Fig 2: JAK3 phosphorylation is dependent on the expression of IL2RG, and associated with the secretion of IL-2 and IL-21. ITK-SYK+ Jurkat cells were transfected with siIL2RG or si-control for 48 h. (A) Western blot analysis of IL2RG and SYK. (B) Exogenous IL-2 (25 ng/ml) or IL-21 (25 ng/ml) was added to the Jurkat cells for 24 h. Cell lysates of si-control and IL2RG-knockdown Jurkat cells, and IL2RG-knockdown Jurkat cells treated with IL-2 or IL-21 were evaluated with western blotting analysis for the expression levels of JAK3, p-JAK3, STAT5 and p-STAT5. Densitometric analysis of the western blotting bands is shown in the graph on the right. Data is expressed as the mean ± SEM of the phosphorylated/total protein ratio. Data is expressed as phospho/total protein ratio. (C) Growth curves in siIL2RG and si-control cells by cell counting. The results are expressed as the mean ± SD of five independent experiments. (D) Flow cytometry analysis with Annexin V-PI staining was performed to evaluate the percentage of apoptotic cells between siIL2RG and si-control cells; the results are quantified in the graph on the right. (E-G) The transduced and transfected Jurkat cells were cultured for 24 h. (E) Cleaved caspase-3 and full-length caspase-3 were evaluated with western blotting analysis. (F) The cells were fixed and stained with PI. Cell cycle profiles were assessed using flow cytometry; the results are quantified in the right panel. (G) p27 and CDK2 expression levels were evaluated with western blotting analysis. (H) The protein concentrations of cytokines (IL-2, IL-4, IL-7, IL-9, IL-15, IL-21) were determined by ELISA (n=3). Quantification of the ELISAs is shown in the panel on the right. (I) Western blotting analysed the expression levels of IL2R, IL7R and IL21R. β-actin was used as the loading control. PI, propidium iodide; AV, Annexin V; IL, interleukin, IL2RG, interleukin 2 receptor γ; ITK-SYK, IL-2-inducible T-cell kinase-spleen tyrosine kinase; JAK, tyrosine-protein kinase JAK; p27, cyclin-dependent kinase inhibitor 1B; CDK2, cyclin-dependent kinase 2; NS, not significant; si-control, scrambled siRNA; si or siRNA, small interfering RNA. *P<0.05 and **P<0.01.

Fig 3: Increased IL-9 production by T cells in the tumoral lung region of NSCLC patients. (A) Representative photographic image of the resected lung of one NSCLC patient as a showcase of defined regions implemented for our human study cohort. Lung tissue samples were dissected from the tumoral area (TU), the peri-tumoral area (PT) surrounding the tumor and from the control area (CTR) consisting out of healthy lung tissue. (B) IL-9 immunohistochemistry (IHC) on paraffin-embedded tissue arrays obtained from the lung of tumor-free control patients (CN) or the CTR and TU lung regions of non-small cell lung cancer (NSCLC) patients (nCN=10; nCTR=15; nTU=17) (scale bar=50 µm). (C, D) Proteins were isolated from lung tissue samples of control patients (CN), the CTR and the TU of NSCLC ADC patients (classified by grading of tumor cell differentiation (G1, G2 and G3) and Western blot was performed. Detected protein levels of IL-9 were normalized on total protein of the samples (CN: n=3, CTR: n=10, TU:n=10). (E) Proteins were isolated from tissue samples of control patients that underwent surgery due to disease unrelated to tumor and from cells of the adenocarcinoma cell line A549. Analysed data are shown as mean ± SEM (nCN=1; nLu-Infl (Lung inflammation)=1; CTR-Lu=CTR, n=2; nA549 = 1) or single values. (F) Flow cytometry analysis of IL-9+ cells (shown in percentage) gated on Epcam+ cells of cells isolated from the CTR, the PT and the TU of a NSCLC patient. (G) Double IHC for IL-9 (brown) and CD3 (blue) on lung tissue obtained from the CTR and TU regions of an adenocarcinoma (ADC) patient (scale bar=50 µm). (H) ELISA analysis of IL-9 levels (pg/ml), (I) IL-21 levels (pg/ml), (J) IFN? (pg/ml) in supernatants obtained from total cells isolated from the control (CTR), peri-tumoral (PT) and tumoral (TU) region of adenocarcinoma (ADC) patients and cultured with anti(a)-CD3/CD28 antibodies for 24h (IL-21: nCTR=6, nPT=4, nTU=5; IFN?: nCTR=3; nPT=3; nTU=3). (K) TNF-alpha mRNA level from total lung cells was normalized on HPRT mRNA level (nCTR=12, nPT=11, nTU=12). N values are given per group. Bar charts indicate mean values +/- s.e.m. using student´s two-tailed t-test *P,0.05; **P,0.01; ***P,0.001.

