Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy Rodrigo Jiménez-Saiz, PhD, Yosef Ellenbogen, BHSc, Kelly Bruton, BSc,

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Presentation transcript:

Human BCR analysis of single-sorted, putative IgE+ memory B cells in food allergy Rodrigo Jiménez-Saiz, PhD, Yosef Ellenbogen, BHSc, Kelly Bruton, BSc, Paul Spill, BHSc, Doron D. Sommer, MD, Hermenio Lima, MD, PhD, Susan Waserman, MD, MSc, Sarita U. Patil, MD, Wayne G. Shreffler, MD, PhD, Manel Jordana, MD, PhD Journal of Allergy and Clinical Immunology Volume 144, Issue 1, Pages 336-339.e6 (July 2019) DOI: 10.1016/j.jaci.2019.04.001 Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Enhanced flow cytometric method for detection of IgE+ MBCs. A, Cytometric detection of IgE+ MBCs. FMO, Fluorescence minus one control. B and C, PBMCs cultured with IL-4 plus anti-CD40 were analyzed by means of ELISpot and ELISA (Fig 1, B) and single-sorted (Fig 1, C). PN, Peanut. D-G, Amplification of IgE transcripts of single-sorted cells from positive control (Fig 1, D and E) or patients with atopic dermatitis (Fig 1, F and G) and alignment to the constant region of IGHE. Data are representative of 2 independent experiments (1-2 donors per experiment and 12-24 single-sorted cells per donor). Journal of Allergy and Clinical Immunology 2019 144, 336-339.e6DOI: (10.1016/j.jaci.2019.04.001) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Methodology to genetically validate the IgE identity of single-sorted cells. A, Schematic of the method. B, Single-sorted human IgE-expressing myeloma cells were assayed by using nested PCR. C, IgE transcripts were shown to align with IGHE alleles. D, Peripheral blood CD20+ B cells that stained negative for IgE were single-sorted. E, IgE transcripts did not amplify, as evidenced by using DNA gel electrophoresis. Journal of Allergy and Clinical Immunology 2019 144, 336-339.e6DOI: (10.1016/j.jaci.2019.04.001) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 BCR analysis of single-sorted, putative IgE+ MBCs stained with commonly used methods demonstrates a non-IgE identity. A, Schematic of the experimental design. B, Gating strategy used with a basic IgE staining (upper) or stepwise exclusion (lower) method. C, BCR amplification with primers specific for IGHE or a mix (IgHGAM). Data are representative of 5 independent experiments (1-2 donors per experiment and 12-24 single-sorted cells per donor). Journal of Allergy and Clinical Immunology 2019 144, 336-339.e6DOI: (10.1016/j.jaci.2019.04.001) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 Assessment of cytotropic (IgG and/or IgA) and negative staining of IgE+ MBCs. A and C, Cytometric detection and sorting of class-switched MBCs from different gates. FMO, Fluorescence minus one control. B and D, BCR amplification with primers specific for IgE (IGHE) or a mix specific of IgG, IgA, and IgM (IgHGAM) of single-sorted cells. Data are representative of 2 independent experiments (2 donors per experiment and 12-18 single-sorted cells per donor). Journal of Allergy and Clinical Immunology 2019 144, 336-339.e6DOI: (10.1016/j.jaci.2019.04.001) Copyright © 2019 American Academy of Allergy, Asthma & Immunology Terms and Conditions