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IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy  Helen A. Brough, MSc, FRCPCH, David J. Cousins,

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Presentation on theme: "IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy  Helen A. Brough, MSc, FRCPCH, David J. Cousins,"— Presentation transcript:

1 IL-9 is a key component of memory TH cell peanut-specific responses from children with peanut allergy  Helen A. Brough, MSc, FRCPCH, David J. Cousins, PhD, Alina Munteanu, MSc, Yuen Fei Wong, PhD, Asha Sudra, MSc, Kerry Makinson, MSc, Alick C. Stephens, PhD, Matthew Arno, PhD, Liviu Ciortuz, PhD, Gideon Lack, FRCPCH, MD, Victor Turcanu, MD, PhD  Journal of Allergy and Clinical Immunology  Volume 134, Issue 6, Pages e10 (December 2014) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Volcano plot of gene expression fold change of significantly differentially expressed genes in peanut-activated memory TH cells from 3 donors with PA and 3 NA donors. Horizontal axis, Fold-change differential gene expression between children with PA and NA children: positive values indicate higher expression in children with PA, and negative values reflect higher expression in NA children. Vertical axis, Statistical significance of differential gene expression between children with PA and NA children, adjusting for gene expression variability within the PA and NA groups, respectively. Points represent individual genes. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Heat map of 48 differentially expressed genes in skin- and gut-homing peanut-activated memory TH cells from donors with PA (n = 3) and NA donors (n = 3; ≥2 fold changes of expression, P < .05). Each column represents TH cell gene expression from an individual donor, and each row shows a single gene. Upregulated genes are red (“hot”), and downregulated genes are blue (“cold”). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Expression of TH2/TH9 subset–specific genes and other genes known to be involved in allergic responses in skin-homing (CLA+) and gut-homing (B7+) peanut-activated memory TH cells from donors with PA (n = 3) and NA donors (n = 3). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 RF algorithm for identifying classifier genes that differentiate between peanut-activated memory TH cells from 3 donors with PA and 3 NA donors. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Differential RT-qPCR gene expression in PBMCs from children with PA (n = 12), children with PS (n = 12), and atopic NA children (n = 6) comparing target gene relative to the endogenous gene 18s (ΔCT) in peanut-stimulated versus unstimulated cultures (ΔΔCT) converted into RQ in log-transformed arbitrary units (AU). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 A, Box plot of FACS-sorted memory TH cells and ICCS of IL-5 and IL-9 expressed in peanut-stimulated (P+) versus unstimulated (P−) memory TH cells (CD4+CD45RO+) from 5 donors with PA and 5 donors with PS. B, Example of FACS analysis of peanut-stimulated memory TH cells from the first donor with PA and PS. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 FACS experiments for 3 donors with PA and 3 NA donors, illustrating the isolation of memory TH cells (expressing CD4 and CD45RO) homing to the skin (CLA+) or the gut (B7+) and either peanut-activated (CD69+) or nonactivated controls (CD69−). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Data normalization and quality checks for microarray hybridization. Box plots of array hybridization signal before and after robust multichip average (RMA) normalization are shown in Fig E2, Ai and Aii, respectively. All arrays had a uniform pattern of hybridization control signal (Fig E2, B), which was at the expected intensity in congruence with their staggered concentration of CreX > BioD > BioC > BioB. Red, CreX; blue, BioD; pink, BioC; and green, BioB. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Principal component analysis (PCA) of gene expression in the Affymetrix GeneChip Human Gene1.0 ST microarray led to clustering of the CLA+ skin-homing TH cells (green centroid), distinguishing them from B7 gut-homing TH cells (yellow centroid). Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Gene expression in peanut-stimulated (Stim) versus unstimulated (Unstim) culture conditions in activated (CD69+) and nonactivated (CD69−) skin-homing (CLA+) and gut-homing (B7+) memory TH cells in 3 children with PA versus 3 NA children. Journal of Allergy and Clinical Immunology  , e10DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions


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