Itk is required for Th9 differentiation via TCR-mediated induction of IL-2 and IRF4

Abstract

Th9 cells produce interleukin (IL)-9, a cytokine implicated in allergic asthma and autoimmunity. Here we show that Itk, a mediator of T cell receptor signalling required for Th2 immune responses and the development of asthma, is a positive regulator of Th9 differentiation. In a model of allergic lung disease, Itk-deficient mice show reduced pulmonary inflammation and IL-9 production by T cells and innate lymphoid type 2 cells (ILC2), despite normal early induction of ILC2s. In vitro, Itk(-/-) CD4(+) T cells do not produce IL-9 and have reduced levels of IRF4 (Interferon Regulator Factor 4), a critical transcription factor for effector T cell function. Both IL-9 and IRF4 expression are rescued by either IL-2 or constitutively active STAT5, but not NFATc1. STAT5 binds the Irf4 promoter, demonstrating one mechanism by which IL-2 rescues weakly activated T cells. Itk inhibition also reduces IL-9 expression by human T cells, implicating ITK as a key regulator of Th9 induction.

Figure 1. Itk is required for Th9 differentiation.

(a–d) Sorted naive CD4⁺ T cells from WT and Itk^−/− mice were differentiated under Th9 conditions (1 μg ml⁻¹ anti-CD3, 3 μg ml⁻¹ anti-CD28, 20 ng ml⁻¹ IL-4, 5 ng ml⁻¹ TGFβ1 with or without 10 ng ml⁻¹ TL1A, in presence of T-depleted splenocytes as APCs) for 3 days, (a) cells were restimulated with PMA and Ionomycin and IL-9 and IFN-γ production were analysed by intracellular staining. Representative flow plots from one out of 10 experiments. (b) Mean±s.e.m. of 10 independent experiments **P<0.01, using two-tailed unpaired Student's t-test. (c) IL-9 was determined by Luminex in supernatants from cells differentiated as in a, before restimulation. Mean±s.e.m of three independent experiments are shown, **P<0.01, using two-tailed unpaired Student's t-test. (d) mRNA of Il9 of cells differentiated as in a were determined by qRT-PCR. (e) Sorted naive CD4⁺ T cells were stained with CSFE, differentiated and stained as in a. (f) Sorted naïve CD4⁺ T cells from WT and Itk^−/− mice were differentiated under Th9 conditions plus TL1A, Th17 or Th2 conditions for 3 days in presence of increasing concentrations of Itk inhibitor BMS-509744 as indicated in the figure. Cells were restimulated with PMA and Ionomycin and stained for IL-9, IL-17A, IL-4 and IFN-γ. Results in panels d,e and f are representative from one of three independent experiments.

Figure 2. IL-9 expression correlates with strength of TCR signals.

(a) Sorted naive WT CD4⁺ T cells were differentiated under Th9 conditions plus TL1A with 0.01, 0.1 or 1 μg ml⁻¹ of anti-CD3, then restimulated with PMA and Ionomycin and IL-9 production analysed by intracellular staining. (b) Itk-deficient CD4⁺ T cells were transduced with retroviruses expressing constitutively active NFATc1 (ca-NFATc1), or a control (MIGR), differentiated under Th9 plus TL1A or Th17 conditions, and cytokine production determined by intracellular staining after PMA and ionomycin restimulation. Results in a and b are from one representative of three independent experiments.

Figure 3. Itk is required for IRF4 expression in CD4⁺ T cells.

(a) Sorted naive WT CD4⁺ T cells were differentiated under Th9 plus TL1A conditions, with 0.01, 0.1 or 1 μg ml⁻¹ of anti-CD3, and then stained for intracellular IRF4 (MFI is indicated). (b) Intracellular levels of IRF4 from WT and Itk^−/− CD4⁺ T cells differentiated under Th9 plus TL1A for 3 days. (c) mRNAs levels of Irf4 and Maf or Spi1, Batf and Gata3 were determined by qRT-PCR on cells differentiated as in b. Data in a-c are representative of one of at least three independent experiments.

Figure 4. IL-2 rescues Th9 differentiation in Itk^−/− CD4⁺ T cells.

(a-c) Sorted naïve CD4⁺ T cells from WT and Itk^−/− mice were differentiated under Th9 conditions plus TL1A for 3 days and pSTAT5 (a) and CD25 (b) were determined by flow cytometry: WT (black), Itk^−/− (grey) lines. (c) Sorted naive CD4⁺ T cells were differentiated as in a or under Th17 conditions and IL-2 in supernatants were determined at 48 and 72 h by Luminex. Th9 conditions: black bars. Th17 conditions: hatched bars. (d,e) Sorted naïve CD4⁺ T cells were differentiated for 3 days under Th9 (d) or Th9 plus TL1A (e) conditions in absence or presence of blocking anti-IL-2 plus hIL-2, restimulated with PMA and Ionomycin and IL-9 analysed by flow cytometry. (f) Sorted naive CD4⁺ T cells from WT and Itk^−/− mice were stained with CSFE, differentiated, and restimulated with PMA and Ionomycin to evaluate IL-9 expression. Data in a-f are representative of one out of at least three independent experiments.

