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IL-10, TGF-β, and glucocorticoid prevent the production of type 2 cytokines in human group 2 innate lymphoid cells  Noriko Ogasawara, MD, PhD, Julie A.

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Presentation on theme: "IL-10, TGF-β, and glucocorticoid prevent the production of type 2 cytokines in human group 2 innate lymphoid cells  Noriko Ogasawara, MD, PhD, Julie A."— Presentation transcript:

1 IL-10, TGF-β, and glucocorticoid prevent the production of type 2 cytokines in human group 2 innate lymphoid cells  Noriko Ogasawara, MD, PhD, Julie A. Poposki, MS, Aiko I. Klingler, PhD, Bruce K. Tan, MD, MS, Ava R. Weibman, BA, Kathryn E. Hulse, PhD, Whitney W. Stevens, MD, PhD, Anju T. Peters, MD, Leslie C. Grammer, MD, Robert P. Schleimer, PhD, Kevin C. Welch, MD, Stephanie S. Smith, MD, David B. Conley, MD, Joseph R. Raviv, MD, Pejman Soroosh, PhD, Omid Akbari, PhD, Tetsuo Himi, MD, PhD, Robert C. Kern, MD, Atsushi Kato, PhD  Journal of Allergy and Clinical Immunology  Volume 141, Issue 3, Pages e8 (March 2018) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Presence of receptors for IL-10 and TGF-β on human ILC2. Representative histograms of flow cytometric plots for IL-10RA (A) and TGFBR2 (B) in ILC2s from a blood sample and an NP are shown. Levels of cell surface expression of receptors on ILC2s from blood (n = 10) and NPs (n = 10) are shown by geometric mean fluorescence intensity (gMFI). PBMCs were stimulated with medium control (filled), 1 ng/mL IL-10 (dashed line), or 10 (solid) ng/mL IL-10 for 15 minutes. The level of intracellular phospho-STAT3 in blood ILC2s was detected by flow cytometry (C, n = 6). APC, Antigen-presenting cell. *P < .05, **P < .01, ****P < .0001, by the paired t test (Fig 1, A and B) and 1-way ANOVA (Fig 1, C). Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 IL-10, TGF-β, and dexamethasone (Dex) inhibit activation of human ILC2. Sorted blood ILC2s were cultured with 10 ng/mL IL-33 and 10 ng/mL TSLP in the presence or absence of 10 ng/mL IL-10 (B, n = 8), 20 ng/mL TGF-β1 (C, n = 6), 0.01% DMSO (vehicle control), and 100 nM Dex (D, n = 6) for 4 days. The morphology of ILC2s was examined by cytospin with Diff-Quik staining (A). The concentrations of IL-4, IL-5, IL-9, and IL-13 were measured by using Luminex (Fig 2, B-D). Cont, Control; DMSO, dimethyl sulfoxide; NS, not significant. *P < .05, **P < .01, ***P < .001, ****P < .0001, by 1-way ANOVA. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Gating strategy to identify ILC2s in human peripheral blood. Representative flow cytometric plots for ILC2s in peripheral blood. We gated on single, live CD45-positive cells and excluded granulocytes (side scatter high). We then assessed ILC2s in the lineage-negative population (CD3, CD11c, CD14, CD16, CD19, CD20, CD56, FcεRIα, CD303) as CD127, CRTH2, and CD161 triple-positive cells. FSC-A, Forward scatter-area; FSC-W, forward scatter-width; SSC-A, side scatter-area; SSC-W, side scatter-width. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Presence of IL-10 receptor on human ILC2s. Representative histograms of flow cytometric plots for IL-10RA (A) and IL-10RB (A-C) in ILC2s from a tonsil (Fig E2, A), a blood sample (Fig E2, B), and an NP (C). Levels of cell surface expression of IL-10 receptors on ILC2s from tonsils (Fig E2, A, n = 6), blood (Fig E2, B, n = 10), and NPs (C, n = 10) are shown by geometric mean fluorescence intensity (gMFI). **P < .01, ***P < .001, by the paired t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 Presence of receptors for IL-10 and TGF-β on human TH2 cells. Representative flow cytometric plots for TH2 cells and ILC2s in NP are shown within the singlets, Aqua- and granulocytes (SSChigh)-excluded population (A). We gated on single, live CD45-positive cells and excluded granulocytes (side scatter high). We then assessed ILC2s as lineage−, CD127+, CRTH2+ CD161+ cells and TH2 cells as CD4+, CRTH2+ cells (Fig E3, A). Levels of cell surface expression of IL-10RA and TGFBR2 on TH2 cells from blood (B, n = 4) and NPs (C, n = 4) are shown by geometric mean fluorescence intensity (gMFI). We normalized the data by calculating the gMFI ratio of receptor to isotype IgG and compared the expression of receptors between TH2 cells and ILC2s in blood (n = 4) and NP tissue (n = 4) (D). SSC-A, Side scatter-area. *P < .05, by the paired t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 Effect of IL-10 and TGF-β on IL-33-stimulated human ILC2s. Sorted blood ILC2s were cultured in the presence or absence of 10 ng/mL IL-33, 10 ng/mL IL-10 (A, n = 8), and 20 ng/mL TGF-β1 (B, n = 5) for 4 days. The concentrations of IL-4, IL-5, IL-9, and IL-13 were measured by Luminex. *P < .05, **P < .01, by 1-way ANOVA. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 RU-486 inhibited dexamethasone (Dex)-mediated suppression in human ILC2s. Sorted blood ILC2s were cultured in the presence or absence of 10 ng/mL IL-33, 10 ng/mL TSLP, 0.01% DMSO (vehicle control), 100 nM Dex, and 100 nM RU-486 (RU) for 4 days (n = 3). The concentrations of IL-5 and IL-13 were measured by Luminex. DMSO, Dimethyl sulfoxide. *P < .05, by the paired t test. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E6 IL-10, TGF-β, and dexamethasone (Dex) inhibit cytokine production in NP ILC2s. Sorted NP ILC2s were cultured in the presence or absence of 10 ng/mL IL-10 (n = 8), 20 ng/mL TGF-β1 (n = 8), and 100 nM Dex (n = 4) for 4 days. The concentrations of IL-5 were measured by Luminex. Four NP donors were excluded from the Dex study because these patients were taking corticosteroids before the surgery. Journal of Allergy and Clinical Immunology  , e8DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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