Fig 4: Anti-IL9 antibody treatment inhibits lung tumor development. (A) Experimental design: BL/6 WT mice were intravenously injected with LL/2 cells on day 0. Mice were then treated intraperitoneally (i.p.) with anti (a)-IL9 antibody or IgG2a isotype control on days 1, 3, 6, 9, 13 and 16 after tumor induction. Overall survival rate of aIL9 antibody or IgG2a isotype control treated mice. Survival rate on the right handside. (B) Monitoring of body weight changes of BL/6 WT mice injected i.v. with LL/2 cells). (C) Tumor load was analyzed via bioluminescence-based imaging system on the indicated days. The experiment ended on day 23. Representative in vivo images of lung tumor load analysis at days 14, 17, 20 and 22 in WT mice treated with aIL9 antibody or IgG2a isotype control (representative of 3 independent experiments) are shown. and lung tumor load (Total flux=photons/second) (Day10-20 nIgG2a+LL/2 = 5, naIL-9+LL/2 = 5; day22 nIgG2a+LL/2 = 5, naIL-9+LL/2 = 4) were quantified. (D) Representative H&E staining of WT mice treated with aIL9 antibody or IgG2a isotype control (scale bar=200 µm). (E) Flow cytometry analysis of TNFa positive cells (%) gated on Tbet and CD4 co-expressing cells (nnaive=3; nIgG2a+LL/2 = 5; naIL-9+LL/2 = 5). (F) Flow cytometry analysis of TNFa positive cells (%) gated on Tbet and CD8 co-expressing cells (nnaive=3; nIgG2a+LL/2 = 5; naIL-9+LL/2 = 5). (G) Experimental design is similar to the A549 cell line culture whereby LL/2 were pre-cultured for 2 days of IL9 (100 ng/ml) and then further cultured for 2 days unstimulated or cultured with either IFN?, or IL9, IL21 or TNFa, respectively. Annexin V(AnnV)/PI flow cytometry analysis of IL9 pre-treated LL/2 cells at the end of the cell culture, A representative dot plot is showed for each group. The data were statistically analyzed with one-way ANOVA, and showed with mean ± SEM. n=3 per group. *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: Targeted deletion of IL-9 resulted in lung tumor rejection Balb/c syngenic mice. (A) Experimental design for the induction of lung tumor development in Balb/c wild-type (WT) and Balb/c IL9-/- mice. Tumor growth was induced via intravenous (i.v.) injection of L1C2 lung carcinoma cells. The experiment ended on day 14. (B) Body weight changes of Balb/c IL9-/- mice and Balb/c control littermates (injected i.v. with/out L1C2 cells) 0 to 14 days after tumor induction (nIL9-/-=4; nbalb/c L1C2 = 3; nbalb/c naive=2). (C) Representative H&E staining of the lung of WT and IL-9-/- mice (top, scale bar=100 µm; bottom, scale bar=50 µm) and (D, E) additional mice were evaluated by a pathologist in a blind manner (n=4 per group). Circles represent mice per group tumor free (sectors in white) and mice with lung bearing tumor (sectors in grey). (F) FACS analysis of Foxp3+ CD25+ T regulatory cells in total lung cells of 4 wt and 4 IL9 ko mice after gating on CD3+CD4+ T cells. (G) Experimental design is similar to the A549 cell line culture whereby L1C2 were pre-cultured or not for 2 days of IL9 (100 ng/ml) and then further cultured for 2 days unstimulated or cultured with either IFN?, or IL9, IL21 or TNFa, respectively. The data were statistically analyzed with two-way ANOVA, and showed with mean ± SEM. n=3 per group. *, P<0.05; **, P<0.01; ***, P<0.001; ****, P<0.0001. (H) Flow cytometry analysis of the percentage of Ki67 expressing L1C2 cells after stimulation with or without IL-9 for 24h and representative histograms (n-unst. = 3; n30 ng/ml=3; n100 ng/ml=3). Annexin V/PI flow cytometry analysis of L1C2 cells after stimulation with or without IL-9 for 24h and representative dot plots (n-unst. = 3; n30 ng/ml=3; n100 ng/ml=3). Bar charts indicate mean values ± SEM using student´s two-tailed t-test (b-g) or ordinary one-way ANOVA (i-j) (*p < 0.05, **p < 0.01, ***p < 0.001).