL-6, IL-21 and increasing concentrations of TGF-β. IL-17 intracellular staining was performed at day 6. (b,c) RT-PCR of RORC and ACTB expression measured in freshly sorted CCR4^−/+CCR6^−/+ adult memory CD4⁺ T cells and in naive CD4⁺ T cells cultivated for 3 days in the presence of various cytokines (b) and in naive cord blood CD4⁺ T cells cultivated with various concentrations of TGF-β (c) (d) Flow cytometry of naive cord blood CD4⁺ T cells transduced with a vector encoding RORγT-IRES-HSA in serum-containing medium with or without anti-TGF-β or in serum-free medium. Intracellular IL-17 and FOXP3 expression was analyzed at day 6. A representative donor with low IL-17 expression following RORγT transduction in the presence of serum is shown. Each panel is representative of at least three independent experiments.

Figure 3. TGF-β, IL-1β and IL-6, IL-21 or IL-23 are required for human T_H-17 cell polarization in serum-free conditions

(a) Naive cord blood CD4⁺ T cells were activated without cytokines or with IL-1β, IL-6 or IL-21 with or without IL-23, alone or with increasing concentrations of TGF-β. IL-2 was added at day 3 and IL-17 expression was analyzed at day 14. (b) Naive cord blood CD4⁺ T cells were activated with no cytokine or with a combination of IL-1β + IL-23 + TGF-β (1 ng/ml), with or without 10 U/ml IL-2 or neutralizing anti-IL-2. IL-17 expression was analyzed at day 6. (c) Naive cord blood CD4⁺ T cells were cultivated with IL-2 and different concentrations of TGF-β in the presence of the listed cytokines. IL-17 expression was analyzed at day 6. (d) Time-course of IL-17 and IFN-γ production in naive cord blood CD4⁺ T cells polarized in the presence of IL-2 + IL-1β + IL-23 + TGF-β (0.1, 1 or 10 ng/ml). (e) Summary of IL-17 expression in day 6 cultures of naive cord blood CD4⁺ T cells from different donors (n = 11) in the presence of IL-2 + IL-1β + IL-23 + 10 ng/ml TGF-β. (f) Naive cord blood CD4⁺ T cells were cultivated in IL-2 alone or IL-23 + IL-1β + IL-2 with increasing concentrations of TGF-β. IL-17 and IL-22 expression were analyzed at day 6. Each panel is representative of at least three independent donors.

Figure 4. Induction of IL26, IL17F, IL17, RORC and IL23R mRNAs during human T_H-17 cell differentiation

(a-c) Naive cord blood CD4⁺ T cells were cultivated with IL-2 alone or with IL-1β + IL-2, IL-23 + IL-2 or IL-23 + IL-1β + IL-2 with increasing concentrations of TGF-β. mRNA levels of ACTB, IL17 (a), IL26 (b) and IL17F (c) were analyzed on day 6 following restimulation with PMA and ionomycin. (d, e) Naive cord blood CD4⁺ T cells were cultivated with IL-2 alone or with IL-1β + IL-2, IL-23 + IL-2 or IL-23 + IL-1β + IL-2 with increasing concentrations of TGF-β. mRNA levels of ACTB, RORC (d) and IL23R (e) were analyzed on day 6. Each panel is representative of three independent donors.

Figure 5. Expression of CCR6 and FOXP3 during human T_H-17 cell differentiation

(a) Naive cord blood CD4⁺ T cells were cultivated with IL-2 alone or with IL-1β + IL-2, IL-23 + IL-2 or IL-23 + IL-1β + IL-2 with increasing concentrations of TGF-β. CCR6 expression was analyzed by surface staining at day 6. (b) Naive cord blood CD4⁺ T cells were cultivated for 6 days in IL-2 + IL-23 + IL-1β + IL-2 + 10 ng/ml TGF-β. CCR6⁺ and CCR6^- cells were sorted and intracellular IL-17 was analyzed. (c) Naive cord blood CD4⁺ T cells were cultivated with IL-2 alone or with IL-1β + IL-2, IL-23 + IL-2 or IL-23 + IL-1β + IL-2 with increasing concentrations of TGF-β. FOXP3 and IL-17 expression were analyzed at day 6. Data are representative of four independent experiments.