Fig 2: EMT–MET PHENOSTAMP serves as a means to assess EMT–MET states in independent NSCLC cell lines.a Projection of NSCLC cell lines onto the EMT–MET PHENOSTAMP (top) and their respective morphological assessment (×10 images, scale bar 200 µm, bottom). HCC827 samples (ctrl, 2-day TGFß) were ran on a different day than the ones used for the map construction for validation (left). A549 and H3255 cells were treated with TGFß for 10 and 13 days, respectively, and analyzed with mass cytometry. b Shown are the respective to each cell line and condition expression profiles of E-Cadherin (top) and Vimentin (bottom) visualized on the EMT–MET PHENOSTAMP. c E-Cadherin/Vimentin and CD44/CD24 mass cytometry plots per projected cell line sample. See Methods and Supplementary Fig. 8 for further details.

Fig 3: A prodrug of selective STAT3 inhibitor suppresses Th17 development.(A) Chemical structure of LLL12b. (B) Splenocytes from naive Va2.3/Vß8.2 TCR transgenic mice that are specific for MBP Ac1-11 were activated with MBP Ac1-11 plus TGF-ß1/IL-6 for 3 days with different concentrations of LLL12b or LLL12. DMSO was used as vehicle control. IL-17 was determined by intracellular staining, gating on CD4+CD44+ T cells. (C and D) Purified CD4+ T cells were activated with aCD3/CD28 plus TGF-ß1/IL-6 for 3 days with different concentrations of LLL12b or vehicle control DMSO. IL-17 and ROR?t were determined by intracellular staining, gating on CD44+CD4+ cells, and compared with 1-way ANOVA (control, n = 16; 0.125 µM LLL12b, n = 5; 0.25 µM LLL12b, n = 8). (E) Adherent cells from spleens of WT/B6 mice were cultured with different concentrations of LLL12b or vehicle control DMSO for 4–5 hours. The cells were then cultured with purified CD4+ T cells from TCR transgenic 2D2 mice that are specific for MOG 35-55 for 3 days, in the presence of MOG 35-55 and TGF-ß/IL-6. IL-17 and ROR?t were determined by intracellular staining, gating on CD44+CD4+ cells, and compared with 1-way ANOVA (control, n = 10; 0.125 µM LLL12b, n = 7; 0.5 µM LLL12b, n = 7). Data are represented as mean ± SEM of 3–5 independent experiments. **P < 0.01; ****P < 0.0001.

Fig 4: LLL12b suppresses IL-23–induced expansion of Th17 cells from EAE mice.(A and B) Splenocytes from immunized SJL/J mice were activated with PLP 139-151 plus IL-23 for 30’ (A) or 3 days (B) with different concentrations of LLL12b. DMSO was used as vehicle control. (A) pSTAT3 was determined by phospho flow cytometry, gating on CD4+ cells, and compared with 1-way ANOVA (n = 3). (B) IL-17 was determined by intracellular staining, gating on CD4+CD44+ T cells. Group means were compared with the control group by 1-way ANOVA (n = 3). (C) Dose response curve of LLL12b concentration and normalized inhibition of % IL-17+CD4+ T cells. Data are represented as mean ± SEM of 3 independent experiments. **P < 0.01; ***P < 0.001; ****P < 0.0001.

Fig 5: IL-10 production by Tr1-like cells is transient and wanes after peak of inflammation.(A) 10BiT reporter mice on a C57Bl/6 background were administered PBS as control or HDM and analysis was performed on groups of mice at the indicated times shown. (B) The total number of cells and (C) total number of eosinophils in the BAL were determined. n=3-5 animals per group per time point. Error bars represent standard deviation of the mean. (D) Representative flow cytometry plots showing the kinetics of IL-10-producing T cell subsets as identified by surface expression of CD90.1 and Foxp3+ regulatory T cell subsets as identified by GFP expression. The frequency of (E) IL-10-producing Foxp3- (CD90.1+Foxp3-), (F) IL-10-producing Foxp3+ (CD90.1+Foxp3+) within all CD4+ T cells is plotted at indicated analysis days. n=3-5 animals per group per time point. Error bars represent standard deviation of the mean. Statistical significance was determined using 2-way ANOVA (post hoc test: Sidaks). (G) Cells from lungs of HDM- treated mice obtained after Day 26 and day 30 were stimulated with PMA, Ionomycin and Brefeldin and surface stained, fixed and permeabilized for detection of cytokines. The frequency of CD90.1+Foxp3- T cells producing IL-10, IFN?, IL-13 and IL-17 is shown for indicated time points. (H) To identify lung resident T cells, intravascular (IV) labeling of cells was performed by retroorbital injection of CD45 antibody and mice were euthanized 2 minutes after injection. The ratio of resident to circulating cells within indicated CD4+ T cells from lungs of HDM-treated mice obtained at day 30 post first challenge is plotted. (I) The frequency of effector (TEM) (CD62l-CD44+), central (TCM) (CD62l+CD44+), and naïve subsets(CD62L+CD44-) within CD90.1+Foxp3- cells in lungs at day 30 post first allergen challenge is plotted. Data representative of three independent experiments ***P=0.001, ****P=0.0001 GFP= green fluorescent protein. PMA= phorbol myristate acetate, IFN?= Interferon gamma.10.7554/eLife.44821.017Figure 5—source data 1.IL-10 production by Tr1-like cells is transient and wanes after peak of inflammation.