Peanut oral immunotherapy transiently expands circulating Ara h 2–specific B cells with a homologous repertoire in unrelated subjects Sarita U. Patil,

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Presentation transcript:

Peanut oral immunotherapy transiently expands circulating Ara h 2–specific B cells with a homologous repertoire in unrelated subjects Sarita U. Patil, MD, Adebola O. Ogunniyi, PhD, Agustin Calatroni, MA, MS, Vasisht R. Tadigotla, PhD, Bert Ruiter, PhD, Alex Ma, BS, James Moon, PhD, J. Christopher Love, PhD, Wayne G. Shreffler, MD, PhD Journal of Allergy and Clinical Immunology Volume 136, Issue 1, Pages 125-134.e12 (July 2015) DOI: 10.1016/j.jaci.2015.03.026 Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 1 PNOIT clinical trial. Patients aged 7 to 21 years (n = 22) with IgE-mediated peanut hypersensitivity (positive specific IgE level, skin prick test response, and history of reaction) underwent PNOIT in a single-center randomized trial (3:1, active/observational arm) with commercially available peanut flour. Patients recruited into the active arm (as above) underwent a 1-day modified rush protocol for a maximum dose of 12 mg, followed by a build-up period with escalating peanut doses every 2 weeks, which included blood draws at every month (arrows), starting at 2 weeks. The build-up period was followed by a maintenance period of 3 months at 4000 mg, followed by an oral food challenge to assess tolerance. The 2 time points used for NGS are noted by asterisks. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 2 Fluorescent Ara h 2 multimer production and validation. A, Four biotinylated Ara h 2 molecules (red = biotin) were conjugated to fluorescent streptavidin. The multimer then could be used to identify B cells expressing a surface BCR specific for Ara h 2 by using flow cytometry. B, For multimer validation, basophils sensitized with serum from a donor with peanut allergy but not a nonallergic donor were specifically stained by the multimer. Gray histogram, Unstained basophils. C, Circulating basophils from patients with peanut allergy undergoing PNOIT (n = 19) and nonallergic subjects (n = 5) were stained with and without the fluorescent Ara h 2 multimer. *P = .05. Fluorescence was standardized with Alexa Fluor 488 MESF beads. D, CD3−CD14−CD16−CD19+ B-cell subpopulations were defined based on their CD27 fluorescence. B-cell subpopulations were defined as naive B cells (CD19+CD27−IgM+), class-switched memory B cells (CD19+CD27intIgM−), and plasmablast cells (CD19+CD27hiIgM−). The multimer gate is determined based on the negative control (stained with the entire panel except for the multimer). Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 3 Multimer-positive B cells and Ara h 2 immunoglobulins during the course of PNOIT. A, The frequency of multimer-positive cells was normalized to the subset (naive, memory, or plasmablast cell) population and described as the number of cells per million subset cells. B, Longitudinal analysis using a generalized, additive, mixed nonlinear model demonstrates that multimer-positive class-switched memory B-cell expansion within the memory B-cell compartment occurs transiently during early PNOIT, peaking at week 7. C, Ara h 2–specific IgG, IgE, IgA, and IgG4 levels were measured longitudinally during PNOIT (n = 22) by using the Phadia ImmunoCAP. Longitudinal sampling begins at pre-PNOIT. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 4 Single-cell antibody cloning of multimer-positive B cells. A, Individual multimer-positive CD19+ B cells were sorted by means of fluorescence-activated cell sorting from 3 patients 1 month after initiation of PNOIT into a 96-well plate (sample shown above). B, Immunoglobulin heavy chain amplification from the 3 patients yielded a total of 135 heavy chains, of which 40% were IgM, 32.6% were IgG, 26.7% were IgA, and less than 1% could not be classified based on identification by primer alignment of sequences. C, Frequency of somatic mutations is higher in heavy chains compared with light chains in the immunoglobulins amplified from sorted multimer-positive B cells. D, In amplified immunoglobulin heavy chain sequences the frequency of nonsilent mutations tends to be higher in the CDRs as opposed to the framework regions (FR) in IgA and IgG sequences compared with IgM sequences, which is suggestive of affinity maturation in the IgA and IgG immunoglobulin heavy chains. E, Distribution of heavy chain V-J gene use is varied in the single-cell amplified immunoglobulins. F, From the 3 patients, a total of 40 recombinant antibodies were produced from paired transfection of HEK293 cells. Although none of the IgM antibodies expressed were Ara h 2–specific, 24 (89%) of the 27 IgA or IgG antibodies were confirmed to be Ara h 2–specific by using ImmunoCAP. