Regulation of IL-13 receptor α1 expression and signaling on human tonsillar B- lymphocyte subsets Oumnia Hajoui, PhD, Huaien Zheng, MD, PhD, Julie Guay,

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Regulation of IL-13 receptor α1 expression and signaling on human tonsillar B- lymphocyte subsets Oumnia Hajoui, PhD, Huaien Zheng, MD, PhD, Julie Guay, BSc, Severine Letuve, PhD, Lama M. Fawaz, PhD, Bruce D. Mazer, MD Journal of Allergy and Clinical Immunology Volume 120, Issue 6, Pages 1425-1432 (December 2007) DOI: 10.1016/j.jaci.2007.07.011 Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Expression of IL-13Rα1 in human B lymphocytes. A and B, Purified B cells were cultured in medium alone (black line), with anti-CD40 antibody (1 μg/mL) and recombinant human IL-4 (400 U/mL; red line), or with αCD40 plus IL-4 plus IL-13 neutralizing antibody (3 μg/mL; blue line). Cells were serially harvested during 14 days of culture. Fig 1, A, shows the kinetics of expression of IL-13Rα1: ∗P < .01 compared with control (n = 5). Fig 1, B, shows the kinetics of expression of IL-4Rα. C through F, B cells grown in slide chambers were stimulated with αCD40/IL-4 for 24 hours, and direct immunofluorescent staining was performed after fixing B cells in slide chambers with phycoerythrin-conjugated anti-CD19 (Fig 1, C), FITC-conjugated anti-IL-13Rα1 (Fig 1, D), or superimposed (Fig 1, E); all yellow cells express both proteins. Fig 1, F, shows the isotype control enhanced to show maximal background. Journal of Allergy and Clinical Immunology 2007 120, 1425-1432DOI: (10.1016/j.jaci.2007.07.011) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Expression of IL-13Rα1 mRNA. A, Analysis of human IL13RA1 mRNA expression detected by means of conventional RT-PCR. Upper panel, RT-PCR of human IL13RA1: lane 1, unstimulated B cells; lane 2, anti-CD40; lane 3, IL-4; lane 4, anti-CD40/IL-4. Lower panel, 18S rRNA under the same conditions. Results are representative of 3 identical experiments. B, Real-time PCR quantitation of IL13RA1 mRNA expressed as a ratio between IL13RA1 and 18S rRNA (n = 3). Journal of Allergy and Clinical Immunology 2007 120, 1425-1432DOI: (10.1016/j.jaci.2007.07.011) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Analysis of phosphorylation of STAT6 by means of flow cytometry. A, Resting B cells were treated with IL-13 (25 ng/mL) or IL-4 (100 U) for 10 minutes. Resting B lymphocytes only respond to IL-4 stimulation. B, After incubation with anti-CD40 for 24 hours, STAT6 phosphorylation is detected after addition of IL-4 or IL-13. Results are representative of 5 identical experiments. C, Graphic representation of mean fluorescence intensity (MFI) showing changes in STAT6 phosphorylation for 5 experiments. Journal of Allergy and Clinical Immunology 2007 120, 1425-1432DOI: (10.1016/j.jaci.2007.07.011) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Induction of STAT6 phosphorylation in mature and memory B cells after addition of IL-13. Purified B cells were stimulated with or without anti-CD40 antibodies for 24 hours and then treated with IL-13 for 10 minutes. The cells were double stained with APC-conjugated CD38 (A) or APC-conjugated CD27 (B) and FITC-conjugated phospho-STAT6 and analyzed by means of flow cytometry (n = 3 experiments). Journal of Allergy and Clinical Immunology 2007 120, 1425-1432DOI: (10.1016/j.jaci.2007.07.011) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 IL-13 induces STAT6 phosphorylation in CD27+ cells. CD27+ cells were purified from tonsils by means of MACS bead separation. Upper panel, Dot plot of STAT6 phosphorylation in CD27+ B cells divided into CD27+CD38−(lower right quadrant) and CD27+CD38+(upper right quadrant) cells. STAT6 phosphorylation is minimally upregulated in unstimulated cells (medium + IL-13) compared with CD40-stimulated cells. Lower panel, Cumulative data for 4 experiments. Journal of Allergy and Clinical Immunology 2007 120, 1425-1432DOI: (10.1016/j.jaci.2007.07.011) Copyright © 2007 American Academy of Allergy, Asthma & Immunology Terms and Conditions