Expression of IL-9 receptor α chain on human germinal center B cells modulates IgE secretion Lama M. Fawaz, PhD, Ehssan Sharif-Askari, PhD, Oumnia Hajoui,

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Expression of IL-9 receptor α chain on human germinal center B cells modulates IgE secretion Lama M. Fawaz, PhD, Ehssan Sharif-Askari, PhD, Oumnia Hajoui, PhD, Abdelilah Soussi-Gounni, PhD, Qutayba Hamid, MD, PhD, Bruce D. Mazer, MD Journal of Allergy and Clinical Immunology Volume 120, Issue 5, Pages 1208-1215 (November 2007) DOI: 10.1016/j.jaci.2007.08.022 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Detection of IL-9Rα on tonsillar B cells. A, Immunocytochemistry for IL-9Rα. a, Isotype control; b, B cells stained for human IL-9R-α. B, FACS for IL-9Rα expression. Representative of 3 individual experiments; mean expression, 21.33% ± 3.21%. C, Immunohistochemistry for IL-9Rα on tonsillar sections. Alkaline-phosphatase antialkaline-phosphatase double-immunostaining for IL-9Rα and CD20 expression of a secondary follicle (magnification ×20). a, Inside the follicle (×40); b, edge of the follicle (×40). D, Immunohistochemistry for IL-9Rα on tonsillar sections. Confocal microscopy. Follicle rim staining IL-9Rα–positive cells (green) and CD19 (red); coexpression of IL-9Ra and CD19 (yellow). Follicle staining shows scant IL-9Rα expression on B cells. M1, Isotype control; M2, IL-9Rα positive population. Journal of Allergy and Clinical Immunology 2007 120, 1208-1215DOI: (10.1016/j.jaci.2007.08.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 IL-9Rα expression on tonsillar B-cell subsets. A, Expression of IL-9Rα on tonsillar B-cell subsets. Percentages indicate IL-9Rα high cells within LD, MD, and HD fractions. Representative of 3 experiments; mean, 36.1% ± 4.8% for LD cells, MD (17.5% ± 4.6%), and HD B cells (18.4% ± 2.2%) Isotype in gray; black represents IL-9Rα+ cells. B, FACS for IL-9Rα/IgD and CD38 expression on LD B cells. Three-color FACS staining of LD cells with anti–IL-9Rα, anti-CD38, and anti-IgD. Results are shown for 1 of 3 individual tonsillars. A representative dot plot is shown for IgD and CD38-positive cells, and histograms for the selected gates are shown: a, FM: IgD+CD38− mean, 29.4% ± SD 6.2%; b, GC: IgD−CD38+ 8.1% ± 4.8%; c, founder GC: IgD+CD38+ 54.3% ± 16.2%; d, memory: IgD−CD38− 19.7% ± 8.5%. IL-9Rα–positive cells in the figure represent 1 of 3 experiments. Journal of Allergy and Clinical Immunology 2007 120, 1208-1215DOI: (10.1016/j.jaci.2007.08.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 IL-9 induces STAT phosphorylation. A, Staining for phosphorylated tyrosine residues associated with activated STAT1, 3, and 5 was performed on unstimulated and IL-9–stimulated LD cells and analyzed by flow cytometry. Representative of 3 independent experiments. B, Mean of 3 experiments + SE. ∗P ≤ .05 and ∗∗P ≤ .01 by paired Student t test. Journal of Allergy and Clinical Immunology 2007 120, 1208-1215DOI: (10.1016/j.jaci.2007.08.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Synergistic effect of IL-9 on IL-4–induced IgE production. IgE measurement from supernatants of cultured LD B lymphocytes. IgE was assayed by ELISA. Values are given in pg/mL and represent the mean of 2 values ± SD. Results shown are for 3 independent tonsils (T1, T2, T3). Journal of Allergy and Clinical Immunology 2007 120, 1208-1215DOI: (10.1016/j.jaci.2007.08.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 A, IL-9Rα expression during 10-day cultures. LD cells were stained for IL-9Rα after culture with anti-CD40/IL-4 and/or IL-9 for the indicated periods. B, IL-9Rα CD27 and CD27 expression on day 1 (n = 3). C, IL-9Rα CD27 and CD27 expression on day 10 (n = 3). D, PBMC staining for IL-9Rα. Staining for IL-9Rα on PBMCs purified from nonatopic donors. IL-9Rα+CD19+ 25.60% of total cells ± 7.34%; IL-9Rα+CD19+CD27+ 25.54% ± 8.32% (n = 3). Journal of Allergy and Clinical Immunology 2007 120, 1208-1215DOI: (10.1016/j.jaci.2007.08.022) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions