Fig 2: Depletion of CD90.1+Foxp3- IL-10 competent Tr1 cells does not influence long-term tolerance to airway allergens.(A) Schematic illustrating the protocol used in experiments A-E. CD90.1+ cells were depleted in the sensitization phase using an anti CD90.1 antibody as depicted. Control animals were given matched isotype. (B) Representative flow cytometry plots showing the efficiency of depletion of CD90.1+ CD4 T cells in the lungs. Frequency of CD90.1+Foxp3- and CD90.1+Foxp3+ cells within CD4+ T cells in lungs of isotype or anti CD90.1 treated mice are plotted. Error bars represent standard deviation of the mean. n = 4 for isotype group, n = 5 for anti-CD90.1 group. Statistical significance was determined using 2-way ANOVA (post hoc test: Sidaks). (C) The total number of cells and (D) total number of eosinophils in the BAL was determined. Each symbol represents a single animal. Error bars represent standard deviation of mean. (E) Lung CD4 cells from isotype or anti-CD90.1 treated mice were restimulated with PMA, Ionomycin and Brefeldin A, surface stained, fixed and permeabilized for detection of intracellular cytokines. The frequency of cells producing IL-13, IL-17, and IFN? within Foxp3- CD90.1- CD4+ T cells are shown. Error bars represent standard deviation of mean. n = 4 for isotype group, n = 5 for anti CD90.1 group. (F) Schematic illustrating the protocol used in experiments F-K. CD90.1+ cells were depleted using an anti CD90.1 antibody as depicted. Control animals were given matched isotype. (G) Representative flow cytometry plots showing the efficiency of depletion of CD90.1+ CD4 T cells in the lungs. Frequency of CD90.1+Foxp3- and CD90.1+Foxp3+ cells within CD4+ T cells in lungs of isotype or anti CD90.1 treated mice are plotted. Error bars represent standard deviation of the mean. n = 7 for isotype group, n = 5 for anti CD90.1 group. Statistical significance was determined using 2-way ANOVA (post hoc test: Sidaks). (H) The total number of cells and (I) total number of eosinophils in the BAL was determined. Each symbol represents a single animal. Error bars represent standard deviation of mean. (J) Lung CD4 cells from isotype or anti-CD90.1 treated mice were restimulated with PMA, Ionomycin and Brefeldin A, surface stained, fixed and permeabilized for detection of cytokines. The frequency of cells producing IL-13, IL-17, and IFN? within Foxp3- CD90.1- CD4+ T cells are shown. Error bars represent standard deviation of mean. n = 7 for isotype group, n = 5 for anti CD90.1 group. (K) The frequency of Tetramer + cells within CD90.1-Foxp3- and CD90.1+Foxp3- cells in the lungs of HDM-treated animals is plotted. Error bars represent standard deviation of the mean. n = 7 for isotype group, n = 5 for anti CD90.1 group. Data representative of at least two independent experiments.10.7554/eLife.44821.039Figure 9—source data 1.Depletion of CD90.1+Foxp3- IL-10 competent Tr1 cells does not influence long-term tolerance to airway allergens.

Fig 3: SFB- and C.-rodentium-Elicited Th17 Cells Have Different Cytokine Profiles(A–D) Representative intracellular staining for IL-17A and IL-22 (A) or IFN-? (C) and corresponding quantifications in (B) and (D) in Th17 cells from the SI of mice colonized with SFB and colon of mice infected with C. rodentium at 1 (n = 9 and 6), 2 (n = 11 and 6), and 4 (n = 14 and 9) weeks after gavage. Lamina propria cells were isolated at the indicated time point, re-stimulated with PMA and ionomycin and Brefeldin A for 2 h, and analyzed by FACS. Bars show the mean ± SEM for the indicated populations, and each symbol represents an individual mouse from two pooled independent experiments.(E) Cytokine concentration in supernatants of FACS-purified Th17 cells from the SI of mice colonized with SFB or colon of mice infected with C. rodentium. In the graphs, bars show the mean ± SEM, and each symbol represents a technical replicate. Cells were FACS-purified from one sample per group, obtained by pooling 3–6 mice. Results are representative of three independent experiments.(F) Fold-change (FC) induction of listed genes quantified by qPCR in the SI (green) and colon (orange) of mice colonized with SFB or infected with C. rodentium, 1 week after gavage. Fold change is calculated on the matching organs from untreated mice and gene expression is normalized against ß-2-microglobulin. Data are from two pooled independent experiments. *p < 0.05, **p < 0.01, ****p < 0.0001 by two-way ANOVA with Sidak’s post-test. Please see also Figure S2.

Fig 4: NO and HIF1a synergistically triggers IL-9 induction in Th9 cells.a–c Naïve CD4+ T cells from WT and Nos2-/- mice were differentiated to Th9 cells followed by a qPCR analysis of Spi1, Irf4, Gata3 and Batf expression. Data are representative of mean ± SEM from three independent experiments. b FACS staining for IL-9 and IL-17 in Th9 cells. c qPCR analysis of Hif1a expression. Data are representative of mean ± SEM from three independent experiments. d Bioinformatics analysis of HIF1a binding motif in Nos2 promoter followed by ChIP analysis of HIF1a binding to Nos2 promoter in Th9 cells represented as enrichment of HIF1a on Nos2 promoter relative to input. Data are representative of mean ± SEM from three independent experiments. e Luciferase assay for Nos2 promoter activity in the presence of HIF1a plasmid at 0, 50, and 100 ng concentrations. Data are representative of mean ± SEM from three independent experiments. f Naïve CD4+ T cells from WT and Hif1akd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 µg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. g Naïve CD4+ T cells from WT mice were differentiated into Th9 cells under normoxic (21% oxygen) or hypoxic (1% oxygen) conditions for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. h Naïve CD4+ T cells from WT and Phd2kd mice were differentiated under Th9 polarizing conditions with daily treatment of 1.0 µg/ml Dox for 3 days followed by qPCR analysis of Nos2 expression. Data are representative of mean ± SEM from three independent experiments. a, c ****P < 0.00001, *P = 0.01, using two-tailed unpaired Student’s t test. d **P = 0.0017, using two-tailed unpaired Student’s t test. e ****P < 0.00001, using one-way ANOVA followed by Tukey’s multiple comparison test. f *P = 0.01, using two-tailed unpaired Student’s t test. g *P = 0.04, using two-tailed unpaired Student’s t test. h **P = 0.0026, using two-tailed unpaired Student’s t test.

Fig 5: TLR2 signaling increases the proliferation of Th17(ß) cells but does not synergize with IL-1ß and IL-23(A) Representative flow cytometry analysis (IL-17 and IFN-?) of TLR2f/f Th17(IL-1/IL-23) cells following 4-day differentiation and transduction of control vector (RV-Mock) or Cre (RV-Cre).(B) Compilation of data from 2 representative experiments.(C–F) Representative proliferation histograms and pooled analysis of TLR2f/f Th17(ß) (C and D) or Th17(IL-1/IL-23) (E and F) cells following 4-day differentiation. n =3 technical replicates per group. Cells were transduced with RV-Mock or RV-Cre 1 day following initial TCR activation and gated on retrovirus (GFP+) for analysis. Data are representative of 3 individual experiments and are presented as means ± SDs. Comparisons between untreated and PAM3CSK4-stimulated cells are shown directly above the bars, while comparisons between RV-Mock and RV-Cre are shown by symbols above the lines. #p < 0.05, *p < 0.01, **p < 0.001, ***p <0.0001 by 2-way ANOVA.