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 5 Stereotyped Ara h 2 clones within the NGS repertoire. In each diagram individual multimer-identified Ara h 2 sequences on the left were mapped to homologous sequences (same V-J gene segment with similar CDR3) within the NGS repertoire represented on the right. Colors on the outer left edge of the circle represent the subject origin of either the Ara h 2 sequence (on the left) or NGS repertoire (on the right). Ribbon width represents the relative frequency of the homologous sequence or clonal group, allowing for a relative comparison of the frequency of homologous sequences in the NGS repertoire to the Ara h 2 single-cell sequence. Ribbon color (by the isotype of the Ara h 2 sequence) highlights those clones shared among unrelated subjects, with gray ribbons showing the subject-specific clonal groups. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig 6 Identity of Ara h 2 homologous sequences within the NGS repertoire to their affinity-selected Ara h 2 single-cell sequence counterparts. A, Ara h 2 similar sequences within the NGS repertoire are shaded by their isotypes, and the multimer-identified Ara h 2 sequences are represented as black dots. The Ara h 2 homologous sequences on the x-axis demonstrate strong nucleotide similarity with the multimer-identified Ara h 2 sequences. Class-switched IgA and IgG sequences are more highly mutated than IgM from germline. These graphs are representative. B, The CDR1, CDR2, and CDR3 regions of homologous NGS sequences from all 3 patients are aligned to a representative Ara h 2 affinity-selected sequence from patient 2. Colored amino acid residues represent disagreements with the Ara h 2 multimer-identified sequence, whereas similar residues are gray. Colors represent polarity of the amino acid residues. Asterisks mark amino acid substitutions from the germline sequence. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E1 Single-cell immunoglobulin amplification. Immunoglobulin amplification of heavy and light chains was performed by using single-cell nested RT-PCR. In this example gel electrophoresis demonstrates recovery of 17 of 24 paired heavy and light chains. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E2 Antibody cloning vectors. Recombinant antibodies were produced after insertion of the multimer-positive single-cell amplified sequences into heavy or light (either kappa or lambda, respectively) vectors, as previously shown.17 HC, Heavy chain; HCMV, human cytomegalovirus; LC, light chain. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E3 NGS analysis pipeline. The NGS analysis pipeline for processing of raw Illumina MiSeq 2x250 reads is used to generate the heavy chain immunoglobulin sequences used for downstream analysis. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E4 Representative results from NGS data analysis. The number of reads retained at each step of the analysis pipeline for one of the subjects' samples is shown. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E5 Frequency of multimer-positive B-cell subsets in allergic and nonallergic subjects. The frequency of multimer-positive cells was normalized to the subset (naive, memory, or plasmablast) population and described as the number of cells per million subset cells or per million CD19+ B cells in patients with peanut allergy from the PNOIT trial before the start of PNOIT compared with nonallergic control subjects. Basophil staining of the nonallergic control subjects is shown in Fig 2, C. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E6 Inference of antigen selection of Ara h 2–positive B-cell IgH. Heavy chain immunoglobulin sequences of Ara h 2 affinity-selected B cells have increased frequency of CDR replacement mutations (RCDR) compared with the total number of mutations in the V region (MV), which is suggestive of antigen selection. The gray area represents the 90% to 95% CI for the probability of random mutations. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E7 Recombinant antibody affinity for Ara h 2. Ara h 2 affinity of recombinant IgG1 antibodies produced from multimer-positive single cells was characterized by using biolayer inferferometry (Octet). The colors of the bars reflect the original multimer-positive single cell isotype from which the recombinant antibody was produced. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions

Fig E8 Ara h 2–specific heavy chain clonal groups. The distribution of clonal groups (same V-J gene use and similar CDR3) demonstrates that 18% of sequences belong to clonal families with related sequences. Journal of Allergy and Clinical Immunology 2015 136, 125-134.e12DOI: (10.1016/j.jaci.2015.03.026) Copyright © 2015 American Academy of Allergy, Asthma & Immunology Terms and Conditions