Research ArticleALLERGY

Origins and clonal convergence of gastrointestinal IgE+ B cells in human peanut allergy

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Science Immunology  06 Mar 2020:
Vol. 5, Issue 45, eaay4209
DOI: 10.1126/sciimmunol.aay4209
  • Fig. 1 Stomach and duodenum are IgE+ B lineage cell reservoirs.

    (A) Gut mucosal and peripheral blood samples were obtained from PA, OA, or NA donors. (B and C) Normalized frequencies of B cell clones containing IgE (B) or IgG4 (C) in biopsies from PA, OA, and NA donors [Wilcoxon-Mann-Whitney (WMW) test: *P < 0.05, **P < 0.01 and ***P < 0.001; ns, not significant]. (D) Spearman’s correlation of peanut allergen–specific serum IgE (y axis) to normalized IgE clone counts in stomach and duodenum (x axis; shaded area = 95% confidence intervals). (E to H) Immunofluorescence of stomach (E and F) and duodenal (G) biopsies from PA donors or from NA stomach (H) [IgE (green), plasma cell marker CD138 (red), and nuclei (DAPI; blue)]. (E) IgE+CD138+ plasma cells (stars) localized singly and in clusters between gastric glands; a white rectangle outlines two IgE+CD138+ plasma cells, for which single-channel staining is shown in (F). (G) IgE+CD138+ plasma cell (arrowhead) and IgE+CD138 putative mast cell (arrow). (H) Representative image from an NA donor. IgE+CD138 putative mast cells were observed (arrows), but IgE+CD138+ plasma cells were absent.

  • Fig. 2 Expanded IgE+ clonal lineages in multiple tissue sites.

    (A) IgE+ clone counts in tissue (squares) and overlapping between tissues (circles). (B) Percentage of clones with IgE members in both indicated tissues, with denominator tissue indicated in the panel upper label. Dots represent PA patients. One outlier value was removed for plotting (42.1% overlap of P107 peripheral blood and stomach). (C) Clonal sharing between tissues (arc segments). Red lines denote clones with IgE+ members (top row) or any isotype (bottom row). Four representative PA patients are shown. (D) Isotype composition of multi-isotype IgE+ clones. (E) Representative clonal lineage from P115. Tissue and isotype are indicated for member nodes. Scale bar indicates IGHV SHM frequency.

  • Fig. 3 Evidence for local IgE CSR in GI tissues.

    (A) Two models for the generation of GI-resident IgE+ B cells. Arrows denote class-switching events (black) or transit of cells to the GI tract (red). (B) Percentage of IgE+ or total clones found in single or multiple tissue sites. (C) Percentage of IgE+ clones that contain members expressing other isotypes in the same tissue. (D) Analysis of tissue distribution of IgE sequences (x axis) and IgM-, IgD-, IgG-, or IgA1-expressing members in the same clone (y axis). Numbers in the boxes represent the mean number of clones identified per PA individual (n = 18 for stomach and duodenum; n = 19 for esophagus and blood). Shading of each box indicates the number of clones, with darker shading for increasing abundance.

  • Fig. 4 SHM patterns in GI tissues are consistent with local IgE CSR.

    (A) Nucleotide sequence alignment of members of an IgE-containing clonal lineage. Red boxes show shared SHM with the illustrated IgE clone member. The sequence with the most shared SHM, 14 in this case, is the nearest neighbor. (B) Collision probabilities that the most closely related clone member to IgE is found in a given tissue (polar axes) indicated by the height of the filled region on the central axis. The panels indicate the nearest neighbor for IgE sequences found in each of the six tissues (indicated by color). Plots show the sum of clones for PA individuals. (C) Nearest isotype neighbors for IgE. Nearest neighbor isotype collision probabilities (top row) or unnormalized clone counts (bottom row) are shown.

  • Fig. 5 Identification of convergent Ara h 2–specific IgE CDR-H3 sequences.

    (A) Panning of scFv phage libraries generated from IgE and IgK or IgL transcripts was used to identify Ara h 2–specific clones. (B and C) Mean number of (B) unique reads or (C) isotypes found in Ara h 2–specific or total clones per donor (WMW test: ***P < 0.001). For plotting, one outlier (P105) with 604 unique reads per Ara h 2–specific clone was excluded. (D) Isotypes expressed in Ara h 2–specific clones containing IgE+ members. (E) Comparison of convergent group CDR-H3 sequences from PA, OA, and NA HTS repertoires to scFv phage sequences. Sequence logos were generated from the most abundant CDR-H3 from each participant. The relative heights of the letters within a stack are proportional to their frequencies and are colored according to chemical properties: polar (green), basic (blue), acidic (red), neutral (purple), or hydrophobic (black). (F) cg3 CDR-H3 sequences, colored by BLOSUM62 scores. The experimental source (indicated by symbol shape) and tissue origin (symbol color) of sequences are illustrated. (G) Isotypes expressed by Ara h 2–binding convergent clone group members. (H) Isotype frequencies for cg1 to cg4, collapsed by unique reads.

Supplementary Materials

  • immunology.sciencemag.org/cgi/content/full/5/45/eaay4209/DC1

    Fig. S1. Overall IGH repertoire properties are similar in allergic and nonallergic individuals.

    Fig. S2. Supplementary material for B cell clone counts in tissues.

    Fig. S3. Immunofluorescence microscopy analysis of IgE+CD138+ cells in tissues.

    Fig. S4. Permutation testing for the frequency of co-occurrence of IgE and clonal precursors in tissue sites.

    Fig. S5. Analysis of multi-isotype IgE+ clones in multiple tissue sites.

    Fig. S6. Supplementary material for nearest neighbor analysis.

    Fig. S7. Analysis of Ara h 2–specific scFv heavy chain sequences.

    Table S1. Patient demographics.

    Table S2. Per-person read and clone counts.

    Table S3. ImmunoCAP serum antibody measurements.

    Table S4. Nearest neighbors for allergen-specific IgE clones.

    Table S5. Phage primers and PCR protocols.

  • Supplementary Materials

    The PDF file includes:

    • Fig. S1. Overall IGH repertoire properties are similar in allergic and nonallergic individuals.
    • Fig. S2. Supplementary material for B cell clone counts in tissues.
    • Fig. S3. Immunofluorescence microscopy analysis of IgE+CD138+ cells in tissues.
    • Fig. S4. Permutation testing for the frequency of co-occurrence of IgE and clonal precursors in tissue sites.
    • Fig. S5. Analysis of multi-isotype IgE+ clones in multiple tissue sites.
    • Fig. S6. Supplementary material for nearest neighbor analysis.
    • Fig. S7. Analysis of Ara h 2–specific scFv heavy chain sequences.
    • Legends for tables S1 and S2
    • Table S3. ImmunoCAP serum antibody measurements.
    • Table S4. Nearest neighbors for allergen-specific IgE clones.
    • Table S5. Phage primers and PCR protocols.

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    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Patient demographics.
    • Table S2 (Microsoft Excel format). Per-person read and clone counts.

    Files in this Data Supplement:

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