Regulation of human Th9 differentiation by type I interferons and IL-21

Abstract

Interleukin (IL)-9-producing CD4(+) T cells are a novel subset of T helper (Th) cells that develops independently of the Th1, Th2, Th17 and regulatory T-cell lineages. Similar to the murine model, transforming growth factor (TGF)-beta and IL-4 directed human naive CD4(+) T cells to produce IL-9. Whereas IL-4 suppressed TGF-beta-induced Foxp3 expression, TGF-beta failed to inhibit IL-4-mediated upregulation of the Th2 transcription factor GATA-3. Addition of IL-1 beta, IL-6, IL-10, interferon (IFN)-alpha, IFN-beta or IL-21 to Th9-polarizing conditions augmented Th9 differentiation, while the Th1-associated cytokines IFN-gamma and IL-27 partially suppressed IL-9 production. Given that T cells are a primary source of IL-21, IL-21 expression was analyzed under Th9-polarizing conditions in the context of inflammatory cytokines. Surprisingly, type I IFNs induced elevated levels of IL-21, and blockade of IL-21 abrogated their ability to enhance Th9 differentiation. Taken together, these data indicate a complex cytokine network in the regulation of human IL-9-producing CD4(+) T cells.

Figure 1

TGF-β in combination with IL-4 induces IL-9 production from human naive CD4⁺ T cells. Naïve CD4⁺ T cells were activated with anti-CD3/CD28 coated beads in the presence or absence of the indicated cytokines for 4 days. (a) ELISA of IL-5, IL-10, IL-13 and IL-9 in cell-free supernatants. Data (mean and s.e.m.) are from four independent experiments with six donors, *P<0.05, **P<0.01 as compared with all other conditions. (b) Intracellular expression of IL-4 and IL-9 after restimulation with PMA and ionomycin in the presence of Brefeldin A (BFA) for an additional 4 h. Data are representative of three independent experiments.

Figure 2

Transcriptional analysis of IL-9-producing cells. Naïve CD4⁺ T cells were activated with anti-CD3/CD28 coated beads in the presence or absence of the indicated cytokines for 3 days. (a, b) Intracellular expression of Foxp3, IL-9 (a) and GATA-3 (b) after restimulation with PMA and ionomycin in the presence of Brefeldin A (BFA) for an additional 4 h. (c) Cells cultured under Th9-polarizing conditions (TGF-β+IL-4) were analyzed for GATA-3 expression (right panel) after gating on IL-9− and IL-9+ cells (left panel). Data are representative of three independent experiments.

Figure 3

Regulation of human Th9 differentiation by inflammatory cytokines. Naïve CD4⁺ T cells were cultured under Th9-polarizing conditions and the indicated cytokines for 4 days. (a) ELISA of IL-9 in cell free supernatants. Data (mean and s.e.m.) are from five independent experiments with seven donors, *P<0.05, **P<0.01, ***P<0.001 as compared with the Th9 condition. (b) Cells were cultured under Th9-polarizing conditions in the presence or absence of increasing concentrations of IFN-γ (left panel) or IL-27 (right panel). ELISA of IL-9 (mean and s.e.m.) are from three independent experiments with three donors, **P<0.01 for decreasing linear trend by repeated-measures one-way analysis of variance (ANOVA) with post-test for linear trend.

Figure 4

Type I IFNs induce elevated levels of intracellular IL-21. Naïve CD4⁺ T cells were cultured under Th9-polarizing conditions and the indicated cytokines. (a) After 4 days, cells were restimulated with PMA and ionomycin in the presence of Brefeldin A (BFA) for an additional 4 h and stained for intracellular expression of IL-9 and IL-21. Data are representative of five independent experiments. (b) Percentage of IL-9+ (left panel) or IL-21+ (right panel) cells as measured by FACS. Data (mean and s.e.m.) are from five independent experiments with five donors.

Figure 5

IL-21 is required for type 1 IFN enhancement of human Th9 differentiation. Naïve CD4⁺ T cells were cultured under Th9-polarizing conditions and the indicated cytokines in the presence of an isotype control (white bars) or an IL-21 receptor-Fc fusion protein (black bars) for 4 days. (a) ELISA measuring IL-9 from cell-free supernatants. Data (mean and s.e.m.) are from three independent experiments with four donors. (b) Intracellular expression of IL-9 and IL-21 after restimulation with PMA and ionomycin in the presence of Brefeldin A (BFA) for an additional 4 h. Data are representative of three independent experiments.

Figure 6

Characterization of IL-9 production in memory CD4⁺ T cells. (a) Freshly isolated memory CD4⁺ T cells were stimulated with PMA and ionomycin in the presence of Brefeldin A (BFA) for 4 h and stained for intracellular expression of IL-4, IL-5, IL-13 and IL-9. Data are representative of three independent experiments. (b) Unfractionated memory CD4⁺ T cells (bulk memory) or memory T cells sorted according to CRTH2 expression were stimulated with PMA and ionomycin for 18 h. Cell-free supernatants were analyzed for expression of IL-4, IL-5, IL-13 and IL-9 by ELISA. Data (mean and s.e.m.) are from three independent experiments with three donors. (c) Memory CD4⁺ T cells were activated with anti-CD3/CD28 coated beads and the indicated cytokines for 4 days. Cells were restimulated with PMA and ionomycin in the presence of Brefeldin A (BFA) for an additional 4 h and stained for intracellular expression of IL-4 and IL-9. Data are representative of three independent experiments.