John-Paul Oliveria, BSc, Brittany M

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Asthmatic subjects with allergy have elevated levels of IgE+ B cells in the airways John-Paul Oliveria, BSc, Brittany M. Salter, PhD, Stephanie Phan, BSc, Caitlin D. Obminski, BSc, Caroline E. Munoz, BSc, Steven G. Smith, PhD, Tara Scime, BSc, Rick M. Watson, BSc, Roma Sehmi, PhD, Gail M. Gauvreau, PhD Journal of Allergy and Clinical Immunology Volume 140, Issue 2, Pages 590-593.e9 (August 2017) DOI: 10.1016/j.jaci.2016.12.981 Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 The levels of B cells, IgE+ B cells, and IgE+ B-cell subsets (memory B cells and plasma cells) in the sputum (A-D) and blood (E-H) of subjects with allergic asthma and nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. In sputum analysis of B cells and IgE+ B cells, the subject numbers were as follows: control, n = 7; allergic asthmatic, n = 10; allergic n = 8. In the sputum analysis of IgE+ B-cell subsets, the subject numbers were as follows: control, n = 6; allergic asthmatic, n = 8; allergic, n = 6. In blood analysis of all cells, the subject numbers were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Significant findings are denoted by *, where P < .05. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 The relationship between sputum IgE+ B cells and eosinophils (A), total IgE (B), and BAFF (C) and the relationship between sputum B cells and eosinophils (D), total IgE (E), and BAFF (F) in subjects with allergic asthma and nonasthmatic controls. Data were correlated with Spearman rank r values and linear regression lines of best fit. For measurements of sputum the subject numbers were as follows control, n = 7; allergic asthmatic, n = 10; allergic, n = 8. Exact P values were denoted, where P < .05 was considered a significant correlation. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Representative sputum sample and flow cytometry gating strategy for enumeration of all cells (A), leukocytes (B), lymphocytes (C), CD19+ B cells (D) with IgE isotype and specific stain (E-F), memory B cells (G) with IgE isotype and specific stain (H-I), and plasma cells (J) with IgE isotype and specific stain (K-L). Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 The number of B cells, IgE+ B cells, and IgE+ B-cell subsets (MBCs and PCs) in the sputum (A-D) and blood (E-H) of subjects with allergic asthma and nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. In sputum analysis of B cells and IgE+ B-cell, the numbers of subjects were as follows: control, n = 7; allergic asthmatic, n = 10; allergic n = 8. In the sputum analysis of IgE+ B-cell subset, the numbers of subjects were as follows: control, n = 6; allergic asthmatic, n = 8; allergic = 6. In blood analysis of all cells, the numbers of subjects were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Significant findings are denoted by *, where P < .05. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 The frequency and number of MBCs and PCs in the sputum (A-D) and blood (E-H) of subjects with allergic asthma and of nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. For measurements in sputum the numbers of subjects were as follows: control, n = 6; allergic asthmatic, n = 8; allergic, n = 6. For measurements in blood, the numbers of subjects were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 The frequency of CD4+ T cells (A) and CD8+ T cells (B) in the sputum of subjects with allergic asthma and of nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. The numbers of subjects were as follows: control, n = 7; allergic asthmatic, n = 10; allergic, n = 8. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 The frequency of lymphocytes in sputum (A) and blood (B) of subjects with allergic asthma and of nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. For measurements in sputum the numbers of subjects were as follows: control, n = 7; allergic asthmatic, n = 10; allergic, n = 8. For measurements in blood the numbers of subjects were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 The frequency and numbers of IgG+ B cells in sputum (A-B) and blood (D-E) and correlation with IgE+ B cells in the sputum (C) and blood (F) of subjects with allergic asthma and of nonasthmatic controls. Data were analyzed using a 1-way ANOVA with Tukey post hoc tests and are presented as means ± SEMs. Correlation data are presented as correlations with Spearman rank r values and linear regression lines of best fit. For measurements in sputum the numbers of subjects were as follows: control, n = 7; allergic asthmatic, n = 10; allergic, n = 8. For measurements in blood the numbers of subjects were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 The relationship between IgE+ B cells and eosinophils (A) and total IgE (B) in blood and the relationship between B cells and eosinophils (C) and total IgE (D) in blood of subjects with allergic asthma and of nonasthmatic controls. Data are presented as correlations with Spearman rank r values and linear regression lines of best fit. The numbers of subjects were as follows: control, n = 11; allergic asthmatic, n = 11; allergic, n = 8. Exact P values were denoted, where P < .05 was considered a significant correlation. Journal of Allergy and Clinical Immunology 2017 140, 590-593.e9DOI: (10.1016/j.jaci.2016.12.981) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions