PU.1 promotes IL-9 production. T_H9 (a,c) or T_H2 (b,c) cultures were transduced with control (MIEG) or PU.1-expressing retroviruses. (a,b) EGFP-positive cells were sorted and stimulated to determine secretion of IL-9 (top) and Il9 expression (bottom). (c) IL-4 and IL-9 production was determined by intracellular staining in the EGFP+ populations of control and PU.1-transduced cells. Percentages in each quadrant are the average of results from 2-3 mice. Data are representative of 2-3 experiments.

PU.1 is required for IL-9 and chemokine expression in allergen sensitized mice. (a) RNA isolated from Ova-stimulated splenocytes as described in (Fig. 6a) was analyzed for expression of Il9 and chemokines associated with allergic inflammation. (b) T_H9 cultures were transduced with control (MIEG) or PU.1 expressing retroviruses. EGFP-positive cells were sorted and stimulated to determine expression of Ccl17 and Ccl22. (c) Total lung RNA was isolated from sensitized and challenged WT and Sfpi1^lck−/− mice, or WT non-challenged (NC) mice, and analyzed for Ccl17 and Ccl22 mRNA by qPCR. *, p<0.05 using two-tailed student t test.

IL-9 and IL-10 are not coordinately regulated in T_H9 cells. (a) Naïve CD4⁺ T cells were cultured under T_H2 or T_H9 conditions for five days before cells were left unstimulated or stimulated with plate-bound anti-CD3 for 6 or 24 hours and RNA was isolated for qPCR analysis of Il9 and Il10. (b) T_H2 and T_H9 cultures derived as in a were stimulated for 5 hours before intracellular cytokine analysis of IL-10 and IL-9. (c) Naïve CD4⁺ T cells were cultured under T_H9 conditions in the presence or absence of anti-IL-10. After five days in culture cells were restimulated with anti-CD3 and supernatants were analyzed for cytokines using ELISA. (d) Naïve CD4⁺ T cells were cultured under T_H9 conditions. After five days in culture, cells were restimulated with anti-CD3 in the presence or absence of TGF-β1 and supernatants were analyzed for cytokines using ELISA. (e) Wild-type and Sfpi1^lck−/− naïve CD4⁺ T cells were cultured under T_H9 conditions for five days, and stimulated with anti-CD3 for 24 hours before supernatants were tested for IL-10 using ELISA. Results are representative of at least three experiments.

Histone modifications at the Il9 locus. (a) VISTA plot of conserved non-coding sequences adjacent to the Il9 locus. (b,c) Naïve CD4⁺ T cells were isolated and analyzed directly or differentiated under T_H1, T_H2, T_H9, T_H17 or iTreg culture conditions for five days. Cells were analyzed for histone modifications using chromatin immunoprecipitation for the indicated histone modifications and primers for Il9 CNS. (d,e) Wild-type and Sfpi1^lck−/− naïve CD4⁺ T cells were cultured under T_H9 conditions and used for ChIP analysis as described in b performed for AcH3 at CNS1 and CNS2 (d), or for AcH4 and me3H3K27 at CNS1 (e). (f) Top, consensus PU.1 binding sites in the Il9 CNS1. Bottom, DAPA analysis of PU.1 binding from T_H2 or T_H9 extracts to site 2. For competition, extracts were incubated with a 5-fold excess of unlabelled PU.1 consensus double-stranded oligonucleotide. Expression of PU.1 in each cell extract is shown as input. Results are representative of three experiments. (g) Naïve CD4⁺ T cells were differentiated under T_H1, T_H2, or T_H9 culture conditions for five days. Cells were analyzed for PU.1 binding by ChIP with primers for Il9CNS1. Results of ChIP experiments are shown as average ± SD of percent input with control IgG subtracted (a-e) or with anti-PU.1 and IgG control ChIP values shown separately (g) and are representative of 2-4 experiments.

