Anti–IL-5 therapy reduces mast cell and IL-9 cell numbers in pediatric patients with eosinophilic esophagitis Iris M. Otani, MD, Arjun A. Anilkumar,

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Anti–IL-5 therapy reduces mast cell and IL-9 cell numbers in pediatric patients with eosinophilic esophagitis Iris M. Otani, MD, Arjun A. Anilkumar, BS, Robert O. Newbury, MD, Monica Bhagat, MD, Lisa Y. Beppu, BS, Ranjan Dohil, MD, David H. Broide, MB, ChB, Seema S. Aceves, MD, PhD Journal of Allergy and Clinical Immunology Volume 131, Issue 6, Pages 1576-1582.e2 (June 2013) DOI: 10.1016/j.jaci.2013.02.042 Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Anti–IL-5 decreases esophageal eosinophil numbers. Peak eosinophil numbers before and after therapy at weeks 0, 12, and 24 by response category (A) and dose (B) are shown. Responders are defined as having less than 15 eosinophils per hpf at week 12. Bars represent median eosinophil counts (Fig 1, A) and means with SEs (Fig 1, B). Fig 1, A: *P < .01, **P < .001, and ***P < .0001. Fig 1, B: *P < .05, **P < .01, ***P < .001, and ****P < .0001. Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Anti–IL-5 decreases esophageal mast cell numbers. Peak mast cell numbers by dose (A) and response (B) at weeks 0, 12, and 24 are shown. Responders are defined as having less than 15 eosinophils per hpf at week 12. Bars represent means with SEs (Fig 2, A) and medians (Fig 2, B). Fig 2, A: *P < .05 and **P < .001. Fig 2, B: *P < .05 and **P < .001. Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Anti–IL-5 decreases eosinophil–mast cell couplet numbers. A, Representative image showing that eosinophils and mast cells are found in close proximity in the esophagi of patients with EoE (red, major basic protein [MBP]; green, tryptase). B, Numbers of eosinophil–mast cell couplets before and after anti–IL-5 therapy by response at weeks 0, 12, and 24. Bars represent means with SEs. Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Anti–IL-5 decreases IL-9+ cell numbers. A, Peak IL-9+ cell numbers by response at weeks 0, 12, and 24. Bars represent median numbers of IL-9+ cells. *P < .05 and **P < .001. B, Individual IL-9 counts among responders. C and D, Representative images of IL-9+ cells before and after anti–IL-5 (week 12) in a responder (Fig 4, C) and of major basic protein (MBP; eosinophils, green), IL-9+ cells (red), and overlapping cells (yellow, arrows; Fig 4, D). E, Mast cells (tryptase, green) do not make IL-9 (red). Arrows show mast cells and IL-9+ cells in proximity. Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Eosinophil and mast cells numbers have a close relationship. The percentage of subjects with varying decreases in numbers of esophageal epithelial eosinophils (open bars) and mast cells (solid bars) in the given eosinophil category. The mean peak numbers of eosinophils and mast cells before (pre) and after (post) anti–IL-5 are indicated above the respective bars. Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 There is a significant decrease in IL-9–producing eosinophil numbers and a nonsignificant decrease in noneosinophilic cell numbers after anti–IL-5 therapy (n = 6 subjects before and after mepolizumab). Journal of Allergy and Clinical Immunology 2013 131, 1576-1582.e2DOI: (10.1016/j.jaci.2013.02.042) Copyright © 2013 American Academy of Allergy, Asthma & Immunology Terms and Conditions