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Mohamed H. Shamji, PhD, Janice A. Layhadi, MSc, Guy W

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Presentation on theme: "Mohamed H. Shamji, PhD, Janice A. Layhadi, MSc, Guy W"— Presentation transcript:

1 Basophil expression of diamine oxidase: A novel biomarker of allergen immunotherapy response 
Mohamed H. Shamji, PhD, Janice A. Layhadi, MSc, Guy W. Scadding, MRCP, Delica K.M. Cheung, MSc, Moises A. Calderon, MD, PhD, Laurence A. Turka, MD, Deborah Phippard, PhD, Stephen R. Durham, MD, FRCP  Journal of Allergy and Clinical Immunology  Volume 135, Issue 4, Pages e9 (April 2015) DOI: /j.jaci Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 RTSSs: SAR group, n = 24; SCIT group, n = 14; SLIT group, n = 12; SLIT-TOL group, n = 6. Data are expressed as medians (interquartile ranges). Mann-Whitney U test: **P < .01 and ***P < .001. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 DAO as a measure of intracellular histamine in CRTh2+ basophils. A, Representative intracellular fluorochrome–labeled DAO immunostaining on CD63+CRTh2+ basophils of 1 allergic subject after ex vivo timothy grass pollen allergen stimulation. B, Percentage of CD63+CRTh2+ and DAO+CRTh2+ basophils after ex vivo allergen stimulation in patients with SAR (n = 12) and NACs (n = 12). C, DAO+CRTh2+ basophils measured in patients with SAR were correlated with basophil histamine release (BHR). Mann-Whitney U test: ***P < Correlation was determined by using the Spearman rank method. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig 3 Grass pollen–specific immunotherapy: ex vivo allergen-induced basophil activation. A, Representative flow cytometric analysis from 3 subjects showing single (CD63) and dual expression of CD63 and CD203cbright on CRTh2+ basophils. Whole-blood basophils obtained from SLIT-treated patients (n = 10), SCIT-treated patients (n = 10), and untreated patients with SAR (n = 10) were stimulated with varying concentrations of P pratense. B-D, Ex vivo allergen-induced basophil responsiveness was measured based on expression of CD203cbright (Fig 3, B), CD63 (Fig 3, C), and CD107a (Fig 3, D) on CRTh2+ basophils.Green circle, SCIT; blue circle, SLIT; orange circle, SAR. Data are expressed as medians (interquartile ranges). Mann-Whitney U test: statistical difference between patients receiving SCIT and patients with SAR (*P < .05) and patients receiving SLIT and patients with SAR (φP< .05). ∗∗P < .01 and ∗∗∗P < .001. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig 4 Persistence of ex vivo allergen-induced basophil hyporeactivity after withdrawal of sublingual immunotherapy. DAO+CRTh2+ (A) and CD63+CRTh2+ (B) basophils were measured in the SAR (n = 24), SCIT (n = 14), SLIT (n = 12), and SLIT-TOL (n = 6) groups. Bars represent medians. ***P < .001, Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig 5 Induction of protective and functional IgG4 antibodies after immunotherapy. A, Serum specific IgG4 levels are represented for all 4 groups: SAR, SCIT, SLIT, and SLIT-TOL. B and C, The ability of IgG-containing serum obtained after immunotherapy to inhibit both CD23- and FcεRI-dependent IgE-mediated events is represented for all 3 immunotherapy-treated groups by the consistently lower levels of IgE-FAB binding to B cells (Fig 5, B) and the lower percentages of basophil histamine release (Fig 5, C), respectively, compared with those in the patients with SAR. Data are expressed as medians (interquartile ranges). *P < .05 and ***P < .001, Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig 6 Suppression of ex vivo allergen-induced basophil reactivity after SCIT and SLIT is serum and FcγRIIb dependent. SCIT (n = 6), SLIT (n = 6), or control serum from a nonatopic subject were incubated with whole blood in the presence of grass pollen allergen (10 ng/mL). Suppression of ex vivo basophil allergen-induced reactivity was measured with or without αCD32 and αCD32/CD16. Data are expressed as medians (interquartile ranges). *P < .05 and **P < .01, Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E1 Ex vivo allergen-induced basophil reactivity is increased in patients with SAR compared with that seen in NACs. Whole-blood basophils obtained from patients with SAR (n = 12) and NAC (n = 12) volunteers were incubated with varying concentrations of P pratense extract. Proportions of CD203cbright (A), CD63+ (B), and CD107a+ (C) CRTh2+ basophils and their relationship with circulating sIgE levels in patients with SAR were assessed. Data are expressed as medians (interquartile ranges). Significance between groups was assessed by using the Mann-Whitney U test as follows: *P < .05, **P < .01, and ***P < .001, respectively. Correlations were obtained by using the Spearman rank method. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

9 Fig E2 Time course of CD203cbright, CD63, and CD107a surface activation markers on CRTh2+ basophils after ex vivo P pratense stimulation. Basophils were stimulated with 100 ng/mL P pratense and incubated for 0, 5, 10, 15, 30, 60, and 120 minutes. Expression of CD203c, CD63, and CD107a on CRTh2+ basophils was measured. Data are shown as median (interquartile range) percentages of CD203cbrightCRTh2+, CD63+CRTh2+, and CD107a+CRTh2+ basophils. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

10 Fig E3 Ex vivo allergen-induced basophil activation is diminished if the cells are stimulated 6 and 24 hours after blood collection. Proportions of CD63+CRTh2+ and CD203cbrightCRTh2+ basophils were assessed by using blood at 0, 6, and 24 hours after venesection. Data are expressed as medians (interquartile ranges). Significance was assessed by using the Mann-Whitney U test. ∗P < .05. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

11 Fig E4 Increased expression of CD203bright, CD63, and CD107a on CRTh2+ basophils is specific for the sensitizing allergen. Subject 1 is allergic and sensitized to P pratense and house dust mite (HDM). Subject 2 was sensitized to P pratense and birch and weakly sensitized to peanut. Ex vivo allergen-specific basophil activation was measured. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

12 Fig E5 Basophil responsiveness after FcεRI cross-linking by anti-IgE mAb. DAO+CRTh2+ and CD63+CRTh2+ basophil numbers were measured in the SAR (n = 24), SCIT (n = 14), SLIT (n = 12), and SLIT-TOL (n = 6) groups. Results for CD63+CRTh2+ (A), CD203cbirghtCRTh2+ (B), and DAO+CRTh2+ (C) basophils after anti-IgE stimulation (0.5 μg/mL) are shown. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

13 Fig E6 Suppression of basophil responsiveness remain unchanged despite variation in pollen counts. A, Pollen counts for summer 2012 and B, DAO+CRTh2+ and CD63+CRTh2+ basophil numbers and RTSSs were measured in subjects recruited during summer 2012 (blue; SAR group, n = 12, SCIT group, n = 7; SLIT group, n = 6; SLIT-TOL group, n = 4) and summer 2013 (red; SAR group, n = 12; SCIT group, n = 7; SLIT group, n = 6; SLIT-TOL group, n = 2). ns, Not statistically significant (P > .05). Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

14 Fig E7 Association between ex vivo allergen-induced basophil responsiveness and RTSSs. DAO+CRTh2+ and CD63+CRTh2+ basophil numbers were measured in the SAR (n = 24, orange circles), SCIT (n = 14, green circles), SLIT (n = 12, blue circles), and SLIT-TOL (n = 6, white circles) groups. DAO+CRTh2+ (A) and CD63+CRTh2+ (B) basophils were correlated with RTSSs by using the Spearman rank method. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

15 Fig E8 Ten- to 100-fold more allergen was required to induce basophil histamine release in patients treated with SCIT and SLIT. A, Ex vivo allergen-induced basophil histamine release was assessed in patients treated with SCIT or SLIT and in patients with SAR (each n = 3). B, Basophil histamine release was also measured before and after SCIT in 6 patients. Data are expressed as medians (interquartile ranges). Significance was assessed by using the Mann-Whitney U test. Journal of Allergy and Clinical Immunology  , e9DOI: ( /j.jaci ) Copyright © 2014 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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