PU.1 promotes IL-9 production in human T cells. (a) Naïve human CD4⁺ T cells were cultured under T_H2 or T_H9 conditions and after five days in culture restimulated to analyze gene expression by qPCR. (b,c) T_H2 and T_H9 cultures were derived as in a and b supernatants from cells stimulated for 24 hours were tested for IL-9 production using ELISA or (c) cells were stimulated with anti-CD3 for 6 hours for analysis of IL-9 and IL-13 by intracellular cytokine staining. (d,e) Th9 cultures derived as in a were transfected with control or SPI1-specific siRNA. Twenty-four hours after transfection cells were analyzed for SPI1 expression by qPCR (d) and IL-9 production using ELISA (e). Results in (a-e) are representative data from 3-5 experiments with different donors. (f) PBMCs from infants (age 18-30 months) classified as atopic (n= 49) or non-atopic (n= 33) based on positive allergen-specific serum IgE, were stimulated with anti-CD3 for 48 hours and supernatants were tested for production of IL-9 using multiplex analysis. *, p<0.04 using an unpaired student t test.

PU.1 is required for optimal IL-9 production in murine T cells. Wild-type and Sfpi1^lck−/− naïve CD4⁺ T cells were cultured under T_H9 conditions. (a) After five days of culture, cells were stimulated with anti-CD3 and supernatants after 1, 2 or 3 days of stimulation were tested for IL-9 using ELISA. (b) RNA was isolated from wild-type and Sfpi1^lck−/− T_H2 and T_H9 cultures after stimulation with anti-CD3. Il9 mRNA was assessed using qPCR. (c) Wild-type and Sfpi1^lck−/– T_H9 cultures were stimulated with PMA-ionomycin for 5 h before intracellular staining for IL-9. Results in (a-c) are the average of 3 mice and representative of more than four experiments. (d) Naïve CD4⁺ T cells cultured under T_H1, T_H2, T_H9 or T_H17 conditions were analyzed for Sfpi1 expression using qPCR. Results are an average of 3-5 experiments. (e) Naïve CD4⁺ T cells were cultured under T_H9 or iTreg conditions for five days and stimulated with anti-CD3 for 6 h before intracellular staining for IL-9 and Foxp3. (f,g) Naïve CD4⁺ T cells were cultured under T_H9 or T_H17 conditions for five days and stimulated with (f) PMA-ionomycin for 6 h before intracellular staining for IL-9 and IL-17 or (g) anti-CD3 for 24 h before supernatants were collected for analysis using ELISA. Results in (e-g) are representative of at least three experiments. (h) T_H2 cultures were separated into IL-4^lo and IL-4^hi populations using magnetic selection. RNA was isolated from separated cells to examine expression of Il4, Il9 and Sfpi1. Results are an average of three experiments. *, P < 0.05 using two-tailed student t test.

PU.1 expression in T cells is required for the development of allergic inflammation. Wild-type and Sfpi1^lck−/− mice were sensitized and challenged intranasally with ovalbumin. Forty-eight hours after the last intranasal challenge mice were sacrificed for analysis. (a) Splenocytes were stimulated with anti-CD3 (left) or Ova (right) for 72 h and supernatants were tested for the amounts of T_H2 cytokines by ELISA. (b) Lungs of challenged (C) or non-challenged (NC) mice were embedded in paraffin and analyzed by H/E staining. Magnification is indicated in the panel. (c) Wild-type and Sfpi1^lck−/− mice were analyzed for airway hyperreactivity using whole body plethysmography 24 h after the last intranasal challenge. Airway function was tested at baseline (B), with saline inhalation (S) and following inhalation of the indicated doses of methacholine. (d) Numbers of cells recovered by bronchoalveolar lavage in challenged or non-challenged mice (left). Numbers of specific cell types in BAL from challenged mice, as determined by flow cytometry, are indicated (right). (e) Amounts of IL-9 and T_H2 cytokines present in BAL fluid were determined using ELISA. (f, g) Wild type mice were sensitized and challenged intranasally with ovalbumin daily for five days. Mice were injected intravenously with control Ig or anti-IL-9, 30 minutes before the first, third and fifth intranasal challenge. Forty-eight hours after the last intranasal challenge, lungs were processed for histological analysis (f) as in b, and BAL cells were harvested and analyzed (g) as in d. *, p<0.05 using two-tailed student t test. All results are the average ± SEM of 5-7 mice and are representative of three experiments.