TGF-beta induces IL-9 production from human Th17 cells

Abstract

The secretion of IL-9, initially recognized as a Th2 cytokine, was recently attributed to a novel CD4 T cell subset termed Th9 in the murine system. However, IL-9 can also be secreted by mouse Th17 cells and may mediate aspects of the proinflammatory activities of Th17 cells. Here we report that IL-9 is secreted by human naive CD4 T cells in response to differentiation by Th9 (TGF-beta and IL-4) or Th17 polarizing conditions. Yet, these differentiated naive cells did not coexpress IL-17 and IL-9, unless they were repeatedly stimulated under Th17 differentiation-inducing conditions. In contrast to the naive cells, memory CD4 T cells were induced to secrete IL-9 by simply providing TGF-beta during stimulation, as neither IL-4 nor proinflammatory cytokines were required. Furthermore, the addition of TGF-beta to the Th17-inducing cytokines (IL-1beta, IL-6, IL-21, IL-23) that induce memory cells to secrete IL-17, resulted in the marked coexpression of IL-9 in IL-17 producing memory cells. The proinflammatory cytokine mediating TGF-beta-dependent coexpression of IL-9 and IL-17 was identified to be IL-1beta. Moreover, circulating monocytes were potent costimulators of IL-9 production by Th17 cells via their capacity to secrete IL-1beta. Finally, to determine whether IL-9/IL-17 coproducing CD4 cells were altered in an inflammatory condition, we examined patients with autoimmune diabetes and demonstrated that these subjects exhibit a higher frequency of memory CD4 cells with the capacity to transition into IL-9(+)IL-17(+) cells. These data demonstrate the presence of IL-17(+)IL-9(+) CD4 cells induced by IL-1beta that may play a role in human autoimmune disease.

FIGURE 1

Both Th9 and Th17 polarizations promote IL-9 expression in human naive CD4 T cells. FACS-sorted naive CD4 T cells were stimulated with anti-CD3, anti-CD28, and IL-2 under Th polarizing conditions: Th0 (anti–IFN-γ/anti–IL-4), Th1 (Il-12/anti–IL-4), Th2 (IL-4/anti–IFN-γ), Th17 (TGF-β/IL-1β/IL-6/IL-21/IL-23), TGF-β/IL-4, or TGF-β. Supernatants and cells were harvested at day 2, 4, and 6 of stimulation. A, Secretion of IFN-γ, IL-10, IL-17, and IL-9 was measured by ELISA and (B) mRNA expression of T Bet, GATA-3, RORC, and FoxP3 was measured by quantitative RT-PCR. Data are represented as mean ± SEM of four different donors.

FIGURE 2

Minimal cytokine requirements for the differentiation of IL-9–producing cells. FACS-sorted naive CD4 T cells were stimulated for 7 d with anti-CD3 and anti-CD28 in the presence of various polarizing cytokines, with (A) or without (B) exogenous IL-2. Intracellular cytokine expression was analyzed by flow cytometry. The frequencies of cells expressing IL-9 or IL-17 are represented as mean ± SEM of five different donors. *p < 0.05; **p < 0.01.

FIGURE 3

Upon differentiation, IL-9–producing cells do not coexpress IFN-γ, IL-4, IL-10, IL-17, or FoxP3. A, FACS-sorted naive CD4 T cells were stimulated for 6 d with anti-CD3, anti-CD28, and IL-2 in the presence of IL-4 (10 ng/ml) and increasing doses of TGF-β. Expression of IL-9 and IL-10 mRNA was measured by quantitative RT-PCR. Data are representative of two different donors. B and C, FACS-sorted naive CD4 T cells were stimulated for 7 d with anti-CD3, anti-CD28, and IL-2 in the presence of various polarizing cytokines, and analyzed for intracellular cytokine expression by flow cytometry. B, The frequencies of FoxP3⁺ cells are represented as mean ± SEM of five different donors. *p < 0.05; **p < 0.01. C, Expression of IL-9 versus IFN-γ, IL-4, IL-10, IL-17, and FoxP3 is shown. Data are representative of five different donors.

