Fig 2: Tr1-like cells contribute to allergen-specific memory T-cells in the lung.(A) Mice were administered either PBS as control or crude HDM intranasally (i.n.) Six times over 2 weeks as shown. The mice were then left unchallenged until day 67 when both PBS (control) and HDM-sensitized animals were challenged with HDM. Analysis was done one day after the rechallenge at day 68. (B) The total number of cells in the BAL was determined. Data are pooled from two experiments. Each symbol represents a single animal. Error bars represent standard error of mean. n=7 for control group, n=13 for HDM group. Statistical significance was determined using an unpaired two tailed students t test. (C) The total number of eosinophils in the BAL is also plotted. Data are pooled from two experiments. Each symbol represents a single animal. Error bars represent standard error of mean. n=7 for control group, n=13 for HDM group. Statistical significance was determined using an unpaired two tailed students t test. **P=0.01, ***P=0.001. (D) The level of IgE in BAL supernatant was determined by ELISA. n=8 for control group n=16 for HDM group. (E) Representative flow cytometry plot showing all IL-10-producing cells within CD4+ T cells in lungs of control or HDM-treated animals at day 68. (F) Composition of IL-10-producing cells is shown by plotting the frequency of indicated cell types within all IL-10-producing (CD90.1+) cells. Data are pooled from two experiments. Each column is an individual animal. (G) Gating scheme showing the HDM specific T cells stained using Derp1 tetramer and the different T cell subsets that constitute the tetramer + CD4+ T cells in the lungs of HDM-treated animals. The frequency of IL-10-producing Foxp3- (CD90.1+Foxp3-), IL-10-producing Foxp3+ (CD90.1+Foxp3+), Foxp3+ cells which do not produce IL-10 (CD90.1-Foxp3+) and CD90.1-Foxp3- within all CD4+ T cells is plotted. (H) 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. (I) PMA, Ionomycin and Brefeldin stimulated CD4 T cells from lungs of HDM-treated mice were surface stained, fixed and permeabilized for detection of cytokines. The frequency of cells producing IL-4, IL-13, IL-10, IFN? and IL-17 within CD90.1+ Foxp3- (Tr1-like) CD4+ T cells is shown. Data are pooled from two experiments. N is between 4-13 mice per cytokine (J). The frequency of cells expressing transcription factors Tbet and GATA3 the within CD90.1+ Foxp3- (Tr1-like) CD4+ T cells is shown. Error bars represent standard deviation of mean. n=13. Data representative of three independent experiments.10.7554/eLife.44821.027Figure 7—source data 1.Tr1-like cells contribute to allergen-specific memory T-cells in the lung.

Fig 3: 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 4: miR-AB-based multicolor-barcoded multiplex RNAiCD8+ T cells were activated for 16 h and then cotransduced with miR-AB retroviruses at 1:1 ratio, which carries a SplashRNA-designed shRNAmir, targeting surface protein CD127, CD90, CD44, or CD8, with Azurite, GFP, Ametrine, or mCherry2 as fluorescent reporters, respectively, for 4 h. miR-AB retroviruses expressing CD19-specific shRNAmirs with same fluorescent reporters were used as controls. Surface staining was performed 72 h after transduction. FACS data were shown as overlaid histograms of control shRNAmirs (shCD19s) and target shRNAmirs (shCD127, shCD90, shCD44, or shCD8). Cells were categorized as 16 populations expressing single, double, triple, or quadruple fluorescent proteins or not (none) and fluorescent reporter-positive plots were highlighted with thick frames and white background. Each population’s surface staining profile was shown on the right. The data are representative of two biologically independent experiments.

Fig 5: Active IL-10 production is associated with Irf1 and Batf expression.CD90.1- and CD90.1+ CD4 T cells were isolated from 10BiT spleens and cultured (A) unstimulated in plain media or (B) with CD3/CD28 stimulation for 5 days to assess kinetics of Thy1.1 surface expression and intracellular IL-10 cytokine staining. (C) Irf1 expression and (D) Batf expression in indicated subsets obtained from in vitro differentiated Tr1 cell cultures. Data was normalized to beta actin as reference gene and is expressed as fold change over 90.1-IL-10- cells using delta Ct method. Expression data are pooled from five independent experiments.10.7554/eLife.44821.021Figure 6—source data 1.Active IL-10 production is associated with Irf1 and Batf expression.