Ramona A. Hoh, PhD, Shilpa A

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Presentation transcript:

Single B-cell deconvolution of peanut-specific antibody responses in allergic patients Ramona A. Hoh, PhD, Shilpa A. Joshi, PhD, Yi Liu, PhD, Chen Wang, PhD, Krishna M. Roskin, PhD, Ji-Yeun Lee, BS, Tho Pham, MD, Tim J. Looney, PhD, Katherine J.L. Jackson, PhD, Vaishali P. Dixit, BA, Jasmine King, BS, Shu-Chen Lyu, MS, Jennifer Jenks, BS, Robert G. Hamilton, PhD, Kari C. Nadeau, MD, PhD, Scott D. Boyd, MD, PhD Journal of Allergy and Clinical Immunology Volume 137, Issue 1, Pages 157-167 (January 2016) DOI: 10.1016/j.jaci.2015.05.029 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 Representative flow cytometric plots of Ara h 1– and Ara h 2–specific B-cell responses in patients with peanut allergy undergoing peanut OIT. Samples are shown from a healthy donor (patient 27, left panels), a patient with peanut allergy at baseline (patient 11, middle panels), and a patient with peanut allergy after 9 months of OIT (patient 5, right panels). A, Ara h 1–specific (top panels) and Ara h 2–specific (bottom panels) B cells (CD19+/CD3−/CD14−/CD16−/CD235a−) were double positive for labeled allergen. B-E, Box plots show frequencies of Ara h 1–specific (top panels) and Ara h 2–specific (bottom panels) cells among total B cells (Fig 1, B), class-switched memory B cells (Fig 1, C), non–class-switched memory B cells (Fig 1, D), and plasmablasts (Fig 1, E) for healthy (n = 9), baseline allergic (n = 18), and OIT (n = 18) samples analyzed by means of flow cytometry. Frequencies of allergen-binding B cells within each subset are plotted as the fraction of total B cells within each subset. P values were calculated by using Wilcoxon tests: *P < .05, **P < .01, and ***P < .001. Journal of Allergy and Clinical Immunology 2016 137, 157-167DOI: (10.1016/j.jaci.2015.05.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Venn diagrams summarizing the results of Ara h 1 and Ara h 2 binding assays with mAbs generated from B cells of allergic patients isolated by using allergen-specific fluorescence-activated cell sorting. Twenty-one Ara h 1–specific mAbs and 36 Ara h 2–specific mAbs were tested. Shown are results of ELISA, Western blotting, and phage display experiments with Ara h 1 (A) and Ara h 2 (B) proteins and results of Western blotting experiments with reduced Ara h 1 (C) and Ara h 2 (D) proteins. Journal of Allergy and Clinical Immunology 2016 137, 157-167DOI: (10.1016/j.jaci.2015.05.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Peptide epitope mapping of patient-derived Ara h 1– and Ara h 2–specific mAbs. Twenty-two mAbs derived from single cells isolated from 5 patients undergoing OIT bound 24-mer peptides from Ara h 1, Ara h 2, or both expressed in a phage display library. Shown are averaged data from at least 2 independent experiments, and the plotted fold enrichment is relative to a mock control containing no antibody (row 1). Antibodies from each person are presented together. Eleven of these 22 antibodies from Ara h 2-binding B cells from 3 patients bound the same Ara h 2 region (peptides 3-5). Positive control anti–Ara h 1 and anti–Ara h 2 antibodies were polyclonal rabbit antibody preparations. Journal of Allergy and Clinical Immunology 2016 137, 157-167DOI: (10.1016/j.jaci.2015.05.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 V, D, and J gene use and isotype frequencies of allergen-specific antibody heavy chains. Data were derived from 21 Ara h 1–specific antibodies (top panels) and 36 Ara h 2–specific antibodies (bottom panels) generated from 6 allergic subjects. V, D, and J gene frequencies (A-C) and V and D gene family frequencies (D-E) are graphed, as well as isotype frequencies (F) and IGHV somatic mutation levels for each isotype (G). Journal of Allergy and Clinical Immunology 2016 137, 157-167DOI: (10.1016/j.jaci.2015.05.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Identification of peanut-specific lineages in deeply sequenced patient antibody heavy chain repertoires. A, Representative heavy chain sequences from a lineage clonally related to AbID 70 were obtained from patient 7 at 9, 21, 25, 34, 36, and 42 months during OIT. The sequence derived from the single-cell AbID 70 is also shown. The germline sequences of the gene segments used in this antibody gene rearrangement are shown for reference. Darker shading indicates a higher percentage identity. CDRs are underlined. Average distance trees were computed by using BLOSUM62 on sequences trimmed to the length of the shortest read. B, Box plots show IGHV somatic mutation levels for members of IgE-containing lineages clonally related to 5 peanut-specific mAbs. C, IGHV mutation levels for IgG4 (left panel) and IgE (right panel) reads in the AbID 70 lineage are plotted over time. Arrows indicate the time points sampled. Points are plotted with horizontal jitter to prevent superposition. The adjusted R2 values are indicated in each panel. Journal of Allergy and Clinical Immunology 2016 137, 157-167DOI: (10.1016/j.jaci.2015.05.029) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions