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Direct monitoring of basophil degranulation by using avidin-based probes  Régis Joulia, PhD, Claire Mailhol, MD, Salvatore Valitutti, MD, Alain Didier,

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Presentation on theme: "Direct monitoring of basophil degranulation by using avidin-based probes  Régis Joulia, PhD, Claire Mailhol, MD, Salvatore Valitutti, MD, Alain Didier,"— Presentation transcript:

1 Direct monitoring of basophil degranulation by using avidin-based probes 
Régis Joulia, PhD, Claire Mailhol, MD, Salvatore Valitutti, MD, Alain Didier, MD, Eric Espinosa, PhD  Journal of Allergy and Clinical Immunology  Volume 140, Issue 4, Pages e6 (October 2017) DOI: /j.jaci Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

2 Fig 1 Fluorescent avidin allows one to monitor basophil degranulation. A, Sequence of snapshots from confocal videomicroscopy of IgE-sensitized basophil stimulated with anti-IgE antibodies. B-D, Flow cytometry analysis of basophils stimulated with anti-IgE antibodies. Gating strategy and representative FACS profiles (Fig 1, B). Percentages of av.A488+ or CD63+ basophils (Fig 1, C) and gMFI of gated basophils following Av.A488 or CD63 stainings (Fig 1, D). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor. Two-tailed paired t test, **P < .01, ***P < .001, ****P <  E, Correlation between avidin or CD63-based assays and histamine release following stimulation with 1 (circles), 2.5 (square), and 5 μg/mL (diamonds) anti-IgE; pooled data from 7 healthy donors, Spearman rank correlation. FACS, Fluorescence-activated cell sorting; FSC-A, forward scatter-area; gMFI, geometric mean fluorescence intensity; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

3 Fig 2 Analysis of basophil degranulation in allergic patients. A-D, A total of 1 × 106 WBCs were stimulated or not with anti-IgE antibodies (2.5 μg/mL) or with indicated allergen preparations. Basophils were gated as in Fig 1, representative fluorescence-activated cell sorting profiles (Fig 2, A), Avidin+ basophil frequency (Fig 2, B), CD63+ basophil frequency (Fig 2, C), and CD203c or avidin fluorescence rMFI following stimulation (Fig 2, D). E, Avidin-A488 rMFI and prick-test results from tested patients. F and G, Correlation analysis between prick-test results and avidin rMFI (Fig 2, F) or CD63 rMFI (Fig 2, G) for 5 grasses mix stimulation, Spearman rank correlation. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

4 Fig E1 Avidin binds to human basophil cell surface following activation. A, Sequence of snapshots from time-lapse analysis of IgE-sensitized basophils stimulated with anti-IgE antibodies. B, Single-cell analysis of Av.SRho integrated fluorescence intensity. Data are from 1 representative experiment out of 3. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

5 Fig E2 Avidin staining is not always correlated with the CD203c upregulation-based assay. A total of 1 × 106 human WBCs were stimulated or not with anti-IgE antibodies (2.5 μg/mL). Basophils were gated as in Fig 1. A, Representative FACS profiles of donors 7 and 1. B and C, gMFI of Av. A488 (Fig E2, B) and CD203c (Fig E2, C) staining on basophils (pooled data, n = 18); arrows indicate discordant results for donors 1 and 6 (yellow circles). D, rMFI of Av. A488+ (red) and CD203c (green) (pooled data, n = 18). Threshold (rMFI = 0.1, dashed line) was determined using nonresponder donors (gray circles). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor. Two-tailed paired t test, **P < .01, ***P < .001. FACS, Fluorescence-activated cell sorting. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

6 Fig E3 IL-3 priming did not affect avidin staining. A total of 1 × 106 human WBCs were stimulated or not with anti-IgE antibodies (2.5 μg/mL) in absence or in presence of IL-3 (3 ng/mL). Basophils were gated as in Fig 1. A and B, Representative FACS profiles of donor 15. C and D, Percentages of Av. A488+ basophil (Fig E3, C) and CD203c gMFI (Fig E3, D) (pooled data, n = 7). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor. Two-tailed paired t test, ns P > .05, *P < .05. FACS, Fluorescence-activated cell sorting; ns, nonsignificant. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

7 Fig E4 pDCs and FcεRIlow monocytes are not stained with avidin upon stimulation. A total of 1 × 106 human PWBCs were stimulated or not with either PMA/ionomycin or anti-IgE mAbs (2.5 μg/mL). A, Gating strategy used to isolate pDCs and FcεRIlow monocytes. Doublets were excluded using FSC-A and FSC-H parameters, and then low SSC-A and FSC-A cells were gated; FcεRIlow monocyte and CD123+ cells were selected. To further separate pDCs from basophils, pDCs were gated as CD203c− cells. Representative FACS profiles of gated CD123+ pDCs (B) or FcεRIlow monocytes (D) for Av.A488 staining following stimulation with PMA/ionomycin or anti-IgE. Percentages of Av.488+ pDCs (C) or FcεRIlow monocytes (E) from pooled data (n = 11). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor; bars represent median. Two-tailed paired t test, ns P > .05. FACS, Fluorescence-activated cell sorting; pDC, plasmacytoid dendritic cell; FSC-A, forward scatter-area; FSC-H, forward scatter-height; ns, nonsignificant; PWBC, peripheral white blood cells; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

8 Fig E5 A small fraction of eosinophils or neutrophils stained dimly positive for avidin following stimulation. A total of 1 × 106 human PWBCs were stimulated or not with either PMA/ionomycin or anti-IgE antibodies (2.5 μg/mL). A, Gating strategy used to isolate neutrophils and eosinophils. Doublets were excluded using FSC-A and FSC-H parameters; CD16high/FcεRI− neutrophils and CD16+/FcεRI+ eosinophils were gated from high SSC-A and FSC-A cells. Representative FACS profiles of gated neutrophils (B) or eosinophils (D) for Avidin-A488 staining following stimulation with PMA/ionomycin or anti-IgE. Percentages of Av.488+ neutrophils (C) or eosinophils (E) from pooled data (n = 14). Percentages in the FACS profiles indicate the frequency of gated cells. Each point represents a donor; bars represent median. Two-tailed paired t test, ns P > .05, ****P <  FACS, Fluorescence-activated cell sorting; FSC-A, forward scatter-area; FSC-H, forward scatter-height; SSC-A, side scatter-area. Journal of Allergy and Clinical Immunology  , e6DOI: ( /j.jaci ) Copyright © 2017 American Academy of Allergy, Asthma & Immunology Terms and Conditions

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