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Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma  Chaoyu.

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Presentation on theme: "Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma  Chaoyu."— Presentation transcript:

1 Increased frequency of dual-positive TH2/TH17 cells in bronchoalveolar lavage fluid characterizes a population of patients with severe asthma  Chaoyu Irvin, MS, Iram Zafar, MS, James Good, MD, Donald Rollins, MD, Christina Christianson, PhD, Magdalena M. Gorska, MD, PhD, Richard J. Martin, MD, Rafeul Alam, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 5, Pages e7 (November 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Expression of TH2, TH17, and TH2/TH17 cells in BAL fluid. A-C, Representative flow cytograms from a TH2-dominant asthmatic patient. BAL cells from an asthmatic patient were gated as shown in Fig E1, A, and then analyzed for CD4 T cells and CD163+ macrophages (Fig 1, A). The CD4+CD163− cell population was then analyzed for expression of IL-17 and IL-4 (Fig 1, B) or IL-5 (Fig 1, C). D and E, Representative flow cytograms from a TH17predominant (patient with recurrent pulmonary aspiration) and TH2/TH17predominant (asthma) patient. BAL cells were processed as per Fig 1, A through C, and then analyzed for IL-4 and IL-17. F, A heat map of the frequency of IL-4+, IL-17+, and dual-positive IL-4/IL-17 CD4 T cells in BAL fluid from 52 asthmatic patients. Each row is a single BAL sample. The embedded number indicates the actual frequency of cells. Based on the dominance of the cell type, asthmatic patients can be divided into TH2predominant, TH2/TH17predominant, and TH2/TH17low. G and H, Expression of IL-4 and IL-17 (Fig 1, G) and coexpression of GATA3 and RORγt (Fig 1, H) in BAL cells by means of immunofluorescence staining. Cytospin preparations of BAL cells were double stained for IL-17 or GATA3 (green) and IL-4 or RORγt (red) and then counterstained with DAPI (blue) for nuclear staining. For GATA3/RORγt, Z-series images of a single lymphocyte were captured by using a confocal microscope. The images from a midsection show coexpression of GATA3 and RORγt in the nucleus of a single cell. Representative images from 3 separate experiments done with BAL cells from 3 different donors are shown. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 BAL TH2/TH17 cells and clinical correlations. A, Comparison of BAL dual-positive TH2/TH17 cells between asthmatic patients and disease control subjects (see the Results for description of disease control subjects). B, Correlation between TH2/TH17 and TH2 cells in BAL from the TH2/TH17 predominant subgroup. C, Correlation of BAL TH2, TH17, and TH2/TH17 cells and clinical parameters. Eos, Eosinophils; Lymph, lymphocytes. D, Comparison of BAL IL-17A levels between asthmatic patients and disease control subjects. E, Correlation between BAL IL-17A levels and FEV1. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Effect of dexamethasone on BAL cell expression of MKP1. BAL cells were cultured overnight (16 hours) with medium alone or dexamethasone (Dex; 10−7 mol/L). The expression of MKP1 in CD4high and CD4low T cells was assessed by using flow cytometry (A and B) and quantified (C and D). Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Effect of dexamethasone on BAL single-positive IL-4 and dual-positive IL-4/IL-17 CD4 T cells. BAL cells were cultured with medium or dexamethasone as in Fig 3 and then analyzed for expression of IL-4 and IL-17 by means of flow cytometry as per Fig 1. A-D, Representative flow cytograms from 2 patients, one with dominant IL-4+ cells (Fig 4, A and B) and the other with both IL-4 and dual-positive IL-4/IL-17 cells (Fig 4, C and D), are shown. E and F, Effect of dexamethasone (Dex) on TH2 and TH2/TH17 cells from 14 asthmatic patients. The numbers on the top of the graphs represent statistical significance. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Preferential expression of dual-positive TH2/TH17 cells in the BAL MEKhigh CD4 T-cell population. A and B, Sensitivity of MEKhigh and MEKlow CD4 populations to dexamethasone-induced cell death. Data are presented as percentages and absolute cell counts (in parentheses) per boxed area. C, Gating strategy for BAL CD4 T cells. D, Gating strategy for separation of MEKhigh and MEKlow cells in CD4-gated cells. E and F, Expression of dual-positive TH2/TH17 cells in MEKhigh and MEKlow BAL CD4 T-cell populations. G and H, Comparison of expression of dual-positive IL-4/IL-17 (Fig 5, G) and pSTAT3/pSTAT6 (Fig 5, H) cells in MEKhigh and MEKlow cell populations. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 TH2predominant, TH2/TH17predominant, and TH2/TH17low subgroups of asthmatic patients and their clinical features. A-C, All asthmatic patients (n = 52) were grouped based on the dominant expression of single-positive IL-4 cells (TH2predominant), dual-positive IL-4/IL-17 cells (TH2/TH17predominant), or 5% or less of either cell types (TH2/TH17low). D-F, Comparison of PC20 for methacholine, FEV1, and blood eosinophilia among the 3 subgroups. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 A, Forward scatter (FS) and side scatter (SS) display of BAL cells presented and analyzed in Fig 1, A through C. The boxed area shows the gating strategy for subsequent analysis. B, Correlation of IL-4 and IL-5 expression by BAL CD4 T cells, as presented in Fig 1, B and C. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 A-D, Flow cytograms of BAL cells stained with single (Fig E2, A-C) and double (Fig E2, D) isotype control antibodies. E, Immunofluorescence staining of a BAL lymphocyte with isotype control antibodies and DAPI. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 A, Forward scatter (FS) and side scatter (SS) display of BAL cells from a different asthmatic patient. The boxed area is gated for further analysis. B, Gated cells were analyzed for expression of CD4 (T-cell marker) and CD68 (macrophage marker). C, The same gated cells as in Fig E3, A, were also analyzed for CD3ε and CD4. D and E, CD3+CD4+ (Fig E3, D) and CD3+CD4− (Fig E3, E) T cells from the flow cytogram in Fig E3, C, were then analyzed for expression of IL-4 and IL-17. Representative flow cytograms from one of 4 different study subjects are shown. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Staining patterns of IL-4, IL-17, and IL-4/IL-17 in BAL CD4 T cells from select TH2predominant (A and B), TH2/TH17predominant (C and D), and TH17predominant (E and F) BAL donors. Flow cytograms presented in this figure are from asthmatic patients, and that in Fig E4, F, is from a patient with chronic pulmonary aspiration. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 A, Confocal Z-series images of a BAL lymphocyte immunofluorescently stained with DAPI and antibodies against GATA3 and RORγt. The bottom panel shows an overlay of the images. B, Images of BAL lymphocytes immunofluorescently stained with DAPI and antibodies against GATA3 and RORγt from 4 different donors. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E6 Coexpression of pSTAT3 and pSTAT6 in CD4 T cells from asthmatic patients. A and B, BAL cells were gated for CD4 T cells and then examined for coexpression of IL-4 and IL-17 (Fig E6, A) and pSTAT3 and pSTAT6 (Fig E6, B). C, Correlation between pSTAT3 and pSTAT6 expression in 9 asthmatic patients. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E7 Coexpression of CCR6 and CRTH2 in BAL CD4 T cells. A and B, BAL cells were immunostained for CD4, CCR6, CRTH2, IL-4, and IL-17. CD4 T cells were gated and analyzed for coexpression of CCR6 and CRTH2 or IL-4 and IL-17. C, Correlation of CCR6/CRTH2+ and IL-4/IL-17+ cells from 6 donors. Journal of Allergy and Clinical Immunology  , e7DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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