FIGURE 4

Th17 cells gain IL-9 expression after multiple rounds of polarization in vitro. FACS-sorted naive CD4 T cells were stimulated for 6 d with anti-CD3, anti-CD28, IL-2, and Th17 polarizing conditions (TGF-β/IL-1β/IL-6/IL-21/IL-23) and analyzed for intracellular expression of IL-17 and IL-9 by flow cytometry (A). B, IL-17⁺ cells were labeled using an IL-17 capture kit (B) and isolated by cell sorting. C, The IL-17⁺ fraction was repeatedly restimulated using Th17-polarizing conditions and analyzed for intracellular expression of IL-17 and IL-9 after second, third, and fourth rounds of polarization. Data are representative of three different donors.

FIGURE 5

IL-9 secretion by memory CD4 T cells is inducible by TGF-β. A, FACS-sorted memory CD4 T cells were stimulated for 5 d with anti-CD3 and anti-CD28 with or without TGF-β or IL-2. The frequencies of cells expressing IL-9 are represented as mean ± SEM of five different donors. B, FACS-sorted memory CD4 T cells were stimulated with anti-CD3, anti-CD28, and IL-2 with or without TGF-β. Supernatants were harvested at day 2, 4, and 6 of stimulation. Secretion of IFN-γ, IL-5, IL-10, IL-17, and IL-9 was measured by ELISA. Data are represented as mean ± SEM of four different donors. *p < 0.05; **p < 0.01.

FIGURE 6

TGF-β promotes the conversion of Th2 cells into IL-9–producing cells. A, FACS-sorted memory CD4 T cells were stimulated for 5 d with anti-CD3, anti-CD28, and IL-2 with or without TGF-β, and analyzed for intracellular expression of IL-9 and GATA-3. Data are representative of two different donors. B, Single-cell clones were derived from memory CD4 T cells. The clones that expressed IL-4 but not IFN-γ or IL-17 (Th2 clones, n = 32/90) were restimulated for 3 d with anti-CD3, anti-CD28, and IL-2 with or without TGF-β. Secretion of IL-5, IL-10, and IL-9 was measured by ELISA.

FIGURE 7

IL-9 secretion by memory Th17 cells requires TGF-β and proinflammatory cytokines. FACS-sorted memory CD4 T cells were stimulated for 5 d with anti-CD3, anti-CD28, IL-2, and various cytokines, and analyzed for intracellular cytokine expression by flow cytometry. Data are represented as mean ± S.E.M. of 5 different donors. A and B, The frequencies of IL-17⁺IL-9⁺ cells, total IL-17⁺, and total IL-9⁺ cells are shown. C, Frequencies of cells expressing IL-17 only, IL-17/IFN-γ, IL-17/IL-9, or IL-17/IL-9/IFN-γ calculated as a percentage of total IL-17⁺ cells. D, Frequencies of IL-9⁺IFN-γ⁺IL-17⁻ cells were calculated as a percentage of total IL-9⁺ cells. E, Intracellular expression of IL-9 versus IL-17 or IFN-γ. *p < 0.05; **p < 0.01.

FIGURE 8

Circulating monocytes are potent costimulators of IL-9 production by Th17 cells. FACS-sorted memory CD4 T cells were stimulated for 6 d with anti-CD3 and allogeneic monocytes, with or without IL-2 or TGF-β. Where indicated, an IL-1RA or neutralizing Abs against IL-6 or TNF-α was added. The frequencies of IL-17⁺IL-9⁺ cells are shown. T cells were isolated from two different donors and monocytes from three different donors. Data are represented as mean ± SEM. *p < 0.05; **p < 0.01.

FIGURE 9

IL-9 secretion by Th17 cells is increased in patients with autoimmune diabetes. Memory CD4⁺ T cells isolated from the blood of T1D patients (n = 11) and healthy controls (n = 11) were stimulated for 5 d with anti-CD3, anti-CD28, and IL-2 in the presence of various cytokines, and analyzed for intracellular expression of IL-9 and IL-17 by flow cytometry. A, The frequencies of IL-17⁺IL-9⁺, total IL-9⁺, and total IL-17⁺ cells are showed. B, The frequencies of IL-17⁺IL-9⁺ cells in diabetic memory CD4 T cells stimulated with TGF-β/IL-1β/IL-6/IL-21/IL-23 are represented versus the frequencies of total IL-9⁺ cells or total IL-17⁺ cells.