Figure 5. IL-2 rescues pSTAT5 and pS6 in Itk^−/− CD4 T cells.

(a-c) Sorted naive CD4⁺ T cells from WT and Itk^−/− mice were differentiated for 3 days under Th9 or Th9 plus TL1A conditions in absence or presence of blocking anti-IL-2 plus hIL-2 and analysed for pSTAT5 (MFI are indicated) (a) CD25 (b) and pS6 (c). (d) Itk-deficient CD4⁺ T cells were transduced with control (MIGR) or constitutively active STAT5-expressing retroviruses, differentiated under Th9 plus TL1A conditions and IL-9 production determined by intracellular staining after PMA and Ionomycin restimulation. Results in a-d are representative of one out of at least 3 experiments.

Figure 6. IL-2 rescues IRF4 expression in Itk^−/− CD4 T cells.

(a) Sorted naive CD4⁺ T cells differentiated for 3 days under Th9 plus TL1A in absence or presence of blocking anti-IL-2 plus hIL-2, were stained for IRF4 and analysed by flow cytometry. (b) mRNA of Irf4 and Maf in cells differentiated as in a evaluated by qRT-PCR. (c) Itk-deficient CD4⁺ T cells were transduced with constitutively active STAT5 (ca-STAT5) or control retroviruses, differentiated under Th9 plus TL1A conditions and intracellular IRF4 determined. (d) Sorted naive WT CD4⁺ T cells were differentiated under Th9 plus TL1A with 0.003 or 0.01 μg ml⁻¹ of anti-CD3, in absence or presence of blocking anti-IL-2 or blocking anti-IL-2 plus hIL-2, then restimulated with PMA and Ionomycin and IL-9 and IRF4 production were analysed by intracellular staining, MFI values for IRF4 are indicated. Data in figures a-d are representative examples from one of at least three independent experiments.

Figure 7. STAT5 binds to the IRF4 promoter.

(a) A cross-species conserved STAT5-binding site in the promoter region of Irf4 gene as predicted by the Mulan software at NCBI DCODE. (b,c) ChIP analysis of Th9 differentiated cells using anti-STAT5 (b), or anti-acetyl Histone H3 (c) antibodies and amplifying a region in the Irf4 promoter ∼1.5 kb from the annotated first exon. (d) ChIP analysis using anti-STAT5 mouse antibodies were performed on Il9 promoter. Data in b-d are representative of one out of two independent experiments.

Figure 8. Itk-deficient mice are resistant to papain-induced lung inflammation.

(a) Outline of papain-induced sensitization. (b) PAS-stained sections of lungs harvested at day 14 from WT and Itk^−/− mice challenged with papain or PBS, airways (aw) and blood vessels (bv) are marked. Scale bar, 50 μm. (c) Absolute numbers of macrophages, neutrophils and eosinophils in the BAL of mice treated with papain or PBS. (d) Airway resistance was measured in response to increasing doses of aerosolized methacholine in mice challenged with papain or PBS. **P<0.01 by two-way ANOVA. (e) Numbers of IL-9, IL-4 and IL-13–producing CD4⁺CD44^hi T cells in lungs are shown from cells harvested at day 14 from mice challenged with papain or PBS. (f) Numbers of activated (CD44^hi) CD4⁺ cells in lungs after papain or PBS treatment. (g,h) mRNA levels of Il9 (g) and Il2 (h) measured by quantitative RT-PCR are shown for lung samples harvested on day 14 after initial challenge with papain or PBS. Values were normalized to the average level of Il9 mRNA in the PBS-treated WT mice. Experiments were performed at least twice, using six female mice for papain-treated group and four female mice for PBS controls. Mean±s.e.m., *P<0.05, **P<0.01 using Mann–Whitney test (c,e,f).

Figure 9. Itk-deficient mice have late defects in ILC2 function.

(a) Outline of 14day papain-induced sensitization. (b) Numbers of ILC2⁺, IL-9- and IL-13-ILC2⁺ producing cells in lungs are shown from cells harvested at day 14 from female mice challenged with papain. (c) Outline of papain sensitization of female mice for 4 days. (d) Numbers of ILC2⁺ and IL-9-producing ILC2⁺ cells from mice treated with papain on day 4. (e) Outline of IL-33-induced sensitization for 4 days. (f) Numbers of ILC2⁺ cells in lungs harvested at day 4 from mice challenged with IL-33. (g) ILC2⁺ cells were sorted from IL-33-treated mice, cultured with 10 μg ml⁻¹ of the indicated cytokines for 3 days and IL-9, IL-5 and IL-13 was measured in the culture supernatants (IL-9 and IL-5 have the same scale for Y axis). Experiments were performed at least twice using six female mice for papain-treated or IL-33 groups and four mice for PBS controls. (b,d,f) Mean±s.e.m. from one representative experiment, *P<0.05, **P<0.01 using Mann–Whitney test.

Figure 10. ITK is required for Th9 differentiation of human cells.

Purified naive human CD4⁺ T cells were differentiated under Th9 conditions for 5 days in presence of increasing concentrations of ITK inhibitor. Cells were restimulated with PMA and Ionomycin and stained for IL-9. Data are representative of one of two independent experiments.